【作者】 马兆峰；

【导师】 刘宝林； 金岩； 宋应亮；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 牙根发育早期,牙囊细胞、上皮根鞘和牙乳头细胞相互作用,形成了牙根牙周组织。经典理论认为,牙周前体细胞—牙囊细胞穿透断裂的上皮根鞘与牙根接触并相互作用,分化成为成牙骨质细胞并形成牙骨质。我们最近的实验结果显示:牙本质非胶原蛋白能够诱导大鼠牙囊细胞向成牙骨质细胞转化。2007年底,Sonoyama等人利用组织工程方法,将发育期根尖牙乳头细胞与牙周膜干细胞顺序复合多孔HA/TCP材料,在小型猪颌骨内自体移植,利用两种细胞的相互作用,成功构建出具有生物活性的牙根牙周结构,并在人工牙根上成功制作义齿修复。在临床实际操作中,牙周炎患牙的牙周洁治与根面平整,以及局部的抗炎处理是促进牙周再生的前提。通过物理或化学的方法进行牙根表面处理,其目的是为了去除牙根表层的玷污层,改善根面周围的局部微环境,暴露新鲜的牙本质或牙骨质相关蛋白,增加局部的趋化刺激因子,以此来诱导牙周细胞的牙根表面贴附性,促进牙周再生9,10。因此可以推断这些暴露的牙本质相关蛋白有可能作为细胞趋化因子诱导牙周细胞牙根表面的贴附、增殖及分化。牙本质胶原与非胶原蛋白(DNCPs)是一组细胞外基质蛋白的总称,包括牙本质涎蛋白,磷蛋白和各种多肽生长因子,部分蛋白已被证实可作为细胞因子诱导牙本质周围的多种胚胎或成体干细胞细胞分化。基于牙周发育学的生理基础与牙周治疗的临床操作实际角度,合并我实验室以前的研究结果,我们认为牙本质基质蛋白可能在牙周再生修复过程中发挥着极其重要的作用。牙周膜干细胞是近两年发现的牙周膜内的多潜能前体细胞,在维持牙周自身内环境稳定,保持牙周膜新陈代谢平衡及牙周损伤修复发挥着重要作用。牙周膜干细胞具有自我更新与多向分化能力,可作为牙周关系中各种不同类型细胞的能源储备细胞,因此现在的观点认为牙周膜干细胞在促进牙周再生方面具有重要的研究与实际意义。发育期根尖牙乳头细胞是成牙本质及牙髓细胞的前体细胞,在细胞增殖、分化并合成基质形成了牙根的牙本质和牙髓组织,因此认为牙根的牙乳头细胞在牙根发育,牙周形成过程中发挥着重要作用。本实验拟探讨牙本质非胶原蛋白(DNCPs)对牙周膜干细胞(HPDLSCs)是否具有定向诱导作用。研究体外DNCPs对HPDLSCs的生物学效应,体内在生物支架材料表面促进再生牙周样结构的能力。本研究是第一次引入牙本质来源的基质蛋白诱导牙周膜干细胞在牙本质表面构建牙周组织结构,此研究策略一旦成功,必将对牙周组织再生拓展新的研究思路,为牙周炎的临床治疗提供新的方法。课题研究目的:探讨以人牙周膜干细胞(hPDLSCs)作为牙周组织工程种子细胞的可行性;分析牙本质相关蛋白(DNCPs)或根尖牙乳头干细胞基质蛋白对hPDLSCs细胞行为的影响。探讨经DNCPs诱导的hPDLSCs复合脱矿、脱蛋白牙本质的方式或根尖牙乳头干细胞与牙周膜干细胞顺序接种生物支架的方式构建组织工程化的牙根牙周组织的可行性。课题研究内容①分离培养人牙周膜干细胞(hPDLSCs)作为牙周再生组织工程的种子细胞,鉴定其干细胞特性及多向分化潜能(成脂、成骨分化)。②分离培养人发育期根尖牙乳头干细胞,鉴定其干细胞特性,观察种子细胞与生物支架材料复合后的生长特点,生成基质的特性;②体外观察DNCPs对牙周膜干细胞(hPDLSCs)增殖与分化能力的影响;③体外观察DNCPs对牙周膜干细胞(hPDLSCs)细胞大体形态的影响,细胞牙本质表面黏附能力的影响;④体内观察DNCPs能否诱导牙周膜干细胞(hPDLSCs)在牙本质表面形成牙周样结构;⑤hPDLSCs与hRAPSCs顺序接种生物支架材料,异位移植观察根尖牙乳头细胞生成的基质对hPDLSCs牙周再生能力的影响;⑥利用明胶微球缓释特性,合成适合hPDLSCs生长分化及组织再生的胶原-羟基磷灰石-明胶微球/缓释TGF-β1的多孔支架材料。主要研究方法1利用酶消化结合组织块法在成人健康牙周膜中培育人牙周膜细胞;通过收集多个克隆化生长的细胞,并将其培育在间充质干细胞培养液内,获得牙周膜干细胞。间充质干细胞标志物STRO-1检测细胞;干细胞成骨诱导与成脂诱导,观察多向分化能力。2分离年轻智齿的根尖部分组织,利用酶消化法培养了根尖牙乳头细胞,对细胞进行了干细胞鉴定与多向诱导分化。细胞接种珊瑚羟基磷灰石支架,观察牙本质基质生成情况,免疫组化染色观察基质特性。3 DNCPs溶解于DMEM培养基,精调pH值制成1ug/ml溶液。以DNCPs溶解的DMEM培养基(2%胎牛血清,50ug/ml维生素C, 2 mmol/L谷胺酰胺, 100 U /ml青霉素, 100μg/ml链霉素)为A液;不添加DNCPs的DMEM培养基为B液。以MTT法,BrdU吸收率,流式细胞仪检测A液与B液孵育的牙周膜干细胞增殖活性。4以倒置显微镜,扫描显微镜观察经A液与B液孵育的牙周膜细胞大体形态变化;以RT-PCR为手段检测成骨标志物COL, OCN, ALP的PCR水平变化;检测诱导前后碱磷酶变化;检测诱导前后对对牙周膜干细胞矿化形成能力的影响。5改良方法制作部分脱矿、脱蛋白牙本质支架材料,干细胞体外支架接种,观察细胞贴附与伸展效果;观察DNCPs诱导下对牙周膜干细胞平皿贴壁速度与能力的影响。6 DNCPs孵育的HPDLSCs接种CCRD支架,裸鼠体内移植,四周后观察牙周组织在牙本质表面再生的情况。7制作Collagen/HA/GM具有缓释支架材料,观察生长因子缓释效果。支架接种牙周膜干细胞,裸鼠体内移植,观察组织再生及因子缓释效果。课题主要研究结果①利用酶消化结合组织块法在成人健康牙周膜和培育了人牙周膜细胞(hPDLSCs);对干细胞特性进行了系统研究:包括主要干细胞标志物检测;克隆形成能力分析;多向分化能力检测。牙周膜细胞的间充质干细胞标志物STRO-1检出细胞群29.01%为阳性;干细胞成骨诱导3周,茜素红染色,细胞周围出现大量、片状矿化基质;成脂诱导液干细胞孵育3周,油红O染色,大量细胞出现脂滴阳性染色。②利用酶消化法在根尖组织中分离、培养了人根尖牙乳头干细胞(hRAPSCs)。③经MTT法检测DNCPs可以促进牙周膜干细胞(hPDLSCs)增殖,DNCPs孵育的hPDLSCs的BrdU吸收率有统计意义的提高;DNCPs处理的hPDLSCs细胞分裂相增加。DNCPs孵育的hPDLSCs细胞贴壁能力明显增强。④经DNCPs诱导的牙周膜干细胞(hPDLSCs)可以增加COL, OCN, ALP的PCR水平,矿化基质形成能力明显增强。形态学检测牙周膜干细胞向成牙骨质细胞分化。⑤DNCPs诱导的牙周膜干细胞(hPDLSCs)可以促进其在牙本质表面牙骨质样结构与牙周纤维样结构再生。⑥根尖牙乳头干细胞(hRAPSCs)在生物支架表面形成了矿化的牙本质基质,这种牙本质基质可诱导牙周膜干细胞在其表面的牙周再生。⑦构建出适合hPDLSCs生长分化的Collagen/HA/GM具有缓释TGF-β1功能的支架材料。结论我们的研究证实,DNCPs是有效的刺激因子可促进牙周膜干细胞增殖及成牙骨质向分化,促使HPDLSCs向牙骨质细胞的形态改变,进而诱导HPDLSCs的牙周组织发生。因此,这些发现对于指导临床如何促进牙周再生是具有重要意义的。但DNCPs有效的促牙周再生因子是什么,以及它与其他促牙周再生因子相互之间的关系如何,仍然是我们研究的课题。更多还原

