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and cell invasion to a tissue engineered approach, whereby the construct
is initially seeded with cells, cultured in vitro, and finally implanted. It is
therefore essential to provide a biological environment in which cells can
readily attach, proliferate and maintain their differentiated phenotype and
to allow deposition of new bone matrix throughout the entire construct.
Scaffold characteristics such as interconnectivity, pore size/curvature,
microporosity, macroporosity and surface roughness influence cellular
responses, but they also collectively control the degree of nutrient
delivery, penetration depth of cells and metabolic waste removal. It is
also important to allow cell-seeded scaffolds to be subjected to a strain
environment, in order to further our understanding of how cells respond
to mechanical stimuli.
A tissue-engineered scaffold must provide a germane environment for
in vitro cell culturing in a bioreactor as well as providing a suitable
environment once implanted in vivo. These two environments differ in
terms of nutrient concentration gradients, pressure gradients and fluid
velocities. In vivo, diffusion is the primary mechanism for transporting
nutrients, whereas fluid flow is the principal mechanism for transport of
nutrients and provision of mechanical stimuli in vitro.
In order for a scaffold to be considered successful, it is essential that
it provide a nutrient rich environment within the scaffold core in order
for cells to lay down new matrix and minimise cell necrosis. Scaffolds
with defined interconnected channels aid in the processes of cell nutrientdelivery, waste removal and vascular invasion (2).
Many of the conventional techniques used yield scaffolds with
random porous architectures which do not necessarily produce a suitable
homogeneous environment for bone formation. Non-uniform micro
environments produce regions with insufficient nutrient concentrations
which can inhibit cellular activity and prevent the formation of new
tissue with a homogeneous quality.
Advanced manufacturing technologies such as rapid-prototyping have
aided in over coming some of these limitations, allowing for greater
control over internal scaffold geometry. However even with these
technological advances, limitations still remain, and need to be resolved
in order to produce the next generation of tissue engineered scaffolds
with suitable chemical and mechanical microenvironments. The authors
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will discuss the advantages and limitations of existing techniques and
present an alternative method to fabricate regular orthogonal architecture
scaffolds for mechanobiology investigations in tissue engineering.
2. Scaffold Properties
Ideally a scaffold should possess the following characteristics to bring
about the desired biologic response (11): (i) three-dimensional and
highly porous with an interconnected pore network for cell/tissue growth
and flow transport of nutrients and metabolic waste, (ii) biodegradable or
bioresorbable with a controllable degradation and resorption rate to
match cell/tissue growth in vitro and/or in vivo, (iii) suitable surface
chemistry for cell attachment, proliferation and differentiation, (iv)
mechanical properties to match those of tissues at the site of
implantation, and (v) be easily processed to form a variety of shapes and
sizes.
2.1 Scaffold Materials
The three main material types which have been successfully investigated
for use in developing scaffolds include (23, 27): (i) Natural polymers,
such as collagens, glycosaminoglycan, starch, chitin and chitosan, (ii)
Synthetic polymers, based on polylactic acid (PLA), polyglycolic acid(PGA) and their co-polymers (PLGA), and (iii) Ceramics, such as
hydroxyapatite (HA) and -tricalcium phosphate (-TCP). While
naturally occurring biomaterials offer the greatest potential in terms of
biocompatibility, large batch-to-batch variations can exist as well as poor
mechanical performance. A concern of material supply limitations has
prompted researchers to investigate the use of synthetic polymers.
Synthetic polymers have been widely used for over 20 years as
surgical sutures, with long established clinical success and many are
approved for human use by the FDA. However, synthetic polymers of
the poly(-hydroxy acids) family release acidic by-products as they
undergo degradation by bulk erosion via hydrolysis when exposed to
aqueous environments (15). Although these degradation products arenaturally present in the human body and are removed by natural
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metabolic pathways (21), the local pH of the surrounding
microenvironment can be reduced below that of the natural physiological
pH and thus elicit an immunological response. The effect of this acidic
environment can cause cell necrosis as well as acting as an autocatalyst,
further accelerating the degradation of the polymer.
Ceramics have also been widely used, due to their high
biocompatibility and resemblance to the natural inorganic component of
bone and teeth (6, 7). Ceramics are inherently brittle and limit their
applicability in tissue engineering/mechanobiology investigations to load
bearing applications, since ceramics are stronger in compression than in
tension.
