Introdução Engenharia Biomédica
Mestrado Integrado Engenharia Biomédica
1º Ano, 1º Semestre 2007/2008
Bone Tissue Engineering: Production of Scaffolds
Carriço, Ana Cláudia1; Farracho, Marta2; Nunes, Cecília3; Ruela, Ana Margarida4; Semedo, João5
[email protected]; [email protected]; [email protected]; [email protected];
[email protected]
Abstract
Tissue Engineering is a relatively recent scientific field which intends to present alternatives to the
conventional treatments for injured organs or body tissues, in a short term future. This paper aims to gather
information related to bone tissue engineering, emphasizing applications of artificially created support systems with
seeded bone cells, named scaffolds. These scaffolds should mimic the host tissue, as well as provide a chemical and
physical environment adequate to cell proliferation and development. Primarily, features of the bone tissue will be
explored in order to understand the properties of the ideal scaffold. Subsequently, the paper focuses on project and
fabrication techniques of scaffolds, namely the ones suitable for bone tissue engineering.
1. Introduction
Tissue Engineering (TE) is a recent, interdisciplinary and multidisciplinary science field that has experienced
intense development in the past few years. Using a combination of biology, engineering and materials science
methods, and providing suitable biochemical and physiochemical factors it aims to improve or replace biological
functions.[2]
This engineering field covers a broad range of applications, mostly associated to the reparation or
replacement of tissue portions or the whole tissue itself (such as bone, cartilage, blood vessels or bladder). These
require certain mechanical and structural properties for function properly. In other words, TE involves attempts to
mimic specific biochemical and physical functions combining cells within artificially-created support systems.
“Regenerative medicine” is often used synonymously to TE, although this area emphasizes the use of stem cells to
produce tissues.[15-16]
When a portion of tissue in the body or an organ is severely injured, largely lost or dysfunctional, it is
clinically treated with either reconstructive surgery or organ transplantation. However, the shortage in tissue or
organ donors is a big obstacle. In addition, patients who undergo surgery require long term administration of
immunosuppressive agents, which cause various negative side-effects, such as viral infections and carcinogenesis.
These problems have recently led to a therapeutic trial to induce the regeneration of patients’ tissues and organs by
Bone Tissue Engineering: Production of scaffolds
making use of their own self-healing potential. TE, a research and development field of biomedical engineering and
technology has been included in this trial. [2]
The idea that tissues and organs can be "engineered" to be used for transplantation is at the same time
revolutionary and stimulating. One of the earliest demonstrations of this possibility was given by Bisceglie in the
1930s, when he encased mouse tumor cells in a polymer membrane and inserted them into a pig’s abdominal cavity.
These studies showed that cells could survive and would not be destroyed by the immune system.
Recent developments in this field have yielded a novel set of tissue replacement parts and implementation
strategies. Scientific advances in biomaterials, stem cells, growth and differentiation factors, and biomimetic
environments have created unique opportunities to fabricate tissues in the laboratory from combinations of
scaffold, cells, and biologically active molecules. One of the main challenges now facing TE is the need for more
complex functionality, as well as both functional and biomechanical stability in laboratory-grown tissues destined for
transplantation. The continued success of investigations in TE and the eventual development of true human
replacement parts will grow from the convergence of engineering and basic research advances in tissue, matrix,
growth factor, stem cell, and developmental biology, as well as materials science and bioinformatics. [1]
In this paper we will mainly focus on TE applied to bone and bone tissue. Primarily focusing in bone
constitution and characteristics, we will approach the production of scaffolds and its necessary properties to be used
in medical treatments.
2. Bone and bone tissue
Bones are the rigid organs that form part of the endoskeleton of
vertebrates that function to move, support, and protect such vital organs
as those in the cranial and thoracic cavities. As their main constituent,
bone tissue supports a fleshy structure that harbors the bone marrow,
located within the medullar cavity of long bones and the interstices of
cancellous bone, where blood cells are formed. Because bones come in a
variety of shapes and have a complex internal and external structure, they
are lightweight, yet strong and hard, in addition to fulfilling their many
other functions - mineral storage, acid-base balance, detoxification and
sound transduction. The tissue that makes up the bone is a mineralized
tissue that gives it the necessary rigidity. Formed mostly of calcium
phosphate in the chemical arrangement termed calcium hydroxyapatite, it
has relatively high compressive strength but poor tensile strength,
meaning it resists pushing forces well, but not pulling them. Collagen
Fig. 1 Bone (From: Wikipedia)
confers it a significant degree of elasticity. [2-3]
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Bone Tissue Engineering: Production of scaffolds
2.1 Compact and spongy bone:
There are two different bone tissues, compact (cortical) bone and spongy (cancellous or trabecular) bone.
