Regenerative Techniques in Periodontology

Salman Aziz                                 FCPS, FICD, MFDS RCPSG, MFGDP, BDS

INTRODUCTION

Regenerative therapy is no doubt a reality! In this era of Hi-tech dentistry it occupies one of the greatly explored aspects of both clinical and laboratory dental medicine. The last few decades have shown an interesting paradigm shift towards procedures directed into conservation, rejuvenation and/or replacement of missing/ lost or endangered oro-dental tissues. It has been quite an intriguing, thought provoking and interesting journey to have moved progressively based upon evidence from xenografts, allografts, alloplasts and autografts alone, to the era of growth factors (GF), proteins and stem cells.

We can broadly categorize the use of ongoing regenerative techniques into three main specialties of dentistry namely periodontics, endodontics/conservative dentistry and oral and maxillofacial surgery. Detailed insight into all specialties would be out of scope for this review however the following discussion will focus on the most practically happening periodontal regenerative techniques at the current time concentrating on the biological mediators of regenerative therapy and stem cells and provide some directives towards future of regeneration in periodontology.

All efforts these days are directed towards either the possibility of re-growing the entire periodontium related to an individual tooth or group of teeth or repairing each individual constituent (periodontal ligaments, cementum, bone or gingiva) as close as possible to the lost natural counterpart. Both require the utility of biomaterials, scaffolds, stem cells, and/or growth factors. Biomaterials and scaffolds have been talked about in many publications however the utilization of the latter two techniques are scarce in literature. The following discussion therefore will be divided into two main parts:

• Advances in Growth factors, signaling molecules and cytokine therapies.
• Advances in Stem cell techniques.

GROWTH FACTORS, SIGNALING MOLECULES, CYTOKINES AND GENE THERAPY

1. Autologous Platelet Products

These products are biological factors extracted from and utilized in the same patients. The main aim behind these techniques is that platelets being a natural source of growth factors are utilized as tools for promotion of regenerative process. These techniques/products could be used solely or in combination with scaffolds like collagen membranes, bone substitutes etc. and include the following:

1.1. Platelet Rich Plasma (PRP)

This is an autologous product derived from patient’s own blood and contains high concentration of platelets suspended in a small amount of plasma. In contrast to Normal platelet count in blood which ranges from 150,000 to 300,000/ µl, PRP by definition contains 1000,000 platelets/µl in 5 ml volume of plasma2. The main idea behind a solution with concentrated platelets is that alpha granules in the platelets contain growth factors which are released by platelet activation initiated by thrombin, calcium chloride or collagen3. The released growth factors include platelet derived growth factor (PDGF), transforming growth factorb1 (TGF-b1), insulin-like growth (IGF), vascular endothelial growth factor (VEGF),fibroblast growth factor and epidermal growth factor (EGF), which promote tissue healing and repair of damaged periodontal tissues, modulate inflammatory process and promote angiogenesis4,5.
The formation of PRP includes collection of patient’s blood in a vacutainer/ collection tube containing anticoagulant6. Blood is mixed with anticoagulant and the vacutainer tube is then subjected to two step centrifugation process. In the first centrifugation RBC’s are separated from plasma and in the second centrifugation the platelets and leukocytes are separated from a cellular plasma also known as platelet poor plasma7. The advantage PRP holds is the ready provision of growth factors (GF) to the site of defect. However as the release is immediate and in high quantities, there is a concern that the GF can be lost without much effect8. Therefore a combination of bone substitutes and PRP is more advantageous in sustaining and retaining GF levels to the site of interest9,10 .

1.2. Plasma Rich in Growth Factors (PRGF)

This technique has been projected as one step ahead of PRP in respect to duration and sustenance of release of GF11,12. Also the concentration of White Blood Cells (WBCs) is nonexistent in PRGF as compared to PRP13,14.
The main difference in the preparation includes onetime centrifugation for PRGF and addition of calcium chloride for activation leading to formation of a gel11. The advantage of this gelatinous substance is that it increases the duration of growth factor release. PRGF can be used in a variety of ways i.e. as supernatant, liquid coating of dental implant surfaces, as scaffold gel or as elastic hemostatic fibrin11.

