In this review, the authors consider the substantial advances that have been made in recent years in stem cell–based periodontal regeneration. These advances involve identifying dental- and nondental-derived stem cells with the capacity to modulate periodontal regeneration, human clinical trials, and emerging concepts, including cell banking, good manufacturing processes, and overall clinical translation.
Key points
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What are stem cells?
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Dental stem cells for periodontal regeneration.
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Extraoral stem cells for periodontal regeneration.
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Stem cell periodontal regeneration in humans.
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Future use of stem cell-based therapies in periodontal regeneration.
Introduction
What Is Periodontal Regeneration?
Periodontal regeneration is the full restoration of a damaged periodontal attachment apparatus (cementum, periodontal ligament, and alveolar bone) to its original architecture and function. It is a biological term defined by its histologic characteristics of restitutio ad integrum (restoration to original condition) of the periodontal architecture, including new cementum, periodontal ligament, and alveolar bone surrounding a tooth that has been deprived of its attachment apparatus usually because of the effects of periodontitis. Clinically, periodontal regeneration is characterized by a gain in clinical attachment, reduction in pocket depth, and radiographic evidence of an increase in alveolar bone levels. However, these observations cannot distinguish between regeneration and reparative healing, especially when various grafting and “regenerative” techniques have been used. Accordingly, these clinical outcomes should be viewed more as a result of periodontal reconstruction than periodontal regeneration per se. Therefore, periodontal reconstruction is viewed as a clinical term used to coin clinical and radiographic improvements, that is, gains in clinical attachment, reduced probing pocket depth, and bone fill, representing reparative and/or partial regenerative healing.
Currently, periodontal regeneration is recognized as being biologically possible but clinically unpredictable and remains an elusive goal. Successful periodontal regeneration is best illustrated within the context of the principles of the “number needed to treat” (NNT) principle, a measure used to assess a health care intervention’s effectiveness. NNT calculates the average number of patients who need to be treated to prevent one additional poor outcome (ie, the number of patients who need to be treated for benefit compared with a control in a clinical trial).
In a recent systematic review, it was noted that the NNT for the current gold-standard periodontal regeneration technique, guided tissue regeneration (GTR), to obtain one extra intrabony defect achieving pocket depth (PD) ≤3 mm or PD ≤4 mm over papilla preservation flaps (control) was 2 and 4, respectively. Although this is an improvement on previous studies indicating the NNT for GTR to achieve one extra site gaining 2 mm or more attachment over open flap debridement was 8, it is still considered far from ideal. Therefore, research efforts are continuing to explore improved ways to achieve predictable periodontal regeneration. To achieve this, several fundamental concepts must be adhered to. First, to fill a defect with a substance that has no relevance to the next functional state of reconstruction is irrational. Furthermore, it has been long recognized that the dental profession has become obsessed with filling holes in bone rather than studying the natural healing processes required for true periodontal regeneration. Clearly, regenerative treatment of periodontal defects with an agent or procedure requires that each functional stage of reconstruction be grounded in a biologically directed process.
Regeneration of the periodontium mimics periodontal development and requires a very complex spatial and hierarchical sequence of events. Critical to this process is the recruitment of precursor (stem) cells that have the potential to differentiate and produce cementum, periodontal ligament, and alveolar bone. In this context, stem cell–based tissue engineering approaches have provided novel, biologically based avenues for enhancing periodontal regeneration.
In this article, the authors consider the substantial advances that have been made in recent years in stem cell–based periodontal regeneration. These advances include identifying dental- and nondental-derived stem cells with the capacity to facilitate periodontal regeneration, human clinical trials, and emerging concepts, including cell banking, good manufacturing processes, and overall clinical translation.
A cellular approach to periodontal regeneration
Following the discovery of numerous subsets and subpopulations of cells residing within the periodontal tissues, it became necessary to study these cells in more detail. , A significant advance was made when it became possible to clone and expand cells from normal, inflamed, and regenerating periodontal tissues. , These studies provided new avenues of investigations and now pave the way for exciting developments in tissue engineering using cell seeding methodologies, gene manipulation, and fabrication of bio-scaffolds for cell delivery.
What are stem cells?