【Abstract】 During onset of root, the interaction of dental follicle cells (DFCs), Hertwig’s epithelial root sheath (HERS) and dental papilla cells (DPCs) gives rise to formation of root and periodontium. Apart from above, it has been well recognized that DFCs which are the periodontal progenitor cells penetrate disintegrated HERS and contact with root dentin surface prior to any cementum formation. Our previous studies have proved that dentin non-collagenous proteins (DNCPs) can stimulate DFCs differentiate into cementoblast lineages. Furthermore, Sonoyama et al have successfully constructed bio-root periodontal complex in miniature pigs by developmental root apical papilla stem cells combining with periodontal stem cells.For true periodontium regeneration, direct contact between the conditioned or denuded root surface with periodontal cells seems to be a prerequisite. Clinically, disinfection and modification of the contaminated root including physical and chemical treatment to restore its biocompatibility and to favor the attachment of periodontal structures become necessity. After the smear layer removal, the new dentin collagenous and non-collagenous proteins are exposed and they are supposed to be a chemo-attractant for periodontal fibroblasts. DNCPs, which compromise glycoproteins/sialoproteins, phosphoroteins, proteoglycans and growth factors have been confirmed to act as an inductive agent in promoting many cells differentiation. Based on the fact of direct dentin to periodontal cell contact and the previous results, we proposed that dentin relative matrix may play an important role in periodontium formation and may benefit periodontal tissue regeneration. Therefore, it would be of great interest to investigate the effects of dentin matrix on periodontal stem cells.Taken into consideration that stem cells are capable of self-renewal and multi-lineage differentiation, which make them very promising in regenerating organs and tissues, these cells are of paramount importance in periodontal morphogenesis. The present study was designed to investigate the biological effects of DNCPs on human periodontal ligament stem cells (HPDLSCs) in vitro, and to determine the potential of reconstructing a periodontal complex with DNCPs treated HPDLSCs on chemical conditioned root dentin (CCRD) in vivo. It is the first time to applying active proteins derived from dentin with periodontal stem cells to construct periodontal structure. The strategy may be shed light on human periodontal tissue regeneration.1 Materials and methods:1.1 Isolation of HPDLSCs and HRAPSCsHPDLSCs and HRAPSCs were isolated from ten healthy donors, cultured, and expanded as previously described. Procedures were performed according to the approval of the institutional review board and the informed consent of the patients. Wisdom and premolar teeth intended for extraction due to orthodontic reasons were used as the cell source. Periodontal ligament tissue and apical tissues were obtained and cultured in MesenPRO RS medium containing 2% FCS, 100 units/mL penicillin, 100μg/mL streptomycin. Multiple colony-derived passage two were used in experiments.1.2 Adipogenetic/osteogenetic differentiation and immunohistochemistry of the stem cellsFor differentiation investigation, 