The bone-ligament interface transitions from a highly organized type I collagen rich matrix to a nonmineralized fibrocartilage region and finally to a mineralized fibrocartilage region that interfaces with the bone. Therefore, engineering the bone-ligament interface requires a biomaterial substrate capable of maintaining or directing the spatially defined differentiation of multiple cell phenotypes. To date the appropriate combination of biophysical and biochemical factors that can be used to engineer such a biomaterial substrate remain unknown. Here we show that microfiber scaffolds functionalized with tissue-specific extracellular matrix (ECM) components can direct the differentiation of MSCs toward the phenotypes seen at the bone-ligament interface. Ligament-ECM (L-ECM) promoted the expression of the ligament-marker gene tenomodulin (TNMD) and higher levels of type I and III collagen expression compared to functionalization with commercially available type I collagen. Functionalization of microfiber scaffolds with cartilage-ECM (C-ECM) promoted chondrogenesis of MSCs, as evidenced by adoption of a round cell morphology and increased SRY-box 9 (SOX9) expression in the absence of exogenous growth factors. Next, we fabricated a multiphasic scaffold by controlling the spatial presentation of L-ECM and C-ECM along the length of a single electrospun microfiber construct, with the distal region of the C-ECM coated fibers additionally functionalized with an apatite layer (using simulated body fluid) to promote endochondral ossification. These ECM functionalized scaffolds promoted spatially defined differentiation of MSCs, with higher expression of TNMD observed in the region functionalized with L-ECM, and higher expression of type X collagen and osteopontin (markers of endochondral ossification) observed at the end of the scaffold functionalized with C-ECM and the apatite coating. Our results demonstrate the utility of tissue-specific ECM derived components as a cue for directing MSC differentiation when engineering complex multiphasic interfaces such as the bone-ligament enthesis.
Keywords: CTGF; biomaterials; biomimetic apatite; cartilage-ECM; ligament-ECM; microfibers.