牙龈间充质干细胞与牙周膜干细胞膜片牙周再生能力的实验研究
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摘要
背景和目的
牙周病治疗的最终目标是能够消除牙周组织局部的炎症,再生和重建因炎症破坏的牙周组织,即再生和重建具有功能学意义的牙周膜、牙骨质和骨组织。但是由于牙周疾病病因学和口腔微环境的复杂性和特殊性,目前尚无疗效满意的治疗方法。近年来,基于干细胞技术的组织工程学治疗方法为牙周组织再生研究提供了新的思路与方法。种子细胞、支架材料、生长因子是传统组织工程学研究领域主要的研究内容,其中,寻找来源丰富、取材方便、具有增殖分化潜能的种子细胞以及合适的细胞移植载体是目前组织再生医学研究中的热点领域。
目前,多种成体干细胞已被应用于牙周再生医学,其中,骨髓基质干细胞(Bone marrow stem cells, BMSCs)和牙周膜干细胞(periodontal ligament stem cells, PDLSCs)被认为是较理想的种子细胞。但BMSCs从骨髓中获得,取材困难,取材过程痛苦,细胞的特性与供者的年龄密切相关,且它的自我更新和分化潜能有限,这些缺点限制了BMSCs的临床应用。PDLSCs是近些年从牙周膜中分离出来的成体干细胞,可以分化为成牙骨质细胞、成骨细胞和牙周韧带细胞,具有很强的分化增殖能力,目前被认为是牙周再生治疗中较理想的种子细胞。但是PDLSCs需要拔除牙齿才能获得,细胞来源少,分离纯化困难。以上这些特点限制了这两种细胞在未来的牙周再生治疗中的广泛应用。
牙龈干细胞(GMSCs)是近几年国外学者从牙龈中分离出来的前体细胞,具有间充质干细胞的特性,具有集落生成能力、自我更新能力、多向分化潜能和体内成骨能力,成为干细胞治疗的又一备选细胞。GMSCs还具有取材容易,来源丰富,抗炎和免疫调节等优点。基于以上特点,将GMSCs应用于牙周组织再生,具有可行性和良好的临床推广应用价值。但目前对于GMSCs促进牙周再生的确切作用及其可能存在的机制尚需要大量深入的研究。
在组织工程研究中,种子细胞的植入载体和移植途径对于组织再生具有至关重要的作用。细胞膜片技术(Cell sheet, CS)是在组织再生领域中具有广阔应用前景的一项移植策略。由于其保存了细胞间自分泌信号分子和细胞外基质,减少了细胞移植后无效移植细胞的数量,有利于组织的重建或再生,细胞膜片技术在牙周组织工程中的应用也逐渐展开并被证实具有显著促进牙周组织再生的作用。
本研究通过体外制备GMSCs和PDLSCs膜片,利用比格犬Ⅲ度根分叉病变模型,探讨并比较GMSCs和PDLSCs修复炎症破坏的牙周组织缺损的能力,为GMSCs在牙周组织工程中的临床应用提供证据。
方法
1.牙龈间充质干细胞和牙周膜干细胞的分离培养和鉴定
1.1GMSCs的分离培养
利用酶消化和组织块混合培养法从临床上需行阻生牙拔除、正畸助萌、牙冠延长术的牙周健康患者的牙龈中获得原代细胞,消化传代后,取P2-P4的细胞供后续实验使用。利用有限稀释法,挑取单克隆获得具有干细胞特性的细胞GMSCs进行传代培养。
1.2PDLSCs的分离培养
利用酶消化和组织块混合培养法从临床阻生拔除的第三磨牙和因正畸需要拔除的健康前磨牙(牙根已发育完成)的牙周膜中获得原代细胞,消化传代后,取P2-P4的细胞供后续实验使用。利用有限稀释法,挑取单克隆获得具有干细胞特性的细胞PDLSCs进行传代培养。
1.3干细胞特性及多向分化能力鉴定
分别对分离纯化的GMSCs和PDLSCs进行克隆形成率(CFU-F)分析,流式细胞术检测细胞表面分子表达。在体外以0.1μ M地塞米松,10mM β-甘油磷酸钠,50mg/ml维生素C-二磷酸盐分别诱导GMSCs和PDLSCs向成骨细胞分化,茜素红染色法检测钙化结节的形成；以0.1μ M地塞米松,60μ M吲哚美辛,50mg/ml维生素C-二磷酸盐分别诱导以上两种细胞向脂肪细胞分化并用油红0(Oil Red0)染色检测脂肪滴的形成。
2. GMSCs和PDLSCs细胞膜片的制备及生物学活性检测
将生长增殖状态良好的P4代GMSCs与PDLSCs以1×104细胞/cm2的密度将细胞分别均匀接种培养皿和6孔板中,分为三组诱导培养。1)膜片诱导培养液组：α-MEM+15%FBS,0.1μM地塞米松,10mM β-甘油磷酸钠,100mg/ml维生素C；2)矿化诱导培养液组：α-MEM+15%FBS,0.1μM地塞米松,10mM β-甘油磷酸钠,50mg/ml维生素C；3)普通培养液组：α-MEM+15%FBS;孵育10-12天后观察三组培养条件下细胞成膜能力。获得的GMSCs和PDLSCs细胞膜片行H&E染色进行组织学观察和ALP活性测定。
3.牙龈间充质干细胞和牙周膜干细胞膜片对牙周再生作用的体内研究
本实验利用包被有eGFP的慢病毒分别转染GMSCs和PDLSCs,经药物筛选后获得稳定表达eGFP的GMSCs和PDLSCs。将这些细胞制备成细胞膜片移植入比格犬牙周炎Ⅲ度根分叉病变模型,8周后处死动物,利用H&E染色、免疫组织化学染色、天狼星红染色及直接荧光显微镜观察,评价GMSCs和PDLSCs对牙周缺损的修复再生能力。
结果
1.牙龈间充质干细胞和牙周膜干细胞的分离培养和鉴定
原代培养3天后,倒置显微镜观察即可见分散的细胞贴壁伸展,当细胞密度达到瓶底的70%-80%时,即可进行传代培养。GMSCs和PDLCs在体外均呈集落样增殖,所形成的克隆形态和大小未见明显差异。克隆形成率分析(CFU-F)结果显示,GMSCs的克隆形成能力略高于PDLSCs。用有限稀释法获得成纤维细胞集落克隆形成单位,将单个克隆扩大培养,获得具有自我更新能力的人GMSCs和PDLSCs。经流式细胞仪检测细胞表面标记发现,该两种细胞均表达CD29、CD105、CD90、 CD73、STRO-1,而不表达CD14、CD45、CD144、CD31以及HLA-DR。GMSCs和PDLSCs经成骨诱导培养基诱导4周后,经茜素红染色后均能观察到大小不一的红染的矿化结节,GMSCs形成的单个矿化结节的面积较PDLSCs的小而分散；GMSCs和PDLSCs向脂肪细胞方向诱导2周后,经油红0染色,可见有红色脂肪小滴形成,两种细胞形成脂滴的形态无明显差别。
2.牙龈间充质干细胞和牙周膜干细胞膜片的制备及生物学活性检测
GMSCs和PDLSCs分别在三种培养液中诱导培养,从诱导第3天开始倒置显微镜下即可观察到大量沉积的细胞外基质,GMSCs的基质分泌量及分布较PDLSCs更为迅速、均匀。诱导培养10-12天后,收集膜片,添加有100mg/ml维生素C的膜片诱导培养液组可形成完整的细胞膜片,能够完整地自培养皿底剥离,可折叠成需要的形状。添加有50mg/ml维生素C的矿化诱导培养液组形成的细胞膜片易破碎,不能完整的自培养皿底剥离。未添加维生素C的普通培养液组的细胞无法形成膜片。膜片诱导培养液组的细胞膜片H&E染色显示膜片由2-3层细胞组成,细胞间富含大量的细胞外基质。ALP活性检测显示两组细胞经诱导培养后均明显增强,50mg/ml维生素C培养组(即矿化诱导培养液组)略低于100mg/ml维生素C培养组(膜片诱导培养液组),两种细胞在相同培养条件下PDLSCs的ALP表达水平均略高于GMSCs,在矿化诱导液组差异有统计学意义,但在膜片诱导培养组两种细胞的ALP活性差异无统计学意义。
3.牙龈间充质干细胞和牙周膜干细胞膜片对牙周再生作用的体内研究
含有eGFP的慢病毒转染P3-P4代GMSCs和PDLSCs,转染后48-72h多数细胞均可发出明亮的绿色荧光,感染效率达90%左右。然后对细胞进行药物筛选纯化,获得稳定表达的eGFP的GMSCS和PDLSCs。扩大培养后,于膜片诱导液中诱导培养10-12天收集细胞膜片,折叠后移植入比格犬Ⅲ度根分叉病变模型。8周后处死动物获取下颌骨标本,切片进行H&E染色、免疫组织化学染色、天狼星红染色及直接荧光显微镜观察。组织形态学计量分析结果显示：GMSCs和PDLSCs膜片移植组的新生牙骨质百分比(PRC)和新生骨面积百分比(PRB)显著高于空白对照组,但两种细胞膜片组间差异无统计学意义；免疫组织化学结果显示GMSCs和PDLSCs移植组新生骨中GFP的表达均呈强阳性,而对照组中GFP染色则为阴性。荧光显微镜下观察,大部分新生骨在显微镜下呈明亮的绿色,在新生牙骨质、牙槽骨中的成牙骨质细胞和成骨细胞中均有绿色荧光的表达。偏振光显微镜观察天狼星红染色标本,在GMSCs和PDLSCs膜片植入组再生的胶原纤维多数呈垂直或斜行插入新生牙骨质,为功能性牙周膜,而空白对照组牙周膜纤维束多平行于牙根面,提示该纤维无正常功能。
结论
1. GMSCs同PDLSCs一样具有自我更新的能力,能够表达干细胞特异性的表面标记,并且具有多向分化的能力。
2.添加100mg/ml维生素C的成骨诱导液既能诱导细胞向成骨细胞方向分化,而且能够诱导细胞大量分泌细胞外基质形成细胞膜片,是一种较简单、有效的膜片制备手段。
3. GMSCs和PDLSCs膜片表达类似的ALP活性,用于犬Ⅲ度根分叉病变模型有类似的促牙周组织再生效果。
4.移植的GMSCs和PDLSCs促进牙周再生的机制包括移植细胞直接分化为牙周再生功能细胞和对宿主细胞的间接调控两种作用。
Background and Objective
The ultimate goal of periodontal therapy is to achieve complete periodontal regeneration, which means the reconstitution of the soft (gingival connective tissue and periodontal ligament) and the hard tissue (cementum and alveolar bone) of the periodontium. Current therapeutics for periodontitis fails to completely and reliably reconstitute all tissues damaged through periodontal inflammation. The cell-based therapeutics has been a promising alternative for clinical trial in periodontal regeneration. Seed cells, scaffold materials, growth factors are the main contents in the traditional tissue engineering research. Looking for an alternative stem cells with abundance and ready access, and an appropriate carrier for cell transplantation is the hotspot in tissue regeneration medicine.
To date, multiple mesenchymal stem cell lineages have been explored in tissue engineering. Bone marrow stem cells (BMSCs) have been extensively studied in contributing to periodontal regeneration, on account of their capacity of self-renewal and multiple differentiation potential. PDLSCs are identified as a promising alternative in periodontal regeneration. However, all of these above stem cells are not easily accessible or cultured, which hampers the utilization for clinical application.
In recent years, gingiva-derived MSCs (GMSCs) have been isolated from gingival tissue and confirmed capable of forming clonogenic colonies, expressing a typical MSC surface marker profile and possessing multiple differentiatial capacity in vitro. Moreover, GMSCs also have been demonstrated to possess the ability to form new bone in vivo, and properties of anti-inflammation and immunomodulation. On account of having properties of stable morphology, uniformly homogenous and faster proliferation, GMSCs is considered being superior to BMSCs and other dental stem cells for cell therapy in regenerative medicine. Moreover, the abundance and ready access of GMSCs is the most fascinating for tissue regeneration. Thus, GMSCs might be potentially a better alternative of cell-based approaches for periodontal regeneration. However, few attempts have been done to explore ability of GMSCs to regenerate periodontal tissue and especially its possible mechanism in tissue regeneration.
