胰岛素样生长因子-Ⅰ和骨形态发生蛋白-2对人牙周膜细胞增殖的作用

目的：重组人胰岛素样生长因子－Ｉ（ｒｈＩＧＦ－Ｉ）、重组人骨形态发生蛋白－２（ｒｈＢＭＰ－２）分别或联合应用对人牙周膜（ＰＤＬ）细胞增殖的影响。方法：采用组织块法体外培养人ＰＤＬ细胞,ＭＴＴ法测定ＰＤＬ细胞在不同生长因子刺激下的增殖情况。结果：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２都可促进人ＰＤＬ细胞的增殖,这种促增殖作用呈一定的浓度依赖性,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对人ＰＤＬ细胞的增殖有协同作用,且与单独应用相比相差显著。结论：ｒｈＩＧＦ－Ｉ、ｒｈＢＭＰ－２可望作为牙周再生的生物活性介质,ｒｈＩＧＦ－Ｉ与ｒｈＢＭＰ－２联合应用对ＰＤＬ细胞的促增殖作用更强。     

Effect of prostaglandin E2 on recombinant human bone morphogenetic protein-2-stimulated osteoblastic differentiation in human periodontal ligament cells

Recombinant human (rh) bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic differentiation in cells isolated from human periodontal ligament (HPLC), and this action of rhBMP-2 may be modulated by prostaglandins (PGs), which are local regulatory factors in the bone metabolism. In the present study, we investigated the effect of prostaglandin E2 (PGE2) on rhBMP-2-stimulated osteoblastic differentiation in cultured HPLC. rhBMP-2 (500 ng/ml)-stimulated alkaline phosphatase (ALPase) activity was enhanced by simultaneous treatment with low concentrations (10(-10)-10(-8) M) of PGE2, whereas a high concentration (10(-6) M) of PGE2 suppressed it. rhBMP-2 did not induce cyclo-oxygenase-2 (COX-2) mRNA expression or subsequent PGE2 production, whereas it remarkably suppressed rhIL-1 beta-induced COX-2 mRNA expression and PGE2 production. The rhBMP-2 action on osteoblastic differentiation in HPLC was also enhanced by co-treatment with 0.25 to 25 ng/ml of rh interleukin-1 beta (IL-1 beta). The ALPase activity stimulated by simultaneous treatment with rhBMP-2 and rhIL-1 beta was partially inhibited by addition of 10(-6) M of indomethacin, which completely inhibited rhIL-1 beta-induced PGE2 production. These results reveal that PGE2 at different concentrations exerts a biphasic effect on BMP-2-stimulated osteoblastic differentiation in HPLC, BMP-2 inhibits IL-1 beta-induced PGE2 production through suppressing COX-2 expression, and the BMP-2-stimulated osteoblastic differentiation may be enhanced by the endogenous PGE2 induced by BMP-2 and IL-1 beta. These suggest that BMP-2 action on osteoblastic differentiation in HPLC may be modulated by PGE2 in autocrine and paracrine fashions.     

In vitro effect of platelet-derived growth factor-BB on collagen synthesis and proliferation of human periodontal ligament cells

OBJECTIVES: Platelet-derived growth factor (PDGF)-BB is a polypeptide growth factor which has been shown to stimulate periodontal regeneration. In this study, we investigated the time- and dose-dependent effect of PDGF-BB on the proliferation and collagen synthesis of human periodontal ligament (PDL) cells. MATERIALS AND METHODS: For the proliferation assay, PDL cells were cultured in 0.01-10 ng ml(-1) of PDGF-BB for 12 or 24 h, and cell numbers were counted. For the collagen synthesis assay, PDL cells were cultured in 0.1-10 ng ml(-1) of PDGF-BB for 1 to 24 h. The ratio of collagen content in total protein was evaluated, and the gene expression of type I collagen was assessed quantitatively by Northern blotting analysis. RESULT AND CONCLUSIONS: PDGF-BB stimulated the proliferation of PDL cells in a time- and dose-dependent manner with the maximum effect at 10 ng ml(-1). PDGF-BB induced the collagen synthesis of PDL cells with the maximum effect for 24-h treatment, and 1 ng ml(-1) of PDGF-BB. PDGF-BB exhibits an inverse dose-dependent effect on proliferation and collagen synthesis by PDL cells. These findings suggest that PDGF-BB is one of the important regulators of the maintenance of the extracellular matrix in PDL, and may play an important role in the regeneration of PDL.     

