Hybrid Biopolymer Nanocomposites for Tissue Engineering Applications

Abstract

This thesis involves study of the biopolymers (natural and synthetic) based scaffolds for tissue engineering applications. Natural polymers were chemically modified for tissue engineering applications leading to cancer targeting and skin tissue regeneration applications. In one approach, synthetic polymer systems were physically modified (blends) and strategically incorporated with lab synthesized calcium phosphate (CAPs) based bio-ceramics for tissue regeneration applications. Scaffolds were physically characterized by Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), Nuclear magnetic resonance spectroscopy (NMR), X-RAY diffraction (XRD), Scanning electron microscopy (SEM), Elemental analysis, Zeta potential measurements, and Tensile mechanical testing. For tissue engineering applications different cell lines were used. Scaffolds for Bone Tissue engineering were tested against normal fibroblasts (NIH3T3), preosteoblast (MC3T3-E1) and bone marrow stem cell (BMSc) cell lines. Scaffold for targeted cancer therapy and wound healing were evaluated against normal fibroblasts (NIH3T3), mouse skin melanoma (B16F10), human epithelial adenocarcinoma (MDA-MB-231) and human breast adenocarcinoma (MCF-7) cell lines. Natural polymer i.e. microcrystalline cellulose (MCC) was modified into aminated cellulose derivatives via tosylation viz. 6-deoxy-6-hydrazide cellulose (Cell Hyd), 6-deoxy-6- diethylamide cellulose (Cell DEA), and 6-deoxy-6-diethyltriamine cellulose (Cell DETA). IC50 values from Presto blue assay and live/dead assay images analysis of amino cellulose were obtained against normal fibroblasts, melanoma, and breast cancer cell lines. MCC was non cytotoxic while Cell Hyd, Cell DEA and Cell DETA exhibited noncytotoxic activity up to 200 μg/ml to normal fibroblast cells NIH3T3 indicating no toxicity against normal cell lines. Cell Hyd, Cell DEA and Cell DETA maintained targeted cytotoxicity against the cancer cells such as mouse skin melanoma (B16F10) followed by human breast adenocarcinoma (MCF-7). In next step natural polymer gelatin was chemically modified into GelMA. MCC, Cell Hyd, Cell DEA and Cell DETA were embedded in methacrylated gelatin (GelMA) by photocrosslinking to obtain hydrogels. Cell Hyd and Cell DETA decreased porosity of GelMA. Tensile strain of GelMA 61.30 % at break was increased to 64.3% in case of GelMA/Cell- HYD. In vitro cytocompatibility and cell proliferation using NIH-3T3 cell lines showed cell density trend on scaffold as GelMA/Cell-DETA>GelMA/Cell-Hyd> GelMA. Scratch assay for wound healing revealed that GelMA/Cell-DETA showed complete wound closure, while GelMA/Cell-Hyd and GelMA exhibited 85.7%, and 66.1% wound healing, respectively in 8 hours. In vivo tests on rats revealed that GelMA/Cell-DETA exhibited 98% wound closure on day 9, whereas GelMA/Cell-Hyd exhibited 97.7% and GelMA 66.1% wound healing on day 14. Our findings revealed that GelMA embedded amine MCC derivatives hydrogels can be applied for achieving accelerated wound healing. Synthetic polymer Poly(L-lactic acid) (PLLA) was blended with COC polymer and nano CAPs such as Hydroxyapatite(nHA) and whitlockite (nWH). Structure, thermomechanical properties and cytocompatibility of these scaffolds were studied for Bone Tissue Engineering (BTE) applications. Prestoblue, LIVE/DEAD assay, Actin and Dapi assay, and Alizarin red assay were carried out for BTE. PLLA/COC blends up to 5-30 wt % of COC were prepared. Structurally, PLLA/COC are miscible, have Vander walls interaction of two polymers, decrease in crystallinity of PLLA, and α′-crystalline phase which coexists with inherent α-phase of PLLA. PLLA/COC blends exhibited superior mechanical properties in relation to the pure PLLA. Compressive modulus values for PLLA/COC 10 wt % increased 117 % as compared to pure PLLA. PLLA/COC blends at 10 wt % has maximum ultimate tensile strength, modulus, and toughness ∼ 123 %, 67.8 % and, 18.87 % respectively. PLLA/COC blends showed increased swelling and degradation results as compared to PLLA. PLLA/COC blends exhibited more than 90 % cytocompatibility over PLLA with preosteoblast (MC3T3-E1) and bone marrow stem cells (BMSc) cell lines suggesting possible candidate for Bone Tissue Engineering. In next step nHA was successfully lab synthesized and characterized. nHA (1, 5, 10, 20 wt %) was added in PLLA/COC10 blend and PLLA for comparative studies and increase in hydrophilicity of the scaffold. nHA has homogeneous distribution up to nHA 10%, whereas agglomeration at nHA 20% in both nanocomposites. nHA addition has effect on interfacial bonding between two polymers in PLLA/COC10. PLLA has α′-crystalline phase which coexists with inherent α-phase alpha form and increase in crystallinity by increasing the concentration of nHA indicated by XRD, DSC, and FTIR. Mechanical properties of PLLA/COC10 with different nHA weight percentage exhibited 140 to 240% increase in toughness as compared to PLLA-nHA. PLLA/COC10-nHA10% showed highest antimicrobial activity in Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes in relation to PLLA-nHA. Invitro MC3T3-E1 and BMSC cell lines showed 2-3 folds enhancement in cell viability and proliferation upon culture on PLLA/COC/HA blends as compared to PLLA/nHA composites. It was clearly observed that ternary system PLLA/COC10-nHA has good dispersion and interfacial interaction resulting in improved thermomechanical and enhanced osteoconductive properties as compared to PLLA-nHA. In another strategy two CAPs namely nanohydroxyapatite (nHA) and nano whitlockite (nWH) were lab synthesized. Different weight percentages (1-5 %) of these nanoparticles were dispersed in PLLA to compare their effects on the structure, mechanical properties, antibacterial potential, and cytotoxicity of PLLA. Homogeneous dispersion of both nanoparticles were achieved in PLLA. Mechanical properties of the materials showed that addition of nanoparticles have made PLLA ductile and strong pertaining to the effective dissipation of loads across the interfaces, enabling them to delay the crack growth by avoiding stress concentration sites. PLLA-nWH10% showed highest antibacterial activity against gram positive bacteria Listeria monocytogenes and Staphylococcus aureus. In-vitro cytocompatibility of PLLA-nWH10% was highest 97.7% at day 7 against fibroblasts NIH3T3 whereas all nanocomposites have enhanced cell viability (94%) as compared to pure PLLA. Alizarin red assay showed 2 folds increase in calcification at day 14 with PLLA-nWH as compared to PLLA-nHA. It was clearly observed that binary system PLLA-nWH at all concentrations has good dispersion and interfacial interaction resulting in improved mechanical and enhanced osteoconductive properties as compared to PLLA-nHA.