By definition, a stem cell refers to an undifferentiated cell capable of self-renewal and multilineage differentiation. These cells can replicate, producing a pool of stem cells with the potential, under specific conditions (in vivo and in vitro), to differentiate and mature along multiple lineages, resulting in a range of tissue-specific cell phenotypes and morphotypes. Importantly, stem cells have the capacity for prolonged self-renewal and/or differentiation controlled by a myriad of intrinsic mechanisms that, in turn, are regulated by the local niche environment of each stem cell.
Three broad categories of stem cells are recognized: (i) embryonic stem cells, (ii) postnatal (adult or somatic) stem cells, and (iii) the genetically modified differentiated cells known as induced pluripotent stem cells (IPSC). Embryonic stem cells have a high pluripotent potential and can differentiate into all 220 types of specialized cells that comprise the human body. Because of the ethical issues surrounding the acquisition of embryonic stem cells, their clinical use is limited. To get around this problem, it is possible to genetically manipulate and reprogram somatic cells into cells with an embryonic stem cell phenotype regarding morphology, gene expression profiles, proliferation, and differentiation capacities, but without the ethical concerns of embryonic stem cells. Just more than a decade ago, the first reports appeared describing the reprogramming of somatic cells into pluripotent cells with a differentiation and self-renewal capacity comparable to embryonic stem cells. These cells were termed “induced pluripotent stem cells” and are considered to provide an ethically viable alternative to embryonic stem cells in regenerative medicine. However, the genetics required for the generation of IPSC are not without concerns, including the potential induction of tumorigenic properties, raising a significant safety challenge in the use of these cells for regenerative therapies. ,
Adult stem cells are responsible for the regeneration of damaged tissues. Initially, these cells were termed stromal precursor cells, but they have been referred to as mesenchymal stem cells (MSC) in recent times. MSC are present in adult tissues and can differentiate into multiple specialized cell types. Their accessibility, high growth capacity, and multipotential make MSC good candidates for applications in tissue regeneration.
MSC are among the most highly studied types of adult stem cells. They include cells derived from nearly all tissues comprising the human body. Initially, these cells were considered to have a limited regenerative capacity, restricted to giving rise only to components of their tissue of origin. However, it is now apparent that as well as maintaining host tissue homeostasis, they can, under specific conditions, differentiate into phenotypes other than their tissue or tissues of origin. , Although MSC have limited differentiation capacity compared with embryonic stem cells, they are easily accessible and immunocompatible and are associated with fewer ethical concerns, making them very good candidates for regenerative medicine applications. Specifically, MSC of dental origin have become an attractive target for use in periodontal regeneration.
Defining properties of mesenchymal stem cells
MSC have several defining features that have been well documented. Specifically, these features include (i) their capacity to differentiate, under appropriate culture conditions, into at least 3 distinct lineages, such as osteoblasts, chondrocytes, and adipocytes; (ii) positive expression of cell surface markers, such as CD73, CD90, CD105, and CD166; (iii) an ability of individual clonal populations to regenerate the stromal microenvironment of their tissue of origin, when implanted into immunocompromised mice; and (4) ability for self-renewal following serial transplantations in vivo. , Another important criterion for defining the function and potency of different MSC-like populations is the assessment of their production of cytokines and growth factors associated with stem cell survival, angiogenesis, and immune cell responses.
Given the complexities and distinctiveness of various stem cell populations, the above fundamental phenotypic and functional features of different MSC populations must be recognized. When studying MSC, the above-described features should be recognized as a minimum set of standards for studying their biology. This minimum set of standards will become increasingly important as studies move from in vitro and animal studies into clinical applications for humans using these unique and highly versatile cells.
Cells with potential for use in periodontal regeneration
Stem cells from both extraoral and intraoral sources have been studied for their potential to be used in periodontal regeneration applications. To date, most studies have investigated MSC derived from a variety of tissue sources in preclinical animal studies for the treatment and regeneration of the periodontium. With ever-increasing evidence that this is a viable technology, reports of human clinical trials are now beginning to appear in the literature.
Multipotent stem cells that can differentiate into tissue-specific cells have been isolated and characterized from all dental tissues (except enamel). Indeed, for some time, the regenerative capacity of the periodontium has been recognized, and this has been attributed to the presence of residual multipotent progenitor cells responsible for the development of the periodontium. Importantly, dental tissue–derived MSC tend to be relatively committed in their regenerative potency because of the fact that dental tissues do not undergo continuous remodeling, as do other tissues with higher turnover such as bone.