Cell sheet engineering is a promising strategy in tissue regeneration. Cell sheet technology may be beneficial for cell transplantation for preserving cellular junctions, endogenous extracellular matrix (ECM). Recent studies have shown that this technique is effective to form new cementum and promote periodontal attachment utilizing periodontal ligament cells and BMSCs.
To estimate the potential of GMSCs and PDLSCs to reconstitute the periodontal tissue and explore their fate and differentiation in vivo, we transplanted GFP labeled GMSCs sheet into inflammed Class III furcation defects in beagle dogs to regenerate periodontium complex.
Methods:
1. Isolation, cultivation and identification of human GMSCs and PDLSCs
1.1Isolation, cultivation of human GMSCs
Human gingival samples were harvested from healthy individuals as discarded tissues following conventional dental procedures such as the extraction of impacted third molars or crown lengthening. GMSCs were isolated, expanded, and cultured. The2rd-4th passage GMSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.
1.2Isolation, cultivation of human PDLSCs
Human periodontal ligament samples were harvested from healthy individuals as discarded tissues following conventional dental procedures. PDLSCs were isolated, expanded, and cultured. The2rd-4th passage PDLSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.
1.3Identification of human GMSCs and PDLSCs
Colony-forming unit-fibroblasts assay and Flow cytometric analysis were performed. GMSCs and PDLSCs plated in24-well plates were cultured respectively in osteogenic medium (a-MEM containing5%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate). Cells cultured in a-MEM containing5%FBS were as control. The mineralized nodules were characterized by2%Alizarin red S four weeks later. Cells were incubated adipogenic medium (a-MEM containing10%FBS,0.1μM dexamethasone,60μM indomethacin, and50mg/ml ascorbate-2-phosphate). Oil Red O staining was performed to identify the oil globules2weeks later.
2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assay
To create the cell sheet, GMSCs and PDLSCs were subcultured in the following medium:1) the cell sheet induction medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and100mg/ml ascorbate-2-phosphate);2) the osteogenic medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate);3) the normal medium (a-MEM with15%FBS). After reaching confluence (approximately at10-12d), the cells were rinsed twice with PBS, and then detached as a cell sheet. To examine the microscopic structure of the cell sheets sections were evaluated via hematoxylin and eosin (H&E) staining. Alkaline phosphatase (ALP) activity were assayed.
3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogs
The lentiviruses encoding eGFP were used to label the3rd passage of GMSCs and PDLSC. GFP expression in transduced cells was evaluated by direct observation of cells using a fluorescence microscope. Hygromycin was used to select the stably transduced GMSCs and PDLSC. GMSC and PDLSC sheets were transplanted into the Class Ⅲ furcation defects in beagle dogs. The animals were sacrificed after8weeks. Tissue sections selected from the most central areas of the defects were used for the H&E staining, Picrosirius red and immunohistochemical staining.
Results
1. Isolation, identification of human GMSCs and PDLSCs
Primary cultures of the single-cell suspensions obtained from the healthy gingiva formed adherent, clonogenic cell clusters, and the cultured GMSCs and PDLSCs retained fibroblastic phenotype. Flow cytometry analysis revealed that both cells were uniformly positive for CD44, CD29, CD73, CD90, CD105markers and Stro-1, and did not express hematopoietic stem markers CD14, and CD45, and endothelial cells markers CD144, CD31and HLA-DR. To investigate the multiple differentiation potential of GMSCs and PDLSCs, the cells were cultured in the osteogenic medium for4weeks. Extensive amounts of mineralized nodules were found in the experimental groups, but the mineralized nodules formed by GMSCs were smaller and scattered than that in PDLSCs group. Moreover, after cultured in the adipogenic medium for2weeks, GMSCs and PDLSCs were found to differentiate towards adipocytes as indicated by the accumulation of lipid droplets. The form of lipid droplets in two groups has no obvious difference. These findings indicates that the single colony-derived human gingival stem cells had the basic characteristics of mesenchymal stem cells.
2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assay
Both GMSCs and PDLSCs, cultured in the cell sheet induction medium, formed a multi-layer structure with abundant ECM. After10-12d of culture, a semi-transparent cell sheet was formed which was tough enough to be completely detached from the culture dish by a scraper. In contrast, cells grown in the osteogenic medium formed a relatively fragile membrane, whereas cells grown in the normal medium without ascorbic acid could not form the multi-layer structure.
In addition, both the cell sheet and osteogenic groups of each cells exhibited markedly increased ALP activity compared with the control group. No statistically significant difference in the ALP activity was detected between the cell sheet group and the osteogenic group. ALP activity under the same conditions of PDLSCs are slightly higher than the GMSCs. But the statistically significant difference between two cells is only in the osteogenic induction group.
