Stem cell‐based tooth and periodontal regeneration

Currently regeneration of tooth and periodontal damage still remains great challenge. Stem cell‐based tissue engineering raised novel therapeutic strategies for tooth and periodontal repair. Stem cells for tooth and periodontal regeneration include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from the dental apical papilla (SCAPs), and stem cells from human exfoliated deciduous teeth (SHEDs), dental follicle stem cells (DFSCs), dental epithelial stem cells (DESCs), bone marrow mesenchymal stem cells (BMMSCs), adipose‐derived stem cells (ADSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). To date, substantial advances have been made in stem cell‐based tooth and periodontal regeneration, including dentin–pulp, whole tooth, bioroot and periodontal regeneration. Translational investigations have been performed such as dental stem cell banking and clinical trials. In this review, we present strategies for stem cell‐based tissue engineering for tooth and periodontal repair, and the translational studies.     

Biologic interaction of three-dimensional periodontal fibroblast spheroids with collagen-based and synthetic membranes

Background: Cell‐based therapy using autologous cells has been suggested as a potential approach for periodontal tissue regeneration. Spheroid systems are a form of three‐dimensional cell culture that promotes cell matrix interaction, which could recapitulate the aspect of cell homeostasis in vivo. The aim of this study is to assess the interaction of periodontal fibroblast spheroids with synthetic and collagen‐based membranes that have been used in guided tissue regeneration. Methods: Commercially available normal human periodontal ligament fibroblasts were grown in spheroid forms using a liquid overlay technique and then transplanted onto a collagen‐based and a polyglycolic acid–based membrane. The biologic interaction of the spheroids with the membranes was assessed using basic histology, Alamar blue tissue viability assay, scanning electron microscopy, and immunohistochemical analysis. Results: Periodontal fibroblast spheroids adhered to both membranes, and the cells were able to proliferate and migrate from the spheroids both horizontally and vertically into the membrane scaffolds. Immunohistochemical analysis showed expression of collagen type I, periostin, and Runx2 by the periodontal fibroblasts. Conclusion: Periodontal fibroblast spheroids were able to grow three‐dimensionally on the biologic membranes and may have the potential to be used together with guided tissue regeneration approaches as an adjunct for periodontal regeneration.     

Sequential process in brain‐derived neurotrophic factor‐induced functional periodontal tissue regeneration

We recently demonstrated that brain‐derived neurotrophic factor (BDNF) promotes periodontal tissue regeneration. The purpose of this study was to establish an essential component of a rational approach for the clinical application of BDNF in periodontal regenerative therapy. Here, we assessed the sequence of early events in BDNF‐induced periodontal tissue regeneration, especially from the aspect of cementum regeneration. Brain‐derived neurotrophic factor was applied into experimental periodontal defects in Beagle dogs. The localization of cells positive for neurotrophic tyrosine kinase, receptor, type 2, proliferating cell nuclear antigen, osteopontin, integrin αVβ3, and integrin α2β1 was evaluated by immunohistochemistry. The effects of BDNF on adhesion of cultured human periodontal ligament cells was examined by an in vitro study. The results suggest that BDNF could induce rapid cementum regeneration by stimulating adhesion, proliferation, and differentiation of periodontal ligament cells in the early regenerative phase, resulting in enhancement of periodontal tissue regeneration.     

Reconstructive periodontal surgery: A challenge for modern periodontology

The overall rationale for the proposed clinical approach in this paper comes from consideration of different regenerative approaches reported in the literature. The protocol proposed is based mainly on evidence and partly on the clinical experience of the author and co‐workers, who have found that patient associated factors such as plaque control, residual periodontal infection, and smoking habits are those of particular relevance. Among the technical/surgical associated factors, lack of primary closure of the interdental space and consequent bacterial contamination of the regenerating wound represent the most significant factors leading to compromised outcomes. Modified flap designs and a micro‐surgical approach have been shown to improve the outcomes. There is now sufficient evidence to suggest that clinicians can incorporate periodontal regeneration in their surgical armamentarium. Periodontal regeneration can be predictably used to treat deep pockets associated with deep intrabony defects in young and old people, aimed at pocket/defect resolution with aesthetic preservation, gain of clinical attachment and bone.     