Dental pulp stem cells (DPSC) were the first dental MSC to be isolated and characterized. Following their discovery, MSCs were identified in exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSC), the stem cells from apical papilla (SCAP), the dental follicle, and gingiva. Each of these MSC populations varies with regards to their growth and differentiation capacities and general gene and protein expression profiles. Whether these differences relate specifically to their specific niche origins remains to be established. Given their specialized sites of origin, these intraoral dental stem cells have been studied for their potential to be used in tissue engineering-based cell therapies for periodontal regeneration in both animals and humans ( Fig. 1 ).
Stem cells of dental origin for periodontal regeneration
Periodontal Ligament Stem Cells
The identification and isolation of PDLSC from the periodontal ligament have made these cells obvious candidates for periodontal regeneration studies. These cells are critical for the maintenance of tissue homeostasis and have considerable regenerative capacity, being able to differentiate into many different cell types, including cementoblasts, osteoblasts, adipocytes, and fibroblasts as well as cementum/ periodontal ligament (PDL)-like structures. , ,
There have been many studies conducted investigating the use of PDLSC for periodontal regeneration. One systematic review concluded that irrespective of the defect type and animal model used, PDLSC implantation can be expected to result in a beneficial outcome for periodontal regeneration. Furthermore, it was concluded that there was sufficient evidence from preclinical animal studies to warrant progression to human studies. Accordingly, human trials using PDLSC for periodontal regeneration have commenced. Two recent reviews have been published assessing the outcomes of these studies. , The evidence to date indicates that although these cells are safe to use, the impact on periodontal regeneration is limited with some improvements noted, but also large heterogeneity was noted most likely because of variations in sample size and study protocols. Until large-scale, high-quality randomized clinical trials can be carried out, evidence to support the use of these cells for periodontal regeneration remains equivocal.
Dental Pulp Stem Cells
DPSC were the first dental stem cells to be identified. Given that dental pulp is similar to bone marrow, being a highly vascularized tissue, it is not surprising that MSC have been found in this tissue. DPSC can form mineralized tissues and have the capacity for colony formation and high proliferation rates in vitro and expression of cell surface markers similar to those found on endothelial cells, smooth muscle cells, osteoblasts, and fibroblasts. More recently, it has been determined that DPSC have higher proliferation rates, a lower rate of senescence, and enhanced osteogenic capacity compared with nondental MSC in vitro. These cells demonstrated good periodontal regenerative capacity in a porcine periodontitis model. DPSC show superior resistance to subculture and inflammation-induced senescence and could be suitable for tissue engineering within an inflammatory environment. The utility of DPSC for periodontal regeneration is now well documented. Nevertheless, a recent systematic review concluded that periodontal regeneration, although DPSC could facilitate periodontal regeneration, was not as effective as PDLSC.
Stem cells from human exfoliated deciduous teeth
Stem cells have been derived from the residual pulp of human exfoliated deciduous teeth. Despite being of dental pulp origin, SHED are unique and distinct from adult DPSC in terms of their appearance, clonal and colony formation capacity, in vivo bone formation, and inability to form dentin-pulp-like tissue. Interestingly, SHED have higher proliferation rates and higher expression of genes involved in producing cytokines responsible for cell proliferation and extracellular matrix synthesis. To date, no studies have been reported on the use of SHED for periodontal regeneration.
Stem Cells from Apical Papilla
MSC have been isolated from the apical papilla, a complex organ comprising a collection of cells involved in the development of the tooth root and dental pulp. The cells have been termed SCAP. Because of their association with tooth root development, these cells appear to have better regenerative capacity than other stem cells of dental origin. , SCAP can form odontoblast-like cells and adipocytes in vitro and also express several neurologic cell markers. , , A good source of SCAP is from extracted third molars with incomplete root formation.
