13

PLAchitosankeratin composites for biomedical applications

Constantin Edi Tanase Iuliana Spiridon

PII S0928-4931(14)00180-5DOI doi 101016jmsec201403054Reference MSC 4544

To appear in Materials Science amp Engineering C

Received date 21 October 2013Revised date 3 March 2014Accepted date 21 March 2014

Please cite this article as Constantin Edi Tanase Iuliana SpiridonPLAchitosankeratin composites for biomedical applications Materials Science ampEngineering C (2014) doi 101016jmsec201403054

This is a PDF file of an unedited manuscript that has been accepted for publicationAs a service to our customers we are providing this early version of the manuscriptThe manuscript will undergo copyediting typesetting and review of the resulting proofbefore it is published in its final form Please note that during the production processerrors may be discovered which could affect the content and all legal disclaimers thatapply to the journal pertain

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PLAchitosankeratin composites for biomedical applications

Constantin Edi Tanase1 Iuliana Spiridon

2

1 Faculty of Medical Bioengineering lsquoGrigore T Poparsquo University of Medicine and Pharmacy 9-

13 Kogalniceanu Street 700454 Iasi Romania

2 ldquoPetru Ponirdquo Institute of Macromolecular Chemistry 41A Grigore Ghica Voda Alley 700487

Iasi Romania

Corresponding author

Constantin Edi Tanase Faculty of Medical Bioengineering ldquoGrigore T Popardquo University of

Medicine and Pharmacy 9-13 Kogalniceanu Street 700454 Iasi Romania

Email etanaselivecom

Abstract

Novel composites based on PLA chitosan and keratin was obtained via blend preparation The

goal of this contribution was to evaluate mechanical and in vitro behaviour of the composites

The results point out composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

Biological assessments using human osteosarcoma cell line on these composites indicate a good

viabilityproliferation outcome Hence preliminary results regarding mechanical behaviour and in

vitro osteoblasts response suggest that these composites might have prospective application in

medical field

Keywords

PLA chitosan keratin biomaterials mechanical properties in vitro studies

1 Introduction

The interest for ldquogreenrdquo materials with medical applications was increased due to both patients

and medical world that search for solutions to their challenges such as the need for substitutes to

replace or repair tissues or organs problems

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The most biodegradable materials comprise synthetic polyesters such as poly (L-lactic acid) and

poly (L-glycolic acid) and natural polymers such as chitosan alginate collagen and fibrin [1]

Also some inorganic materials eg hydroxyapatite or certain glasses have been used to obtain

materials for hard tissue applications [2] The interest for polyesters is due to their hydrolysable

ester bonds and one of the most important constituent of this class is poly lactic acid (PLA)

which is derived fully from renewable resources It has been used in biomedical applications but

its application is somewhat limited by its inherently poor properties such as reduced impact

strength and low thermal stability [3]

Poly (L-lactic acid) has been widely studied for use in biomedical applications such as sutures

scaffolds for tissue engineering orthopaedic devices or drug delivery systems due to its

biocompatibility and bioresorbability [45]

Chitosan is a polysaccharide obtained by the deacetylation of chitin and has many applications

due to its price excellent oxygen barrier properties antimicrobial effects biodegradability

biocompatibility antimicrobial activity and non-toxicity [67] Due to easy of processing method

one may obtain films fibres gels and foams as well as beads of different sizes and morphology

with medical applications [8] One important property is the fact that chitosan interacts with cells

and cellular lysozyme degrades chitosan in vivo [9] In the same time various kinds of chitosan

derivatives have medicine applications eg bone cartilage skin nerve and blood vessel [1011]

Literature data reported a good biocompatibility of PLA [912] That is why it is used in

biomedical applications as internal body components for implants and drug delivery systems

[13] Keratin is the major component of feathers It is a structural protein characterized by high

cystine content and a significant amount of hydroxyl amino acids especially serine [14] Itrsquos

characterized by the presence of a range of noncovalent interactions (electrostatic forces

hydrogen bonds hydrophobic forces) and covalent interactions (disulphide bonds) which are

difficult to be damaged

The recent trends in biodegradable polymers indicate new development strategies and

engineering to achieve polymeric materials with great interest both in the academic and industrial

fields It was found that the incorporation of functional fillers in the PLA matrix could improve

the physical properties as well as the surface characteristics of the matrix that are important for

tissue engineering and artificial bone reconstruction Motivated by our preliminary results [15]

the purpose of this work is to investigate PLA chitosan keratin composites as biomaterials with

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potential applications in medicine by means mechanical and in vitro studies Preliminary results

regarding mechanical behaviour and in vitro osteoblasts response confirmed their potential for

medical applications

2 Materials and methods

21 Preparation of the composite film

Before blend preparation PLA ((type 2002D supplied by NatureWorks) pallets chitosan

(produced by VansonInc with an average molecular weight of 1200 KDa and acetylation degree

of 34) and featherfibres were dried in a vacuum oven for 6 h at 50ordmC Components

compounding was performed at 175 ordmC for 10 min at a rotor speed of 60 rpm using a fully

automated laboratory Brabender station Literature data [16] skow that the chitosan addition did

no influence thermal properties and degree of crystallinity of PLA Specimens for the mechanical

characterization were prepared by compression molding using a Carver press The compression

moulding was carried out at 175 ordmC with a pre-pressing step of 3 min at 50 atm and a pressing

step of 2 min at 150 atm A neat PLA sheet was prepared in the same conditions and acted as a

reference Samples composition and preparation are as follows A111 70PLA and 30

chitosan A121 68 PLA 30 chitosan and 2 keratin A131 66 PLA 30 chitosan and

4 keratin

22 DSC Analysis

Thermal characterization of composites has been performed with a TA Instruments Q20 Dynamic