3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogs
The lentiviruses encoding eGFP were transduced into the3rd passage of GMSCs and PDLSC. Bright green fluorescence was observed at48-72h using a fluorescence microscope, and infection efficiency was up to90%. The cells were purified by hygromycin and eGFP+GMSCS and eGFP+PDLSCs were harvested. Then they were expanded and cultured10to12days to fabricate the cell sheets, and then transplanted into Class Ⅲ furcation defects created in beagle dogs. The dogs were sacrificed8weeks after transplantation. The specimen slices were observed by H&E staining, immunohistochemical staining, Sirius red staining and fluorescence microscopy observation directly. Histomorphometric analysis was performed to evaluate the amount of periodontal tissue regeneration. Alveolar bone regeneration was observed in both cell sheets groups. Our results showed enhanced new bone and new cementum formation in both GMSCs and PDLSCs group when compared with that in the control group. Immunohistochemical results show the expression of GFP was strong positive in the new bone in both GMSCs and PDLSCs groups, while GFP were negative in control group. Most of the new bone are bright green under a fluorescence microscope. GFP protein was detected in the cementoblast-like cells, the periodontal ligament fibroblasts and osteoblasts. These results indicate that the transplanted GMSCs are capable of differentiating into the osteoblasts, cementoblasts and periodontal ligament fibroblasts, and therefore directly participate in the reconstitution of the periodontal tissues. To examine the periodontal ligament regeneration, Picrosirius red stained sections were observed by both light and polarizing light microscope. In both GMSCs and PDLSC groups, newly formed Sharpey's fibers anchored into the newly regenerated cementum were arranged perpendicularly to the root surface. In contrast, the newly formed fibers were parallel to the root surface in the control group.
Conclusion
1. Both GMSCs and PDLSCs share the similar characteristics with other stem cells, including self-renewability, expression of specific surface marker of stem cells and multi-lineage differentiation potential.
2. The osteogenic medium supplemented with100mg/ml ascorbic acid can not only induce osteogenic differentiation but also effectively fabricate cell sheets with highly biological activity.
3. Both GMSCs and PDLSC sheets express similar ALP activity and have the similar potential to regenerate bone, cementum/periodontal ligament complex in Class III furcation defects created in beagle dogs.
4. The mechanism of transplanted GMSCs and PDLSCs to promote periodontal regeneration might be directly differentiating into periodontal functional cells and indirectly induction effects on host cells.
牙周病治疗的最终目标是能够消除牙周组织局部的炎症,再生和重建因炎症破坏的牙周组织,即再生和重建具有功能学意义的牙周膜、牙骨质和骨组织。但是由于牙周疾病病因学和口腔微环境的复杂性和特殊性,目前尚无疗效满意的治疗方法。近年来,基于干细胞技术的组织工程学治疗方法为牙周组织再生研究提供了新的思路与方法。种子细胞、支架材料、生长因子是传统组织工程学研究领域主要的研究内容,其中,寻找来源丰富、取材方便、具有增殖分化潜能的种子细胞以及合适的细胞移植载体是目前组织再生医学研究中的热点领域。
目前,多种成体干细胞已被应用于牙周再生医学,其中,骨髓基质干细胞(Bone marrow stem cells, BMSCs)和牙周膜干细胞(periodontal ligament stem cells, PDLSCs)被认为是较理想的种子细胞。但BMSCs从骨髓中获得,取材困难,取材过程痛苦,细胞的特性与供者的年龄密切相关,且它的自我更新和分化潜能有限,这些缺点限制了BMSCs的临床应用。PDLSCs是近些年从牙周膜中分离出来的成体干细胞,可以分化为成牙骨质细胞、成骨细胞和牙周韧带细胞,具有很强的分化增殖能力,目前被认为是牙周再生治疗中较理想的种子细胞。但是PDLSCs需要拔除牙齿才能获得,细胞来源少,分离纯化困难。以上这些特点限制了这两种细胞在未来的牙周再生治疗中的广泛应用。
牙龈干细胞(GMSCs)是近几年国外学者从牙龈中分离出来的前体细胞,具有间充质干细胞的特性,具有集落生成能力、自我更新能力、多向分化潜能和体内成骨能力,成为干细胞治疗的又一备选细胞。GMSCs还具有取材容易,来源丰富,抗炎和免疫调节等优点。基于以上特点,将GMSCs应用于牙周组织再生,具有可行性和良好的临床推广应用价值。但目前对于GMSCs促进牙周再生的确切作用及其可能存在的机制尚需要大量深入的研究。
在组织工程研究中,种子细胞的植入载体和移植途径对于组织再生具有至关重要的作用。细胞膜片技术(Cell sheet, CS)是在组织再生领域中具有广阔应用前景的一项移植策略。由于其保存了细胞间自分泌信号分子和细胞外基质,减少了细胞移植后无效移植细胞的数量,有利于组织的重建或再生,细胞膜片技术在牙周组织工程中的应用也逐渐展开并被证实具有显著促进牙周组织再生的作用。
本研究通过体外制备GMSCs和PDLSCs膜片,利用比格犬Ⅲ度根分叉病变模型,探讨并比较GMSCs和PDLSCs修复炎症破坏的牙周组织缺损的能力,为GMSCs在牙周组织工程中的临床应用提供证据。
方法
1.牙龈间充质干细胞和牙周膜干细胞的分离培养和鉴定
1.1GMSCs的分离培养
利用酶消化和组织块混合培养法从临床上需行阻生牙拔除、正畸助萌、牙冠延长术的牙周健康患者的牙龈中获得原代细胞,消化传代后,取P2-P4的细胞供后续实验使用。利用有限稀释法,挑取单克隆获得具有干细胞特性的细胞GMSCs进行传代培养。
1.2PDLSCs的分离培养
利用酶消化和组织块混合培养法从临床阻生拔除的第三磨牙和因正畸需要拔除的健康前磨牙(牙根已发育完成)的牙周膜中获得原代细胞,消化传代后,取P2-P4的细胞供后续实验使用。利用有限稀释法,挑取单克隆获得具有干细胞特性的细胞PDLSCs进行传代培养。
1.3干细胞特性及多向分化能力鉴定
分别对分离纯化的GMSCs和PDLSCs进行克隆形成率(CFU-F)分析,流式细胞术检测细胞表面分子表达。在体外以0.1μ M地塞米松,10mM β-甘油磷酸钠,50mg/ml维生素C-二磷酸盐分别诱导GMSCs和PDLSCs向成骨细胞分化,茜素红染色法检测钙化结节的形成；以0.1μ M地塞米松,60μ M吲哚美辛,50mg/ml维生素C-二磷酸盐分别诱导以上两种细胞向脂肪细胞分化并用油红0(Oil Red0)染色检测脂肪滴的形成。
2. GMSCs和PDLSCs细胞膜片的制备及生物学活性检测
将生长增殖状态良好的P4代GMSCs与PDLSCs以1×104细胞/cm2的密度将细胞分别均匀接种培养皿和6孔板中,分为三组诱导培养。1)膜片诱导培养液组：α-MEM+15%FBS,0.1μM地塞米松,10mM β-甘油磷酸钠,100mg/ml维生素C；2)矿化诱导培养液组：α-MEM+15%FBS,0.1μM地塞米松,10mM β-甘油磷酸钠,50mg/ml维生素C；3)普通培养液组：α-MEM+15%FBS;孵育10-12天后观察三组培养条件下细胞成膜能力。获得的GMSCs和PDLSCs细胞膜片行H&E染色进行组织学观察和ALP活性测定。
3.牙龈间充质干细胞和牙周膜干细胞膜片对牙周再生作用的体内研究
本实验利用包被有eGFP的慢病毒分别转染GMSCs和PDLSCs,经药物筛选后获得稳定表达eGFP的GMSCs和PDLSCs。将这些细胞制备成细胞膜片移植入比格犬牙周炎Ⅲ度根分叉病变模型,8周后处死动物,利用H&E染色、免疫组织化学染色、天狼星红染色及直接荧光显微镜观察,评价GMSCs和PDLSCs对牙周缺损的修复再生能力。
结果
1.牙龈间充质干细胞和牙周膜干细胞的分离培养和鉴定
原代培养3天后,倒置显微镜观察即可见分散的细胞贴壁伸展,当细胞密度达到瓶底的70%-80%时,即可进行传代培养。GMSCs和PDLCs在体外均呈集落样增殖,所形成的克隆形态和大小未见明显差异。克隆形成率分析(CFU-F)结果显示,GMSCs的克隆形成能力略高于PDLSCs。用有限稀释法获得成纤维细胞集落克隆形成单位,将单个克隆扩大培养,获得具有自我更新能力的人GMSCs和PDLSCs。经流式细胞仪检测细胞表面标记发现,该两种细胞均表达CD29、CD105、CD90、 CD73、STRO-1,而不表达CD14、CD45、CD144、CD31以及HLA-DR。GMSCs和PDLSCs经成骨诱导培养基诱导4周后,经茜素红染色后均能观察到大小不一的红染的矿化结节,GMSCs形成的单个矿化结节的面积较PDLSCs的小而分散；GMSCs和PDLSCs向脂肪细胞方向诱导2周后,经油红0染色,可见有红色脂肪小滴形成,两种细胞形成脂滴的形态无明显差别。
2.牙龈间充质干细胞和牙周膜干细胞膜片的制备及生物学活性检测
GMSCs和PDLSCs分别在三种培养液中诱导培养,从诱导第3天开始倒置显微镜下即可观察到大量沉积的细胞外基质,GMSCs的基质分泌量及分布较PDLSCs更为迅速、均匀。诱导培养10-12天后,收集膜片,添加有100mg/ml维生素C的膜片诱导培养液组可形成完整的细胞膜片,能够完整地自培养皿底剥离,可折叠成需要的形状。添加有50mg/ml维生素C的矿化诱导培养液组形成的细胞膜片易破碎,不能完整的自培养皿底剥离。未添加维生素C的普通培养液组的细胞无法形成膜片。膜片诱导培养液组的细胞膜片H&E染色显示膜片由2-3层细胞组成,细胞间富含大量的细胞外基质。ALP活性检测显示两组细胞经诱导培养后均明显增强,50mg/ml维生素C培养组(即矿化诱导培养液组)略低于100mg/ml维生素C培养组(膜片诱导培养液组),两种细胞在相同培养条件下PDLSCs的ALP表达水平均略高于GMSCs,在矿化诱导液组差异有统计学意义,但在膜片诱导培养组两种细胞的ALP活性差异无统计学意义。
