latest technology in regeneration

1. TISSUE
ENGINEERING
DR. RINISHA SINHA
PART III POSTGRADUATE TRAINEE
DEPARTMENT OF PERIODONTOLOGY
See the hole, fill it, with anything
Perio 2000; Vol. 50
JOURNAL CLUB

2. INTRODUCTION
TISSUE ENGINEERING IN PERIODONTOLOGY
FROM PASSIVE TO ACTIVE APPROACH
CRITERIA FOR SUCCESS
CELLS
SCAFFOLDS
SIGNALS
BONE MORPHOGENETIC PROTEIN-2
POTENTIAL CHALLENGES
REFERENCES

3. INTRODUCTION
• Regeneration of the lost periodontal tissue is the ultimate goal of a successful periodontal therapy.
• There are two strategies for periodontal regeneration: Guided Tissue Regeneration and TISSUE
ENGINEERING approaches.
• The field of tissue engineering/regenerative medicine has emerged with the goal of replacing and
regenerating lost or damaged tissues to restore normal function and structure.
Fig: Apical migration of the supracrestal tissue
attachment, formation of periodontal pockets, and
destruction of PDL and alveolar bone are sequalae of
periodontitis. After decontamination of the defect,
periodontal tissue engineering based treatment
strategies can be implemented to regenerate the
periodontium, and restore structure and function.
Reference: Brouwer KM, Lundvig DM, Middelkoop E, Wagener FA, Von den Hoff JW. Mechanical cues in
orofacial tissue engineering and regenerative medicine. Wound Repair Regen 2015;23:302-11.
Craniofacial tissue engineering
conduction and
induction of host cells
cell transplantation gene therapy
• involves the reconstitution of cementum, periodontal
ligament (PDL) and alveolar bone around teeth
Periodontal Regeneration
Reference: Alsberg E, Hill EE,
Mooney DJ. Craniofacial
tissue engineering. Crit Rev
Oral Biol Med 2001;12:64-
75.
Reference: Wang HL, Greenwell H, Fiorellini J, et al. Research,
Science and Therapy Committee. Periodontal regeneration. J

4. TISSUE ENGINEERING IN PERIODONTOLOGY
• The concept of Tissue Engineering was proposed by Langer and Vacanti in 1993.
• As defined by Langer and Vacanti, tissue engineering 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.”
Sixteenth century Tagliacozzi of Bologna, Italy “De Custorum Chirurigia per
Insitionem”
1970 WT Green (Orthopaedic
surgeon)
created cartilage
1986 Karl Meyer created a skin substitute by
using a collagen matrix
1993 Dr. Langer defined the appropriate
concept
Reference: Kaban LB, et al. The engineering of craniofacial tissues in the laboratory: A review of
biomaterials for scaffolds and implant coatings. Dent Clin North Am 2006;50:205-16.

5. FROM “PASSIVE TO ACTIVE” – THE TISSUE
ENGINEERING
APPROACH
Periodontal healing : most complex process in the human body
Whether the damaged tissues heal by regeneration or repair depends upon two crucial factors:
• The availability of cell types needed; and
• The presence or absence of cues or signals necessary to recruit and stimulate these cells.
EX-VIVO APPROACH
In this approach, the tissue is created in a laboratory by culturing the cells on a biodegradable
scaffold in the presence of molecular factors required for growth and then it is transferred into the
body.
Various body organs have been developed by this technique.
Reference: Pandit N, Malik R, Philips D. Tissue engineering: A new vista in
periodontal regeneration. J Indian Soc Periodontol 2011;15:328-37.