Scanning Calorimeter All the samples were heated from 25 oC up to 200 oC with 10 ordmCmin

kept for 2 minutes and then cooled down to 25 oC with a cooling rate of 5 ordmCmin An empty

crucible was used as a reference material All measurements were performed under N2

atmosphere The degree of crystallinity of the PLA samples was obtained by dividing the melting

enthalpy of the sample by 937 Jg [17] which is the estimated melting enthalpy of a pure PLA

The crystallinity of the composite materials was estimated as function of PLA fraction in the

composite and the melting enthalpy

23 Characterization of composites

Mechanical tests in terms of tensile and impact strength were performed

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Thus tensile strength measurements were carried out following ISO 527-2000 standard method

using an Instron 5 kN test machine operated at a crosshead speed of 30 mmmin The unnotched

Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus

provided with a hammer of 15J Seven specimens were tested for each material

The Vickers hardness tests were performed with a Shimadzu microhardness tester A constant

load of 4903 N was applied and 12 sec for all composite samples Ten tests have been carried out

for each sample and the average value is given

24 Biological tests

241 Cell culture

MG63 osteoblast-like cells (ATCCreg ndeg CRL-1427trade Rockville MD-USA) were cultured in

Dulbeccorsquos modified Eaglersquos medium with 4500 mg L-1

glucose (DMEM) from Gibco

supplemented with 10 foetal bovine serum (FBS) 2 mM Glutamax I (Life Technologies) and

100 IUmL-1

penicillin 100 μg mL-1

streptomycin Cell cultures were sustained at 37degC under a

humidified atmosphere of 5 CO2 and 95 air The samples were sterilized using gamma

irradiation according with ISO 11137-20 kGy at room temperature [18] and before using them in

cell culture studies the samples were submerge in a diluted ethanol solution (70 vv) for 15

minutes followed by intensive washing in sterile phosphate buffer saline (PBS) solution The

cells were seeded over the sterile samples at a density of 30 times 104 cells per cm

2 in a 48-well plate

(Cellstarreg Greiner Bio-one) up to 7 days Standard 48-well tissue culture plates (polystyrene)

were used as control surface Cells were maintained under standard cell culture conditions (5

CO2 95 humidity and 37degC) The medium was changed every 2ndash3 days Control cultures and

seeded material samples were evaluated at days 1 2 and 3 for cell viabilityproliferation and at

day 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM Leica

SP2)

242 Cytotoxicity assay

CellTiter 96regAqueous One Solution Cell Proliferation (Promega Madison WI) assay was used

to study cell viability The metabolic cell activity (an indirect measure of cytotoxicity) was

measured by the conversion of MTS to formazan which can be photometrically detected MTS

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was mixed with fresh medium at the ratio 110 and added to the cells for 15 h The cells were

placed in a CO2 incubator at 37degC After incubation time supernatants were transferred to a new

microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-

plate reader All experiments were performed in triplicate and were treated and represented by

their mean value and standard deviation parameters

The percentage cell viability was calculated according to the following equation

cell viability = 100 x (AbssampleAbscontrol)

where Abssample is absorbance of cells tested with various formulation and Abscontrol is the

absorbance of reference cells (incubated on the culture media only)

243 Immunofluorescence analysis

The cells were seeded 30 times 104 cells per cm

2 in a 48-well plate After preset time intervals

immunocytochemical staining was performed on whole samples Briefly the samples with cells

were washed with PBS and fixed using a solution of 37 vv paraformaldehyde The fixed cells

were permeabilized with buffered 05 vv Triton X-100 Subsequently the cells were stained

for nuclei with DAPI (04 μg mL-1

) and cytoskeletal organization was revealed by actin labelling

F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated

phalloidin 02 μg mL-1

(Sigma St Louis MO USA) Labelled samples were examined by

CLSM

25 Statistics

Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software

Inc) statistical software package One-way analysis of variance (ANOVA) was employed to

assess the statistical significance of results at a probability of error of 5 () 1 () and 01

() All experiments were repeated at least three times and the results are presented as mean plusmn

standard deviation (SD)

3 Results and discussions

31 DSC results

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The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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The most biodegradable materials comprise synthetic polyesters such as poly (L-lactic acid) and

poly (L-glycolic acid) and natural polymers such as chitosan alginate collagen and fibrin [1]

Also some inorganic materials eg hydroxyapatite or certain glasses have been used to obtain

materials for hard tissue applications [2] The interest for polyesters is due to their hydrolysable

ester bonds and one of the most important constituent of this class is poly lactic acid (PLA)

which is derived fully from renewable resources It has been used in biomedical applications but

its application is somewhat limited by its inherently poor properties such as reduced impact

strength and low thermal stability [3]

Poly (L-lactic acid) has been widely studied for use in biomedical applications such as sutures

scaffolds for tissue engineering orthopaedic devices or drug delivery systems due to its

biocompatibility and bioresorbability [45]

Chitosan is a polysaccharide obtained by the deacetylation of chitin and has many applications

due to its price excellent oxygen barrier properties antimicrobial effects biodegradability

biocompatibility antimicrobial activity and non-toxicity [67] Due to easy of processing method

one may obtain films fibres gels and foams as well as beads of different sizes and morphology

with medical applications [8] One important property is the fact that chitosan interacts with cells

and cellular lysozyme degrades chitosan in vivo [9] In the same time various kinds of chitosan

derivatives have medicine applications eg bone cartilage skin nerve and blood vessel [1011]

Literature data reported a good biocompatibility of PLA [912] That is why it is used in

biomedical applications as internal body components for implants and drug delivery systems

[13] Keratin is the major component of feathers It is a structural protein characterized by high

cystine content and a significant amount of hydroxyl amino acids especially serine [14] Itrsquos

characterized by the presence of a range of noncovalent interactions (electrostatic forces

hydrogen bonds hydrophobic forces) and covalent interactions (disulphide bonds) which are

difficult to be damaged

The recent trends in biodegradable polymers indicate new development strategies and

engineering to achieve polymeric materials with great interest both in the academic and industrial

fields It was found that the incorporation of functional fillers in the PLA matrix could improve

the physical properties as well as the surface characteristics of the matrix that are important for

tissue engineering and artificial bone reconstruction Motivated by our preliminary results [15]

the purpose of this work is to investigate PLA chitosan keratin composites as biomaterials with

