GITAM DENTAL COLLEGE & HOSPITAL

DEPARTMENT OF

ORAL & MAXILLOFACIAL SURGERY

SEMINAR ON

Tissue engineering in oral and maxillofacial surgery

Presented By:

Dr. Sambhav K Vora

III MDS

Contents

1. Overview

2. Cells as building blocks

a.  Extraction

b. Types of cells

3. Scaffolds

a. Materials

b. Synthesis

4.  Assembly methods

5.  Tissue culture

a. Bioreactors

6. Organotypic And Histiotypic Models Of Engineered Tissues

7. Fiber bonding

8.  References

Tissue engineering is considered as a field in its own right. It is the use of a

combination of cells, engineering and materials methods, and

suitable biochemical and physio-chemical factors to improve or replace

biological functions. While most definitions of tissue engineering cover a broad

range of applications, in practice the term is closely associated with applications

that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood

vessels, bladder, skin etc.). Often, the tissues involved require certain

mechanical and structural properties for proper functioning. The term has also

been applied to efforts to perform specific biochemical functions

using cells within an artificially-created support system (e.g. an artificial

pancreas, or a bioartificial liver). The term regenerative medicine is often used

synonymously with tissue engineering, although those involved in regenerative

medicine place more emphasis on the use of stem cells to produce tissues.

Definition –

It is "an interdisciplinary field that applies the principles of engineering and life

sciences toward the development of biological substitutes that restore, maintain,

or improve tissue function or a whole organ".

Tissue engineering has also been defined as "understanding the principles of

tissue growth, and applying this to produce functional replacement tissue for

clinical use

Biologic tissues consist of the cells, the extracellular matrix (made up of a

complex of cell secretions immobilized in spaces continuous with cells), and the

signaling systems, which are brought into play through differential activation of

genes or cascades of genes whose secreted or transcriptional products are

responsible for tissue building and differentiation.

The triad of tissue engineering, which is based on the three basic components of

biologic

tissues. The principal components of scaffolds (into which the extracellular

matrix is organized in actual tissues) are collagen biopolymers, mainly in the

form of fibers and fibrils. Other forms of polymer organization have also been

used (gels, foams, and membranes) for engineering tissue substitutes. The

various forms can be combined in the laboratory to create imitations of

biopolymer organization in specific tissues. Scaffolds can be enriched with

signaling molecules, which may be bound to them or infused into them. The

focus of the triad is the prosthesis.

The incorporation of cells in reconstituted prosthetic tissue devices often can

provide the signals needed for tissue building, but the repertoire of feats of

differentiation may be limited (see section on stem cells below). For example,

although cultivated allogeneic keratinocytes and dermal fibroblasts, plus a

collagen scaffold, can be assembled into a graftable organ that differentiates a

fully formed epidermis, having a stratum corneum with barrier properties and a

basal lamina, the secondary derivatives such as hair follicles and sebaceous and

sweat glands do not develop. Improving the quality and functionality of tissue-

engineered skin will mean the introduction of new versions of skin that address

the clinical needs in a way better than their precursors have addressed them.

Oversimplified materials used for the scaffolding component (the extracellular

matrix of the tissue being engineered) may be limiting. If the scaffold cannot

provide the developmental signals for tissue building needed by the cells that

are seeded into it in vitro, or mobilized by it in vivo, tissue building might fail,

as it does when a Dacron sleeve is used in vivo to replace a segment of artery.

Man-made biopolymers such as poly(L-lactic acid), poly(glycolic acid),

polyglycolide, and poly(L-lactide) have built-in ranges of degradation times that

may not be

in tune with the required rate of remodeling characteristic of regeneration,

because the polymers are not susceptible to breakdown by metalloproteinases

and tryptic enzymes, which function normally in the remodeling of collagen-

based scaffolds. If they are out of tune with the remodeling activities of cells

that occupy the transient scaffolds, including matrix biosynthesis, the process of

matrix renewal may be compromised. A potentially valuable

attribute of acellular materials installed in vivo as precursors of tissue

replacements is their ability to mobilize appropriate cells from contiguous

tissues, circulating body fluids, or stem cell sources, making it unnecessary to

populate the prosthesis with cells before implantation. Because acellular

implants of man-made biopolymers are information poor, cell mobilization and

vascularization may fail, and so may the organization of the mobilized cells and

their secretory output, needed to regenerate a replacement matrix and a

functional tissue.

STEM CELLS

Scaffolds can be populated with adult-derived cells that are capable of

undergoing subsequent differentiation after being cultivated in vitro. In this

category are cells of the skin, cartilage, muscle, tendon, ligament, bone, adipose

tissue, endothelium, and many others. Aside from skin, the foregoing cell types

are harbored as stem cell populations in the marrow, in addition to those of the

hematopoietic and immune systems, but the diversity of mesenchymal

and possibly other cell types in the marrow still needs to be probed. Stimulating

factors, the cytokines, which move some of the cells into the circulation, will be

important for engineering acellular scaffolds. Other stem cells are available to

tissue engineering, such as the satellite cells found in striated muscle and to

some degree keratinocytes

of the skin. Where host cells are available, an acellular scaffold, particularly one

enhanced with signals and possessing the binding sites needed for cell

attachment, can mobilize host cells that will populate the prosthesis. Already,

new sources of stem cells, particularly neuronal stem cells, have been

discovered in the adult brain and are opening the door to the reconstitution of

nerve tissue for tissue engineering. In addition to the striatum, which harbors

extracellular growth factor-responsive stem cells, other central nervous system

sources of stem cells in adult vertebrates include the hippocampus and the

periventricular subependymal zone. Stem cells giving rise to neuronal and glial

phenotypes, from the adult rat hippocampus, are isolated with the help of

fibroblast growth factor 2 and are stimulated to differentiate with the help of

retinoic acid. Stem cells from these sources are also

present in the adult human brain. The discovery that embryonic stem cells can

be recovered from human fetal tissue and propagated for long periods without

losing their toti- or pluripotency is of huge importance for tissue engineering.