Cellular therapy in periodontal regeneration

Periodontitis is a chronic inflammatory condition leading to destruction of the tooth supporting tissues, which if left untreated may cause tooth loss. The treatment of periodontitis mainly aims to arrest the inflammatory process by infection control measures, although in some specific lesions a limited periodontal regeneration can also be attained. Current regenerative approaches are aimed to guide the cells with regenerative capacity to repopulate the lesion and promote new cementum and new connective tissue attachment. The first phase in periodontal tissue regeneration involves the differentiation of mesenchymal cells into cementoblasts to promote new cementum, thus facilitating the attachment of new periodontal ligament fibers to the root and the alveolar bone. Current regenerative approaches limit themselves to the confines of the lesion by promoting the self‐regenerative potential of periodontal tissues. With the advent of bioengineered therapies, several studies have investigated the potential use of cell therapies, mainly the use of undifferentiated mesenchymal cells combined with different scaffolds. The understanding of the origin and differentiation patterns of these cells is, therefore, important to elucidate their potential therapeutic use and their comparative efficacy with current technologies. This paper aims to review the in vitro and experimental studies using cell therapies based on application of cementoblasts and mesenchymal stem cells isolated from oral tissues when combined with different scaffolds.     

Growth and differentiation factors for periodontal regeneration: a review on factors with clinical testing

Stavropoulos A, Wikesjö UME. Growth and differentiation factors for periodontal regeneration: a review on factors with clinical testing. J Periodont Res 2012; 47: 545–553. © 2012 John Wiley & Sons A/S Background and Objective: A large body of evidence implies that growth and differentiation factors, based on their ability to regulate various functions of cells originating in the periodontal tissues, may support periodontal wound healing/regeneration, creating an environment conducive to and/or immediately inducing de novo tissue formation. This study presents a short systematic overview on growth and differentiation factor technologies evaluated in the clinic for their potential to enhance periodontal wound healing/regeneration. Material and Methods: Reports on growth and differentiation factor technologies evaluated in the clinic for their potential to enhance periodontal wound healing/regeneration were selected for review. Results: Growth and differentiation factor technologies intended for periodontal wound healing/regeneration and evaluated clinically included platelet‐derived growth factor, insulin‐like growth factor‐I and ‐II, basic fibroblast growth factor, bone morphogenetic protein‐3 and growth differentiation factor‐5; platelet‐derived growth factor was the only Food and Drug Administration‐approved commercially available growth and differentiation factor technology. In general, enhanced periodontal regeneration was observed in sites receiving growth and differentiation factors compared with control(s). However, improvements of relatively limited clinical magnitude have been shown thus far. Conclusion: Although growth and differentiation factors project considerable appeal as candidate technologies in support of periodontal wound healing/regeneration, current candidate and commercially available technologies enhance treatment outcomes only to a limited extent in clinical settings.     

牙周韧带细胞膜片技术用于牙周组织再生的研究进展

为了解决中重度牙周炎引发的牙周组织破坏后无法自主再生的问题,细胞膜片工程根据引导性组织再生术的基本原理,通过将使用温敏培养皿等方法获取的完整细胞片层直接移植贴附到缺损部位的方法,实现获取牙骨质、牙槽骨及嵌入二者的牙周韧带同时再生的目标.近年来,细胞膜片技术不断发展,逐步克服了单层细胞膜片厚度偏低、血管化不良及自体细胞来源不足等问题,其中运用牙周韧带细胞膜片进行牙周组织再生已经正式进入临床试验阶段.本文对近年来牙周韧带细胞膜片及其他常用细胞膜片在牙周组织再生方向应用的新进展作一综述.     