ACC

EPTE

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IPT

ACCEPTED MANUSCRIPT

potential applications in medicine by means mechanical and in vitro studies Preliminary results

regarding mechanical behaviour and in vitro osteoblasts response confirmed their potential for

medical applications

2 Materials and methods

21 Preparation of the composite film

Before blend preparation PLA ((type 2002D supplied by NatureWorks) pallets chitosan

(produced by VansonInc with an average molecular weight of 1200 KDa and acetylation degree

of 34) and featherfibres were dried in a vacuum oven for 6 h at 50ordmC Components

compounding was performed at 175 ordmC for 10 min at a rotor speed of 60 rpm using a fully

automated laboratory Brabender station Literature data [16] skow that the chitosan addition did

no influence thermal properties and degree of crystallinity of PLA Specimens for the mechanical

characterization were prepared by compression molding using a Carver press The compression

moulding was carried out at 175 ordmC with a pre-pressing step of 3 min at 50 atm and a pressing

step of 2 min at 150 atm A neat PLA sheet was prepared in the same conditions and acted as a

reference Samples composition and preparation are as follows A111 70PLA and 30

chitosan A121 68 PLA 30 chitosan and 2 keratin A131 66 PLA 30 chitosan and

4 keratin

22 DSC Analysis

Thermal characterization of composites has been performed with a TA Instruments Q20 Dynamic

Scanning Calorimeter All the samples were heated from 25 oC up to 200 oC with 10 ordmCmin

kept for 2 minutes and then cooled down to 25 oC with a cooling rate of 5 ordmCmin An empty

crucible was used as a reference material All measurements were performed under N2

atmosphere The degree of crystallinity of the PLA samples was obtained by dividing the melting

enthalpy of the sample by 937 Jg [17] which is the estimated melting enthalpy of a pure PLA

The crystallinity of the composite materials was estimated as function of PLA fraction in the

composite and the melting enthalpy

23 Characterization of composites

Mechanical tests in terms of tensile and impact strength were performed

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Thus tensile strength measurements were carried out following ISO 527-2000 standard method

using an Instron 5 kN test machine operated at a crosshead speed of 30 mmmin The unnotched

Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus

provided with a hammer of 15J Seven specimens were tested for each material

The Vickers hardness tests were performed with a Shimadzu microhardness tester A constant

load of 4903 N was applied and 12 sec for all composite samples Ten tests have been carried out

for each sample and the average value is given

24 Biological tests

241 Cell culture

MG63 osteoblast-like cells (ATCCreg ndeg CRL-1427trade Rockville MD-USA) were cultured in

Dulbeccorsquos modified Eaglersquos medium with 4500 mg L-1

glucose (DMEM) from Gibco

supplemented with 10 foetal bovine serum (FBS) 2 mM Glutamax I (Life Technologies) and

100 IUmL-1

penicillin 100 μg mL-1

streptomycin Cell cultures were sustained at 37degC under a

humidified atmosphere of 5 CO2 and 95 air The samples were sterilized using gamma

irradiation according with ISO 11137-20 kGy at room temperature [18] and before using them in

cell culture studies the samples were submerge in a diluted ethanol solution (70 vv) for 15

minutes followed by intensive washing in sterile phosphate buffer saline (PBS) solution The

cells were seeded over the sterile samples at a density of 30 times 104 cells per cm

2 in a 48-well plate

(Cellstarreg Greiner Bio-one) up to 7 days Standard 48-well tissue culture plates (polystyrene)

were used as control surface Cells were maintained under standard cell culture conditions (5

CO2 95 humidity and 37degC) The medium was changed every 2ndash3 days Control cultures and

seeded material samples were evaluated at days 1 2 and 3 for cell viabilityproliferation and at

day 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM Leica

SP2)

242 Cytotoxicity assay

CellTiter 96regAqueous One Solution Cell Proliferation (Promega Madison WI) assay was used

to study cell viability The metabolic cell activity (an indirect measure of cytotoxicity) was

measured by the conversion of MTS to formazan which can be photometrically detected MTS

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was mixed with fresh medium at the ratio 110 and added to the cells for 15 h The cells were

placed in a CO2 incubator at 37degC After incubation time supernatants were transferred to a new

microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-

plate reader All experiments were performed in triplicate and were treated and represented by

their mean value and standard deviation parameters

The percentage cell viability was calculated according to the following equation

cell viability = 100 x (AbssampleAbscontrol)

where Abssample is absorbance of cells tested with various formulation and Abscontrol is the

absorbance of reference cells (incubated on the culture media only)

243 Immunofluorescence analysis

The cells were seeded 30 times 104 cells per cm

2 in a 48-well plate After preset time intervals

immunocytochemical staining was performed on whole samples Briefly the samples with cells

were washed with PBS and fixed using a solution of 37 vv paraformaldehyde The fixed cells

were permeabilized with buffered 05 vv Triton X-100 Subsequently the cells were stained

for nuclei with DAPI (04 μg mL-1

) and cytoskeletal organization was revealed by actin labelling

F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated

phalloidin 02 μg mL-1

(Sigma St Louis MO USA) Labelled samples were examined by

CLSM

25 Statistics

Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software

Inc) statistical software package One-way analysis of variance (ANOVA) was employed to

assess the statistical significance of results at a probability of error of 5 () 1 () and 01

() All experiments were repeated at least three times and the results are presented as mean plusmn

standard deviation (SD)