How to direct their

differentiation is a subject of high current interest.

EXTRACELLULAR MATRIX STRUCTURE AND FUNCTION

COMPOSITION AND ORGANIZATION

One of the most critical elements of tissue engineering is the ability to mimic

the ECM scaffolds that normally serve to organize cells into tissues. ECMs are

composed of different collagen types, large glycoproteins (e.g., fibronectin,

laminin, entactin, osteopontin), and proteoglycans that contain large

glycosaminoglycan side chains (e.g., heparan sulfate, chondroitin sulfate,

dermatan suflate, keratan sulfate, hyaluronic acid). Although all ECMs share

these components, the organization, form, and mechanical properties of ECMs

can vary widely in different tissues depending on the chemical composition and

three-dimensional organization of the specific ECM components that are

present. For example, interstitial collagens (e.g., types I and III) self-assemble

into a three-dimensional lattice, which, in turn, binds fibronectin and

proteoglycans. This type of native ECM hydrogel forms the backbone of loose

connective tissues, such as dermis. In contrast, basement membrane collagens

(types IV and V) assemble into planar arrays; when these collagenous sheets

interact with fibronectin, laminin, and heparan sulfate proteoglycan, a planar

ECM results (i.e., the “basement membrane”). The ability of tendons to resist

tension and of cartilage and bone to resist compression similarly result from

local differences in the organization and composition of the ECM.

Matricellular proteins-

THROMBOSPONDIN-1 AND THROMBOSPONDIN-2

Thrombospondin-1 is a 450,000-Da glycoprotein with seven modular domains.

At least five different extracellular matrix-associated proteins are able to bind to

thrombospondin-1: collagens I and V, fibronectin, laminin, fibrinogen, and

SPARC . One cytokine known to interact with thrombospondin-1 is scatter

factor/hepatocyte growth factor (HGF), a known angiogenesis-promoting factor.

Thrombospondin-1 has also been shown to interact specifically with another

cytokine, transforming growth factor (TGF- ). Thrombospondin-1 is also able

to influence cell adhesion and cell shape. For example, it will diminish the

number of focal adhesions of bovine aortic endothelial cells and thus will

promote a migratory phenotype. Thrombospondin-1, therefore, has been

proposed to modulate cell–matrix interaction to allow for cell migration when

necessary. Thrombospondin-1 and -2 can act as negative regulators of cell

growth. In particular, endothelial cells are susceptible to an inhibition of

proliferation by both proteins and, as such, have been classified as inhibitors of

angiogenesis

TENASCIN-C

Tenascin-C is a matricellular protein with a widespread pattern of

developmental expression

in comparison to a restricted pattern of expression in adult tissues

OSTEOPONTIN

Osteopontin associates with the extracellular matrix, in that it binds to

fibronectin and to collagens I, II, III, IV, and V. Osteopontin also affects

cellular signaling pathways by virtue of its capacity to act as a ligand for

multiple integrin receptors as well as CD44 (Denhardt and Noda, 1998; Weber

et al., 1996). Thus osteopontin, like most of the matricellular proteins, is able to

act as a bridge between the extracellular matrix and the cell surface. The

promotion of cell survival is another property ascribed to osteopontin. Finally,

osteopontin appears to be involved in inflammatory responses. Expression of

osteopontin was found to increase during intradermal macrophage infiltration,

and purified osteopontin injected into the rat dermis led to an increase in the

number of macrophages at the site of administration.

SPARC

SPARC (also known as BM-40 and osteonectin) was first identified as a

primary component

of bone but has since been known to have a wider distribution. SPARC is found

in the gut epithelium, which normally exhibits rapid turnover, and in healing

wounds. Another significant effect of SPARC on cells in culture is its capacity

to elicit changes in cell shape.

Role of growth factors in bone healing:5

Growth factors that can help in bone healing are

Platelet derived growth factor

Transforming growth factor

Insulin deriver growth factor

Fibroblast derived growth factor

Platelet derived growth factor-

It is released from platelet alpha granule, macrophages or

monocytes,endothelial cells & as well as from osteoblast cells.

The specific activities of PDGF includes mitogenesis(increase in the cell

population of healing cells), angiogenesis(endothelial mitosis into

functional capillaries), & macrophage activation(debridement of the

wound site &second phase source of growth factors for continued repair

& bone regeneration).

Transforming growth factor:

It is present in abundance in the bone matrix, with bone representing the

major site for the storage of the TGF –beta in the body.the primary effect

of TGF –beta is on the bone formation, particularly in the early phase of

the osteoblast development.it stimulates matrix protein synthesis by

human osteoblasts.the most important function of TGF beta 1 & TGF

beta 2seems to be chemotaxis &mitogenesis of osteoblast precursors, &

they also have the ability to stimulate osteoblast deposition of the

collagen matrix of wound healing & of the bone. In addition they also

inhibit osteoclast formation & also bone resorption, thus favoring bone

formation than resorption.

