TISSUE ENGINEERING

PRECEPTOR- DR.VISHWAS BHATIA

PRESENTED BY- DR.SURABHI VASHISTHA

CONTENTS

• INTRODUCTION
• DEFINITION
• NEED FOR TISSUE ENGINEERING
• FUNDAMENTAL ELEMENTS OF TISSUE ENGINEERING
• STRATEGIES OF TISSUE ENGENEERING

CONDUCTIVE APPROACH

TISSUE INDUCTION

CELL TRANSPLANTATION APPROACH

• ENGINEERED OROFACIAL TISSUES
• CONCLUSION
• REFERENCES

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INTRODUCTION

• Term tissue engineering covers a broad range of applications.
• In practice term is closely associated with applications that repair or replace portion of or whole tissues i.e.

-Bone

-Cartilage

-Blood vessels

-skin

• Tissues involved require certain mechanical and structural properties for proper functioning.

DEFINITION

• In 1987, term “tissue engineering” was coined at a National Science Foundation (N.S.F) bioengineering meeting in Washington D.C

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• SHALAK & FOX 1988,

“ The application of principles & methods of engineering & life sciences, to obtain a fundamental understanding of structural and functional relationships in novel and pathological mammalian tissues, & the development of biological substitutes to restore, maintain or improve tissue function”

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Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

NEED FOR TISSUE ENGINEERING

• Tissue engineering holds promise of producing better organs for transplant.

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• Using TE techniques & gene therapy it may be possible to correct many otherwise incurable genetic defects.

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• A major goal of TE is in-vitro construction of transplantable vital tissues.

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• Artificial tissues can revoltionize healthcare by providing a supply of soft & hard CT on demand.

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

FUNDAMENTAL ELEMENTS OF TISSUE ENGINEERING

TRIAD OF TISSUE ENGINEERING

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Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

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1. The cells- responsible for synthesis of the new tissue matrix.

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(II) Growth factors -that promote and facilitate cell function.

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(III) scaffolds- that act as an extracellular matrix, allowing cell differentiation, proliferation and biosynthesis.

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

CELLS

SOURCES

 Autologous cells (the host’s own cells)

 Allogenic cells (cells from a donor)

 Xenogenic cells (cells from a different species)

 Stem cells: either allogenic (fetal or adult derived) or autologous (adult derived).

AUTOLOGOUS CELLS -are obtained from same individual to which they will be re-implanted.

-Have fewest problems with rejection & pathogen transmission, however in some cases might not be available (like genetic disease suitable autologous cells are not available).

-These cells can differentiate into a variety of tissue types, including bone, cartilage, fat, & nerve.

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ALLOGENIC CELLS come from body of a donor of same species.

-Employment of dermal fibroblasts from human foreskin has been demonstrated to be immunologically safe & thus a viable choice for TE of skin.

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• STEM CELLS:- are classified into

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

• ADULT STEM CELLS :-may be found in bone marrow, adipose tissue, umbilical cord and in dental tissues.

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ADULT STEM CELLS( DENTAL ORIGIN)

(Dental mesenchymal stem cells –MSCs)

These cells are denominated according to their tissue of origin, such as stem cells from human exfoliated deciduous teeth – SHED)

Dental pulp stem cells – DPSCs);

Periodontal ligament stem cells – PDLSCs)

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

STEM CELLS

PRIMARY TOOTH PULP

PERMANENT TOOTH PULP

PERIDONTAL LIGAMENT STEM CELLS

Present high potential differentiation into odontoblasts,

osteoblasts,

adipocytes chondrocytes

found in third molar pulp, may differentiate into odontoblasts, adipocytes, chondrocytes and myoblasts

differentiate into osteoblasts, cementoblasts and fibroblasts, and can be used in regenerating both periodontal ligaments and bone tissues

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

2) SCAFFOLDS OR EXTRACELLULAR MATRIX

• Scaffolds are biomaterials with two-dimensional or three-dimensional architecture that provide the cells with an adequate environment, making it possible for them to migrate, proliferate and differentiate. In addition, they allow the transportation of nutrients, oxygen and cellular metabolic residues, making them a crucial element for tissue regeneration.