3 Results and discussions

31 DSC results

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The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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potential applications in medicine by means mechanical and in vitro studies Preliminary results

regarding mechanical behaviour and in vitro osteoblasts response confirmed their potential for

medical applications

2 Materials and methods

21 Preparation of the composite film

Before blend preparation PLA ((type 2002D supplied by NatureWorks) pallets chitosan

(produced by VansonInc with an average molecular weight of 1200 KDa and acetylation degree

of 34) and featherfibres were dried in a vacuum oven for 6 h at 50ordmC Components

compounding was performed at 175 ordmC for 10 min at a rotor speed of 60 rpm using a fully

automated laboratory Brabender station Literature data [16] skow that the chitosan addition did

no influence thermal properties and degree of crystallinity of PLA Specimens for the mechanical

characterization were prepared by compression molding using a Carver press The compression

moulding was carried out at 175 ordmC with a pre-pressing step of 3 min at 50 atm and a pressing

step of 2 min at 150 atm A neat PLA sheet was prepared in the same conditions and acted as a

reference Samples composition and preparation are as follows A111 70PLA and 30

chitosan A121 68 PLA 30 chitosan and 2 keratin A131 66 PLA 30 chitosan and

4 keratin

22 DSC Analysis

Thermal characterization of composites has been performed with a TA Instruments Q20 Dynamic

Scanning Calorimeter All the samples were heated from 25 oC up to 200 oC with 10 ordmCmin

kept for 2 minutes and then cooled down to 25 oC with a cooling rate of 5 ordmCmin An empty

crucible was used as a reference material All measurements were performed under N2

atmosphere The degree of crystallinity of the PLA samples was obtained by dividing the melting

enthalpy of the sample by 937 Jg [17] which is the estimated melting enthalpy of a pure PLA

The crystallinity of the composite materials was estimated as function of PLA fraction in the

composite and the melting enthalpy

23 Characterization of composites

Mechanical tests in terms of tensile and impact strength were performed

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Thus tensile strength measurements were carried out following ISO 527-2000 standard method

using an Instron 5 kN test machine operated at a crosshead speed of 30 mmmin The unnotched

Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus

provided with a hammer of 15J Seven specimens were tested for each material

The Vickers hardness tests were performed with a Shimadzu microhardness tester A constant

load of 4903 N was applied and 12 sec for all composite samples Ten tests have been carried out

for each sample and the average value is given

24 Biological tests

241 Cell culture

MG63 osteoblast-like cells (ATCCreg ndeg CRL-1427trade Rockville MD-USA) were cultured in

Dulbeccorsquos modified Eaglersquos medium with 4500 mg L-1

glucose (DMEM) from Gibco

supplemented with 10 foetal bovine serum (FBS) 2 mM Glutamax I (Life Technologies) and

100 IUmL-1

penicillin 100 μg mL-1

streptomycin Cell cultures were sustained at 37degC under a

humidified atmosphere of 5 CO2 and 95 air The samples were sterilized using gamma

irradiation according with ISO 11137-20 kGy at room temperature [18] and before using them in

cell culture studies the samples were submerge in a diluted ethanol solution (70 vv) for 15

minutes followed by intensive washing in sterile phosphate buffer saline (PBS) solution The

cells were seeded over the sterile samples at a density of 30 times 104 cells per cm

2 in a 48-well plate

(Cellstarreg Greiner Bio-one) up to 7 days Standard 48-well tissue culture plates (polystyrene)

were used as control surface Cells were maintained under standard cell culture conditions (5

CO2 95 humidity and 37degC) The medium was changed every 2ndash3 days Control cultures and

seeded material samples were evaluated at days 1 2 and 3 for cell viabilityproliferation and at

day 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM Leica

SP2)

242 Cytotoxicity assay

CellTiter 96regAqueous One Solution Cell Proliferation (Promega Madison WI) assay was used

to study cell viability The metabolic cell activity (an indirect measure of cytotoxicity) was

measured by the conversion of MTS to formazan which can be photometrically detected MTS

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was mixed with fresh medium at the ratio 110 and added to the cells for 15 h The cells were

placed in a CO2 incubator at 37degC After incubation time supernatants were transferred to a new

microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-

plate reader All experiments were performed in triplicate and were treated and represented by

their mean value and standard deviation parameters

The percentage cell viability was calculated according to the following equation

cell viability = 100 x (AbssampleAbscontrol)

where Abssample is absorbance of cells tested with various formulation and Abscontrol is the

absorbance of reference cells (incubated on the culture media only)

243 Immunofluorescence analysis

The cells were seeded 30 times 104 cells per cm

2 in a 48-well plate After preset time intervals

immunocytochemical staining was performed on whole samples Briefly the samples with cells

were washed with PBS and fixed using a solution of 37 vv paraformaldehyde The fixed cells

were permeabilized with buffered 05 vv Triton X-100 Subsequently the cells were stained

for nuclei with DAPI (04 μg mL-1

) and cytoskeletal organization was revealed by actin labelling

F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated

phalloidin 02 μg mL-1

(Sigma St Louis MO USA) Labelled samples were examined by

CLSM

25 Statistics

Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software

Inc) statistical software package One-way analysis of variance (ANOVA) was employed to

assess the statistical significance of results at a probability of error of 5 () 1 () and 01

() All experiments were repeated at least three times and the results are presented as mean plusmn

standard deviation (SD)

3 Results and discussions

31 DSC results

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The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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Thus tensile strength measurements were carried out following ISO 527-2000 standard method

using an Instron 5 kN test machine operated at a crosshead speed of 30 mmmin The unnotched

Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus

provided with a hammer of 15J Seven specimens were tested for each material

The Vickers hardness tests were performed with a Shimadzu microhardness tester A constant

load of 4903 N was applied and 12 sec for all composite samples Ten tests have been carried out

for each sample and the average value is given

24 Biological tests

241 Cell culture

MG63 osteoblast-like cells (ATCCreg ndeg CRL-1427trade Rockville MD-USA) were cultured in

