Preparation and characterization of collagen/PLA, chitosan/PLA,and collagen/chitosan/PLA hybrid scaffolds for cartilage tissueengineering

Anne-Marie Haaparanta • Elina Jarvinen •

Ibrahim Fatih Cengiz • Ville Ella • Harri T. Kokkonen •

Ilkka Kiviranta • Minna Kellomaki

Received: 11 September 2013 / Accepted: 15 December 2013 / Published online: 28 December 2013

� Springer Science+Business Media New York 2013

Abstract In this study, three-dimensional (3D) porous

scaffolds were developed for the repair of articular carti-

lage defects. Novel collagen/polylactide (PLA), chitosan/

PLA, and collagen/chitosan/PLA hybrid scaffolds were

fabricated by combining freeze-dried natural components

and synthetic PLA mesh, where the 3D PLA mesh gives

mechanical strength, and the natural polymers, collagen

and/or chitosan, mimic the natural cartilage tissue envi-

ronment of chondrocytes. In total, eight scaffold types were

studied: four hybrid structures containing collagen and/or

chitosan with PLA, and four parallel plain scaffolds with

only collagen and/or chitosan. The potential of these types

of scaffolds for cartilage tissue engineering applications

were determined by the analysis of the microstructure,

water uptake, mechanical strength, and the viability and

attachment of adult bovine chondrocytes to the scaffolds.

The manufacturing method used was found to be applica-

ble for the manufacturing of hybrid scaffolds with highly

porous 3D structures. All the hybrid scaffolds showed a

highly porous structure with open pores throughout the

scaffold. Collagen was found to bind water inside the

structure in all collagen-containing scaffolds better than the

chitosan-containing scaffolds, and the plain collagen scaf-

folds had the highest water absorption. The stiffness of the

scaffold was improved by the hybrid structure compared to

plain scaffolds. The cell viability and attachment was good

in all scaffolds, however, the collagen hybrid scaffolds

showed the best penetration of cells into the scaffold. Our

results show that from the studied scaffolds the collagen/

PLA hybrids are the most promising scaffolds from this

group for cartilage tissue engineering.

1 Introduction

Articular cartilage injuries are common, but the treatment

of such injuries remains challenging due to the lack of

spontaneous tissue regeneration and the nature of the tis-

sue. Articular cartilage tissue is avascular and there is

relatively low cell density and low mitotic activity of

chondrocytes [1]. There are various treatment options

depending on the injury, but with current repair techniques

it is not possible to repair large lesions [1–5]. After treat-

ment, lesions often form fibrous tissue that has poor quality

and durability [6–8].

Autologous chondrocyte implantation (ACI) is a prom-

ising method for the repair of cartilage defects [9]. The

major limitation, however, is the lack of appropriate bio-

material scaffolds that can be used in combination with

ACI. The main focus thus far has been on developing three-

dimensional (3D) scaffolds that have a highly porous

structure and an interconnected pore network that supports

chondrocyte proliferation and cartilage matrix production

[10, 11]. Sufficient mechanical strength is also required to

A.-M. Haaparanta (&) � I. F. Cengiz � V. Ella � M. Kellomaki

Department of Electronics and Communications Engineering,

Tampere University of Technology, Korkeakoulunkatu 3,

33720 Tampere, Finland

e-mail: anne-marie.haaparanta@tut.fi

A.-M. Haaparanta � V. Ella � M. Kellomaki

BioMediTech, Institute of Biosciences and Medical Technology,

Tampere, Finland

E. Jarvinen � I. Kiviranta

Department of Orthopaedics and Traumatology, University of

Helsinki and Helsinki University Central Hospital,

Haartmaninkatu 8, 00290 Helsinki, Finland

H. T. Kokkonen

Department of Applied Physics, University of Eastern Finland,

Yliopistonranta 1F, 70211 Kuopio, Finland

123

J Mater Sci: Mater Med (2014) 25:1129–1136

DOI 10.1007/s10856-013-5129-5

withstand the high loads to which the forming repair tissue

is subjected.