SCAP have the potential for use in the regeneration of the dentin/pulp complex and bioroot engineering. , An early study demonstrated that SCAP together with PDLSC in a prefabricated hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold carrier regenerated a functional root/periodontal complex that could support a porcelain crown in miniature pigs. More recently, a study has demonstrated that in a porcine model, local injection of SCAP into periodontal defects resulted in periodontal regeneration, indicating that these cells could be a useful source of dental-derived stem cells for periodontal regeneration. No human studies have been carried out to date.
Dental Follicle Stem Cells
The dental follicle is a critical structural component of tooth development and is also a good source of MSC termed dental follicle stem cells (DFSC). DFSC can be sourced from unerupted or impacted third molars. Although some studies have demonstrated the capacity of these cells to regenerate bone when implanted subcutaneously or into bony defects, there are no studies to date investigating the use of DFSC in periodontal regeneration of human periodontal defects.
Gingival Mesenchymal Stem Cells
Although the multipotent potential of gingival fibroblasts has been recognized for a long time, it is not until recently that mesenchymal stem cells from gingival tissues have been studied. These cells demonstrate high proliferation rates, a stable morphology, and osteogenic capacity in vitro. , Gingival mesenchymal stem cells (GMSC) can promote bone formation in vivo when implanted into calvarial critical-size defects and periodontal defects in animals. , GMSC appear to have properties that are very similar to PDLSC. GMSC have been studied for their capacity to assist in periodontal regeneration in several animal models (pig and dog). These studies have demonstrated that GMSC possess good periodontal regenerative potential, facilitating newly formed bone, cementum, and periodontal ligament fibers. , Although these cells have been proposed to be a good source of MSC-like cells for periodontal regeneration because of their easy availability and robust in vitro characteristics, no human studies using these cells for periodontal regeneration have been carried out to date.
Extraoral stem cells for periodontal regeneration
Bone Marrow–Derived Mesenchymal Stem Cells
There are 3 major stem cell populations residing within the bone marrow: hematopoietic stem cells, angioblasts, and bone marrow stromal stem cells (BMSSC). ,
These cells have considerable capacity to proliferate and expand in vitro and can differentiate into multiple cell lineages. , , Accordingly, they have been studied extensively for their regenerative capacity in a wide variety of tissues and diseases. BMSSC have been used for transplantation into periodontal defects in animal models and have been shown to have good potential to differentiate into cementoblasts, periodontal ligament fibroblasts, and alveolar bone osteoblasts in vivo . Human trials using these cells for periodontal regeneration have been carried out. An early study demonstrated good clinical results when autologous, expanded bone marrow-derived MSC mixed with atelocollagen were transplanted into periodontal osseous defects at the time of periodontal surgery. The results showed a 4-mm reduction in probing depths and reduction in intrabony defect depth as well as resolution of bleeding and interdental papilla regeneration.
A recent systematic review has indicated that BMSSC do support periodontal regeneration, but the evidence to date is still low quality, and further larger studies are needed to confirm their clinical utility.
Adipose Tissue Stem Cells
Adipose tissue is emerging as an excellent source of easily accessible multipotent MSC with properties very similar to BMSSC. Emerging studies are now suggesting that ASC may be superior to BMSCC for use in regenerative medicine because they are easier to harvest, demonstrate less senescence in vitro, and produce a vast array of growth and immunomodulatory factors conducive for tissue regeneration.
The osteogenic capacity of adipose stem cells has been studied in considerable detail and has led to the suggestion that these cells are among the most promising for use in cell-based bone regeneration. The osteogenic effect may arise due to their ability to differentiate into osteoblasts as well as through paracrine mechanisms, facilitating the migration and differentiation of other precursor osteoprogenitors. ,
Adipose tissue stem cells (ASC) have been studied for periodontal regeneration. An early study demonstrated that, in a mouse model, ASC could promote regeneration of a periodontal ligament-like structure along with the alveolar bone.
A recent systematic review assessed 15 publications reporting on the in vivo use of adipose tissue/cell for periodontal regeneration. These preclinical studies reported a general improvement of bone and periodontal healing when adipose tissue cells were delivered in a variety of carrier vehicles. It was concluded that adipose-derived cells might contribute to bone and periodontal regeneration; however, no meta-analyses could be carried out because of the heterogeneity of the studies. There is an ongoing need for more definitive, well-controlled, and large-scale studies before any definitive conclusions can be made. To date, no human clinical trials for periodontal regeneration using ASC have been reported.
