tissue engineering

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GOOD MORNING

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Department of periodontics

By :-Prateek irwin gargpg 1st year

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TISSUE ENGINEERING

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CONTENTS

Introduction Definition Historical Background Need for Tissue Engineering Tissue engineering Triad Strategies to engineer tissue Basic Principles

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Cells Scaffolds Signalling Molecules

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Gene Therapy Soft tissue Augmentation Platelet Rich Plasma Distraction Osteogenesis Conclusion References

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INTRODUCTION

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Ð Term TE covers a broad range of applications. Ð In practice-term is closely associated with

applications that repair or replace portions of or whole tissues i.e. Bone cartilage blood vessels bladder Skin

Ð Tissues involved require certain mechanical & structural properties for proper functioning. 

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TISSUE ENGINEERING

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

VACANTI & LANGER,

“A combination of the principles & methods of life sciences with that of engineering, to develop materials & methods to repair damaged or diseased tissues, & to create entire tissue replacements”

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DEFINITION

SHALAK & FOX 1988,

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

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HISTORICAL BACKGROUND

In 1970 W.T. Green, an orthopedic surgeon conducted 1st research related to TE.

suggested that by

implanting chondrocyte cells into spicule of bone, where cell multiplication & growth of bone continues →cartilage formation

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In the Mid-1980’s Dr. Vacanti and Dr. Langer devised a method that would attempt to create scaffoldings for cell delivery instead of using naturally occurring scaffoldings that could not be replicated.

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In 1994, TES was founded by Charles & Vacanti officially in Boston.

By 2005, TERMIS which included both Asian & European Societies, was created.

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NEED FOR TISSUE ENGINEERING

Tissue engineering holds promise of producing better organs for transplant. Using tissue engineering techniques & gene therapy it may be possible to correct many otherwise incurable genetic defects.

A major goal of tissue engineering is in-vitro construction of transplantable vital tissue.

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

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Major shortcoming autografts & allografts in achieving regeneration

humans don’t have significant stores of excess tissue for transplantation

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TRIAD

CELLS

SCAFFOLDS SIGNALLING MOLECULES

REGENERATEDTISSUE

Time

AppropriateEnvironment

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Successful tissue engineering requires interplay among three components:

Implanted & cultured cells that will create new tissue;

Biomaterial to act as scaffold or matrix to hold cells;

Biological signaling molecules that instruct cells to form desired tissue type.

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STRATEGIES TO ENGINEER TISSUES

Characterised in 3 major classes

Conductive

Inductive

Cell transplantation approaches

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TECHNIQUES

2 methods In Vitro In vivo

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IN VITRO

Construction in laboratory of vital tissue & its subsequent implantation into host body.

Advantage is ability to examine tissues as they are formed, & to perform specific tissue measurements.

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By in-vitro TE of tissues such as bone, need for recruitment of specific cells to site is negotiated & predictability of regeneration is enhanced,

overcoming many of limitations with conventional therapies.

Disadvantage is absence of a physiologic

environment Implanted tissue has to be incorporated

with the surrounding bone.

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IN VIVO

Indicates obvious advantage of tissue regeneration in-vivo in which incorporation occurs as tissues are formed.

This has formed basis for tissue engineering, which now includes implantation of porous

matrices, seeded with appropriate cells & signalling molecules, to facilitate tissue regeneration in-vivo.

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Disadvantage of in-vivo approach

regenerating tissues may get dislodged or degraded by mechanical forces acting normally at site, before regenerated tissue is fully formed & incorporated

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CELLS

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ORIGIN OF CELLS

Osteogenic cells could be obtained through an atraumatic biopsy & amplified in an appropriate 3-D carrier in-vitro.

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MODES OF SUPPLY

There are two modes for supplying exogenous cells into defect:

Cell seedingCell suspension

Cell incorporation into implantable matrices, which ensures their localization at treatment site - concept being referred to as cell seeding.An alternative is to inject a cell suspension into sealed compartment containing defect.

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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).

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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.Example   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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Allogeneic 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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Xenogenic cells are these isolated from individuals of another species. In particular animal cells have been used quite extensively in experiments aimed at construction of CV implants.

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STEM CELLS

Undifferentiated cells with ability to divide in culture & give rise to different forms of specialized cells.

Characteristic Features: They are capable of dividing & renewing

themselves for long periods  They are unspecialized  They can give rise to specialized cell types.

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Stem cells could be: Adult stem cells Embryonic stem cells

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Adult stem cells Also known as somatic (from Greek "of the body") stem cells & germline (giving rise to gametes) stem cells, they can be found in children, as well as adults.

