Stem Cell Transplantation and Cell-Free Treatment for Periodontal Regeneration

Increasing attention has been paid to cell-based medicines. Many in vivo and in vitro studies have demonstrated the efficacy of stem cell transplantation for the regeneration of periodontal tissues over the past 20 years. Although positive evidence has accumulated regarding periodontal regeneration using stem cells, the exact mechanism of tissue regeneration is still largely unknown. This review outlines the practicality and emerging problems of stem cell transplantation therapy for periodontal regeneration. In addition, possible solutions to these problems and cell-free treatment are discussed.


Introduction
Periodontal disease is a chronic inflammatory state of periodontal tissues caused mainly by the colonization of Gram-negative bacteria [1,2]. In the local area affected by periodontitis, chronic inflammation over a long period of time leads to progressive destruction of the tooth-supporting tissues, and eventually, the affected teeth are extracted due to the loss of supporting tissues [3].
Periodontal disease is one of the primary causes of tooth extraction worldwide [4], and it is widely recognized that periodontal treatment is important in maintaining oral functions including mastication and pronunciation. In addition, recent studies have reported that periodontal disease is related to the pathogenesis of various systemic diseases such as diabetes, cardiovascular disease, low birth weight, osteoporosis, etc. [5,6]. The importance of periodontal treatment has been emphasized from the perspective of maintaining systemic health.
The ultimate treatment goal of periodontal disease is to regenerate lost periodontal tissues including cementum, periodontal ligament, and alveolar bone. However, there is still no complete periodontal regenerative treatment, and thus, research is being conducted to develop a new treatment for periodontal regeneration. In this context, attempts to regenerate periodontal tissues by stem cell transplantation have been made over the past 20 years. A number of studies have examined whether various stem cells can regenerate periodontal tissues in multiple animal models. This review provides an overview of periodontal regeneration using stem cells and summarizes the characteristics, problems, and new research developments in periodontal regeneration using stem cells.
Human clinical studies have already begun based on these animal studies. The results of the controlled clinical trial in human patients are summarized in Table 2 [59][60][61][62][63]. All of these studies confirmed the safety of stem cell transplantation and reported that clinical parameters such as pocket depth, clinical attachment level, and radiographic bone volume tended to improve by stem cell transplantation [59][60][61][62][63]. However, of the five meta-analyses, two found statistically significant differences between control and cell treatment. The results from human clinical studies seems to be less impactful than those from preclinical studies. Further controlled clinical studies with larger sample size and long term observation period are needed.

Problems in Periodontal Regenerative Therapy Using Stem Cell Transplantation
As mentioned above, many animal studies have demonstrated through histologic evaluation that periodontal tissue regeneration can be achieved by stem cell transplantation, suggesting that stem cell transplantation may be a promising new treatment. However, there are still some problems that remain unresolved.

Mechanism of Tissue Regeneration
In most stem cell-based tissue regeneration procedures, transplanted stem cells are expected to localize or migrate, proliferate, differentiate, and form new tissues at the transplanted site. However, many cell transplantation studies have reported that the actual engraftment of transplanted cells is very low [64][65][66][67] [40]. In addition, they attempted to detect human-specific DNA using genomic DNA recovered from sections prepared 3, 7, and 28 days after surgery in the same experiment. The results showed that human DNA detection did not increase significantly in the tissues but rather decreased compared with immediately after the cell transplantation. These results suggest that transplanted stem cells are partially engrafted in regenerated tissues; however, their direct contribution to new tissue formation is limited. The local conditions after transplantation surgery are difficult for the survival of transplanted cells due to ischemia caused by vascular disruption and inflammatory reactions related to wound healing, which may limit the viability of the transplanted cells. In addition, in almost all animal experiments, regenerated tissues were formed in continuity with the surrounding tissues. This indicates that the regeneration of periodontal tissues by stem cell transplantation may be the result of accelerated spontaneous wound healing. If the transplanted stem cells are unlikely to engraft, proliferate, and differentiate to form tissue directly, the mechanism by which transplanted stem cells lead to tissue regeneration remains unclear.

Limitations of Autologous Stem Cells
In animal experiments, allogenic and xenogeneic cells are often used to examine their regenerative potential. However, when considering cell therapy in humans, the first choice is to transplant autologous cells. Autologous stem cell transplantation in patients with periodontal disease requires several considerations. The first is the limitation of tissue retrieval for harvesting stem cells. In order to culture stem cells, tissues such as the periodontal ligament, dental pulp, gingiva, bone marrow, and fat need to be harvested; however, these tissues are sometimes difficult to collect due to their scarcity. Second, the majority of patients with periodontal disease are middle-aged or older, and the effects of aging on tissues and cells need to be taken into account when transplanting autologous cells. Aging is known to negatively affect the number and quality of tissue stem cells [69,70]. Animal experiments are usually performed using stem cells from relatively young adult animals or young human volunteers. Animal experiments mimicking periodontal patients in age have not yet been conducted. Furthermore, ex vivo expansion of stem cells is necessary to ensure the number of cells required for cell transplantation. Stem cells from middle-aged and older donors are susceptible to replicative aging in culture, which may significantly impair their stem cell properties [71]. When considering autologous stem cell transplantation in patients with periodontal disease, available cell sources may be limited.