The native extracellular matrix (ECM) of articular carti-

lage is a complex structure composed of a collagen fiber

network filled with proteoglycans. Therefore, a structural

protein such as collagen is a potential scaffold material for

application in cartilage tissue engineering [12]. Chitosan, a

widespread polysaccharide, is a natural polymer derived

from chitin that possesses a similar structure to the naturally

present glycosaminoglycans found in articular cartilage [13,

14]. However, collagen and chitosan alone cannot be used in

load bearing applications because of their low mechanical

strength. As a result, composite or hybrid structures con-

taining a synthetic polymer, such as polylactide (PLA) or

poly(lactide-co-glycolide) (PLGA) with good mechanical

properties have been developed [15]. Many of the hybrid

scaffolds developed have a two-dimensional synthetic fiber

component [16, 17], or a synthetic component that is formed

into a 3D structure using a non-fibrous method, for example

salt leaching [18, 19]. The biomechanical properties of

articular cartilage rely on the ability to restore its shape and

regain lost liquids into the structure after loading, which is an

essential feature for a scaffold [1, 10]. Therefore, a hybrid

scaffold comprising a highly hydrophilic natural component

together with a fibrous synthetic component with higher

strength could present an optimal scaffold structure for

cartilage tissue engineering.

The aim of this study was to manufacture novel colla-

gen/PLA, chitosan/PLA, and collagen/chitosan/PLA hybrid

scaffolds for use in cartilage tissue engineering. The hybrid

scaffolds were consequently compared with plain freeze-

dried collagen or chitosan, or collagen–chitosan blend

scaffolds. The manufactured scaffolds were studied by

determining the microstructure (SEM, microCT), water

uptake abilities, mechanical properties, and cell viability

and attachment with adult bovine chondrocytes in order to

ascertain the suitability of these scaffold types for cartilage

tissue engineering applications.

2 Materials and methods

2.1 Scaffold fabrication

Eight scaffold types (Table 1) were manufactured as

described below.

2.1.1 Fabrication of plain and blend solutions

Type I bovine dermal collagen (PureCol�, Nutacon B.V.,

Leimuiden, the Netherlands) fibril formation was carried

out as described earlier [20]. Briefly, the collagen–HCl

solution was mixed with fibrillogenesis buffer at a ratio of

1:10. The pH of the solution was adjusted to 7.20 and the

solution was incubated at room temperature (RT) over-

night. The initial collagen concentration of the collagen

solution was 0.3 wt% and the final concentrations of 0.5 or

1.0 wt% were achieved by centrifuging and then concen-

trating the solution.

Medical grade chitosan (Protasan UP B 90/500, FMC

Biopolymer d/b/a NovaMatrix, Sandvika, Norway) with a

deacetylation degree of 91 % and a molecular weight of

460,000 g/mol was dissolved in an acetic acid solution at a

ratio of 1:1 (w/v), as concentrations of 0.5 or 1.0 wt%.

For collagen–chitosan blends, the initial collagen and

chitosan solutions were gently mixed together in ratios of

1:1 (v/v) or 2:1 (v/v), respectively.

2.1.2 Fabrication of PLA mesh

For hybrids, a PLA 96/4 mesh was manufactured. Medical

grade polymer poly(L/D)lactide 96/4 with an intrinsic vis-

cosity of 2.18 dl/g (Purac Biochem, Gorinchem, The

Netherlands) was used for fiber manufacture. The polymer

was melt-spun into multi-filament fibers (16-ply, average

diameter of single fiber *20 lm), using a Gimac mic-

roextruder (Gimac, Gastronno, Italy) with a screw diameter

12 mm. The fibers were online oriented using godets. The

fibers were cut to staple fibers, at a length of *10 cm, and

carded into mesh. The mesh was then cut with a puncher to

produce samples with a radius of 8 mm.

2.1.3 Fabrication of different scaffold types

The manufactured solutions were then loaded into custom-

made Teflon sample moulds (diameter 8 mm, height 4 mm).

For the plain collagen, chitosan, or collagen–chitosan blend

scaffolds, the 1.0 wt% solutions were used. For the hybrids,

the 0.5 wt% collagen, chitosan, or collagen–chitosan blend

solution was used and the mesh was loaded at the bottom and

at the top of the moulds. The samples were then frozen for

24 h at -30 �C prior to freeze-drying for 24 h. All the

samples were held under vacuum at RT for a minimum of

48 h before cross-linking of the collagen containing samples

or the neutralization of chitosan samples.