1.3. Platelet Rich Fibrin (PRF)

This is a fibrin membrane consisting of a collection of immune and platelet concentrate15. The PRF membrane has three fold benefits in that it provides a platform for development of angiogenesis, wound coverage and immune support at the site of interest16-18. The technique for PRF generation is much easier as compared to the previously mentioned methods. It has the advantage of exclusion of addition of any sort of activator or anticoagulant and therefore is less technique sensitive. There is a handsome constituency of leukocytes in PRF which release cytokines, and therefore PRF acts as a double edge sword by regulating immune process on one hand and promoting bone and tissue regeneration on the other19. The biggest advantage PRF has is that the duration of release of growth factors is enhanced up to several days as compared to PRP and PRGF20. In addition to this the PRF membrane has better mechanical properties than PRGF membrane21.

2. Enamel Matrix Derivatives

These proteins are available in the market by the name Emdogain since the past nineteen years. It is mainly used for periodontal regeneration. The main constituents of Emdogain include enamel matrix derivatives (mainly amelogenins isolated from developing porcine teeth), water and propylene glycol alginate (carrier)22,23. Its use and effectiveness is backed by considerable evidence24 however it faces some religious issues in countries which do not use porcine products for health care purposes. Moreover in a fairly recent Cochrane review it was summarized that, although there are less post-operative complications, post one year application Emdogain has neither been able to save more compromised teeth as compared to the other regenerative methods nor It has been able to demonstrate Patient’s perceived esthetic improvement one year post procedure25.
The techniques of using Emdogain is simple. After open flap debridement and root surface conditioning, the gel is applied directly to the denuded root and flap is repositioned26,27.

3. Recombinant / Synthetic Products Produced by Virtue of Gene Therapy

As opposed to their autologous counterparts there are a few commercially available products manufactured using recombinant (genetic) technologies. One of the uses of gene therapy is to introduce vectors into the target cells which are then programmed to yield the required protein or growth factor28. These provide a source of one growth factor/protein in high potency to fulfill specific regenerative needs.

3.1. Recombinant Human Platelet Derived Growth Factor (rhPDGF)

PGDF is a potent cytokine with the ability to mediate wound healing and regeneration29. This is also released by platelets as mentioned above, however the concentration available varies depending upon the platelet product and its generation technique used. Commercially a genetically engineered Food and drug Administration (FDA) approved product available in the market is GEM-21S (growth-factor enhanced matrix) which contains 0.5 ml (at a concentration of 0.3mg/ml i.e. 0.15mg) of PDGF along with beta tri calcium phosphate (β-TCP) 0.5 cc and is indicated for use in intra bony defects, furcation defects and gingival recession associated with periodontal defects30

3.2. Recombinant Human Bone Morphogenetic Protein 2 (rhBMP 2)

This is one of the most happening and researched upon product these days in my humble opinion. It belongs to the transforming growth factor β (TGF β) family31. It regulates the differentiation of stem cells located in the bone tissues. The main reason for its popularity is its osteoinductive nature32. Recombinant human BMP-2 (rhBMP-2) is available in the market by the name of Infuse an FDA approved product from Medtronic. The Infuse kit is available in various sizes from 0.7 cc to 8.0 cc and consists of sterile water vial/vials, absorbable collagen sponge, and vial/vials of lyophilized rhBMP-2. The sterile saline is mixed with lyophilized rhBMP-2 and the absorbable collagen sponge is dipped in the resulting solution. The BMP dipped collagen sponge is then placed in the defect. This product has provided with more than satisfactory results to be considered a promising solution for bone regeneration33.

3.3. Recombinant Human Fibroblast Growth Factor 2 (rhFGF 2)

Recombinant human Fibroblast growth factor (rhFGF-2) has been reported to exert potent angiogenic and mitogenic effects on mesenchymal cells34. To date very few randomized control trials have been carried out utilizing rhFGF-2. A phase II trial previously conducted did not reveal any significant findings35. However a recently published phase III study provides some more promising results, however post marketing stage of this product in the near future will enlighten us more about rhFGF-2’s efficacy and capability in periodontal regeneration36.

