高强度纳米羟基磷灰石/胶原仿骨材料研究

【作者】 林晓艳；

【导师】 张兴栋；

【作者基本信息】 四川大学 ， 生物医学工程， 2005， 博士

【摘要】 随着现代交通、体育事业发展带来的中青年创伤以及人口老龄化造成的骨质疏松症的增加,对骨修复材料的需求不断增长,综观骨修复材料的研究现状不难发现,现有的骨修复材料:自体骨或异体骨、医用金属或合金、陶瓷及高分子材料等存在许多不足,没有一种材料能完全满足当前临床治疗的需求。虽然自体骨移植临床效果令人满意,但骨源数量有限,且需进行两次手术。异体骨移植容易带来疾病并存在免疫反应等问题。金属植入材料因与周围的骨弹性模量不匹配,可能引起应力遮挡和骨吸收。高分子材料的低弹性模量限制了它们在骨修复中的广泛应用。尽管生物活性的陶瓷材料如羟基磷灰石与骨形成骨性结合,但其易碎性和难降解性使其不适宜用于承重骨的修复。因此,单一的材料难以满足骨修复的需要,应用现代高新技术研制新的骨替代复合材料刻不容缓。 纳米羟基磷灰石/胶原复合材料,因其组成结构类似于自然骨,具有良好的生物相容性、生物降解吸收性、生物活性等特点,而倍受人们的关注。然而,目前报道的各种方法所制备出的纳米羟基磷灰石/胶原复合材料都存在力学强度低、溶胀度高、降解快等缺点,限制了材料的临床应用。上述材料的不足之处主要归咎于胶原纤维热稳定性差、易降解,纳米羟基磷灰石团聚、难于在胶原基质中均匀分布,无机相的含量难于提高,相对含量低,与胶原的界面结合差等。针对这些问题,本研究从胶原的提取及其特性表征、胶原纤维形成特性、灭菌条件改进、复合材料的合成方法及工艺条件优化、干燥方式、交联方法等多方面进行系统研究,研究出低温酸溶-胃蛋白酶消化、盐析、胶原纤维重建形成水凝胶的提取方法和25kGy⁶⁰Co低温辐照的灭菌方法。根据胶原受热易降解的特性和胶原纤维形成特性研究出低温原位合成、自然干燥、戊二醛原位交联更多还原

【Abstract】 With the growth of the aged population and the development of modern transportation and athletics, the increment of osteoporosis and the middle-age and youth wound, the need to bone repairing materials increased continuously. The bone substitutes currently available have specific disadvantages and none of them is entirely suitable for the clinical applications including autograft or allograft, metals or metal alloy, polymer and ceramics. While autografts and allografts usually achieve good results, their applications are restricted due to requiring two surgical procedures and donor shortage for autograph and immunologic response and risk of transmitting diseases for allograft. A mismatch of mechanical properties between metals and surrounding bone may cause stress-shielding and bone absorption. Low elastic modulus of polymer limited its application in bone reconstruction. Although hydroxyapatite bioactive ceramics can bond to bone tissue, it is not proper for them to be used for the repair of bearing-bone due to its brittleness and undegradability. Therefore, it is unlikely that a single materials woule be capable of meeting the functional requirements of bone.Nanohydroxyapatite/collagen (NanoHA/COL) composites have been extensively investigated due to their composition and structure similar to natural bone. However, nano-HA/COL composites synthesized by various current methods demonstrate low mechanical strength, high swelling degree and fast degradation. These inadequacies limit their clinical application. Those shortcomings of the composite are arrtributed to instability and fast degradation of collagen fibers, conglomeration of nanohydroxyapatite, inhomogeneous dispersion and low content of hydroxyapatite in collagen matrix and weak interface bond between hydroxyapatite and collagen. Therefore, a systematic study, from collagen extraction, sterilization and in vitro fibrillogenesis, synthesis and drying of the composite, optimization of preparation conditions and crosslinkage of the composite, was performed with the aim to improve the mechanical strength and swelling degree of the composites.The extraction method and conditions of acid dissolution-pepsin digestion,reconstruction of collagen fibrils and 60Co irradiation sterilization at low temperature were developed to obtain medical type I collagen. After implanted subcutaneously in rat 12 weeks, collagen degradation was observed. Collage shape was still intact at 20 weeks. Degradation of collagen in subcutaneous was found much slower than the others reported. Fibrillogenesis of collagen at low temperature is favorable to form the uniform and stable collagen fibers and nano-H A/COL composites. The degree of cross-linking and stability of collagen after y-irradiation were improved. In a range of less than 25kGy irradiation dose, no significant differences in cytocompatibility of collagen irradiated by y-ray were observed. However, when irradiation doses were beyond 25 kGy, the cytocompatibility of collagen was influenced by y-radiation to some degree. Collagen sterilized by 25kGy 60Co at low temperature had passed hygienic evaluationIn this paper, a method of in situ synthesis, crosslinking by glutaraldehyde and air-drying of the composite at low temperature was explored to fabricate the nanoHA/COL composite with high bending-strength, low swelling degree and proper degradation rate. Comparison of the characteristics of the composites prepared by three methods revealed that the low temperature in situ synthesis and air-drying was an effective way to obtain biomimetic nanoHA/COL composites with good homogeneity and mechanical strength.L9(3)4 orthogonal array design was implemented to optimize experimental conditions for preparation of the composite using in situ synthesis. Content of hydroxyapatite of the composite, synthesis temperature and pH were chosen as main parameters. As a result of the orthogonal analysis in this study, content of hydroxyapatite and synthesis temperature were the most influencing parameter on bending and compressive strength. Proper content of HA and low synthesis temperature were beneficial to high bending strength of the composite. The optimum experimental conditions were achieved by orthogonal array. The mean bending-strength of the composite synthesized by in situ synthesis and air-dry at the optimum synthesis condition was 90Mpa which is two times higher than that reported.The composite were crosslinked by immersing them in aqueous solutions containing glutaraldehyde (GA) and in situ crosslinking methods. GA in situ crosslinkage was more effective to decrease the swelling degree and to improve the mechanical stability of the composite than the other.FTIR, XPS, DSC and SEM were employed to investigate the mechanism of synthesis, drying and crosslinkage of the composite with high mechanical strength. Air-drying and GA crosslinking of the composite induced the conformation changes of the collagen molecules and the formation of bonding between collagen fibers and HA crystals, as indicated by the shifts of amide A, B, I and HI bands to lower wave numbers and changes in chemical state and binding energy of Ols, Cls, Nls, Ca2p, and P2p. The formation of network structures and enhanced interfacial bond in lie crosslinked HA/COL composite resulted to the decrease of swelling degree and increment of mechanical strength of the composites.Scanning electron microscopy and energy-dispersive X-ray analysis before and after immersion of the composite in SBF confirmed that the composite had bioactivity. Biocompatibility and degradability of the composite were evaluated via tests of cytotoxity, sensitization, genotoxicity and implantation in bone and subcutaneous of the composite, according to ISO 10993 and GB/T16886 guidelines. New bone formation was observed in a boundary layer between the composite and recipient bone after 4 weeks. Lager quantitative of newly bones was found in the region around the composite after 12 weeks. After implanted subcutaneously in rat 44 weeks, the composite degraded into small fragments and tissues grew into the region resulted from the degradation of the composite. The results of biological evaluation suggested that the composites had excellent biocompatibility, satisfactory biodegradability and bone-formation ability. The composite did not induce genotoxicity, cytotoxicity and sensitization.更多还原