Compact bone corresponds to the dense areas without cavities. This tissue gives bones their smooth, white, and
solid appearance, and accounts for 80% of the total bone mass of an adult skeleton. Spongy bone corresponds to the
areas with the lacunae and accounts for the remaining 20% of total bone mass, but has nearly ten times the surface
area of compact bone. [2-3]
Fig. 2 Bone tissues structure
(From: Wikipedia)
2.1 Constitution:
All bones consist of living cells, making up a specialized connective tissue composed of intercellular calcified
material, the bone matrix, and different types of cells. The matrix is the major constituent of bone, surrounding the
cells. The inorganic matter in the matrix represents about 50% of its dry weight. Mainly crystalline mineral salts and
calcium, it is almost constituted of hydroxyapatite. The organic part of matrix is type I collagen. The main difference
that distinguishes the matrix of a bone from that of another cell is its hardness. The matrix is initially laid down as
non-mineralized osteoid (manufactured by osteoblasts). Mineralization involves the secretion of alkaline
phosphatase by osteoblasts’ vesicles. Regarding to the cells, there are three different types: osteocytes (internal
bone cells), osteoblasts (bone creation) and osteoclasts (bone resorption). The first ones, which synthesize the
organic components of the matrix (type I collagen, proteoglycans, and glycoproteins), are exclusively located at the
surfaces of bone tissue. Some of them, when surrounded by newly formed matrix become osteocytes and create an
empty space, named lacuna. These osteocytes, found each one in a different lacuna, have a kind of extensions – a
network of thin canaliculi – able to pass molecules from cell to cell. Osteoclasts, which are multinucleated giant cells
involved in the resorption and remodeling of bone tissue, are derived from the fusion of bone marrow-derived cells,
which secret specific enzymes that promote the digestion of collagen and dissolution of calcium salt crystals. [2-3, 25]
2.2 Histogenesis and bone growth:
Bone can be formed in two different ways: by direct mineralization of matrix secreted by osteoblasts
(intramembranous ossification) or by deposition of bone matrix on a preexisting cartilage matrix (endochondral
ossification). In both processes, the bone tissue that appears first is primary, or woven. It is a temporary tissue and is
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Bone Tissue Engineering: Production of scaffolds
soon replaced by the definitive lamellar secondary tissue, in a process that allows maintaining the bone shape while
it grows. The rate of bone remodeling (bone turnover) is very active in young children, where it can be 200 times
faster than that in adults. Bone remodeling in adults is a dynamic physiologic process that occurs simultaneously in
multiple locations of the skeleton, not related to bone growth.[3]
The relevance of Bone Tissue Engineering lies on replacing the organism in a function it can’t naturally
perform. The body’s bone regenerative capacity is insufficient to heal severely injured bone portions.
3. Scaffolds
Our body is continuously creating new cells. However, when it comes to repair, cell growth is often
undirected, which inhibits healing. One approach to TE is to use structures to help guide the body in its reparation
and regeneration. Cells can be implanted or 'seeded' into an artificial structure capable of supporting threedimensional tissue formation, named scaffold – porous structures for cell colonization and subsequent formation of
new tissue, which tries to reproduce the bone matrix.
TE’s scaffolds must serve three functions also called properties:
 Geometric (to define the volume that will shape the regenerating tissue);
 Mechanical (to provide temporary function in a defect while tissue regenerates);
 Biologic (to enhance ingrowth of tissue and allow inclusion of seeded cells, proteins and/or
genes to accelerate tissue regeneration).
These porous matrices where the cells are seeded should ideally show specific characteristics that make
them accomplish these properties, as is referred subsequently in this paper. As each bone has different tissue types
in different areas, a large range of scaffolds should ideally be produced. For example, cortical bone demands
scaffolds with low empty spaces, and spongy bone requires high porous and resistant scaffolds, when facing the
same stresses as the bone. Once inserted in the body, the scaffold provides a physical structure for directing the
growth of new replacement cells. Eventually, the scaffold can disintegrate and all that is left is the body's natural
tissue. In addition, scaffolds offer the opportunity to introduce growth factors into the body. In image 3 we see the
process of tissue regeneration using scaffolds, simultaneously to the degradation of the construct.
Figure 3: Regeneration of bone tissue using a scaffold.