3.牙龈间充质干细胞和牙周膜干细胞膜片对牙周再生作用的体内研究
含有eGFP的慢病毒转染P3-P4代GMSCs和PDLSCs,转染后48-72h多数细胞均可发出明亮的绿色荧光,感染效率达90%左右。然后对细胞进行药物筛选纯化,获得稳定表达的eGFP的GMSCS和PDLSCs。扩大培养后,于膜片诱导液中诱导培养10-12天收集细胞膜片,折叠后移植入比格犬Ⅲ度根分叉病变模型。8周后处死动物获取下颌骨标本,切片进行H&E染色、免疫组织化学染色、天狼星红染色及直接荧光显微镜观察。组织形态学计量分析结果显示：GMSCs和PDLSCs膜片移植组的新生牙骨质百分比(PRC)和新生骨面积百分比(PRB)显著高于空白对照组,但两种细胞膜片组间差异无统计学意义；免疫组织化学结果显示GMSCs和PDLSCs移植组新生骨中GFP的表达均呈强阳性,而对照组中GFP染色则为阴性。荧光显微镜下观察,大部分新生骨在显微镜下呈明亮的绿色,在新生牙骨质、牙槽骨中的成牙骨质细胞和成骨细胞中均有绿色荧光的表达。偏振光显微镜观察天狼星红染色标本,在GMSCs和PDLSCs膜片植入组再生的胶原纤维多数呈垂直或斜行插入新生牙骨质,为功能性牙周膜,而空白对照组牙周膜纤维束多平行于牙根面,提示该纤维无正常功能。
结论
1. GMSCs同PDLSCs一样具有自我更新的能力,能够表达干细胞特异性的表面标记,并且具有多向分化的能力。
2.添加100mg/ml维生素C的成骨诱导液既能诱导细胞向成骨细胞方向分化,而且能够诱导细胞大量分泌细胞外基质形成细胞膜片,是一种较简单、有效的膜片制备手段。
3. GMSCs和PDLSCs膜片表达类似的ALP活性,用于犬Ⅲ度根分叉病变模型有类似的促牙周组织再生效果。
4.移植的GMSCs和PDLSCs促进牙周再生的机制包括移植细胞直接分化为牙周再生功能细胞和对宿主细胞的间接调控两种作用。
Background and Objective
The ultimate goal of periodontal therapy is to achieve complete periodontal regeneration, which means the reconstitution of the soft (gingival connective tissue and periodontal ligament) and the hard tissue (cementum and alveolar bone) of the periodontium. Current therapeutics for periodontitis fails to completely and reliably reconstitute all tissues damaged through periodontal inflammation. The cell-based therapeutics has been a promising alternative for clinical trial in periodontal regeneration. Seed cells, scaffold materials, growth factors are the main contents in the traditional tissue engineering research. Looking for an alternative stem cells with abundance and ready access, and an appropriate carrier for cell transplantation is the hotspot in tissue regeneration medicine.
To date, multiple mesenchymal stem cell lineages have been explored in tissue engineering. Bone marrow stem cells (BMSCs) have been extensively studied in contributing to periodontal regeneration, on account of their capacity of self-renewal and multiple differentiation potential. PDLSCs are identified as a promising alternative in periodontal regeneration. However, all of these above stem cells are not easily accessible or cultured, which hampers the utilization for clinical application.
In recent years, gingiva-derived MSCs (GMSCs) have been isolated from gingival tissue and confirmed capable of forming clonogenic colonies, expressing a typical MSC surface marker profile and possessing multiple differentiatial capacity in vitro. Moreover, GMSCs also have been demonstrated to possess the ability to form new bone in vivo, and properties of anti-inflammation and immunomodulation. On account of having properties of stable morphology, uniformly homogenous and faster proliferation, GMSCs is considered being superior to BMSCs and other dental stem cells for cell therapy in regenerative medicine. Moreover, the abundance and ready access of GMSCs is the most fascinating for tissue regeneration. Thus, GMSCs might be potentially a better alternative of cell-based approaches for periodontal regeneration. However, few attempts have been done to explore ability of GMSCs to regenerate periodontal tissue and especially its possible mechanism in tissue regeneration.
Cell sheet engineering is a promising strategy in tissue regeneration. Cell sheet technology may be beneficial for cell transplantation for preserving cellular junctions, endogenous extracellular matrix (ECM). Recent studies have shown that this technique is effective to form new cementum and promote periodontal attachment utilizing periodontal ligament cells and BMSCs.
To estimate the potential of GMSCs and PDLSCs to reconstitute the periodontal tissue and explore their fate and differentiation in vivo, we transplanted GFP labeled GMSCs sheet into inflammed Class III furcation defects in beagle dogs to regenerate periodontium complex.
Methods:
1. Isolation, cultivation and identification of human GMSCs and PDLSCs
1.1Isolation, cultivation of human GMSCs
Human gingival samples were harvested from healthy individuals as discarded tissues following conventional dental procedures such as the extraction of impacted third molars or crown lengthening. GMSCs were isolated, expanded, and cultured. The2rd-4th passage GMSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.
1.2Isolation, cultivation of human PDLSCs
Human periodontal ligament samples were harvested from healthy individuals as discarded tissues following conventional dental procedures. PDLSCs were isolated, expanded, and cultured. The2rd-4th passage PDLSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.
1.3Identification of human GMSCs and PDLSCs
Colony-forming unit-fibroblasts assay and Flow cytometric analysis were performed. GMSCs and PDLSCs plated in24-well plates were cultured respectively in osteogenic medium (a-MEM containing5%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate). Cells cultured in a-MEM containing5%FBS were as control. The mineralized nodules were characterized by2%Alizarin red S four weeks later. Cells were incubated adipogenic medium (a-MEM containing10%FBS,0.1μM dexamethasone,60μM indomethacin, and50mg/ml ascorbate-2-phosphate). Oil Red O staining was performed to identify the oil globules2weeks later.
2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assay
To create the cell sheet, GMSCs and PDLSCs were subcultured in the following medium:1) the cell sheet induction medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and100mg/ml ascorbate-2-phosphate);2) the osteogenic medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate);3) the normal medium (a-MEM with15%FBS). After reaching confluence (approximately at10-12d), the cells were rinsed twice with PBS, and then detached as a cell sheet. To examine the microscopic structure of the cell sheets sections were evaluated via hematoxylin and eosin (H&E) staining. Alkaline phosphatase (ALP) activity were assayed.