Tissue engineered periodontal products

Attainment of periodontal regeneration is a significant clinical goal in the management of advanced periodontal defects arising from periodontitis. Over the past 30 years numerous techniques and materials have been introduced and evaluated clinically and have included guided tissue regeneration, bone grafting materials, growth and other biological factors and gene therapy. With the exception of gene therapy, all have undergone evaluation in humans. All of the products have shown efficacy in promoting periodontal regeneration in animal models but the results in humans remain variable and equivocal concerning attaining complete biological regeneration of damaged periodontal structures. In the early 2000s, the concept of tissue engineering was proposed as a new paradigm for periodontal regeneration based on molecular and cell biology. At this time, tissue engineering was a new and emerging field. Now, 14 years later we revisit the concept of tissue engineering for the periodontium and assess how far we have come, where we are currently situated and what needs to be done in the future to make this concept a reality. In this review, we cover some of the precursor products, which led to our current position in periodontal tissue engineering. The basic concepts of tissue engineering with special emphasis on periodontal tissue engineering products is discussed including the use of mesenchymal stem cells in bioscaffolds and the emerging field of cell sheet technology. Finally, we look into the future to consider what CAD/CAM technology and nanotechnology will have to offer.     

不同干细胞来源外泌体在牙周再生领域的研究进展

牙周炎是由牙菌斑中的微生物所引起的慢性感染性疾病,可引起牙周支持组织的炎症和破坏,是成年人失牙的最主要原因。良好的菌斑控制可有效抑制炎症进展,然而目前在临床诊疗中,尚难以获得稳定的、令人满意的牙周组织再生。外泌体是真核细胞分泌的一种细胞外膜性微囊泡,可作为干细胞旁分泌活动的一种重要形式参与多种组织的再生。外泌体的应用为牙周组织再生提供了一种新策略,本文对不同干细胞来源外泌体在牙周再生领域的研究进展进行综述。     

Stromal cell-derived factor-1 significantly induces proliferation, migration, and collagen type I expression in a human periodontal ligament stem cell subpopulation

Background: The pivotal role of chemokine stromal cell–derived factor‐1 (SDF‐1) in bone marrow mesenchymal stem cells recruitment and tissue regeneration has already been reported. However, its roles in human periodontal ligament stem cells (PDLSCs) remain unknown. PDLSCs are regarded as candidates for periodontal tissue regeneration and are used in stem cell–based periodontal tissue engineering. The expression of chemokine receptors on PDLSCs and the migration of these cells induced by chemokines and their subsequent function in tissue repair may be a crucial procedure for periodontal tissue regeneration. Methods: PDL tissues were obtained from clinically healthy premolars extracted for orthodontic reasons and used to isolate single‐cell colonies by the limited‐dilution method. Immunocytochemical staining was used to detect the expression of the mesenchymal stem cell marker STRO‐1. Differentiation potentials were assessed by alizarin‐red staining and oil‐red O staining. The expression of SDF‐1 receptor CXCR4 was evaluated by real‐time polymerase chain reaction (PCR) and immunocytochemical staining. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay and bromodeoxyuridine incorporation assay were used to determine the viability and proliferation of the PDLSC subpopulation. Expression of collagen type I and alkaline phosphatase was detected by real‐time PCR to determine the effect of SDF‐1 on cells differentiation. Results: Twenty percent of PDL single‐cell colonies expressed STRO‐1 positively, and this specific subpopulation was positive for CXCR4 and formed minerals and lipid vacuoles after 4 weeks induction. SDF‐1 significantly increased proliferation and stimulated the migration of this PDLSC subpopulation at concentrations between 100 and 400 ng/mL. CXCR4 neutralizing antibody could block cell proliferation and migration, suggesting that SDF‐1 exerted its effects on cells through CXCR4. SDF‐1 promoted collagen type I level significantly but had little effect on alkaline phosphatase level. Conclusion: SDF‐1 may have the potential of promoting periodontal tissue regeneration by the mechanism of guiding PDLSCs to destructive periodontal tissue, promoting their activation and proliferation and influencing the differentiation of these stem cells.     

