Significance of Stem Cells in Periodontal Regeneration: An Overview

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ABSTRACT

Significance of Stem Cells

in Periodontal Regeneration:

An Overview

Copyright©2015

Stem cell refers to a clonogenic, undifferentiated cell that is capable of self-renewal and multi-lineage differentiation. They are unique and form the basis of development, growth and survival of a living organism. Stem cells are the foundation cells of every organ and tissue in the body including the periodontium. Numerous clinical techniques, including bone grafts, root surface conditioning, barrier membranes and various growth factors, have been utilised over the years in an attempt to achieve periodontal regeneration. The recent identification and isolation of stem cells from the periodontal ligament represents a significant development in the progress towards predictable periodontal regeneration. This review is an overview on the role of stem cells and tissue engineering in periodontal regeneration.

INTRODUCTION

Stem cells are unspecialised cells in the human body that are capable of becoming specialised cells, each with new specialised cell functions1. Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialised function, such as a muscle cell, a red blood cell or a brain cell2.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialised cells capable of renewing them-selves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Given their unique regenerative abilities, stem cell research has the potential to impact not just one disease but numerous ones, e.g., diabetes, cancer, cardiovascular disease, Alzheimer’s disease, burn victims, leukemia, tooth regeneration, bone regeneration and many more.

DISCUSSION OF STEM CELL

Properties of stem cells

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Stem cells are different from the other types of cells found in the human body. Although they can be harvested from various sources, they all share some of the same properties like the following:

• Stem cells can divide and renew themselves • Stem cells are unspecialised

• Stem cells can give rise to specialised cells

It is thought that a cell’s genes regulate the internal signals that trigger this process. External signals are those outside of the cell, which include

Rosaiah Kanaparthy1*, Aruna Kanaparthy2, Richa Jain3

1 Assistant Professor, Department of Periodontics, College of Dentistry, Jizan University, Jizan, Saudi Arabia 2 Assistant Professor, Department of

Operative Dentistry, College of Dentistry, Jizan University, Jizan, Saudi Arabia 3 Sr. Lecturer, Department of Periodontology,

Peoples Dental Academy, Bhopal Peoples University, India

 Address reprint requests to *Dr. Rosaiah Kanaparthy, Assistant Professor, Department of Periodontics, College of Dentistry, Jizan University, Jizan, Saudi Arabia

E-mail: drrosaiah@gmail.com

 Article citation: Kanaparthy R, Kanaparthy A, Jain R. Significance of stem cells in periodontal regeneration: an overview.

J Pharm Biomed Sci2015;05(12): 954–960.

Available at www.jpbms.info

Statement of originality of work: The manuscript has been read and approved by all the authors, the requirements for authorship have been met, and that each author believes that the manuscript represents honest and original work.

Source of funding: None.

Competing interest / Conflict of interest:

The author(s) have no competing interests for financial support, publication of this research, patents, and royalties through this collaborative research. All authors were equally involved in discussed research work. There is no financial conflict with the subject matter discussed in the manuscript.

Disclaimer: Any views expressed in this

paper are those of the authors and do not reflect the official policy or position of the Department of Defense.

NLM Title J Pharm Biomed Sci CODEN JPBSCT

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chemicals released from other cells, physical connec-tions with nearby cells and various other molecules in the surrounding area.

Stem cells have the capacity of performing three important functions: (1) plasticity, (2) homing, and (3) engraftment.

Plasticity: It is the potential to change into other cell types. Under special conditions tissue-specific adult stem cells can generate a whole spectrum of cell types of other tissues, even crossing germ layers. This phenom-enon is referred to as stem cell transdifferentiation. It can be induced by modifying the growth medium when stem cells are cultured in vitro or transplanting them to an organ of the body different from the one they were originally isolated from. Four explanations for the phe-nomenon of plasticity in post natal stem cells have been proposed: which are listed:

• There might be persistent stem cells from embryonic development with broad develop-mental potentials which are maintained within the adult bone marrow4. When transplanted into other organs, these cells are instructed to differ-entiate into tissue specific cells under inductive signals from that specific tissue.

