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.     

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.     

Dose- and time-dependent effects of recombinant human bone morphogenetic protein-2 on the osteogenic and adipogenic potentials of alveolar bone-derived stromal cells

Park J‐C, Kim J.C, Kim B‐K, Cho K‐S, Im G‐I, Kim B‐S, Kim C‐S. Dose‐ and time‐dependent effects of recombinant human bone morphogenetic protein‐2 on the osteogenic and adipogenic potentials of alveolar bone‐derived stromal cells. J Periodont Res 2012; 47: 645–654. © 2012 John Wiley & Sons A/S Background and Objective: Recombinant human bone morphogenetic protein‐2 (rhBMP‐2) is a well‐known growth factor that can induce robust bone formation, and recent studies have shown that rhBMP‐2‐induced osteogenesis is closely related to adipogenesis. The aim of the present study was to determine the dose‐ and time‐dependent effects of rhBMP‐2 on the osteogenic and adipogenic differentiation of human alveolar bone‐derived stromal cells (hABCs) in vivo and in vitro. Material and Methods: hABCs were isolated and cultured, and then transplanted using a carrier treated either with or without rhBMP‐2 (100 μg/mL) into an ectopic subcutaneous mouse model. Comprehensive histologic and histometric analyses were performed after an 8‐wk healing period. To further understand the dose‐dependent (0, 10, 50, 200, 500 and 1000 ng/mL) and time‐dependent (0, 3, 5, 7 and 14 d) effects of rhBMP‐2 on osteogenic and adipogenic differentiation, in vitro osteogenic and adipogenic differentiation of hABCs were evaluated, and the expression of related mRNAs, including those for alkaline phosphatase, osteocalcin, bone sialoprotein, peroxisome‐proliferator‐activated receptor gamma‐2 and lipoprotein lipase, were assessed using quantitative RT‐PCR. Results: rhBMP‐2 significantly promoted the osteogenic and adipogenic differentiation of hABCs in vivo, and gradually increased both the osteogenic and adipogenic potential in a dose‐ and time‐dependent manner with minimal deviation in vitro. The expression of osteogenesis‐ and adipogenesis‐associated mRNAs were concomitantly up‐regulated by rhBMP‐2. Conclusion: The findings of the present study showed that rhBMP‐2 significantly enhanced the adipogenic as well as the osteogenic potential of hABCs in dose‐ and time‐dependent manner. The control of adipogenic differentiation of hABCs should be considered when regenerating the alveolar bone using rhBMP‐2.     

釉基质衍生物对人牙周膜干细胞成骨分化的影响

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。     

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.     

人脐带Wharton's Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton's Jelly来源间充质干细胞(human umbilical cord Wharton's Jelly-derived mesechymal stem ceils,hUCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,hPDLSCs)成骨分化能力.方法:体外培养hUC-WJMSCs和hPDLSCs.MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-timePCR分析OPN和Runx2基因的表达.结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCs ALP表达、矿化结节形成高于hUCWJMSCs(P＜0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P ＜0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P＜0.05).结论:hUCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强.     

The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells

Lee Y‐M, Shin S‐I, Shin K‐S, Lee Y‐R, Park B‐H, Kim E‐C. The role of sirtuin 1 in osteoblastic differentiation in human periodontal ligament cells. J Periodont Res 2011; 46: 712–721. © 2011 John Wiley & Sons A/S Background and Objective: Activation of sirtuin 1 (SIRT1) promotes the differentiation of keratinocytes and mesenchymal stem cells, but inhibits the differentiation of muscle and fat cells. However, the involvement of SIRT1 in the differentiation of human periodontal ligament cells into osteoblast‐like cells remains unclear. To identify the role of SIRT1 in human periodontal ligament cells, we measured SIRT1 mRNA and SIRT1 protein levels during the osteoblastic differentiation of human periodontal ligament cells. Additionally, we investigated the effects of overexpressing and underexpressing SIRT1 on the differentiation of human periodontal ligament cells, and the signaling mechanisms involved. Material and Methods: Expression of SIRT1 and osteoblastic differentiation markers was assessed by RT‐PCR, real‐time PCR, Alizarin red staining and western blotting. Results: Marked upregulation of SIRT1 mRNA and SIRT1 protein was observed in cells grown for 3 d in osteogenic induction medium (OM). Activation of SIRT1 using resveratrol and isonicotinamide stimulated osteoblastic differentiation in a dose‐dependent manner, as assessed by the expression of mRNAs encoding alkaline phosphatase, osteopontin, osteocalcin, osterix and Runx2, and induced calcium deposition. In contrast, inhibition of SIRT1 using sirtinol, nicotinamide and gene silencing by RNA interference suppressed mineralization and the expression of osteoblast marker mRNAs. Further mechanistic studies revealed that resveratrol treatment increased the phosphorylation of Akt, adenosine monophosphate kinase (AMPK), Smad 1/5/8 and c‐Jun N‐terminal kinase, but reduced OM‐induced activation of nuclear factor‐κB. Conversely, application of sirtinol suppressed the phosphorylation of Akt, AMPK, Smad 1/5/8, p38, ERK and c‐Jun N‐terminal kinase, and enhanced nuclear factor‐κB activity, in OM‐stimulated cells. Conclusion: These data suggest that SIRT1 is a potent regulator of differentiation of human periodontal ligament cells and may have clinical implications for periodontal bone regeneration.     