It directly inhibits both proliferation & differentiation of the osteoclast

precursor cells & inhibits the function of the mature osteoclasts with

reduction in reactive e oxygen radicals.

Insulin like growth factor:

It consists of two proteins-IGF 1(somatomedin c) & IGF2 (skeletal

growth factor) which are secreted by osteoblasts,both the factors induce

preosteoblast proliferation & differentiation, osteoblast collagen

synthesis,& inhibit collagen breakdown.IGF bound to the protein in the

matrix may be released in the active form following osteoclastic

resoeption.locaaly produced IGF1secreted by fibroblast& cells in the

bone & cartilage is controlled by variety of factors. Corticosteroids

reduces IGF1 synthesis.

Fibroblast growth factor:

The matrix proteins, acidic FGF & basic FGFare produced by

osteoblast,bind heparin & are angiogenic factors. But there effects on

bone invivo are not known.in vitro they cause proliferation of osteoblast

progenitor cells but inhibit differentiation, & do not appear to effect the

osteoclast.FGFs stimute new bone formation.

BONE MORPHOGENETIC PROTEIN:

One group of cytokines, bone morphogenetic proteins (BMPs), has been

demonstrated to have true osteoinductive properties. BMPs have been proven to

stimulate new bone formation in vitro and in vivo. In addition, they play critical

roles in regulating cell growth, differentiation, and apoptosis a variety of cells

during development, particularly in osteoblasts and chondrocytes.

There are currently 16 identified BMPs, although only a subset have been found

to be expressed

in fracture healing. BMPs were initially characterized by Urist; their

identification was based on the capacity of demineralized bone powder to

induce de novo bone formation in an intramuscular pouch, demonstrating the

ability to directly induce mesenchymal connective tissue to become bone-

forming osteoprogenitor cell.

During fracture repair & graft healing, BMP-2, BMP-3 (also known as

osteogenin), BMP-4, and BMP-7 (OP-1) have been found to be expressed to

varying degrees. BMPs are initially released in low levels from the extracellular

matrix (ECM) of fractured bone. Osteoprogenitor cells in the cambium layer of

the periosteum may respond to this initial BMP presence by differentiating into

osteoblast. Immunolocalization demonstrates an increase in detectable BMP-

2=4 in the cambium region of the periosteum. BMP receptor IA and IB

expression is dramatically increased in osteogenic cells of the periosteum near

the ends of the fracture in the early postfracture period or post grafting period.

Approximately 1–2 weeks postfracture or graft placement, BMP-2=4 expression

is maximal in chondroid precursors, while hypertrophic chondrocytes and

osteoblasts show moderate levels of expression. It is hypothesized that the role

of BMPs in fracture repair is to stimulate differentiation in osteoprogenitor and

mesenchymal cells that will result in osteoblasts and chondrocyte. As these

primitive cells mature, BMP expression decreases rapidly. BMP expression

temporarily recurs in chondrocytes and osteoblasts during matrix formation, and

eventually decreases during callus remodeling.

TYPES OF BMPs THEIR PROPERTIES, LOCATION & ROLES:

BMP-1: functions as procollagen C- proteinase responsible for removing

carboxyl propeptides from procollagen I, 2 ,3 . It activates bmp but not

osteoinductive

BMP-2: osteoinductive , embryogenesis, differentiation of osteoblasts ,

adipocytes, chondrocytes & also may influence osteoclast activity , may inhibit

bone healing

It is located in the bone, spleen, liver, brain, kidney, heart, placenta.

BMP-3:(osteogenin)- osteoinductive , promotes chondrogenic phenotype

It is located in the lung, kidney, brain, intestine.

BMP-4: osteoinductive, embryogenesis, fracture repair, gastrulation &

mesoderm formation (mouse).

It is located in the apical ectodermal ridge, meninges, lung, kidney, liver.

BMP-5:-osteoinductive, embryogenesis. It is located in lung, kidney, and liver.

BMP-6:- not osteoinductive, embryogenesis, neuronal maturation, regulates

chondrocyte differentiation. It is located in the lung, brain, kidney, uterus,

muscle, skin.

BMP-7:-(osteogenic protein-1) osteoinductive, embryogenesis, repair of long

bones, alveolar bone, differentiation of osteoblasts,chondroblasts & adipocytes.

It is located in the adrenal glands, bladder, brain, eye, heart, kidney, lung,

placenta, spleen & skeletal muscles.

BMP-8(osteogenic protein-2) osteoinductive, embrogenesis,

spermatogenesis(mouse).

BMP-8B(osteogenic protein-3)initiation & maintainance of

spermatogenesis(mouse).

BMP-9:-osteoinductive, stimulates hepatocyte proliferation, hepatocyte growth

& function.

BMP-12 & BMP-13:-inhibition of terminal differentiation of myoblasts.

Cells as building blocks

Tissue engineering utilizes living cells as engineering materials. Examples

include using living fibroblasts in skin replacement or repair, cartilage repaired

with livingchondrocytes, or other types of cells used in other ways.