3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogs
The lentiviruses encoding eGFP were used to label the3rd passage of GMSCs and PDLSC. GFP expression in transduced cells was evaluated by direct observation of cells using a fluorescence microscope. Hygromycin was used to select the stably transduced GMSCs and PDLSC. GMSC and PDLSC sheets were transplanted into the Class Ⅲ furcation defects in beagle dogs. The animals were sacrificed after8weeks. Tissue sections selected from the most central areas of the defects were used for the H&E staining, Picrosirius red and immunohistochemical staining.
Results
1. Isolation, identification of human GMSCs and PDLSCs
Primary cultures of the single-cell suspensions obtained from the healthy gingiva formed adherent, clonogenic cell clusters, and the cultured GMSCs and PDLSCs retained fibroblastic phenotype. Flow cytometry analysis revealed that both cells were uniformly positive for CD44, CD29, CD73, CD90, CD105markers and Stro-1, and did not express hematopoietic stem markers CD14, and CD45, and endothelial cells markers CD144, CD31and HLA-DR. To investigate the multiple differentiation potential of GMSCs and PDLSCs, the cells were cultured in the osteogenic medium for4weeks. Extensive amounts of mineralized nodules were found in the experimental groups, but the mineralized nodules formed by GMSCs were smaller and scattered than that in PDLSCs group. Moreover, after cultured in the adipogenic medium for2weeks, GMSCs and PDLSCs were found to differentiate towards adipocytes as indicated by the accumulation of lipid droplets. The form of lipid droplets in two groups has no obvious difference. These findings indicates that the single colony-derived human gingival stem cells had the basic characteristics of mesenchymal stem cells.
2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assay
Both GMSCs and PDLSCs, cultured in the cell sheet induction medium, formed a multi-layer structure with abundant ECM. After10-12d of culture, a semi-transparent cell sheet was formed which was tough enough to be completely detached from the culture dish by a scraper. In contrast, cells grown in the osteogenic medium formed a relatively fragile membrane, whereas cells grown in the normal medium without ascorbic acid could not form the multi-layer structure.
In addition, both the cell sheet and osteogenic groups of each cells exhibited markedly increased ALP activity compared with the control group. No statistically significant difference in the ALP activity was detected between the cell sheet group and the osteogenic group. ALP activity under the same conditions of PDLSCs are slightly higher than the GMSCs. But the statistically significant difference between two cells is only in the osteogenic induction group.
3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogs
The lentiviruses encoding eGFP were transduced into the3rd passage of GMSCs and PDLSC. Bright green fluorescence was observed at48-72h using a fluorescence microscope, and infection efficiency was up to90%. The cells were purified by hygromycin and eGFP+GMSCS and eGFP+PDLSCs were harvested. Then they were expanded and cultured10to12days to fabricate the cell sheets, and then transplanted into Class Ⅲ furcation defects created in beagle dogs. The dogs were sacrificed8weeks after transplantation. The specimen slices were observed by H&E staining, immunohistochemical staining, Sirius red staining and fluorescence microscopy observation directly. Histomorphometric analysis was performed to evaluate the amount of periodontal tissue regeneration. Alveolar bone regeneration was observed in both cell sheets groups. Our results showed enhanced new bone and new cementum formation in both GMSCs and PDLSCs group when compared with that in the control group. Immunohistochemical results show the expression of GFP was strong positive in the new bone in both GMSCs and PDLSCs groups, while GFP were negative in control group. Most of the new bone are bright green under a fluorescence microscope. GFP protein was detected in the cementoblast-like cells, the periodontal ligament fibroblasts and osteoblasts. These results indicate that the transplanted GMSCs are capable of differentiating into the osteoblasts, cementoblasts and periodontal ligament fibroblasts, and therefore directly participate in the reconstitution of the periodontal tissues. To examine the periodontal ligament regeneration, Picrosirius red stained sections were observed by both light and polarizing light microscope. In both GMSCs and PDLSC groups, newly formed Sharpey's fibers anchored into the newly regenerated cementum were arranged perpendicularly to the root surface. In contrast, the newly formed fibers were parallel to the root surface in the control group.
Conclusion
1. Both GMSCs and PDLSCs share the similar characteristics with other stem cells, including self-renewability, expression of specific surface marker of stem cells and multi-lineage differentiation potential.
2. The osteogenic medium supplemented with100mg/ml ascorbic acid can not only induce osteogenic differentiation but also effectively fabricate cell sheets with highly biological activity.
3. Both GMSCs and PDLSC sheets express similar ALP activity and have the similar potential to regenerate bone, cementum/periodontal ligament complex in Class III furcation defects created in beagle dogs.
4. The mechanism of transplanted GMSCs and PDLSCs to promote periodontal regeneration might be directly differentiating into periodontal functional cells and indirectly induction effects on host cells.
引文
[1]INTINI G. Future approaches in periodontal regeneration:gene therapy, stem cells, and RNA interference [J]. Dental clinics of North America,2010,54(1):141-55.
[2]NUNN M E. Understanding the etiology of periodontitis:an overview of periodontal risk factors [J]. Periodontology 2000,2003,32(1):11-23.
[3]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[4]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[5]AKIYAMA K, CHEN C, GRONTHOS S, et al. Lineage differentiation of mesenchymal stem cells from dental pulp, apical papilla, and periodontal ligament [J]. Methods in molecular biology,2012, 887(111-21.
[6]SONOYAMA W, LIU Y, FANG D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine [J]. PLoS One,2006,1(1):e79.
[7]MORSCZECK C, G TZ W, SCHIERHOLZ J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth [J]. Matrix biology,2005,24(2):155-65.
[8]GRONTHOS S, MANKANI M, BRAHIM J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo [J]. Proceedings of the National Academy of Sciences,2000,97(25):13625-30.
[9]STENDERUP K, JUSTESEN J, CLAUSEN C, et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells< sup>, [J]. Bone,2003,33(6): 919-26.
[10]NAGATOMO K, KOMAKI M, SEKIYA I, et al. Stem cell properties of human periodontal ligament cells [J]. Journal of periodontal research,2006,41(4):303-10.
[11]MARCHESAN J T, SCANLON C S, SOEHREN S, et al. Implications of cultured periodontal ligament cells for the clinical and experimental setting:a review [J]. Archives of oral biology,2011,56(10): 933-43.
[12]LIU Y, ZHENG Y, DING G, et al. Periodontal Ligament Stem Cell-Mediated Treatment for Periodontitis in Miniature Swine [J]. Stem cells,2008,26(4):1065-73.
[13]SEO B-M, MIURA M, SONOYAMA W, et al. Recovery of stem cells from cryopreserved periodontal ligament [J]. J Dent Res,2005,84(10):907-12.
[14]NU EZ J, SANZ-BLASCO S, VIGNOLETTI F, et al. Periodontal regeneration following implantation of cementum and periodontal ligament-derived cells [J]. Journal of periodontal research,2012,47(1): 33-44.
[15]SUAID F F, RIBEIRO F V, GOMES T R, et al. Autologous periodontal ligament cells in the treatment of Class III furcation defects:a study in dogs [J]. Journal of clinical periodontology,2012,39(4):377-84.
[16]FOURNIER B P, FERRE F C, COUTY L, et al. Multipotent progenitor cells in gingival connective tissue [J]. Tissue engineering Part A,2010,16(9):2891-9.
[17]SU W R, ZHANG Q Z, SHI S H, et al. Human gingiva-derived mesenchymal stromal cells attenuate contact hypersensitivity via prostaglandin E2-dependent mechanisms [J]. Stem cells,2011,29(11): 1849-60.
[18]TANG L, LI N, XIE H, et al. Characterization of mesenchymal stem cells from human normal and hyperplastic gingiva [J]. Journal of cellular physiology,2011,226(3):832-42.
[19]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[20]WADA N, WANG B, LIN N H, et al. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts [J]. Journal of periodontal research,2011, 46(4):438-47.
[21]WANG F, YU M, YAN X, et al. Gingiva-derived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration [J]. Stem Cells Dev,2011,20(12):2093-102.
[22]YANG H, GAO L N, AN Y, et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions [J]. Biomaterials,2013,34(29):7033-47.
[23]ZHANG Q Z, SU W R, SHI S H, et al. Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing [J]. Stem cells,2010,28(10): 1856-68.
[24]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[25]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[26]KELM J M, FUSSENEGGER M. Scaffold-free cell delivery for use in regenerative medicine [J]. Adv Drug Deliv Rev,2010,62(7-8):753-64.
[27]ELLOUMI-HANNACHI I, YAMATO M, OKANO T. Cell sheet engineering:a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine [J]. Journal of internal medicine,2010,267(1):54-70.