3.4. Recombinant Human Growth Differentiation Factor 5 (rhGDF 5) or BMP 14, rhBMP 7 and rhBMP

These are other potential products undergoing animal trials and would be commercially available in the near future for oral and maxillofacial purposes37.

ADVANCES IN STEM CELL TECHNIQUES

In the quest for approaches to restore oral structures to their normal function and form, and possibly to engineer the entire tooth and periodontium, novel technologies have come up in the form of stem cell tissue engineering techniques.

1. Stem Cells Types and Utility

These are unspecialized and immature cells with selfrenewal programmability potential and the ability to differentiate into a variety of cell lines38,39. Due to their unique traits, stem cells have gained popularity in tissue engineering to regenerate or replace missing or damaged tissues and/or organs.

The two main sources of stem cells in dentistry are adult stem cells and pluripotent stem cells. The latter includes embryonic stem cells and induced pluripotent stem cells (iPS) Pluripotent variant are the most promising, having the ability to develop into all cell types from all germ layers in contrast to adult stem cells which can only differentiate into limited cell types40.

1.1. Adult Stem Cells
There are many variants of these somatic or postnatal stem cells depending upon the specific ability of differentiation and include:

• Mesenchymal stem cells or mesenchymal stromal cells (MSCs)
• Bone marrow derived MSC (BMSCs)
  From iliac crest
  From orofacial bones
• Dental tissues derived stem cells
• Epithelial stem cells

MSC-like cells: These are called MSC-like because they have similar phenotypic characteristics of BMSCs and include:

• • Dental Pulp Stem Cells (DPSC)
  • Stem cells from human exfoliated deciduous teeth (SHED)
  • Periodontal ligament stem cells (PDLSCs)
  • Dental Follicle Stem Cells (DFSCs)
  • Tooth Germ Progenitor Cells (TGPCs)
  • Stem cells from apical papilla (SCAP)
    Oral Mucosa derived stem cells/ Gingiva derivedMesenchymal Stem Cells (OMSCs/GMSCs)
  • Periosteum derived stem/progenitor cells
  • Salivary gland derived stem cells
  • Adipose tissue derived stem cells (ASCs)

A detailed account of each of the above would require another review as it is not possible to accommodate the details they deserve, in current manuscript. Moreover the above mentioned adult stem cell techniques are in their infancy and it will take considerable amount of time for all to reach the clinical settings. Currently only periodontal .

ligament stem cells (PDLSCs) have been introduced in the clinics and are discussed below.
1.2. Pluripotent Stem Cells
1.2.1. Embryonic Stem Cells (ES Cells

ES are extracted from undifferentiated inner mass of blastocyst, which is an early stage of embryonic development post fertilization41. This is the main reason for major ethical and moral concerns whenever the topic of ES cell extraction from human beings comes up. Therefore ES cell use in regenerative dentistry is still a grey area with unpredictable future.
1.2.2. Induced Pluripotent SC (iPS Cells

When human or mouse somatic cells undergo nuclear reprogramming by introduction of certain genetic transcription factors, they can be transformed to embryonic state thus unlocking pluripotency. These induced cells then act similarly to ES cells42. Ever since their discovery, iPS cells have been the research focus of regenerative medicine and dentistry. To the dentists advantage luckily the oral cavity is a unique and rich source of stem cells43. iPS have been successfully generated from SCAP44, DPSCs45, gingival fibroblasts and Periodontal ligament fibroblasts46.

Induced pluripotent stem cells have great potential in regenerative dentistry in the upcoming future however their clinical utility currently is limited to animal models46.