Dulbeccorsquos modified Eaglersquos medium with 4500 mg L-1

glucose (DMEM) from Gibco

supplemented with 10 foetal bovine serum (FBS) 2 mM Glutamax I (Life Technologies) and

100 IUmL-1

penicillin 100 μg mL-1

streptomycin Cell cultures were sustained at 37degC under a

humidified atmosphere of 5 CO2 and 95 air The samples were sterilized using gamma

irradiation according with ISO 11137-20 kGy at room temperature [18] and before using them in

cell culture studies the samples were submerge in a diluted ethanol solution (70 vv) for 15

minutes followed by intensive washing in sterile phosphate buffer saline (PBS) solution The

cells were seeded over the sterile samples at a density of 30 times 104 cells per cm

2 in a 48-well plate

(Cellstarreg Greiner Bio-one) up to 7 days Standard 48-well tissue culture plates (polystyrene)

were used as control surface Cells were maintained under standard cell culture conditions (5

CO2 95 humidity and 37degC) The medium was changed every 2ndash3 days Control cultures and

seeded material samples were evaluated at days 1 2 and 3 for cell viabilityproliferation and at

day 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM Leica

SP2)

242 Cytotoxicity assay

CellTiter 96regAqueous One Solution Cell Proliferation (Promega Madison WI) assay was used

to study cell viability The metabolic cell activity (an indirect measure of cytotoxicity) was

measured by the conversion of MTS to formazan which can be photometrically detected MTS

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was mixed with fresh medium at the ratio 110 and added to the cells for 15 h The cells were

placed in a CO2 incubator at 37degC After incubation time supernatants were transferred to a new

microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-

plate reader All experiments were performed in triplicate and were treated and represented by

their mean value and standard deviation parameters

The percentage cell viability was calculated according to the following equation

cell viability = 100 x (AbssampleAbscontrol)

where Abssample is absorbance of cells tested with various formulation and Abscontrol is the

absorbance of reference cells (incubated on the culture media only)

243 Immunofluorescence analysis

The cells were seeded 30 times 104 cells per cm

2 in a 48-well plate After preset time intervals

immunocytochemical staining was performed on whole samples Briefly the samples with cells

were washed with PBS and fixed using a solution of 37 vv paraformaldehyde The fixed cells

were permeabilized with buffered 05 vv Triton X-100 Subsequently the cells were stained

for nuclei with DAPI (04 μg mL-1

) and cytoskeletal organization was revealed by actin labelling

F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated

phalloidin 02 μg mL-1

(Sigma St Louis MO USA) Labelled samples were examined by

CLSM

25 Statistics

Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software

Inc) statistical software package One-way analysis of variance (ANOVA) was employed to

assess the statistical significance of results at a probability of error of 5 () 1 () and 01

() All experiments were repeated at least three times and the results are presented as mean plusmn

standard deviation (SD)

3 Results and discussions

31 DSC results

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The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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was mixed with fresh medium at the ratio 110 and added to the cells for 15 h The cells were

placed in a CO2 incubator at 37degC After incubation time supernatants were transferred to a new

microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-

plate reader All experiments were performed in triplicate and were treated and represented by

their mean value and standard deviation parameters

The percentage cell viability was calculated according to the following equation

cell viability = 100 x (AbssampleAbscontrol)

where Abssample is absorbance of cells tested with various formulation and Abscontrol is the

absorbance of reference cells (incubated on the culture media only)

243 Immunofluorescence analysis

The cells were seeded 30 times 104 cells per cm

2 in a 48-well plate After preset time intervals

immunocytochemical staining was performed on whole samples Briefly the samples with cells

were washed with PBS and fixed using a solution of 37 vv paraformaldehyde The fixed cells

were permeabilized with buffered 05 vv Triton X-100 Subsequently the cells were stained

for nuclei with DAPI (04 μg mL-1

) and cytoskeletal organization was revealed by actin labelling

F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated

phalloidin 02 μg mL-1

(Sigma St Louis MO USA) Labelled samples were examined by

CLSM

25 Statistics

Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software

Inc) statistical software package One-way analysis of variance (ANOVA) was employed to

assess the statistical significance of results at a probability of error of 5 () 1 () and 01

() All experiments were repeated at least three times and the results are presented as mean plusmn

standard deviation (SD)

3 Results and discussions

31 DSC results

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The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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ACCEPTED MANUSCRIPT

where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

ACC

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ACCEPTED MANUSCRIPT

The thermal behaviour of the studied materials is presented in Figure 1 and Table 1

Pure chitosan does not have melting properties and hence no endothermic peaks associated to

melting process were detected as other authors reported [19] The thermogram of neat PLA

revealed a glass transition temperature at 594 degC followed by a cold crystallization process

peak at 1278 degC and then melting peak at 15297degC with an enthalpy of fusion of 1362

Jmol It was found that the addition of chitosan determined an increase of Tg to 602oC

which can be explained by chitosan hindering movements of PLA chains

The presence of chitosan which is a semicrystalline polymer determines a loss of

crystallinity in the PLA matrix which drops from 2061 to 1805 The presence of

keratin in PLA chitosan system decreased Tg to 58 9oC while crystallinity increased to

1917 Apparently the keratin decreases the Tg of PLA in PLAchitosan system and

facilitates crystallization of PLA Probably crosslinking structures from keratin interfere with

the crystallization process many imperfect crystallites are formed and thus keratin serves as

nucleating agents

Insert Figure 1

Insert Table 1

32 Mechanical properties

Mechanical properties are important to the design biomaterials for medical applications Chitosan

incorporation into the PLA matrix improved Young modulus and decreased the tensile strength

of PLA (Figure 1) Polar interactions between ester functional groups of PLA and amine groups

of chitosan were expected but it is possible that processing conditions applied in our study may

not have been sufficient for a reaction to occur between the chitosan and PLA with significant

effects on the mechanical properties The addition of keratin resulted in an increase of impact

strength and decrease of tensile properties compared to PLAchitosan composite The feather

fibers appear to act as stress concentrators in the polymer matrix thus reducing the crack

initiation energy and consequently enhancing the impact strength of the composites The structure

of keratin the primary constituent of chicken feathers affects the chemical durability Because of

extensive cross-linking and strong covalent bonding within its structure keratin provides high

