成牙骨质细胞研究现状

牙骨质是覆盖在牙根表面的一种矿化组织,其发生和形成对牙周组织的发生、发育和再生起着非常重要的作用.目前这方面的研究主要着重于对成牙骨质细胞的研究上,本文就成牙骨质细胞在来源和分化、矿化相关蛋白和特异表达因子、体外培养以及在正畸学中的研究现状作一综述.     

成牙骨质细胞研究现状

牙骨质是覆盖在牙根表面的一种矿化组织，其发生和形成对牙周组织的发生、发育和再生起着非常重要的作用。目前这方面的研究主要着重于对成牙骨质细胞的研究上，本文就成牙骨质细胞在来源和分化、矿化相关蛋白和特异表达因子、体外培养以及在正畸学中的研究现状作一综述。     

牙囊细胞条件培养液在大鼠脂肪间充质干细胞向成牙骨质分化中的作用

目的：探讨牙囊细胞条件培养液（DFCCM）在诱导大鼠脂肪间充质干细胞（ADSCs）向成牙骨质样细胞分化中的作用。方法：分离培养大鼠脂肪间充质干细胞、牙囊细胞,制备DFCCM。用DFCCM诱导ADSCs,通过MTT检测ADSCs增殖能力的变化;免疫荧光及实时定量PCR方法检测成骨关键蛋白一骨钙素（OCN）、碱性磷酸酶（ALP）及成牙骨质关键蛋白一牙骨质附着蛋白（CAP）,牙骨质蛋白（CP23）的情况。结果：牙囊细胞条件培养液诱导ADSCs后可抑制ADSCs的增殖能力。DFCCM诱导7d后,ADSCs细胞浆内表达OCN及CAP。同时DFC—CM诱导ADSCs后CAP,CP23,ALP及OCNmRNA表达水平显著高于对照组。结论：牙囊细胞条件培养液构建的微环境可使ADSCs向成牙骨质样细胞分化,为于细胞介导的牙骨质再生提供新的思路。     

牙骨质相关细胞的研究进展

到目前为止，人们对牙骨质的了解仍较少。人牙骨质再生被认为是牙周病暴露的根面上牙周组织再生的关键。本文回顾了学者们对牙骨质及其相关细胞的研究，阐述了形成牙骨质前体细胞的位置以及成牙骨质细胞的起源。     

牙囊和上皮根鞘细胞成无细胞牙骨质的机制

无细胞牙骨质是锚定牙周纤维和牙根的重要组织结构,牙囊细胞（DFC）通过自身分化和上皮根鞘（ERS）通过上皮-间质转化（EMT）形成的成牙骨质细胞参与无细胞牙骨质形成。牙囊细胞体外移植后可形成纤维样和牙骨质样组织,而牙乳头和上皮根鞘可以诱导牙囊细胞从基因及其蛋白质水平高表达碱性磷酸酶、骨涎蛋白等无细胞牙骨质相关标志物。其中钙离子、骨形态发生蛋白、Wnt／β-连环蛋白通路等目前被认为是介导牙囊细胞成牙骨质分化的信号转导通路。目前发现,调控无机磷酸盐胞内外浓度的转运蛋白对无细胞牙骨质形成调节的敏感性更高。ERS亦可通过EMT形成间质细胞来参与牙周组织的发生。钙结合蛋白D28k、末端较小的同源异形盒-2等在无细胞牙骨质、ERS以及牙囊细胞表达差异都说明了ERS具有成牙骨质细胞特性。在此过程中,TGFβ激活下游蜗牛蛋白后,通过磷脂酰肌醇-3-激酶和蛋白激酶B信号转导通路来介导ERS的无细胞牙骨质分化过程。     

釉基质蛋白影响牙周组织再生的研究进展

众多研究表明由Hertwig上皮根鞘内层的成釉细胞分泌的釉基质蛋白和釉基质蛋白样因子可以调节参与牙周组织再生细胞的活性,参与诱导形成新的牙骨质和牙周支持组织,本文就近年来釉基质蛋白诱导牙周组织再生方面的研究进展做一综述。     

釉基质蛋白影响牙周组织再生的研究进展

众多研究表明由Hertwig上皮根鞘内层的成釉细胞分泌的釉基质蛋白和釉基质蛋白样因子可以调节参与牙周组织再生细胞的活性,参与诱导形成新的牙骨质和牙周支持组织,本文就近年来釉基质蛋白诱导_牙周组织再生方面的研究进展做一综述。     

牙骨质活性蛋白的组成、提取及其作用机理

牙骨质基质中有机成分主要由胶原和糖蛋白组成,其中糖蛋白对牙骨质周围牙龈组织和牙周组织的形成及再生起着非常重要的作用。近年来,国外不少学者对牙骨质基质中活性蛋白的组成和提取以及牙骨质活性蛋白对成纤维细胞的生成、移行、粘附等作用进行了深入的研究,本文就目前牙骨质活性蛋白的组成、来源、提取、活性测定、作用和作用机理研究状况作一综述。     