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Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

• These biomaterials should be:-

 Biocompatible

Biodegradable

Good mechanical properties

A porous structure

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• The choice of material for fabricating the scaffold:-

Ceramics

Metals

Polymers

Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

Polymers have gained emphasis because they have biodegradability and great flexibility in their processing . Polymers may be of natural origin, and be composed of collagen, fibrin and hyaluronic acid; synthetic, made of polymer compounds such as Poly lactic acid (PLA), polyglycolic acid (PGA) and its poly copolymer (Poly(Lactide-co-Glycolide) acid) (PLGA); or hybrids (natural and synthetic)

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• Techniques for fabrication of scaffolds:-

Lyophilizing

Phase separating

Foaming

Rapid prototyping

Electrospinning

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Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

Tissues are composed of

• Cells
• Insoluble extracellular matrix (E.C.M.)
• Soluble molecules that serve as regulators of cell function

Ensanya ali abou Neel et al. Tissue Engineering in dentistry. J Dent. Aug 2014

E.C.M. usually composed of 3 components:

• Collagen
• Glycoprotein
• Proteoglycan

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The E.C.M. is important for

• Growth
• Function - various cell types involved.

Ensanya ali abou Neel et al. Tissue Engineering in dentistry. J Dent. Aug 2014

Langer R, Vacanti JP (May 1993). "Tissue engineering". Science 260 (5110)

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Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

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SYNTHETIC CERAMICS

Implemented as matrix materials for facilitating regeneration in-vivo (Bucholtz et al 1987). 2 most widely used forms are:

• Tricalcium phosphate
• Hydroxyapatite.

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1. Tricalcium Phoshphate:

• Porous form of calcium phosphate
• ß-TCP
• Problem -physiochemical dissolution after implantation

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

2. Synthetic Hydroxyapatite:

• Development - second form of bioceramic.

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• Rationale - mineral naturally occurring in bone is hydroxyapatite.

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Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

SYNTHETIC POLYMERS

• PTFE (Polytetrafluoroethylene) – synthetic fluoropolymer of tetrafluoroethylene that finds numerous applications. well known brand name of PTFE is Teflon by DuPont Co.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

SYNTHETIC POLYMERS

• Degradation by hydrolysis

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• Polyglycolic acid - degrades fast
• Polylactic acid (L-lactide) - most stable in-vitro

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• Thus, modification of poly (L-lactide) by crosslinking or addition of D-lactide more rapid degradation.
• Polyglactin 910, a co-polymer of glycolide and Llactide – 90/10 molar ratio.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

NATURAL POLYMERS

• Collagen - protein with 3 polypeptide chains, known as α-chains, each containing at least 1 stretch of repeating AA sequence

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• Collagen constitutes almost 1/3 of all protein in body, & accounts for almost 60% of gingival connective tissue & 90% of total protein in bone.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Collagen - medical devices,
• Derived from animal sources,- bovine skin, tendon, intestine or sheep intestine.
• Collagen based sutures & hemostatic sponges have also been used.
• Resorbable collagen barriers have been used clinically for G.T.R. procedures, although their combination with biologic modifiers has not been explored.
• Also, absorbable collagen sponge (ACS) has been used as a carrier for rhBMP-2

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

NATURAL MINERALS

• HA skeleton (Bio-Oss®, Osteograf®) - retains microporous & microporous structure of cortical & cancellous bone.

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• Remaining after chemical or low heat extraction of the organic component.

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• Usually bovine bone mineral is used

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• Currently available - deproteinated, which supports cell-mediated resorption.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

3) GROWTH FACTORS

• Growth factors are extracellularly secreted proteins that stimulate cell growth and bind to specific receptors in the cell membranes.