• True precursors of post natal stem cells with embryonic stem cells like properties persist in the adult bone marrow, such as the multipotent adult progenitor cells5 may be that the nuclei of the transplanted stem cells undergo reprogramming of the existing genetic information, expressing new genes and proteins that are consistent with the novel lineage and this might be a result of de-differentiation and re differentiation.

• When cell fusion accuracy which is a rare phenomenon reported in vitro and in vivo in tissues where polyploidy is common, such as hepatocytes, skeletal muscles, cardiac muscles and Purkinje cells of the cerebellum6 as a result, the genetic information of both the fused donor and host cells is partially changed7.

Homing: These cells migrate to site of tissue damage.

Engraftment: To unite with other tissues.

Potential implications of stem cells in dentistry

1. Regeneration of dental hard tissues

• Dentine regeneration

• Cementum regeneration

• Enamel formation

2. Regenerating of bone from autologous stem cells 3. Regenerative endodontics

4. Bone implant associated bone regeneration 5. Condyle regeneration

6. Periodontal tissue regeneration 7. Stem cells in sinus augmentation 8. Repair of cleft lip and palate defects 9. Regeneration of irradiated salivary glands

10. Peripheral nerve regeneration 11. Management of oral cancer

Sources of stem cells in oral tissues

1. Dental pulp stem cells (DPSCs)

2. Stem cells from human exfoliated deciduous teeth (SHED)

3. Periodontal ligament stem cells (PDLSCs) 4. Stem cells from apical papilla (SCAP) 5. Dental follicle progenitor cells (DFPCs) 6. Dental epithelial stem cells

7. Cementoblasts like stem cells

Dental pulp stem cells (DPSCS)8: Regenerative capacity of human dentin pulp complex is not well understood but it is known that upon injury reparative dentin is formed as a protective barrier for the pulp. So we can anticipate that dental pulp contains the dentinogenic progenitors that are responsible for dentin repair. Potential deriva-tion of stem cells during reparative dentinogenesis may be from cell rich layer of pulp adjacent to odontoblasts fibroblast perivascular cells, and presence of unique pop-ulation of post natal dental pulp stem cells.

Studies by Gronthos et al. demonstrated that human DPSC cultures are negative for odontoblastic-specific markers, such as dentin sialophosphoprotein (DSPP) and dentin sialoprotein (DSP), suggesting an undifferentiated phenotype of these cells. When 12 single -colony-derived DPSCs strains were transplanted with hydroxiapatite/tricalcium phosphate (HA/TCP) parti-cles subcutaneously into immunocompromised mice, two-thirds of the single-colonies developed an abun-dant typical dentin/pulp-like complex consisting of a layer of odontoblastic like cells, aligned around miner-alised dentin, while only a limited amount of dentin was detected in the remaining one-third. These results imply that single-colony-derived DPSC strains differ from each other with respect to their rate of odontogenesis9.

However, independent of the density formed, all DPSCs transplants were capable of expressing DSPP identified by immunohistological analysis, indicating that this population of cells might respond to specific environmental signals and differentiate into cells with specific phenotype, in this case, the odontoblasts10.

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Miura et al. (2003) found that SHEDs were capa-ble of differentiating into neural cells, adipocytes and odontoblasts when cultured in vitro with specific medium such as DPSC. After 12 single-colony-derived SHED clones were transplanted into inmmunocompro-mised mice, 25% of the clones demonstrated a potential to differentiate into odontoblast-like cells and to form ectopic dentin-like tissue. However, SHEDs were unable to regenerate the dentin-pulp-like complex completely. In addition, transplanted single-colony-derived SHED clones were capable of inducing murine host cells to dif-ferentiate into osteoblasts and osteocytes, and to form a significant amount of new bone.