牛牙骨质来源细胞的分离、培养和鉴定

目的 探索体外分离培养牙骨质来源细胞（cementum-derived cells,CDC)的方法，并研究其生物学特性。方法 用组织法联合酶消化法，分离培养新生牛牙根表面的细胞；免疫细胞化学方法检测CDC中与骨相关的标志物如骨钙素、碱性磷酸酶和特异性牙骨质附着蛋白的表达；改良钙钴法检测碱性磷酸酶的活性。结果 培养增殖的细胞具有成牙骨质细胞的形态特征，经5次传代后细胞形态无明显改变；该细胞骨钙素、碱性磷酸酶免疫细胞化学染色均为阳性，并有碱性磷酸酶活性表达；牙骨质附着蛋白呈阳性着色。结论 初步确定本实验首次成功分离培养了牛牙骨质来源的、具成牙骨质细胞特性的成牙骨质细胞。为进一步研究成牙骨质细胞的生物学特性及牙骨质生成和修复提供了条件。     

甲状旁腺激素相关蛋白与成牙骨质细胞

成牙骨质细胞主要位于牙骨质表面,是由牙囊细胞分化而来。牙骨质覆盖于牙根表面,具有保持牙周组织平衡的作用。能否促进成牙骨质细胞形成新的牙骨质是评价牙周疾病治疗效果的重要方面。近年来一些研究对多肽类生长分化因子调控成牙骨质细胞生物活性的作用和机制进行了探讨,取得了新的进展,该文对甲状旁腺激素相关蛋白对成牙骨质细胞的调控作用进行综述。     

Emdogain诱导人牙周膜细胞群向成牙骨质细胞分化的研究

目的探讨人牙周膜细胞群（human periodontal ligament cell populations,hPDLPs）在Emdogain（EMD,商品化的猪釉基质蛋白）诱导下生物学特性的改变。方法组织块法分离培养牙周膜细胞,用含100 mg/L EMD的培养液诱导6 d,光镜下观察细胞形态改变,扫描电镜观察细胞在牙骨质片上的形态变化。免疫细胞化学检测成牙骨质细胞相关矿化蛋白：骨涎蛋白（bone sialoprotein,BSP）、Ⅰ型胶原蛋白（collagenⅠ,COLⅠ）的表达。结果EMD诱导后细胞由长梭形向多角形的类成牙骨质细胞分化。免疫组织化学检测结果显示诱导后BSP、COLⅠ的表达增强。结论EMD可以诱导人牙周膜细胞群向类成牙骨质细胞方向分化。     

Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed.     

Biology of periodontal regeneration: a review and a hypothesis for future researches

Since the end of the seventies several studies have been carried out about the possibility of regeneration of periodontal osseous defects. The results of such researches supported by histological tests allowed to establish which surgical techniques could yield periodontal regeneration in a predictable way. In spite of a quite large diffusion of these sugical techniques in the treatment of periodontal osseous defects, some biological aspects of periodontal regeneration are still unknown. The most important among them is the origin and the differentiation pathway of the synthetic cells that shall provide for the reconstruction of the deep periodontium. Particularly, the phenotypic pattern and the origin of cementoblasts are not clear. Our researches focused on pericytes, a mesenchyma-derived cell population with remarkable differentiation capacities which have a microvascular location. We considered the hypothesis that pericytes could play an important role in regeneration of the alveolar bone, of the cementum and of the periodontal ligament. Should pericytes be actually the precursors of osteoblatsts, fibroblasts and cementoblasts involved in periodontal regeneration, new therapies, such as cultivated cells reimplantation or the targeted use of growth factors could be feasible. Future researches shall verify these observations.     

Cementoblast delivery for periodontal tissue engineering

BACKGROUND: Predictable periodontal regeneration following periodontal disease is a major goal of therapy. The objective of this proof of concept investigation was to evaluate the ability of cementoblasts and dental follicle cells to promote periodontal regeneration in a rodent periodontal fenestration model. METHODS: The buccal aspect of the distal root of the first mandibular molar was denuded of its periodontal ligament (PDL), cementum, and superficial dentin through a bony window created bilaterally in 12 athymic rats. Treated defects were divided into three groups: 1) carrier alone (PLGA polymer sponges), 2) carrier + follicle cells, and 3) carrier + cementoblasts. Cultured murine primary follicle cells and immortalized cementoblasts were delivered to the defects via biodegradable PLGA polymer sponges, and mandibulae were retrieved 3 weeks and 6 weeks post-surgery for histological evaluation. In situ hybridization, for gene expression of bone sialoprotein (BSP) and osteocalcin (OCN), and histomorphometric analysis were further done on 3-week specimens. RESULTS: Three weeks after surgery, histology of defects treated with carrier alone indicated PLGA particles, fibrous tissue, and newly formed bone scattered within the defect area. Defects treated with carrier + follicle cells had a similar appearance, but with less formation of bone. In contrast, in defects treated with carrier + cementoblasts, mineralized tissues were noted at the healing site with extension toward the root surface, PDL region, and laterally beyond the buccal plate envelope of bone. No PDL-bone fibrous attachment was observed in any of the groups at this point. In situ hybridization showed that the mineralized tissue formed by cementoblasts gave strong signals for both BSP and OCN genes, confirming its nature as cementum or bone. The changes noted at 3 weeks were also observed at 6 weeks. Cementoblast-treated and carrier alone-treated defects exhibited complete bone bridging and PDL formation, whereas follicle cell-treated defects showed minimal evidence of osteogenesis. No new cementum was formed along the root surface in the above two groups. Cementoblast-treated defects were filled with trabeculated mineralized tissue similar to, but more mature, than that seen at 3 weeks. Furthermore, the PDL region was maintained with well-organized collagen fibers connecting the adjacent bone to a thin layer of cementum-like tissue observed on the root surface. Neoplastic changes were observed at the superficial portions of the implants in two of the 6-week cementoblast-treated specimens, possibly due in part to the SV40-transformed nature of the implanted cell line. CONCLUSIONS: This pilot study demonstrates that cementoblasts have a marked ability to induce mineralization in periodontal wounds when delivered via polymer sponges, while implanted dental follicle cells seem to inhibit periodontal healing. These results confirm the selective behaviors of different cell types in vivo and support the role of cementoblasts as a tool to better understand periodontal regeneration and cementogen-     