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• They have the capacity to regulate cell growth, development proliferation and migration

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Growth factors have been used in tissue engineering

-Bone morphogenetic proteins (BMP)-applied for dental regeneration 

- Hedgehog proteins (HHS)

-Fibroblast growth factor (FGF)

- Interleukins

-Tumor necrosis factor (TNF)

-Vascular endothelial growth factor (VEGF)

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• These proteins play an important role in tooth development, and are associated with the differentiation of odontoblasts and ameloblasts, and in the development of cement and alveolar bone

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

SIGNALLING MOLECULES

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

CLASSIFICATION

3 groups

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1. Growth & Differentiation Factors

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• Extracellular Matrix Proteins & Attachment Factors

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• Mediators of Bone Metabolism

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

GROWTH AND DIFFERENTIATION FACTORS

Growth factors - play important role in regeneration are:

1. Platelet derived growth factor (P.D.G.F.),
2. Insulin-like growth factor (I.G.F.),
3. Transforming Growth Factor- β (T.G.F.-β),
4. Fibroblast Growth Factor,
5. Bone Morphogenetic Proteins (B.M.P.s).

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• CHEMISTRY: 2 disulphide bonded poly-peptide chains that encoded by 2 different genes-P.D.G.F.- A & P.D.G.F.-B.
• FORMS: exist either as
• heterodimer (AB) or
• homodimer (AA, AB).
• 3 isoforms of PDGF have unique binding properties for PDGF receptor sub-units, α & β, found on cell membrane.

PLATELET DERIVED GROWTH FACTOR

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• PRODUCTION: Several cell types produce PDGF, including
• Degranulating platelets,
• Smooth muscle cells,
• Fibroblasts,
• Endothelial cells,
• Macrophages & keratinocytes.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Recombinant proteins are produced from one of several cellular expression systems:
• Bacteria,
• Insect cells or mammalian cells.
• rh BMP-2 is produced using mammalian cell expression system, which allows for proficient execution of post-translational modifications that are present in human BMPs.

RECOMBINANT BMP-2 PRODUCTION

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Chinese Hamster Ovary (CHO) cells are host of choice. Because mammalian cells synthesize a variety of GF, they are capable of synthesizing & secreting active BMP.

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• Includes many steps:
• Synthesizing of precursor polypeptide chains.
• Correct refolding & demineralization of these chains,
• Glycosylation of protein.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

 Peptide growth factors with biochemical & functional similarities to insulin.

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 Bone cells produce & respond to IGF’s, and bone is a storage house for these factors in their inactive form.

INSULIN-LIKE GROWTH FACTORS (IGF-I,II):

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Multifactorial growth factor, structurally related to B.M.P.s, but functionally quite different.

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• Chemotactic for bone cells, & may increase or decrease their proliferation depending upon the differentiation state of the cells, culture conditions and concentration of TGF-β applied.

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• In-vivo, produces new cartilage and / or bone, if injected in proximity to bone; however, it does not induce new bone formation when implanted

away from a bony site.

TRANSFORMING GROWTH FACTOR-β:

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

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• Urist in 1965, reported that protein extracts from bone, implanted into animals at non- bone sites induced formation of new cartilage & bone tissue.

BONE MORPHOGENETIC PROTEINS (BMPS):

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• 2 modes of preparation have been used:
• Preparations derived from bovine or human bone, which contains complex mixture of BMP molecules & possibly other factors & proteins

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• RECOMBINANT DNA METHODS-
• when recombined with DNA of cloning vector, can be replicated, transcribed & translated. Used for production of (rh BMP-2)

MODES OF PREPARATION

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

cDNA CODING FOR rh BMP-2

STORED IN ALIQUOTS & FROZEN

TRANSFECTED INTO HOST CELL (CHO CELL)

rh BMP-2 SECRETED

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rh BMP2 PRODUCTION

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

PUT IN GROWTH MEDIUM & HARVESTED

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rh BMP-2 REMOVED BY FILTRATION

PURIFIED BY COLUMN CHROMATOGRAPHY

PLACED IN VIALS & LYOPHILIZED

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Several agents which affects the growth of bone:

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• PROSTAGLANDINS: Result of cyclo-oxygenation of precursors derived from arachnoid acid. Found – in variety of tissues. Effect varies considerably from stimulating inflammation & bone resorption to enhance bone formation

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• GLUCOCORTICOIDS: Such as dexamethasone have prostaglandins, complex direct & indirect effects on bone formation. Chronic glucocorticoids administration results in bone loss.