ACC

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ACCEPTED MANUSCRIPT

resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

ACC

EPTE

D M

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SCR

IPT

ACCEPTED MANUSCRIPT

where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

ACC

EPTE

D M

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IPT

ACCEPTED MANUSCRIPT

of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

ACC

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IPT

ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

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17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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resistance to degradation [20]The possible increase of the toughness may be due to microplastic

deformation created around the feather particles

Insert Figure 2

It is well-known that hardness and modulus of a polymeric material depend on its structure As

can see in Figure 2 a significant rise in hardness has been obtained by adding of chitosan to PLA

matrix This suggests that the fibre surface becomes rougher According with hardness values the

Young modulus of PLA chitosan composite is higher than that of PLA matrix The incorporation

of chitosan with or without keratin stabilized PLA that became consequently less brittle The

rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as

the content of keratin increases A131 material reflecting a solid-like behavior [21] Keratin fibers

serve as nucleation sites and probably polymer chains flexibility The polymer volume

immediately surrounding the keratin fibers has properties different from the bulk polymer [22]

During hardness test the stress transferability inside the biocomposite may be reduced due to the

distance between the fibers decreasesie work done will be gone to the deformation of matrix

rather than fibers

Insert Figure 3

33 Water uptake

The diffusion is a process by which small molecules are transported from one side of a system to

another one as a result of random molecular motions The water sorption is a complex

mechanism which is influenced by swelling molecular interactions accessibility of sorption sites

and material crystallinity and crosslinking as well as of the presence of fillers Using the above

boundary conditions Fickrsquos law has been used to give the time dependence of concentration as

shown below

2

22)12(

2212

181 l

tnD

t enM

M

(1)

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where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

ACC

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ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

ACC

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

where t is the time measured from when the concentration is changed l is the plate thickness Mo

is the initial equilibrium mass and M is the change in the mass from Mo to the new equilibrium

mass Mt The equation (1) describes the change in mass due to the movement of the diffusing

species responding to the sudden change in pressurehumidity around the sample The half

thickness (l2) is used as the diffusion length above since in a typical sorption experiment the

entire plate is measured and the substance is assumed to diffuse uniformly to the central plane

For MtMinfin lt05

tD

lM

M t

4 (2)

and for MtMinfin gt05 2

2

2

81 l

tD

t eM

M

(3)

Insert Table 2

Literature data reported that the PLA degradation occurs principally at the surface because of the

absorption gradient of water[23] the evolution of this process being explained either via a bulk

erosion mechanism starting at the surface [24] or as a heterogeneous process[25] It is well

known that the presence of chitosan influences water uptake and diffusion coefficients [26]

The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a

new pathway for the water vapour to diffuse Thus in material comprising PLA and chitosan

both D2 and K2 ( MtMinfingt05) were less affected while D1 and K1 were slowly affected

Addition of 4 keratin has improved the K1 value as well as D1 (for for MtMinfinlt05) while for

D2 and K2(for MtMinfingt05)slowly decreased

Our results suggest that water uptake process is more intense at the surface of material We

suppose that water molecule penetrates with difficulty the core of material due to the strong

linkages between components of the studied materials confirmed by rheological studies[27]

Literature data show that PLA has advantage to be processing in shapes for orthopaedic

applications and fabricated into scaffolds for replacement and regeneration of tissues or devices

for controlled delivery of biomolecules [2829] Also it must be mentioned that in vivo the

performance of the biomaterial-based structure is not only derived from the mechanical resistance

ACC

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IPT

ACCEPTED MANUSCRIPT

of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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of the biomaterial itself but from the complex mixture of biomaterial [30] surface properties

being important to induce cellular attachment differentiation and proliferation [31]

34 Behaviour of MG63 osteoblast-like cells

The MTS tetrazolium compound is reduced by cells into a colored formazan product that is

soluble in tissue culture medium The quantity of the formazan product as measured at 492 nm is

directly proportional to the number of living cells in a culture The results of viability assay are

represented in Figure 3

Insert Figure 4

As it can be seen the cell viability percent is comparable with the control (cell viability 100) at

24h respectively 48h After the second day when the cultures reached confluence and formed a

dense cell layer this layer was easily lost during routine medium change This phenomenon

explains the decrease in MTS reduction values observed from at day 3 Even in this case the cell

viabilityproliferation reveals values appropriated to the control From these results we can

conclude that the cell viabilityproliferation of the MG63 osteoblast-like cells seeded on the

samples containing PLA-chitosan-keratin is similar to the control with a slightly variation which

might be influenced by the material composition Therefore we can conclude that PLA-chitosan-

keratin composites do not affect cells viability indicate the potential use in bone tissue

engineering The ratio of components in composite materials used in this study was appropriate

to support the MG63 growth and offer proper space and support for cell proliferation

On the other hand to evaluate the proliferation distribution and cell adhesion of the cells was

necessary to use CLSM The CLSM images of the samples seeded with MG63 cells can be

visualized in Figure 4 at various time points 72h and respectively 168h The cytoskeletal

organization is generally determined by actin staining with fluorescence labeled-phalloidin and is

used to evaluate the motility spreading and shape of the cells [32] Regarding the cells

distribution and proliferation after 72h on the samples it can be observed that for samples A111

(Figure 4 A) and for samples A131 (Figure 4 C) the MG63 cells cover almost the entire sample

surface For the same time point (72h) regarding sample A121 (Figure 4 B) it can be observed

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that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

ACC

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References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