1×104 cells/well were plated in a 12-well dish and cultured in DMEM supplemented with 2% FCS, 100 units/ml penicillin, 100μg/ml streptomycin, 50μg/ml anti ascorbic acid (medium A) for 1 day. Then, the cells were incubated with adipogenic or osteogenetic medium for another 21 days, and detected by Oil red O solution or Alizarin red staining respectively.16 Isolated putative stem cells were incubated with STRO-1 antibody, and subsequently incubated with FITC-conjugated anti-mouse secondary antibody. The surface marker STRO-1 positive cells were also analyzed by flow cytometry.1.3 Preparation of CCRDThe teeth for culturing cells were prepared for CCRD. Roots were sliced in longitudinal section. A series of physical and chemical procedures were performed according to reported protocols with minor modifications. In brief, dentinal materials were treated for 3 min with Chloroform-methanol (1:1), 0.6 M HCl for 5 min, 0.5M EDTA for 3 min, CaCl2 2M for 3 min, LiCl for 5 min, and absolute ethanol for 10 min at room temperature. Thereafter, the freeze-dried process was performed.1.4 Assessment of the effect of DNCPs on HPDLSCs1.4.1 Cell growth rate assay, flow cytometry and BrdU incorporationTo evaluate the proliferation potential and viability of the HPDLSCs treated or untreated with DNCPs, growth rate MTT assay, cell cycle analysis and BrdU incorporation into DNA were conducted. In the present study, DNCPs was dissolved in acetic acid solution. Two groups were set, to the test group, 1ug/ml DNCPs were added to medium A (medium B), and to the control group, only the medium A. The MTT assay was carried out for 5 days according to the cell proliferation kit protocol (Sigma). Flow cytometry analysis was respectively treated as previous report. The fractions of cells in the G1, S, and G2 phases of cell cycle were analyzed. DNA synthesis of the HPDLSCs in different medium was assessed by measuring bromodeoxyuridine incorporation (BrdU). Immunodetection of the incorporated BrdU into cells was performed as described previously. The BrdU-labelling index was determined as the percentage of BrdU-positive cells to the total cell number.1.4.2 Clonogenic assays and cells morphology observationTo assess colony-forming efficiency, single-cell suspensions within medium A or medium B (1×103 cells) were seeded into 6-well dishes. Day 7th cultures were stained with 0.1% toluidine blue. Cells morphology was examined under phase-contrast microscopy and scanning electron microscope.1.4. 3 Cell adhesion assayA cell adhesion assay was performed using the methods of Rodrigues. Briefly, 24-well culture plates were incubated with DNCPs, Nonspecific binding sites were blocked with bovine serum albumin. Cells suspension (5×104/ml) was plated into plates. Attached cells were harvested with trypsin, and counted in a haemocytometer. Data are expressed as the percentage of attached cells compared with negative control.1.4. 