Cells became available as engineering materials when scientists at Geron Corp.

discovered how to extend telomeres in 1998, producing immortalized cell lines

Before this, laboratory cultures of healthy, noncancerous mammalian cells

would only divide a fixed number of times, up to the Hayflick limit.

Extraction

From fluid tissues such as blood, cells are extracted by bulk methods,

usually centrifugation or apheresis. From solid tissues, extraction is more

difficult. Usually the tissue is minced, and then digested with

the enzymes trypsin or collagenase to remove the extracellular matrix that holds

the cells. After that, the cells are free floating, and extracted using

centrifugation or apheresis.

Digestion with trypsin is very dependent on temperature. Higher temperatures

digest the matrix faster, but create more damage. Collagenase is less

temperature dependent, and damages fewer cells, but takes longer and is a more

expensive reagent.

Types of cells

Cells are often categorized by their source:

Autologous  cells are obtained from the same individual to which they will

be reimplanted. Autologous cells have the fewest problems with rejection

and pathogen transmission, however in some cases might not be available.

For example in genetic disease suitable autologous cells are not available.

Also very ill or elderly persons, as well as patients suffering from severe

burns, may not have sufficient quantities of autologous cells to establish

useful cell lines. Moreover since this category of cells needs to be harvested

from the patient, there are also some concerns related to the necessity of

performing such surgical operations that might lead to donor site infection or

chronic pain. Autologous cells also must be cultured from samples before

they can be used: this takes time, so autologous solutions may not be very

quick. Recently there has been a trend towards the use of mesenchymal stem

cells frombone marrow and fat. These cells can differentiate into a variety of

tissue types, including bone, cartilage, fat, and nerve. A large number of

cells can be easily and quickly isolated from fat, thus opening the potential

for large numbers of cells to be quickly and easily obtained.

Allogeneic cells come from the body of a donor of the same species. While

there are some ethical constraints to the use of human cells for in

vitro studies, the employment of dermal fibroblasts from human foreskin has

been demonstrated to be immunologically safe and thus a viable choice for

tissue engineering of skin.

Xenogenic cells are these isolated from individuals of another species. In

particular animal cells have been used quite extensively in experiments

aimed at the construction of cardiovascular implants.

Syngenic or isogenic cells are isolated from genetically identical organisms,

such as twins, clones, or highly inbred research animal models.

Primary cells are from an organism.

Secondary cells are from a cell bank.

.

Scaffolds

Cells are often implanted or 'seeded' into an artificial structure capable of

supporting three-dimensional tissue formation. These structures, typically

called scaffolds, are often critical, bothex vivo as well as in vivo, to

recapitulating the in vivo milieu and allowing cells to influence their own

microenvironments. Scaffolds usually serve at least one of the following

purposes:

Allow cell attachment and migration

Deliver and retain cells and biochemical factors

Enable diffusion of vital cell nutrients and expressed products

Exert certain mechanical and biological influences to modify the behaviour

of the cell phase

This animation of a rotating Carbon nanotubeshows its 3D structure. Carbon

nanotubes are among the numerous candidates for tissue engineering scaffolds

since they arebiocompatible, resistant to biodegradation and can be

functionalized with biomolecules. However, the possibility of toxicity with non-

biodegradable nano-materials is not fully understood.

To achieve the goal of tissue reconstruction, scaffolds must meet some specific

requirements. A high porosity and an adequate pore size are necessary to

facilitate cell seeding and diffusion throughout the whole structure of both cells

and nutrients. Biodegradability is often an essential factor since scaffolds should

preferably be absorbed by the surrounding tissues without the necessity of a

surgical removal. The rate at which degradation occurs has to coincide as much

as possible with the rate of tissue formation: this means that while cells are

fabricating their own natural matrix structure around themselves, the scaffold is

able to provide structural integrity within the body and eventually it will break

down leaving the neotissue, newly formed tissue which will take over the

mechanical load. Injectability is also important for clinical uses.

Materials

Many different materials (natural and synthetic, biodegradable and permanent)

have been investigated. Most of these materials have been known in the medical

field before the advent of tissue engineering as a research topic, being already

employed as bioresorbable sutures. Examples of these materials

are collagen and some polyesters.

New biomaterials have been engineered to have ideal properties and functional

customization: injectability, synthetic manufacture,biocompatibility, non-

immunogenicity, transparency, nano-scale fibers, low concentration, resorption

rates, etc. PuraMatrix, originating from the MIT labs of Zhang, Rich,

Grodzinsky and Langer is one of these new biomimetic scaffold families which

has now been commercialized and is impacting clinical tissue engineering.

A commonly used synthetic material is PLA - polylactic acid. This is a

polyester which degrades within the human body to form lactic acid, a naturally

occurring chemical which is easily removed from the body. Similar materials

are polyglycolic acid (PGA) and polycaprolactone(PCL): their degradation

mechanism is similar to that of PLA, but they exhibit respectively a faster and a

slower rate of degradation compared to PLA.

Scaffolds may also be constructed from natural materials: in particular different

derivatives of the extracellular matrix have been studied to evaluate their ability

to support cell growth. Proteic materials, such as collagen or fibrin, and

polysaccharidic materials, like chitosan or glycosaminoglycans (GAGs), have

all proved suitable in terms of cell compatibility, but some issues with potential

immunogenicity still remains. Among GAGs hyaluronic acid, possibly in

combination with cross linking agents (e.g.glutaraldehyde, water soluble

carbodiimide, etc...), is one of the possible choices as scaffold material.

