Adult human bone marrow stromal cells regulate expression of their MMPs and TIMPs in differentiation type-specific manner

Previously, we described a profound impact of structural conformation of collagen matrix on osteogenic and adipogenic differentiation of bone marrow stromal cells. Thus, a marginal p38-independent adipogenesis on native collagen I matrix contrasts with an efficient p38-dependent differentiation on denatured collagen I. An efficient Hsp90-dependent osteogenesis occurs on native collagen I matrix but not on its denatured counterpart where it is insignificant and proceeds in an Hsp90-independent manner. Whereas only marginal osteogenesis and no detectable adipogenesis of bone marrow stromal cells occur on native collagen IV, the same matrix supports a highly efficient adipogenesis in denatured structural state. The present study addresses the opposite direction in the flow of cell–matrix interaction, namely the cells' influence on structural state of collagen matrix, and tests the possibility that differentiating bone marrow stromal cells may adjust the expression phenotype of MMP and TIMP in such a way that, if translated into matrix modification, would facilitate the maintenance of collagen matrix in or its modification into structural state optimal for the ongoing differentiation process. The results obtained indicate that this is indeed the case. In bone marrow stromal cells stimulated to undergo adipogenesis the expression of MMP increases and that of TIMP decreases. In cells induced to undergo osteogenesis the opposite is true: MMP/TIMP expression is adjusted in a manner that, if translated into matrix modification, could promote the native structural conformation optimal for this type of differentiation. The results obtained also indicate that the observed adjustment in MMP/TIMP expression phenotype might be an early differentiation event and that differentiation stimulation alone might be sufficient to trigger it even on matrices not favorable to a given type of differentiation. The findings of the present study raise significant questions and indicate directions for further experimentation.     

大戟苷抑制大鼠骨髓间充质干细胞成骨分化

目的:观察泽漆主要活性成分大戟苷(euphornin)对大鼠骨髓间充质干细胞(rMSC)成骨分化的影响。方法:从大鼠股骨中分离培养rMSC,并诱导其成骨分化。用MTT法检测细胞增殖情况,通过茜素红染色,碱性磷酸酶(ALP)活性检测和钙含量测定分别定性、定量地判断其在成骨分化中的效果。实时定量PCR(Q-PCR)检测主要成骨标志因子骨桥蛋白(OPN)和一型胶原蛋白(COL-Ⅰ)及主要转录因子骨形成蛋白2(BMP2)、Runt相关转录因子2(Runx2)和Osterix(Osx)mRNA的表达。结果:大戟苷能剂量依赖性地抑制rMSC成骨分化,并一定程度地抑制其细胞增殖。COL-Ⅰ和OPN的表达在第4、8天分别有显著下降。BMP2、Runx2和Osx等关键转录因子的表达也被明显抑制。结论:大戟苷能抑制rMSC成骨分化,其作用主要是通过抑制BMP通路相关因子的表达而实现的。     

Collagen type I promotes osteogenic differentiation of amniotic membrane-derived mesenchymal stromal cells in basal and induction media

Collagen has been widely shown to promote osteogenesis of bone marrow mesenchymal stromal cells (BM-MSCs). Due to the invasive procedure of obtaining BM-MSCs, MSCs from other tissues have emerged as a promising alternative for regenerative therapy. MSCs originated from different sources, exhibiting different differentiation potentials. Therefore, the applicability of collagen type I (COL), combining with amniotic membrane (AM)-MSCs was examined through proliferation and differentiation assays together with the expression of surface markers and genes associated with stemness and differentiation under basal or induction conditions. No increase in cell growth was observed because AM-MSCs might be directed toward spontaneous osteogenesis. This was evidenced by the calcium deposition and elevated expression of osteogenic genes when AM-MSCs were cultured in collagen plate with basal media. Under the osteogenic condition, reciprocal expression of OCN and CEBPA suggested a shift toward adipogenesis. Surprisingly, adipogenic genes were not elevated upon adipogenic induction, although oil droplets deposition was observed. In conclusion, our findings demonstrated that collagen causes spontaneous osteogenesis in AM-MSCs. However, the presence of exogenous inductors could shift the direction of adipo-osteogenic gene regulatory network modulated by collagen.     

Temporal and spatial gene expression of major bone extracellular matrix molecules during embryonic mandibular osteogenesis in rats

It is not known how gene expression of bone extracellular matrix molecules is controlled temporally and spatially, or how it is related with morphological differentiation of osteoblasts during embryonic osteogenesis in vivo. The present study was designed to examine gene expressions of type I collagen, osteonectin, bone sialoprotein, osteopontin, and osteocalcin during mandibular osteogenesis using in situ hybridization. Wistar rat embryos 13–20 days post coitum were used. The condensation of mesenchymal cells was formed in 14-day rat embryonic mandibles and expressed genes of pro-(I) collagen, osteonectin, bone sialoprotein and osteopontin. Cuboidal osteoblasts surrounding the uncalcified bone matrix were seen as early as in 15-day embryonic mandibles, while flat osteoblasts lining the surface of the calcified bone were seen from 16-day embryonic mandibles. Cuboidal osteoblasts expressed pro-1(I) collagen, osteonectin and bone sialoprotein intensely but osteopontin very weakly. In contrast, flat osteoblasts expressed osteopontin very strongly. Osteocytes expressed the extracellular matrix molecules actively, in particular, osteopontin. The present study demonstrated the distinct gene expression pattern of type I collagen, osteonectin, bone sialoprotein, osteopontin and osteocalcin during embryonic mandibular osteogenesis in vivo.     

Mg2+ in β-TCP/Mg–Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg–Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg–Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and β-tricalcium phosphate (β-TCP; manifesting process not shown), prepared Mg, Zn, and Mg–Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg–Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg–Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg–Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg²⁺ in β-TCP/Mg–Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.     

骨形态发生蛋白2通过Smad途径上调Osterix的表达

Osterix（Osx）是一种重要的调控成骨细胞分化的具有锌指结构的转录因子．骨形态发生蛋白2（bone morphogenetic protein 2, BMP2）能够上调Osx的表达,但其分子机制并不清楚．采用实时定量RT-PCR方法检测到BMP2诱导成骨相关细胞C3H10T1/2, MC3T3-E1, C2C12中Osx的转录水平显著上调,并且与成骨分化指标Col1a1, osteocalcin具有相似的表达动力学特征．而且,在C3H10T1/2细胞中过表达负显性（dominant negative, DN）Osx基因,能够有效抑制BMP2诱导的成骨分化．过表达BMP/Smad信号通路抑制蛋白Smad6,能够抑制Osx转录水平的上调．但是通过荧光素酶报告载体对Osx的启动子-1254～+85区域进行分析后未发现接受BMP通路调控的启动子区域．上述结果表明,BMP2能够通过Smad途径上调Osx的表达,并对成骨分化的过程具有十分重要的作用．