CURRENT PRACTICAL SUITABILITY OF STEM CELLS IN CLINICAL DENTISTRY

Apart from the ability of differentiation into specific target tissues, and ease of collection and preparation, in order to ensure patient safety in regenerative dentistry, complete control of cellular fate is a mandatory requirement for all stem cells as well. Based upon this only adult MSCs currently seem to have a convincing clinical potential47,48. Initial case reports and randomized control trials (RCTs) utilizing stem cells for periodontal regeneration are coming up48,49.

The autologous periodontal ligament stem cell technique reported in very recent literature involves prior extraction of third molars from subjects scheduled to receive stem cell treatment for periodontal defects. Post Extraction, production of single cell suspensions is carried out by cell isolation process, which involves separation of Periodontal ligament (PDL) cells from the roots followed by digestion, straining and centrifugation processes. The cells then undergo culture and cell characterization procedures, which are followed by creation of cell sheets. Once the PDLSC sheets are ready for use, freshly prepared Bone particles e.g. Bio-Oss® etc. (in a concentration of 0.25g/sheet) are sprinkled over each sheet to be used. The periodontal ligament stem cell sheets are then rolled up to pack the bone particles and introduce into the defect49.

Although the results of Chen and co-worker’s RCT49 showed no significant difference between the control group and stem cell group for pocket depth fill, the emergence of such clinical trials are affirming and encouraging for us to start preparing for the future of stem cell use in clinics.

We must therefore not hesitate to incorporate the above mentioned techniques in our daily practices as soon as they are available, but after achieving adequate skills through continuing education programs. The utility of regenerative techniques might, not only, serve to provide the patients with comparatively non-invasive treatment plans but also bring more and more promising results as the use becomes wide spread. We are therefore looking towards a future that would be the beginning of the end of material based therapies and start of stem cell based regenerative therapies.

CONFLICT OF INTEREST

Declared none.