[28]AKIZUKI T, ODA S, KOMAKI M, et al. Application of periodontal ligament cell sheet for periodontal regeneration:a pilot study in beagle dogs [J]. Journal of periodontal research,2005,40(3):245-51.
[29]HASEGAWA M, YAMATO M, KIKUCHI A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model [J]. Tissue engineering,2005,11(3-4): 469-78.
[30]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[31]ISHIKAWA I, IWATA T, WASHIO K, et al. Cell sheet engineering and other novel cell-based approaches to periodontal regeneration [J]. Periodontology 2000,2009,51(1):220-38.
[32]IWATA T, YAMATO M, TSUCHIOKA H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model [J]. Biomaterials,2009,30(14):2716-23.
[33]TSUMANUMA Y, IWATA T, WASHIO K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model [J]. Biomaterials,2011,32(25): 5819-25.
[34]VAQUETTE C, FAN W, XIAO Y, et al. A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex [J]. Biomaterials,2012,33(22):5560-73.
[35]WEI F, QU C, SONG T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity [J]. Journal of cellular physiology, 2012,227(9):3216-24.
[36]ZHOU Y, LI Y, MAO L, et al. Periodontal healing by periodontal ligament cell sheets in a teeth replantation model [J]. Archives of oral biology,2012,57(2):169-76.
[37]IWATA T, WASHIO K, YOSHIDA T, et al. Cell sheet engineering and its application for periodontal regeneration [J]. Journal of tissue engineering and regenerative medicine,2013,
[38]ZHAO Y H, ZHANG M, LIU N X, et al. The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation [J]. Biomaterials, 2013,34(22):5506-20.
[1]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[2]刘娟,赵红宇,轩东英,et al.人牙周膜细胞群多向分化潜能的实验研究[J].华西口腔医学杂志,2010,28(2)：185-9.
[3]GRONTHOS S, ZANNETTINO A C, HAY S J, et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow [J]. J Cell Sci,2003,116(9):1827-35.
[4]李纾,汪说之.人牙龈和牙周韧带成纤维细胞的体外培养及生物学特性研究—细胞形态,生长曲线及碱性磷酸酶活性测定[J].口腔医学纵横,2001,17(2)：95-7.
[5]SOMERMAN M, ARCHER S, IMM G, et al. A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro [J]. J Dent Res,1988,67(1):66-70.
[6]BOYKO G, MELCHER A, BRUNETTE D. Formation of new periodontalligament by periodontal ligament cells implanted in vivo after culture in vitro [J]. Journal of periodontal research,1981,16(1): 73-88.
[7]NAGATOMO K, KOMAKI M, SEKIYA I, et al. Stem cell properties of human periodontal ligament cells [J]. Journal of periodontal research,2006,41(4):303-10.
[8]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[9]LALLIER T E, SPENCER A, FOWLER M M. Transcript profiling of periodontal fibroblasts and osteoblasts [J]. Journal of periodontology,2005,76(7):1044-55.
[10]NEELEY W W, CARNES D L, COCHRAN D L. Osteogenesis in an in vitro coculture of human periodontal ligament fibroblasts and human microvascular endothelial cells [J]. Journal of periodontology,2010,81(1):139-49.
[11]CARNES D L, MAEDER C L, GRAVES D T. Cells with osteoblastic phenotypes can be explanted from human gingiva and periodontal ligament [J]. Journal of periodontology,1997,68(7):701-7.
[12]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[13]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[14]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[1]WEI F, QU C, SONG T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity [J]. Journal of cellular physiology, 2012,227(9):3216-24.
[2]VAQUETTE C, FAN W, XIAO Y, et al. A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex [J]. Biomaterials,2012,33(22):5560-73.
[3]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[4]IWATA T, YAMATO M, TSUCHIOKA H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model [J]. Biomaterials,2009,30(14):2716-23.
[5]ZHOU Y, LI Y, MAO L, et al. Periodontal healing by periodontal ligament cell sheets in a teeth replantation model [J]. Archives of oral biology,2012,57(2):169-76.
[6]ZHAO Y H, ZHANG M, LIU N X, et al. The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation [J]. Biomaterials, 2013,34(22):5506-20.
[7]HASEGAWA M, YAMATO M, KIKUCHI A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model [J]. Tissue engineering,2005,11(3-4): 469-78.
[8]ZHOU W, HAN C, SONG Y, et al. The performance of bone marrow mesenchymal stem cell-implant complexes prepared by cell sheet engineering techniques [J]. Biomaterials,2010,31(12): 3212-21.
[9]TSUMANUMA Y, IWATA T, WASHIO K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model [J]. Biomaterials,2011,32(25): 5819-25.
[10]REZWAN K, CHEN Q, BLAKER J, et al. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering [J]. Biomaterials,2006,27(18):3413-31.
[11]JENSEN S S, BROGGINI N, HJ RTING-HANSEN E, et al. Bone healing and graft resorption of autograft, anorganic bovine bone and β-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs [J]. Clinical oral implants research,2006,17(3):237-43.
[12]SOGAL A, TOFE A. Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications [J]. Journal of periodontology,1999,70(9):1053-63.
[13]SCULEAN A, NIKOLIDAKIS D, SCHWARZ F. Regeneration of periodontal tissues:combinations of barrier membranes and grafting materials-biological foundation and preclinical evidence:a systematic review [J]. Journal of clinical periodontology,2008,35(s8):106-16.
[14]REYNOLDS M A, AICHELMANN-REIDY M E, BRANCH-MAYS G L, et al. The efficacy of bone replacement grafts in the treatment of periodontal osseous defects. A systematic review [J]. Annals of periodontology,2003,8(1):227-65.
[15]KELM J M, FUSSENEGGER M. Scaffold-free cell delivery for use in regenerative medicine [J]. Adv Drug Deliv Rev,2010,62(7-8):753-64.
[16]KLEINMAN H K, PHILP D, HOFFMAN M P. Role of the extracellular matrix in morphogenesis [J]. Curr Opin Biotechnol,2003,14(5):526-32.
[17]HOWLETT A, BISSELL M. The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium [J]. Epithelial cell biology,1993, 2(2):79-89.
[18]ROZARIO T, DESIMONE D W. The extracellular matrix in development and morphogenesis:a dynamic view [J]. Dev Biol,2010,341(1):126-40.
[19]ADAMS J C, WATT F M. Regulation of development and differentiation by the extracellular matrix [J]. DEVELOPMENT-CAMBRIDGE-,1993,117(1183-.
[20]STONE N, MEISTER A. Function of ascorbic acid in the conversion of proline to collagen hydroxyproline [J]. Nature,1962,194(555.
[21]BARNES M. Function of ascorbic acid in collagen metabolism [J]. Ann N Y Acad Sci,1975,258(1): 264-77.
[22]HUTTON JR J J, TAPPEL A, UDENFRIEND S. Cofactor and substrate requirements of collagen proline hydroxylase [J]. Archives of Biochemistry and Biophysics,1967,118(1):231-40.
[23]CHOI K-M, SEO Y-K, YOON H-H, et al. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation [J]. J Biosci Bioeng,2008,105(6):586-94.
[24]JI A-R, KU S-Y, CHO M S, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage [J]. Experimental & molecular medicine,2010, 42(3):175-86.
[25]POTDAR P D, D'SOUZA S B. Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2 [J]. Human cell,2010,23(4):152-5.
[26]YANG Z, JIN F, ZHANG X, et al. Tissue engineering of cementum/periodontal-ligament complex using a novel three-dimensional pellet cultivation system for human periodontal ligament stem cells [J]. Tissue Engineering Part C:Methods,2009,15(4):571-81.
[1]HYNES K, MENICANIN D, GRONTHOS S, et al. Clinical utility of stem cells for periodontal regeneration [J]. Periodontology 2000,2012,59(1):203-27.
[2]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[3]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[4]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[5]WANG F, YU M, YAN X, et al. Gingiva-derived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration [J]. Stem Cells Dev,2011,20(12):2093-102.
[6]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[7]詹以安,王共先.骨髓间充质干细胞作为基因运载细胞在基因治疗中的应用[J].中国组织工程研究与临床康复,2007,
[8]胡运生,马保安,李文海,et al绿色荧光蛋白标记基因体内示踪骨髓间充质干细胞的分化[J].中国修复重建外科杂志,2007,21(6)：
[9]TANI G, USUI N, KAMIYAMA M, et al. In vitro construction of scaffold-free cylindrical cartilage using cell sheet-based tissue engineering [J]. Pediatric surgery international,2010,26(2):179-85.
