构建一种新型牙周膜牙囊复合细胞膜片的研究

目的：拟构建一种牙周膜牙囊复合细胞膜片,并比较其与单一膜片的性能差异,以期寻找一种更加优越的牙周缺损修复材料。方法：将第三代牙周膜干细胞及牙囊干细胞直接混合共同培养,将牙周膜牙囊混合细胞、牙周膜干细胞及牙囊干细胞分别制备成细胞膜片,应用HE染色,扫描电镜比较三种膜片形态学差异,应用茜素红染色,RT—PCR比较三种膜片成骨能力差异。结果：扫描电镜结果显示,牙囊牙周膜混合细胞膜片分泌更多的细胞外基质。HE染色结果也显示牙囊牙周膜混合细胞膜片细胞层数更多,基质分泌量更大。同时,牙囊牙周膜混合细胞膜片AIJP、Runx2、OCN表达显著高于单一细胞膜片。成骨诱导21天后,茜素红染色结果显示,与单一细胞膜片相比,牙囊牙周膜混合细胞膜片染色更深。结论：牙周膜牙囊复合细胞膜片较单一细胞膜片,细胞层数更多,基质分泌量更大,骨向分化能力更强,为牙周再生提供了新的思路。     

Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein-2 on periodontal regeneration

Song D‐S, Park J‐C, Jung I‐H, Choi S‐H, Cho K‐S, Kim C‐K, Kim C‐S. Enhanced adipogenic differentiation and reduced collagen synthesis induced by human periodontal ligament stem cells might underlie the negative effect of recombinant human bone morphogenetic protein‐2 on periodontal regeneration. J Periodont Res 2011; 46: 193–203. © 2010 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a potent inducer for the regeneration of mineralized tissue, but has a limited effect on the regeneration of cementum and periodontal ligament (PDL). The aim of the present study was to determine the effects of rhBMP‐2 on the in vitro and in vivo biologic activity of well‐characterized human PDL stem cells (hPDLSCs) and to elucidate the underlying mechanism of minimal periodontal regeneration by rhBMP‐2. Material and Methods: hPDLSCs were isolated and cultured, and then transplanted into an ectopic subcutaneous mouse model using a carrier treated either with or without rhBMP‐2. Comprehensive histologic, histometric and immunohistochemical analyses were performed after an 8‐wk healing period. The effects of rhBMP‐2 on the adipogenic and osteogenic/cementogenic differentiation of hPDLSCs were also evaluated. The effect of rhBMP‐2 on both soluble and insoluble collagen synthesis was analyzed, and the expression of mRNA and protein for collagen types I, II, III and V was assessed. Results: In the present study, rhBMP‐2 promoted both adipogenic and osteogenic/cementogenic differentiation of hPDLSCs in vitro, and the in vivo potential of hPDLSCs to form mineralized cementum and organized PDL tissue was down‐regulated following treatment with rhBMP‐2. Collagen synthesis, which plays a crucial role in the regeneration of cementum and the periodontal attachment, was significantly reduced, with associated modification of the relevant mRNA and protein expression profiles. Conclusion: In summary, the findings of the present study suggest that enhanced adipogenic differentiation and inhibition of collagen synthesis by hPDLSCs appear to be partly responsible for the minimal effect of rhBMP‐2 on cementum and PDL tissue regeneration by hPDLSCs.     