Safety Issues in Stem Cell Transplantation
The safety of stem cell transplantation for periodontal regeneration has been reported by many clinical studies [59][60][61][62][63]72]. Safety considerations regarding autologous stem cell transplantation include the transmission of infection during ex vivo expansion, and tumor or unintended tissue formation after transplantation. The number of cells required for cell transplantation may vary depending on the degree of periodontal tissue destruction; however, it is believed that approximately 10 6 to 10 7 cells are required per deep periodontal defect. Therefore, ex vivo expansion of cells is unavoidable in the stem cell treatment of periodontal disease. The contamination of pathogenic factors in the process of cell culture must be avoided. Therefore, GMP-compliant facilities are required to ensure a high level of safety, and at the same time, a variety of quality control inspections are essential. The required safety level for cell treatment in dentistry is the same as that of general cell therapy in the medical field. Cell production methods for human clinical use have been largely established, and as long as they are followed, there may be no concerns about safety.
In general, MSCs are thought to have very low tumorigenicity; however, it is assumed that, through cell culture and passaging, the cells are greatly affected by cellular stress, which increases the risk of tumorigenesis. Since most animal experiments were completed in a relatively short period of time, from several weeks to months, there is a paucity of data showing the long-term safety of MSC transplantation. Dannan et al. reported the development of squamous epithelial-cell carcinoma after transplantation of human periodontal ligament-derived stem cells into periodontal defects in athymic rats [73]. They found that, after transplantation, tumors were found in two out of four rats at 6 weeks and in all four mice at 8 weeks, and human mitochondria and karyotypic variation were observed in the tumors. These results may be very rare cases of animal experiments; however, the safety of MSC transplantation is not completely guaranteed. Stem cells can differentiate into various types of cells, and after transplantation into the human body, we cannot control their spontaneous growth and differentiation. Therefore, there is a possibility of unintended tissue formation by transplanted stem cells. Safety has not been established in this regard because of the lack of detailed long-term histological studies of the fate of transplanted cells.

Cost of Cell Therapy
It is not clear what the reasonable treatment cost is for cell-based dental treatments. Considering the case of autologous cell transplantation, the cost of tissue harvesting and preparation of clinical-grade products, including cell culture, various tests for infections, and labor costs, is expected to be at least several thousand dollars. In the US, the cost of stem cell therapy is USD 4000-8000, and the price of cultured cells is reported to be USD 15,000-30,000 [74]. On the contrary, the average treatment fee for a single dental implant in the US for general dentistry is approximately USD 3000 to USD 4000 [75]. The cost of dental treatments, such as bridges, dentures, and periodontal tissue regeneration, including guided tissue regeneration and bone grafting, is approximately a few thousand dollars, and treatment costs exceeding USD 10,000 are not realistic in dentistry. This compares to diseases that severely affect life and quality of life, such as spinal cord injury, graftversus-host disease, stroke, and myocardial infarction, in which it may be worth spending over USD 10,000. Currently, stem cell therapy is very expensive compared with regular dental treatments.

Allogeneic Cell Transplantation
The problems of qualitative and quantitative limitations of stem cells and the cost of treatment considered in autologous cell transplantation may be solved to some extent by allogeneic cell transplantation. The use of cells from young donors allow for the transplantation of a large number of cells with a high therapeutic efficacy. In the case of using stem cells from dental tissues, tissue donations are readily available because most patients with premolar or wisdom teeth extraction are young, and these teeth are often discarded as medical waste. The safety issues associated with allogeneic cell transplantation need to be confirmed in clinical studies in humans. Dhote et al. transplanted allogenic umbilical cord-derived MSCs into human periodontal defects and reported improved periodontal tissue formation and safety after 6 months [59]. Tsumanuma et al. also reported in dogs that transplantation of allogenic MSC from periodontal ligament resulted in the regeneration of cementum and periodontal ligament without any side effects at 8 weeks post transplantation [39]. Further safety verification may solve this safety issue in allogeneic cell transplantation. It is necessary to accumulate sufficient evidence to determine whether the benefits outweigh the drawbacks of autologous cell treatment.