Pluripotent adult stem cells are rare & generally small in number but can be found in a number of tissues including umbilical cord blood

Most adult stem cells are lineage-restricted & are generally referred to by their tissue origin 

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Embryonic stem cell lines are cultures of cells derived from epiblast tissue of inner cell mass of a blastocyst or earlier morula stage embryos — approximately 4 to 5 days old in humans & consisting of 50–150 cells. ES cells are pluripotent & give rise during development to all derivatives of 3 primary germ layers: ectoderm, endoderm & mesoderm.

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Based on potency the cells are divided into:1. Totipotent cells.2. Pluripotent cells.3. Multipotent cells.4. Oligopotent cells.5. Unipotent cells.

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Totipotent stem cells can differentiate into embryonic & extraembryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from fusion of an egg & sperm cell. Eg: Fertilized egg

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Pluripotent stem cells are descendants of totipotent cells & can differentiate into nearly all cells, but cannot give rise to an entire organism. i.e. cells derived from any of three germ layers

Multipotent stem cells give rise to a limited range of cells within a tissue type. Eg: Hematopoietic stem cells.

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Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.

Unipotent cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells. E.g. muscle stem cells.

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APPLICATION

Cell Replacement Therapies Cells could be stimulated to develop into

specialized cells that represent renewable sources of cells & tissue for transplantation.

Cell replacement therapy could treat injuries & various genetic & degenerative conditions including muscular dystrophies, retinal degeneration, Alzheimer disease, Parkinson's disease, arthritis, diabetes, spinal cord injuries, & blood disorders such as hemophilia.

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SCAFFOLDS

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SCAFFOLDS

Used to guide organization, Growth & differentiation of cells in process of

forming functional tissue provide both physical & chemical signals.

Tissues are composed of cells, insoluble extracellular matrix (E.C.M.) soluble molecules that serve as regulators of cell

function.

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E.C.M. usually composed of 3 components: Collagen Glycoprotein Proteoglycan

The E.C.M. is important for Growth Function - various cell types involved.

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TYPES OF SCAFFOLDS

ABSORBABLE NON-ABSORBABLE

SYNTHETIC POLYMERS•P.L.A.•P.G.A

NATURAL MINERALS•Anorganic Bone

NATURAL POLYMERS•Collagen•Fibrin•Chitosan

SYNTHETIC CEREMICS

•Calcium Phosphate

SYNTHETIC POLYMERS

•Polytetra flouroethylene

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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.

1. Tricalcium Phoshphate: Porous form of calcium phosphate ß-TCP Problem -physiochemical dissolution after implantation

2. Synthetic Hydroxyapatite: development - second form of bioceramic. Rationale - mineral naturally occurring in bone is

hydroxyapatite.

NON-ABSORBABLE

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SYNTHETIC POLYMERS

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

NON-ABSORBABLE

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SYNTHETIC POLYMERS

degradation by hydrolysis Polyglycolic acid - degrades fast Polylactic acid (L-lactide) - most stable in-

vitro Thus, modification of poly (L-lactide) by

cross-linking or addition of D-lactide more rapid degradation, thus diminishing poly L-lactide disadvantage of slow degradation.

polyglactin 910, a co-polymer of glycolide and L-lactide – 90/10 molar ratio

ABSORBABLE

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NATURAL POLYMERS

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

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

ABSORBABLE

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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

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NATURAL MINERALS

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

remaining after chemical or low heat extraction of the organic component.

Usually bovine bone mineral is used Currently available - deproteinated,

which supports cell-mediated resorption.

ABSORBABLE

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SIGNALLING MOLECULES

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SIGNALLING MOLECULES

Signalling molecules or biologic modifiers - materials or proteins & factors that have potential to alter key cellular events in host tissue, by stimulating or regulating the wound healing process.

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MODE OF ACTION

INTRACRINE(PTHrp)

PARACRINE(PDGF, TGF-β)

JUXTACRINE(Stem cell

factor)

AUTOCRINE(BMPs, TGF-

α)

SYSTEMIC(ENDOCRINE)(PTH,GH,LH)

LOCAL

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CLASSIFICATION

 3 groups1. Growth & Differentiation Factors

2. Extracellular Matrix Proteins & Attachment Factors

3. Mediators of Bone Metabolism

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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).

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PLATELET DERIVED GROWTH FACTOR

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.

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

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RECOMBINANT BMP-2 PRODUCTION

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.

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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.

Includes many steps: Synthesizing of precursor polypeptide chains. Correct refolding & demineralization of these

chains, Glycosylation of protein.

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Ð Protein is then secreted out of cell into conditioned medium, in process of which propeptide is removed from mature portion of protein atspecific AA sequences.

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

factors with biochemical & functional similarities to insulin.

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

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TRANSFORMING GROWTH FACTOR-β:

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

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.

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.