Functionalized groups of scaffolds may be useful in the delivery of small

molecules (drugs) to specific tissues.

Synthesis

A number of different methods has been described in literature for preparing

porous structures to be employed as tissue engineering scaffolds. Each of these

techniques presents its own advantages, but none is devoid of drawbacks.

Nanofiber Self-Assembly: Molecular self-assembly is one of the few

methods to create biomaterials with properties similar in scale and chemistry

to that of the natural in vivo extracellular matrix (ECM). Moreover, these

hydrogel scaffolds have shown superior in vivo toxicology and

biocompatibility compared with traditional macroscaffolds and animal-

derived materials.

Textile technologies: these techniques include all the approaches that have

been successfully employed for the preparation of non-woven meshes of

different polymers. In particular non-woven polyglycolide structures have

been tested for tissue engineering applications: such fibrous structures have

been found useful to grow different types of cells. The principal drawbacks

are related to the difficulties of obtaining high porosity and regular pore size.

Solvent Casting & Particulate Leaching (SCPL): this approach allows the

preparation of porous structures with regular porosity, but with a limited

thickness. First the polymer is dissolved into a suitable organic solvent (e.g.

polylactic acid could be dissolved into dichloromethane), then the solution is

cast into a mold filled with porogen particles. Such porogen can be an

inorganic salt like sodium chloride, crystals of saccharose, gelatin spheres

or paraffin spheres. The size of the porogen particles will affect the size of

the scaffold pores, while the polymer to porogen ratio is directly correlated

to the amount of porosity of the final structure. After the polymer solution

has been cast the solvent is allowed to fully evaporate, then the composite

structure in the mold is immersed in a bath of a liquid suitable for dissolving

the porogen: water in case of sodium chloride, saccharose and gelatin or an

aliphatic solvent like hexane for paraffin. Once the porogen has been fully

dissolved a porous structure is obtained. Other than the small thickness range

that can be obtained, another drawback of SCPL lies in its use of organic

solvents which must be fully removed to avoid any possible damage to the

cells seeded on the scaffold.

Gas Foaming: to overcome the necessity to use organic solvents and solid

porogens a technique using gas as a porogen has been developed. First disc

shaped structures made of the desired polymer are prepared by means of

compression molding using a heated mold. The discs are then placed in a

chamber where are exposed to high pressure CO2 for several days. The

pressure inside the chamber is gradually restored to atmospheric levels.

During this procedure the pores are formed by the carbon dioxide molecules

that abandon the polymer, resulting in a sponge like structure. The main

problems related to such a technique are caused by the excessive heat used

during compression molding (which prohibits the incorporation of any

temperature labile material into the polymer matrix) and by the fact that the

pores do not form an interconnected structure.

Emulsification/Freeze-drying: this technique does not require the use of a

solid porogen like SCPL. First a synthetic polymer is dissolved into a

suitable solvent (e.g. polylactic acid in dichloromethane) then water is added

to the polymeric solution and the two liquids are mixed in order to obtain

an emulsion. Before the two phases can separate, the emulsion is cast into a

mold and quickly frozen by means of immersion into liquid nitrogen. The

frozen emulsion is subsequently freeze-dried to remove the dispersed water

and the solvent, thus leaving a solidified, porous polymeric structure. While

emulsification and freeze-drying allows a faster preparation if compared to

SCPL, since it does not require a time consuming leaching step, it still

requires the use of solvents, moreover pore size is relatively small and

porosity is often irregular. Freeze-drying by itself is also a commonly

employed technique for the fabrication of scaffolds. In particular it is used to

prepare collagen sponges: collagen is dissolved into acidic solutions of acetic

acid or hydrochloric acid that are cast into a mold, frozen with liquid

nitrogen then lyophilized.

Thermally Induced Phase Separation (TIPS): similar to the previous

technique, this phase separation procedure requires the use of a solvent with

a low melting point that is easy to sublime. For example dioxane could be

used to dissolve polylactic acid, then phase separation is induced through the

addition of a small quantity of water: a polymer-rich and a polymer-poor

phase are formed. Following cooling below the solvent melting point and

some days of vacuum-drying to sublime the solvent a porous scaffold is

obtained. Liquid-liquid phase separation presents the same drawbacks of

emulsification/freeze-drying.

CAD/CAM Technologies: since most of the above described approaches are

limited when it comes to the control of porosity and pore size, computer

assisted design and manufacturing techniques have been introduced to tissue

engineering. First a three-dimensional structure is designed using CAD

software, then the scaffold is realized by using ink-jet printing of polymer

powders or through Fused Deposition Modeling of a polymer melt.

ORGANOTYPIC AND HISTIOTYPIC MODELS OF

ENGINEERED TISSUES-

THE COLLAGEN GEL MODEL-The model uses a collagen gel scaffold prepared by combining, in the cold, a

neutralized 0.3–1.0 mg/ml solution of acid extracted collagen with medium,

serum, and mesenchymal cells (Bell et al., 1979)—dermal fibroblasts, for

example, if the goal is to fabricate a skin equivalent. The bacteriological petri

plate or other vessel, to which cells do not attach, into which the mix is poured,

is incubated at 37"C in a 5% CO2 incubator. The collagen polymerizes, when

neutralized and warmed, forming a lattice of fine fibrils, 10–20 nm in size,

which trap fluid. The result is a gel in which the previously added cells are

distributed. The mesenchymal cells in the gel extend and attach podial processes

to the collagen fibrils and withdraw the processes with the attached fibrils

toward the cell body. As the fibrils are bundled by the cells, fluid is squeezed

out of the lattice. The process of gel contraction, known as syneresis, can reduce

the size of the collagen lattice 30- to 40- fold, depending on the cell and

collagen concentration used. The condensed gel is tissuelike in its

consistency, providing a substrate on which epithelial, endothelial, or

mesothelial cells may be plated.