As synthetic polymers are deemed to be ductile with insufficient
rigidity, some researchers have developed composite materials (e.g.
polymers with ceramic particles embedded within the polymer matrix) to
improve mechanical performance and render the material more suitable
for load bearing applications. The added advantage of this is that the
embedded ceramic particles act as a buffer to the degradation of by-
products produced (13). The development of materials for tissue
engineering scaffolds presents many challenges in obtaining specific
mechanical and bioresorbable properties, as well as developing materials
suitable for various fabrication processes.
2.2 Pore Size and Curvature
Many investigators have defined scaffold pores based on size as either
micro (diameter < 100m) or macro (diameter > 100m). For
colonisation of macropores to occur, the minimum pore size in which
bone will form is claimed to be approximately 100m (6). Other
researchers have created scaffolds with pore sizes of between 150-300m
and 500-710m to promote bone formation (12, 18). However many of
these pore sizes were determined using random pore geometries, and
hence do not define optimum pore sizes accurately; rather they define the
range of pore sizes in which bone formation was observed.
The pore size employed may also be dependent on the tissue-type
desired. For example scaffolds with pore sizes less than 150m have
been successfully used for regeneration of skin in burn patients (20).
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Osteoblasts appear to exhibit greater cellular response when pore sizes of
between 200 and 400m are employed (6). This may be due to the
curvature of the pore which may provide optimum compression and
tension on the cells mechanoreceptors and allows them to migrate into
openings of such a size (4).
2.3 Interconnectivity, Macroporosity and Microporosity
A scaffold should provide an open porous networked structure allowing
for easier vascularisation, which is important for the maintenance of
penetrating cells from surrounding tissues and the development of new
bone in vivo. The higher the macroporosity the easier it is for
vascularisation to occur. Failure to develop an adequate vascular network
will mean that only peripheral cells may survive or differentiate,
supported by diffusion. Chang et al. (6) proposed that the degree of
interconnectivity rather than the actual pore size has a greater influence
on osteoconduction. Interconnectivity is a physical characteristic that
aids in the delivery of nutrients and removal of metabolic waste products.
Studies have shown that bone normally forms in the outer 300m
periphery of scaffolds and that this may be explained by the lack of
nutrient delivery and waste removal (12). When the pore size used is too
small, pore occlusion can occur by cells preventing further cell
penetration and bone formation (14). It is pertinent to note that muchhigher rates of mass transfer exist at the periphery of a scaffold, and that
these higher rates promote mineralisation, further limiting the mass
transfer of nutrients to the core of a scaffold (17). It is essential that a
scaffold possess a high degree of interconnectivity in conjunction with a
suitable pore size, in order to minimize diffusion limitations and pore
occlusion.
The incorporation of microporosity within the scaffold material may
have added advantages with regard to nutrient delivery and cellular
response. Taboas et al. (22), successfully incorporated microporosity
(Fig. 1) within a scaffold material (PLA) consisting of interconnected
plate structures, yielding 5-11m void openings, through an emulsion-
solvent diffusion technique. The microporosity of a scaffold gives it the
potential to be preconditioned with bone morphogenetic proteins (BMPs)
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(1), anti-inflammatory drugs (Dexamethasone) (29) and oxygen release
agents (ORAs) such as perfluorocarbons.
Tancred et al. (24) assessed the fluid-retention characteristics (Fig. 2)
of ceramic based scaffolds produced by identically replicating the
architecture of bovine cancellous bone and observed that these scaffolds
were capable of retaining water at the level of at least 50 wt% of the
mass of the mineral, at less than 65% porosity, to about 150 wt% of the
mass of the mineral at 80-85% porosity, indicating that these -TCP
replicated structures could also be useful as a carrier for osteogenic
agents such as BMP.
Fig. 1. Interconnected plate structures (7x5m average) yielding 5-11m void openings
within PLA material (22).
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Fig. 2. Water retention characteristics of porous -TCP replicas of bovine cancellous
bone showing the increase in water retention (wt% of matrix mineral) with increasing
construct porosity (24).
3. Traditional Scaffold Fabrication Techniques
Several techniques have been developed to fabricate scaffolds. Theseinclude solvent-casting and particulate-leaching, gas foaming, fibre
meshes/fibre bonding, phase separation, melt moulding, emulsion freeze
drying, solution casting and freeze drying (Table 1). Traditional methods
of fabricating scaffolds, through material processing and casting, have
largely been unsuccessful in controlling the internal architecture to a high
degree of accuracy or homogeneity (Fig. 3), since the resulting interior
architectures are determined by the processing technique. For example,
particulate leaching is a process whereby the internal architecture is
determined by embedding a high density of salt crystals into a dissolved
polymer or ceramic matrix. The dissolved mixture is then poured into a
mould and treated under heat and pressure to form the external shape.
