(162ad) Effect of Incorporation Strategy on BMP-2 Release from Chitosan Nanoparticles and Osteoblastic Differentiation

Functional regeneration of complex three-dimensional tissues necessitates advanced scaffolds possessing spatial organization of architectures, cell types, and bioactive factors. For example, a scaffold intended for regenerating the anterior cruciate ligament (ACL) should possess a region that will develop into ligament tissue flanked by regions that will readily integrate with bone. To this end, we envision that the packaging of bioactive factors into nanoparticles can facilitate their spatially-controlled incorporation into 3D scaffolds during the fabrication process.

In this project we examined different strategies for incorporating the osteogenic factor bone morphogenic protein (BMP)-2 into chitosan nanoparticles. Chitosan is a polycationic glycosaminoglycan that has seen application in bone regeneration, and can be formed into nanoparticles with the addition of tripolyphosphate (TPP). In this study, 500 nm particles were fabricated and 280 ng BMP-2 (with 28 μg bovine serum albumin, BSA) was loaded by either adsorption or covalent conjugation. In addition, BMP-2 (together with BSA) was encapsulated within nanoparticles by combining the proteins with chitosan prior to the addition of TPP. Results indicated 92-98% incorporation of BMP-2 with the three approaches. However, release kinetics over 28 days differed with the incorporation strategy. Adsorption produced a burst release followed by a sustained release, encapsulation exhibited negligible release, and conjugation demonstrated a burst followed by negligible release. Here, the negligible release from encapsulation and conjugation may reflect the low rate of chitosan dissolution or degradation. In addition, it was noted that the conjugation strategy led to particle aggregation. Next, bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in the presence of the different particles. While cells proliferated over 21 days in all conditions, only those cultured with BMP-2 adsorbed to chitosan nanoparticles exhibited elevated alkaline phosphatase activity (an early marker of osteoblastic differentiation).

Together, these data show that BMP-2 can be incorporated into chitosan nanoparticles by a variety of strategies that impact the rate of release and osteogenic activity. We are currently examining strategies to accelerate the release rates of BMP-2 from the different nanoparticles. Our next step will be to incorporate these nanoparticles within a hydrogel phase to evaluate the osteogenicity of 3D printed scaffolds.