Fabrication and Characterization of Multifunctional Chitosan Microspheres and Their Incorporation Into Composite Scaffolds for Enhanced Bone Regeneration

Insufficient fracture healing affects hundreds of thousands of people every year in the United States, resulting in devestating economic and social impacts. To treat these severe fractures, our laboratory has developed chitosan-nano-hydroxyapatite microspheres and scaffolds. The purpose of this research was to improve these scaffolds by increasing their degradation rate to allow better bone ingrowth and to increase and extend the elution of bone morphogenetic protein-2 (BMP-2) from the constructs. We hypothesized that a composite approach combining chitosan and carboxymethylchitosan microspheres would result in scaffolds with improved degradation and elution properties while maintaining good cytocompatibility and sufficient mechanical properties. The scaffold constructs are prepared by fusing chitosan microspheres together using an acid wash to make the beads adherent. This approach promotes flexibility by allowing multiple microsphere types to be incorporated into the scaffolds. Chitosan microspheres with 80% degree of deacetylation were selected as the first component of the composite scaffolds. These microspheres demonstrated good mechanical properties (compressive modulus of 1.6 ± 0.3 MPa) and excellent cytocompatibility. The second bead type was optimized for degradation and drug delivery. The carboxymethylation of chitosan microspheres was performed using monochloroacetic acid. The carboxymethylchitosan microspheres were crosslinked using two different approaches: amine-amine crosslinking using genipin (Gen-X CMCS beads) and amide bone formation using carbodiimide chemistry (X-CMCS). The Gen-X CMCS beads displayed poor degradation and elution properties; whereas, the X-CMCS beads displayed extensive degradation (82.7 ± 1.2%) and extended elution of BMP-2 for at least forty-five days. Composite X-CMCS/CS scaffolds were prepared and demonstrated improved degradation and drug delivery compared to CS-only scaffolds while maintaining sufficient mechanical characteristics and cytocompatibility. This research demonstrated the advantages of using a composite approach and supported our hypothesis. By optimizing each bead type for a specific purpose, the overall properties of the scaffolds were improved. The combination of CS and X-CMCS microspheres resulted in composite scaffolds that demonstrated exellent potential for enhancing bone regeneration in severe fractures