Chitosan/Hydroxyapatite Scaffolds with P28 as a Promising Osteoinductive Scaffold for Bone Healing Applications

Abstract

Despite bone’s inherent ability to heal, large bone defects remain a major clinical concern. This study proposes an off-the-shelf treatment combining chitosan/hydroxyapatite (CS/HAp) scaffolds, covalently linked with either bone morphogenetic protein-2 (BMP-2) or its related peptide P28 via a UV crosslinking process. Although covalently binding the growth factors was reported as a great alternative to the conventionally physical adsorption and encapsulation methods, this method presents the risk of altering the molecular activity and interaction of the growth factors. Therefore, alkaline phosphatase (ALP) activity and alizarin red staining (ARS) with a quantitative cetylpyridinium chloride (CPC) assay were conducted to validate that our photo-crosslinking fabrication method did not interfere with the functionality of the growth factors. The ALP activity of C2C12 with 100 µg/mL P28 was found to be comparable to 0.5 µg/mL BMP-2 after two weeks, where 0.001 U/mL was recorded for both treatments. The C2C12 cultured with CS/HAp/BMP-2 and CS/HAp/P28 scaffolds also showed an increased ALP activity compared to the negative control. ARS-CPC assay presented the highest optical density in 0.3 µg/mL BMP-2 and 50 µg/mL P28, while the highest intensity of ARS was observed in C2C12 cultured with CS/HAp/BMP-2 and CS/HAp/P28 scaffolds compared to the negative controls. The osteoconductive capability of this delivery system was then investigated through a rat femoral condyle defect model, where the new bone mineral density and the bone volume increased for all CS/HAp scaffolds compared to the collagen sponge control treatment. The histological assessment showed a favourable bone regeneration efficacy of the CS/HAp/P28 compared to the CS/HAp/BMP-2 treatment, thus showing the use of CS/HAp scaffolds with P28 as a promising osteoinductive scaffold for bone healing applications.