TISSUE ENGINEERING INTRODUCTION Periodontal regeneration is a laudable but often elusive goal.

The way forward in the field of periodontal regeneration is through application of current knowledge in the fields of molecular and cell biology, developmental biology and tissue engineering principles as applied to tissue regeneration. Through a combination of

transplanted biomaterials containing appropriately selected and primed cells, together with an appropriate mix of regulatory factors to allow growth and specialization of the cells and matrix, a new risk for periodontal regeneration is becoming possible. Definition Tissue engineering is a term originally used to describe tissue produced in culture by cells seeded (grown) in various porous absorbable matrices. Langer. R., Vacanti J.P. 1993. Tissue engineering is a relatively new field of reconstructive biology that utilize mechanical, cellular and biologic mediators to facilitate reconstruction, regeneration of a particular tissue.

The term tissue engineering was originally coined to denote the construction in the laboratory of a device containing viable cells and biologic mediators (eg. Growth factors and adhesions) in a synthetic or biologic matrix that could be implanted in patients to facilitate regeneration of particular tissues. Tissue engineering is the emerging field of science aimed at developing techniques for the science aimed at developing

techniques for the fabrication of new tissue to replace damaged tissues and is based on principles of cell biology, developmental biology and biomaterials science. Narem R, Sambams 1995, Reddi. A.A.1998., The terms tissue engineering and biomimetics are often use interchangeably. Biomimetics however is the science of reconstructing or mimicking nature processes or tissues, with the expectation that regeneration will follow. Periodontal regeneration The events associated with periodontal regeneration are extraordinarily complex and require the participation of many, if

not all cellular components of the periodontium. These include an initial inflammatory cells component, recruitment of connective tissue cell populations, their proliferation and differentiation and synthesis of matrix constituents. Bartold & Naryanan 1998. Whether the damaged tissues heal by regeneration or are repaired by scar tissue depends upon atleast three factors. i) ii) Availability of the appropriate cell type(s). Soluble mediators of cell function that activate these cells. Mc. Kay.R. 1997 and iii) An evolving (developing extracellular matrix.

Cell selection, differentiation and maturation. The cell types required for periodontal regeneration include i) ii) Epithelial cells to seal the wound area Fibroblastic cells for the soft connective tissues of the gigniva and periodontal ligament. iii) Osteoblastic cells osteoblasts, alveolar bone cells, cementoblasts. iv) Endothelial cells for forming blood vessels.

During early stages of regeneration, inflammatory cells are also necessary, since they secrete a wide variety of molecules required for the appropriate recruitment and functioning of the connective tissue cells that participate in healing and regeneration. The molecules include Growth factors : Fibroblast GF1 and 2, insuline coke GFI & II, bone morphogenetic proteins, epiodermal growth factor, PDGF. Adhesion molecular: Fibronectin, laminin, osteopontin, bone

sialoprotein, collagens, cementum, attachment protein. Structural protein: Types I, III, V, XII and XIV collagen

proteglycans, hyalusonan, osteocalcin. Non collagenous proteins, tenaxin, osteonectin, dentin / enamel matrix proteins. Sufficient number of cells responsible for each process must be produced to participate at the right location and in an appropriate temperature sequence.

Soluble mediators and regulators of cells functions Critical messages for cell activity are provided by substances present in the local environment and mediated their effects through specific cell surface receptors. Binding of soluble mediators to the cell surface receptors lead to cell responses such as cell migration, changes in cell shape and synthesis of extracellular matrix macro molecules. Soluble mediators of importance in regeneration include growth factors such as PDGF,, TGF- and FGF; cytokines including IL-1, TNF-, JL-6, lymphokines such as interferon - , fibronectin and other adhesion molecules. Growth factors play different roles depending upon the healing stage, target cell type and available of matrix components. A growth factor may have primarily a proliferative function during early stage of healing and prior to extracellular matrix formation. H however, with the formation of an extracellular matrix, the same molecule may promote cellular differentiation. This concept that the presence of a mature and functional extracellular matrix permits the major action of growth factors to be

