Smart pH and Thermosensitive Injectable Hydrogels:Chitosan – Hydroxyapatite – Heparin Based Functionalised Biomaterials for Bone Regeneration

Abstract

Hard and soft tissue repair and regeneration require multiple surgical interventions, as very often-repeated clinical procedures are required. In addition, insufficient vascularisation remains as the main challenge for bone repair and regeneration. Therefore, bone regeneration via minimal invasive biomaterials promoting angiogenesis by bioactive agents are very much considered at present. Hereof, among new generation biomaterials, in-situ forming stimuli responsive injectable hydrogels have been of interest both in clinical and non-clinical communities. In this study, novel pH and thermosensitive in-situ formed injectable hydrogel compositions were developed by a heparin (Hep) functionalised chitosan (CS) polymer matrix integrated with a nonsintered hydroxyapatite (HA) system neutralised with sodium bicarbonate (NaHCO3). Due to designed bioactive features, CS/HA/Hep hydrogels are desired to ensure strong chemical anchorage and biological bridging by promoting vascular network formation at bone tissue defects due to the potential binding capacity of Hep for endogenous angiogenic growth factors and proteins. The production and optimisation of hydrogel compositions and their modification for pre-sterile synthesis technique with glycerol additive for enhancing mechanical properties meanwhile were investigated. The versatile properties of hydrogels were investigated for many aspects, such as injectability, gelation, rheology, mechanical properties, degradability, bioactivity, biocompatibility and angiogenesis. Hydrogel compositions showed facile injectability allowing suitable manual injection by 21 gauge (G) minimum needle thickness, and fast gelation at average body temperature (37 °C). In modified hydrogels, slight pH increment at solutions above 6.4 provided interpenetrating cross-linked morphology starting gelation in 2-3 minutes with enhanced elasticity, and mechanical strength. Hydrogels showed great bioactivity confirmed in Simulated Body Fluid (SBF) by forming mineralised carbonated apatite layer. The gradual hydrogel degradation was confirmed over 6-weeks up to 60% and 70% in PBS and Lysozyme-PBS media respectively. All developed hydrogels were cytocompatible and ensured proliferation of osteoblast-like cells. Optimal CS/HA/Hephydrogel compositions showed excellent pro-angiogenic capacity in ex-ovo chick embryo’s CAM model. Novel tuneable injectable pH and thermosensitive hydrogels (CS/HA/Hep) have tremendous clinical potential for minimal invasive surgery for bone repair and regeneration and targeted drug delivery applications.