MEDIATORS OF BONE FORMATION

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• BISPHOSPHONATES: A class of pharmacuetical agents, which are structurally similar to pyrophosphates, natural product of human metabolism. Bisphosphonates binds to HA crystal of bone & prevent their growth & dissolution

CLASSIFIED AS:

• 1^st Generation : alkyl side chains

Eg: Endronate

• 2^nd Generation : amino terminal grp.

Eg: Alendronate & Pamidronat

• 3^rd Generation : cyclic side chains

Eg: Risedronate

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BISPHOSPHONATES

PYROPHOSPHATES

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Family of at least 9 related gene products of which 2 major members are a-FGF or FGF-1 & b-FGF or FGF-2.

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• Stimulate endothelial cells & PDL cell migration & proliferation, as well as stimulation of bone c replication.

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• b-FGF is more potent than a-FGF & may act via stimulation of other growth factors like TGF-β.

FIBROBLAST GROWTH FACTORS:

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• PDGF & TGF-β are well-established wound healing

―hormones.

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• One of highest concentrations of PDGF & TGF-β in body are found within α-granules of blood platelets

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• Thus, concentrating platelets would result in concentration of growth factors, enhancing wound healing on application.

PLATELET RICH PLASMA

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

Dhurat R. Principles and methods of preparations platelet rich plasma: A Review; JOCAS oct-Dec 2014,vol 7 issue 4

Apheresis

Procurement from one unit blood

Procurement on a small scale

• Autologous platelet rich plasma (PRP) was developed in the 1970’s as a by-product of multiple component apheresis.

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• Today, there are 3 main techniques available for procurement of PRP:

PROCESSING OF P.R.P.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• The process of apheresis basically involves removal of whole blood from a patient or donor.

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• Within an instrument that is essentially designed as centrifuge components of whole blood are separated.

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• One of components is then withdrawn & remaining components

are re-transfused into patient or donor.

APHERESIS

APHERESIS

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Uses one unit (350 ml) of the patient’s blood, but instead of using an apheresis apparatus, it uses a temperature-controlled centrifuge (cold centrifuge).

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• Whole blood is obtained in a transfusion bag & subjected to a low spin cycle of 1100 rpm for 15 minutes, which results in separation of 3 basic fractions

PROCUREMENT FROM ONE UNIT BLOOD

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Recent studies focussed on using minimal amount of blood (10-50 ml) depending upon procedure involved, & common laboratory centrifuge for procurement of PRP.

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• This procedure uses double-spin centrifugation (2,400 rpm for 10 minutes, & then after discarding RBC fraction, 3,600 rpm for 15 minutes), & 3 components are obtained in test-tube.

PROCUREMENT ON A SMALL SCALE

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Safe as it is autologous preparation.

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• Promotes adhesiveness & tensile strength for clot stabilization.

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• Biologically acceptable.

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• Contains growth factors (PDGF & TGF-β) released by platelets.

ADVANTAGES OF USE OF AUTOLOGOUS P.R.P.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

 Promotes angiogenesis.

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 Haemostatic properties.

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 Dense fibrin net that is highly osteoconductive.

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 High concentrations of leukocytes, which act as

―autologous antibiotic‖, reducing risk of infection.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• A problem with current delivery of growth factors to wounds is extremely short half-lives of these factors. This can be attributed to:
• Proteolytic breakdown.
• Receptor mediated endocytosis.
• Solubility of delivery vehicle.

GENE THERAPY

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• Genes are specific portions of DNA that code for proteins.