6. IN-VIVO APPROACH
In this technique, all the components which are required for regeneration are placed in the tissue
defect and an environment which is conducive to maximum regeneration is created to achieve
favourable regeneration.
Stem/ Progenitor cells
Conductive scaffolds /
Extracellular matrix
Signalling molecules
The tissue engineering approach to bone and periodontal regeneration combines three key elements to
enhance regeneration.
Reference: Kao RT, Murakami S, Beirne OR. The use of biological mediators and
tissue engineering in dentistry. Periodontol 2000 2009;50:127-53

7. CRITERIA FOR SUCCESSFUL TISSUE
ENGINEERING
BIOMECHANICA
L PROPERTIES
Scaffold
Architectural geometry
Space-maintaining
properties
BIOLOGICAL
FUNCTIONS
Cell recruitment,
proliferation, survival in
culture and at the site of
implantation
Neovascularization
Delivery of
morphogenetic
regulatory and growth
factors
Events and processes required for periodontal regeneration:
1. Regeneration depends on availability of appropriate cell types and signals that activate
the cells
2. Wrong cell types may have to be excluded
3. Local environment plays a major role in the recruitment of the right cells and preventing
the wrong cells. Local environment includes cementum matrix and cementodentinal junction
4. Substances in the local environment affect cell migration, adhesion, proliferation, and
differentiation.
Reference: Malgikar S, Akula U. Bone morphogenetic proteins in periodontal tissue
regeneration. J Dent Allied Sci 2017;6:74-7.

8. CELLS
• Stem cells are immature progenitor cells capable of self renewal and multi-lineage
differentiation through a process of asymmetric mitosis that leads to two daughter cells, one
identical to the stem cell (daughter stem cell) and one capable of differentiation into more
mature cells (progenitor cells).
• Stem cells may be:
1. Totipotent
2. Pluripotent
3. Multipotent
Reference: Nadig RR. Stem cell therapy- hype or hope. A review. J Conserv Dent
2009;12:131-8.
Reference: Krampera M, Franchini M, Pizzolo G, Aprili G. Mesenchymal stem cells: From biology to clinical use. Blood
Transfus 2007;5:120-9.
Stem Cells
Embryonic
Stem cells
Derived from
blastocysts
Totipotent
cells
Adult Stem
Cells
Multipotent
cells
Hemopoetic
stem cells
Mesenchymal
Stem Cells
Reference: Friedenstein AJ. Precursor cells
of mechanocytes. Int Rev Cytol
1976;47:327-59.

10. SCAFFOLDS
• Provides a 3D substratum on to which the cells can proliferate and migrate, produce a
matrix and form a functional tissue with a desired shape.
Naturally derived Synthetic
• Ceramics
• HA [Lee YK et al. 2008]
• TCP [Stavropoulos A et al. 2010]
• Natural polymers like hyaluronic acid (Ballini A et al. 2009), alginate
(Lin HR 2004), agarose (Tabata M et al. 2005), collagen (d’Aquino R et al. 2009),
albumin (Chan WD et al. 2009), chitosan (Lee YM et al. 2000)
Synthetic
•Polymers
•Synthetic polyesters, such as PGA, PLA and polycaprolactone. e.g. PGA
meshes [Gunatillake PA. 2003]
•Co polymers of polyethylene oxide, and polypropylene oxide known as
“pluronics” [Xu XL et al. 2005]
•PLGA a copolymer of PGA and PLA.
•PLGA copolymer foam.[Abukawa H et al. 2003]
•PLGA/HA matrices.[Kim S et al. 2008]
•Polyphosphazenes.[Laurencen CT et al. 2003]
•Nano calcium sulphate.[Mohan et al. 2008]

11. SIGNALS
• Signaling molecules are proteins that may act locally or
systemically to affect the growth and function of cells in various
manners.
• The two types of signaling molecules that have received the
greatest attention are growth factors and morphogens that act by
altering the cell phenotype i.e. by causing the differentiation of
stem cells into bone forming cells - a process commonly known as
osteoinduction.
• Growth factors act on the external cell membrane receptors of a
target cell, provide the signal to local mesenchymal and epithelial
cells to migrate, divide, and increase matrix synthesis.
• The growth factor that has received the most attention in hard and
soft tissue wound healing is platelet derived growth factor.

