Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro
Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro
复制标题
DOI：
10.1902/jop.1997.68.4.320
复制
发表时间：
1997-04-01
影响因子：
4.3
通讯作者：
Somerman, MJ
中科院分区：
文献类型：
--
作者：
Nohutcu, RM;McCauley, LK;Somerman, MJ
牙周再生是一个复杂的过程，需要牙周组织内几种细胞类型的协调反应。人们普遍认为，牙周膜（PDL）具有异质性细胞群，其中一些细胞可能能够分化成成牙骨质细胞或成骨细胞。因此，有人假设PDL细胞在促进牙周再生中起作用。然而，缺乏支持这一概念的确切证据。以前，我们报告说，PDL细胞诱导生物矿化确定Von Kossa组织化学和透射电子显微镜。为了进一步确定PDL细胞的成骨细胞样特性，将人PDL细胞暴露于地塞米松（DEX）以促进成骨细胞表型，然后在体外矿物质结节形成期间监测细胞活性。对于矿化研究，将细胞在含有10%FBS和a）仅赋形剂; B）抗坏血酸（50 μ g/ml）和β-甘油磷酸盐（10 mM）;或c）抗坏血酸、β-甘油磷酸盐和DEX（100 nM）的DMEM中培养30天。此外，DEX对PDL细胞在非矿化培养基中的影响进行了测定。使用Von Kossa方法，每周对细胞进行染色以评价矿物质样结节。几种骨相关蛋白的mRNA稳态水平的北方印迹分析，骨桥蛋白（OPN）、骨涎蛋白（BSP）、碱性磷酸酶（ALP）、骨钙素（OCN）、α（2）（1）（1型）胶原和骨连接素（ON）。在30天的矿化期内也测定了DNA水平。在相差显微镜下，PDL细胞在非矿化介质与DEX处理表现出更多的纺锤形形态相比，类似的细胞没有暴露于DEX。需要有成矿条件才能诱发矿物结核的形成。然而，在这种情况下，矿物质诱导是独立的DEX，而且，DEX处理的细胞没有表现出不同的形态模式时，与非DEX处理的细胞。矿物质样结节在第15天首次出现，与DEX处理和未处理培养物中I型胶原和ON mRNA表达的增加和随后的减少相一致。使用北方印迹分析检测特异性蛋白，我们发现PDL细胞在本研究中使用的任何条件下均不表达OPN、BSP、OCN或ALP。DEX没有改变DNA含量的文化在矿化期间。这些结果证实，人牙周韧带细胞可以在体外诱导矿化，并表明地塞米松不会显着改变矿化的程度和模式。
PERIODONTAL REGENERATION IS A COMPLEX PROCESS that requires coordinated responses from several cell types within the periodontium. It is generally accepted that the periodontal ligament (PDL) has a heterogeneous cell population, where some of the cells may be capable of differentiating into either cementoblasts or osteoblasts. Thus, it has been hypothesized that PDL cells play a role in promoting periodontal regeneration. However, definitive evidence to support this concept is lacking. Previously, we reported that PDL cells induce biomineralization as determined by Von Kossa histochemistry and transmission electron microscopy. To further determine the osteoblast-like properties of PDL cells, human PDL cells were exposed to dexamethasone (DEX) in order to promote an osteoblast phenotype, and then cell activity monitored during mineral nodule formation in vitro. For mineralization studies, cells were cultured in DMEM containing 10% FBS and a) vehicle only; b) ascorbic acid (50 mu g/ml) and beta-glycerophosphate (10 mM); or c) ascorbic acid, beta-glycerophosphate and DEX (100 nM) for 30 days. In addition, the effects of DEX on PDL cells in non-mineralizing media were determined. Cells were stained weekly to evaluate mineral-like nodules, using the Von Kossa method. Northern blot analyses for mRNA steady state levels for several bone-associated proteins, i.e., osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (ALP), osteocalcin (OCN), alpha(2)(1)(type 1) collagen and osteonectin (ON), were performed. DNA levels were also determined during the 30-day mineralization period. Under phase contrast microscopy, PDL cells in non-mineralizing media treated with DEX exhibited a more spindle-shaped morphology when compared with similar cells not exposed to DEX. Mineralizing conditions were required to induce mineral nodule formation. However, in this situation, mineral induction was independent of DEX; and furthermore, DEX-treated cells did not exhibit a different morphological pattern when compared with non-DEX treated cells. Mineral-like nodules wen first seen al day 15, in concert with an increase followed by a decrease in expression of type I collagen and ON mRNA in both DEX-treated and non-treated cultures. Using Northern blot analysis for detection of specific proteins, we found that PDL cells did not express OPN, BSP, OCN, or ALP under any of the conditions used in this study. DEX did not alter DNA content in the cultures during the mineralization period. These results confirm that human periodontal Ligament cells can be induced to mineralize in vitro and indicate that dexamethasone does not significantly alter the extent and pattern of mineralization.