Periodontal ligament stem cells (PDLSCS)12: Earlier evidence has shown that PDL contains cell populations that can differentiate into either cementum-forming cells (cementoblasts) or bone-forming cells (osteo-blasts). The presence of multiple cell types within PDL suggests that this tissue contains progenitor cells that maintain tissue homeostasis and regeneration of peri-odontal tissue. Enzyme digestion treatment of PDL releases a population of clonogenic cells with character-istics of postnatal stem cells. The successful isolation and characterization of PDLSCs have led to the identification of tendon mesnchymal stem cells (MSCs) by the same approaches.

i. In vitro characterisation of PDLSCs—multi-lineage differentiation potential: PDLSCs express the MSC-associated markers STRO-1, CDs and scleraxis - a tendon-specific transcription factor, which is expressed at higher levels in PDLSCs than in bone marrow MSCs and DPSCs. Immuno-histochemical staining and Western blot analysis showed that cultured PDLSCs expressed an array of cementoblastic/osteoblastic markers. Similar to the other dental stem cells described above, PDLSCs exhibit osteogenic, adipogenic and chon-drogenic characteristics under defined culture conditions13–15.

ii. In vivo characterisation of PDLSCs—formation of cementum—and periodontal ligament (PDL) like tissue: Typical cementum/PDL-like structure can be regenerated after transplantation of ex vivo expanded PDLSCs into immunocompromised mice. A thin layer of cementum-like tissue is formed along with condensed collagen fibers with sparse cells resembling PDL structures. The cementum/PDL-like structures are totally different from typical bone/marrow structures generated by BMMSCs and dentin/pulp-like structures generated by DPSCs.

Stem cells from apical papilla (SCAP): Apical papilla refers to the soft tissue at the apices of devel-oping permanent teeth. Apical papilla is more apical to the epithelial diaphragm, and there is an apical cell-rich zone lying between the apical papilla and the pulp16–18.

i. In vitro characterization of SCAP—multiline age differentiation potential: Similar to DPSCs and SHED, ex vivo expanded SCAP can undergo odontogenic differentiation in vitro. SCAP express lower levels of DSPP, matrix extracellular phosphoglycoprotein (MEPE), transforming growth factor β receptor II (TGFβRII), fibro-blast growth factor (FGFR3), Flt-1 (VEGF receptor 1), (FGFR1) and melanoma-associated glycoprotein (MUC18) in comparison with DPSCs. Significantly, CD24 is expressed by SCAP which is not detected on DPSCs or BMMSCs. The expression of CD24 by SCAP is down-regulated in response to osteogenic stimulation. However, the biological significance of this finding requires further investigation.

SCAP also demonstrate the capacity to undergo adipogenic differentiation following induction in vitro. Interestingly, without neurogenic stimulation, cultured SCAP show positive staining for several neural mark-ers19. After stimulation, additional neural markers are also expressed by SCAP, including glutamic acid decar-boxylase (GAD), neuronal nuclear antigen (NeuN), neurofilament M (NFM), neuron-specific enolase (NSE) and glial markers 2′,3′-cyclic nucleotide 3′ -phosphodi-esterase (CNPas).

Dental follicle precursor cells (DFPCs): Dental folli-cle is an ectomesenchymal tissue surrounding the enamel organ and the dental papilla of the developing tooth germ prior to eruption. This tissue contains progenitor cells that form the periodontium, i.e., cementum, periodontal liga-ment and alveolar bone. Precursor cells have been isolated from human dental follicles of impacted third molars. Similar to other dental stem cells, these cells form low numbers of adherent clonogenic colonies when released from the tissue following enzymatic digestion20.

The dental follicle has long been considered as a multipotent tissue because of its ability to generate cementum, bone and POL from the homogeneous-like ectomesenchymal-derived fibrous tissue. Bovine dental follicle cells show a differentiation ability to form cementoblasts when transplanted into immunodeficien-cymice21,22. Human dental follicle progenitor cells can be obtained from human third molars and are charac-terised by their plastic attachment in culture and expres-sion of makers such as Nestin and Notch-1. These cells are considered to be able to differentiate into POL-like structures, bone and cementum.20

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Cementoblast-like stem cells: Satio et al. (2005)24 have achieved success in developing bovine cemen-toblast progenitor line. These cells were transplanted subcutaneously into nude mice on a hydroxyapatite or tricalcium phosphate scaffold. Histological analysis indicated that a bone-like tissue was formed containing cementocyte-like cells in a mineralised matrix. Finally, it is worth noting that periodontal ligament may itself serve as a source of cells for cementum formation.