Table 1 Manufactured scaffold types and compositions

Scaffolds Hybrid scaffolds

Col Collagen ColH Collagen ? PLA mesh

Chi Chitosan ChiH Chitosan ? PLA mesh

C1C1 Collagen–

chitosan 1:1

C1C1H Collagen–chitosan

1:1 ? PLA mesh

C2C1 Collagen–

chitosan 2:1

C2C1H Collagen–chitosan

2:1 ? PLA mesh

1130 J Mater Sci: Mater Med (2014) 25:1129–1136

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The scaffolds containing collagen were cross-linked with

95 % ethanol solution with 14 mM EDC (N-(3-dimethyl-

aminopropyl)-N0-ethylcarbodiimide hydrochloride, Sigma-

Aldrich, Helsinki, Finland) and 6 mM NHS (N-Hydroxy-

succinimide, Sigma-Aldrich, Helsinki, Finland) for 4 h at

RT. The chitosan scaffolds were neutralized with 99.5 and

70 % ethanol steps for 30 min each. All the samples were

washed afterwards with deioniced water, placed into the

sample moulds, and re-freeze-dried as described earlier.

2.1.4 Scaffold microstructure analysis

Scanning electron microscope (SEM) observation was done

for the scaffolds at d0 and for the cell-cultured samples at d7.

Samples containing cultured chondrocytes were fixed at d7,

critical point dried by Bal-Tec CPD 030 (Bal-Tec Union Ltd.,

Liechtenstein), platinum coated (Quorum Q150TS, Quorum

Technologies, UK) and imaged with a scanning electron

microscope (SEM, FEI Quanta 250 Field Emission Gun) at a

magnification of 5009. At d0, the scaffolds were platinum

coated and imaged with SEM at the Electron Microscope

Unit, Institute of Biotechnology, Helsinki, Finland.

MicroCT analysis of the scaffolds was carried out with a

MicroCT scanner (SkyScan 1172, SkyScan, Kontich, Bel-

gium). The tube voltage and voxel size were 40 kV and

30.2 9 30.2 9 30.2 lm3, respectively. The image was

averaged 50 times during scanning and no filters were used.

2.1.5 Water uptake

The water uptake was measured by immersing the scaf-

folds (n = 6) into 5 ml phosphate buffered saline (PBS) for

24 h at 37 �C.

First, the ability of the scaffold structure as a whole (the

material itself with the pore system) to bind water was

measured by removing the scaffold from the PBS, shaken

gently, and weighed without dripping. Next, the ability of

the scaffold material itself (no excess water inside the pore

system) to bind water was measured after drying the

scaffolds between filter papers to remove the water from

the porous structure of the scaffold [21].

The percentage of water uptake was calculated by using

the equation:

Water uptake %ð Þ ¼ ½ðWw �WdÞ=Wd� � 100%;

where Ww is the wet weight and Wd is the dry weight of the

scaffold.

2.1.6 Mechanical tests

The compression tests were done for both dry and wet scaffolds

by using a Lloyd LR30K mechanical tester (Lloyd Instruments

Ltd, Hampshire, UK). Before testing, the wet scaffolds were

immersed in PBS for 24 h at 37 �C. Each sample was com-

pressed at a rate of 0.5 mm/min and a cell load of 1 kN. The

corresponding Young’s modulus was determined from the

linear elastic region (from 7 to 9 % strain). The stiffness values

of each scaffold group were also determined (n = 6).

2.1.7 Isolation of bovine primary articular cartilage

chondrocytes, seeding, and culture of chondrocytes

in the scaffolds

Adult bovine primary chondrocytes were isolated from the

femoral condyle of the knee of 5–6 month-old male cows (Bos

taurus), as described previously [22]. One million chondro-

cytes were seeded into the 0.5 cm2 scaffold by pipetting. First,

50 ll of cell culture medium containing 500,000 cells were

pipetted on top of the scaffold. After 5 min of incubation at RT,

the scaffolds were turned around and another 50 ll of cell

culture medium containing 500,000 cells were pipetted on the

other side of the scaffold. The scaffolds were cultured in the

common proliferation medium DMEM/F12 (21331-020 Gib-

co, Invitrogen, USA) that contains 1 % L-glutamin (25030-

024, Gibco, Invitrogen, USA), 1 % Penicillin/Streptomycin

(15070-063, Gibco, Invitrogen, USA), 1 % Fungizone (15290-

026, Gibco, Invitrogen, USA), 10 % FBS (CH30160.03, Hy-

clone, Biofellows, Finland), and 50 lg/ml ascorbic acid

(A4034, Sigma, USA) at 37 �C in 5 % CO2 for up to one week.

2.1.8 Immunocytochemistry

The chondrocytes were fixed with 4 % paraformaldehyde

(PFA) for 10 min at RT and washed with PBS. Blocking was

done in 1 % BSA ? 0.1 % TritonX in PBS for 1 h at RT.

