Construction And Bioproperty Study Of Two-level Three-dimensional Networked Citosan/Hydroxyapatite Composite Scaffold Materials For Bone Tissue Engineering

In the modern world there are increasingly urgent demands for various biomedical bone implants to repair bone defects caused by bone fractures, congenital malformation, osteoarthritis, osteoporosis or bone cancers. However, bone substitute including autografts, allografts, and an assortment of synthetic or biomimetic materials and device, all have some drawbacks. Although bone autografts are recognized as gold standard, their supply is limited and there are many potential drawbacks associated with their use including donor site morbidity, danger of infection, and pain. Allografts have the risk of introducing infectious agents or immune rejection. Though the traditional synthetic bone substitute including hydroxyapatite or calcium phosphate ceramics do not have the drawbacks from autografts and allografts, they only possess the functions of filling, supporting and osteoconduction, and do not have high bioactivity, especially osteoinduction. Therefore, it is difficult to achieve the functions of natural bone. Thus, the requirement for complete regeneration of bone and restoration of its function is a major clinical need.Bone tissue engineering has been regarded as an important strategy to repair and regenerate bone tissue. Bone scaffolds as synthetic extracellular matrix materials are significant for bone tissue engineering. According to the structure and composition of natural bone, biodegradable polymer/hydroxyapatite (HAp) composites have been widely studied. Now, chitosan as an important biodegradable natural polymer, due to its biocompatibility, biodegradability, cationic nature, ready availability, and anti-bacteria, has been the most promising natural polymer next to collagen for bone tissue engineering.Presently, the fabrication techniques for porous HAp/chitosan composite bone scaffolds are based on either mixing or in situ co-precipitation methods. Though the mixing and in situ co-precipitation approaches are simple processes with low cost, it is difficult to control the surface chemistry and geometry within a large and complex structure. In addition, these methods for chitosan/HAp scaffold preparation normally use glutaraldehyde (GTA) as a crosslinker, a substance that is toxic when it is released in the host during the biodegradation process. In order to develop ideal bone-like composites with enhanced mechanical properties and improved bioactivity, biomimetic mineralization has become an effective strategy to assemble bone-like apatite that is close to natural bone with low crystallinity and nanoscale size. However, it is difficult to achieve the controlled nucleation and growth of HAp nano-crystals on a chitosan-based framework. Thus, it is critical to control and attain desirable bioactive mineral surface structures within a chitosan-based framework through a nontoxic and controllable preparation method, and it is still a great challenge for bone tissue engineering.Bone marrow-derived mesenchymal stem cells (BMSCs) as one of adult stem cells have attracted particular attention in bone tissue engineering, because they are readily accessible, with low immunogenicity and multilineage differentiation potential. Recent studies have indicated the surface characteristics of biomaterials including chemical composition, roughness, and topography, especially nanotopography, can profoundly affect cell functions. Thus, understanding BMSCs response to the microenvironment in scaffold can identify the effect of the microenvironment in scaffold on BMSCs behaviors at the molecular level. It can provide the basis of the theory for the application of BMSCs in bone tissue engineering and exploring tissue-inducing biomaterials that have special bioacitivity. Recently, with increased knowledge of the interactions of stem cells with biomaterial surfaces, tissue engineering is becoming increasingly oriented toward designing and engineering biomaterial surfaces that promote specific cellular phenotypes. Moreover, the surface characteristics of bone scaffold, especially regarding the sustained delivery of growth factors (such as bone morphogenetic protein-2,BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis.Based on the backgrounds mentioned above, the objective of this study was to control and attain desirable bioactive hydroxyapatite structures within a chitosan-based scaffold through a nontoxic and controllable preparation method, and investigate the effect of surface microenvironment of the obtained composite scaffolds on the osteogenic differentiation potential of seeded BMSCs in vitro and sustained delivery of bioactive molecules (such as BMP-2). The main topics of this dissertation are as follows:1. Construction of two-level three-dimensional networked chitosan/hydroxyapatite composite scaffold for bone tissue engineering and its cell biocompatibilityUsing a nontoxic cross-linker (genipin), a nano-crystallon induced biomimetic mineralization method, and hydrothermal synthesis, in situ co-precipitation and lyophilization technology, we have demonstrated the construction of a2-level3D networked chitosan/hydroxyapatite (HGCCS) by assembling HAp nanostructures on the chitosan framework for bone tissue engineering. The first level of the network is the chitosan3D interconnected macroporous framework (-150μm) that is favorable for cell immigration and mass transport; the second level is the nano-network and nanostructure of HAp self-assembled on the channel surface of the chitosan framework (-150nm) that provides the special microenvironment for cell growth and function.X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and fourier transform infrared spectroscopy (FTIR) analysis confirm that the continuous network-like nanostructure on the channel surface of the HGCCS is composed of crystalline HAp. Non-toxic genipin is used as a crosslinker and it endows high strength and intrinsic fluorescence to the chitosan framework. The nano-crystalline HAp seeds play a determining role in the construction of HAp nanostructure on the channel surface, and they reinforce the produced scaffold.The compressive elastic modulus (EM) of the HGCCS obtained increases to50.49±2.23MPa. We further explore the novel fluorescence properties of genipin-cross-linked chitosan that suggest a promising application for3D scaffold imaging and tracking, and structural observation of the degradation process. In addition, the intrinsic fluorescence may provide an effective way to image the cell-scaffold interaction and effectively monitor the adhesion, localization and migration of cells on the surface of the scaffolds.The results of SEM observation of cell morphology, and CLSM observation of MC3T3-E1pre-osteroblasts cultured for3days on GCF and HGCCS after nuclei and F-actin staining confirmed their good cell compatibility. Rat BMSCs were extracted and seeded in scaffolds, the results of SEM observation of cell morphology, and CLSM observation of cytoskeleton organization further confirmed that HGCCS possess good cell compatibility.Fetal bovine serum (FBS) as a model protein was used to evaluate the protein adsorption capacity of HGCCS. Compared to GCF and GCGF, the special surface properties of HGCCS, including increased surface area, nanocrystallinity and microporosity with nanosized pores enhanced increased adsorption of FBS for easier access to nutrients by BMSCs. The results of adsorption experiments of FBS further confirmed the results of cell proliferation assay.2. In vitro assessment of the osteogenic differentiation potential of BMSCs on HGCCSWe examined the osteogenic differentiation potential of BMSCs on HGCCS in vitro. Given the same culture conditions, cell shape and cytoskeleton organization showed significant differences between cells cultured on GCF and those cultured on HGCCS after7days of incubation. The result of specific alkaline phosphatase (ALP) activity as an indicator of osteogenic differentiation showed that the ALP activity of rat BMSCs was higher on HGCCS. Based on quantitative real time PCR (RT-PCR), HGCCS induced highest mRNA expression of osteogenic differentiation markers, runt-related transcription factor2(Runx2) by7days, osteopontin (OPN) by7days and osteocalcin (OCN) by14days, respectively. The enhanced ability of BMSCs on HGCCS to produce mineralized extracellular matrix and nodules was also assessed on day14with Alizarin red staining. The results of this study suggest that in the same culture conditions, compared to GCF, the surface HAp nano-network structure of HGCCS acts as a critical signal cue promoting osteogenic differentiation in vitro.3. Enhanced osteogenic differentiation of BMSCs in vitro on two-level three-dimensional networked HGCCS based on surface microstructure for sustained delivery of BMP-2We evaluated the effect of the surface special HAp nano-network structure of HGCCS on the BMP-2adsorption and release ability, and the resulting osteogenic differentiation of rat BMSCs in vitro. HGCCS exhibited a loading efficiency of65%, which is significantly higher than28%for GCF, as quantified by an enzyme-linked immunosorbent assay (ELISA). We also found that the release of BMP-2from HGCCS was sustained for at least14days in vitro, compared to that from GCF, which consisted of burst releases on days1and3. Moreover, the BMP-2release from HGCCS induced an increase in alkaline phosphatase activity of BMSCs for14days in vitro. Based on quantitative real time PCR, HGCCS also stimulated the highest mRNA expression of osteogenic differentiation markers, Runx2for14days, OPN for3days and OCN for14days. The results of this study suggest that the surface HAp nano-network structure of two-level3D HGCCS used as a delivery system for BMP-2is capable of promoting osteogenic differentiation in vitro. As a result, HGCCS is a promising scaffold for bone tissue engineering.4. Study of repairing of rat cranial critical size defect with porcine accellular dermal matrix (PADM)-HAp composite scaffold in a cranial modelIn previous studies, we first demonstrated the construction of a2-level3D PADM-HAp composite scaffold. Further, critical bone defects of rat cranial about4mm in diameter were used for investigation of the bioactivity of PADM and PADM-HAp. After15weeks, the results of histochemical stainings and Micro-CT showed that compared to PADM, the defect was well repaired with use of PADM-HAp.