In vitro effects of enamel matrix derivative on microvascular cells
In vitro effects of enamel matrix derivative on microvascular cells
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DOI：
10.1902/jop.2007.060111
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Cochran, David L.
中科院分区：
文献类型：
--
作者：
Schlueter, Scott R.;Carnes, David L., Jr.;Cochran, David L.
背景资料：牙周膜再生需要一系列协调的事件，不仅包括牙周膜（PDL）特异性细胞的招募，而且还包括血管细胞。釉基质衍生物（EMD）的作用机制知之甚少，其对血管细胞的影响尚不清楚。本研究的目的是研究在何种程度上EMD影响血管生成和PDL cell recruitment.Methods：EMD对人微血管内皮细胞（HMVECs）的影响，通过检查增殖，趋化性，血管生成，和迁移。使用水溶性四唑盐（WST）-1试剂测定增殖。使用微孔培养孔插入物测定趋化性。在含有基质胶的平板上评估血管生成。HMVEC对PDL细胞迁移的影响通过评价PDL细胞从胶原凝胶的生长来评估，所述胶原凝胶在HMVEC存在下在纤维蛋白基质上培养并在24小时被含纤连蛋白的纤维蛋白凝块包围。采用逆转录-聚合酶链反应（RT-PCR）检测EMD对血管内皮细胞（VEGF）A、B、C和D型及其亚型PDL表达的影响。采用特异性酶联免疫吸附试验（ELISA）定量测定EMD刺激PDL细胞产生VEGF、血小板衍生生长因子（PDGF）-AA、PDGF-BB、PDGF-AB和转化生长因子（TGF）-β 1的情况。当PDL细胞存在时，EMD也刺激HMVEC趋化性增加100%（P < 0.05）。所有剂量的EMD测试（25，50，和100 μ g/ml）增加血管生成在体外。HMVECs，在与EMD在100 μ g/ml的浓度组合，刺激750%的PDL细胞从胶原凝胶迁移到纤维蛋白凝块相比，当两者都不存在。RT-PCR结果表明，PDL细胞表达VEGF-A、-B和-C以及多种VEGF-A亚型，包括VEGF（121）、VEGF（165）和VEGF（189），无论培养基中是否存在EMD。ELISA测定了在EMD刺激的条件培养基中PDL C细胞的VEGF浓度增加了400%，并且在TGF-β 1刺激的培养基中也有类似的增加。结论：EMD可能通过刺激内皮细胞直接刺激血管生成，并通过刺激PDL细胞产生血管生成因子（VEGF）间接刺激血管生成。重要的是，这些数据与EMD在与愈合相关的血管生成期间增强HMVEC和PDL细胞之间的双向通信的概念一致。
Background: Periodontal regeneration requires a coordinated series of events that includes not only the recruitment of periodontal ligament (PDL)-specific cells, but vascular cells as well. The mechanisms of action of enamel matrix derivative (EMD) are poorly understood, and its effects on vascular cells are unknown. The objective of this study was to examine the extent to which EMD affects angiogenesis and PDL cell recruitment.Methods: The effects of EMD on human microvascular endothelial cells (HMVECs) were determined by examining proliferation, chemotaxis, angiogenesis, and migration. Proliferation was determined using water-soluble tetrazolium salt (WST)-1 reagent. Chemotaxis was determined using micro-porous-culture well inserts. Angiogenesis was assessed on plates containing matrigel. The effects of HMVECs on the migration of PDL cells were assessed by evaluating PDL cell outgrowth from Collagen gels cultured in the presence of HMVECs on fibrin matrix and surrounded by fibronectin-containing fibrin clots at 24 hours. Effects of EMD on PDL expression of vascular endothelial cell (VEGF) types (A, B, C, and D) and isoforms were determined using reverse transcription-polymerase chain reaction (RT-PCR). Production of VEGF, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and transforming growth factor (TGF)-beta 1 by EMD-stimulated PDL cells was assessed quantitatively in conditioned media using specific enzyme-linked immunosorbent assays (ELISAs).Results: EMD at concentrations < 50 mu g/ml resulted in significant (P < 0.05) stimulation of HMVEC proliferation. Compared to baseline, EMD also stimulated a 100% increase in HMVEC chemotaxis when PDL cells were present (P < 0.05). All doses of EMD tested (25, 50, and 100 mu g/ml) increased angiogenesis in vitro. HMVECs, in combination with EMD at a concentration of 100 mu g/ml, stimulated a 750% increase in migration of PDL cells from collagen gels into fibrin clots compared to controls when neither was present. RT-PCR results indicated that PDL cells expressed VEGF-A, -B, and -C and multiple isoforms of VEGF-A, including VEGF(121), VEGF(165), and VEGF(189), whether or not EMD was present in the culture media. ELISAs determined a 400% increase in VEGF concentration by PDL C cells in EMD-stimulated conditioned media and a similar increase in TGF-beta(1)-stimulated media.Conclusions: It is likely that EMD stimulates angiogenesis directly by stimulating endothelial cells and indirectly by stimulating the production of angiogenic factors (VEGF) by PDL cells. Importantly, the data are consistent with the concept that EMD enhances bidirectional communication between HMVEC and PDL cells during angiogenesis associated with healing.