rhBMP-2对人牙龈成纤维细胞体外矿化能力的影响

目的 探讨重组人骨形成蛋白-2(rhBMP-2)对人牙龈成纤维细胞(GF)体外矿化能力的影响。方法 以组织块培养法培养人牙龈成纤维细胞，分别以含或不含rhBMP-2的矿化培养液长期培养，以倒置显微镜观察细胞生长状况。于培养第7天检测碱性磷酸酶(ALP)活性。于培养第30天行von Kossa染色观察矿化结节。结果 rhBMP-2提高GF的ALP水平，呈剂量依赖性，并能够刺激GF在体外形成矿化结节。结论 rhBMP-2能够诱导人GF向成骨细胞表型分化。     

兔骨髓基质干细胞骨向诱导实验研究

目的:研究兔骨髓基质干细胞(bone marrow stromal cells,BMSC)的体外培养以及骨向诱导分化情况,进一步探讨其作为骨组织工程种子细胞的可行性。方法:无菌条件下抽取兔的股骨骨髓,然后进行骨髓基质干细胞的分离和体外培养,并向成骨细胞定向诱导分化。结果:体外分离的骨髓基质细胞呈贴壁生长,改良MTT法测定显示骨髓基质细胞于第7.8天左右可达到增殖高峰;诱导后BMSC可向成骨细胞分化,细胞呈成骨细胞形态为梭形和多角形并可形成钙化结节。改良钙钴染色法检测诱导细胞碱性磷酸活性呈强阳性。扫描电镜观察细胞呈较典型的成骨细胞状态,并可见钙盐沉积。结论:骨髓基质细胞具有来源充足,取材方便,创伤小无明显并发症。骨髓基质干细胞分化增殖能力较强,成骨能力确定。所以采用骨髓基质细胞作为种子细胞构建组织工程骨有比较可靠的依据。     

The effects of enamel matrix derivative (EMD) on osteoblastic cells in culture and bone regeneration in a rat skull defect

OBJECTIVE: Enamel matrix derivative (EMD) has been clinically used to promote periodontal tissue regeneration. The purpose of the present study is to clarify EMD affects on osteoblastic cells and bone regeneration. MATERIALS AND METHODS: Mouse osteoblastic cells (ST2 cells and KUSA/A1 cells) are used in culture experiments. After cells were treated with EMD, cell growth was evaluated with DNA measurement, 5-bromo-2'-deoxyurydine (BrdU) incorporation assay. Measurement of alkaline phosphatase (ALP) activity and mineralized-nodule (MN) formation, Northern blotting analysis and zymography are also performed. In addition, EMD was applied to a rat skull defect and the defect was radiographically and histologically evaluated 2 weeks after the application. RESULTS: EMD did not stimulate ST2 cell growth; however, it enhanced KUSA/A1 cell proliferation. Although EMD stimulated ALP activity in both the cells, ALP activity in KUSA/A1 cells was affected to a much greater degree. Corresponding to the increase in ALP activity, MN formation in KUSA/A1 cells was enhanced by EMD. EMD stimulated osteoblastic phenotype expression of KUSA/A1 cells such as type I collagen, osteopontin, transforming growth factor beta 1 and osteocalcin. EMD treatment also stimulated matrix metalloproteinase production in KUSA/A1 cells. Although the effects of EMD on osteoblastic cells depend on cell type, the overall effect of EMD on osteoblastic cells is stimulatory rather than inhibitory. Finally, EMD application to a rat skull defect accelerated new bone formation. CONCLUSION: These results indicate that EMD affects osteoblastic cells and has potential as a therapeutic material for bone healing.     

In vitro biologic response of human bone marrow stromal cells to enamel matrix derivative

BACKGROUND: In vitro investigations suggest that enamel matrix derivative (EMD) may affect the biologic response of periodontal-related cells, including osteoblasts and their precursors, the bone marrow stromal cells (BMSCs), which could play a crucial role in the regenerative process. In this study, we investigated the effects of EMD on human BMSCs. METHODS: Primary cultures of BMSCs were obtained from bone marrow samples of healthy donors. Cell proliferation and osteogenic marker expression in response to serial dilutions of EMD (12.5, 25, and 50 microg/ml) were assessed. Cell growth was measured by 3H-thymidine incorporation and type I collagen synthesis by immunoblotting. Alkaline phosphatase (AP)-specific activity in the early phase (7 days), in vitro mineralization by von Kossa staining and calcium quantification, and osteocalcin levels at prolonged times (3 weeks) also were evaluated. RESULTS: EMD stimulated BMSC growth in a dose-dependent manner. When EMD 50 microg/ml was followed over time, the highest proliferative effect was evident at 24 hours (3.4-fold of the control). Type I collagen level was significantly lower than the control after a 7-day incubation with EMD 50 microg/ml. AP activity was reduced in a dose-dependent manner down to 55% of the control. Also, the extracellular matrix mineralization decreased in EMD-treated cells with respect to the control, whereas only a slight, not significant, decrease in osteocalcin levels was found. CONCLUSIONS: EMD significantly increased BMSC growth and simultaneously decreased their osteogenic differentiation. The clinical efficacy of EMD in regenerating periodontal tissues can be attributed, in part, to the biologic effects exerted on the bone marrow stromal component of resident cells.     