Induced Pluripotent Stem Cells
Reprogramming adult somatic cells into a pluripotent stem cell state has been a landmark development in stem cell biology and regenerative medicine. These cells are termed IPSC. Using this technology, large populations of adult-derived stem cells can be generated without the ethical concerns of using embryonic stem cells ( Fig. 2 ). IPSC can be generated from cells obtained from any tissue, and because of their unlimited growth capacity, that can provide an everlasting supply of stem cells. Although there has been considerable interest in using these cells in regenerative medicine, some features, such as potential for tumorigenicity and instability, significantly diminish their clinical utility.
IPSC have been generated from several dental-derived cells, including gingival, pulp, and periodontal ligament. IPSC have been studied for their potential use in periodontal regeneration. , When implanted into periodontal defects, IPSC can significantly enhance regeneration and newly formed mineralized tissue formation. Interestingly, IPSC have been found to have the capacity to control acute and chronic inflammatory responses associated with the destruction of periodontal tissue when injected locally or systemically.
Cell sheets for periodontal regeneration
Rather than using suspensions of cells embedded with various carrier scaffolds, the use of “cell sheets” has emerged as a novel alternative approach for periodontal tissue engineering ( Fig. 3 ). , This technology uses temperature-responsive cell culture dishes modified to facilitate the removal of multilayered cell sheets. At normal culture conditions, the culture plate substrate is in a solid format, but it becomes more fluid when cooled and permits easy removal of the cell sheet from the culture plate. This eliminates the need for enzymes to release the cells from the culture plate and is thus a noninvasive gentle process that maintains an intact extracellular matrix necessary for the viability of the cells at the time of transplantation. Using this technology, the implanted cells presented to the defect site are less disrupted, allowing for critical cell-cell functions to continue operating. Several preclinical trials in animal models have demonstrated good periodontal regenerative outcomes with bone, periodontal ligament, and cementum confirmed histologically to have regenerated. , Transplants of cell sheets containing allogeneic cells in miniature swine have also demonstrated good periodontal tissue regenerative with no evidence of any adverse effects. A recent study demonstrated favorable periodontal regeneration following the use of BMMSC cell sheets combined with multiphasic scaffolds in an ovine periodontal defect model.
Human clinical trials for cell-based periodontal regeneration
With the demonstration that MSC can result in safe and efficacious periodontal regeneration in animal models, the time has come for the field to move on to human clinical trials. Early case reports on the use of autologous periodontal cells and other MSC began to appear in the literature around 2005 ( Table 1 ). An early single-arm and single-institute clinical study that commenced in 2011 investigated the potential use of autologous PDLSC sheets for periodontal regeneration. They reported that the therapeutic effects were sustained over 4.5 years with no serious side effects. The first human clinical trials using BMSSC and PDLSC were reported in 2016. To date, cells from a variety of sources, including bone marrow, dental pulp, and periodontal ligament, have been studied with mixed results. A systematic review of these studies concluded that there is only low-quality evidence demonstrating that MSC use for periodontal regeneration results in generally small gains. Because of the heterogeneity of the studies and low sample sizes, conclusions are difficult to make, and more definitive studies are needed.