骨形成蛋白-2和碱性成纤维细胞生长因子对牙周膜细胞碱性磷酸酶活性的联合效应

目的 :了解重组人骨形成蛋白 - 2 (rhBMP - 2 )和碱性成纤维细胞生长因子 (bFGF)单独和联合作用对人牙周膜细胞 (PDLC)碱性磷酸酶 (ALP)活性的影响。方法 :体外培养人PDLC ,分别用不同浓度的rhBMP- 2和bFGF单独或联合作用 ,用酶动力学方法检测PDLC的ALP活性。结果 :5 0～ 2 0 0 μg/L浓度的rhBMP - 2可显著增强人PDLC的ALP活性 (P <0 .0 1) ,而 10 μg/L浓度的bFGF可显著抑制人PDLC的ALP活性(P <0 .0 1) ,rhBMP - 2和bFGF联合作用仍可较明显地增强人PDLC的ALP活性 (P <0 .0 5 )。结论 :rhBMP - 2和bFGF联合应用可增强人PDLC的ALP活性     

血小板衍化生长因子-BB和碱性成纤维细胞生长因子对人牙周膜细胞增殖的影响

目的探讨重组人血小板衍生生长因子（rhPDGF）-BB和重组人碱性成纤维细胞生长因子（rh—bFGF）对人牙周膜细胞（PDLC）增殖的影响。方法原代培养人PDLC,应用甲噻唑四唑氮（MTT）比色和流式细胞仪检测rh-PDGF-BB和rh-bFGF单独及联合作用下细胞的增殖能力和增殖指数。结果rhPDGF-BB和rh-bFGF联合作用后,PDLC增殖能力提高,活性增强。其中,10μg&#183;L^-1的rhPDGF—BB加10μg&#183;L^-1的bFGF为最佳显效质量浓度,第3天为最佳显效时间。结论PDGF—BB和bFGF联合能更显著促进人PDLC的增殖并且具有明显的协同效应。     

Subculture affects the phenotypic expression of human periodontal ligament cells and their response to fibroblast growth factor-2 and bone morphogenetic protein-7 in vitro

Background and Objective: Although periodontal ligament cells display several osteoblastic traits, their phenotypic expression is still not well established. It remains a matter of debate whether they resemble a terminally differentiated cell type or an intermediate maturation state that potentially can be directed towards a fibroblastic or an osteoblastic phenotype. Material and Methods: To explore the characteristics of periodontal ligament cells in greater detail, fourth‐passage, sixth‐passage and eighth‐passage human periodontal ligament cells were cultured for up to 3 wk. Ki‐67, alkaline phosphatase, osteocalcin, osteoprotegerin and receptor activator of nuclear factor‐κB ligand (RANKL) mRNA expression was quantified by real‐time polymerase chain reaction. Furthermore, the cellular response to fibroblast growth factor‐2 and bone morphogenetic protein‐7 was examined in first‐passage and fourth‐passage cells. Dermal fibroblasts (1BR.3.G) and osteoblast‐like cells (MG63) served as reference cell lines. Results: Proliferation decreased over time and was highest in fourth‐passage cells. The expression of differentiation parameters, osteoprotegerin and RANKL increased with culture time and was higher in fourth‐passage cells than in cells of later passages. The RANKL/osteoprotegerin ratio increased steadily until day 21. Administration of fibroblast growth factor‐2 enhanced cell numbers in both passages, whereas alkaline phosphatase and osteocalcin production remained unchanged. By contrast, exposure of periodontal ligament cells to bone morphogenetic protein‐7 resulted in a reduction of cell number in the first and fourth passages, whereas the production of alkaline phosphatase and osteocalcin was enhanced. In dermal fibroblasts, differentiation parameters did not respond to both stimuli. MG63 cells behaved similarly to periodontal ligament cells. Conclusion: These results indicate that subculture affects the phenotypic expression of human periodontal ligament cells with respect to the characteristics that these cells share with osteoblasts. Furthermore, the periodontal ligament cell phenotype can be altered by fibroblastic and osteoblastic growth factors.     