The salt particles are subsequently leached out to leave interconnecting
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Fig. 3. Random porous architecture of PLGA created via liquid-liquid phase separation
(16).
interior channels. Running the salt crystals through a sieve to obtain a
specific range of pore size can control the pore diameters; although the
agglomeration of salt particles can alter the eventual pore size and pore
distribution during leaching (14). Particulate leaching techniques are
limited to producing thin membranes (2-3mm), due to the difficulty in
ensuring complete removal of the embedded particles. Also, there is littlecontrol over the orientation and the degree of interconnectivity. However
the degree of interconnectivity can be improved by having a high density
of salt particles (8, 12) and also by fusion of the salt crystals prior to
infiltration (19). Creation of scaffolds with identical internal
architectures for mass transfer and mechanobiology investigations is
essential. Previous researchers have demonstrated that control over the
interior architecture is crucial to ensure scaffold vascularisation and bone
deposition (7, 18).
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Table 1. Conventional scaffold processing techniques for tissue engineering (14, 27).
Process Advantages Disadvantages
Solvent casting
and particulate
leaching
Large range of pore sizes
Independent control of
porosity and pore size
Crystallinity can be tailored
Highly porous structures
Limited membrane thickness
(3mm)
Limited interconnectivity
Residual porogens
Poor control over internal
architecture
Fibre bonding High porosity Limited range of polymers
Residual solvents
Lack of mechanical strength
Phase separation Highly porous structures
Permits incorporation of
bioactive agents
Poor control over internal
architecture
Limited range of pore sizes
Melt moulding Independent control of
porosity and pore size
Macro shape control
High temperature required for
nonamorphous polymer
Residual porogens
Membrane
Lamination
Macro shape control
Independent control of
porosity and pore size
Lack of mechanical strength
Limited interconnectivity
Polymer/ceramicfibre composite
foam
Independent control ofporosity and pore size
Superior compressive strength
Problems with residual solvent
Residual porogens
High-pressure
processing
No organic solvents Nonporous external surface
Closed-pore structure
Freeze drying Highly porous structures
High pore interconnectivity
Limited to small pore sizes
Hydrocarbon
templating
No thickness limitation
Independent control of
porosity and pore size
Residual solvents
Residual porogens
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3.1 Solid Free Form (SFF) Fabrication Technologies
Rapid prototyping (RP) or solid free form (SFF) fabrication technologies
are currently being used by investigators to manufacture scaffolds for use
in tissue engineering (3, 7, 9, 10, 25, 26). SFF methods are based on the
premise that a material in either powdered or liquid form is solidified one
layer at a time. It is thus an additive process unlike traditional methods of
manufacturing which are subtractive based. Each layer is created as
defined by a computer-generated file. Once the layer is complete, the
build platform is indexed downward by one layer thickness and the
process is repeated. The main systems that fall under this category are:
(1) stereolithography (SLA), (2) selective laser sintering (SLS), (3) fuseddeposition modelling (FDM) and (4) three-dimensional printing (3-DP).
Each SFF fabrication process has its own advantages and disadvantages
in fabricating scaffolds as summarised in Table 2.
Table 2. Advantages and limitations of SFF fabrication techniques.
Technique Advantages Limitations
SLA Relatively easy to remove
support materials
Accurate small features
Limited by the development of
photopolymerizeable andbiocompatible, biodegradable
liquid polymer material
SLS Good compressive strengths
Greater material choice
Solvent free
High processing temperatures
Materials trapped in small
inner features is difficult toremove
FDM No material trapping withinsmall features
Solvent free
Good compressive strengths
Requires support material forirregular structures
Anisotropy between XY and Z
direction
3D-P Greater material choice
Low heat effect on raw material
Materials trapped in small
inner features is difficult to
remove
Use of toxic organic solvents
Lack of mechanical strength
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The main limitations include the use of toxic binders, poor feature
symmetry (Fig. 4c) and materials. Due to these material limitations,
researchers have also used SFF techniques to indirectly cast scaffolds
with controlled internal and external architecture by means of a lost
mould process (7, 22). Manufacturing of these scaffolds consists of three
different types of development and optimisation work. They include: (a)
mould design (b) sacrificial mould fabrication (c) material casting and
(d) thermal or chemical removal of mould.