Cells were incubated in the primary antibody overnight at

?4 �C in blocking buffer. The primary antibodies used were

Collagen type II 1:200 (ab34712, Abcam, UK) and Collagen

type II 1:200 (ab34712, Abcam, UK). A secondary antibody,

alexa-fluor anti-rabbit 488 (A11008, Invitrogen, USA), was

diluted in blocking buffer (1:200) and incubated at RT for 1 h.

Cells were counterstained in 2 lg/ml Hoechst 33342 (H3570,

Invitrogen, USA) for 10 min at RT. Cells were imaged at the

Light Microscope Unit, Institute of Biotechnology, Helsinki

with a Leica TCS SP5II HCS A confocal microscope using

109 or 209 air objectives. For the imaging of the cross-

section, the scaffolds were cut in half with a scalpel and the

cross-section was imaged at an approximate depth of 150 lm.

3 Results

3.1 Scaffold morphology and porosity

Figure 1 shows the highly porous structure of the manu-

factured freeze-dried scaffolds. As the microCT images

show, all the scaffolds had interconnected pores and a

J Mater Sci: Mater Med (2014) 25:1129–1136 1131

123

relatively homogenous matrix structure (collagen, chitosan,

or collagen–chitosan blend). The matrix component in the

hybrids also shows highly similar porous structure, as

observed in the plain scaffolds (Fig. 1). The SEM images

supported the microCT results and the high interlocking

between the PLA fibers and the freeze-dried collagen/

chitosan can be seen in the hybrids in Fig. 2.

All the samples had high porosity varying from 66.2 to

92.8 % (Table 2) detected from the microCT studies. C1C1

showed only 66.2 % porosity and all the other scaffolds had

porosity higher than 85 %. The highest porosity value was in the

ColH (92.8 %). The open porosity values of the scaffolds varied

from 66.2 to 92.8 %. In all scaffolds, the pores were highly

interconnected as the open porosity values of different scaffolds

varied maximally only 0.05 % from the total porosity values.

3.2 Water uptake

In general, water intake was the highest for collagen

component in the scaffolds (Fig. 3). Therefore, the higher

amount of collagen increased the water uptake of the

blends. For chitosan, on the other hand, the water intake

was much lower and the scaffolds with chitosan suffered

from shrinking when wet. The hybrids followed the same

trend as the plain scaffolds in water uptake, but the PLA in

the scaffolds lowered the water uptake abilities of the

scaffolds. As shown in Fig. 3, the water uptake of the

whole scaffold structure (the material itself with the pore

system) was much higher than the water uptake of the

scaffold material itself (no excess water inside the pore

system).

3.3 Mechanical properties

The Young’s modulus and stiffness values are listed in

Table 3. For all of the scaffolds, elastomeric foam com-

pression curves with an initial linear elastic region, a

middle collapse plateau region, and a final densification of

the material could be noticed (Fig. 4), as described by

Harley et al. [23]. The stress–strain curves (Fig. 4) show

Fig. 1 Cross-section views and 3D reconstructions of microCT images of different scaffold types

1132 J Mater Sci: Mater Med (2014) 25:1129–1136

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that the stress values of the dry scaffolds rose steadily after

the linear elastic region and the final densification occurred

after 75 % of strain. Also, the stress stayed at a relative low

level with the wet scaffolds until 60 % of strain for ChiH,

C2C1H and C1C1, and 70 % of strain for Col, Chi, C2C1,

ColH, and C1C1H. The hybrids showed significantly

higher stress values than the Col, Chi and C2C1 after 70 %

of strain. C1C1 showed the highest stress values for wet

scaffolds after 70 % of strain. The corresponding stiffness

of dry scaffolds between the plain and hybrids was over

50 % higher for the hybrids. Also, in wet conditions the

stiffness of hybrids was over 70 % higher than for plain

scaffolds, except for the C1C1 that had a much higher

stiffness than other plain scaffolds. The collagen compo-

nent in the studied Col and ColH recovered their shapes

after the compression test studies almost completely after

re-immersing the scaffolds into the PBS for 1 h (data not

shown).