ACKNOWLEDGEMENTS

Declared none

REFERENCES

1. Murray PE, Garcia-Godoy F, Hargreaves KM. Regenerative endodontics: a review of current status and a call for action. J Endod. 2007; 33: 377-90.
2. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant dent. 2001;10:225-8.
3. Wang H-L, Avila G. Platelet rich plasma: myth or reality? Europ J Dent. 2007;1:192.
4. Senzel L, Gnatenko DV, Bahou WF. The platelet proteome. Curr Open Hematol. 2009;16:329.
5. Inchingolo F, Tatullo M, Marrelli M, Inchingolo A, Inchingolo A, Dipalma G, et al. Regenerative surgery performed with platelet-rich plasma used in sinus lift elevation before dental implant surgery: an useful aid in healing and regeneration of bone tissue. Eur Rev Med Pharmacol Sci. 2012;16:1222-6.
6. Tsay RC, Vo J, Burke A, Eisig SB, Lu HH, Landesberg R. Differential growth factor retention by platelet-rich plasma composites. J Oral Maxillofac Surg. 2005;63:521-8.
7. Bielecki T, M Dohan Ehrenfest D. Platelet-rich plasma (PRP) and Platelet-Rich Fibrin (PRF): surgical adjuvants, preparations for in situ regenerative medicine and tools for tissue engineering. Curr Pharm Biotech. 2012;13:1121-30.
8. Alsousou J, Thompson M, Hulley P, Noble A, Willett K. The biology of platelet-rich plasma and its application in trauma and orthopaedic surgery. Bone Joint J. 2009; 91:987-96.
9. Varkey M, Gittens SA, Uludag H. Growth factor delivery for bone tissue repair: an update. Expert opinion on drug delivery. 2004;1:19-36.
10. Amini AR, Laurencin CT, Nukavarapu SP. Bone tissue engineering: recent advances and challenges. Crit Rev Biomed Eng. 2012;40(5).
11. Anitua E, Sánchez M, Orive G, Andía I. The potential impact of the preparation rich in growth factors (PRGF) in different medical fields. Biomater. 2007; 28:4551-60.
12. Anitua E. Plasma rich in growth factors: preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants. 1999;14:529-35.
13. Tinsley BA, Ferreira JV, Dukas AG, Mazzocca AD. Platelet-rich plasma nonoperative injection therapy—a review of indications and evidence. Operative Techniques Sports Med. 2012;20:192-200.
14. Weibrich G, Kleis WK, Hitzler WE, Hafner G. Comparison of the platelet concentrate collection system with the plasma-rich-in-growth-factors kit to produce platelet-rich plasma: a technical report. Int J Oral Maxillofac Implants. 2005; 20(1).
15. Choukroun J, Diss A, Simonpieri A, Girard M-O, Schoeffler C, Dohan SL, et al. Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol. 2006; 101: e56-e60.
16. Dvorak HF, Harvey V, Estrella P, Brown L, McDonagh J, Dvorak A. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Laboratory investigation. J Technical Methods Pathol. 1987; 57: 673-86.
17. Tuan T-L, Song A, Chang S, Younai S, Nimni ME. In VitroFibroplasia: Matrix Contraction, Cell Growth, and Collagen Production of Fibroblasts Cultured in Fibrin Gels. Experimental Cell Res. 1996;223:127-34.
18. Loike JD, Sodeik B, Cao L, Leucona S, Weitz JI, Detmers PA, et al. CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen. Proc Natl Acad Sci. 1991;88:1044-8.
19. Inchingolo F, Tatullo M, Marrelli M, Inchingolo A, Scacco S, Inchingolo A, et al. Trial with Platelet-Rich Fibrin and Bio-Oss used as grafting materials in the treatment of the severe maxillar bone atrophy: clinical and radiological evaluations. Eur Rev Med Pharmacol Sci. 2010;14:1075-84.
20. Dohan Ehrenfest DM, de Peppo GM, Doglioli P, Sammartino G. Slow release of growth factors and thrombospondin-1 in Choukroun’s platelet-rich fibrin (PRF): a gold standard to achieve for all surgical platelet concentrates technologies. Growth Factors. 2009;27:63-9.
21. Khorshidi H, Raoofi S, Bagheri R, Banihashemi H. Comparison of the Mechanical Properties of Early Leukocyte-and Platelet-Rich Fibrin versus PRGF/Endoret Membranes. Int J Dent. 2016;2016:1849207
22. Gestrelius S, Lyngstadaas S, Hammarström L. Emdogain–periodontal regeneration based on biomimicry. Clin Oral Investig. 2000;4:120-5.
23. Bosshardt DD. Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels. J Clin Periodontol. 2008;35:87-105.
24. Sculean A, Alessandri R, Miron R, Salvi GE, Bosshardt DD. Enamel matrix proteins and periodontal wound healing and regeneration. Clinic Adv Periodontics. 2011;1:101-17.
25. Esposito M, Grusovin MG, Papanikolaou N, Coulthard P, Worthington HV. Enamel matrix derivative (Emdogain®) for periodontal tissue regeneration in intrabony defects. The Cochrane Library. 2009.
