The optimisation of biomimetic scaffold fabrication for the controlled release of Osteogenic factors for bone tissue regeneration

Abstract

Bone tissue defect is a rising global concern and is one of the leading causes of morbidity and disability in patients. Bone regeneration treatments that demonstrate all key characteristics for successful bone healing: osteoinductivity, osteoconductivity, osteogenicity and mechanical stability, are not yet available. Also, mitigation against bacterial infections, an increasing complication associated with impeded bone tissue repair, is urgently required. This report presents the development of a biomimetic antimicrobial scaffold that capable of inducing bone tissue-specific regeneration. The tunable biodegradability feature for a sustained bio-agent release was facilitated using a tailored crosslinking formulation that maintained the scaffolds' mechanical performance. In this work, biodegradable chitosan (CS) scaffolds were prepared with combinations of bioactive ceramics, namely hydroxyapatite (HAp), tricalcium phosphate-α (TCP-α), and fluorapatite (FAp), crosslinked using a UV curing system. The efficacy of the crosslinking reaction was assessed using swelling and compression testing, SEM and FTIR analysis, and biodegradation studies in simulated body fluid. Consequently, various benzophenone concentrations were added to CS/HAp formulations to determine their effect on the degradation rate. The results presented indicate that incorporating bioceramics with a suitable photoinitiator concentration can tailor the biodegradability and load-bearing capacity of the scaffolds. Subsequently, a combination of CS/HAp scaffold was UV crosslinked with either bone morphogenetic protein-2 (BMP-2) or its related peptide P28. Alkaline phosphatase (ALP) activity and alizarin red staining (ARS) were conducted to validate that the photo crosslinking fabrication method did not interfere with the functionality of the growth factors. The C2C12 cultured with CS/HAp/BMP-2 and CS/HAp/P28 scaffolds showed an increased ALP activity compared to the negative control. The in vivo osteoconductive of the treatment was then investigated through a rat femoral condyle defect model. The ex vivo 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 P28 as a promising osteoinductive treatment. Further work was carried out to improve the degradation rate in vivo, utilising CS, HAp and FAp at different ratios. Various P28 concentrations were incorporated into the CS/HAp/FAp scaffolds for implantation in vivo. H&E staining shows minimal scaffold traces in most of the defects induced after eight weeks, showing that the combination of HAp and FAp in the chitosan-based delivery system has enhanced the biodegradability of the scaffolds in vivo. Histological assessments indicated ongoing bone formation throughout the in vivo duration of the study. These results show the ability of this tailored formulation to improve the scaffold degradation for bone regeneration and presenting a cost-effective alternative to BMP-2. In conclusion, the CS/ceramic scaffold with the presence of P28 peptide appears to have potential in bone defect healing due to its potent osteogenicity, biocompatibility and non toxicity features.