| More and more patients are suffered from bone defects caused by traffic accident, construction safety, work injury, tumor and various diseases. So far, autografts can repair bone defects fast, but they may result in some other diseases. Allogenic demineralized bone matrix, the primary alternative in skeletal reconstructive surgery lacks the osteoactive capacity of autografts, and carries the risk of introducing infectious agents or immune rejection. In addition, traditional biomaterials, including bioceramic, bioglass, metal and polymers don't have bioactivity. If they were used as bone grafts, some intrinsic problems such as weak connection, wear and tear, and erosion would appear. Moreover, they are bio-inert. Therefore, it is difficult to alleviate the pains of patients and achieve the function of natural bone. Tissue engineering (TE) provides a solution method for repair of bone defects.Bone TE scaffold materials have been focusing on by researchers, and some have been successfully used in clinical treatment. However, large amounts of problems still need to be resolved. Ceramic is difficult to be degraded; the degraded resultants of polylactic (PLA) acid and poly(L-lactic acid-co-glycolic acid) (PLGA) are acidic, which are not beneficial for new tissue formation; collagen-hydroxyapatite composite scaffolds have good biocompatibility and bioactivity, which can achieve the reconstruction of bone tissue. Collagen-hydroxyapatite composite is a promising bone TE scaffold, but it is expensive and difficult to be widely used. Moreover, the telopeptides that can cause antigenicity after implantation should be removed if they are used in clinical treatment. In addition, glutaraldehyde (GTA), which is widely used as cross-linker to prepare the collagen scaffold due to its abundant content, low price and high crosslinking effect for collagenous tissues, probably causes toxicity when it is released into the host due to the in vivo biodegradation of collagen. Therefore, there is an urgent need in bone TE to build a very strong three dimensional (3D) collagen framework with low price, high bioactivity, high mechanical strength and easy reconstruction in vivo.Porcine acellular dermal matrix (PADM) has been successfully used in covering full thickness of burn wounds and other skin defects. Moreover, PADM is mainly composed of type?and?collagen, which is similar to the organic part of bone matrix. In this paper, we proposed a new method including pre-calcification and biomimetic mineralization to prepare bone TE scaffolds from PADM. Using PADM as an organic matrix and hydroxyapatite as an inorganic component, we prepared PADM-HAp composite scaffolds with two-level 3D porous structures. The preparation method is low-cost, simple and controllable, and the obtained composite scaffold is very tough, flexible and can easily be tailored to a certain size and shape without a mold. We also studied its bioactivity. The main topics of this dissertation are as follows: 1. Extraction and characterization of PADM We explored a new route for the extraction and purification of PADM by basic treatment, enzymic treatment, SDS and sodium chloride treatment. H&E staining and scanning electron microscopy (SEM) analysis prove that there are no remaining cells in PADM, and natural collagen structure is well preserved. The morphology and size of interconnected pores can be adjusted by changeing pH value of the solution. FTIR spectrum of PADM is consistent with commercial type I collagen (Sigma Co., Ltd). The test of total content of amino acids shows that the content of amino acids in PADM is decreased with increasing the concentration of enzymic concentration, while the content of hydroxyproline increases accordingly, the mechanical strength decreases, and the in vitro biodegradation rate increases. 2. In vitro construction and characterization of PADM-HAp composite scafollds with two-level 3D porous structure.There is a small amount of amino acid residues on PADM due to the nature origin of collagen and the mild extraction and purification method. Therefore, there are few active sites for HAp nucleation on collagen molecular surface. We proposed a two-step biomineralization method including precalcification and biomineralization in simulated body fliud (SBF). Precalficaiton process plays a key role in promoting the nucleation of HAp, while biominerzlization in SBF benefits the formation of HAp nanostructures on channel wall of PADM. The obtained PADM-HAp composite scaffolds have two-level pores, with large channels (approximately 100?m in diameter) inherited from the purified PADM microstructure and small pores (less than 100 nm in diameter) formed by self-assembling HAp on the channel surfaces. The mechanical strength of the obtained PADM-HAp scaffolds increases with biomineralization time in SBF and PADM biomineralized for 15 days (S15D) has a compressive elastic modulus as high as 600 kPa, which is much higher than collagen/HAp scaffolds prepared by traditional methods. The presence of HAp in sample S15D reduces the degradation rate of PADM in collagenase solution at 37?. After the culture of MC3T3-E1 pre-osteroblasts for 7 days, MTT data shows no statistically significant difference between pure PADM framework and HAp-PADM scaffold (S15D) indicating its good cell compatibility. 3. Studies on the formation mechanism of two-level 3D PADM-HAp network structurePADM-HAp composite scaffolds are prepared through assembling HAp nanostructures on PADM scaffolds by a two-step biomineralization process. FTIR and EDS results show that large amounts of Ca2+ and HPO42- ions are adsorbed by collagen molecules. They are not precipitated to form calcium phosphate crystals but adsorbed by collagen molecules. SEM results show that there are flake-like structures formed on the collagen fibers. These flake-like HAp nanostructures are proved to grow along their C-axis. Moreover, the two-step biomineralization process is successfully mimicked, and the electric double layer (EDL) theory is employed to explain the mechanism of the adsorption of PO43- (HPO42-) and Ca2+ on collagen molecule surfaces during precacification process, which further promote the fast formation of HAp nanostructures on channel wall of PADM. 4. Protein adsorption capacity of PADM-HAp composite scaffolds and their influence on cell viability. Through changing the biomineralization time in SBF, we prepared PADM-HAp composite scaffolds with different HAp content (S10D:PADM biomineralized in SBF for 10 days, S15D:PADM biomineralized in SBF for 15 days). The porosities of PADM, S10D and S15D are measured to be 87.5%,87.5%and 80%, respectively. Adsorption experiments of fibrinogen and bovine serum albumin (BSA) show that the adsorption capacity of PADM is much higher than that of S10D and S15D. For all the samples, the largest protein adsorption amounts can be achieved between 4-12 hours. Protein adsorption and desorption capacities obviously affect the MC3T3-E1 cell viability seeded on and around FBS-SCAFFOLDs. After cell seeding for 48 h, cells seeded on PADM and S10D show significant proliferation, while on S15D shows no proliferation. Results indicate that the adsorption capacity of scaffolds decreases with the increasing of HAp content. 5. Biocompatibility and bioactivity studies of PADM-HAp composite scaffoldsMicrostructures of scaffolds with different biomineralization periods are reconstructed by micro-CT, and then transplanted into rat muscle to study their in vivo biocompatibility. H&E staining results show that all three kinds of scaffolds (PADM, S10D and S15D) have good biocompatibility. After transplantation into muscle for 30 days, vascular permeability is obvious in the three scaffolds, while large blood vessel can be observed after 60 days. In vivo degradation rates are decreased with increasing biomineralization time in SBF. After intramuscular implantation in rat for 60 days, PADM is completely degraded, most of S10D is degraded, while S15D lost large amounts of its weight after 120 days. It can be observed that both scaffolds and their degraded residues are biocompatible and there are very low immune rejections in muscle. Critical bone defects of rat mandibles about 4 mm in diameter are used for investigation of the bioactivity of PADM and S15D. Micro-CT results show that the bone defects are repaired form the periphery of the defect edges. S15D can promote the repair of bone mandible defect in comparison with PADM. After 15 weeks, H&E, X-ray and micro-CT results show that the defect is well repaired by S15D. |
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