�������ʸ�ϸ����Դ��������ٽ����������������о�

??Objective    To study the influence of exosome secreted by the human bone marrow mesenchymal stem cells ??hBMMSCs?? on proliferation and differentiation of periodontal ligament stem cells ??hPDLSCs????and to provide experimental basis and evidence of the mechanism of hBMMSCs in promoting periodontal tissue regeneration. Methods    hPDLSCs were cultured by enzyme digestion method and hBMMSCs were cultured by centrifugation method?? and the immunophenotype was identified by flow cytometry. Supernatant of hBMMSCs in P3 was collected?? exosome was extracted by kit and observed by SEM??and CD63 level was tested by WB. The experiment group was treated by exosome of hBMMSCs and control group was treated by DD water. hPDLSCs proliferation was tested by MTT and colony unite forming was tested in 7 days. After osteogenetic induction?? ALP and Alizarin Red staining were performed and quantitative analysis was carried. Meanwhile osteogenesis-related gene expression was tested by qPCR. Results    hPDLSCs positively expressed CD29?? CD44?? CD90?? CD146 and Stro-1?? hBMMSCs positively expressed CD29?? CD44?? CD90 and CD106??both negatively expressed CD14?? CD34 and CD45. Exosome secreted by hBMMSCs were small ball-shaped under SEM and expressed CD63 strongly by WB. The proliferation and colony unite forming of experimental group was significantly higher than control group ??P < 0.05??. The quantitative expression of ALP and alizarin red staining after osteogenetic induction were significantly stronger than the control group ??P < 0.05??. The osteogenetic gene expression??including Runx2?? OCN?? ALP?? BSP and Col - ?? was significantly higher than that of control group by qPCR ??P < 0.05??. Conclusion    The exosome secreted by hBMMSCs can promote??proliferation and osteogenesis of hPDLSCs in vitro.     

骨髓间充质干细胞来源的外泌体促进牙周再生的体外研究

目的    研究人骨髓间充质干细胞（human bone marrow mesenchymal stem cells, hBMMSCs）分泌的外泌体（exosome）对人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）增殖和分化功能的影响,为进一步明确hBMMSCs促进牙周组织再生的机制提供实验基础和证据。方法    分别用酶消化法和离心法培养hPDLSCs和hBMMSCs,并用流式细胞术检测其间充质表面标志物。收集培养的第3代hBMMSCs上清液,利用试剂盒提取exosome,电镜下观察其结构,Western Bolt法检测其CD63表达水平。用hBMMSCs分泌的exosome处理hPDLSCs设为实验组,四甲基偶氮唑盐（MTT）法检测hPDLSCs的增殖情况,克隆形成率检测多克隆形成能力,成骨诱导后碱性磷酸酶（ALP）染色和茜素红染色并定量,同时实时荧光定量PCR（qPCR）检测成骨相关基因表达。结果    流式细胞术检测,hPDLSCs阳性表达CD29、CD44、CD90、CD146和Stro-1,hBMMSCs阳性表达CD29、CD44、CD90和 CD106,两者均阴性表达CD14、CD34和CD45。hBMMSCs分泌的exosome电镜下呈小球状,Western Bolt法检测其强表达CD63。实验组hPDLSCs经MTT法检测其增殖速率和克隆形成能力显著高于对照组（P < 0.05）。实验组hPDLSCs成骨诱导后ALP染色、茜素红染色定量表达显著强于对照组（P < 0.05）。qPCR检测成骨基因Runx2、OCN、ALP、BSP和Col-Ⅰ的表达,实验组显著高于对照组（P < 0.05）。结论    hBMMSCs分泌的exosome可促进牙周膜干细胞的体外增殖和成骨分化能力。     

Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats

Aim: The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue. Material and Methods: To obtain the cell sheet, human PDL cells were cultured on temperature‐responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing. Results: Most of the experimental group exhibited a new cementum‐like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed. Conclusions: Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.     