THE SKIN EQUIVALENT AS A DEVELOPMENTAL MODEL-

The first skin consisting of “living” dermis and epidermis reconstituted from

cultivated cells and collagen scaffolding (Bell et al., 1979, 1981, 1983) was

shown to undergo virtually complete differentiation in vitro, lacking, however,

pigment, sweat glands, neurogenic elements, a micro-circulation, and hair

follicles. The model can be reproduced faithfully and be kept alive in vitro for

months, at least. Although collagenolytic activity is high in young dermal

equivalents (Nusgens et al., 1984; Rowling et al., 1990), possibly associated

with tissue remodeling, it has been observed that the resistance of dermal

equivalents to breakdown by collagenase is greatly enhanced by 30 days of

cultivation in vitro, suggesting that extensive cross-linking (probably by cell-

secreted lysyl oxidase) of the collagen fibrils has occurred, as shown by

Rowling et al. (1990). Continued differentiation of the model in vitro and the

resemblance of cells in the matrix to their in vivo counterparts, rather than to

cells grown on plastic in two dimensions, are distinguishing feature.

THE SKIN EQUIVALENT AS AN IMMUNOLOGICAL MODEL-

The skin equivalent can be constituted with cultivated parenchymal cells free of

any subsets of immune cells normally found in the dermis and epidermis. Using

the X chromosome as a genetic marker, female cells are used to make up skin

equivalents, which are then transplanted to male hosts across a major

histocompatibility barrier, e.g., from Brown Norway to Fisher rats. Sher

et al. (1983) demonstrated in the rat model, by karyotyping cells grafted in skin

equivalents, that allogeneic fibroblasts were not rejected. It has been reported

that clinical trials of skin equivalents made up with human allogeneic

keratinocytes as well as allogeneic fibroblasts do not provoke an

immune reaction in recipients (Parenteau et al., 1994). The model should be a

valuable tool for determining the roles played by cells of the immune system

and the microcirculation in allograft rejection of actual skin. It should allow use

of cells of any genotype and of human origin to study genetic abnormalities, as

well as the contribution of specific genetic loci to skin development by

transplanting skin equivalents to immunodeficient rodents.

THE SKIN EQUIVALENT AS A DISEASE MODEL-

A psoriasis model was fabricated to test the contribution of psoriatic dermal

fibroblasts to the expression of features of the disease in vitro (Saiag et al.,

1985). A button of normal keratinocytes suspended in medium was plated in the

centers of dermal equivalent disks constituted with normal human or psoriatic

dermal fibroblasts, and the rate of spreading of keratinocytes over the dermal

substrate was measured. It was observed that the psoriatic fibroblasts induced

hyperproliferation and greater spreading of keratinocytes compared with the

growth and spreading induced by control fibroblasts, suggesting that dermal

cells may play a role in the progress of the disease. In addition to the study of

psoriasis, and other epidermal diseases such as epidermolysis bullous,

the model should provide an in vitro basis for studying dermal connective tissue

disorders, including dermatosparaxiis and sclerosis. It is obvious that any pair of

populations of mesenchymal cells and epithelial cells, of which one or both is

diseased or aberrant, can be used in the threedimensional coculturing system for

studying the expression of features of the disease and testing modalities of

treatment.

THE SKIN EQUIVALENT AS A WOUND HEALING MODEL-

Two-tissue skin models can be used in vitro to analyze the role of dermis in

epidermal wound healing (Bell and Scott, 1992). After constituting a

differentiated skin equivalent in a 24-mm multiwall well-plate insert, a central

disk of the skin is removed with a punch. The acellular layer of collagen in

contact with the membrane of the insert is replaced and the remainder of the gap

is filled with a collagen scaffold to the level of the interface between the dermis

and epidermis. The rate of overgrowth of the neodermis by keratinocytes and

the development of the epidermis can be taken as measures of the effectiveness

of the design of neodermis as an interacting substrate. The wound healing

model can accommodate acellular dermal scaffolds with or without signals to

test their effectiveness in attracting dermal fibroblasts from the surrounding

matrix.

VASCULAR MODELS WITH CELLS ADDED-

Vascular models that examine the effect of shear and other forces on

monolayers of endothelial cells in vitro have been developed by Nerem et al.

(1993), who have shown that the rate of endothelial cell proliferation is

decreased by flow and that entry of cells into a cycling state is inhibited. They

suggest that a coculture system in which the endothelium is supported on

smooth muscle tissue would be superior for providing a more physiologic

environment. Such a system was developed by Weinberg and Bell (1986), who

showed that a basal lamina was laid down between the endothelium and the

contiguous smooth muscle tissue, cast in the form of a small-caliber tube

in vitro. Fabricating the vessel was a three-step procedure. The first tissue layer

cast around a small caliber mandrel was a smooth muscle cell media, whose

ends were anchored in a velcro cuff or held fast by ridges and grooves in the

mandrel until radial contraction made space for bands that were

secured around the ends. Hence the mechanical restraint imposed on the

contracting tube allowed contraction to occur radially but not longitudinally,

because each end of the vessel equivalent was held fast. The second tissue layer

cast was the adventitia surrounding the smooth muscle media.