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Bone Tissue Engineering: Production of scaffolds
3.1 Scaffolds’ characteristics
In order to be used in TE, scaffolds’ production needs extended investigation around the ideal material and
necessary characteristics, which confine them the adequate properties
As far materials are concerned, they should present:
Biocompatibility – scaffolds and their degradation products must not provoke an inflammatory response or
toxicity in the organism.[4, 14]
Biodegradability – scaffolds must be capable of breaking and dispersing in vivo, even thought there is no
proof of elimination from the body yet, due to macromolecular degradation – so they can vanish, leaving space for
the tissue to grow. [4, 12, 14]
Controlled degradation rate – strength decreases with the degradation of the material over time, so, as
different tissues have different rates of regeneration, it is important that the degradation rate matches the rate of
tissue regeneration. Tissues with a smaller rate of regeneration must grow in a scaffold with a smaller degradation
rate and vice-versa. [4, 14]
Out of materials reach, are scaffolds’ specific characteristics such as:
Appropriate pore size and morphology - porosity (percentage of open spaces), pore size and pore structure
play an essential role in the design of scaffolds. They are responsible for the nutrient supply to transplanted and
regenerated cells. In order to facilitate the nutrient supply, the scaffold must be permeable and the pores must have
interconnectivity, because it increases the diffusion rates and allows a better vascularisation (improves the exchange
of oxygen, nutrient supply and waste with the exterior of the scaffold). However, for large scaffolds volumes,
interconnected pores are not enough to provide the necessary oxygen and nutrients to the seeded cell, so a
vascularised bed has to be created to allow these exchanges. (There is no consensus about the pore size of the
scaffold for bone regeneration, but they should measure between 100µm and 400µm.) [4, 6, 14]
Appropriate surface chemistry for cell attachment, proliferation and differentiation – The surface needs to
have a suitable substrate that permits cell adhesion on biomaterials. As referred in the first section of this paper,
some cells – osteocytes are exclusively located on the surface of bone matrix. This cell adhesion is essential, because
it will allow further cellular function (spreading, proliferation, migration and biosynthetic activity). [14]
Sufficient strength and stiffness to support in vivo stresses (mechanical properties) – The scaffold must
possess sufficient strength and stiffness to support the tissue ingrowth until the time it has sufficient strength to
support itself. For this to happen, material must have high interatomic and intramolecular bonding, this is possible
by using Tensigrity (distributes and balances mechanical stresses), the walls, layers or struts are connected into
triangles, pentagons or hexagons, and each of them can bear tension or compression. [4, 14]
Three-dimensional shape – is the most appropriate design to sustain cell adhesion and proliferation.
[4, 14]
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Bone Tissue Engineering: Production of scaffolds
Strategies in TE
The polymer selection is based on two different strategies and the strategy chosen depends mainly on the
tissue to be restored and the location of the defect to be treated.
Strategy I
The cells are seeded in a polymeric scaffold in a static culture (Petri dish), from this moment, the scaffold
starts losing molecular weight. After that, the scaffold is placed in a dynamic environment and premature tissue
starts growing (spinner flask). Then the premature tissue is placed in a physiological environment (bioreactor) and
mature tissue starts to grow. However, when the surgical transplant is performed the tissue implanted is still
premature. Finally, the transplant is assimilated and remodelled, while the scaffold starts gradually losing mass.
In this strategy the mechanical properties of the scaffold remain constant for six months (four months for
cell culturing and two months in situ), after this period the scaffold starts losing its physical properties and generally,
after 12-18 months, is metabolized by the body. It should maintain its strength and stiffness until the transplanted
tissue has replaced the vanishing scaffold matrix. The mechanical properties of the implanted tissue and of the host
tissue should be as similar as possible. [4, 11, 14]
Fig. 4. Graphical illustration of the complex interdependence of molecular weight loss and mass loss of a strategy I
3D scaffold matrix plotted against the time frame for TE a cartilage/bone transplant.
(A) Fabrication of bioresorbable scaffold; (B) Seeding of the osteoblast/cartilage populations into the polymeric
scaffold in a static culture (petri dish); (C) Growth of premature tissue in a dynamic environment (spinner flask);
(D) Growth of mature tissue in a physiologic environment (bioreactor); (E) Surgical transplantation; (F) tissueengineered transplant assimilation/ remodeling.
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Bone Tissue Engineering: Production of scaffolds
Strategy II
As in strategy I, the cells are seeded in a polymeric scaffold in a static culture (Petri dish) and the scaffold is
placed in a dynamic environment and premature tissue starts to grow (spinner flask). Then the premature tissue is
placed in a physiological environment (bioreactor) and mature tissue starts to grow. In this strategy the molecular
weight loss starts in this phase. Once the mature tissue is formed the surgical implantation is performed. Finally, the
transplant is assimilated and remodelled, while the scaffold starts gradually losing mass.