directed primarily towards the recruitment and differentiation of cells is fundamental to future progress in the field of periodontal regeneration. Role of the evolving and developing extracellular matrix. Matrix synthesis Gene expression Nucleus Cytoskeleton Cell surface (Integrim, Receptors) Matrix (Macromolecules, Bound growth factors) Components located within the extracellular matrix either matrix molecules or bound growth factors can interact with specific cell surface receptors. This activates a number of intercellular

signaling mechanisms which ultimately lead to specific gene expression for matrix synthesis which ultimately can lead to a self regulatory feedback loop. Thus interactions with the ECM will i) ii)
iii)

lead to recruitment of appropriate cell types. Determine whether cells divide or differentiate And thus determine whether a wound heal by

regeneration or repair. Bartold, S.Narayanan, S.Pital 2000. In tissue engineering, a prefabricated extracellular matrix with the appropriate cell types and growth factors makes

regeneration more predictable. Periodontal tissue engineering The main requirements for producing an engineered tissue are i) Appropriate levels and sequencing of regulatory signals (signaling molecules). ii) The presence and numbers of respounine progenitor cells (cells) and

iii)

An appropriate ECM or carried construct (Scaffolds or supporting matrices)

Signaling molecules: These are two main approaches involving preparations containing biological mediator to selectively enhance the cells that populate the periodontal wound. 1) semipurified preparation such as enamel matrix derivative and antagons platelet rich plasma preparation. 2) Recombinant growth factors such as recombinant human platelet derived growth factor BB, recombinant human bFGF, and morphogus such as recombinant human bone morphogenetic protein. The advantages of recombinant growth factors are i) that adequate quantities can be produced for clinical application ii) the tissue engineering device is of consistent quality. The variations in the regenerative response is then limited to the individuals healing response and surgical techniques. All these ecological mediators are commercially available with the reception of PRP, which is prepared prior to usage.

PLATELET RICH PLASMA PREPARATION In this approach, autologus blood is drawn and separated into three fractions : i) ii) iii) Platelet poor plasma (fibrin flue or adhesive) Platelet rich plasma and Red blood cells

PRP contains 338% of platelts, 41% ng/ml of PDGF and 45.9 ng/ml of TGF - . Landersberg. R. 2000. PRP also contains IGF, bFGF 2, EGF and VEGF Kantarc et al 2007. This mixture of growth factors stimulate i) ii) iii) Proliferation of fibroblasts and periodontal ligament cells ECM formation and Nervous culriation

Additionally PRP may suppress cytokine release and limit inflammation, thereby promoting tissue regeneration kantarc A et al 2007.

PRP also contains a high concentration of fibrinogen. In clinical use, calcium and thrombin are added to activate the proteolytic cleavage of fibrinogen into fibrin. Fibrin formation initiates clot formation which in turn imitates wound healing and stabilizes the wound healing matrix. Kantarci A. 2007 Enhanced healing is a results of synergistric effect between various growth factors in combination with the anti inflammatory effect of PRP. Howell T h et al 1997 The level of PDGF in PRP is 3000 fold less than the concentration needed for periodontal

regeneration. Dori. F. et al, Subagioglu et al 2007 PRP may be ineffective for periodontal regeneration, but enhances early soft tissue healing. Enamel C matrix derivative EMD harvested from developing povine teeth has been reported to induce periodontal regeneration. YU KNA R.A., Mellonig JT. 2000. Hagewald et al 2004, Jiang J. 2004, Dischou et al 2006 EMD contans a mixture of low molecular weight

proteins that stimulate cell growth and the differentiation of mesenchymal cells including osteoblasts. Jiang. J. 2004, Suzuki 2005 EMD contains TGF - and also stimulates BMP, TGF - , and CTGF expression in osteoblastic cells. Schlueter SR 2007 EMD stimulates angiogenesis directly by stimulating endothelial cell proliferation and chemotaxis, and stimulates CEGF production by periodontal ligament cells. When applied to root surfaces, the proteins are absorbed into the hydroxyapatite and collagen fibers of the root surface, where they induce cementum formation followed by periodontal

regeneration. Chong CH et al 2006 When PDL cells are exposed to EMD, the cells exhibit enchanced protein production, cell proliferation and mineral nodule formation. A knowledge of how cells respond to EMD, can make it useful in tissue engineering devices.

