(496d) BMP-2 Conjugated Micro-Fiber/Hydrogel Composites for Bone Integration to Engineered Ligament Tissue

While the bone-patellar tendon-bone autograft is considered the gold standard material for anterior cruciate ligament (ACL) reconstruction, alternative materials such as the hamstring or quadriceps tendon reveal that directly fixing the soft tissue graft to the native bone via bone screws risks mechanical instability and pullout of the graft. This is intrinsically a materials problem whereby micromotions and stress concentrations between the hard bone tissue and soft implant impede bone integration. In view of this challenge, the purpose of this work is to develop an engineered bone regeneration structure – as a portion of an engineered ligament tissue – that could be easily infiltrated by new bone tissue to facilitate integration of the implant within the bone tunnels.

To this end, we applied a two-step approach. First, we immobilized bone morphogenic protein (BMP)-2 to the surface of electrospun microfibers and characterized its ability to stimulate osteoblastic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Specifically, we examined the effect of incorporating 0.5 and 1% wt% heparin into polycaprolactone (PCL) fibers on BMP-2 adsorption and covalent conjugation (using EDC/NHS bioconjugation). Here, preliminary data show a five-fold increase of immobilized BMP-2 concentration at the highest heparin content when covalently tethered to the fibers. In addition, in vitro culture of BMSCs demonstrated that the micro-fiber meshes support cell viability and proliferation. In addition, alkaline phosphatase activity – a marker of osteoblastic differentiation –was significantly higher upon the binding of BMP-2 to the meshes. PCR assays to assess other osteogenic markers are underway.

Second, to form a robust 3D framework for bone-regeneration, we embedded this osteogenic fiber mesh into a cross linked 3 % gelatin methacrylate (GelMA) hydrogel. Here, a simple multilayered fiber/hydrogel composite system was created and the migration of MSCs through the hydrogel and onto the fiber layer in response to BMP-2 was assessed by confocal imaging. In the same system, the expression of markers of osteoblastic differentiation will be analyzed by real-time PCR. We postulate that the porosity provided by the GelMA phase, coupled with the mechanical properties and localized bioactive signals of the BMP-2 conjugated fibers will facilitate bone formation both in vitro and ultimately in vivo.