4 Analysis of alkaline phosphatase (ALP) activity, quantitative real time PCR and MineralizationTo investigate the potential of HPDLSC treated with DNCPs to differentiate into mineralizing cementoblast-like lineages in vitro, the ALP activity, real time RT-PCR and mineralization behavior were analyzed. The quantitative ALP activity was measured at 1, 7 and 14 days. The mRNA expressions of several osteoblastic markers, including COL ?, ALP and OCN were evaluated by real-time quantitative RT-PCR analysis following stimulated by DNCPs. cells were maintained in medium A or medium B and supplemented with osteogenetic media for 21 days. Mineralized formation was identified by Alizarin red staining. Amount of Alizarin red bound to the mineral in each dish was quantified according to Jo.1.4.5 In vitro Cells attachment to CCRDPrepared and sterilized CCRD were soaked in medium A or medium B for 24h. HPDLSCs were seeded on each CCRD surface. The morphologies of the CCRD and CCRD-cells composite were examined under SEM.1.4. 6 Statistical analysisData were analysed using SPSS version 10.0 (Chicago, IL, USA). Statistical analysis of the data was performed by student’s t-test. For all tests, significance level was set at P < 0.05 for all tests.1. 5 In vivo studyHigh density of HPDLSCs and ECM were harvested by TrypLETM Express and were seeded on each CCRD which having infiltrated with medium A or medium B. All the CCRD-cells composite were implanted into dorsal subcutaneous area of athymic mice. CCRD were also implanted into dorsal subcutaneous area of athymic mice as negative control. The specimens were harvested at 4 weeks post-transplantation, and stained with H&E.1.6 Preparation of Collagen/HA/GM controlled release TGF-β1 scaffoldsPreparation of gelatin microsphere TGF-β1 composite were fabricated according to the method described by Sha Huang and Faming Chen in our lab. The HA powders were prepared by a simple aqueous precipitation method as described in our previous reports. An 5%(w/w) equal volume of both nano-CDHA and gelatin microsphere- TGF-β1 was put into solution and stirred for 1 hour, Every 1 ml of composite solution was poured into one well of 24-well plate, and then kept at -200C overnight. These complexes were then frozen by immersion into -800C for 2 h and transferred into a freezedrying vessel (OHRIST BETA 1-15, Germany) for 24 h until dry. HPDLSCs were seeded onto scaffolds composite and cultured for 3 days. Then the scaffolds and cells compounds were observed under SEM and also traplanted into athymic mice for 2 weeks.2 Results2.1 Characteristics of HPDLSCs and HRAPSCsWe isolated postnatal stem cells from periodontium and apical tissues, and the isolated cells formed single-cell-derived colonies and most of the cells retained their fibroblastic spindle shape. Ex-vivo expanded HPDLSCs and HRAPSCs expressed the cell surface molecules STRO-1 by immunohisto- chemical staining. After 3 weeks of culture with an adipogenic inductive cocktail, stem cells developed into oil red O-positive lipid fat cells. Small round alizarin red-positive nodules formed in the cultures after 3 weeks of osteoblastic induction, indicating calcium accumulation in vitro.2.2 SEM observation of CCRDWhen the specimens were examined under SEM, CCRD demonstrated clean surface which was eliminated smear layer, opened and widened dentin tubules. Cells showing well growth were seen on all specimens. In dense cell area, cell sheets were formed and covered dentin surface. In sparse cell area which CCRD and cells treated with DNCPs, cells showed polygonal or cubical shape in stead of spindly shape. 