3.4 Cell culture studies

Seeded cells were evenly distributed on the surfaces of the

scaffolds. The cells were viable after one week of culture

Fig. 2 SEM micrographs of the scaffolds at d0 (without cells) and

scaffolds containing bovine chondrocytes at d7 of culture. Scale bars

100 lm

Table 2 Porosity values of different scaffold types collected from

microCT data

Sample Total

porosity (%)

Open

porosity (%)

Total porosity - open

porosity (%)

Col 85.92 85.91 0.01

Chi 89.41 89.41 0.00

C1C1 66.15 66.13 0.02

C2C1 86.64 86.63 0.01

ColH 92.76 92.75 0.01

ChiH 88.40 88.38 0.02

C1C1H 85.44 85.39 0.05

C2C1H 88.48 88.47 0.01

Fig. 3 Water uptake properties of the scaffolds after immersion in

PBS for 24 h in 37 �C

J Mater Sci: Mater Med (2014) 25:1129–1136 1133

123

and attached well to the freeze-dried collagen and chitosan

networks and the PLA fibers (Fig. 2). The cells expressed

collagen type II in the cytoplasm, confirming their chon-

drogenic phenotype (Fig. 5).

There was a high variation in the penetration of the cells

into the different scaffolds. As cells were pipetted on top of

the dense scaffolds, most cells remained at the top in all

scaffold types. In ColH and C2C1H scaffolds, however, the

penetration of the cells was good and cells were detected

throughout the scaffold. In Col, Chi, C1C1, and C2C1

scaffolds, the penetration of the cells was weak. In ChiH

and C1C1H scaffolds, some penetration was detected.

4 Discussion

In earlier studies, neither PLA nor collagen has been an

optimal scaffold for articular cartilage, as the PLA matrix

has shown to be too hard [24], and collagen gels, even

though being a fairly good option for cartilage tissue

engineering, often suffer from contraction [22]. Therefore,

in our present study, we used synthetic PLA to give the

scaffolds mechanically a more stable skeleton. The highly

hydrophilic components, collagen and chitosan were used

to give the scaffolds a better water absorbing ability and to

mimic the native ECM components in the cartilage. The

studied scaffolds are suturable in order to achieve the

mechanical locking of the scaffold in situ. The freeze-

drying and cross-linking of the collagen component

Table 3 Mechanical test results (Young’s modulus and stiffness

values ± SD) of dry and wet scaffolds

Samples Young’s modulus (E, kPa) Stiffness (N, mm)

Dry Wet Dry Wet

Col 115.1 ± 22.6 5.0 ± 0.5 32.3 ± 2.4 7.8 ± 1.9

Chi 86.0 ± 16.5 4.5 ± 1.4 26.1 ± 2.0 20.2 ± 3.2

C1C1 60.3 ± 14.3 10.0 ± 3.5 35.2 ± 5.9 76.0 ± 6.1

C2C1 130.8 ± 27.6 5.2 ± 1.0 33.0 ± 4.3 17.3 ± 3.8

ColH 22.8 ± 7.8 3.3 ± 1.5 81.0 ± 13.1 65.1 ± 10.9

ChiH 42.5 ± 9.5 9.0 ± 3.6 75.3 ± 5.1 85.7 ± 15.2

C1C1H 48.4 ± 8.5 5.9 ± 2.2 86.8 ± 9.3 95.1 ± 13.6

C2C1H 52.3 ± 6.3 5.3 ± 1.6 83.9 ± 13.6 99.4 ± 10.9

Fig. 4 Stress–strain curves for a dry and b wet scaffolds showing linear elastic, collapse plateau, and densification regimes

Fig. 5 Evenly distributed and

viable chondrocytes show

collagen II expression in the

scaffolds after one week in

culture, indicating that the cells

have retained their

chondrogenic phenotype in the

scaffolds. Green collagen II,

blue nuclei of the cells. Scale

bar 50 lm (Color figure online)

1134 J Mater Sci: Mater Med (2014) 25:1129–1136

123

prevents the possible undesired contraction of the collagen

component. Blends with collagen/chitosan ratio between

2:1 to 1:1 was expected to be ideal as the number of

organic components of collagen and the proteoglycans in

native articular cartilage are 15–20 wt% of collagen and

10 wt% of proteoglycans [25].

The highly porous structure of a cartilage tissue engi-

neering scaffold is essential for cell migration and the

diffusion of oxygen and nutrients, and it also improves the

mechanical interlocking of the scaffolds to the surrounding

tissue [11]. The structure of the studied scaffolds was

highly porous (Fig. 2) and the vast majority of the pores

were interconnected, as seen in the microCT images

(Fig. 1). The PLA fibers in the hybrids were well inter-

locked between the matrix polymer. The geometry of each

hybrid component remained very stable and did not alter

the shape of the meshs or the pore structure of the matrix

component. The difference between the hybrid and plain

scaffolds varied only 1.0 % for the Chi/ChiH and 1.8 % for

the C2C1/C2C1H scaffolds, but the porosity of ColH

compared to Col increased by 6.8 % and C1C1H compared

to C1C1 by 19.3 %. All scaffolds had a high and similar

porosity with the exception of C1C1 that had relatively low

porosity values because of the more dense structure. The

microCT studies confirmed the highly interconnected pore

structure.