In this strategy the mechanical properties of the scaffold remain until the moment when the premature
tissue is placed in the bioreactor. The degradation and resorption kinetics are designed to allow the seeded cells
proliferation and extracellular matrix secretion in the growth phase (static or dynamic) while the scaffold starts
vanishing allowing the tissue to grow and fulfil the empty spaces left by the scaffold. However, there isn’t a material
that offers a degradation and resorption rate compatible with this strategy yet. [4, 11, 14]
Fig. 5. Graphical illustration of the complex interdependence of molecular weight loss and mass loss of a strategy
II 3D scaffold matrix plotted against the time frame for TE a cartilage/bone transplant.
(A) Fabrication of bioresorbable scaffold; (B) Seeding of the osteoblast/cartilage populations into the polymeric
scaffold in a static culture (petri dish); (C) Growth of premature tissue in a dynamic environment (spinner flask);
(D) Growth of mature tissue in a physiologic environment (bioreactor); (E) Surgical transplantation; (F) tissueengineered transplant assimilation/remodeling.
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Bone Tissue Engineering: Production of scaffolds
3.2 Development of Scaffolds: Materials, Design and Fabrication
3.2.1 Materials
In order to develop scaffolds, it is firstly necessary to choose an appropriate material. A material is
considered appropriate if, in first place, it is biocompatible. As an example of biocompatible materials we have
metals, ceramics and polymers. However, as biocompatibility isn’t the only thing to attend, it is not possible to use
all of them. They also have to be biodegradable, which automatically excludes all metals and most ceramics although
some ceramics have been used with success, like tri-calcium phosphate and sea coral. [4, 14]
The advantages of using polymers instead of ceramics are that polymers are ductile, easily formed to any
shape and have similar characteristics to the biological materials; on the contrary, ceramics are less malleable and
present unsatisfactory degradation rates. There are three material categories for polymer-based scaffolds design
and fabrication, naming: [14]
I – Polymers which have been used for clinically established products (approved polymers), like collagen,
hydrogells, polyglicolide (PGA), polylactides (PLLA, PDLA), poly caprolactone (PCL). PGA and PLA are the most
common biodegradable polymers known and have been used in drug delivery, bone osteosynthesis and tissue
engineering of skin. PCL has two more two more carbon atoms than PGA. Its degradation rate is much slower than
PGA and PLA and has a high thermal stability (low freezing point and high melting point).
II – Polymers which are under clinical investigation (non-approved polymers), like polyorthoester (POE),
polyanhydrides, polyhydroxyalkanoate (PHA).
III – The syntesis of entrepreneurial polymeric biomaterials, like poly(lactic acid-co-lysine).
These polymers can also be divided in natural (collagen, inorganic hydroxyapatite, etc.) and synthetic (PGA, PLA,
PLGA, PCL, etc.). PCL is the polymer more frequently used as a scaffold material. [4, 5, 11, 14]
3.2.2 Design phase[7, 24, 28]
The design bounds are the relations between a certain scaffold characteristic and a property. For instance
porosity is related to mass transport, in a proportional reason. Design bounds define which properties are
theoretically expected using certain characteristics. A single effective bound is a known relation between one
characteristic and one or more property.
The design phase relies on theoretically combining, through mathematical, engineering and computational
means, known bounds to predict what the obtained properties and new bounds will be. Therefore, each time single
bounds are combined is important to study the resultant properties to establish new effective bounds for combined
characteristics. That way, design study will evolve, allow researchers to combine more and more bounds. For
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Bone Tissue Engineering: Production of scaffolds
instance, in theory, a hybrid composite made of to distinct phases, in solid and liquid physical stated, has zero
stiffness. However, there are stiff composites made of these two phases.
Summarily, the design phase of scaffold processing consists of coming up with a set of combined
characteristics that will give rise to the desired properties. To accomplish this, cause-effect relations between
characteristics and properties (design bounds) are studied and applied.
Scaffold properties: As was previously mentioned scaffold properties are the functions scaffolds should
perform. Optimized scaffolds should be able to:

Define tissue’s anatomic shape and volume;

Provide mechanical support;

Assure enhanced tissue regeneration i.e. cell delivery (from the pores or from release after
degradation).
These target properties are quantified in different ways, corresponding to the scaffolds characteristics that
cause them, in order to be studied. The desired shape is known through geometrical data obtained from the injured
host tissue. Mechanical support is quantified through linear/non linear elasticity, viscoelasticity, poroelasticity and
essentially, material distribution, though the relative importance of each of these parameters is not yet certain. As
for cell delivery, it is related to mass transport characteristics, such as permeability or diffusion, and cell material
interactions like surface chemistry and roughness.