人牙周膜干细胞的分离鉴定及骨形态发生蛋白-2对其趋化效应的研究

目的研究人牙周膜干细胞（PDLSCs）对骨形态发生蛋白-2（BMP-2）的趋化反应。方法通过有限稀释法分离、培养人PDLSCs,利用免疫荧光染色检测人PDLSCs波形丝蛋白及干细胞表面标志物STRO-1的表达,检测人PDLSCs多向分化能力,通过克隆形成实验和5-溴-2-脱氧尿嘧啶核苷（BrdU）共培养的方法检测其干细胞特性。利用24孔的Transwell细胞培养室来检测人PDLSCs对BMP-2的趋化反应,光镜下计迁移至滤膜下侧面的不同视野的细胞数。结果人PDLSCs抗波形丝蛋白染色阳性,表达干细胞表面标志物STRO-1,体外诱导培养的人PDLSCs能够向成骨细胞和成脂细胞分化,具有较高的自我更新能力,并在体外呈克隆状生长。在100、200 ng·mL-1 BMP-2实验组,Transwell细胞培养室中迁移的细胞数目显著多于空白对照组（P<0.01）。结论BMP-2对人PDLSCs有趋化效应。     

骨形成蛋白—2和成纤维细胞生长因子对人牙周膜细胞增殖的联合效应

目的：探讨重组人骨形成蛋白－2（rhBMP-2)和碱性成纤维细胞生长因子(bFGF) 单独或联合作用对人牙周膜细胞（PDLCs)增殖的影响。方法：体外培养人PDLCs,分别用不同浓度的rhBMP-2和bFGF单独或联合作用,用四唑盐比色法（MTT法）进行观察。结果：rhBMP-2和bFGF单独作用后PDLCs的增殖较对照组有明显的升高;而rhBMP-2与bFGF联合作用后PDLCs的增殖较各自单独作用有更明显的升高（P＜0．05）。结论：rhBMP-2与bFGF联合应用对于促进人PDLCs的增殖具有协同作用。     

rhBMP-2对人牙周膜细胞骨桥蛋白表达的影响

目的　深入了解牙周膜细胞 (periodontalligamentcells,PDLC)的成骨样细胞特性 ,以及非胶原蛋白在牙周组织矿化中的作用。方法　在体外培养条件下 ,观察重组人骨形成蛋白 2(recombinanthumanbonemorphogeneticprotein 2 ,rhBMP 2 )作用前后是否对矿化相关蛋白之一的骨桥蛋白 (osteopontin ,OPN)在人PDLC中的存在及表达情况产生影响。在小玻片上培养第 5代PDLC ,分为加rhBMP 2 (5 0 μg/L)刺激的实验组和不加任何因子的空白对照组 ,以地高辛标记的OPNcDNA探针 ,采用原位杂交技术对PDLC中OPN的表达情况进行检测 ;同时做PBS替代探针和RNA酶预处理的方法学对照。结果　对照组、替代对照和RNA酶预处理的PDLC均显示为阴性反应 ,没有显示出OPN的阳性表达信号 ;实验组PDLC的胞浆中可见明显的OPN阳性反应 ,表达信号较强。结论　rhBMP 2的刺激作用可使PDLC表达出较强的OPN阳性信号 ;表明在一定条件和外界因子的诱导下 ,PDLC具有向成骨特性方向转变的潜能 ,对牙周组织再生有促进效应。     

牛血小板衍化生长因子对人牙周膜成纤维细胞的生物学效应

目的：观察在牛血小板衍化生长因子作用下人牙周膜成纤维细胞ＤＮＡ和胶原蛋白合成的情况。方法：采用体外细胞培养技术和核素掺入法。结果：２０ｎｇ／ｍｌ～６０ｎｇ／ｍｌ牛血小板衍化生长因子可明显促进人牙周膜成纤维细胞ＤＮＡ合成，４０ｎｇ／ｍｌ浓度使细胞ＤＮＡ合成在２４ｈ达最高峰；对细胞的胶原蛋白合成无明显促进作用。结论：牛血小板衍化生长因子对人牙周膜成纤维细胞ＤＮＡ合成有促进作用，在牙周组织的创伤修复中可能起重要作用。     

Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage.     

