Putative stem cells in regenerating human periodontium

Background and Objective:  Human postnatal stem cells have been identified in periodontal ligament, with the potential to regenerate the periodontium in vivo . However, it is unclear if periodontal ligament stem cells are present in regenerating periodontal tissues. The aim of this study was to identify and localize putative stem cells in block biopsies and explant cultures of regenerating human periodontal tissues.
Material and Methods:  Guided tissue regeneration was carried out on the molars of three human volunteers. After 6 wk, the teeth with the surrounding regenerating tissues and bone were surgically removed and processed for immunohistochemistry. The mesenchymal stem cell-associated markers STRO-1, CD146 and CD44 were used to identify putative stem cells. Cell cultures established from regenerating tissue explants were analysed by flow cytometry to assess the expression of these markers. Mineralization, calcium concentration and adipogenic potential of regenerating tissue cells were assessed and compared with periodontal ligament stem cells, bone marrow stromal stem cells and gingival fibroblasts.
Results:  STRO-1⁺, CD44⁺ and CD146⁺ cells were identified in the regenerating tissues. They were found mainly in the paravascular and extravascular regions. Flow cytometry revealed that cultured regenerating tissue cells expressed all three mesenchymal stem cell associated markers. The regenerating tissue cells were able to form mineral deposits and lipid-containing adipocytes. However, the level of mineralization in these cells was lower than that of periodontal ligament stem cells and bone marrow stromal stem cells.
Conclusion:  Cells with characteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration.     

牙周膜维持结构和功能稳定的分子机制

牙周膜中存在有多向分化潜能的成体干细胞，能够分化形成成骨细胞和成牙骨质细胞，参与牙周组织骨改建、损伤修复及牙周再生，同时，牙周膜还具有抑制矿化、维持其结构与功能稳定的调节机制。本文就牙周膜细胞的分化能力和参与维持牙周膜结构与功能稳定的分子机制作简要综述。     

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.     

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.     

炎症微环境对牙周组织再生的影响

慢性牙周炎是一种以牙周组织破坏为特征的慢性炎症性疾病。牙周病的最终治疗目的是牙周组织再生。而牙周炎时形成的局部炎症微环境,不仅造成牙周组织的破坏,而且会影响牙周组织再生过程,比如影响牙周膜干细胞的增殖分化以及生长因子的作用等。本文就炎症微环境对牙周组织再生的影响作一综述。     

牙周膜干细胞的研究进展

牙周病是一种由菌斑微生物引起的慢性感染性疾病,可引起牙周支持组织的破坏和丧失,最终导致牙齿松动脱落。牙周病治疗的最终目标是修复和重建受损的牙周支持组织。从牙周膜中分离获取的间充质干细胞具有成体干细胞的特性及多重分化潜能,可以分化为骨组织和牙周支持组织等多种类型的组织,这对牙周组织修复再生和牙周组织工程具有重大意义,因而备受关注。本文就牙周膜干细胞、牙周膜干细胞的生物学特性、牙周膜干细胞的影响因素及其调控机制等研究进展作一综述。     

经典Wnt信号通路与牙周膜干细胞成骨分化

牙周炎是口腔最常见的疾病之一,累及牙周支持组织,随着疾病的进展将引起附着丧失、牙周袋形成、牙槽骨吸收,最终导致牙齿松动脱落。因被牙周炎破坏吸收的牙槽骨自愈能力十分有限,所以牙周炎的治疗目标是在彻底清除菌斑生物膜的基础上,争取获得较多的牙周组织再生。牙周膜干细胞作为最适宜进行牙周组织再生的细胞,被广泛研究。Wnt信号通路分为经典Wnt通路和非经典Wnt信号通路,为十分复杂而高度保守的通路传导途径。该通路与牙周膜干细胞成骨分化的关系十分密切,牙周膜干细胞的成骨分化又对牙周组织再生有重要意义。该文对经典Wnt信号通路与牙周膜干细胞成骨分化的研究概况作一综述。     

牙周膜干细胞的研究进展

牙周膜干细胞可分化为成骨细胞或成牙骨质细胞、脂肪细胞和胶原形成细胞,是牙周组织工程中新的种子细胞。笔者就牙周膜干细胞的生物学特性、牙周膜干细胞的多向分化潜能、牙周膜干细胞的移植及其潜在的临床应用价值作一综述。     

Location of putative stem cells in human periodontal ligament

BACKGROUND AND OBJECTIVE: The origin of cells in the mature periodontium, and the location of their progenitors, are still unknown. It is also unknown whether inflammation influences the number and distribution of these cells within the periodontium. Molecules such as STRO-1, CD146 and CD44 have been identified on a variety of mesenchymal stem cells. The aim of this study was to identify and localize putative stem cells in diseased and healthy human periodontal ligament using cell-surface markers for mesenchymal stem cells. MATERIAL AND METHODS: Healthy and periodontitis-affected teeth were collected, fixed in 10% neutral-buffered formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry. Antibodies against STRO-1, CD146 and CD44 were used to identify putative stem cells in the periodontal ligament. RESULTS: Putative stem cells were identified in both healthy and diseased periodontal ligament. They were mainly located in the paravascular region and small clusters of cells were also found in the extravascular region. Wider distributions of these cells were detected in sections of diseased ligament. CONCLUSION: Within the periodontal ligament of both healthy and diseased teeth, cells have been identified consistent with their identification as putative stem cells. The presence of an inflammatory reaction associated with periodontitis may enhance the number of these cells.     