2.3 Effect of DNCPs on HPDLSCsMTT assays showed that cells cultured with DNCPs demonstrated a statistically significant increase in proliferation at 3d and 5d as compared to control group (P < 0.05). In addition, the former showed a significantly higher rate of BrdU uptake than the later. The higher proliferation activity of induced HPDLSCs was further confirmed by flow cytometry.Both group of HPDLSCs showed the ability to form adherent clonogenic cell clusters. These results showed no statistically significant difference between the two groups. Observed under phase-contrast microscope, untreated cells were fibroblastic and bipolar in shape. While treated cells became flatter and most of them were cuboidal or polygonal after the induction of DNCPs for 7 days. The SEM results were coincident with above light microscope findings, there were mineralized secretary matrix granules can be found on the surface of the treated cellsCell adhesion assay showed both groups have proper adhesive ability. But increased cells attachment presented statistically in the group whose plates and cells were incubated with DNCPs (Fig. 4H; P<0.05).ALP activity of hPDLSCs in response to different DNCPs concentrations was increased. ALP activity of HPDLSCs was obviously higher under the induction of DNCPs with respect to non-induced group at 7 and 14 days (P<0.05), After incubation in mineralized culture for 21 days, HPDLSCs cultured in presence of DNCPs produced extensive sheets of calcified deposits whereas deposits of the HPDLSCs in absence of DNCPs were sparsely scattered. Quantification of the amount of Alizarin red also showed significantly difference.The mRNA expressions of COL I, ALP and OCN in presence of DNCPs caused a 1.7, 2.2, 1.2 folds increase compared with those in absence of DNCPs respectively for 2 day incubation.2.4 Historical observations of tissue samples in vivoIn 2 out of 6 DNCPs treated specimens for 4-week transplantation, new cementum-like tissue with cell-rich fibrous tissue adjacent or inserted into it formed along the CCRD surface. In 3 out of 6 DNCPs treated specimens, there are obvious separations between neo-formed tissues with dentin surface, only showing monostratified or stratified cubical cementoblast-like cells regularly aligned on dentin surface. While to the all DNCPs untreated group and one DNCPs treated specimen, no obvious cementum-like structure or periodontal fibrous tissue formed along the CCRD surface. To the negative control of naked CCRD transplantation, there was no host cells attachment or tissue regeneration on all CCRD surface.2.5 characteristics of Collagen/HA/GM controlled release TGF-β1 scaffoldsScaffolds exhibited macroporous microstructure. The pores were interconnected with pore size about 100-200 mm. scaffolds demonstrated superior biocompatibility.The amount of TGF-β1 was detected for 2 weeks in vitro and in vivo.3 Discussion:Natural dentin contains numerous collagenous protein and non-collagenous signaling molecular protein, sequestered in mineralized matrix, which includes BMPs, TGF-β, DSP, DPP, DMP-1 and others. Accordingly, we hypothesized that dentin molecules bound to matrix components or to hydroxyapatite crystals may be exposed or released as a consequence of injury to the periodontal ligament. 