In vivo differentiation of human periodontal ligament cells leads to formation of dental hard tissue

Objective

Following trauma, periodontal disease, or orthodontic tooth movement, residual periodontal ligament (PDL) cells at the defect site are considered mandatory for successful regeneration of the injured structures. Recent developments in tissue engineering focus, as one pillar, on the transplantation of PDL cells to support periodontal regeneration processes. Here, we examined the ability of osteogenically predifferentiated PDL cells to undergo further osteoblastic or cementoblastic differentiation and to mineralize their extracellular matrix when transplanted in an in vivo microenvironment.

Materials and methods

Using collagen sponges as carriers, osteogenically predifferentiated human PDL cells were transplanted subcutaneously into six immunocompromised CD-1® nude mice. Following explantation after 28 days, osteogenic and cementogenic marker protein expression was visualized immunohistochemically.

Results

After 28 days, transplanted PDL cells revealed both cellular, cytoplasmatic and extracellular immunoreactivity for the chosen markers alkaline phosphatase, osteopontin, PTH-receptor 1, and osteocalcin. Specific osteogenic and cementoblastic differentiation was demonstrated by RUNX2 and CEMP1 immunoreactivity. Early stages of mineralization were demonstrated by calcium and phosphate staining.

Conclusion

Our results reinforce the previously published reports of PDL cell mineralization in vivo and further demonstrate the successful induction of specific osteogenic and cementogenic differentiation of transplanted human PDL cells in vivo. These findings reveal promising possibilities for supporting periodontal remodeling and regeneration processes with PDL cells being potential target cells with which to influence the process of orthodontically induced root resorption.     

乏氧微环境下机械应力对人牙周膜干细胞成骨分化的影响

人牙周膜干细胞是牙周组织中具有自我更新和多向分化潜能的一类成体干细胞,在特定诱导条件下可以成骨分化。牙周膜处于一个相对乏氧的环境,人牙周膜干细胞在牙周膜中承载着多种机械应力。探讨乏氧环境下机械应力对人牙周膜干细胞成骨分化的影响更接近于体内环境,更有助于获得真实的牙周组织改建的实验室资料。该文对乏氧微环境下机械应力对人牙周膜干细胞成骨分化的影响做一系统性回顾。     

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.     

低强度高频振动对人牙周膜干细胞成骨分化能力的影响

目的 研究低强度高频振动（low-magnitude high frequency vibration,LMHFV）对人牙周膜干细胞（human periodontal ligament stem cells,hPDLSCs）增殖、迁移、成骨分化能力的影响。方法 体外分离培养hPDLSCs;流式细胞术检测间充质干细胞表面标志物;加载LMHFV（加速度=0.3 g,频率=40 Hz,时间=15 min/24 h）刺激后,采用CCK-8试剂盒检测细胞增殖能力,通过细胞划痕实验检测细胞迁移能力;通过qRT-PCR、Western免疫印迹检测成骨相关基因、蛋白表达水平,通过茜素红染色检测细胞成骨分化能力。采用SPSS 21.0软件包对数据进行统计学分析。结果 加载振动刺激后,hPDLSCs的增殖能力增强,迁移能力上升;RUNX2、ALP、Col-1、OCN的mRNA表达量和蛋白表达量均上升,茜素红染色结果与qRT-PCR、Western免疫印迹结果一致。结论 LMHFV可提高hPDLSCs的增殖、迁移能力和成骨分化能力。     

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.     

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。     

Isolation and characterization of multipotent human periodontal ligament stem cells

BACKGROUND: Periodontal ligament (PDL) repair is thought to involve mesenchymal progenitor cells capable of forming fibroblasts, osteoblasts and cementoblasts. However, full characterization of PDL stem cell (SC) populations has not been achieved. OBJECTIVE: To isolate and characterize PDLSC and assess their capability to differentiate into bone, cartilage and adipose tissue. METHODS: Human PDL cells were stained for STRO-1, FACS sorted and expanded in culture. Human bone marrow SC (BMSC) served as a positive control. PDLSC and BMSC were cultured using standard conditions conducive for osteogenic, chondrogenic and adipogenic differentiation. Osteogenic induction was assayed using alizarine red S staining and expression of alkaline phosphatase (ALP) and bone sialoprotein (BSP). Adipogenic induction was assayed using Oil Red O staining and the expression of PPAR gamma 2 (early) and LPL (late) adipogenic markers. Chondrogenic induction was assayed by collagen type II expression and toluidine blue staining. RESULTS: Human PDL tissue contains about 27% STRO-1 positive cells with 3% strongly positive. In osteogenic cultures ALP was observed by day-7 in BMSC and day-14 in PDLSC. BSP expression was detectable by day-7; with more intense staining in PDLSC cultures. In adipogenic cultures both cell populations showed positive Oil Red O staining by day-25 with PPAR gamma 2 and LPL expression. By day-21, both BMSC and PDLSC chondrogenic induced cultures expressed collagen type II and glycosaminoglycans. CONCLUSIONS: The PDL contains SC that have the potential to differentiate into osteoblasts, chondrocytes and adipocytes, comparable with previously characterized BMSC. This adult PDLSC population can be utilized for potential therapeutic procedures related to PDL regeneration.