Significance of stem cells in periodontics: The con-cept that stem cells may reside in the periodontal tissues was first proposed about 20 years ago by Melcher, who queried whether the three cell populations of the odontium (cementoblasts, alveolar bone cells and peri-odontal ligament fibroblasts) were ultimately derived from a single population of ancestral cells or stem cells. For many decades, periodontists have been inter-ested in regenerating tissues destroyed by periodontitis. Periodontitis is a disease of the periodontium character-ised by irreversible loss of connective tissue attachment and the supporting alveolar bone.

Periodontal regeneration can be defined as the complete restoration of the lost tissues to their original architecture and function by recapitulating the crucial wound healing events associated with their devolop-ment19. Conventional open flap debridement falls short of regenerating tissues destroyed by the disease, and current regenerative procedures offer a limited potential towards attaining complete periodontal restoration. Recently, the isolation of adult stem cells from human periodon-tal ligament has presented new opportunities for tissue engineering. Clearly, in order for such therapies to be suc-cessful, a thorough understanding of stem cells and their role in regenerating periodontal tissues is required.

Generations in periodontal regenerative

therapies:

i. First generation regenerative therapies

These therapies were clinically applied in the dental regenerative therapy before any other medical field. Ex: guided tissue regeneration, Emdogain.

ii. Second generation regenerative therapies

This approach is making surprisingly rapid progress and has expanded from its original application in the medical field to various related disciplines in which the introduction of biologic and engineering knowledge and skills are allowing the development of new approaches. Ex: Tissue engineering (regenerative medicine).

iii. Third generation regenerative therapies

In the near future these therapies will involve nanoscale science and mouldless manufacturing tech-nology commonly known as rapid prototyping (RP) or solid free formed fabrication (SFF). Identification of stem cells in postnatal dental tissues has presented excit-ing possibilities for the application of tissue engineerexcit-ing as well as gene and cell-based therapies in reconstruc-tive dentist. The use of stem cells with these technologies

may constitute novel strategies for regenerative peri-odontal therapy.

This strategy eliminates some of the limitations associated with conventional regenerative procedures because the direct placement of growth factors and pro-genitor cells into the defect site overcomes the normal lag phase of progenitor cell recruitment to the site25.

TISSUE ENGINEERING

Tissue engineering is a specialised field of science based on principles of cell biology, developmental biology and biomaterials science to fabricate new tissues to replace lost or damaged tissues. Successful tissue engineering requires an appropriate extracellular matrix or carrier construct which contains regulatory signals and respon-sive progenitor cells. A potential tissue engineering approach to periodontal regeneration involves incor-poration of progenitor cells and instructive messages in a prefabricated three-dimensional construct, which is subsequently implanted into the defect site.

Definition

Langer and Vacanti26 defined tissue engineering as, “an inter disciplinary field that applies the principles of engineering and life sciences towards the develop-ment of biological substitutes that restore, maintain, or improve tissue function”.

MacArthur and Oreffo defined tissue engineering as “understanding the principles of tissue growth and applying this to produce functional replacement tissue for clinical use”27.

Principles of tissue engineering

• Conductive approaches

• Inductive approaches

• Cell transplantation

Conductive approach: This approach makes use of a barrier membrane to exclude connective tissue cells that will interfere with the regenerative process, while enabling the desired host cells to populate the regen-eration site. Examples are dental implants and guided tissue regeneration (GTR) membranes. GTR membranes are used to regenerate the periodontal tooth supporting structures and they are used as a material barrier to create a protected compartment for selective wound healing.