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• Their role in protein synthesis can be illustrated as follows:
• Activation of transcription via cell surface receptors.
• Transcription of DNA code into mRNA.
• Processing of mRNA in preparation for transportation to cytoplasm.
• Transport of mRNA to cytoplasM

GENE EXPRESSION & PROTEIN SYNTHESIS

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• RNA translation & peptide synthesis.
• Polypeptide elongation.
• Post-translational modifications.
• Transport to & across cell membrane.
• At each stage of gene expression, there is an opportunity for control & regulation of protein synthesis.

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

• 2 general ways to transfer genes:
• Virus mediated vectors: - ex-vivo approach - in-vivo approach
• Naked DNA using Plasmids.
• Transduction (i.e. transfer of genetic fragment) to appropriate target cells (i.e. osteoblasts) represents first critical step in gene therapy.

GENE TRANSFER

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

NON VIRAL METHODS

Beverly E. Chaignaud Robert Langer Joseph P. Vacanti, The History of Tissue Engineering Using Synthetic Biodegradable Polymer Scaffolds and Cells; 1997 Birkhiiuser Boston

STRATEGIES OF TISSUE ENGINEERING

1. CONDUCTIVE APPROACHES
2. TISSUE INDUCTION
3. CELL TRANSPLANTATION APPROACHES

Tyagi P, Dhindsa Mk; Tissue engineering and its implication in dentistry,Indian J Dent Res, 20(2), 2009

CELL INJECTION THERAPY

• The tissue formation resulted from cellular action, injection of stem cells into the defect have been suggested to regenerate tissues.

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LIMITATION OF THIS THERAPY:-

• Inadequate localization of injected cells particularly in areas showing continuous movement e.g., beating heart.
• Requirement of delivery vehicle.(for adequate localization and prevention of direct contact with the immune system, using a delivery vehicle to carry and deliver the material has been attempted.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

CONDUCTIVE APPROACHES�

• Dental implant
• Guided tissue regeneration. ( used to regenerate the periodontal supporting structures and uses a material barrier to create a protected compartment for selective wound healing)

TISSUE INDUCTION�

• The inductive approach uses activation of cells situated close to the damaged or deficient tissue with specific signals. New bone could be formed at a non mineralizing site after implantation of powdered bone.

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• Exogenous factors are utilized in the form of injecting the signaling molecules

Fibroblasts growth factors-2 and 9 (FGFs-2 and -9),

Transforming growth factors b1 (TGF-b1)

Vascular endothelial growth factors (VEGFs),

Recombinant human growth/differentiation factor- 5 (rhGDF-5)

Bone morphogenetic protein.

Tissue engineering strategies. Three different tissue engineering approaches: conductive, inductive, and cell transplantation. (From Alsberg E, Hill E, Mooney DJ. Craniofacial tissue engineering. Crit Rev Oral Biol Med 2001;12(1):64–7

CELL TRANSPLANTATION APPROACHES�

Cell transplantation is an extremely attractive option when the inductive for a specific tissue factors are not known, when a large tissue mass or organ is needed, or when tissue replacement must be immediate.

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EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

cell-matrix tissue engineering strategy. Different methods used to produce cellular suspensions from a tissue biopsy are described in details by Tomlinson.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

ENGINEERED OROFACIAL TISSUES

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Orofacial structures are very unique in their development and function.

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• Orofacial bones are derived from both neural crest and paraxial mesoderm.

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• The skeletal bones are derived from mesoderm. Furthermore, orofacial bones undergo significant stress and strain produced from different muscles of mastication and respond differently to growth factors and mechanical stimuli.

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• Unlike alveolar bones, cementum has a very slow regenerative capacity. Unlike enamel, dentine can regenerate.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Dental implants have been advocated as tooth replacement; lack of adequate bone support and the proximity to anatomic structures e.g., maxillary sinus and inferior alveolar canal are the most frequently encountered problems.