The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model

Kim Y‐T, Park J‐C, Choi S‐H, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. The dynamic healing profile of human periodontal ligament stem cells: histological and immunohistochemical analysis using an ectopic transplantation model. J Periodont Res 2012; 47: 514–524. © 2012 John Wiley & Sons A/S Background and Objective: Human periodontal ligament stem cells (hPDLSCs) have been reported to play the pivotal role in periodontal regeneration. However, the dynamic cellular healing process initiated by hPDLSCs still remains to be elucidated. In the present study, the sequence of regeneration by hPDLSCs was assessed using histological and immunohistochemical observation in an ectopic transplantation model, which is a well‐standardized assessment tool that excludes the innate healing factors from the animals. Material and Methods: Human periodontal ligament stem cells that were isolated and characterized from teeth (n = 12) extracted for the purpose of orthodontic treatment were transplanted with carriers into ectopic subcutaneous pouches in immunocompromised mice (n = 20). Animals were killed after several different healing periods: 3 d (n = 4), 1 (n = 4), 2 (n = 4), 4 (n = 4) and 8 wk (n = 4). Histological analysis for regenerated tissues formed by hPDLSCs was conducted using hematoxylin and eosin, Masson’s trichrome and picrosirius red staining. In addition, immunohistochemical staining was performed to observe the sequential expression of osteogenic/cementogenic and periodontal ligament tissue‐specific markers associated with periodontal regeneration. Results: The whole healing process by transplanted hPDLSCs could be broadly divided into four distinctive phases. In the first phase, proliferated hPDLSCs migrated evenly all over the carrier, and collagenous tissues appeared in the form of amorphous collagen matrices. In the second phase, collagen fibers were well arranged among the carriers, and cementoid‐like tissues were observed. In the third phase, the formation of mature collagen fibers, resembling Sharpey’s fibers, was associated with active mineralization of cementum‐like tissues, and in the fourth phase, the maturation of cementum‐like tissues was observed on carrier surfaces. Various osteogenic/cementogenic markers related to the regeneration processes were expressed in a well‐orchestrated time order. Interestingly, well‐organized cementum‐like and periodontal ligament fiber‐like tissues and cells with early and late osteogenic/cementogenic markers were frequently observed in the secluded area of carrier surfaces. We termed this area the cell‐rich zone. Conclusion: The results from this study clearly demonstrated the sequential histological changes during periodontal tissue regeneration by hPDLSCs. Understanding of this process would potentially enable us to develop better cell‐based treatment techniques.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.     

Effect of humoral factors from hPDLSCs on the biologic activity of hABCs

Oral Diseases (2012) 18, 537–547 Objective: The human periodontal ligament stem cells (hPDLSCs) and human alveolar bone–derived stromal cells (hABCs) seem to be closely involved in the maintenance of alveolar bone in an anatomically indirect manner; however, there is little study on this matter. Therefore, the effect of hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was evaluated, focusing on the humoral factors released by hPDLSCs. Materials and methods: Human periodontal ligament stem cells and hABCs were isolated and characterized. hPDLSCs were indirectly cocultured to observe the in vitro effect of humoral factors released from hPDLSCs on the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs. Human gingival fibroblasts (hGFs) were utilized as positive control. Results: Isolated cells demonstrated the presence of stem cells within. Indirect coculture of hPDLSCs greatly inhibited osteoclastogenesis by hABCs. Osteogenesis/adipogenesis of hABCs was also inhibited by indirect coculture with hPDLSC. The magnitude of regulatory effect from hPDLSCs was significantly greater than that of hGFs. Conclusions: Humoral factors released from hPDLSCs seemed to modulate the differentiation of hABCs, and the osteoclastogenic, osteogenic, and adipogenic differentiation of hABCs was all inhibited, suggesting the potential role of hPDLSCs in the maintenance of the alveolar bone.     

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.     

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.     

基质细胞衍生因子-1对炎症和正常来源的 人牙周膜干细胞成骨分化能力的影响

目的 通过体外培养炎症来源的人牙周膜干细胞（iPDLSCs）和正常来源的人牙周膜干细胞（hPDLSCs）,比较基质细胞衍生因子-1（SDF-1）对于两种来源细胞的成骨分化作用。方法 采用组织块酶消化法原代培养iPDLSCs 和hPDLSCs,经有限稀释法纯化,通过流式细胞仪对干细胞表面标记物检测鉴定后,对其进行成骨诱导;MTT法检测并比较SDF-1对两种来源的细胞增殖能力的影响;茜素红染色检测SDF-1作用于两种来源的细胞后钙化骨量的表达;碱性磷酸酶法比较SDF-1作用于两者的成骨分化能力;逆转录聚合酶链反应（RT-PCR）法检测SDF-1作用于两种牙周膜干细胞前后成骨相关基因表达水平的变化。结果 两种来源的牙周膜细胞经纯化后均阳性表达干细胞标记物。hPDLSCs较iPDLSCs增殖能力高;两种细胞经SDF-1成骨诱导培养后,成骨相关基因的表达水平均较诱导前明显上调（P<0.05）,SDF-1在50、200 ng·mL ^-1时分别对iPDLSCs和hPDLSCs细胞成骨分化作用最明显（P<0.05）。结论 正常来源和炎症来源的人牙周膜干细胞均具有成骨分化能力,SDF-1可增强两种来源的牙周膜干细胞的成骨分化能力。     