12. BONE MORPHOGENETIC PROTEINS (BMP)
• Bone morphogenetic proteins (BMPs) are the members of transforming growth factor-β (TGF-β)
superfamily.
• They possess the unique ability to induce cartilage and bone formation.
• The name BMP was given in 1965 by Urist and colleagues to the active components in demineralizing
bone and bone extracts, which are capable of inducing bone formation at ectopic sites (intramuscular).
• Reddy and Huggins (1972) found that bone extracts induce bone formation in ectopic sites
(subcutaneous).
• In 1988, the first BMPs were isolated and their cDNAs were cloned by Wozney et al.
• At least seven BMPs have been isolated from bovine and human sources.
• In the field of periodontal regeneration, much of the research interest has focused on BMP-2 (OP-2),
BMP-3 (osteogenin), and BMP-7 (OP-1).
Reference: Hou LT, Liu CM, Liu BY, Chang PC, Chen MH, Ho MH, et al. Tissue engineering bone formation in novel
recombinant human bone morphogenetic protein-2 atellocollagen sponge composite scaffolds. J Periodontol
2007;78:335-43.
Reference: King GN, Cochran DL. Factors that modulate the effects of bone morphogenetic protein-induced periodontal
regeneration: a critical review. J Periodontol. 2002 Aug;73(8):925-36. doi: 10.1902/jop.2002.73.8.925. PMID: 12211503.

13. STRUCTURE AND CLASSIFICATION
• The human genome encodes 20 BMPs.
• BMPs are dimeric molecules critically dependent on the single intermolecular disulfide bond
for biological activity. The monomeric subunit has about 120 amino acids, including seven
conserved cysteine residues.
• Divided into four distinct subfamilies:
1st group: BMP-2 and BMP-4
2nd group: BMP-3, BMP-3B (growth differentiating factor 10 or GDF-10)
3rd group: BMP-5, BMP-6, BMP-7, BMP-8
4th group: GDF-5, GDF-6, GDF-7 (cartilage-derived morphogenetic protein
1, 2, 3)

14. FUNCTIONS OF BMP
It regulates various
mesenchymal/osteoblasti
c activities like the
following:
Chemotaxis
Anchorage-dependent cell attachment (fibronectin)
Cell replication (mitosis)
Differentiation of osteoblasts
Alkaline phosphatase activity
Osteocalcin synthesis/mineralization[10]
BMPs also play a critical role in
tooth morphogenesis.
BMPs 2, 4, and 7 are expressed in dental epithelium.
Recombinant BMPs 2 and 4 can be used as a substitute for dental
epithelium in inducing mesenchyme differentiation.
BMP-3 and BMP-7 have been “immune” localized to developing
periodontal ligament, cementum, and alveolar bone.
On the other hand, BMP-2 was localized only in alveolar bone
during root morphogenesis.
A role of BMP-3 in the cementoblast lineage has been suggested
Reference: Wozney JM. The potential role of
bone morphogenetic proteins in periodontal
reconstruction. J Periodontol. 1995
Jun;66(6):506-10. doi:
10.1902/jop.1995.66.6.506. PMID:
7562340.
Reference: Hughes FJ, Turner W, Belibasakis G,
Martuscelli G. Effects of growth factors and cytokines
on osteoblast differentiation. Periodontol 2000.
2006;41:48-72. doi: 10.1111/j.1600-
0757.2006.00161.x. PMID: 16686926.
Reference: Wozney JM. The potential role of bone
morphogenetic proteins in periodontal
reconstruction. J Periodontol. 1995 Jun;66(6):506-
10. doi: 10.1902/jop.1995.66.6.506. PMID:
7562340.

15. BONE MORPHOGENETIC PROTEIN CARRIERS
• One of the major obstacles to the clinical use of BMPs is the challenge to define the optimal delivery
system.
• As BMP is soluble in extracellular solution, it must have a carrier, without which it is phagocytized
within 10 days.
• The carrier material can be in the form of blocks, granules, paste, and solution or as self-setting
cement.
Reference: Bartold PM, Xiao Y, Lyngstaadas SP, Paine ML, Snead ML. Principles and applications of cell delivery systems for periodontal regeneration.
Periodontol 2000 2006;41:123-35.
Although a matrix
carrier is not essential
to promote bone
formation, there are a
number of advantages
to an appropriate
carrier including:
localization and retention of BMP to the site,
providing a 3D extracellular matrix scaffold for mesenchymal cell infiltration,
a shape that may help and define the resulting new bone, and
providing a substrate for cell growth and differentiation.