Effect of recombinant human bone morphogenetic protein-2 on bone regeneration and osseointegration of dental implants

Recombinant human bone morphogenetic protein-2 (rhBMP-2) induced bone regeneration and osseointegration was evaluated in bony defects created within the hollow chamber of endosseous dental implants in 14 foxhound dogs. Bilateral extractions of mandibular premolars were performed and surgical implantation of 104 hollow cylinder implants followed after 8 weeks of healing. Experimental implants had their hollow chamber filled with 20 microg of rhBMP-2 delivered with a bovine collagen carrier, whereas the control implants had their apical chamber left empty. Dogs were followed for 2, 4, 8 and 12 weeks. Histomorphometric evaluation and immunohistochemical analysis were performed. Minimal bone was regenerated at 2 weeks for both groups. At 4 weeks, bone fill averaged 23.48% for the rhBMP-2 and 5.98% for the control group (P<0.05). At 8 weeks, mean bone fill was 20.94% and 7.75% for the rhBMP-2 and the controls, respectively (P<0.05). At 12 weeks, mean bone fill was 31.39% and 24.31% for the rhBMP-2 and control implants, respectively (P>0.05). Bone-implant contact (BIC) increased for both groups over time and at 8 weeks the rhBMP-2 BIC value was 18.65% and for the control 7.22% (P<0.05). At 12 weeks, the BIC was 43.78% and 21.05% for the rhBMP-2 and the control group, respectively (P<0.05). Immunohistochemical staining for type II collagen was positive only for parts of the collagen carrier and formation of cartilaginous intermediate was not observed in any of the specimens. The results suggest that, in confined defects adjacent to dental implants, rhBMP-2 can induce bone regeneration in close apposition to the implant surface.     

牛血小板衍化生长因子对人牙周膜成纤维细胞超微结构的影响

目的：对经牛血小板衍化生长因子作用的人牙周膜成纤维细胞超微结构进行观察。方法：透射电镜观察。结果：发现细胞超微结构发生了明显的变化，包括粗面内质网扩张和增多，发达的线粒体与大量的多聚核糖体形成，胞核增大，核内常染色质增多等。结论：牛血小板衍化生长因子可促进人牙周膜成纤维细胞ＤＮＡ、ＲＮＡ、蛋白质合成旺盛，代谢、增殖活跃。提示这些变化可能与细胞在该因子作用下生物学功能增强有密切关系。     

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

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

Modulation of osteogenic potential by recombinant human bone morphogenic protein-2 in human periodontal ligament cells: effect of serum, culture medium, and osteoinductive medium

BACKGROUND AND OBJECTIVE: Bone morphogenic proteins are known, in animal models, to promote many developmental processes, including osteogenesis. Clinical trials are currently underway to evaluate the potential of bone morphogenic proteins to promote bone and periodontal regeneration in humans. The aim of this study was to establish an optimal cell culture condition for using to study the biological effects of recombinant human bone morphogenic protein-2 on periodontal ligament cells. MATERIAL AND METHODS: The roles of serum concentration, types of culture medium (alpha-modified essential medium or Dulbecco's modified Eagle's medium), the presence of osteoinductive medium (including dexamethasone, ascorbic acid and beta-glycerophosphate), and timing of addition of the osteoinductive medium and recombinant human bone morphogenic protein-2, on the expression of alkaline phosphatase were investigated in cultured periodontal ligament cells. Cytochemical stainings and biological assay of alkaline phosphatase were also demonstrated. RESULTS: Our results suggested that an increased concentration of serum might mask the effect of recombinant human bone morphogenic protein-2 on the expression of alkaline phosphatase in periodontal ligament cells. alpha-Modified essential medium was found to induce a stronger cytochemical staining of the alkaline phosphatase than Dulbecco's modified Eagle's medium under similar culture conditions. Pre-incubation of cells with osteoinductive medium before the addition of various concentrations of recombinant human bone morphogenic protein-2 enhanced greater alkaline phosphatase expression than the simultaneous presence of both osteoinductive medium and recombinant human bone morphogenic protein-2. CONCLUSION: The findings of this study suggest that the effect of recombinant human bone morphogenic protein-2 on periodontal ligament cells could be efficiently investigated after the proper selection of culture variables and temporal sequence of adding bioactive factors. The optimal culture condition identified in this study might be useful in further studies to elucidate the regulatory mechanism of periodontal ligament cells in periodontal regeneration after stimulation with recombinant human bone morphogenic protein-2.     