To make room for it, the fluid expressed from the collagen gel scaffold was

drawn out of the casting tube, and the mixture of adventitial fibroblasts

suspended in medium containing neutralized collagen was introduced into the

space between the media and the wall of the cylindrical casting chamber. After

the adventitia had contracted radially but not longitudinally, because the media

provided a frictional surface that prevented it, the mandrel was extracted,

leaving a lumen of the tissue tube that was filled with a suspension of

endothelial cells. The cells came to rest on the inner surface of the media as the

tube was rotated.

VASCULAR MODELS WITHOUT CELLS-

Vascular prostheses constructed from Dacron and other synthetics have been in

use for many years but are known to elicit persistent inflammatory reactions and

to become occluded. The thermosetting polymers are not biodegradable and do

not integrate with host tissues, but some successes have been reported under

limited conditions in experimental animals. For the foregoing reasons, other

acellular materials have been proposed and tried as arterial substitutes.

Animal tissues that resemble arteries have been used with some success, in

particular, the porcine small intestine (Sandusky et al., 1992). The mucosal cells

are scraped off the luminal side and the muscular layers are removed from the

abluminal side, leaving the stratum compactum, a dense, highly organized

fibrillar collagen matrix and the looser connective tissue of the mucosa. The

material can be used as a scaffold for cells in vitro and has been used in animal

experiments. In vivo, it is invaded by capillaries that contribute cells that

provide an intima, whereas smooth muscle cells migrating from the

anastomoses provide a media.

SOFT LITHOGRAPHY

As the need of biologists to control and manipulate materials on the micrometer

scale has increased, so has the need for new microfabrication techniques. Our

laboratory has developed a set of microfabrication techniques that are useful for

patterning on the scale of 0.5 m and larger. We call these techniques “soft

lithography” because they use elastomeric (that is, soft) stamps, molds,

membranes, or channels (Xia and Whitesides, 1998). Many other techniques

can and have been used to pattern cells and their environment (Hammarback

et al., 1985; López et al., 1993a; Park et al., 1998; Vaidya et al., 1998). The

most commonly used method has been photolithography. This technique has, of

course, been highly developed for the microelectronics industry; it has also been

adapted, with varying degrees of success, for biological studies (Hammarback et

al., 1985; Kleinfeld et al., 1988; Ravencroft et al., 1998). As useful and

powerful as photolithography is (it is capable of mass production at 200-nm

resolution of multilevel, registered structures), it is not always the best or only

option for biological studies. It is an expensive technology; it is poorly suited

for patterning nonplanar surfaces; it provides almost no control over the

chemistry of the surface and hence is not very flexible in generating

patterns of specific chemical functionalities or proteins on surfaces; it can

generate only two dimensional microstructures; and it is directly applicable to

patterning only a limited set of photosensitive materials (e.g., photoresists).

Soft lithographic techniques are inexpensive, are procedurally simple, are

applicable to the complex and delicate molecules often required in biochemistry

and biology, can be used to pattern a variety of different materials, are

applicable to both planar and nonplanar substrates ( Jackman et al., 1995), and

do not require stringent control (such as a clean room environment) over

the laboratory environment beyond that required for routine cell culture (Xia

and Whitesides, 1998). Access to photolithographic technology is required only

to create a master for casting the elastomeric stamps or membranes, and even

then, the requirement for chrome masks—the preparation of which is one of the

slowest and most expensive steps in conventional photolithography—can often

be bypassed (Deng et al., 1999; Duffy et al., 1998; Grzybowski et al., 1998; Qin

et al., 1996). Soft lithography offers special advantages for biological

applications, in that the elastomer most often used (PDMS) is optically

transparent and permeable to gases, is flexible and seals conformally

to a variety of surfaces (including petri dishes), is biocompatible, and can be

implanted if desired. The soft lithographic techniques that we will discuss

include microcontact printing, patterning with microfluidic channels, and

laminar flow patterning.

SELF-ASSEMBLED MONOLAYERS

Because many of the studies involving the patterning of proteins and cells using

soft lithography have been carried out on self-assembled monolayers (SAMs) of

alkane thiolates on gold, we give a brief discussion of SAMs (Bain and

Whitesides, 1988b; Bishop and Nuzzo, 1996; Delamarche and Michel, 1996;

Dubois and Nuzzo, 1992; Merritt et al., 1997; Ostuni et al., 1999;

Prime and Whitesides, 1993; Ulman, 1996). SAMs are organized organic

monolayer films (Fig. 18.1A) that allow control at the molecular level over the

chemical properties of the interface by judicious design and fabrication of

derivatized alkane thiol(s) adsorbed to the surface of films of gold or silver. The

ease of formation of SAMs, and their ability to present a range of chemical

functionality at their interface with aqueous solution, make them particularly

useful as model surfaces in studies involving biological components.