16. RECEPTOR FOR BMPS
• A comparison of other growth factors reveals that BMPs are (34-38%) related to the
transforming growth factor beta (TGF-β) family. BMPs and TGFβ ligands have cognate
BMP type I and II receptors and TGF-β type I and II receptors, respectively, which
function as protein kinases.
Reference: Fiorellini JP, Howell TH, Cochran D, Malmquist J, Lilly LC, Spagnoli D, Toljanic J, Jones A, Nevins M. Randomized study evaluating recombinant human
bone morphogenetic protein-2 for extraction socket augmentation. J Periodontol. 2005 Apr;76(4):605-13. doi: 10.1902/jop.2005.76.4.605. PMID: 15857102.
The BMP type I receptor
protein kinase
phosphorylates
intracellular signaling
substances, Smads (a
fusion of the Sma gene
in Caenorhabditis.
elegans and Mad gene
in Drosophila) 1, 5, and
8.
The TGF-β type I
receptor kinase
specifically
phosphorylates Smads 2
and 3.
The phosphorylated
Smads 1, 5, and 8
(BMP-signaling Smads)
and Smads 2 and 3
(TGFβ-signaling
Smads) partner with
Smads 4 to form
signaling complexes in
the cytosol that enter
the nucleus to initiate
the transcription of
downstream targets.
Although BMPs and
TGFβ ligands signal via
distinct receptors, they
act in collaboration
during bone and tooth
morphogenesis.
MECHANISM OF ACTION

17. PRODUCTION OF RECOMBINANT HUMAN BONE
MORPHOGENETIC PROTEIN-2
• Recombinant proteins are produced from one of several cellular expression systems of bacteria, insect
cells, or mammalian cells.
• Recombinant human (rhBMP-2) is produced using a mammalian cell expression system.
Reference: Lynch SE, Genco RJ, Marx RE. Tissue Engineering, Applications in Maxillofacial Surgery and Periodontics. Chicago: Quintessence
Books; 1996. p. 25-75.
A Strong
Promote
r
A
Selectable
Marker

18. BMP IN PERIODONTAL REGENERATION
• In the field of periodontal regeneration, much of the research interest has focused on BMP-2 (OP-2),
BMP-3 (osteogenin), and BMP-7 (OP-1).
• The first human study using a bmp to promote periodontal regeneration utilized a single application of
BMP-3 (osteogenin) combined with demineralized bone allograft in a submerged tooth model.
• Crude preparations of BMP-2 and BMP-3 applied in surgically induced furcation defects appeared to
stimulate periodontal regeneration.
• Recent studies have utilized recombinant human BMP to determine their potential for correcting
intrabony, supra-alveolar, furcation, and fenestration defects. Histologic analysis revealed periodontal
regeneration with areas of ankylosis.
• Contrary to these findings, BMP-7 augmentation resulted in a significant increase in periodontal
regeneration without any ankylosis.
Reference: Bowers G, Felton F, Middleton C, Glynn D, Sharp S, Mellonig J, et al. Histologic comparison of regeneration in human
intrabony defects when osteogenin is combined with demineralized freeze-dried bone allograft and with purified bovine
collagen. J Periodontol. 1991;62:690–702.
Reference: Ripamonti U, Renton L. Bone morphogenetic protein and the induction of periodontal tissue regeneration. Periodontol
2000. 2006;41:73–87
Reference: Saito A, Saito E, Handa R, Honma Y, Kawanami M. Influence of residual bone on recombinant human bone
morphogenetic protein-2-induced periodontal regeneration in experimental periodontitis in dogs. J Periodontol. 2009;80:961–8.