Fig. 4. (A) & (B) Solid free form (SFF) fabricated scaffold produced using selective laser
sintering (SLS) technique, material is Duraform polyamide. (C) Threshold image of asingle pore showing poor symmetry formed through selective laser sintering.
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Lost mould processes are mainly suited to ceramic infiltrates as ceramics
are typically sintered to temperatures in excess of 1000C, which ensures
complete removal of a polymer mould. When attempting to fabricate
polymer scaffolds though indirect fabrication methods, an extra step of
creating a ceramic-type mould is required. This ceramic mould is
infiltrated through melt or solvent casting depending on the desired
polymer. Once cured, the ceramic mould can be removed through solvent
dissolution. The choice of solvent for mould dissolution is dependent on
the cast and mould material. This iteration further reduces the quality of
the final scaffold in terms of pore symmetry, and material properties due
to polymer exposure to solvents. An advantage of indirect casting is the
production of discrete composite scaffolds in which material regions are
mechanically interdigitated (Fig. 5), as well as the incorporation of
microporosity within the scaffold material which allows for
preconditioning with bioactive agents and manipulation of the surface
roughness.
Fig. 5. (A) Biphasic PLA/HA scaffold (top=PLA, bottom=HA). PLA global pores are
600m, HA global pores are 500m. (B) Biphasic PLA/PGA scaffold (top=PGA,
bottom=PLA), 800m orthogonal pores (22).
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3.2 Mechanobiology/Tissue Engineering Scaffold Criteria
With regard to tissue engineering investigations, it is necessary to
fabricate scaffolds which meet the following criteria; (i) regular internal
geometries, (ii) complete interconnectivity, (iii) independent control over
pore size and porosity, (iv) scaffolds for use within strain environments,
(v) facilitate the creation of scaffolds from a broad range of materials
without loss of pore definition, and (vi) allow for the incorporation and
manipulation of microporosity within the scaffold material.
Some of these criteria cannot be easily met with the existing
techniques as outlined previously.
Regular architecture scaffolds permit cells to be seeded in the coremuch more readily than random architecture scaffolds and create
environments which encourage uniform conditions for promoting cell
viability. The added advantage of developing regular architecture
scaffolds is that they permit parametric analyses to be conducted, which
is essential in scientific investigations of how scaffolds perform as a
function of their physical characteristics. Finite element
diffusion/perfusion studies are more feasible when regular architecture
scaffolds are employed. As a first step in addressing these issues, we
have developed a regular orthogonal fibre stacking (ROFS) technique to
fabricate scaffolds.
4. Process Overview
The process is essentially a lost mould process, in which layers with
parallel unidirectional nylon fibres of equal spacing create the mould.
The layers are created by winding nylon fibre around a plate with defined
notches of equal spacing. The mould architecture forms a 3D mesh-like
structure. This mould is infiltrated with either a ceramic slip or polymer
solution. Once the infiltrate has been cured/sintered, the nylon fibre
mould is removed either by pyrolisation (ceramic infiltrate) or by
physical extraction of the fibres (polymer infiltrate) (Fig. 6).
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Fig. 6. Process flow chart for creation of regular orthogonal polymer/ceramic scaffold.
4.1 Materials and Methods
Poly(dimethylsiloxane) (PDMS) is widely used in microfabrication for
biological applications and offers biocompatibility, optical transparency,
permeability to gases, flexibility, and durability. PDMS produced by
Dow Corning, Sylgard 184 was obtained from R W Greef (Glasgow,
UK). The notched recessed plate (Fig. 7) was fabricated using a diamondsaw (STRUERS ACCUTOM 50) and 400 m blade (METPREP 10-
12-50), with a notch spacing of 300 m. Nylon fibre of diameter 250,
300 and 350 m (Climax, Germany) was used in this study. PDMS
infiltrate was mixed using a 10:1 weight ratio of base to curing agent,
and degassed under vacuum for 1hr. The polymer solution was then
infiltrated using a 10ml surgical syringe into the fibre mesh matrix and
cured for 4hrs at 60C. After curing, the fibres were then extracted from
the polymer to reveal a slab (50mm x 50mm x 3mm) with orthogonal
oriented channels (Fig 9.).