Comparison of gene expression profile of cementoblasts with periodontal ligament cells in mouse mandible with laser capture microdissection

Cementum is an essential tissue to maintain tooth function and should be closely correlated to tooth root development and periodontal tissue regeneration. However, detailed features of the periodontium including cementum and specific markers for cementoblasts are unknown. Moreover, the molecular mechanism of periodontal tissue development, homeostasis and regeneration remains unknown. Previous studies have usually examined cementum or periodontalligament (PDL) tissue obtained by manual curettage, resulting in difficulties in isolating pure cementum or PDL. We employed laser capture microdissection (LCM) to isolate cementoblasts and PDL cells from undecalcified frozen sections of murine mandible and to obtain RNA of good quality for subsequent genetic analysis. Over 500 cementoblasts and PDL cells were separately laser captured under microscopy. A bioanalyzer detected peaks of 18S and 28S rRNA both in the laser-dissected cementoblasts and in PDL cells, suggesting that the RNA was of sufficient quality. The RNA samples were amplified due to their small amount and a comparative analysis of mRNA expression by GeneChip showed that about 2,000 genes were differentially expressed between cementoblasts and PDL cells. Both cementoblast-positive and PDL cell-negative genes were serially analyzed by quantitative RT-PCR using RNA samples obtained from mandibles and femurs. Several genes were expressed at higher levels in the mandible than in the femur, suggesting that some might be cementoblast-specific markers. We established a novel experimental system with which to isolate target tissues from single cells in undecalcified frozen sections and to obtain intact RNA. These methodologies could be useful for further investigation of mineralized tissues and to explore tissue-specific factors.     

Employing a transgenic animal model to obtain cementoblasts in vitro

BACKGROUND: Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. METHODS: In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. RESULTS: The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. CONCLUSIONS: This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.     

Expression of functional Toll-like receptors and nucleotide-binding oligomerization domain proteins in murine cementoblasts and their upregulation during cell differentiation

Background and Objective:  While the primary role of cementoblasts is to synthesize the components of cementum, we have reported that immortalized murine cementoblasts (OCCM-30) express functional Toll-like receptor (TLR)-2 and -4, and these receptors are involved in the alteration of gene expression associated with cementum formation and in the upregulation of osteoclastogenesis-associated molecules, such as receptor activator of nuclear factor-κB (NF-κB) ligand. We hypothesized that cementoblasts express a wide range of pattern recognition receptors in a manner comparable to osteoblasts, which are known to express various functional TLRs and nucleotide-binding oligomerization domain (NOD) proteins.
Material and Methods:  Murine cementoblasts and pre-osteoblasts were used. The gene and protein levels of TLRs/NODs were analyzed using real-time polymerase chain reaction and flow cytometry. Interleukin-6 (IL-6) and activated NF-κB were measured using enzyme-linked immunosorbent assay.
Results:  The expressions of TLR-1, -2, -4, -6 and -9, CD14, NOD-1 and -2 were detected in cementoblasts and were upregulated upon differentiation induced by ascorbic acid. Similar patterns were observed in the mouse MC3T3-E1 osteoblast cell line. Synthetic ligands, Pam3CSK4 (TLR-1/2 agonist), Pam2CGDPKHPKSF (TLR-2/6 agonist), lipid A (TLR4 agonist), CpG DNA (TLR-9 agonist), FK565 (NOD1 agonist) and muramyldipeptide (NOD2 agonist), effectively induced NF-κB activation in cementoblasts and/or ascorbic acid-treated cementoblasts. Furthermore, these ligands induced IL-6 production in a NF-κB-dependent manner in cementoblasts and/or ascorbic acid-treated cementoblasts.
Conclusion:  These results indicate that cementoblasts possess functional TLR and NOD signaling systems and have a similar capacity to osteoblasts in responding to a wide variety of pathogens.