The target properties can be achieved through distribution of the scaffold’s materials ranging four different
length scales, from less than a micron to several millimetres. At a larger scale it should be rough, as at a micro scale
it is ideally smooth.
The main design issue is to combination optimized porosity/pore size and stiffness. In order to fulfill the
mechanical properties scaffolds should be strong and stiff, which is obtained through higher material mass and less
empty space inside the construct. However, empty space provides better cell regeneration. Optimization of these
characteristics through ideal microstructure is required.
Scaffold architecture: The scale of ten hundreds of microns is termed microstructure (or mesostructure)
and, engineering at this level is called scaffold architecture, (examples in figure 6). Architecture level is controlled by
fabrication processes (rapid prototyping techniques) that will be mentioned in this paper. A determinant factor is the
smoothness of the microstructure.
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Bone Tissue Engineering: Production of scaffolds
Figure 6: examples of several scaffold architectures
The main aim of the design phase in producing a scaffold is to project an architecture that will result in the desired
properties, because the architecture is were the most properties rely. There are two ways in which this can be done
and each of them creates a volumetric 3D design by replicating unit cells periodically in 3D space.

The first consists of coming up with an architecture first and this design properties are subsequently
computed.
 In the second approach, a topology computed optimization method is used to design the architecture
based on the desired properties.
3.2.3 Fabrication of scaffolds
Many techniques have been applied or developed for scaffold fabrication, involving materials methods.
Some specific characteristics are that they don’t allow high control over
porosity or pore size and scaffolds are not produced layer by layer. Some of
them are Fibre Bonding, Solvent Casting and Porogen Leaching, Melt
Moulding, Gas Foaming, Phase Separation and Emulsion Freeze Dying, which
are summarily approached in this section: [14, 26, 27]
Fibre Bonding - Methods based in fibre bonding consist of individual
fibres that are bonded to form a 3D network or mesh, with a specific pattern
and size. They are often the first step of the scaffold fabrication and involve
Fig. 7 Example of a nonwoven
material microstructure - filter
different techniques involving weaving, knitting or physical bonding of the
fibres.[14] As examples:
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Bone Tissue Engineering: Production of scaffolds

Fibres of a polymer, PGA are mixed in a PLA solution. Solvent is removed, fibres are bonded through heating
and PLA is removed.

PLA is sprayed over PLA fibres, bonding them.
Scaffolds produced by Fibre Bonding of the polymers have a large surface area and high interconnectivity, which
allows cell attachment and facilitated diffusion of ECM. However it is difficult to control the porosity and toxic
solvents are required. When several layers are bonded it’s called membrane lamination (also obtained through other
processes).
Nonwoven meshes - Polymers fibres such as PGA fibres are folded, cut, stretched and relaxed to finally form
a nonwoven mesh, in other words meshes that are neither woven nor knitted. These nonwoven meshes, despites
having been commonly used in TE don’t provide an interconnected 3D structure for cell development. Therefore,
they require subsequent modification. Fibre bonding methods have been agreed to overcome these drawbacks,
bonding the fibres of the nonwoven mesh polymer.
Melt Moulding - Melt moulding is often combined with other methods as it produces fibres. This method
involves a mixture of a polymer powder with gelatine microspheres, or another leachable substance, that is loaded
into a mould with a desired shape. After removal from the mould, the porogen is leached through high temperature
to leave a porous scaffold. Biomolecules can be incorporated, such as drug delivery systems, because no toxic
solvents are needed for porogen removal. High temperatures are required, excluding the incorporation of proteins.
Particle Leaching - Particle Leaching is a materials method that involves porogens. Porogens are particles of a
soluble substance, such as NaCl or other salts, which are mixed with the matrix material. They are subsequently
solved and removed from the matrix leaving blank spaces that consist of pores. Other techniques involve different
types of porogens. The main advantage of this technique is that pore size and interconnectivity are easily controlled
by controlling the size of the porogen particles and their mass in the mixture.
Compression moulding and Particle Leaching - The combination of these methods involves joining a
porogen with a polymer and submitting the mixture to a specific mould compression device that physically mixes
them. This device combines several temperatures and pressures in order to obtain a mould of the desired scaffold.
The resultant structure is subsequently immersed in a solvent to remove the porogen particles. This method allows
solvent casting and particle leaching.
Solvent Casting and Particle Leaching - A mixture of a polymer solution with a porogen is casted through
freeze drying, in order to remove the polymer solvent resulting in a compact structure. It is subsequently immersed
in a porogen solvent, such as distilled water in many cases, leaving a polymeric porous scaffold. Disadvantages
include usage of toxic solvents to dissolve the polymer e.g. chloroform, low mechanical properties obtained and low
grand scale reproducibility due to intensive manual labour required. (Figure 8).