Effect of recombinant human bone morphogenetic protein-12 (rhBMP-12) on regeneration of periodontal attachment following tooth replantation in dogs

OBJECTIVES: Subcutaneous and intramuscular implants of bone morphogenetic protein-12 (BMP-12) have been shown to induce formation of tendon and ligament tissue. BMP-12 induced a new attachment with a distinct fibrocartilaginous zone at the tendon-bone interface in the rat tendon-bone attachment model. Surgical controls showed poor healing and failure to reform the appropriate tendon-bone attachment morphologically. Application of recombinant human BMP-12 (rhBMP-12) to periodontal defects suggests that rhBMP-12 has the potential to support regeneration of the periodontal ligament (PDL). The objective of this pilot study was to evaluate this effect of rhBMP-12 in a tooth replantation model. METHODS: Six, young adult, male Hound Labrador mongrel dogs were used. Maxillary and/or mandibular incisor and premolar teeth were extracted and the PDL was either left "intact" or removed by root planing. rhBMP-12 (1.0 mg/ml) or a buffer control was topically applied to teeth with "intact" PDL in contralateral jaw quadrants in each of 3 animals. The teeth were immersed in 1.0 ml of the rhBMP-12 or the buffer solution for 10 min and then replanted. The remaining three animals received rhBMP-12 (1.0 mg/ml) and the buffer control in a similar fashion applied to teeth instrumented to remove the PDL and cementum, and surface demineralized with citric acid. The animals were euthanized at 8 weeks postsurgery and block sections were collected and processed for histopathologic analysis. RESULTS: No dramatic differences were found between teeth receiving topical rhBMP-12 and the buffer control. Application of rhBMP-12 did not have an apparent effect on new cementum and PDL formation in the tooth replantation model. Moreover, application of rhBMP-12 did not increase nor did it decrease the apparent presence and extent of ankylosis along the root surface compared to the control. CONCLUSIONS: The observations from this study do not support the use of topical rhBMP-12 to support the reestablishment of the PDL including regeneration of cementum and functionally oriented fibers, and to prevent ankylosis and root resorption following replantation of teeth.     

Human periodontal ligament cell responses to recombinant human bone morphogenetic protein-2 with and without bone allografts

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been found to promote the osteoblastic differentiation of human periodontal ligament cells. Its effect depends on the delivery system used. In this study we examined the effect of rhBMP-2 on the proliferation and osteoblastic differentiation of human periodontal ligament cells cultured alone or with 3 different bone allografts. METHODS: The rhBMP-2 effect on cell proliferation and osteoblastic differentiation was examined by measuring [3H] thymidine incorporation and ALPase activity, respectively, on human periodontal ligament (hPDL) cells. Two human demineralized freeze-dried allografts of cortical (DFDBAco) and cancellous (DFBDAca) bone origin and 1 non-demineralized freeze-dried allograft (FDBA) of cancellous bone origin, derived from different tissue banks, were used to evaluate the rhBMP-2 effect on cell osteoblastic differentiation. The measurements were taken on various days. RESULTS: rhBMP-2 decreased hPDL cell proliferation. rhBMP-2 acted on the third day of the process of cell differentiation, had a specific time of action, achieved its peak effect on the fourth and fifth days, and then did not provoke any further effects. The 3 bone allografts were efficiently combined with rhBMP-2. The combination of rhBMP-2 and DFDBAco showed the effect with the longest duration. rhBMP-2, on day 4, made the inactive bone allograft more active while, on the other days, its effect was dependent on the allograft alone. CONCLUSIONS: rhBMP-2 promotes the osteoblastic differentiation of human periodontal ligament cells and decreases cell proliferation. In this study rhBMP-2 in the presence of the bone allografts tested resulted in hPDL cell differentiation.     