Efficacy of stem cells on periodontal regeneration: Systematic review of pre‐clinical studies

This systematic review aims to evaluate mesenchymal stem cells (MSC) periodontal regenerative potential in animal models. MEDLINE, EMBASE and LILACS databases were searched for quantitative pre‐clinical controlled animal model studies that evaluated the effect of local administration of MSC on periodontal regeneration. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses statement guidelines. Twenty‐two studies met the inclusion criteria. Periodontal defects were surgically created in all studies. In seven studies, periodontal inflammation was experimentally induced following surgical defect creation. Differences in defect morphology were identified among the studies. Autogenous, alogenous and xenogenous MSC were used to promote periodontal regeneration. These included bone marrow‐derived MSC, periodontal ligament (PDL)‐derived MSC, dental pulp‐derived MSC, gingival margin‐derived MSC, foreskin‐derived induced pluripotent stem cells, adipose tissue‐derived MSC, cementum‐derived MSC, periapical follicular MSC and alveolar periosteal cells. Meta‐analysis was not possible due to heterogeneities in study designs. In most of the studies, local MSC implantation was not associated with adverse effects. The use of bone marrow‐derived MSC for periodontal regeneration yielded conflicting results. In contrast, PDL‐MSC consistently promoted increased PDL and cementum regeneration. Finally, the adjunct use of MSC improved the regenerative outcomes of periodontal defects treated with membranes or bone substitutes. Despite the quality level of the existing evidence, the current data indicate that the use of MSC may provide beneficial effects on periodontal regeneration. The various degrees of success of MSC in periodontal regeneration are likely to be related to the use of heterogeneous cells. Thus, future studies need to identify phenotypic profiles of highly regenerative MSC populations.     

基质细胞衍生因子-1对炎症和正常来源的人牙周膜干细胞成骨分化能力的影响

目的 通过体外培养炎症来源的人牙周膜干细胞（iPDLSCs）和正常来源的人牙周膜干细胞（hPDLSCs）,比较基质细胞衍生因子-1（SDF-1）对于两种来源细胞的成骨分化作用。方法 采用组织块酶消化法原代培养iPDLSCs 和hPDLSCs,经有限稀释法纯化,通过流式细胞仪对干细胞表面标记物检测鉴定后,对其进行成骨诱导;MTT法检测并比较SDF-1对两种来源的细胞增殖能力的影响;茜素红染色检测SDF-1作用于两种来源的细胞后钙化骨量的表达;碱性磷酸酶法比较SDF-1作用于两者的成骨分化能力;逆转录聚合酶链反应（RT-PCR）法检测SDF-1作用于两种牙周膜干细胞前后成骨相关基因表达水平的变化。结果 两种来源的牙周膜细胞经纯化后均阳性表达干细胞标记物。hPDLSCs较iPDLSCs增殖能力高;两种细胞经SDF-1成骨诱导培养后,成骨相关基因的表达水平均较诱导前明显上调（P<0.05）,SDF-1在50、200 ng·mL ^-1时分别对iPDLSCs和hPDLSCs细胞成骨分化作用最明显（P<0.05）。结论 正常来源和炎症来源的人牙周膜干细胞均具有成骨分化能力,SDF-1可增强两种来源的牙周膜干细胞的成骨分化能力。     

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.     

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.     