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Bone Tissue Engineering: Production of scaffolds
Figure 8: Microstructure obtained through
Solvent Casting a Particle Leaching (from
http://mitr.p.lodz.pl/biomat/overview.html Division of applied radiation chemistry,
Technical University of Lodz)
Ice particles as porogens (Figure 9) - Particle Leaching has been tried using particles of iced water. Water is
sprayed over liquid nitrogen causing it to instantaneously freeze forming round particles. These are mixed with a
chloroform solution of the polymer, at a below zero temperature, commonly -20ºC. Then the mixture is submitted to
a freeze drier to remove chloroform. Subsequently, it is left at room temperature so than the icy particles melt,
resulting in a porous scaffold. Pore size and interconnectivity are easily controlled by managing spraying speed,
travel distance and mass of the sprayed water, as well as the concentration of the polymer. A high degree of porosity
and interconnectivity are obtained, as well as a even ore distribution
through the construct. The porogen removal is easier then the
conventional porogens.[11]
Gas Foaming - Supercritical fluids are liquid or gas substances
used in a state above the critical point, gathering properties if both
these physical states. The critical point is the state of a substance that is
above the critical temperature and the critical pressure, in which liquid
and gas states can coexist. A solid mass of a polymer is put in a chamber
and exposed o the expansion of a gas in supercritical state, commonly
CO2. The gas solved in the matrix expands creating pores. High porosity
is obtained though interconnectivity lacks.
Phase Separation - Polymers are solved in a low melting
temperature solvent. The mixture is frozen rapidly and goes through a
freeze drying process to remove the solvent. Scaffolds obtained have a
large surface area and high porosity.
Emulsion Freeze Drying - An emulsion in created by mixing
polymer (PLGA) with a solvent solution and distilled water. After being
Figure 9: Particle leaching (icy
porogens)
deposited in an appropriate mould, the emulsion undergoes
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Bone Tissue Engineering: Production of scaffolds
temperature lowering and freeze drying to remove both water and polymeric solvents. A PLGA small pore sized
scaffold is obtained.
Rapid Prototyping techniques
The scaffold processing techniques referred previously (fiber bonding, solvent casting, particulate leaching,
melt molding, temperature-induced phase separation, and gas foaming) were unable to respond to the demand of
characteristics such as controlled porosity, interconnectivity and pore size. To surpass this problem, investigators
have turned their attention to rapid prototyping (RP) techniques. RP are a subset of mechanical processing
techniques which allows highly complex, but reproducible structures, to be constructed one layer at a time, via
computer-aided design models and computer-controlled tooling processes. Still, after being fabricated through RP
techniques, scaffolds undergo treatment with those and other techniques to gain extra properties such as richer
surface chemistry to enhance cell adhesion.[20]
RP methodologies include stereolithography, selective laser
sintering, ballistic particle manufacturing, 3D printing (3-DP) and
fused deposition modeling (FDM). However, among the different RP
technologies, stereolithography cannot be used for scaffold
production due to used toxic resins. Laser sintering applies high
temperatures which are not suitable for biodegradable materials.
Then, the most adequate techniques are 3-DP and FDM [4,20,27].
3D Printing
3-DP consists in the application of ink-jet technology,
Fig. 10. The 3-DP deposition device consists of five
printing on sequential layers. A default device for this technique is
main components: (1) a thermostatically controlled
described below:
heating jacket; (2) a molten co-polymer dispensing
unit consisting of a syringe and nozzle; (3) a force-
The heating jacket (1) evenly conducts heat through the
controlled plunger to regulate flow of molten co-
syringe (2). Co-polymer granules are placed in the syringe (2), which
polymer (4); a stepper motor driven x-y-z table; and
is filled with nitrogen gas that allows it to melt. The plunger (3)
(5) a positional control unit consisting of stepper-
applies pressure to the molten polymer, pushing it out through the
nozzle. Therefore, by controlling pressure in the plunger, an
motor drivers linked to a personal computer
containing software for generating fiber deposition
paths.
accurate control over the flow rate is achieved. The x-y-z table (4) is
controlled by a custom deposition program (5). The program
requires inputs of the overall scaffold dimensions, the spacing
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Bone Tissue Engineering: Production of scaffolds
between deposited fibers, the number of fiber layers, and the speed at which the x-y-z table translated. By lowering
the x-y table one layer-step in the z-direction, successive layers of rapidly solidifying fibers were laminated to
previous layers in a 0º–90º pattern creating a consistent pore size and 100% interconnecting pore volume [20].