Cell-Free Treatment
The basic idea of tissue regeneration by MSC transplantation is to utilize the differentiation ability of stem cells and to expect that the transplanted stem cells will grow and differentiate locally to regenerate the tissues. However, in many stem cell transplantation studies, the local retention of transplanted cells has been much lower than expected, and it is clear that tissue regeneration and wound healing promotion are mediated by factors secreted from the cells [64][65][66][67]. Secreted factors produced by MSCs are known to have antiinflammatory, immunoregulatory, angiogenic, and anti-apoptotic functions [76,77]. Based on the potential of secreted factors, tissue regeneration using secreted factors/substances from stem cells is being investigated as a cell-free treatment.

Conditioned Medium
A method has been reported in which secreted factors released from stem cells are collected in the form of culture supernatant and transplanted instead of the cells themselves. Nagata et al. showed that the transplantation of a conditioned medium (CM) obtained from PDLSCs resulted in the regeneration of periodontal tissues. This study demonstrated that PDLSC-CM contained various growth factors and angiogenic factors and that the gene expression of tumor necrosis factor (TNF)-alpha was suppressed at the CM implantation site [78]. Qiu et al. transplanted the CM from gingival MSCs and PDLSCs into periodontal defects in rats, observed that both CMs enhanced periodontal regeneration, and showed that the expression levels of interleukin-1 and TNF-alpha were reduced by CM transplantation using immunostaining [79]. Similarly, periodontal tissue regeneration by the transplantation of CM from bone marrow-MSCs has also been reported [80]. These results indicate that the transplantation of MSC-derived CM induces periodontal regeneration mainly through its anti-inflammatory effect, suggesting the possibility of cell-free treatment using stem cell-derived CM. Animal studies of cell-free treatment for periodontal regeneration are listed in Table 3.

Exosomes/Extracellular Vesicles
Exosomes/extracellular vesicles are small particles (<100 nm) from cells with a lipid bilayer structure. Exosomes contain various cellular components, such as proteins, mRNAs, DNA, and miRNAs. Exosomes released from cells have recently been recognized as new tools for cell-cell communication, as exosomes can be taken up by distant cells and can exert their functions within those cells [81,82]. MSC-derived exosomes contain molecules that modulate wound healing and are believed to be responsible for many of the actions of MSC humoral factors, including angiogenesis, anti-inflammation, anti-apoptosis, and immune regulation [83,84]. Two reports have shown that periodontal tissues can be regenerated by transplantation of exosomes from MSCs. Chew et al. showed that the transplantation of bone marrow MSC-derived exosomes into rat periodontal defects enhanced regeneration of periodontal tissues [85]. Wu et al. reported that the implantation of SHED-derived exosomes induced periodontal regeneration in rats [86].

Features of Cell-Free Treatment
Cell-free treatment, consisting of a CM and exosomes, has features that cover some of the disadvantages of cell transplantation. In CM and exosome transplantation, the possibility of tumor formation and immune rejection is considered to below. In addition, because cells are not transplanted, problems with donor aging and cell sources can be avoided. CMs and exosomes can be stored in freezers, which makes them more practical than cells, as they are inexpensive and easy to manage and use. However, since tissue regeneration with cell-free treatment improves the patient's wound healing process, the amount of tissue regeneration may be lower than that with cell transplantation. The number of regenerative studies using cell-free treatment is still small compared with that of cell transplantation, and further extensive research is needed.

Conclusions
Recently, clinical studies on periodontal tissue regeneration by stem cell transplantation in humans have been reported. In three of these studies, there was a trend toward improvement of clinical parameters in the stem cell transplantation group; however, no statistically significant differences were observed [60,62,63]. Autologous cells were used in these studies, and the results of the studies differed from the results of previous animal studies. The quality of transplanted stem cells due to patient aging and chronic inflammation or changes in the transplantation site may have affected the results of human clinical studies. The efficacy of stem cell transplantation for periodontal regeneration has been shown mainly in animal experiments, and we need to find a way to solve the remaining problems to establish the stem cell therapy as a new periodontal regenerative strategy in human patients. Considering the cost and safety issues, much more research needs to be conducted to make stem cell therapy practical for periodontal regeneration. To achieve this, it is necessary to optimize the technique, including the appropriate cell type, number of cells, and graft carrier, and to verify the therapeutic effect and safety through rigorous clinical studies with appropriate controls. Furthermore, the mechanism of periodontal tissue regeneration by stem cell transplantation, including the fate of transplanted cells, needs to be elucidated. Cell-free treatment also requires detailed mechanisms, verification of therapeutic effects, and clinical studies in humans.
Stem cell transplantation studies over the past two decades have provided a great deal of useful information on periodontal regeneration. The information revealed by many stem cell transplantation studies is expected to result in the development of new periodontal tissue regeneration therapies.

Conflicts of Interest:
The authors declare no conflict of interest.