Creation ofRegular
OrthogonalStacked mesh
Ultrasonic Bath
Treatment
Infiltration of
mesh with
ceramic slip
Thermal mould
removal
Physicalextraction of
fibers
Infiltration of
mesh with
polymer viasolvent/melt
casting
Sintering
Regular
ArchitectureScaffold sheet
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Fig. 7. 400m notched aluminium plate, with notch spacing of 300m for creation of the
3D orthogonal stacked mesh.
4.2 Results
Each layer of the unidirectional fibres is stacked orthogonal to the
previous layer creating a 3D mesh-type structure (Fig. 8). Once the
mould is infiltrated and the fibres are removed, macro pores with non-
random interconnectivity are created in the x, y andzplanes. The pores
created in thezdirection are a direct result of the contact points between
the fibres in thex andy directions. The pores generated in thezdirection
are typically around 100m (when 350m fibres are employed), but this
is dependent on the viscosity of the polymer infiltrate.
Fig. 8. (A) Layer with fibres of 200m diameter and spacing of 300m. (B) Stacked
layers showing orthogonal mesh structure.
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Fig. 9. (A) Approx 5cm square slab of PDMS scaffold (B) & (C) Magnified structure of
regular PDMS scaffold 350m pore size and 300m pore spacing.
5. Discussion
Scaffolds have been successfully fabricated according to the method of
preparation described. The resulting scaffold slab is approximately 5cm
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square, with regular orthogonal geometry in all three directions. Figure 9
shows images of the regular geometrical structure obtained with a PDMS
infiltrate. As can been seen the structure is highly regular and repeatable.
A limitation with this technique, is that the resulting scaffold is
anisotropic (between xy and z direction), and this is due to penetration of
the polymer around the point of contact of the fibres. A higher infiltrate
viscosity results in larger sized pores in the z direction. However when
viscous solutions are employed, higher pressures are required to
successfully infiltrate the mesh mould, which can result in spreading of
the fibres.
Thermal treatment of the mould prior to infiltration may improve
contact area, but this may only be applicable when fabricating ceramic
scaffolds, since fusion of the fibres may occur, and make the process of
extracting fibres form a polymer matrix difficult. Another possibility in
improving the contact area includes using fibres of square or hexagonal
cross sectional area, to create the mesh mould. However this may not
provide an optimum substrate for cells, in terms of pore curvature.
Since the notched plate was created using a 400m blade and the
fibre employed was 350m in diameter, it is possible for the resulting
pore spacing to vary between 300m and 400m. Using fibres with
diameters closer to the notch width will further reduce the variation
between pore spacing. Other methods of creating a notched plate can be
employed in order to increase the range of pore sizes, and increasemacroporosity. Possible methods include the use of electro-discharge
machining (EDM) or SFF techniques to fabricate notched plates for the
creation of the orthogonal mesh through fibre winding. This technique
also allows independent control over pore size and porosity, which is
essential for parametric analyses.
Indirect casting techniques allow for a much wider range of materials
to be employed, and also facilitates the incorporation of microporosity
into the scaffold material, which may have added advantages with regard
to nutrient delivery and cellular response. Traditional fabrication
methods such as particulate leaching, emulsion-solvent diffusion and gas
foaming could technically impart microporosity to scaffolds. However,
control over the actual micro-pore size may be difficult to achieve and
control. Further studies will determine the optimum technique to be used.
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Another advantage of developing regular architecture scaffolds is that
they permit parametric analyses to be conducted in terms of nutrient
concentration gradients and local strain environments, which is essential
in identifying and predicting optimal cell environments in order to
develop scaffolds for preliminary analysis and eventual implantation.
6. Conclusions and Outlook
A process has been described which allows regular orthogonal repeating
unit scaffolds from a wide range of materials to be produced in large
quantities with independent control over pore size and porosity, with
complete interconnectivity. Although PDMS is a non-bioresorbable
material, it is biocompatible and could serve as a delivery vehicle as well
as a model for future work on bioresorbable scaffolds such as PLA, PGA
and their copolymers. Regular architectures allow mechanical and
diffusion/perfusion finite element analyses to be carried out, which
permit quantification of suitable mechanical and chemical
microenvironments for bone cells, aiding in the development of the next
generation of tissue engineered scaffolds.
Acknowledgments
This work was supported by the HEA under the Programme for Researchin Third Level Institutions (PRTLI) Cycle III. The authors would also
like to thank Mr. Tony Tansey of the Department of Mechanical
Engineering, IT Tallaght, Dublin 24 for producing the RP scaffolds.
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