DEVELOPMENT OF CHITOSAN BASED POROUS COMPOSITES FOR BONE TISSUE ENGINEERING

Abstract

Impaired fracture healing during post-traumatic orthopedic condition is one of the major health related issues worldwide arising at an exponential pace. The most commonly prevalent diseased states like diabetes, age-related conditions like osteoporosis and gender-specific conditions like menopause diminish the healing mechanism. Morbidity, loss of functionality, increased chances of infection and compliance issues in conventional techniques (allograft or artificial implants) make the treatment less effective, henceforth there arises a need for reconstructing the lost tissue by stimulating the body’s innate healing mechanism. The limitation of the bone repair mechanism to hairline fractures necessitates the need for a new strategy. Stimulating the regeneration process with the assistance of cell proliferation template, cells and growth factors is one of the potential strategies of bone tissue engineering which builds a sort of hope of obtaining the lost tissue without losing its functionality. An interconnected porous matrix with optimum pore size, mechanical strength, mineralization tendency, synchronized degradation rate, osteoinductive and osteogenic differentiation potential is a characteristic of an ideal three-dimensional construct, however a single material is not sufficient to cover all these aspects so a combination of two or more hybrid materials is generally taken to attain desired properties. In order to overcome these shortcomings we selected xylan/chitosan conjugate as a parent matrix for fabricating three-dimensional construct owing to its inherent antioxidant, antimicrobial and immunomodulatory tendencies of the matrix which makes it an ideal candidate for bone healing application. In the current research, we developed porous composites of xylan/chitosan conjugate by incorporating various fillers with aim of enhancing the desired properties for bone construct. In the first study we fabricated xylan/chitosan/MMT (Montmorillonite) based composite by the freeze drying process. Addition of MMT brought improvement in microstructural properties with refined porous architecture. The pore size significantly increased from 102 to 290 µm on increasing filler content from 1 to 4%. All the composite matrices also exhibited significant mineralization tendency, however, apatite deposited on samples with 2% MMT content had Ca/P ratio of 1.67, which matches the stoichiometric ratio of natural bone apatite. Needle-like morphology of deposited apatite crystals were observed in samples with 5% MMT content. The average length and thickness of apatite needles were calculated to be 140 µm and 1.2 µm respectively. MTT assay results demonstrated the non-cytotoxic nature of the scaffold with good cell viability. ALP assay revealed increase in osteogenic differentiation potential on increasing MMT content. The second step was to analyze the effect of bioactive ceramics on the properties of xylan/chitosan composite matrix and optimize the composition for further studies. Different concentrations of HAp (hydroxyapatite) and BG (bioactive glass) nanopowder were incorporated in xylan/chitosan conjugate solution and the matrix was fabricated by freeze-drying samples at -72°C. A parallel comparison in properties of two bioactive nanoceramic nanocomposites was performed. The samples were analyzed for their microstructural properties, mechanical strength, mineralization tendency and osteoinductive potential to obtain the best possible combination. 30% HAp incorporated nanocomposite proved to be the best suitable material of choice for bone repair. The third study analyzes the effect of GO (graphene oxide) and rGO (reduced graphene oxide) on the xylan/chitosan/HAp matrix to be used for bone healing. The results revealed a synergistic increase in mineralization potential, mechanical properties, cell viability and cell differentiation capability at a definite filler loading of both fillers, however, mineralization tendency and osteogenic differentiation potential was comparatively higher in a rGO-based composite. In fourth study we fabricated xylan/chitosan/nanoβ-TCP/CNC-based scaffolds and analyzed the effect of varying concentrations of CNC on the properties of the composite. The results revealed a linear decline in swelling and degradation rate on increasing filler concentration from 1-10%. Increase in mechanical properties was observed up to 1% CNC content. Variations in mineralization, protein adsorption, and osteogenic differentiation was observed on tuning CNC concentration. Thus it can be stated that xylan/chitosan porous composites with different combination of nanofillers (rGO/GO-nHAp, MMT and CNC-TCP) can be a futuristic approach in bone regeneration therapy.