Yang Weidong, Gomes Ronald R, Brown Anissa J, Burdett Ashley R, Alicknavitch Michael, Farach-Carson Mary C, Carson Daniel D
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.
Tissue Eng. 2006 Jul;12(7):2009-24. doi: 10.1089/ten.2006.12.2009.
Extracellular matrix (ECM) molecules in cartilage cooperate with growth factors to regulate chondrogenic differentiation and cartilage development. Domain I of perlecan (Pln) bears heparan sulfate chains that bind and release heparin binding growth factors (HBGFs). We hypothesized that Pln domain I (PlnDI) might be complexed with collagen II (P-C) fibrils to improve binding of bone morphogenetic protein-2 (BMP-2) and better support chondrogenesis and cartilage-like tissue formation in vitro. Our results showed that P-C fibrils bound more BMP-2 than collagen II fibrils alone, and better sustained BMP-2 release. Polylactic acid (PLA)-based scaffolds coated with P-C fibrils immobilized more BMP-2 than either PLA scaffolds or PLA scaffolds coated with collagen II fibrils alone. Multipotential mouse embryonic mesenchymal cells, C3H10T1/2, were cultured on 2-dimensional P-C fibrils or 3-dimensional P-C/BMP-2-coated (P-C-B) PLA scaffolds. Chondrogenic differentiation was indexed by glycosaminoglycan (GAG) production, and expression of the pro-chondrogenic transcription factor, Sox9, as well as cartilaginous ECM proteins, collagen II, and aggrecan. Immunostaining for aggrecan, perlecan, tenascin, and collagen X revealed that both C3H10T1/2 cells and primary mouse embryonic fibroblasts cultured on P-C-B fibrils showed the highest expression of chondrogenic markers among all treatment groups. Safranin O-Fast Green staining indicated that cartilage-like tissue was formed in the P-C-B scaffolds, while no obvious cartilage-like tissue formed in other scaffolds. We conclude that P-C fibrils provide an improved biomimetic material for the binding and retention of BMP-2 and support chondrogenic differentiation.
软骨中的细胞外基质（ECM）分子与生长因子协同作用，以调节软骨形成分化和软骨发育。基底膜聚糖（Pln）的结构域I带有硫酸乙酰肝素链，可结合并释放肝素结合生长因子（HBGFs）。我们推测，Pln结构域I（PlnDI）可能与II型胶原（P-C）原纤维复合，以改善骨形态发生蛋白-2（BMP-2）的结合，并在体外更好地支持软骨形成和类软骨组织形成。我们的结果表明，P-C原纤维比单独的II型胶原原纤维结合更多的BMP-2，并且能更好地持续释放BMP-2。涂有P-C原纤维的聚乳酸（PLA）基支架比单独的PLA支架或涂有II型胶原原纤维的PLA支架固定更多的BMP-2。将多能小鼠胚胎间充质细胞C3H10T1/2培养在二维P-C原纤维或三维P-C/BMP-2包被（P-C-B）的PLA支架上。通过糖胺聚糖（GAG）的产生、促软骨形成转录因子Sox9以及软骨细胞外基质蛋白II型胶原和聚集蛋白聚糖的表达来衡量软骨形成分化。对聚集蛋白聚糖、基底膜聚糖、腱生蛋白和X型胶原的免疫染色显示，在所有治疗组中，培养在P-C-B原纤维上的C3H10T1/2细胞和原代小鼠胚胎成纤维细胞均显示出最高的软骨形成标志物表达。番红O-固绿染色表明，P-C-B支架中形成了类软骨组织，而其他支架中未形成明显的类软骨组织。我们得出结论，P-C原纤维为BMP-2的结合和保留提供了一种改良的仿生材料，并支持软骨形成分化。