Mesenchymal stem cells derived from dental tissues

Regeneration of tissues occurs naturally due to the existence of stem cells with the capacity to self-regenerate and differentiate; however, regenerative capacity decreases with age, and in many cases, regeneration is not sufficient to repair the damage produced by degenerative, ischaemic, inflammatory, or tumour-based diseases. In the last decade, advances have been made in the understanding of stem cells, the genes that control the alternative fates of quiescence and differentiation, and the niches that provide specific signals that modulate cell fate decisions. Embryonic stem-cell research is shedding light on the secrets of development. Adult stem cells (AS cells) are available from several sources. Bone marrow and connective tissue have been used in preliminary clinical trials for regenerative therapy. Recently, several types of AS cells have been isolated from teeth, including dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor stem cells and stem cells from apical papilla. Preliminary data suggest that these cells have the capacity to differentiate into osteoblasts, adipocytes, chondrocytes and neural cells. If confirmed, these data would support the use of these cells, which are easily obtained from extracted teeth, in dental therapies, including in regenerative endodontics, providing a new therapeutic modality.     

影响牙周膜干细胞成骨分化能力的因素

牙周膜干细胞(PDLSC)是维持牙周组织动态平衡和缺损修复的关键细胞,被认为是牙周组织工程的关键种子细胞之一.近年来的研究证明其有较强的的增殖和分化能力,然而在不同因素影响下其功能差异也较大.认识并研究这些影响因素不仅有助于深入了解和发掘PDLSC的生物学功能,而且对于今后基于PDLSC的牙周修复与再生治疗具有指导意义.该文就影响PDLSC成骨分化的多种因素作一叙述,展望其在牙周缺损修复治疗中的应用前景.     

Up-regulation of estrogen receptor-beta expression during osteogenic differentiation of human periodontal ligament cells

Background and Objective: Estrogen has been shown to up‐regulate the expression of osteoblastic phenotypes of human periodontal ligament cells via binding to estrogen receptors and may also help periodontal tissue regeneration. However, which subtype of estrogen receptor (α or β) is predominately expressed in human periodontal ligament cells, and how estrogen receptor expression is regulated during the osteogenic differentiation of human periodontal ligament cells, is still unclear. This study aimed to explore the expression and regulation of estrogen receptor subtypes in human periodontal ligament cells and during their osteogenic differentiation. Material and Methods: Human periodontal ligament cells derived from 10 individual age‐matched donors (five male and five female donors) were cultured. Human periodontal ligament cells under osteogenic induction (group M) and the corresponding controls (group C) were harvested on days 7, 14 and 21 for estrogen receptor detection. Results: Both estrogen receptor‐α and estrogen receptor‐β mRNAs were expressed in human periodontal ligament cells from all of the 10 donors. Protein only of estrogen receptor‐β (not of estrogen receptor‐α) was detected and was shown to be located in the nuclei of human periodontal ligament cells. The expression levels of estrogen receptor‐β mRNA and protein from both male and female donors in group M were significantly higher compared with group C during the 21‐d study period. In comparison, the expression level of estrogen receptor‐α mRNA of the donors was not significantly different from that of the controls during osteogenic differentiation and no estrogen receptor‐α protein was detected. Conclusion: The results suggest that estrogen receptor‐β may be the predominant subtype expressed in human periodontal ligament cells and may actively participate in the osteogenic differentiation process of human periodontal ligament cells, both in male and in female subjects.     

A Synthetic Oligopeptide Derived From Enamel Matrix Derivative Promotes the Differentiation of Human Periodontal Ligament Stem Cells Into Osteoblast‐Like Cells With Increased Mineralization

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs. Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC‐derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO‐1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition. Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO‐1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization. Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast‐like cells and is a useful material for periodontal tissue regeneration.     

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。     

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.     

人牙周膜干细胞与牙周膜非干细胞差异表达microRNA靶基因及基因功能分析

目的 研究人牙周膜干细胞与牙周膜非干细胞之间差异表达的microRNA（miRNA）的靶基因并对其功能进行预测分析。方法 本研究于2011年7月至2013 年10月在福建医科大学附属口腔医院进行。采用基因芯片技术筛选人牙周膜干细胞与牙周膜非干细胞之间差异性表达的miRNA,并通过生物信息学对差异表达的miRNA进行靶基因预测及靶基因功能富集分析。 结果 与人牙周膜非干细胞相比,人牙周膜干细胞中明显上调的关键miRNA 25个,调控的关键靶基因21个;明显下调的关键miRNA 5个,调控的关键靶基因9个。结论 特异性表达的miRNA主要调控的靶基因及其基因功能与人牙周膜干细胞的干性维持密切相关。     

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目的研究人牙周膜干细胞与牙周膜非干细胞之间差异表达的microRNA（miRNA）的靶基因并对其功能进行预测分析。方法本研究于2011年7月至2013年10月在福建医科大学附属口腔医院进行。采用基因芯片技术筛选人牙周膜干细胞与牙周膜非干细胞之间差异性表达的miRNA,并通过生物信息学对差异表达的miRNA进行靶基因预测及靶基因功能富集分析。结果与人牙周膜非干细胞相比,人牙周膜干细胞中明显上调的关键miRNA25个,调控的关键靶基因21个;明显下调的关键miRNA5个,调控的关键靶基因9个。结论特异性表达的miRNA主要调控的靶基因及其基因功能与人牙周膜干细胞的干性维持密切相关。     

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.     

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.