12,13,26,27,28,29 We hypothesized that dentin matrix produced by odontoblasts could migrate through and diffuse to periodontal tissues, which is similar to the well-known phenomenon of diffusion of enamel matrix proteins through the pre-mineralizing mantle dentin into the odontoblast layer.Based on previous studies and theory of root onset, we managed to determine whether the DNCPs derived from dentin provided additional advantages for the cellular events associated with periodontal regeneration. The present study indicated that DNCPs can promote proliferation potential and viability of HPDLSCs. In addition, induced HPDLSCs presented several features of cementoblast differentiation, as indicated by morphologic changes, enhanced alkaline phosphatase (ALP) activity, increased matrix mineralization, and up-regulated the expressions of mineralization associated genes such as Collagen I, OCN and ALP. Furthermore, our adhesion assay showed that DNCPs can slightly promote HPDLSCs attachment ability, compared with control treatment. It is suggested that, although the underlying mechanisms are still not well understood, cemento/osteoblastic differentiation of HPDLSCs may be affected by the dose of DNCPs according to our results.Clinically, organic or inorganic acid characterized as partly demineralization and deproteinization which is frequently used as root conditionding. They may partly demineralize the planed root surfaces, eliminate the smear layer, and expose some components of the extracelular matrix of dentin or cementum. Our SEM observations of CCRD demonstrated elimination of smear layer, opening and widening the tubules. Cell spreading was already evident and the penetration of cytoplasmic process into dentinal tubules was frequently observed.Ex-vivo-expanded HPDLSCs aggregate treated or untreated by DNCPs combining with CCRD were transplanted into immunocompromised mice. 4 weeks post-transplantation, histological findings demonstrated that new cementum-like tissue formed on CCRD surfaces. In light of these in vivo evidence, we testified again that DNCPs have provided additional advantages for the cellular events associated with periodontal regeneration.We developed a novel three-dimensional special scaffold consists of collagen, nano-calcium deficient hydroxyapatite and gelatin microsphere combining with controlled release growth factors, which provided structural support and stimulated repair, and within certain parameters, the ability to use it to promote periodontal tissue regeneration.In conclusion, our study has demonstrated that DNCPs is an effective stimulator of HPDLSCs proliferation, morphological changes, attachment as well as cenmento/osteoblastic differentiation. DNCPs facilitate HPDLSCs forming cementum-like adjacent fibrous tissue on CCRD. These findings therefore provide convincing evidence and useful data for DNCPs as a potent tool to facilitate periodontal regeneration.更多还原