Furthermore, SAMs can be easily patterned by simple

methods such as microcontact printing ( CP) with features down to 500 nm in

size and smaller (Xia and Whitesides, 1998). These features of SAMs make

them the best structurally defined substrates for use in patterning proteins and

cells. SAMs on gold are used for the majority of experiments requiring the

patterning of proteins and cells, because they are biocompatible, easily handled,

and chemically stable [for example, silver oxidizes relatively rapidly, and Ag(I)

ions are cytotoxic].

POLYMER SCAFFOLD PROCESSING-

Restoration of organ function by utilizing tissue engineering technologies often

requires the use of a temporary porous scaffold. The function of the scaffold is

to direct the growth of cells migrating from surrounding tissue (tissue

conduction) or the growth of cells seeded within the porous structure of the

scaffold. The scaffold must therefore provide a suitable substrate for cell

attachment, proliferation, differentiated function, and, in certain cases, cell

migration. These critical requirements can be met by the selection of an

appropriate material from which to construct the scaffold, although the

suitability of the scaffold may also be affected by the processing technique.

Many biocompatible materials can be potentially used to construct scaffolds.

However, a biodegradable material is normally desired because the role of the

scaffold is usually only a temporary one. Many natural and synthetic

biodegradable polymers, such as collagen, poly(2-hydroxyesters),

and poly(anhydrides), have been widely and successfully used as scaffold

materials due to their versatility and ease of processing (Thomson et al., 1995).

Many researchers have used poly(2-hydroxyesters) as starting materials from

which to fabricate scaffolds using a wide variety of processing techniques.

These polymers have proved successful as temporary substrates for a

number of cell types, allowing cell attachment, proliferation, and maintenance

of differentiated function. Poly(2-hydroxyesters), such as poly(L-lactic acid)

(PLLA), poly lactic–co-glycolic acid(PLGA) copolymers, and poly(glycolic

acid) (PGA), are linear, uncross-linked polymers. These materials are

biocompatible, degradable by simple hydrolysis, and are Food and Drug

Administration (FDA) approved for certain clinical applications. The

mechanical properties of the scaffold are often of critical importance especially

when regenerating hard tissues such as cartilage and bone. Although the

properties of the solid polymer and the porosity of the scaffold have a profound

effect on its mechanical properties, polymer processing can also be influential in

this respect. The tensile strength may, for example, be enhanced due to the

crystallization of polymer chains. Alternatively, the manufacturing process may

cause a reduction in the molecular weight of the polymer, resulting in a

deleterious effect on mechanical properties. The shape of a hard tissue is often

important to its function and in such cases the processing technique must allow

the preparation of scaffolds with irregular three-dimensional geometries.

FIBER BONDING-

Fibers provide a large surface area: volume ratio and are therefore desirable as

scaffold materials. One of the first biomedical uses of PGA was as a degradable

suture material, which is why it is commercially available in the form of long

fibers. PGA fibers in the form of tassels and felts were utilized as scaffolds in

organ regeneration feasibility studies (Cima et al., 1991). However, these

scaffolds lacked the structural stability necessary for in vivo use. A fiber

bonding technique was therefore developed to prepare interconnecting fiber

networks with different shapes for use as scaffolds in organ regeneration (Mikos

et al., 1993a). PLLA is dissolved in methylene chloride, a nonsolvent for PGA,

and the resulting polymer solution is cast over a nonwoven mesh of PGA fibers

in a glass container. The solvent is allowed to evaporate and residual amounts

are removed by vacuum drying. A composite material is thus produced

consisting of nonbonded PGA fibers embedded in a PLLA matrix. The PLLA–

PGA composite is then heated to a temperature above the melting point of PGA

for a given time period. During heating the PGA fibers join at their cross-points

as melting commences, but the two polymers do not join due to their

immiscibility in the melt state. The composite is quenched to prevent any

further melting of the PGA fibers during cooling. After heat treatment, the

PLLA matrix of a PLLA–PGA composite membrane is selectively dissolved in

methylene chloride and the resulting bonded PGA fibers are vacuum dried.

Using this technique, the fibers are physically joined without any surface or

bulk modification and retain their initial diameter. The PLLA matrix

is required to prevent collapse of the PGA mesh and to confine the melted PGA

to a fiberlike shape (Fig. 21.1). The heating time is also of critical importance

because prolonged exposure to the elevated temperature results in the gradual

transformation of the PGA fibers into spherical domains.

References

1. Text books of Principles of tissue engineering. (Second edition). Robert . P. Lanza, Robert Langer, Joseph Vacanti.

2. Accuracy Of Three Techniques To Determine Cell Viability In 3D Tissues Or Scaffolds. Benjamin Gantenbein-Ritter, Ph.D.,1 Esther Potier, Ph.D.,1,2 Stephan Zeiter, D.V.M.,1 Marije Van Der Werf, M.Sc.,1,2 Christoph M. Sprecher, Dipl-Ing.,1 And Keita Ito, M.D., Sc.D. TISSUE ENGINEERING: Part C Volume 14, Number 4, 2008.

3. REPAIR OF MANDIBLE DEFECT WITH TISSUE ENGINEERING BONE IN RABBITS. ANZ J. Surg.2005;75: 1017–1021.

4. Proteins and Their Peptide Motifs in Acellular Apatite Mineralization of Scaffolds for Tissue Engineering. TISSUE ENGINEERING: Part B Volume 14, Number 4, 2008.