Development and characterization of calcium phosphate and α/β chitosan biocomposites for bone tissue engineering

Abstract

Since bone is the second most replaced tissue in the human body after blood hence, tissue engineering has been an active research field to investigate possible alternatives for natural bone. Bone is a living tissue that consists of organic and inorganic materials. The organic phase is mainly collagen, and the calcium phosphate compounds that comprise the inorganic phase are known as apatite. This research investigated the potential use of marine waste, such as, mussel shells and squid pens, for the production of biocomposites for tissues engineering. It is envisaged that this approach will add-value to these waste streams as well as improve the environmental impact in terms of the waste management of these materials. With a unique macro and micro structure, to some extent, natural bone has the ability to repair and renew itself. Therefore, bone substitutes with specific pore size, porosity, and biocompatibility that can mimic the natural bone physicochemical properties are required for bone grafting. Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are biocompatible calcium phosphate (CaP) materials which can mimic the inorganic phase of the bone, and so extensive research has taken place to reveal the potential of CaP with respect to bone grafting applications. There is a constant demand for producing high purity CaP with the physicochemical properties that are close to the apatite crystals in natural bone. Firstly, this thesis investigated the feasibility of using microwave irradiation to convert waste Green mussel shells (GMS) (Perna canaliculus) to CaP compounds for biomedical application. Therefore, HA and β-TCP were synthesised from the shells and were chemically, compositionally and structurally characterized and compared with commercial samples of HA and β-TCP. The synthesized β-TCP powders were observed to have a spherical morphology with a length in the range of 100-150 nm while the HA powders showed a rod-like crystalline with length in the range of 30-70 nm. Even though the apatite (CaP) phase of the bone provides the inorganic framework, the bone also has an organic phase therefore the second component of the thesis is related to the synthesis and characterization of the two different chitosan from various sources (crab chitosan (CHC) and arrow squid pen chitosan (CHS)) as the proposed organic phase. Compared to CHC, squid pen chitosan (CHS) had a significantly (P < 0.05) higher viscosity, water and fat binding capacity. Compared to CHC, CHS also had a low packing crystallographic structure and a lower crystallinity index which makes it more susceptible to irradiation. Consequently, this may result in a different structure and different functional properties. In the third phase, the chitosans and the CaP compounds were incorporated in the fabrication of biocomposites. Three biocomposites were prepared using each chitosan and the synthesised CaP from mussel shells, with weight ratios of 20/10/70 to 30/20/50 and 40/30/30 (chitosan/HA/β-TCP wt%). These biocomposites were chemically cross-linked using tripolyphosphate (TPP) and were frozen at -20°C, -80°C or -196°C. The physicomechanical properties, including structural morphology, biodegradation rate, and bioactivity of the biocomposites were investigated. The samples that were frozen at -196°C were not suitable for further investigation as they were too fragile. It was found that with an increase in the weight ratio of HA to β-TCP from 30% to 70%, the Young’s modulus of the biocomposites was increased while the porosity of the biocomposites was decreased. After 28 days of incubation in a physiological solution, the biocomposite with the highest weight ratio of HA to β-TCP showed lower biodegradation (13%) compared with the weight losses of 15% and 17% of the two biocomposites with a lower weight ratio of HA to β-TCP. The CHS composites were found to have a higher porosity (62%) compared to the CHC composites (porosity 42%) and better mechanical properties. The results of this study indicated that the biocomposites produced at -20°C had higher mechanical properties and lower degradation rate compared with -80°C. The experimental results obtained in this study suggest that CaP compounds (HA and β-TCP) with proper physiochemical properties suitable for bone tissue engineering application can be produced from the GMS using the microwave irradiation technique. In addition, it was observed that the squid pen chitosan is a potential alternative for crab shell chitosan for bone regeneration applications. In vitro investigations showed that the biocomposites were cytocompatible and supported the growth of L929 and Saos2 cells. The biocomposites are potential candidates for bone-tissue regeneration.