The effects of enamel matrix derivative (EMD) on osteoblastic cells

Enamel matrix derivative (EMDOGAIN, EMD) has been clinically used to promote regeneration of periodontal tissue, including cementum, periodontal ligament (PDL), and alveolar bone. However, it has not been clear whether EMD directly affects osteoblastic cells. To answer this question, we examined EMD effects on bovine PDL cells, rat and mouse bone marrow cells (RBM cells and MBM cells, respectively), and mouse osteoblastic cells (Kusa/A 1 cells). EMD was dissolved in 10 mM acetic acid and added to the culture medium at a final concentration of 50 micrograms/ml. EMD stimulated mineralized-nodule formation of PDL cells, RBM cells, and Kusa/A 1 cells. In Kusa/A 1 cells, EMD enhanced ALP activity, together with DNA content. Northern blotting analysis on Kusa/A 1 cells demonstrated stimulatory effects of EMD on the gene expression of type I collagen and osteopontin. Further, application of EMD on MBM cell culture, under 1,25(OH)2 vitamin D3 supplementation, stimulated osteoclast-like cell formation. These results indicate that osteoblastic cells respond to EMD, and that EMD would be potentially useful for bone regeneration.     

Enamel matrix derivative promotes osteoclast cell formation by RANKL production in mouse marrow cultures

OBJECTIVES: Enamel matrix derivative (EMD) is used clinically to promote periodontal tissue regeneration, however, there are few reports regarding effects of EMD on bone metabolism. We evaluated the influence of EMD on osteoclast formation using in vitro bone marrow culture. METHODS: Bioactive fractions were purified from EMD by reverse-phase HPLC on a C18 hydrophobic support, then mouse bone marrow cells were cultured with EMD or its purified fractions for 8 days. Following tartrate resistant acid phosphatase (TRAP) staining, TRAP-positive multinucleated cells were counted. The expression of receptor activator of NF-kappaB ligand (RANKL) in osteoblastic cells was detected using immunoblotting. RESULTS: EMD was dissolved in 0.1% (vol/vol) trifluoroacetic acid and applied to a C18 column for HPLC. Two major peaks were obtained of which the second (fraction numbers 21-25) was found to induce the formation of osteoclasts in mouse marrow cultures. Further, osteoprotegerin completely inhibited osteoclast formation in mouse marrow cultures with or without osteoblastic stromal cells, when being cultured with EMD or its purified fractions. In addition, Western blot analysis revealed the presence of RANKL in mouse osteoblastic cells stimulated with EMD or its purified fractions. CONCLUSION: Our results indicate that EMD induces the formation of osteoclasts through RANKL expressed by osteoblastic cells, and suggest that EMD may regulate both bone formation and bone resorption during periodontal tissue regeneration.     

Effects of enamel matrix derivative and transforming growth factor-beta1 on human periodontal ligament fibroblasts

AIM: The objective of this study was to evaluate the effects of enamel matrix derivative (EMD), transforming growth factor-beta1 (TGF-beta1), and a combination of both factors (EMD+TGF-beta1) on periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS: Human PDL fibroblasts were obtained from three adult patients with a clinically healthy periodontium, using the explant technique. The effects of EMD, TGF-beta1, or a combination of both were analysed on PDL cell proliferation, adhesion, wound healing, and total protein synthesis, and on alkaline phosphatase (ALP) activity and bone-like nodule formation. RESULTS: Treatment with EMD for 4, 7, and 10 days increased cell proliferation significantly compared with the negative control (p<0.05). At day 10, EMD and EMD+TGF-beta1 showed a higher cell proliferation compared with TGF-beta1 (p<0.01). Cell adhesion was significantly up-regulated by TGF-beta1 compared with EMD and EMD+TGF-beta1 (p<0.01). EMD enhanced in vitro wound healing of PDL cells compared with the other treatments. Total protein synthesis was significantly increased in PDL cells cultured with EMD compared with PDL cells treated with TGF-beta1 or EMD+TGF-beta1 (p<0.05). EMD induced ALP activity in PDL fibroblasts, which was associated with an increase of bone-like nodules. CONCLUSION: These findings support the hypothesis that EMD and TGF-beta1 may play an important role in periodontal regeneration. EMD induced PDL fibroblast proliferation and migration, total protein synthesis, ALP activity, and mineralization, while TGF-beta1 increased cellular adhesion. However, the combination of both factors did not positively alter PDL fibroblast behaviour.     