Interactions of regenerative, inflammatory and biomechanical signals on bone morphogenetic protein-2 in periodontal ligament cells

Nokhbehsaim M, Deschner B, Winter J, Bourauel C, Rath B, Jäger A, Jepsen S, Deschner J. Interactions of regenerative, inflammatory and biomechanical signals on bone morphogenetic protein‐2 in periodontal ligament cells. J Periodont Res 2011; 46: 374–381.© 2011 John Wiley & Sons A/S Background and Objective: Regeneration of periodontal tissues by EMD remains a major challenge because a number of modifying factors are as yet unknown. The effects of EMD seem to be mediated, at least in part, by bone morphogenetic protein‐2 (BMP‐2). This in vitro study was performed to examine whether the effects of EMD on BMP‐2 activity are modulated by inflammatory and/or biomechanical signals. Material and Methods: Periodontal ligament cells were seeded on BioFlex^® plates and exposed to EMD under normal, inflammatory or biomechanical loading conditions for 1 and 6 d. In order to mimic proinflammatory or biomechanical loading conditions in vitro, cells were stimulated with interleukin‐1β (IL‐1β), which is increased at inflamed periodontal sites, and cyclic tensile strain of various magnitudes, respectively. The synthesis of BMP‐2, its receptors (BMPR‐1A, BMPR‐1B and BMPR‐2) and its inhibitors (follistatin, matrix gla protein and noggin) were analyzed using real‐time RT‐PCR and ELISA. Results: In EMD‐treated cells, BMP‐2 synthesis was increased significantly at 1 d. EMD also induced the expression of all BMP receptors, and of the BMP inhibitors follistatin and noggin. In general, IL‐1β and biomechanical loading neither down‐regulated BMP‐2 nor up‐regulated BMP inhibitors in EMD‐stimulated cells. However, IL‐1β and biomechanical loading, when applied for a longer time period, caused a down‐regulation of EMD‐induced BMP receptors. Conclusion: EMD induces not only BMP‐2, but also its receptors and inhibitors, in PDL cells. IL‐1β and biomechanical forces may counteract the beneficial effects of EMD on BMP‐2 activity via the down‐regulation of BMP receptors.     

两种体外培养人牙周韧带成纤维细胞方法比较

目的探索一种在体外短时间内简便、可靠获取大量人牙周韧带成纤维细胞(human periodontal ligament fibroblast,HPLF)、建立稳定的体外培养体系的方法。方法采用酶消化法和组织贴块法进行HPLF体外原代培养及传代培养的对比研究。通过细胞形态学、超微结构观察及波形蛋白和角蛋白免疫组化染色等对细胞进行定性研究;测定细胞生长曲线了解细胞生长基本规律及其增殖能力。结果采用酶消化法和组织贴块法均可成功的进行HPLF连续传代培养。最高传代数为30代。培养的细胞具有成纤维细胞的典型形态,波形蛋白染色阳性,角蛋白染色阴性,生长稳定期倍增时间为48~72h。组织块培养法需培养时间长,原代培养获取的细胞量较少,较难传代。酶消化法可短时间内获取大量细胞,细胞产量高,但操作手续复杂易污染,细胞易受损伤。结论成功建立了一个稳定的HPLF体外培养体系。除常用的组织块法外,胰蛋白酶及胶原酶联合消化法不失为一种简便、快速、可靠的组织原代分离培养方法。     

茶多酚对牙周膜细胞的影响

茶多酚(TP)是从茶叶中提取的多酚类物质,是茶叶药效的主要活性成分,具有抗氧化、抗脂质过氧化、清除自由基、抗基因突变和抗肿瘤形成等生物学活性和药理效应.此外,TP还有较强的抗炎和抑制免疫炎症反应的作用,对牙周炎有较强的抑制作用.牙周膜细胞(PDLC)是牙周组织中重要的细胞成分,能合成胶原并将其释放于细胞外基质进一步形成胶原纤维,而胶原纤维对牙体组织有重要的作用.笔者下面就TP对PDLC的作用作一综述,以进一步探讨其对PDLC附着和增殖的影响.