非编码RNA调控人牙周膜干细胞成骨向分化的研究进展

牙周炎是一种常见的炎症性疾病,以牙齿支持结构的进行性损伤为特点,是我国成人牙齿丧失的主要原因。治疗牙周炎的目的不仅是通过控制炎症来阻止疾病发展,更重要的是获得牙周再生。人牙周膜干细胞具有成骨向分化潜能,有望在牙周组织修复再生的临床应用上发挥重要作用。非编码RNA(ncRNA)一般是指不编码蛋白质的RNA。伴随高通量检测技术的不断发展,发现了大量的种类丰富的ncRNA,其生物学功能也被不断揭示。越来越多证据显示,ncRNA在分子机制及细胞组织层面对疾病的发生、发展起重要调控作用,因此探索ncRNA调控机制可为牙周再生的研究提供新思路。本综述主要阐述了目前研究较多的几种ncRNA在人牙周膜干细胞成骨向分化中的调控机制。     

Experimental and clinical studies on regenerative periodontal therapy

The recognition of a periodontal therapy as a regenerative procedure requires the demonstration of new cementum, periodontal ligament, and bone coronal to the base of the defect. A diversity of regenerative strategies has been evaluated, including root surface conditioning, bone grafts and bone substitute materials, guided tissue regeneration, enamel matrix proteins, growth/differentiation factors, combined therapies and, more recently, tissue‐engineering approaches. The aim of this chapter of Periodontology 2000 is to review the research carried out in Latin America in the field of periodontal regeneration, focusing mainly on studies using preclinical models (animal models) and randomized controlled clinical trials. This review may help clinicians and researchers to evaluate the current status of the therapies available and to discuss the challenges that must be faced in order to achieve predictable periodontal regeneration in clinical practice.     

Enamel matrix derivative,inflammation and soft tissue wound healing

Over 15 years have now passed since enamel matrix derivative (EMD) emerged as an agent capable of periodontal regeneration. Following thorough investigation, evidenced‐based clinical application is now established for a multitude of clinical settings to promote regeneration of periodontal hard tissues. Despite the large number of studies and review articles written on this topic, no single review has compiled the influence of EMD on tissue inflammation, an area of research that merits substantial attention in periodontology. The aim of the present review was to gather all studies that deal with the effects of EMD on tissue inflammation with particular interest in the cellular mechanisms involved in inflammation and soft tissue wound healing/resolution. The effects of EMD on monocytes, macrophages, lymphocytes, neutrophils, fibroblasts and endothelial cells were investigated for changes in cell behavior as well as release of inflammatory markers, including interleukins, prostaglandins, tumor necrosis factor‐α, matrix metalloproteinases and members of the OPG‐RANKL pathway. In summary, studies listed in this review have reported that EMD is able to significantly decrease interleukin‐1b and RANKL expression, increase prostaglandin E₂ and OPG expression, increase proliferation and migration of T lymphocytes, induce monocyte differentiation, increase bacterial and tissue debris clearance, as well as increase fibroplasias and angiogenesis by inducing endothelial cell proliferation, migration and capillary‐like sprout formation. The outcomes from the present review article indicate that EMD is able to affect substantially the inflammatory and healing responses and lay the groundwork for future investigation in the field.     