Inductive approaches: This approach uses a biode-gradable polymer scaffold as a vehicle to deliver growth factors and genes to the host site. The growth factors or genes can be released at a controlled rate, based on the breakdown of the polymer. One limitation of the induc-tive approach is that the inducinduc-tive factors for a particular tissue may not be known.

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alteration or changing gene expression and transplanted back to patient. The cell transplantation strategy truly reflects the multidisciplinary nature of tissue engineer-ing that requires a clinician, a bioengineer and a cell biologist.

• Clinician - takes a biopsy of the tissue sample, contain ing the cells of interest.

• Cell biologist - multiplies the cells and maintains their function.

• Bioengineer - the manufacturer of the tissue, the bioreactor and the material onto which the cells will be placed for transplantation.

Finally, the clinician transplants the engineered tissue polymer scaffold degrades and is remodelled by host and transplanted cells resulting in complete natural tissue.

The technical requirements for successful cell-based tissue engineering can be divided into two main cat-egories: engineering issues related to maintenance of an in vivo cell culture in the defect (e.g., biomechanical properties of the scaffold) and biological functions of the engineered matrix (including cell recruitment, neo-vascularisation and bioavailability of growth factors)28.

Scaffold

With respect to the biochemical features of the matrix scaffold, these compounds should act in a manner con-sistent with the principles of membrane-based-guided tissue regeneration and have similar design features29.

In particular, these properties should include: ease of handling, rigidity to withstand soft tissue collapses into the defect, and ability to maximise cell colonisation and tissue in growth of desired type. It is also important that unwanted epithelium is not totally excluded, but rather encouraged to form a biological seal over the scaf-fold and onto the tooth in the vicinity of the cemento- enamel junction, protecting the regenerating events occurring beneath30.

Ex vivo expanded PDLSCs co-transplanted with hydroxyapatite/tricalcium phosphate ceramic (HMCP) particles into nude rats are capable of forming cemen-tum/periodontalligament-like structures. One novel report has shown that stem cells isolated from the root apical papilla of human teeth and PDLSC can be com-bined to regenerate the root/periodontal structure, respectively. In this study, a root-shaped scaffold struc-ture was prepared into which stem cells isolated from the root apical papilla from porcine teeth were seeded. Gelfoam containing porcine PDLSC was then wrapped around the artificial root construct and then placed into a prepared bony socket in the mandible of mini-pigs. After a 3-month healing phase, this biologically created “root” was restored with a porcelain crown. Collectively, these findings demonstrate the feasibility (and poten-tial) of using a combination of mesenchymal stem cell-like cell populations for functional tooth regeneration.

Tissue engineering triad

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Tissue engineering is the employment of biological thera-peutic strategies which are aimed at the replacement, repair, maintenance and/or the enhancement of tissue function.

Tissue engineering is generally considered to consist of three key elements

1. Stem cells/progenitor cells 2. Scaffolds or extra cellular matrix 3. Signalling molecules

Bioengineered scaffolds

The basic role of scaffolds in tissue engineering is to act as carriers for cells, to maintain the space and to create an environment in which the cells can proliferate and produce the desired tissue matrix.

Types of scaffolds:

1. Natural scaffolds 2. Mineral scaffolds 3. Synthetic scaffolds 4. Hybrid scaffolds

Signaling molecules: These are the molecules that transmit signals between cells, functioning as stimula-tors/inhibitors of growth, as well as the modulators of differentiation. These consist of growth factors (PDGF, TGF), differentiation factors, bone morphogenic pro-teins (BMPs) and stimulating factors32.

Ideal scaffolds would allow the delivery of signalling molecules capable of coordinating cellular proliferation and differentiation. Each of the scaffolds has their own advantages and disadvantages.

1. Natural scaffolds

Ex: Collagen, Hyaluronic acid and Chitosan

• Advantages of naturally derived scaffolds include the ability to support cellular invasion and proliferation.

• Disadvantage is that they lack the desired structural rigidity for independent use in load - bearing regions.

