Biomimetic Materials and Tissue Engineering Laboratories, Department of Chemical Engineering, University of South Carolina, Columbia, South Carolina 29208, USA.
Langmuir. 2012 Mar 27;28(12):5387-97. doi: 10.1021/la205005h. Epub 2012 Mar 12.
The objective of this work was to investigate the combined effect of grafting the peptide corresponding to amino acid residues 162-168 of osteopontin (OPD peptide) and the peptide corresponding to amino acid residues 73-92 of bone morphogenetic protein-2 (BMP peptide) to an RGD-conjugated inert hydrogel on osteogenic and vasculogenic differentiation of bone marrow stromal (BMS) cells. RGD-conjugated three-dimensional (3D) porous hydrogel scaffolds with well-defined cylindrical pore geometry were produced from sacrificial wax molds fabricated by fused deposition modeling rapid prototyping system. Propargyl acrylate and 4-pentenal were conjugated to the hydrogel for orthogonal grafting of BMP and OPD peptides by click reaction and oxime ligation, respectively. The OPD peptide was grafted by the reaction between aminooxy moiety of aminooxy-mPEG-OPD (mPEG = mini-poly(ethylene glycol)) and the aldehyde moiety in the hydrogel. The BMP peptide was grafted by the reaction between the azide moiety of Az-mPEG-BMP and the propargyl moiety in the hydrogel. The hydrogels seeded with BMS cells were characterized by biochemical, immunocytochemical, and mRNA analyses. Groups included RGD control hydrogel (RGD), RGD and BMP peptides without OPD (RGD+BMP), RGD and BMP peptides with mutant OPD (RGD+BMP+mOPD), and RGD and BMP peptides with OPD (RGD+BMP+OPD) grafted hydrogels. The extent of mineralization of RGD, RGD+BMP, RGD+BMP+mOPD, and RGD+BMP+OPD groups after 28 days was 650 ± 70, 990 ± 30, 850 ± 30, and 1150 ± 40 mg/(mg of DNA), respectively, indicating that the BMP and OPD peptides enhanced osteogenic differentiation of the BMS cells. The BMS cells seeded on RGD+BMP+OPD grafted hydrogels stained positive for vasculogenic markers α-SMA, PECAM-1, and VE-cadherin while the groups without OPD peptide (RGD+BMP and RGD+BMP+mOPD) stained only for α-SMA but not PECAM-1 or VE-cadherin. These results were consistent with the significantly higher PECAM-1 mRNA expression for RGD+BMP+OPD group after 21 and 28 days, compared to the groups without OPD. These findings suggest that the RGD+BMP+OPD peptides provide a favorable microenvironment for concurrent osteogenic and vasculogenic differentiation of progenitor marrow-derived cells.
本工作旨在研究将骨桥蛋白（OPD 肽）的 162-168 位氨基酸和骨形态发生蛋白-2（BMP 肽）的 73-92 位氨基酸对应的肽段同时接枝到 RGD 偶联惰性水凝胶上对骨髓基质（BMS）细胞成骨和血管生成分化的联合作用。通过熔融沉积建模快速原型系统制造的牺牲蜡模，制备了具有明确定义的圆柱孔几何形状的 RGD 接枝三维（3D）多孔水凝胶支架。丙烯酰基丙烯酸酯和 4-戊烯醛分别与水凝胶偶联，通过点击反应和肟连接分别将 BMP 和 OPD 肽接枝到水凝胶上。OPD 肽通过氨基氧基 -mPEG-OPD（mPEG= 小聚（乙二醇））的氨基氧基部分与水凝胶中的醛基部分之间的反应接枝。BMP 肽通过 Az-mPEG-BMP 的叠氮部分与水凝胶中的炔基部分之间的反应接枝。用 BMS 细胞接种水凝胶，通过生化、免疫细胞化学和 mRNA 分析进行表征。各组包括 RGD 对照水凝胶（RGD）、无 OPD 的 RGD 和 BMP 肽（RGD+BMP）、有突变 OPD 的 RGD 和 BMP 肽（RGD+BMP+mOPD）和有 OPD 的 RGD 和 BMP 肽（RGD+BMP+OPD）接枝水凝胶。28 天后，RGD、RGD+BMP、RGD+BMP+mOPD 和 RGD+BMP+OPD 组的矿化程度分别为 650±70、990±30、850±30 和 1150±40mg/（mg DNA），表明 BMP 和 OPD 肽增强了 BMS 细胞的成骨分化。接种 RGD+BMP+OPD 接枝水凝胶的 BMS 细胞对血管生成标志物α-SMA、PECAM-1 和 VE-cadherin 呈阳性染色，而不含 OPD 肽的组（RGD+BMP 和 RGD+BMP+mOPD）仅对α-SMA 呈阳性染色，而对 PECAM-1 或 VE-cadherin 不呈阳性染色。这些结果与第 21 天和第 28 天 RGD+BMP+OPD 组的 PECAM-1mRNA 表达明显更高一致，与无 OPD 组相比。这些发现表明，RGD+BMP+OPD 肽为祖细胞骨髓来源细胞的成骨和血管生成分化提供了有利的微环境。