Osteoblast differentiation of human bone marrow cells under continuous and discontinuous treatment with dexamethasone

Dexamethasone (Dex) has been shown to induce osteoblast differentiation in several cell culture systems. This study investigated the effect of continuous and discontinuous treatment with Dex on osteoblast differentiation of human bone marrow stromal cells (BMSC). Primary culture and first passage were cultured in media with or without Dex 10(-7) M. During the culture period, cells were incubated at 37 degrees C in humidified atmosphere of 5% CO2 and 95% air. At 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity and bone-like formation were evaluated. Data were compared by two-way analysis of variance. Dex did not affect cell viability and total protein content, but reduced cell number. ALP activity and bone-like formation increased when only first passage or both primary culture and first passage were treated with Dex, in comparison to the groups that did not have contact with Dex after first passage. The results of this study indicate that, for human BMSC, continuous presence of Dex did not appear to be required for development of the osteoblast phenotype, but Dex must be present after first passage to allow osteoblast differentiation expressed by reduced cell proliferation and increased ALP activity and bone-like formation.     

In vitro wound healing responses to enamel matrix derivative

BACKGROUND: Enamel matrix derivative (EMD) contains a variety of hydrophobic enamel matrix proteins and is extracted from developing embryonal enamel of porcine origin. EMD has been associated with the formation of acellular cementum and it has been found to stimulate periodontal regeneration. The present study was established to investigate the influence of EMD on human periodontal ligament (PDL) cells, gingival fibroblasts (GF), and osteosarcoma (MG-63) cells on wound-fill rates using an in vitro wound model. METHODS: Wounds were created by making 3 mm incisions in cell monolayers across the length of tissue culture plates. The wounded PDL, GF, and MG-63 cell monolayers were treated with media containing EMD over a concentration range of 5 to 100 microg/ml, platelet-derived growth factor (PDGF-BB) at 20 ng/ml as a positive control and insulin-like growth factor (IGF-I) at 100 ng/ml as a negative control. PDL cell wounded monolayers also were treated in EMD coated tissue culture plates. After an incubation period (up to 9 days), the cells were fixed and stained and cellular fill was measured across the width of the wound by computer-assisted histomorphometry. RESULTS: When PDL, GF, and MG-63 cells were exposed to EMD in culture medium, an enhanced wound-fill was observed for all cells compared to untreated conditions. At early time points, PDL wound-fill rates in the presence of EMD were statistically greater than the rates of GF and MG-63 treated with EMD (P<0.001). There were no significant differences in wound-fill rates of PDL cells treated with EMD in medium versus EMD coated on culture plates. At days 3 and 6 post-wounding, PDL cells showed a significantly greater response to EMD than to PDGF-BB (P <0.001). EMD also had a greater effect on GF wound-fill rates than PDGF-BB at days 6 and 9. MG-63 cells were less responsive to PDGF-BB and EMD than PDL cells and GF. All 3 cell types treated with IGF-I showed no significant increase of wound-fill rates. CONCLUSION: The present data support the concept that clinical application of enamel matrix derivative may enhance periodontal wound regeneration by specifically modifying periodontal ligament cell proliferation and migration.     

Coverage of gingival recession defects using guided tissue regeneration with and without adjunctive enamel matrix derivative in a dog model

The aim of this study was to clarify the adjunctive effect of enamel matrix derivative (EMD) on expanded polytetrafluoroethylene membrane guided tissue regeneration (GTR)-based root coverage by creating gingival recessions in beagle dogs. Recessions were treated with GTR + EMD, GTR alone, or neither GTR nor EMD (control). The control group was characterized by long junctional epithelium and little bone formation. The GTR + EMD group showed a statistically significant increase (P < .01) in new bone and cementum formation compared with the GTR group. The results of the present investigation suggest that the adjunctive use of EMD with GTR promotes formation of new bone and cementum without root resorption in recession-type defects in dogs.     