[10]LALLIER T E, MINER JR Q W, SONNIER J, et al. A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts [J]. Cell Tissue Res,2007,328(2):339-54.
[11]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[12]XIE H, LIU H. A novel mixed-type stem cell pellet for cementum/periodontal ligament-like complex [J]. Journal of periodontology,2012,83(6):805-15.
[13]AKIZUKI T, ODA S, KOMAKI M, et al. Application of periodontal ligament cell sheet for periodontal regeneration:a pilot study in beagle dogs [J]. Journal of periodontal research,2005,40(3):245-51.
[14]NUNN M E. Understanding the etiology of periodontitis:an overview of periodontal risk factors [J]. Periodontology 2000,2003,32(1):11-23.
[15]武影,张秀丽,束蓉EDTA对人牙周膜细胞在病变根面附着和增殖影响[J].实用口腔医学杂志,2005,21(4)：554-7.
[16]何勇.牙周膜干细胞膜片在牙周再生中的实验研究[D]；第四军医大学,2010.
[17]LIECHTY K W, MACKENZIE T C, SHAABAN A F, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep [J]. Nat Med,2000, 6(11):1282-6.
[18]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[19]SEO B-M, MIURA M, SONOYAMA W, et al. Recovery of stem cells from cryopreserved periodontal ligament [J]. J Dent Res,2005,84(10):907-12.
[2]NUNN M E. Understanding the etiology of periodontitis:an overview of periodontal risk factors [J]. Periodontology 2000,2003,32(1):11-23.
[3]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[4]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[5]AKIYAMA K, CHEN C, GRONTHOS S, et al. Lineage differentiation of mesenchymal stem cells from dental pulp, apical papilla, and periodontal ligament [J]. Methods in molecular biology,2012, 887(111-21.
[6]SONOYAMA W, LIU Y, FANG D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine [J]. PLoS One,2006,1(1):e79.
[7]MORSCZECK C, G TZ W, SCHIERHOLZ J, et al. Isolation of precursor cells (PCs) from human dental follicle of wisdom teeth [J]. Matrix biology,2005,24(2):155-65.
[8]GRONTHOS S, MANKANI M, BRAHIM J, et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo [J]. Proceedings of the National Academy of Sciences,2000,97(25):13625-30.
[9]STENDERUP K, JUSTESEN J, CLAUSEN C, et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells< sup>, [J]. Bone,2003,33(6): 919-26.
[10]NAGATOMO K, KOMAKI M, SEKIYA I, et al. Stem cell properties of human periodontal ligament cells [J]. Journal of periodontal research,2006,41(4):303-10.
[11]MARCHESAN J T, SCANLON C S, SOEHREN S, et al. Implications of cultured periodontal ligament cells for the clinical and experimental setting:a review [J]. Archives of oral biology,2011,56(10): 933-43.
[12]LIU Y, ZHENG Y, DING G, et al. Periodontal Ligament Stem Cell-Mediated Treatment for Periodontitis in Miniature Swine [J]. Stem cells,2008,26(4):1065-73.
[13]SEO B-M, MIURA M, SONOYAMA W, et al. Recovery of stem cells from cryopreserved periodontal ligament [J]. J Dent Res,2005,84(10):907-12.
[14]NU EZ J, SANZ-BLASCO S, VIGNOLETTI F, et al. Periodontal regeneration following implantation of cementum and periodontal ligament-derived cells [J]. Journal of periodontal research,2012,47(1): 33-44.
[15]SUAID F F, RIBEIRO F V, GOMES T R, et al. Autologous periodontal ligament cells in the treatment of Class III furcation defects:a study in dogs [J]. Journal of clinical periodontology,2012,39(4):377-84.
[16]FOURNIER B P, FERRE F C, COUTY L, et al. Multipotent progenitor cells in gingival connective tissue [J]. Tissue engineering Part A,2010,16(9):2891-9.
[17]SU W R, ZHANG Q Z, SHI S H, et al. Human gingiva-derived mesenchymal stromal cells attenuate contact hypersensitivity via prostaglandin E2-dependent mechanisms [J]. Stem cells,2011,29(11): 1849-60.
[18]TANG L, LI N, XIE H, et al. Characterization of mesenchymal stem cells from human normal and hyperplastic gingiva [J]. Journal of cellular physiology,2011,226(3):832-42.
[19]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[20]WADA N, WANG B, LIN N H, et al. Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts [J]. Journal of periodontal research,2011, 46(4):438-47.
[21]WANG F, YU M, YAN X, et al. Gingiva-derived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration [J]. Stem Cells Dev,2011,20(12):2093-102.
[22]YANG H, GAO L N, AN Y, et al. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions [J]. Biomaterials,2013,34(29):7033-47.
[23]ZHANG Q Z, SU W R, SHI S H, et al. Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing [J]. Stem cells,2010,28(10): 1856-68.
[24]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[25]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[26]KELM J M, FUSSENEGGER M. Scaffold-free cell delivery for use in regenerative medicine [J]. Adv Drug Deliv Rev,2010,62(7-8):753-64.
[27]ELLOUMI-HANNACHI I, YAMATO M, OKANO T. Cell sheet engineering:a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine [J]. Journal of internal medicine,2010,267(1):54-70.
[28]AKIZUKI T, ODA S, KOMAKI M, et al. Application of periodontal ligament cell sheet for periodontal regeneration:a pilot study in beagle dogs [J]. Journal of periodontal research,2005,40(3):245-51.
[29]HASEGAWA M, YAMATO M, KIKUCHI A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model [J]. Tissue engineering,2005,11(3-4): 469-78.
[30]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[31]ISHIKAWA I, IWATA T, WASHIO K, et al. Cell sheet engineering and other novel cell-based approaches to periodontal regeneration [J]. Periodontology 2000,2009,51(1):220-38.
[32]IWATA T, YAMATO M, TSUCHIOKA H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model [J]. Biomaterials,2009,30(14):2716-23.
[33]TSUMANUMA Y, IWATA T, WASHIO K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model [J]. Biomaterials,2011,32(25): 5819-25.
[34]VAQUETTE C, FAN W, XIAO Y, et al. A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex [J]. Biomaterials,2012,33(22):5560-73.
[35]WEI F, QU C, SONG T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity [J]. Journal of cellular physiology, 2012,227(9):3216-24.
[36]ZHOU Y, LI Y, MAO L, et al. Periodontal healing by periodontal ligament cell sheets in a teeth replantation model [J]. Archives of oral biology,2012,57(2):169-76.
[37]IWATA T, WASHIO K, YOSHIDA T, et al. Cell sheet engineering and its application for periodontal regeneration [J]. Journal of tissue engineering and regenerative medicine,2013,
[38]ZHAO Y H, ZHANG M, LIU N X, et al. The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation [J]. Biomaterials, 2013,34(22):5506-20.
[1]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[2]刘娟,赵红宇,轩东英,et al.人牙周膜细胞群多向分化潜能的实验研究[J].华西口腔医学杂志,2010,28(2)：185-9.
[3]GRONTHOS S, ZANNETTINO A C, HAY S J, et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow [J]. J Cell Sci,2003,116(9):1827-35.
[4]李纾,汪说之.人牙龈和牙周韧带成纤维细胞的体外培养及生物学特性研究—细胞形态,生长曲线及碱性磷酸酶活性测定[J].口腔医学纵横,2001,17(2)：95-7.
[5]SOMERMAN M, ARCHER S, IMM G, et al. A comparative study of human periodontal ligament cells and gingival fibroblasts in vitro [J]. J Dent Res,1988,67(1):66-70.
[6]BOYKO G, MELCHER A, BRUNETTE D. Formation of new periodontalligament by periodontal ligament cells implanted in vivo after culture in vitro [J]. Journal of periodontal research,1981,16(1): 73-88.
[7]NAGATOMO K, KOMAKI M, SEKIYA I, et al. Stem cell properties of human periodontal ligament cells [J]. Journal of periodontal research,2006,41(4):303-10.
[8]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[9]LALLIER T E, SPENCER A, FOWLER M M. Transcript profiling of periodontal fibroblasts and osteoblasts [J]. Journal of periodontology,2005,76(7):1044-55.
[10]NEELEY W W, CARNES D L, COCHRAN D L. Osteogenesis in an in vitro coculture of human periodontal ligament fibroblasts and human microvascular endothelial cells [J]. Journal of periodontology,2010,81(1):139-49.
[11]CARNES D L, MAEDER C L, GRAVES D T. Cells with osteoblastic phenotypes can be explanted from human gingiva and periodontal ligament [J]. Journal of periodontology,1997,68(7):701-7.