Fused Deposition Modeling
The FDM process forms 3D objects from CAD files or
digital data produced by an imaging source such as computer
tomography or magnetic resonance imaging.[4]
The process begins with the design of a conceptual
geometric model on a CAD workstation. The design is imported
into software, which mathematically slices the conceptual
model into horizontal layers. The FDM extrusion head operates
in the X- and Y -axes while the platform lowers in the Z-axis for
each new layer to form. Effectively, the process draws the
scaffold one layer at a time.[5]
FDM uses a small temperature controlled extruder to
force out a thermoplastic filament material and deposit the
semi-molten polymer onto a platform in a layer by layer
process. The monofilament is moved by two rollers and acts as
a piston to drive the semi-molten extrude. At the end of each
finished layer, the base platform is lowered and the next layer
is deposited. In comparison with other SFF techniques, the
FDM method does not require any solvent and offers great
ease and flexibility in material handling and processing.[26]
Unfortunately, the FDM technique requires pre-formed
fibers with specific size and material properties to feed
through the rollers and nozzle which narrows the processing
Fig. 11. The FDM device.
window and its use with biodegradable polymer systems other than poly(e-caprolactone) (PCL).[20]
Example of a successful scaffold application outside TE: Hip prosthetic operation
An interesting scaffold application is the one combined with prostheses technologies, outside TE.
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Bone Tissue Engineering: Production of scaffolds
In a hip prosthetic operation, to avoid the prosthesis failure, debris due to wear on the implant must be
minimized. Debris is normally produced due to the friction between the implant and the bone. Therefore, the metal
must correctly attached to the bone.
Solutions for this issue are the usage of UHMWPE (ultra-high molecular weight polyethylene) or the stem
cementing with PMMA (polymethylmethacrylate – cement used as a prostheses/bone interface). Although these
techniques are physically well formed they face the problem of bio-incompatibility, forcing then to fail. This is where
scaffolds play their part.
The idea is to use scaffolds to make the bridge between bone and metal. This would occur by attaching the
scaffolds to the prosthesis. Bone cells would grow in the scaffold, making a physical and 100% bio-compatible link
between the prosthetic and the femur.[25]
4. Discussion:
In this paper we focused on bone tissue. Unlike other tissues, its low regenerative capacity and functional
importance make the bone tissue an important target for TE. In order to perfectly reproduce a natural tissue,
scaffolds must define the injured tissue’s shape, provide temporary mechanical support and deliver biologic
components to enhance regeneration, as previously explored. The main difference between this and other tissues
are the mechanical properties. From our perspective, mechanical properties suitable for bone TE are easily obtained
through rapid prototyping techniques. This method allows a high control over the scaffolds’ architecture, permitting
the definition of mechanical attributes and biologic release. This is where the previously used materials techniques
fail because both porosity and architecture are low controllable. (For instance, Porogen Leaching depends on the
size and amount of particles, and they can be more concentrated in certain areas. A fact that is neither controllable
nor identifiable after production.) Also, a high porosity is achieved, over 90%. This means RP techniques lead us to
functional applications. However, they are not transferable to grand scale production. As far as used materials are
concerned, the most adequate ones are polymers because they are biodegradable, malleable and similar to biologic
tissues. Because PCL has a much lower degradation rate than the other common polymers (PGA and PLA), this
polymer would be our choice as a material.
5. Conclusion:
Tissue Engineering, namely Bone Tissue Engineering, is a scientific area taking the first steps, as not many
tissue engineered portions have been accomplished and implanted, and certainly not applied as a common medical
procedure. Because Scaffold production, varying accordingly to the techniques and materials used, requires
extended laboratory work and labour. The high number of factors that have to be attended in the production of a
scaffold conflicts with an industrial production and commercial distribution perspective. The latest developed
techniques, namely FDM and 3-DP seem to us as the as the way through which bone TE will evolve.
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Bone Tissue Engineering: Production of scaffolds
In a short term period of time scaffolds are expected to be used extensively, replacing current techniques to heal
damaged tissue portions. The complexity of this work, such as the combination of many characteristics, the
unknown bounds between several characteristics and respective resultant properties, and the simple fact that we
want to resemble nature in its best work, producing life, is a huge obstacle to the success of this vanguard science
field tissue engineering.
Acknowledgments:
We would like to thank professor Paulo R. Fernandes for the support, attention and information provided for this
work, as well as professor Fátima Vaz for the information given.
References
[1] - http://www.nsf.gov/pubs/2004/nsf0450/start.htm
[2] - http://en.wikipedia.org/wiki/Tissue_engineering
[3] - Basic Histology, 11th edition, Junqueira e Carneiro, McGraw-Hill, 2005
[4] - Hutmacher, D. (2000). Scaffolds in Tissue Engineering bone and cartilage. Biomaterials, 21, 2529-2543.