Influence of Enamel Matrix Derivative on Human Epithelial Cells In Vitro

Background: In periodontal therapy enamel matrix derivative (EMD) has been successfully used for tissue regeneration by altering activity of various cells involved in periodontal regeneration. Studies have focused primarily on clinical parameters and outcome. Effects of EMD on oral epithelial cells are of crucial importance in order to understand the biology of regeneration. Aims of this study are to investigate proliferative and cytotoxic effects of EMD on oral epithelial cells and their possible influences on epithelial barrier function. Methods: SCC‐25 cells, a human squamous cell carcinoma cell line, and primary keratinocytes were either treated with EMD dissolved in culture medium or added to wells/inserts precoated with EMD. Cells were incubated for 24, 48, and 72 hours. Proliferation rate was analyzed measuring the 5‐bromo‐2’‐deoxyuridine nucleotide uptake. Cytotoxic effects of EMD treatment were sampled by lactate dehydrogenase release. Alterations of the epithelial barrier function induced by EMD were investigated by analysis of transepithelial electrical resistance (TER). Results: Statistically significant inhibitory effects of both malignant and primary cell proliferation could be demonstrated by precoating culture plate wells with EMD. No cytotoxic effects caused by EMD were detected. Precoating of inserts with EMD induced a significant increase of TER and barrier function. Conclusions: This investigation compares applying EMD in solution to cells with precoating of wells with EMD. When precoating of wells was used solely, inhibition of cell proliferation was evident. Precoating may represent more suitable clinical usage. Furthermore, prelayering EMD induced an increase of TER of primary cells. These results suggest EMD may enhance barrier function.     

An Autologous Platelet‐Rich Plasma Stimulates Periodontal Ligament Regeneration

Background: Regeneration of periodontal tissues is one of the most important goals for the treatment of periodontal disease. The technology of plasma rich in growth factors provides a biologic approach for the stimulation and acceleration of tissue healing. The purpose of this study is to evaluate the biologic effects of this technology on primary human periodontal ligament fibroblasts. Methods: The authors studied the response of periodontal ligament cells to this pool of growth factors on cell proliferation, cell migration, secretion of several biomolecules, cell adhesion, and expression of α2 integrin. Cell proliferation and adhesion were evaluated by means of a fluorescence‐based method. Cell migration was performed on culture inserts. The release of different biomolecules by periodontal ligament fibroblasts was quantified through enzyme‐linked immunosorbent assay. The α2 integrin expression was assessed through Western blot. Results: This autologous technology significantly stimulated cell proliferation, migration, adhesion, and synthesis of many growth factors from cells including vascular endothelial growth factor, thrombospondin 1, connective tissue growth factor, hepatocyte growth factor, and procollagen type I. The α2 integrin expression was lower in plasma rich in growth factor–treated cells compared to non‐stimulated cells, although no statistically significant differences were observed. Conclusion: This plasma rich in growth factors exerts positive effects on periodontal ligament fibroblasts, which could be positive for periodontal regeneration.     

Identification of multipotent stem cells from adult dog periodontal ligament

Periodontal diseases, which are characterized by destruction of the connective tissues responsible for restraining the teeth within the jaw, are the main cause of tooth loss. Periodontal regeneration mediated by human periodontal ligament stem cells (hPDLSCs) may offer an alternative strategy for the treatment of periodontal disease. Dogs are a widely used large-animal model for the study of periodontal-disease progression, tissue regeneration, and dental implants, but little attention has been paid to the identification of the cells involved in this species. This study aimed to characterize stem cells isolated from canine periodontal ligament (cPDLSCs). The cPDLSCs, like hPDLSCs, showed clonogenic capability and expressed the mesenchymal stem cell markers STRO-1, CD146, and CD105, but not CD34. After induction of osteogenesis, cPDLSCs showed calcium accumulation in vitro. Moreover, cPDLSCs also showed both adipogenic and chondrogenic potential. Compared with cell-free controls, more cementum/periodontal ligament-like structures were observed in CB-17/SCID mice into which cPDLSCs had been transplanted. These results suggest that cPDLSCs are clonogenic, highly proliferative, and have multidifferentiation potential, and that they could be used as a new cellular therapeutic approach to facilitate successful and more predictable regeneration of periodontal tissue using a canine model of periodontal disease.     