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BONE MORPHOGENETIC PROTEINS (BMPS):

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

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MODES OF PREPARATION:

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

RECOMBINANT DNA METHODS-when recombined with DNA of cloning vector,

can be replicated, transcribed & translated. Used for production of (rh BMP-2) & (rh BMP-7).

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rh BMP2 PRODUCTION

cDNA CODING FOR rh BMP-2

TRANSFECTED INTO HOST CELL (CHO CELL)

rh BMP-2 SECRETED

STORED IN ALIQUOTS & FROZEN

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PUT IN GROWTH MEDIUM & HARVESTED

rh BMP-2 REMOVED BY FILTRATION

PURIFIED BY COLUMN CHROMATOGRAPHY

PLACED IN VIALS & LYOPHILIZED

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MEDIATORS OF BONE FORMATION Several agents which affects the growth of bone:

PROSTAGLANDINS: Result of cyclo-oxygenation of precursors derived

from arachnoid acid. Found - variety of tissues. Effect varies considerably from stimulating inflammation & bone resorption to enhance bone formation

GLUCOCORTICOIDS:Such as dexamethasone have prostaglandins, complex

direct & indirect effects on bone formation. Chronic glucocorticoids administration results in bone loss, through depression in osteoblast function

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: 1st Generation : alkyl side chains

Eg: Endronate 2nd Generation : amino terminal grp.

Eg: Alendronate & Pamidronate 3rd Generation : cyclic side chains

Eg: Risedronate

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BISPHOSPHONATES

PYROPHOSPHATES

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FIBROBLAST GROWTH FACTORS:

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

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

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

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PLATELET RICH PLASMA

PDGF & TGF-β are well-established wound healing “hormones”.

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

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

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PROCESSING OF P.R.P.

ApheresisProcurement 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.

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

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APHERESIS

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

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

One of components is then withdrawn & remaining components are re-transfused into patient or donor.

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PROCUREMENT FROM ONE UNIT BLOOD:

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).

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

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PROCUREMENT ON A SMALL SCALE Recent studies focussed on using minimal

amount of blood (10-50 ml) depending upon procedure involved, & common laboratory centrifuge for procurement of PRP.

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.

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ADVANTAGES OF USE OF AUTOLOGOUS P.R.P.

Safe as it is autologous preparation.

Promotes adhesiveness & tensile strength for clot stabilization.

Biologically acceptable.

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

Promotes angiogenesis.

Haemostatic properties.

Dense fibrin net that is highly osteoconductive.

High concentrations of leukocytes, which act as “autologous antibiotic”, reducing risk of infection.

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FUTURE PERSPECTIVE

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GENE THERAPY

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.

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GENE EXPRESSION & PROTEIN SYNTHESIS

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

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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.

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GENE TRANSFER

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.

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VIRAL METHODS• Introduce RNA with two enzymes- reverse transcriptase &

integrase• Enable productin of DNS from RNA – latter add DNA copy to

target cell DNARetroviruses

• DNA is transferred into target cell nucleus, but not integrated with host DNA.

• Infects dividing & non-dividing cellsAdenoviruses

• Small viruses with single stranded DNA that cause no human disease.

• Infects dividing & non-dividing cells

Adeno Associated

Viruses

Herpes Virus

Lentivirus

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NON VIRAL METHODS• Direct injection of therapeutic DNA into target

cells using a gene gunMicro seeding gene therapy

• Creation of artificial lipid spheres with an aqueous core

• Carries therapeutic DNA, capable of passing DNA through target cell membrane

Cationic Liposomes

• Therapeutic DNA gets inside target cells by chemically linked DNA to molecule that bind to special cell receptor

Macromolecular Conjugate

• Delivers naked DNA via polymer matrix sponges.

Gene Activated Matrices

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SOFT TISSUE AUGMENTATION

Most commonly used applications of tissue engineering is in field of dermatology, where possibility of obtaining a large amount of dermal-epidermal tissue from a small portion of skin of same patient in a short period of time, has allowed treatment of extensive burns.

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CONCLUSION

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Future developments in fields of molecular & cell biology, developmental biology & tissue engineering, will have significant impact on managing anatomic changes due to disease process.

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REFERENCES

Vacanti, Charles A. "The history of tissue engineering." Journal of Cellular and Molecular Medicine 10 (2006): 569-76.

Lynch SE, Genco RJ, Marx RE. Tissue Engineering: applications in maxillofacial surgery and periodontics.

Tissue engineering - Wikipedia, the free encyclopedia.

Langer R, Vacanti JP (May 1993). "Tissue engineering". Science 260 (5110): 920–6. doi:10.1126/science.8493529.

Stem cell - Wikipedia, the free encyclopedia. Stem Cells: General Features and

Characteristics. Hongxiang Hui.

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