[5] - Hutmacher, D. (2001). Scaffold design and fabrication technologies for engineering tissues – state of the art and future
perspectives. J Biomaster. Sci. Polymer Edn, Vol. 12, (1), 107-124.
[6] - Grant, P., James S., Mikhalovsky S. e Tomlins, P. Measurement of pore size and porosity of tissue scaffolds.
[7] - Hollister, S., Maddox, R, e Taboas, J. (2002). Optimal design and fabrication of scaffolds to mimic tissue properties and
satisfy biological constraints. Biomaterials, 23 ,4095-4103.
[8] - Hutmacher, D., Kaps C., Kläring Svea, Ng Kee e Sittinger, M. (2003). Elastic cartilage engineering using novel scaffold
architectures in combination with a biomimetic cell carrier. Biomaterials, 23, 4445-4458.
[9] - Cheng, T., Hammer, B., Hutmacher, D., Oberholzer, M. Rohner, D. (2003). In vivo efficacy of bone-marrow-coated
polycaprolactone scaffolds for the reconstruction of orbital defects in the pig. Wiley periodicals, Inc J Biomed Master Res Part B:
Appl Biomater 66B, 574-580.
[10] - Cao, T.,Ho, K., Hutmacher, D., Rai e B., Teoh (2005). Novel PCL-based honeycomb scaffolds as drug delivery systems for rh
BMP-2. Biomaterials, 26, 3739-3748.
[11] - Chen, G., Ushida, T. e Tateisshi, T. (2001) Development of biodegradable porous scaffolds for Tissue Engineering. Materials
Science and Engineering, C 17, 63-69.
[12] - Barbanti, S., Duek, E. e Zavaglia, C. (2005). Polímeros bioreabsorvíveis na engenharia de tecidos. Polímeros: Ciência e
Tecnologia15, (1), 13-21.
[13] - Beer, B., Larcher Y., Schnabelrauch,M , Vogt, S. e Wilke, I. ( ). Fabrication of highly porous scaffold materials based on
functionalizes oligolactides and preliminary results on their use in bone Tissue Engineering.
[14] – Gomes, Maria Manuela Estima (2004). A Bone Tissue Engineering strategy based on starch scaffolds and bone marrow
cells cultured in a flow perfusion bioreactor
16/17
Bone Tissue Engineering: Production of scaffolds
[15] - Coelho, P. G., Fernandes, P. R., Guedes, J. M., Reis, N., Rodrigues, H. C.(2005). A Hierarchical model for boné remodelling –
application to scaffold design
[16] – Heo, Su-Jin; Hyun, Young-Taek; Kim, Seung-Eon; Shin, Jung-Woog (2007). Characterization of PCL/HA composite scaffolds
for bone Tissue Engineering.
[17] - P. X. Ma, J. H. Elisseeff, “Scaffolds in tissue engineering”, Taylor&Francis, pp. 27-46
[18] - V. Griffith, “The growth of tissue engineering”, 2006
[19] - P. G. Coelho, P. R. Fernandes, J. M. Guedes, N. Reis, H. C. Rodrigues, “A hierarchical model for bone remodeling –
applications to scaffold design”, pp. 2
[20] - T.B.F. Woodfield, J. Malda, J. de Wijn, F. Peters, J. Riesle, C.A. van Blitterswijk, “Design of porous scaffolds for cartilage
tissue engineering using a three-dimensional fiber-deposition technique”, Biomaterials, pp. 4149-4150
[22] - Hollister, JH, Porous scaffold design for tissue engineering, Nature materials, Vol. 4, pp.512-524., 2005
[23] - Shimko, DA, Shimko VF, Sander, EA, Dickson, KF and Nauman, EA, Effect of Porosity on the Fluid Flow Characteristics and
Mechanical Properties of Tantalum Scaffolds, Journal of Biomedical Material Research part B: Applied Biomaterials, 73B, pp.315324, 2005
[24] - Lin, CY, Kikuchi, J, Hollister, SJ, A novel method for biomaterial scaffold internal architecture design to match bone elastic
properties with desired porosity, Journal of Biomechanics 37, pp.623-636, 2004
[25] - http://web.mit.edu/filip/Public/3.082/index.html
[26] – Fátima Vaz, Apresentação Scaffolds para os alunos
[27] - www.springerlink.com
[28] - Scott J. Hollister, Cheng Yu Lin, Computational design of tissue engineering scaffolds, Computer methods in applied
mechanics and engineering.
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