The therapeutic role of bone marrow stem cell local injection in rat experimental periodontitis

Mesenchymal stem cell therapy brings hope for regenerating damaged periodontal tissues. The present study aimed to investigate the therapeutic role of local bone marrow stem cell (BMSC) injection in ligation-induced periodontitis and the underlying mechanisms. Alveolar bone lesion was induced by placing ligatures subgingivally around the bilateral maxillary second molars for 28 days. The alveolar bone lesion was confirmed by micro-CT analysis and bone histomorphometry. Allogeneic BMSC transplantation was carried out at 28 day after ligation. The survival state of the transplanted BMSC was observed by bioluminescent imaging. The implantation of the BMSC into the gingival tissues and periodontal ligament was confirmed by green fluorescent protein (GFP) immunohistochemical staining. The expression level of pro-inflammatory, tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and receptor activator of nuclear factor-κ B ligand (RANKL) and osteoprotegerin (OPG) in periodontal tissues were evaluated by immunohistochemical staining and real-time PCR. Significant reverse of alveolar bone lesion was observed after BMSC transplantation. The expression of TNF-α and IL-1β was down-regulated by BMSC transplantation. The number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the periodontal ligament was reduced, and the increased RANKL expression and decreased OPG expression were also reversed after BMSC transplantation. It is concluded that allogeneic BMSC local injection could inhibit the inflammation of the periodontitis tissue and promote periodontal tissue regeneration.     

兔骨髓基质细胞的体外成骨定向诱导培养

目的:体外分离培养兔骨髓基质细胞(bonemarrowstromalcells,BMSCs),并向成骨定向诱导培养,为骨组织工程提供适宜的种子细胞。方法:抽取新西兰白兔的骨髓,体外分离纯化得到BMSCs,并利用条件培养液将其向成骨定向诱导培养、扩增。采用倒置相差显微镜观察细胞增殖分化情况;使用碱性磷酸酶(ALP)和VonKossa染色的方法,鉴定其成骨性能;用MTT法描绘标准细胞浓度-OD值曲线。结果:BMSCs在培养皿中贴壁生长、增殖;经ALP和VonKossa染色证实其有成骨潜能;标准BMSCs细胞浓度-OD值曲线显示细胞浓度和OD值呈线性正相关。结论:可以通过在体外分离纯化骨髓并诱导培养,获得足够数量、有成骨潜能的BMSCs作为骨组织工程的种子细胞。     

组蛋白甲基化促进AP2a基因表达及骨髓间充质干细胞成骨分化

目的研究转录因子AP2a的组蛋白甲基化状态对骨髓间充质干细胞成骨分化功能的影响。方法利用染色质免疫沉淀技术检测AP2a启动子甲基化状态。利用慢病毒载体的AP2ashRNA基因敲除AP2a进行丧失性功能研究。通过ALP活性、茜素红染色及钙离子定量分析研究骨髓间充质干细胞体外成骨分化能力。结果与牙源性间充质干细胞相比,AP2a在骨髓间充质干细胞中表达明显升高,AP2a启动子区域H3K4me3和H3K36me2明显增强。基因敲除AP2a能抑制骨髓间充质干细胞ALP活性及体外矿化能力。结论AP2a启动子区域H3K4me3和H3K36me2升高可以促进AP2a的表达和骨髓间充质干细胞成骨分化能力。     

富血小板血浆诱导骨髓基质细胞碱性磷酸酶活性的研究

目的：体外观察不同浓度富血小板血浆（PRP）对自体骨髓基质细胞碱性磷酸酶活性,对PRP诱导骨髓基质细胞向成骨细胞分化的生物学效应进行评价.方法：抽取狗静脉血及骨髓,采用改进Appel方法制备不同浓度的PRP;采用传代培养筛选法,获得足够数量稳定的骨髓基质细胞（BMSC）,酶动力学法检测碱性磷酸酶活性.结果：（1）原代培养的骨髓基质细胞24h开始贴壁,12d后细胞融汇成单层,细胞多呈成纤维梭形细胞形态,经诱导液传代培养后,细胞形态渐变为方形、多角形.（2）酶动力学法测定各实验组细胞第三、六、九、十二天检测碱性磷酸酶活性,经过配对t检验,各组第六、九、十二天的ALP活性水平均较第三天明显升高,低浓度PRP组较高浓度组碱性磷酸酶活性升高,差异具有统计学意义（P＜0.05）.结论：一定浓度的PRP能促进骨髓基质细胞碱性磷酸酶的活性,PRP能有效的促使BMSC向前期成骨细胞分化.     

The effect of enamel matrix derivative on gene expression in osteoblasts

Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro , causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo , at least partly, involves stimulation of bone remodelling.     