Optimal Medium Formulation for the Long‐Term Expansion and Maintenance of Human Periodontal Ligament Stem Cells

Background: Human periodontal ligament stem cells (hPDLSCs) are promising mesenchymal stem cells that are readily accessible. However, there is as yet no consensus as to the optimal culture medium for hPDLSCs. Thus, the purpose of the present study is to determine the optimal culture medium for long‐term expansion of hPDLSCs. Methods: hPDLSCs were isolated from healthy third molars, and the most widely used medium formulations in previous studies were used: 1) an α minimum essential medium–based medium formulation (MBM); and 2) a Dulbecco's minimum essential medium‐based medium formulation. Passage 5 (P5) and P8 were evaluated with the two media for cell proliferation, differentiation, and immunophenotype. Results: hPDLSCs that were primarily cultured in MBM were far more proliferated than those grown in DBM. In general, application of the MBM for longer periods produced greater cell growth and osteogenic differentiation. Furthermore, MBM‐precultured hPDLSCs exhibited a greater degree of cell proliferation and a greater production of mineralized tissue and alkaline phosphatase (ALP) activity in vitro, although the levels of both were dependent on the culture medium used. With respect to long‐term expansion, the P5 hPDLSCs grew and produced the largest amount of mineralized nodules faster than the P8 hPDLSCs, but both passages exhibited a similar phenotype for stemness and ALP activity. Conclusion: The present study indicates that the inherent capacity of hPDLSCs could be maintained until a later passage, P8 in MBM, and MBM appears to be an optimal choice for manipulating the finest and most stable hPDLSCs.     

淫羊藿苷促进人牙周膜干细胞增殖及骨向分化的作用

目的 采用体外和体内方法研究淫羊藿苷（ICA）对人牙周膜干细胞（hPDLSCs）增殖及骨向分化的影响。方法 采用体外酶消化组织块法培养hPDLSCs,有限稀释克隆法分离纯化,检测细胞表面标志物以进行鉴定。体外实验：hPDLSCs与1×10-7 mol•L-1 ICA共同培养,用四甲基偶氮唑盐法检测其增殖情况,经碱性磷酸酶（ALP）染色后采用酶联免疫仪检测其骨向分化情况,逆转录聚合酶链反应（RT-PCR）检测其成骨基因的表达情况,茜素红染色检测其骨量表达。体内实验：将hPDLSCs和纳米羟磷灰石（nHAC）复合物经1×10-7 mol•L-1 ICA共同诱导培养后,将其植入免疫缺陷小鼠,术后30 d采用组织切片法观察成骨状况。结果 体外实验：1×10-7 mol•L-1 ICA作用于hPDLSCs后,与不含ICA的对照组相比,实验组从培养的第2天开始,hPDLSCs增殖明显;培养3、5、7 d,实验组ALP活性明显升高;培养7 d,成骨基因的相对表达量明显增高;培养14、21、28 d,实验组钙化结节的面积明显大于对照组。体内实验：实验组骨量较对照组明显增加。结论 1×10-7 mol•L-1 ICA可以促进hPDLSCs的增殖及骨向分化,提示采用ICA代替常规生长因子应用于牙周组织工程具有一定可行性。     

The effects of enamel matrix derivative (EMD) on osteoblastic cells

Enamel matrix derivative (EMDOGAIN, EMD) has been clinically used to promote regeneration of periodontal tissue, including cementum, periodontal ligament (PDL), and alveolar bone. However, it has not been clear whether EMD directly affects osteoblastic cells. To answer this question, we examined EMD effects on bovine PDL cells, rat and mouse bone marrow cells (RBM cells and MBM cells, respectively), and mouse osteoblastic cells (Kusa/A 1 cells). EMD was dissolved in 10 mM acetic acid and added to the culture medium at a final concentration of 50 micrograms/ml. EMD stimulated mineralized-nodule formation of PDL cells, RBM cells, and Kusa/A 1 cells. In Kusa/A 1 cells, EMD enhanced ALP activity, together with DNA content. Northern blotting analysis on Kusa/A 1 cells demonstrated stimulatory effects of EMD on the gene expression of type I collagen and osteopontin. Further, application of EMD on MBM cell culture, under 1,25(OH)2 vitamin D3 supplementation, stimulated osteoclast-like cell formation. These results indicate that osteoblastic cells respond to EMD, and that EMD would be potentially useful for bone regeneration.