ACC

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D M

ANU

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IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

that MG63 cells presents a good distribution but not similar with the other samples At this time

of culture CLSM shows a suggestive proliferative cell population (figure 4-B- circled cells)

These data are correlated with the results obtained in MTS assays At 168h after culture the cells

formed multilayerrsquos of flattened sheets covering completely the material surface for all samples

(D-sample A111-figure 4 E-sample A121-figure 4 and F-sample A131-figure 4) From Figure 4

it can be seen that the cells have a wide connection with each other throughout cytoplasmic

extensions presenting an elongated shape thereby emphasizing the distribution and proliferation

of cells at different time point The physicalndashchemical and topographical features of the

biomaterial surface could influence the distribution of focal contact and cytoskeleton organization

[3331] PLA-chitosan-keratin samples showed increased cell attachment and maintenance of cell

numbers over the time periods (up to 168h)

As a final point the MTS viabilityproliferation assays and CLSM analysis highlights a good cell

viability proliferation and distribution pointing out a good biocompatibility of the PLA-chitosan-

keratin samples in contact with MG63 osteoblast-like cells Hence these composite materials

reveal estimable biocompatibility features demonstrating their potential use as substitute

materials for bone tissue engineering

This preliminary investigation shows that the composition of PLAchitosankeratin materials is

important to obtain materials with appropriate properties and careful tailoring of the keratin

should modify mechanical properties of material and cellular adhesion In the future follow up

study more investigations will be done to further enhance the efficiency of cell bone formation in

PLAchitosankeratin materials

Insert Figure 5

4 Conclusions

In summary new biomaterials based on PLA chitosan and keratin composites were designed and

evaluated These composites with improved Young modulus and decreased tensile strength

significant increase in hardness (compared to PLA) and a good uptake of the surface properties

were evaluated with regards of in vitro behaviour

This study showed that PLA chitosan and keratin composites support osteoblasts attachment and

proliferation during short-term culture indicating that these composites might be promising

materials for medical application

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

References

1R Langer D A Tirrell Designing materials for biology and medicine Nature 428(6982)

(2004) 487ndash492 2AR Boccaccini J J Blaker Bioactive composite materials for tissue engineering scaffolds

Expert Rev Med Devices 2(3) (2005) 303ndash317 3R Bhardwaj A K Mohanty Advances in the properties of polylactides based materials a

review J Biobased Mater Bioenergy 1(2) (2007) 191ndash209 4Saiz-Arroyo C Wang Y Rodriguez-Perez M A Alves N M and Mano J F In vitro

monitoring of surface mechanical properties of poly(L-lactic acid) using microhardness J Appl

Polym Sci 105 (2007) 3858ndash3864 5Qizhi Chen Chenghao Zhu and George A Thouas Progress and challenges in biomaterials used

for bone tissue engineering bioactive glasses and elastomeric composites Progress in

Biomaterials 12 (2012) 1-22 6R Jayakumar M Prabaharan P T Sudheesh Kumar S V Nair and H Tamura Biomaterials

based on chitin and chitosan in wound dressing applications Biotechnology Advances 29

(2011) 322ndash337 7In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications

Biotechnology Advances 26 (2008) 1ndash21 8Scott P Noel Harry Courtney Joel D Bumgardner Warren O Haggard Chitosan Films A

Potential Local Drug Delivery System for Antibiotics Clin Orthop Relat Res 466 (2008) 1377ndash

1382 9Tomihata K Ikada Y In vitro and in vivo degradation of films of chitin and its deacetylated

derivatives Biomaterials 18 (1997) 567ndash575

10

In-Yong Kim Seog-Jin Seo Hyun-Seuk Moon Mi-Kyong Yoo In-Young Park Bom-Chol

Kim Chong-Su Cho Chitosan and its derivatives for tissue engineering applications Biotechnol

Adv 261(2008) 1-21 11

Tanase CE Popa MI Verestiuc L Biomimetic chitosan-calcium phosphate composites

with potential applications as bone substitutes Preparation and characterization Journal of

Biomedical Materials Research - Part B Applied Biomaterials 100 B (3) (2012) 700-708 12

Rokkanen PU Bostman O Hirvensalo E Makela EA Partio EK Patiala H Vainionpaa SI

Vihtonen K Tormala P Bioabsorbable fixation in orthopaedic surgery and traumatology

Biomaterials 21 (2000) 2607ndash2613 13

Mills CA Navarro M Engel E Martinez E Ginebra MP Planell J Errachid A Samitier J

Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications Journal of

Biomedical Materials Research - Part A 76 (2006) 781ndash787 14

Arai KM Takahashi R Yokote Y Akahane K Amino acid sequence of feather keratin from

fowl J Biochem 132 (1983) 501ndash507 15

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 16

J Bonilla E Fortunati M Vargas A Chiralt JM Kenny Effects of chitosan on the

physicochemical and antimicrobial properties of PLA films Journal of Food Engineering 119

236ndash243(2013)

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

17

[Liao RG Yang B Yu W Zhou CX Isothermal cold crystallization kinetics of polylactidenucleating agents J

Appl Polym Sci 2007104(1)310ndash317 18

M Bernkopf Sterilisation of Bioresorbable Polymer Implants Medical Device Technology vol 18 (3) May-

June 2007 19

C Swapna Joseph K V Harish Prashanth N K Rastogi A R Indiramma S Yella Reddy and K S M S

Raghavarao Optimum Blend of Chitosan and Poly-(ε-caprolactone) for Fabrication of Films for Food Packaging

Applications Food Bioprocess Technol 2011 41179ndash1185 20

Mishra S C Nayak N B Satapathy A Investigation on Biowaste Reinforced Epoxy

Composites J Reinf Plast Compos 2010 29 (19) 3016 21

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 22

J R Barone Polyethylenekeratin fiber composites with varying polyethylene crystallinity