犬骨髓基质细胞体外成骨的实验研究

目的:研究犬骨髓基质细胞(bonemarrowderivedstromacell,BMSC)定向分化为成骨样细胞(osteoblasts-like,OBL)后的体外成骨能力和细胞内钙离子浓度的变化。方法:分离、培养犬骨髓基质细胞,并使用诱导培养液进行成骨诱导,取第3代成骨样细胞。通过碱性磷酸酶(alkalinephosphatase,ALP)染色和vonKossa染色检测BMSC分化为OBL的程度。检测细胞增殖率,比较细胞与多孔支架材料β-磷酸三钙(β-TCP)黏附前后的生长情况,用对BMSC分化前后、OBL黏附多孔β-TCP前后细胞内钙离子浓度荧光值进行统计学分析。结果:ALP染色和vonKossa染色均显示为阳性。细胞增殖率曲线结果显示,细胞黏附多孔β-TCP后增殖率没有明显差异。统计学分析发现,BMSC分化前后和OBL黏附β-TCP前后细胞内钙离子浓度荧光值均有明显的差异,P<0.05,有统计学意义。结论:犬OBL有很好的体外增殖能力和成骨能力,黏附多孔β-TCP之后,细胞的生长状况没有差别,多孔β-TCP材料对BMSC细胞内钙离子浓度有很大的影响。     

大鼠骨髓基质细胞与胶原-壳聚糖复合后修复牙槽骨缺损的研究

目的：以大鼠骨髓基质细胞为种子细胞，胶原一壳聚糖复合物为支架材料，通过骨组织工程的原理和方法修复牙槽骨缺损。方法：将28只SD大鼠分为实验组和对照组，实验组取大鼠骨髓基质细胞进行体外培养并矿化诱导为成骨样细胞，植入胶原一壳聚糖复合物培养1周后，植入大鼠下颌骨缺损处，2周、4周后取材。对照组细胞未进行诱导。结果：骨髓基质细胞矿化诱导后表现出与成骨细胞相似的形态学与功能表现，ALP染色阳性，并形成矿化结节，细胞在胶原一壳聚糖复合物内伸展良好，组织学观察：实验组2周后可见有少量新骨形成，4周后新骨形成面积增大对照组无明显新骨形成，材料周围有炎细胞浸润。并可见部分纤维结缔组织包绕。结论：利用胶原一壳聚糖复合物与诱导后的BMSC复合有良好的新骨形成能力。对临床修复唇腭裂骨缺损并进行正畸治疗有良好的应用前景。     

BCOR基因敲除促进骨髓间充质干细胞成骨分化的研究

目的研究BCL6共抑制因子-BCOR对骨髓间充质干细胞成骨定向分化能力的影响。方法利用慢病毒载体的BCORshRNA基因敲除BCOR,进行丧失性功能研究。通过检测ALP活性、茜素红染色、钙离子定量分析、成骨分化相关基因的表达,观察骨髓间充质干细胞体外成骨分化能力。结果BCOR在牙源性间充质干细胞和骨髓间充质干细胞的表达无明显差异。基因敲除BCOR促进了骨髓间充质干细胞ALP活性、体外矿化能力及成骨分化相关基因骨涎蛋白、骨钙素的表达。结论BCOR基因表达降低能促进骨髓间充质干细胞成骨分化,证实BCOR是骨髓间充质干细胞成骨分化的抑制基因。     

Inhibitory effect of enamel matrix derivative on osteoblastic differentiation of rat calvaria cells in culture

Background and Objective:  The effect of enamel matrix derivative (EMD) on bone differentiation remains unclear. Transforming growth factor β1 (TGF - β1) is reported to be contained in EMD. The aim of this study was to clarify the effect of EMD on osteoblastic cell differentiation and the possible role of TGF - β1.
Material and Methods:  Fetal rat carvarial cells were treated with 10, 50 or 100 µg/ml EMD for 5–17 days. Alkaline phosphatase (ALP) activity and bone nodule formation were measured, and mRNA expressions of bone matrix proteins and core binding factor were analysed.
Results:  Enamel matrix derivative inhibited ALP activity from the early stage of culture (29–44% inhibition) on days 5 and 10 and decreased bone nodule formation by 37–67% on day 17. These effects of EMD were concentration dependent. Enamel matrix derivative inhibited mRNA expression of osteocalcin and core binding factor. A high level of the active form of TGF - β1 protein was detected in the conditioned medium treated with 100 µg/ml EMD. Treatment with TGF - β1 antibody partly restored the inhibitory effect of EMD on ALP activity.
Conclusion:  Enamel matrix derivative inhibited the osteoblastic differentiation of rat carvarial cells and this was partly mediated by an increase in the activated form of TGF - β1, suggesting that EMD may function initially to inhibit osteoblastic differentiation to allow a predominant formation of other periodontal tissues.