19. • Healing through ankylosis has been a concern, so most of the recent research utilizing recombinant
human BMPs has involved in the preparation of implant site for osseointegration.
• BMPs also show much promise in promoting dental implant wound healing.
• A pilot study in non-human primates tested the single application of BMP-7 (OP-1) around
immediate extraction socket implants and found increased bone growth as measured histologically
at 3 weeks.
• In a recent study, combined adenovirus mediated human BMP-2 (Adv-hBMP-2) gene-modified bone
marrow stromal cells (BMSCs) with allograft enhanced the defect healing and improved the
strength of implant fixation with osseointegration in 3-mm bone defect around a titanium alloy
implant.[12]
Reference: Recombinant Human Bone Morphogenetic Protein-2 for Peri-Implant Bone Regeneration: A Case Report. J
Periodontol. 2011;82:1212–8.
Reference: Rutherford RB, Sampath TK, Rueger DC, Taylor TD. The use of bovine osteogenic protein to promote rapid
osseointegration of endosseous dental implants. Int J Oral Maxillofac Implants. 1992;7:297–301.
Reference: Lan J, Wang Z, Wang Y. The effect of combination of recombinant human bone morphogenetic protein-2 and basic
fibroblast growth factor or insulin-like growth factor-I on dental implant osseointegration by confocal laser scanning
microscopy. J Periodontol. 2006;77:357–63.

20. • Bovine BMP (bBMP) tested in a dog model has shown to increase the rate of osseointegration
around cylindrical uncoated endosseous implants as evidenced histomorphometrically 4
weeks after implantation.
• The tissue reactions to titanium implants coated with bBMP were further assessed by
scanning electron microscopy (SEM) for 12 weeks in the same dog model.
• The results revealed abundant lamellar bone formation around bBMP-coated implants. This
bone was found adjacent to the implant threads and frequently entered the implant holes.
Reference: Wang X, Baolin L, Yan J, Yang X. The effect of bone morphogenetic protein on osseointegration of titanium implants. J
Oral Maxillofac Surg. 1993;51:647–51.
Reference: Wang X, Jin Y, Liu B, Zhou S, Yang L, Yang X, et al. Tissue reactions to titanium implants containing bovine bone
morphogenetic protein: A scanning electron microscopic study. Int J Oral Maxillofac Surg. 1994;23:115–9.

21. POTENTIAL CHALLENGES YET TO OVERCOME
• Notwithstanding these significant advances, there are still numerous biological, technical and
clinical hurdles to be overcome:
1. Complete understanding about the molecular and signaling pathways that form the
ground for tissue engineering and underlie cell renewal and differentiation is yet to
obtain.
2. As cell culture medium often requires xenogenic products (such as fetal bovine serum or
mouse feeder layers), cell cultures may not be completely free of pathogens and
infectious risks are a concern.
3. Culture conditions are not sufficiently developed to mimic the cell microenvironment in
vivo. The ideal matrix scaffold should mimic native extracellular matrix, support cell
attachment, allow controlled release of bioactive factors, be conducive to tissue in growth
and facilitate laboratory handling.
4. Cell based periodontal therapy relate to immune rejection after administration of stem
cells into the host. A potential solution to this problem lies in the use of autologous stem
cells to overcome immune rejection.
5. Recently, it was hypothesized that bone-marrow-derived mesenchymal stem cells could
Reference: Bartold PM, Xiao Y, Lyngstaadas SP, Paine ML, Snead ML. Principles and applications of cell delivery systems for periodontal regeneration.
Periodontol 2000. 2006;41:123–35.
Reference: Choumerianou DM, Dimitriou H, Kalmanti M. Stem cells: Promises versus limitations. Tissue Eng Part B
Rev. 2008;14:53–60.
Reference: Mishra PJ, Mishra PJ, Glod JW, Banerjee D. Mesenchymal stem cells: Flip side of the coin. Cancer Res.
2009;68:1255–8.

22. CONCLUSION
• Several preclinical studies have shown that rhBMP-2 induces normal physiological bone in
clinically relevant defects in the craniofacial skeleton.
• Recombinant protein morphogens recently approved by the FDA, such as BMP-2 and
BMP-7, could be applied in dentistry to facilitate the repair of craniofacial
structures.
• Further, studies are needed for the development of carrier materials that have mechanical
properties and surgical practicality appropriate for controlled release of BMPs.
We need to look beyond before we can achieve the dream.
Tissue engineering has enlarged our vision and thus made the fascination of being able to achieve regeneration of
periodontal complex in its entirety a reality. Though the task has been arduous, but the promise still remains…

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