[12]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[13]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[14]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[1]WEI F, QU C, SONG T, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity [J]. Journal of cellular physiology, 2012,227(9):3216-24.
[2]VAQUETTE C, FAN W, XIAO Y, et al. A biphasic scaffold design combined with cell sheet technology for simultaneous regeneration of alveolar bone/periodontal ligament complex [J]. Biomaterials,2012,33(22):5560-73.
[3]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[4]IWATA T, YAMATO M, TSUCHIOKA H, et al. Periodontal regeneration with multi-layered periodontal ligament-derived cell sheets in a canine model [J]. Biomaterials,2009,30(14):2716-23.
[5]ZHOU Y, LI Y, MAO L, et al. Periodontal healing by periodontal ligament cell sheets in a teeth replantation model [J]. Archives of oral biology,2012,57(2):169-76.
[6]ZHAO Y H, ZHANG M, LIU N X, et al. The combined use of cell sheet fragments of periodontal ligament stem cells and platelet-rich fibrin granules for avulsed tooth reimplantation [J]. Biomaterials, 2013,34(22):5506-20.
[7]HASEGAWA M, YAMATO M, KIKUCHI A, et al. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model [J]. Tissue engineering,2005,11(3-4): 469-78.
[8]ZHOU W, HAN C, SONG Y, et al. The performance of bone marrow mesenchymal stem cell-implant complexes prepared by cell sheet engineering techniques [J]. Biomaterials,2010,31(12): 3212-21.
[9]TSUMANUMA Y, IWATA T, WASHIO K, et al. Comparison of different tissue-derived stem cell sheets for periodontal regeneration in a canine 1-wall defect model [J]. Biomaterials,2011,32(25): 5819-25.
[10]REZWAN K, CHEN Q, BLAKER J, et al. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering [J]. Biomaterials,2006,27(18):3413-31.
[11]JENSEN S S, BROGGINI N, HJ RTING-HANSEN E, et al. Bone healing and graft resorption of autograft, anorganic bovine bone and β-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs [J]. Clinical oral implants research,2006,17(3):237-43.
[12]SOGAL A, TOFE A. Risk assessment of bovine spongiform encephalopathy transmission through bone graft material derived from bovine bone used for dental applications [J]. Journal of periodontology,1999,70(9):1053-63.
[13]SCULEAN A, NIKOLIDAKIS D, SCHWARZ F. Regeneration of periodontal tissues:combinations of barrier membranes and grafting materials-biological foundation and preclinical evidence:a systematic review [J]. Journal of clinical periodontology,2008,35(s8):106-16.
[14]REYNOLDS M A, AICHELMANN-REIDY M E, BRANCH-MAYS G L, et al. The efficacy of bone replacement grafts in the treatment of periodontal osseous defects. A systematic review [J]. Annals of periodontology,2003,8(1):227-65.
[15]KELM J M, FUSSENEGGER M. Scaffold-free cell delivery for use in regenerative medicine [J]. Adv Drug Deliv Rev,2010,62(7-8):753-64.
[16]KLEINMAN H K, PHILP D, HOFFMAN M P. Role of the extracellular matrix in morphogenesis [J]. Curr Opin Biotechnol,2003,14(5):526-32.
[17]HOWLETT A, BISSELL M. The influence of tissue microenvironment (stroma and extracellular matrix) on the development and function of mammary epithelium [J]. Epithelial cell biology,1993, 2(2):79-89.
[18]ROZARIO T, DESIMONE D W. The extracellular matrix in development and morphogenesis:a dynamic view [J]. Dev Biol,2010,341(1):126-40.
[19]ADAMS J C, WATT F M. Regulation of development and differentiation by the extracellular matrix [J]. DEVELOPMENT-CAMBRIDGE-,1993,117(1183-.
[20]STONE N, MEISTER A. Function of ascorbic acid in the conversion of proline to collagen hydroxyproline [J]. Nature,1962,194(555.
[21]BARNES M. Function of ascorbic acid in collagen metabolism [J]. Ann N Y Acad Sci,1975,258(1): 264-77.
[22]HUTTON JR J J, TAPPEL A, UDENFRIEND S. Cofactor and substrate requirements of collagen proline hydroxylase [J]. Archives of Biochemistry and Biophysics,1967,118(1):231-40.
[23]CHOI K-M, SEO Y-K, YOON H-H, et al. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation [J]. J Biosci Bioeng,2008,105(6):586-94.
[24]JI A-R, KU S-Y, CHO M S, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage [J]. Experimental & molecular medicine,2010, 42(3):175-86.
[25]POTDAR P D, D'SOUZA S B. Ascorbic acid induces in vitro proliferation of human subcutaneous adipose tissue derived mesenchymal stem cells with upregulation of embryonic stem cell pluripotency markers Oct4 and SOX 2 [J]. Human cell,2010,23(4):152-5.
[26]YANG Z, JIN F, ZHANG X, et al. Tissue engineering of cementum/periodontal-ligament complex using a novel three-dimensional pellet cultivation system for human periodontal ligament stem cells [J]. Tissue Engineering Part C:Methods,2009,15(4):571-81.
[1]HYNES K, MENICANIN D, GRONTHOS S, et al. Clinical utility of stem cells for periodontal regeneration [J]. Periodontology 2000,2012,59(1):203-27.
[2]SILVERIO K G, BENATTI B B, CASATI M Z, et al. Stem cells:potential therapeutics for periodontal regeneration [J]. Stem cell reviews,2008,4(1):13-9.
[3]ZHANG Q, SHI S, LIU Y, et al. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis [J]. Journal of immunology,2009,183(12):7787-98.
[4]TOMAR G B, SRIVASTAVA R K, GUPTA N, et al. Human gingiva-derived mesenchymal stem cells are superior to bone marrow-derived mesenchymal stem cells for cell therapy in regenerative medicine [J]. Biochemical and biophysical research communications,2010,393(3):377-83.
[5]WANG F, YU M, YAN X, et al. Gingiva-derived mesenchymal stem cell-mediated therapeutic approach for bone tissue regeneration [J]. Stem Cells Dev,2011,20(12):2093-102.
[6]FAWZY EL-SAYED K M, PARIS S, BECKER S T, et al. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study [J]. Journal of clinical periodontology,2012, 39(9):861-70.
[7]詹以安,王共先.骨髓间充质干细胞作为基因运载细胞在基因治疗中的应用[J].中国组织工程研究与临床康复,2007,
[8]胡运生,马保安,李文海,et al绿色荧光蛋白标记基因体内示踪骨髓间充质干细胞的分化[J].中国修复重建外科杂志,2007,21(6)：
[9]TANI G, USUI N, KAMIYAMA M, et al. In vitro construction of scaffold-free cylindrical cartilage using cell sheet-based tissue engineering [J]. Pediatric surgery international,2010,26(2):179-85.
[10]LALLIER T E, MINER JR Q W, SONNIER J, et al. A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts [J]. Cell Tissue Res,2007,328(2):339-54.
[11]FLORES M G, YASHIRO R, WASHIO K, et al. Periodontal ligament cell sheet promotes periodontal regeneration in athymic rats [J]. Journal of clinical periodontology,2008,35(12):1066-72.
[12]XIE H, LIU H. A novel mixed-type stem cell pellet for cementum/periodontal ligament-like complex [J]. Journal of periodontology,2012,83(6):805-15.
[13]AKIZUKI T, ODA S, KOMAKI M, et al. Application of periodontal ligament cell sheet for periodontal regeneration:a pilot study in beagle dogs [J]. Journal of periodontal research,2005,40(3):245-51.
[14]NUNN M E. Understanding the etiology of periodontitis:an overview of periodontal risk factors [J]. Periodontology 2000,2003,32(1):11-23.
[15]武影,张秀丽,束蓉EDTA对人牙周膜细胞在病变根面附着和增殖影响[J].实用口腔医学杂志,2005,21(4)：554-7.
[16]何勇.牙周膜干细胞膜片在牙周再生中的实验研究[D]；第四军医大学,2010.
[17]LIECHTY K W, MACKENZIE T C, SHAABAN A F, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep [J]. Nat Med,2000, 6(11):1282-6.
[18]SEO B-M, MIURA M, GRONTHOS S, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament [J]. The Lancet,2004,364(9429):149-55.
[19]SEO B-M, MIURA M, SONOYAMA W, et al. Recovery of stem cells from cryopreserved periodontal ligament [J]. J Dent Res,2005,84(10):907-12.