Hemopoiesis Research Laboratory, School of Medicine, University of Crete, Heraklion, Greece.
Department of Materials Science and Technology, University of Crete, Heraklion, Greece.
Biomed Mater. 2020 Oct 3;15(6):064101. doi: 10.1088/1748-605X/aba325.
In the present study we explore the extracellular matrix (ECM) produced by human bone marrow mesenchymal stem/stromal cells (BM-MSCs) induced to undergo osteogenic differentiation within porous chitosan/gelatin (CS:Gel) scaffolds by investigating their multiple gene expression profile and mechanical behavior. Initially, the efficiency of the BM-MSCs osteogenic differentiation within the constructs was confirmed by the significant rise in the expression of the osteogenesis associated genes DLX5, RUNX2, ALP and OSC. In line with these findings, OSC and Col1A1 protein expression was also detected in BM-MSCs on the CS:Gel scaffolds at day 14 of osteogenic differentiation. We then profiled, for the first time, the expression of 84 cell adhesion and ECM molecules using PCR arrays. The arrays, which were conducted at day 14 of osteogenic differentiation, demonstrated that 49 genes including collagens, integrins, laminins, ECM proteases, catenins, thrombospondins, ECM protease inhibitors and cell-cell adhesion molecules were differentially expressed in BM-MSCs seeded on scaffolds compared to tissue culture polystyrene control. Moreover, we performed dynamic mechanical analysis of the cell-loaded scaffolds on days 0, 7 and 14 to investigate the correlation between the biological results and the mechanical behavior of the constructs. Our data demonstrate a significant increase in the stiffness of the constructs with storage modulus values of 2 MPa on day 7, compared to 0.5 MPa on day 0, following a drop of the stiffness at 0.8 MPa on day 14, that may be attributed to the significant increase of specific ECM protease gene expression such as MMP1, MMP9, MMP11 and MMP16 at this time period.
在本研究中，我们通过研究多孔壳聚糖/明胶（CS：Gel）支架内诱导人骨髓间充质干细胞/基质细胞（BM-MSCs）向成骨分化产生的细胞外基质（ECM），来探索其多种基因表达谱和力学行为。首先，通过检测与成骨相关基因 DLX5、RUNX2、ALP 和 OSC 的表达显著升高，证实了 BM-MSCs 在构建物内成骨分化的效率。与这些发现一致，在第 14 天的成骨分化中，也在 CS：Gel 支架上的 BM-MSCs 中检测到 OSC 和 Col1A1 蛋白的表达。然后，我们使用 PCR 阵列首次对 84 种细胞黏附分子和 ECM 分子的表达进行了分析。在第 14 天的成骨分化中进行的阵列显示，49 个基因（包括胶原蛋白、整合素、层粘连蛋白、ECM 蛋白酶、连环蛋白、血小板反应蛋白、ECM 蛋白酶抑制剂和细胞-细胞黏附分子）在接种于支架的 BM-MSCs 中表达不同，与组织培养聚苯乙烯对照相比。此外，我们在第 0、7 和 14 天对细胞加载支架进行了动态力学分析，以研究生物结果与构建物力学行为之间的相关性。我们的数据表明，与第 0 天的 0.5 MPa 相比，第 7 天的存储模量值为 2 MPa 的构建物的刚度显著增加，而第 14 天的刚度下降至 0.8 MPa，这可能归因于特定 ECM 蛋白酶基因表达的显著增加，如 MMP1、MMP9、MMP11 和 MMP16 在这段时间内的表达。
