Preparation and Characterization of Chitosan-natural Nano-Hydroxyapatite-Fucoidan Biomaterial based Nanocomposite for Bone Tissue Engineering

다국어 초록 (Multilingual Abstract)

Tissue engineering processes involve the design and development of functional biomaterial substitutes which can serve as carbon copy of the extracellular matrix and produce the essential information for cells to capacitate tissue development processes. Bone tissue engineering provides us with the option to repair damage bone tissues emanating from injuries sustained as a result of bone fracture, osteoarthritis, tumor, trauma and other congenial diseases. As a result of growing demand for bone grafts, research in the area of artificial organ development using functional biomaterials as suitable prostheses in reconstructive surgery for the treatment of loss or damage tissue or organs is gaining momentum.
In this respect, Nano-Hydroxyapatite (nHA) was isolated from salmon bone via an alkaline hydrolysis. The resulting nHA was characterized using several analytical tools, including thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), to determine the purity of the nHA sample. The removal of organic matter from the raw fish was confirmed by TGA. FT-IR confirmed the presence of a carbonated group and the similarities to synthetic Sigma HA. XRD revealed that the isolated nHA was amorphous in nature. Microscopic examination further revealed that the isolated nHA possessed a nanostructure with a size range of 6–37 nm. The obtained nHA interacted with mesenchymal stem cells (MSCs) was non-toxic. Increased mineralization was observed for nHA treated MSCs compared to the control group.
Further, a solid three dimensional (3D) composite scaffold with a high potential usage for bone tissue engineering was prepared using freeze drying method composed of chitosan (C), natural nano-hydroxyapatite (nHA) isolated from salmon bones and fucoidan (F), (C-nHA-F). Fourier transform infrared spectroscopy (FT-IR), Thermal Gravimetric Analysis (TGA), X-ray Diffraction Analysis (XRD), Scanning Electron Microscopy (SEM) and Optical Microscopy (OM) were used to determine physiochemical constituents and morphology of the scaffold. The addition of nHA in the C-F composite scaffold reduce the water uptake and water retention ability. FT-IR analysis shows the presence of carbonated group in the scaffold which is due the presence of nHA that was isolated via alkaline hydrolysis from salmon fish bones. Microscopic results indicated that dispersion of nHA and fucoidan in the chitosan matrix were uniform and showed the pore size (10 to 400 µm) of the composite revealing a suitable micro architecture for cell growth and nutrient supplementation. This was further elucidated in vitro using Periosteum-derived Mesenchymal Stem cells (PMSCs) and revealed profound biocompatibility and excellent mineralization. Thus, we suggest that with further in vivo and clinical investigations, Chitosan-nHA-Fucoidan nanocomposite will be a promising biomaterial for use in bone tissue regeneration applications.

목차 (Table of Contents)

• Abstract 1
• Chapter 1 14
• 1.1 Introduction 14
• 1.2 Nano-hydroxyapatite from natural source 18
• 1.3. Preparation of nHA by synthetic methods 20
• 1.4. Stem cells 22
• 1.5. Stem cell interactions with nHA 23
• 1.6. nHA as delivery vehicles 25
• 1.7. Future directions 31
• 1.8. Conclusion 32
• Chapter 2. 33
• Isolation and characterization of nanohydroxyapatite from salmon fish bone 33
• 2.1. Introduction 34
• 2.2. Materials and methods 36
• 2.2.1. Preparation of salmon fish bone 36
• 2.2.2. Isolation of hydroxyapatite from salmon bone 37
• 2.3. General characterization 37
• 2.3.1 Thermogravimetric analysis 37
• 2.3.2. Fourier transform infrared spectroscopy 37
• 2.3.3. X-ray diffraction analysis 37
• 2.3.4. Microscopic analyses 37
• 2.3.5. Cell culture studies 38
• 2.3.6. Cytotoxicity assessment 38
• 2.3.7. Optical microscopy 38
• 2.3.9. Statistical analysis 39
• 2.4. Results and Discussion 39
• 2.4.1. General observations 39
• 2.4.2. Thermal gravimetric results of salmon HA 39
• 2.4.3. FT-IR spectra results 40
• 2.4.4. X-Ray Diffraction results 42
• 2.4.5. Microscopic results 42
• 2.4.6. Cell culture results 44
• 2.4.7. Morphological studies with Optical microscopy 45
• 2.4.8. Mineralization results 45
• 2.5. Conclusions 46
• Chapter 3. 47
• Preparation and characterization of chitosan-natural nano hydroxyapatite-fucoidan nanocomposite for bone tissue engineering 47
• 3.1. Introduction 47
• 3.2. Materials and Methods 51
• 3.3. Fabrication procedure of scaffolds 52
• 3.3.1. Preparation of chitosan scaffold 52
• 3.3.2. Chitosan-Fucoidan Scaffold 52
• 3.3.3. Chitosan-natural nano hydroxyapatite Scaffold 52
• 3.3.4. Chitosan- natural nHA-Fucoidan Scaffold: 53
• 3.4. Experimental Section 53
• 3.4.1. Thermogravimetric analysis 53
• 3.4.2. Fourier transform infrared spectroscopy 54
• 3.4.3. X-ray diffraction analysis 54
• 3.4.4. Porosity 54
• 3.4.5. Water uptake and retention 54
• 3.4.6. Scanning Electron Microscopy 55
• 3.4.7. Optical microscopy 55
• 3.4.8. PMSC cell culture 55
• 3.4.9. Cytotoxicity assessment 55
• 3.4.10. Optical Microscopy of scaffolds with cells 56
• 3.4.11. Mineralization study 56
• 3.5. Results and Discussion 57
• 3.5.1. General observation 57
• 3.5.2. Porosity of the scaffolds 58
• 3.5.3. Water Absorption and Retention 59
• 3.5.4. Thermogravimetric Analysis results 59
• 3.5.5. FT-IR Analysis Results 59
• 3.5.6. X-Ray Diffraction Results 61
• 3.5.7. Scanning electron microscopy and Optical microscopy 62
• 3.5.8. Cytotoxicity assessment 63
• 3.5.9. Mineralization Results 65
• 3.6. Conclusion 67
• Chapter 4. 68
• 4.1. Cumulative Summary and Conclusion 68
• 5.0. References 69
• 5.1 Acknowledgement 107