| Reference | Study Format | Stem Cell Type | Number of Defects/Subjects | Carrier Vehicle | Defect | Outcome | Complications/Safety Issues |
|---|---|---|---|---|---|---|---|
| Akbay et al, 2005 | Randomized clinical trial | Periodontal ligament graft Autologous |
20/10 No controls |
PDL scraped from tooth root; no carrier used | Mandibular class II furcation | Improved CAL, pocket, bone fill Increased gingival recession |
No foreign body response noted |
| Yamada et al, 2006 | Case report | Bone marrow stromal stem cells Autologous |
1/1 | Platelet-rich plasma | Intrabony | Improved CAL, pocket depth, bone fill | Not assessed |
| Feng et al, 2010 | Pilot study/case report | Periodontal ligament stem cells Autologous |
16/3 | Hydroxyapatite/tricalcium phosphate | Intrabony | Improved CAL, pocket depth Increased gingival recession |
Nil |
| McAlliser, 2011 | Case report | Bone marrow stromal stem cells Allogeneic |
2/2 | Commercial cellular allograft bone matrix (Osteocel; ACE Surgical Supply) | 1 × intrabony 1 × grade II furcation |
Improved pocket depth, bone fill | Not assessed |
| Koo et al, 2012 | Case report | Bone marrow stromal stem cells Allogeneic |
1/1 | Osteocel; NuVasive, San Diego, CA, USA covered with a resorbable membrane (DynaMatrix; Keystone Dental, Burlington, MA, USA) | Intrabony | Improved CAL, pocket depth | Not assessed |
| Yamada et al, 2013 | Case series | Bone marrow stromal stem cells Autologous |
17/17 | Platelet-rich plasma | Intrabony | Improved CAL, pocket depth | Nil |
| Rosen, 2013 | Case report | Bone marrow stromal stem cells Allogeneic |
1/1 | Osteocel; NuVasive, covered with an amnion-chorion membrane (BioXclude; Snoasis Medical, Denver, CO, USA) | Mandibular grade III furcation | Improved pocket depth Increased gingival recession |
Not assessed |
| Aimetti et al, 2014 | Case report | Dental pulp Autologous |
1/1 | Dental pulp cell suspension from dissociated third molar pulp mixed into collagen sponge | Intrabony | Improved pocket depth, CAL, bone fill | Not assessed |
| Aimetti et al, 2015 | Case series | Dental pulp Autologous |
4/4 | Dental pulp cell suspension from dissociated third molar pulp mixed into collagen sponge | Intrabony | Improved CAL, pocket depth, bone fill | Not assessed |
| Baba et al, 2016 | Phase I/II clinical trial | Bone marrow stromal stem cell Autologous |
10/10 No treatment controls |
Platelet-rich plasma | Intrabony | Improved CAL, pocket depth, bone fill | Nil |
| Li et al, 2016 | Case report | Inflamed dental pulp stem cells Autologous |
2/2 | β-Tricalcium phosphate | Intrabony | Improved pocket depth | Nil |
| Chen et al, 2016 | Randomized clinical trial | Periodontal ligament stem cells Autologous |
41/30 Test = 21 Control = 20 Bio-Oss with GTR membrane (no cells) |
Bovine-derived bone mineral materials (Bio-Oss) | Intrabony | Improved CAL, pocket depth, bone fill No difference with the control group |
Nil |
| Kl et al, 2017 | Case report | Periodontal ligament and cementum scraping Autogenous |
1/1 | Gelatin sponge (Abgel) | Intrabony | Improved CAL, pocket depth | Not assessed |
| Aimetti et al, 2018 | Case series | Dental pulp Allogeneic |
11/11 | Dental pulp cell suspension from dissociated third-molar pulp mixed into collagen sponge | Intrabony | Improved CAL, pocket depth, bone fill | Not assessed |
| Ferraroti et al, 2018 | Randomized clinical trial | Dental pulp stem cells Autologous |
29/29 Test = 15 Control = 14 (collagen sponge without cells) |
Collagen sponge (Condress; Istituto Gentili, Milano, Italy) | Intrabony | Improved CAL, pocket depth, bone fill, increased gingival recession | Not assessed |
| Hernandez-Monjaraz et al, 2018 | Case report Allogeneic |
Deciduous tooth pulp Allogeneic |
1/1 | Lyophilized Collagen-polyvinylpyrrolidone sponge |
Intrabony | Improved pocket depth, bone fill | Nil |
| Iwata et al, 2018 | Single-arm and single-institute clinical study | Autogenous periodontal cell sheets | 10/10 No controls |
Cell sheets laid adjacent to root surface and defect filled with β-tricalcium phosphate | Intrabony | Improved CAL, probing depth, bone fill | Nil |
| Shalini & Vandana, 2018 | Randomized clinical trial | Autologous periodontal ligament cells | 28 subjects: 14 control 14 test |
PDL scraped from tooth root | Infrabony | Improved CAL, probing depth, bone fill, bone density | Not assessed |
| Hernandez-Monjaraz et al, 2020 | Case/control clinical study | Deciduous tooth pulp Allogeneic |
22/22 Test = 11 Control = 11 (carrier with no cells) |
Lyophilized polyvinylpyrrolidone Sponge |
Nil |
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