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• Using bone grafts to provide bone support has been attempted; the success however was limited.

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• Tissue engineering, therefore, found an interest as the clinically relevant approach to regenerate dental tissues as well as the whole tooth.

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• The first attempt involved the application of calcium hydroxide for regeneration of dentine and pulp in traumatically exposed teeth.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• The field of tissue engineering has then grown tremendously to the development of fully functional bioengineered tooth and also replaces the soft tissues (skin, mucosa, muscles and salivary glands), bone and temporomandibular joints (TMJ).

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

Dentine-pulp complex

The regeneration of the dentine-pulp complex obtained

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By using pulp capping materials (e.g., calcium hydroxide, mineral trioxide aggregates, Biodentine),

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The stimulation of differentiation of the pulp progenitor cells into odontoblast-like cells or secretion of TGF-b1

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Angiogenesis, recruitment of progenitor cells, cell differentiation.

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finally mineralisation of the injured area.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Also Encapsulated stem cells were used for dentine-pulp regeneration.(Eg: Gelfoam-encapsulated dental stem cells )

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• Examples of materials employed for cell encapsulation :-

-Enzyme-cleavable,

-Customised self-assembled peptide hydrogels,

-Biodegradable lactide and glycolide.

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• Gelfoam-encapsulated dental stem cells :-stimulated the formation of the dentine-pulp complex in pulpless root canals in young permanent incisors.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Cell-free scaffolds e.g., Emdogain gel or combination of Emdogain and platelet rich plasma stimulated the regeneration of the dentine-pulp complex.

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• Growth factors [e.g., fibroblast growth factor basic (FGF), transforming growth factor b1 (TGF-b1) and endothelial growth factor (EGF)] have been also included within the scaffolds to modulate the function of stem cells.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

Periodontium

• Periodontitis is a widespread condition of inflammation that causes destruction of tooth supporting connective tissues (gingiva, alveolar bone, periodontal ligament and root cementum) and eventually teeth loss.

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• Guided bone/tissue regeneration to the most recent advances in tissue engineering employed to replace the lost tooth supporting structures in an attempt to maintain natural dentition.

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EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

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Guided tissue/bone regeneration membrane (GTR/GBR) utilises occlusive membranes to maintain the defective space (periodontal and alveolar defect), by encourage the appropriate cells to regenerate the lost tissues and support the newly formed tissues.

• SYNTHETIC POLYMERS used as GTR/GBR membranes:-

Polytetrafluoroethylene (PTFE, Gore- Tex),

Polylactide (e.g., Vivosorb & Epi-Gide),

Polylactide/glycolide.

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(The synthetic polymer’s degradation can be controlled by adjusting the molecular weights and the ratio of polylactide to polyglycolide segments )

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Biomimetic materials, collagen in particular, has been advocated as alternative to synthetic polymers

Eg:- Ossixt, Bio-Gide, Neomem, Biomend, Biomend Extendt.

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• Alkaline phosphatase or bioactive glass on collagen membranes has been also attempted:- To control the degradability & enhance the osteogenic potential of collagen membranes.

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• Growth factors and cytokines – also used for periodontal regeneration.

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EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

• Bacterial infection is a common problem with GTR/ GBR membranes. Incorporation of tetracycline, chlorhexidine and zinc could overcome this problem.

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• Recently, carbon nanotubes or CN-based composites (i.e. CNT associated with different biological molecules or polymers) have been identified as a innovative biomaterial for oral tissue regeneration.

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• In case of large defect:-

The application of multilayered membranes combining a layer of flexible synthetic polymer (e.g., polylac- tide-co-glycolide dimethacrylate) encased between two layers of natural polymers (e.g., collagen).

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

Endogenous regenerative technology�

• ERT depends on key endogenous resources (e.g., cells or growth factors and proteins) for regeneration of functional tissues.

