C.T. Buckley and K.U. O’Kelly- Regular Scaffold Fabrication Techniques for Investigations in...

8/3/2019 C.T. Buckley and K.U. OKelly- Regular Scaffold Fabrication Techniques for Investigations in Tissue Engineering

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Topics in Bio-Mechanical Engineering148

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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Regular Scaffold Fabrication Techniques 149

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


Macro shape control

High temperature required for

nonamorphous polymer

Residual porogens

Membrane

Lamination

Macro shape control




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



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



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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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