Composites Part A 36 (2005) 1518ndash1524 23

C Saiz-ArroyoY WangM A Rodriguez-PerezN M AlvesJ F Mano In Vitro Monitoring

of Surface Mechanical Properties of Poly(L-Lactic Acid) Using Microhardness Journal of

Applied Polymer Science Vol 105 3858ndash3864 (2007) 24

Tsuji H Mizuno A Ikada Y J Appl Polym Sci 2000 771452 25

Li S M Garreau H Vert M J Mater Sci Mater Med 1990 1 131 26

Vitor M Correlo Elisabete D Pinho Iva Pashkuleva Mrinal BhattacharyaNuno M Neves

Rui L Reis Water Absorption and Degradation Characteristics of Chitosan-Based Polyesters and

Hydroxyapatite Composites Macromol Biosci 2007 7 354ndash363 27

Iuliana Spiridon Oana Maria Paduraru Mirela Fernanda Zaltariov and Raluca Nicoleta Darie

Influence of keratin on Polylactic AcidChitosan composite properties Behavior upon accelerated

weathering Ind Eng Chem Res 52 (29) (2013) 9822ndash9833 28

JM Anderson MS Shive Biodegradation and biocompatibility of PLA and PLGA

microspheres Adv Drug Deliv Rev 28 (1997) 5ndash24 29

M Denti P Randelli D Lo Vetere M Moioli M Tagliabue Bioabsorbable interference

screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction clinical and

computerized tomography results of four different models A prospective study J Orthop

Traumatol 5 (2004)151ndash155 30

Y Luo G Engelmayr DT Auguste LS Ferreira JM Karp R Saigal R Langer Three-

Dimensional Scaffolds R Lanza R Langer JP Vacanti (Eds) Principles of Tissue

Engineering (3rd ed) Elsevier Amsterdam (2007) 359ndash373 31

Anselme K Osteoblast adhesion on biomaterials Biomaterials 21(7) (2000) 667-681 32

DE Ingber Tensegrity II How structural networks influence cellular information processing

networks J Cell Sci 116 (2003) 1397ndash1408 33

K Anselme M Bigerelle B Noel E Dufresne D Judas A Iost P Hardouin Qualitative and

quantitative study of human osteoblast adhesion on materials with various surface roughnesses J

Biomed Mater Res 49 (2000) 155ndash166

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Figure captions

Figure 1 DSC thermograms (second run) of the studied materials

Figure 2 Mechanical properties of composite materials Data are presented as the mean plusmn SD

n=7 p lt 0001 compared with PLA (PLA served as control)

Figure 3 Hardness as function of materials composition

Figure 4 Mitochondrial activity measured via the MTS assay Data are depicted as percentage of

the untreated control Triplicates were performed and the data represent means plusmn SD p lt 005

p lt 001 and p lt 0001 compared to the untreated control

Figure 5 CLSM images (DAPI and TRITC) at 72h (A- samples A111 B- samples A121 and C-

samples A131) respectively 168h (D- samples A111 E- samples A121 and F- samples A131)

after MG63 cells cultured on PLA-chitosan-keratin samples

Table 1 DSC parameters of PLA and composite materials

Table 2 Diffusion coefficient values calculated form normalized mass changing vs time

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Table 1 DSC parameters of PLA and composite materials

Sample Tg (oC)

Tm (oC)

ΔHm (Jg)

X ()

PLA 594 15297 1932 2061

A111 602 15345 2417 1805

A123 589 15067 2698 1917

Tm melting temperature ΔHm melting enthalpy X crystallinity index Tg transition temperature

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Table 2 Diffusion coefficient values calculated form normalized mass changing vs time

K1

lt05

K2

gt05

l

cm

D1

MtMinfinlt05

Eq (2)

cm2s

D2

MtMinfingt05

Eq (3)

cm2s

A131 129E-03 -000075520 0098 24321E-06 73515E-07

A121 724E-04 -000091237 0101 14508E-06 74336E-07

A111 984E-04 -000081704 0093 16704E-06 71626E-07

PLA 714E-04 -000088931 0089 11095E-06 71400E-07

K1 K2 - is slope of linearized equations (2) and respective (3)

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Table 1 DSC parameters of PLA and composite materials

Sample Tg (oC)

Tm (oC)

ΔHm (Jg)

X ()

PLA 594 15297 1932 2061

A111 602 15345 2417 1805

A123 589 15067 2698 1917

Tm melting temperature ΔHm melting enthalpy X crystallinity index Tg transition temperature

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Table 2 Diffusion coefficient values calculated form normalized mass changing vs time

K1

lt05

K2

gt05

l

cm

D1

MtMinfinlt05

Eq (2)

cm2s

D2

MtMinfingt05

Eq (3)

cm2s

A131 129E-03 -000075520 0098 24321E-06 73515E-07

A121 724E-04 -000091237 0101 14508E-06 74336E-07

A111 984E-04 -000081704 0093 16704E-06 71626E-07

PLA 714E-04 -000088931 0089 11095E-06 71400E-07

K1 K2 - is slope of linearized equations (2) and respective (3)

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Table 2 Diffusion coefficient values calculated form normalized mass changing vs time

K1

lt05

K2

gt05

l

cm

D1

MtMinfinlt05

Eq (2)

cm2s

D2

MtMinfingt05

Eq (3)

cm2s

A131 129E-03 -000075520 0098 24321E-06 73515E-07

A121 724E-04 -000091237 0101 14508E-06 74336E-07

A111 984E-04 -000081704 0093 16704E-06 71626E-07

PLA 714E-04 -000088931 0089 11095E-06 71400E-07

K1 K2 - is slope of linearized equations (2) and respective (3)

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Figure 3

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Figure 4

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Figure 5

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour

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Highlights

PLA chitosan and keratin composites are prepared by blend preparation

PLA chitosan and keratin composites present improved mechanical properties and water uptake

compare to PLA

PLA chitosan and keratin composites present good in vitro behaviour