Collagen showed the best water uptake abilities in the

scaffolds. Scaffolds containing chitosan showed the

weakest water uptake abilities that lead to the shrinkage of

the Chi scaffolds (data not shown). In addition, C1C1

showed considerable shrinking in wet conditions (data not

shown), indicating an ineffective neutralization of the

chitosan component in C1C1. The PLA was not able to

bind the water inside its structure during the wetting period

of 24 h and therefore the percentual water uptake of the

hybrids was much lower than the values for the plain

scaffolds.

The compressive modulus of native cartilage varies

between the different layers of cartilage [26]. It is known

that the stiffness of the scaffold influences the mechanical

environment of the cells, which in turn can influence cell

differentiation and tissue growth. However, the optimal

mechanical properties of a plain scaffold for cartilage tis-

sue engineering are not known, and the engineered tissue

may not necessarily be an exact copy of the natural tissue

[11]. The cellular microenvironment changes in the scaf-

fold during tissue development in vivo and the mechanical

properties of the scaffolds are found to improve compared

to in vitro cultivation [22]. In our study, the dry C1C1

showed the lowest Young’s modulus, which could be

explained by much lower porosity values (shown in the

microCT studies). The lower porosity values were possibly

due to a partially-collapsed pore structure during

processing that lead to a faster pore collapse. The Young’s

modulus for the dry hybrids was lower than for the cor-

responding plain scaffolds. The difference between the wet

hybrid scaffolds and the plain scaffolds was more moder-

ate. The lower Young’s modulus of the hybrid scaffolds

could be explained by the structure, where the more

mechanically stable PLA mesh is at the top and at the

bottom of the sandwich structure. Therefore, the initial

elastic properties may only be the properties of the softer

middle component of the collagen, chitosan, or collagen–

chitosan blend. The C1C1 had the highest Young’s mod-

ulus for plain wet scaffolds, but this was due to the denser

structure of the scaffold, as described earlier. Prominent

shrinking of the C1C1 was detected in wet conditions (data

not shown), leading to a higher Young’s modulus as well as

higher stiffness for C1C1. The hybrid structure improved

the stiffness of the scaffolds giving the scaffolds more

mechanical strength compared to the plain scaffolds. Even

when wet, the hybrids retained their structure after the

mechanical loading. Moreover, the ability to reabsorb the

removed water after loading is an essential characteristic

for articular cartilage tissue engineering scaffolds, as the

mechanical loading and unloading is a biological phe-

nomenon in the native tissue. As a consequence, the liquid

and nutrition exchange is implemented in the native

avascular tissue via the phenomenon [6]. The ColH scaf-

folds recovered well from the mechanical load after com-

pression, representing the ideal characteristics for a

mechanically compatible scaffold. This phenomenon can

also be seen in the stress–strain curves of wet scaffolds

(Fig. 4) as the ColH scaffolds showed no densification

before the compression of 70 %, indicating that no plastic

deformation yet exist.

Chondrocytes were cultured in the scaffolds to study the

viability and attachment of the cells in the scaffolds

in vitro. Our results show that cell viability and attachment

was good in all scaffolds. The addition of chitosan lowered

the penetration of the cells, and the addition of a loose PLA

fiber network increased the penetration indicating the

positive effect of the used hybrid structure. However, other

cell seeding methods, such as injection by needle may be

considered as an additional way to improve the even dis-

tribution of the cells inside the scaffolds.

5 Conclusion

In the present study, we showed the ability of this fabri-

cation method to be used for processing novel hybrid

structures with natural and synthetic components. The

hybrid collagen scaffolds showed desirable properties for

articular cartilage tissue engineering applications, although

the composition of the hybrid should be studied more

J Mater Sci: Mater Med (2014) 25:1129–1136 1135

123

detailed to find out an optimal scaffold structure. These

scaffolds had high porosity and interconnected pores,

improved mechanical strength compared to plain collagen

scaffolds, good water uptake, and penetration of chondro-

cytes indicating this kind of collagen/PLA hybrids to be

potential scaffolds for cartilage tissue engineering.

Acknowledgments We would like to thank Antti Aula for microCT

image editing. The financial support of the Finnish Funding Agency

for Technology and Innovation (TEKES) is greatly appreciated (Grant

3110/31/08).

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