Morphogens proteins

or

differentiation

factors

: bon

morphogens

Bone morphogenetic proteins are a group of regulatory glycoproteins that are members of the TGF - superfamily. These molecules primarily stimulate differentiation of

mesenchymal stem cells into chondroblasts and osteoblats. At last 7 BMPs have been identified from bovine and human sources. In periodontal regeneration BMP 2 (OP-2) BMP-3 (osteogenin) and BMP 7 (OP-1) are important Massague 1998. It has been proved by numerous studies that BMPs have osteoinductive properties Schwartz et al. Approximately 10kg of bovine bone fields only 2g of BMP. Hence now BMPs are synthetically produced using recombination DNA technology. King GW 1997, Wikesjo et al 1995. Recombination human BMPs have been used for correcting intrabony, suprabony, furcation and fenestration defects. When a human BMP L was used histologic analysis revealed periodontal regeneration without

ankylosis. With concerns about ankylosis recent research is now utilizing recombination human BMPs in implant site preparation. Cochran DL et al 2000, GROWTH FACTORS Growth factors are naturally occurring proteins that regulate various aspects of cell growth and development. Lind M. 1996, AAP 1996. During proliferation, functions. wound healing, ECM factors growth factors and modulate other cell Grsenneld 2000.

migration, growth

formation may

cellular as cell

Some

also

function

differentiation factors. In periodontal regeneration, the focus is on PDGF and bFGF2. PDGF is a potent mitogenic and chemotactic factors for mesenchymal cells in cell culture. Lynch 1989, 1991 when PDGF and insulin like growth factor -1 were topically applied to periodontology diseased root surfaces in beagle dogs, there was formation of substantial amounts for new bone, cementum and periodontal ligament after 2 weeks. The results of this were

confirmed in three other studies in beagle dogs and non human primitive. Giannobile 1994, 1196, Rutherford et al 2003. Basic fibroblast growth factor 2 in one of the most potent mitogens for cells of mesodermal + neuroectodermal origin and induces angiogenesis. Cheng J. 1986. Mixazaki K. 2001 B-FGF has the ability to promote mesenchymal cell proliferation and still maintain the multipotent properties of these cells. Takayama et al When FGF 2 was applied to experimental class II furcation defects, there was improved periodontal

regeneration in the defect Lites without epithelial down growth, ankylosis on root resorption. Takeiyama et al 1997, Ledouse et al 1992 FGF2 not only promotes proliferation of periodontal ligament cells, but also induces these cells to produce a regenerative environment that would support regeneration and angiogenesis. Terashima. M. et al 2008 These cells re induced to produce an ECM containing hyaluronan, heparin sulfate and osteopontin.

Thus FGF 2 in strongly suggested to be efficacious in human periodontal regeneration. Scaffold or supporting matrices : Matrices form the framework for the engineered tissues to form. Material to used for the fabrication of matrices to engineer tissue in vitro, or to facilitates regeneration in vivo, must have the micro structure and chemical composition required for normal cell growth and function. Role in the regeneration process : 1) To provide physical support for the healing area so that there is no collapse of the surrounding tissue into the wound site. 2) Serve as a barrier to the ingrowth of surrounding tissue that may impede the process of regeneration.
3) Serve as a scaffold for migration and proliferation of cells in

vivo or for cells seeded in vitro 4) To potentially serve as a time release mechanism for signaling molecules.

Commercially available scaffold materials available for oral tissue engineering application bone grafts : Allografts 1) Demineralised freeze dried bone allograft (DFDBA)
2) Free- dried bone allograft (FDBA)

Xenografts 1) Anorganic bovine bone 2) Hydroxyapatite Alloplasts

1) Tricalcium phosphate

2) Hydroxy apatite Bioactive glass polymers 1) Hand tissue replacement polymer 2) Coraline calcium carbonate Matrices (Porous structures) Absorbable Synthetic polymers Polylactice acid Polyglycolic acid

Natural polymers Collagen types I, II, III, IV Collagen, glycosaminoglycan copolymer Fibrin Chitosan Non resorbable Synthetic polymer Polytetrafluoroethylene Synthetic ceramics Calcium phosphate Of the allografts, tricalcium phosphate (TCP) is used in combination with recombinant human PDGF BB. TCP physically fills bone defects, provides a scaffold for new bone formation and prevents soft tissue collapse into the wound site. Froum. S. Stahl S.S. 1987. It is osteoconduction and supports only healing. TCP resorbs at varying rates depending on the chemicals stutures, porosity and particle size.