基因治疗在牙周组织再生中的应用

安全有效的牙周组织再生疗法一直是牙周领域研究的热点,但是现有的疗法都存在着各自的缺陷。由于基因治疗和组织工程技术的联合应用模仿了牙周自然生物发育过程,因此在牙周组织再生中已经有了不少的应用研究。本文就基因治疗在牙周组织工程领域方面的应用作一综述。     

Immunomodulatory effects of stem cells

Adult‐derived mesenchymal stem cells have received considerable attention over the past two decades for their potential use in tissue engineering, principally because of their potential to differentiate into multiple stromal‐cell lineages. Recently, the immunomodulatory properties of mesenchymal stem cells have attracted interest as a unique property of these cells that may be harnessed for novel therapeutic approaches in immune‐mediated diseases. Mesenchymal stem cells have been shown to inhibit the proliferation of activated T‐cells both in vitro and in vivo but to stimulate T‐regulatory cell proliferation. Mesenchymal stem cells are also known to be weakly immunogenic and to exert immunosuppressive effects on B‐cells, natural killer cells, dendritic cells and neutrophils through various mechanisms. Furthermore, intravenous administration of allogeneic mesenchymal stem cells has shown a marked suppression of host immune reactions in preclinical animal models of large‐organ transplant rejection and in various autoimmune‐ and inflammatory‐based diseases. Some clinical trials utilizing human mesenchymal stem cells have also produced promising outcomes in patients with graft‐vs.‐host disease and autoimmune diseases. Mesenchymal stem cells identified from various dental tissues, including periodontal ligament stem cells, also possess multipotent and immunomodulatory properties. Hence, dental mesenchymal stem cells may represent an alternate cell source, not only for tissue regeneration but also as therapies for autoimmune‐ and inflammatory‐mediated diseases. These findings have elicited interest in dental tissue mesenchymal stem cells as alternative cell sources for modulating alloreactivity during tissue regeneration following transplantation into human leukocyte antigen‐mismatched donors. To examine this potential in periodontal regeneration, future work will need to assess the capacity of allogeneic periodontal ligament stem cells to regenerate periodontal ligament in animal models of periodontal disease. The present review describes the immunosuppressive effects of mesenchymal stem cells on various types of immune cells, the potential mechanisms through which they exert their mode of action and the preclinical animal studies and human clinical trials that have utilized mesenchymal stem cells, including those populations originating from dental structures.     

Cementum-periodontal ligament complex regeneration using the cell sheet technique

Background and Objective: In the present study we evaluated if a multilayered human periodontal ligament cell sheet could reconstruct the physiological architecture of a periodontal ligament–cementum complex. Material and methods: Human periodontal ligament cells were isolated and then cultured in dishes coated with a temperature‐responsive polymer to allow cell detachment as a cell sheet. In the control group, human periodontal ligament cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium containing 10% fetal bovine serum and 1% antibiotics. In the experimental group, human periodontal ligament cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium and osteodifferentiation medium containing dexamethasone, ascorbic acid and β‐glycerophosphate. After 3 wk, scanning electron microscopy was carried out, in addition to staining for alkaline phosphatase activity and for calcium (using the Von Kossa stain). Then human periodontal ligament cell sheets were multilayered and placed onto dentin blocks. The constructs were transplanted subcutaneously into the back of immunodeficient rats. At 1 and 6 wk after transplantation, the animals were killed. Demineralized tissue sections were stained using hematoxylin and eosin, and Azan, and then analyzed. Results: After 3 wk of culture in osteodifferentiation medium, human periodontal ligament cells produced mineral‐like nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and mRNA expression of type I collagen. By contrast, in the control group only weak alkaline phosphatase staining was observed, the Von Kossa stain was negative and there was no mRNA expression of type I collagen. Six weeks after transplantation with human periodontal ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a newly immature cementum‐like tissue formation and periodontal ligament with perpendicular orientation inserted into the newly deposited cementum‐like tissue. Conclusion: This study suggests that the multilayered temperature‐responsive culture system can be used as a novel strategy for periodontal regeneration. The human periodontal ligament cell sheet technique may be applicable for regeneration of the clinical periodontal ligament–cementum complex.