26. Hoffmann T, Al-Machot E, Meyle J, Jervøe-Storm PM, Jepsen S. Three-year results following regenerative periodontal surgery of advanced intrabony defects with enamel matrix derivative alone or combined with a synthetic bone graft. Clin Oral Investig. 2016; 20: 357-64.
27. Froum SJ, Weinberg MA, Rosenberg E, Tarnow D. A comparative study utilizing open flap debridement with and without enamel matrix derivative in the treatment of periodontal intrabony defects: a 12- month re-entry study. J Periodontol. 2001; 72:25-34.
28. Gupta K, Singh S, Garg KN. Gene therapy in dentistry: Tool of genetic engineering. Revisited. Arch Oral Biol. 2015;60:439-46.
29. Deuel TF, Kawahara RS, Mustoe TA, Pierce GF. Growth factors and wound healing: platelet-derived growth factor as a model cytokine. Annu Rev Med. 1991;42:567-84.
30. Nevins ML, Reynolds MA, Camelo M, Schupbach P, Kim DM, Nevins M. Recombinant human plateletderived growth factor BB for reconstruction of human large extraction site defects. Int J Periodontics Restorative Dent. 2014; 34(2):157-63.
31. Kirker-Head CA. Potential applications and delivery strategies for bone morphogenetic proteins. Advanced drug delivery reviews. 2000; 43(1): 65-92.
32. Lieberman JR, Le LQ, Wu L, Finerman GA, Berk A, Witte ON, et al. Regional gene therapy with a BMP‐2‐producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. J OrthopRes. 1998; 16:330-9.
33. McKay WF, Peckham SM, Badura JM. A comprehensive clinical review of recombinant human bone morphogenetic protein-2 (INFUSE® Bone Graft). Int Orthop. 2007;31:729-34.
34. Kitamura M, Akamatsu M, Machigashira M, Hara Y, Sakagami R, Hirofuji T, et al. FGF-2 stimulates periodontal regeneration results of a multi-center randomized clinical trial. J Dent Res. 2011;90:35-40.
35. Kitamura M, Nakashima K, Kowashi Y, Fujii T, Shimauchi H, Sasano T, et al. Periodontal tissue regeneration using fibroblast growth factor-2: randomized controlled phase II clinical trial. PloS one. 2008;3:e2611.
36. Kitamura M, Akamatsu M, Kawanami M, Furuichi Y, Fujii T, Mori M, et al. Randomized Placebo‐Controlled and Controlled Non‐Inferiority Phase III Trials Comparing Trafermin, a Recombinant Human Fibroblast Growth Factor 2, and Enamel Matrix Derivative in Periodontal Regeneration in Intrabony Defects. J Bone Miner Res. 2016;31:806-14.
37. Moore YR, Dickinson DP, Wikesjö UM. Growth/differentiation factor‐5: a candidate therapeutic agent for periodontal regeneration? A review of pre‐clinical data. J Clin Periodontol. 2010;37:288-98.
38. Slack J. Origin of stem cells in organogenesis. Sci.2008; 322:1498-501.
39. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005; 52:2521-9.
40. Armstrong L, Tilgner K, Saretzki G, Atkinson SP, Stojkovic M, Moreno R, et al. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem cells. 2010;28:661-73.
41. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Sci. 1998;282:1145-7.
42. Patel M, Yang S. Advances in reprogramming somatic cells to induced pluripotent stem cells. Stem Cell Rev Reports. 2010;6:367-80.
43. Volponi AA, Pang Y, Sharpe PT. Stem cell-based biological tooth repair and regeneration. Trends Cell Biol. 2010;20:715-22.
44. Yan X, Qin H, Qu C, Tuan RS, Shi S, Huang GT-J. iPS cells reprogrammed from human mesenchymallike stem/progenitor cells of dental tissue origin. Stem Cells Dev. 2010;19:469-80.
45. Tamaoki N, Takahashi K, Tanaka T, Ichisaka T, Aoki H, Takeda-Kawaguchi T, et al. Dental pulp cells for induced pluripotent stem cell banking. J Dent Res.2010;89:773-8.
46. Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts. J Periodont Res.2011;46:438-47.
47. Liu Y, Zheng Y, Ding G, Fang D, Zhang C, Bartold PM, et al. Periodontal ligament stem cell‐mediated treatment for periodontitis in miniature swine. Stem Cells. 2008;26:1065-73.
48. Yamada Y, Ueda M, Hibi H, Baba S. A novel approach to periodontal tissue regeneration with mesenchymal stem cells and platelet-rich plasma using tissue engineering technology: a clinical case report. Int J Periodontics Restorative Dent. 2006; 26:363-9.
49. Chen F-M, Gao L-N, Tian B-M, Zhang X-Y, Zhang Y-J, Dong G-Y, et al. Treatment of periodontal intrabony defects using autologous periodontal ligament stem cells: a randomized clinical trial. Stem Cell Res Ther. 2016;7:1-11.

Consultant in Restorative Dentistry, Institute for Advanced Dental Sciences and Research and Ministry of Higher Education,

Al Jouf University, Saudi Arabia < drsalmanaziz@gmail.com >