Absorption, release and bioactivity studies indicate that TCP or calcium sulfate can be an effective carrier for PDGF BB 45% of PDGF BB is released after 10 days, Bateman et al 2005. Clinical studies of TCP with recombination human PDGF, have shown that the material is resorbed and was replaced with regenerated periodontium. One commercially available tissues engineering system

involving the use of PDGF BB and TCP is now available (GEM21; Osteohealth, Shinely NY, USA). Collagen carriers JUNCOSA Melvin. N. et al 2005 Collagen has been widely used in tissue engineering for seeding mesenchymal cells and for incorporation of growth factors Howell 1997. Because most collagen are derived from bovine dermal or skeletal tissue, concerns related to purity, quality and

immunogenicioty have been raised. Boyne R. et al 2007. For these purposes, the antigenic and terminal tetopeptides of bovine, collagen have been removed Hou. L. et al 2007.

Collagen scaffolds are particularly effective in supporting recombinant human. BMP induced bone formation. A tissue engineering system Infuse, metronic soufamouse danek, Memphis USA containing both BMP and type I collagen sponge is now commercially available. Calcium sulfate Calcium sulfate is one of the oldest bone graft materials. Clinical and animal studies indicate that calcium sulfate. 1) In biocompatible 2) Degrades over time 3) Is subsequently replaced with granulation tissue. 4) May be used in infected areas with no complications. Peltier 2001. 5) Has barrier properties KAO. RT. 2004, KIM CK 1995. 6) Enhances angiogenesis Strucchi et al 2002. 7) May be an effective delivery vehicle for antibiotics as well as for growth factors Beardmore AA et al 2005.

Rosenblum et al 1993 FGF released at a rate directly proportional to the rate of calcium sulfate dissolution. Walsh WR et al 2003 Defects filled with calcium sulfate showed increased immunostaining for BMP 2, BMP 7, TGF - and PDGF BB. Lynch SE et al 2005- Calcium sulfate was found to be a suitable carrier for recombinant human PDGF BB with a longer release kinetic profile ( = 16 days). Other carriers Bio restorable polymers polylactide and polyglycolic acid as scaffolding agents, have biodegradable and tissue compatible properties. Various tissue responses like foreign body reactions, inflammation and local acid accumulation during polymer

degradation has made clinical application of these materials problematic King GN et al 2002. Substrate modification to enhance cell selections Scaffolds are coated with various cell binding peptide sequences of cell adhesions or integrins to mimic the extracellular

components of bone Heaby KE. et al 1999. This type of scaffold permits cell binding, osteoblastic phenotypic expression and differentiation. However they are still in the experimental phase. Exogenous cells for engineering tissues : In many tissues such as bone, the number and activity of precursor cells is so high that there is normally an ample sources of endogenous cells to populate implanted scaffold for the

regeneration of tissue. In most instances, matrices alone can serve to facilitate regeneration. Exogenous and mitogenic factors may be necessary only in special cases, when the proliferation of the precursor cells is impeded or their pool has been greatly diminished by previous surgery or concomitant disease. When these are indications for exogenous cells, their incorporation into implanted matrix is generally required. An alternative is to inject a cells suspension into a sealed compartment containing the defect. Currently allogenic and antologous cells digested in vitro are being used. When allogenic cells are used, the potential for immune response and disease transmission must be considered.

Issues related to the use of antologus cells include the requirements for harvesting the cells and donor site morbidity. Recently it has been proposed that marrow stromal cells. Caplan AI. Dink 1993 can be used as precursor for many connective tissues. Requirements for successful tissue engineering These have been divided into two main areas Brekke 1998. 1) Engineering issues related to maintaining an invivo cell culture substratum such as biomechanical properties of the scaffold, properties. 2) Biological function of the engineered matrix including cell recruitment, permission of neovascularization and delivery of the requisite morphognatic, regulatory and growth factors for tissue engineering. Biomechanical features Space maintenance Barrier or exclutrary features architectural geometry and space maintaining

Biological functions Biocompatibility Incorporation of cells Incorporation of instructive message Space maintenance within the defect site 1) The bioengineered matrix should be of sufficient form to be placed into a defect and prevent collapse of the reposition tissues into the defect site. 2) This results in a sufficient wound space to permit the cascade of molecular events needed to drive the regeneration process. 3) Ability to be easily cut or molded into a desired shape and be of a consistency compatible with easy handling. 4) The scaffold should be a sufficient rigidity to withstand soft tissue collapse. 5) The internal architecture of the scaffold should be such as to maximize rapid colonization by cells of the desired phenotype and ingrowth of tissue compatible with those to be

regenerated. Whang 1999.