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• For cell homing, a material niche (e.g., autogenic growth factors in combination with fibrin and Emdogain and Bio-Oss) is required to recruit the host stem cells to regenerate the periodontium.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

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For example:-

1. injection of autogenic gingival stem cells encapsulated within collagen or deproteinized bovine cancellous bone scaffold showed a significant improvement in periodontal tissue regeneration.

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2. Injection of autogenic fibroblasts is found to be safe and effective in restoring the interdental papillae.

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3. Combination of PRP with either human cultured periosteum/ hydroxyapatite or with patient’s own mesenchymal stem cells is effective in periodontal regeneration.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

The third generation of periodontal regeneration strategies, following GBR/GTR and ERT, involves the use of enamel matrix derivatives (EMD, Emdogain), that contains >90% amelogenin and <10% other protein.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

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These cells processed the amilogenin into 2 isoform

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TRAP LRAP

EMD( enamel matrix derivatives)

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stimulatory effect on the proliferation and differentiation of human periodontal ligament cells (HPDLCs) (90 percent amilogenin taken up by these cells)

(Tyrosin rich amilogenin peptide)suppressed the osteogenic differentiation of bone precursor cells

(leucin rich amilogenin peptide) enhanced terminal differentiation of bone-forming cells.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

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The differential effect of TRAP and LRAP can be employed to limit the pathological bone growth or to enhance bone formation as in the treatment of periodontal and orthopaedic diseases.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

Bioengineered teeth

Tooth development, odontogenesis, is a complex process involving a series of reciprocal epithelial–mesenchymal interactions and coordination between the crown and the root with its associated periodontium.

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Accordingly, cells dissociated from epithelium and mesenchymal tissues of prenatal or postnatal tooth germ were used to reconstitute a ‘‘bioengineered tooth germ’’ in vitro.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

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Test:- transplantation of bioengineered tooth germ into the oral environment or an organ culture has been then attempted to produce a whole tooth.

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Implantation of biodegradable polyglycolic/polylactide scaffolds, having the shape of a tooth and seeded with cells isolated from dissociated postnatal porcine third molar tooth buds, into rat hosts for 20–30 weeks successfully produced recognisable tooth structures (dentine, well defined pulp chamber, putative Hertwig’s root sheath epithelia, putative cementoblasts and dental organ with fully formed enamel).

The size of bioengineered tooth however was very small and did not conform to the shape and size of the scaffolds.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

Skin, oral mucosa, facial muscles

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• Initially, The keratinocytes sheet was used.

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• Recently, few composite allografts produced from decellularised collagen are commercially available e.g., Apligraf

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• Bio-Col or Mucograft, silk fibroin, collagen-elastin (Matriderm) is also used.

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• For engineering the facial muscles:- SATELLITE CELLS is used.

EAA NEEL, tissue engineering in dentistry:jop2014,8(42),915-928

CONCLUSION

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Tissue engineering provides a new era for therapeutic medicine; it is progressing very rapidly and extends to involve all tissues in our body.

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Tissue engineering brings the power of modern biological,chemical, and physical science to solve real clinical problems. This should yield numerous clinical benefits in dentistry, e.g., improved treatment for intraosseus periodontal defects; enhanced maxillary and mandibular grafting procedures, possibly even allowing lost teeth to be regrown; use of devices such as an artificial salivary gland and muscle (tongue) or mucosal grafts to replace tissues lost through surgery or trauma

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REFERENCES

• Tyagi P, Dhindsa Mk; Tissue engineering and its implication in dentistry,Indian J Dent Res, 20(2), 2009

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• Tissue engineering perspectives in dentistry: review of the literature RGO, Rev. Gaúch. Odontol. vol.66 no.4 Campinas Oct./Dec. 2018

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• Ensanya ali abou Neel et al. Tissue Engineering in dentistry. J Dent. Aug 2014

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• Alizadeh E, “A review on the applications of tissue engineering in branches of dentistry, Int J Contemp Dent Med Rev, vol. 2017, Article ID: 010617, 2017.

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• Langer R, Vacanti JP (May 1993). "Tissue engineering".Science 260 (5110)