• Type I collagen has been used specifically to promote bone formation in rat mandibular defects.

• Sculean et al. reported that the application of bovine derived xenograft and bioresorbable collagen in patients with periodontal defects resulted in significant improvement, with reduction of periodontal probing depth and clinical attachment levels observed 1 year post-treatment33.

• Chitosan has emerged for using craniofacial defect.

2. Mineral-based scaffolds

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• These have been engineered to reproduce the molecular environment of bone.

• By reproducing 3–0 structure of bone, these mineral lattices confer an osseoinductive signal to promote the maturation of progenitor cells down a path toward bone

.

3. Synthetic polymers

Ex: Polymers in use include polyglycolic acid, polylactic acid, polydioxanone, polycaprolactone or various com-binations of the above

Advantages are (1) offer ease of processing and mechanical strength, (2) or use in load bearing areas and (3) reat durability.

Disadvantage is that they typically lack the osteoin-ductive properties of natural and mineral based scaf-folds.

Recently two scaffold systems polyglycolic acid and polycaprolactone have been applied to chondrogenesis of mesenchymal stem cells with success29.

4. Hybrid scaffolds

Ex: Apatite-coated macroporous scaffolds

• These combine the osteoconductive properties of apatites with the strength and versatility of degradable polymers.

• Studies by Kokubo et al.34 have demonstrated the ability to coat polymer scaffolds with a uniform, dense, nano-crystalline apatite coating.

In vitro studies have already shown biomimetic apa-tites coated onto synthetic polymers to promote mat-uration of osteogenic precursors, with upregulation of osteocalcin and bone sialoprotein in MC3T3-E1 cells cultured on such surfaces.

5. Ceramic scaffolds

Ex: Natural or synthetic hydroxyapatite, 13-tri calcium phosphate

• These are biocompatible, osteoconductive and may directly bind to bone

• They are protein-free and, thus, stimulate no immunological reaction

• Long degradation times (many years) in vivo

• Disadvantage is that they are not strong enough scaffolds to provide mechanical strength when replacing load bearing skeletal structures.

6. Polymers: Natural

Ex: Collagen, Hyaluronic acid, Alginate, Agarose

7. Synthetic polyesters

Ex: Polyglycolic acid, Polylactic acid and Polycaprolactone

8. Pluronics

Ex: Copolymers of polyethylene oxide and poly-propylene oxide

These are recently developed in the form of injectable hydrogels with attached adheshion peptide.

Advantages:

• Ease and control of synthesis

• Unlimited supply

• Non cell mediated degradation

• Desirable pore features and shape

Disadvantages:

• Lack of mechanical strength

• Difficulty in 3D fabrication

• Uncontrollable shrinkag.

• Possible local toxicity resulting from acidic degradation products

• Temperature sensitive hydrogels

• Photopolymerizing hydrogels

Ex: exploit growth factor (BMPs and TGF), signaling and controlled degradation characteristics to improve tissue formation35.

CONCLUSION

From a biological perspective, in order for periodon-tal regeneration to occur, the availability of appropriate cell types, together with a favourable local environment promoting cell migration, adhesion, proliferation and differentiation, all need to be precisely coordinated both temporally and spatially. Unfortunately, current therapeu-tic measures are unable to achieve predictable regenera-tion, thus underscoring the importance of restoring or providing the cells and microenvironment capable of ini-tiating and promoting. Thus, a tissue engineering strategy for periodontal regeneration that exploits the regenerative capacity of stem cells residing within the periodontium, grown in a three-dimensional construct and subsequently implanted into the defect may help to overcome many limitations with current regeneration modalities36. In light of these findings, the identification of stem cells within the periodontal ligament with the ability to achieve new attachment formation in vivo is a significant development toward a tissue engineering approach to treating peri-odontal regeneration. Furthermore, the ready availability of periodontal ligament tissue from redundant teeth such as third molars may provide a supply of stem cells that may be utilised for regenerating other body parts.

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