Barrier or Exclusionary functions

a. Engineered tissues should act as a barrier to the ingrowth of

unwanted tissues gingival epithelium and connective tissue, yet permit selective in growth of regenerating tissues cementum, PDL and bone.
b. To achieve this, design features will have to be incorporated

wherely the external surface may be exclusionary, get the internal scaffold conducive to new tissue in growth. Biocompatibility and design features 1) The scaffold material should be either biocompatible with the tissue to be regenerated or biodegradable allowing for gradual replacement with regenerated tissue.
2) Design features need to include considerations of pore size

for both cell attachment in vivo and incorporation of cell in vitro. 3) Materials should be free from transmissible disease,

immunologically inert and not induce an inflammatory response. Omstead PR 1998.

4) In the case of the periodontium, a biodegraded material that would degrade slowly overtime and be replaced with a soft CT compatible with PDL, together with new alveolar bone and root surface cementum with appropriately oriented and inserted collagen fibres would be desirable. Incorporation of cells with appropriate phenotype for ongoing periodontal regeneration. 1) It should be possible to culture cells with a periodontal regeneration phenotype and incorporate them into a suitable biodegradable scaffold for immediate

introduction into a periodontal defect. 2) Likely sources of cells for periodontal tissues

engineering are to be from the host site (cementum, PDL and Gone). Incorporation of cells with appropriate phenotype for ongoing periodontal regeneration 1) It should be possible to culture cells with a periodontal regeneration phenotype and incorporate them into a suitable

biodegradable scaffold for immediate introduction into a periodontal defect. 2) Likely sources of cells for periodontal tissue engineering are to be from the host site (cementum, PDL, Gone). Incorporation and bioavailability of instructive messages 1) The biomaterials should have a suitable affinity for the absorption of appropriate growth or differentiation factors as well as integrins, cell receptors and other instructive molecules normally found in regenerative tissue. 2) One major obstacle to be overcome in the control of delivery. Because the particular inductive agent is released from the biomatrix, it may be capable of inducing ectopic tissue formation in the neighbouring regions. King GN et al 1997. Thus control and containment of the agent is paramount for its effectiveness and safety. CONCLUSION Development in the field of molecular and cell biology, developmental biology and tissue engineering have had a

significant impact on managing the anatomic changes due to the disease process. But many questions remain does the regenerated tissue behave and act like the original tissue? Studies comparing the chemical qualities of regenerated bone with native bone in a vertical bone grafting study, ensured they were similar Rocchietta et al 2007. Are the regenerated tissues sustainable ? 5 year date of positive results for EMD and 2 year date for recombinant human PDGF are available. The ultimate test for implants would be whether the survival rates of implants placed in tissue engineered bone are the same as those for implants placed in native bone. Will the added expense be justified by the final healing results? Can the level of response for tissue engineering be increased by adding multiple signaling molecules, improving the scaffold or with cells therapy ?

The potential projects for researches are almost limitless. Clinicians can help direct these investigations by being good observer of both positive and negative therapeutic responses. These observations can help to determine the design of randomized clinical trials that can improve our current therapeutic approach. Reference : 1) Richard T. Kao, Shinya Murakami, O.Ross Beirue The use of biologic mediators and tissue engineering in density. Perio 2000. Vol50, 2009, 127 153. 2) P. Mark Bartold, Chirstopher. A.G. Mc Culloch, A. Sampath

Narayanan and Sandu Pitaru Perio 2000, Vol.24, 2000, 253 269. 3) Samuel E. Lynch, Robert J. genco, Robert. E. Marx Tissue Engineering Applications in Maxillofacial Surgery and Periodontics.