摘要
软骨组织内缺乏血管化作用和神经末梢,所以受损的软骨自修复能力有限。这使软骨缺损的治疗成为外科一大难题。用成熟的软骨进行同种异体移植术缺少移植物与周围组织的横向结合,而尖端的自体移植术又不能赋予植入体早期的机械强度。
水凝胶能被用于人工软骨是由于它具有好的润滑性和粘弹性。在各种用于制备水凝胶的人工合成亲水性高分子中,聚乙烯醇(polyvinyl alcohol,简PVA)水凝胶因具有无毒、生物相容性良好及机械性能优良等特性而被广泛用于生物医学领域。纳米羟基磷灰石(nano-hydroxyapatite,简n-HA)具有良好的生物相容性和骨传导性,期望它能诱导钙化和在植入体与软骨下骨间形成生物性键合。明胶(gelatin,简Gel)是一种结缔组织蛋白,无毒,有好的生物相容性和生物降解性。
基于上述特性,本研究制备了一种n-HA/PVA/Gel人工软骨材料和一种潜在的组织工程支架材料。本论文主要包含如下研究内容和结果:
1.用溶液共混法制备了纳米羟基磷灰石/聚乙烯醇/明胶三元复合水凝胶材料。测定了材料的含水率,采用TEM、SEM、EDAX、IR、XRD和燃烧实验对材料的结构及组分进行了表征和分析,此外,还研究了材料的吸水性。结果表明,制备的复合水凝胶组成均一,各组分间存在相互作用,并具有良好的吸水和保水性能。本研究制备的纳米羟基磷灰石/聚乙烯醇/明胶复合水凝胶可望作为一种人工软骨材料。
2.采用FT-IR和XRD技术对n-HA/PVA/Gel复合材料进行了分析,详细讨论了其内部的化学键合作用。测定了不同组分比例的复合水凝胶的拉伸强度和压缩强度,并分别测定了干胶的吸水率。结果表明,n-HA/PVA/Gel复合材料内部有较强的氢键和配位键作用;吸水率随PVA含量增加而增加;其机械强度与自然软骨相近。
3.用乳化法制备了n-HA/PVA/Gel多孔支架材料。SEM观察表明该支架材料孔间贯通性良好,富含100~500μm的大孔,大孔壁上有小于20μm的小孔,其孔隙率达78%,而吸水率高达312.7%。材料浸入水中由于明胶的降解其重量不断下降。当其浸入模拟体液后,由于Ca2+和PO43-离子在材料的内外表面不断沉积使其重量不断增加。通过TF-XRD, FT-IR和EDAX等表征手段证明沉积物是碳酸根替代的羟基磷灰石。该材料由MTT法证明无细胞毒性。同时,n-HA/PVA/Gel复合材料被皮下植入成年雌性大鼠的背部,12周后,可观察到多孔材料与周围组织紧密结合,成纤维组织长入孔隙内部,并有部分材料发生降解。
4.本文合成了PVA/Gel水凝胶,并用IR及XRD对其进行表征,结果表明PVA和Gel之间通过氢键发生交联。以吐温20为乳化剂采用乳化发泡法制备了PVA/Gel多孔支架。用SEM观察表明支架中孔隙相互连通,孔隙率达76%,孔径大小适于组织长入。该支架在去离子水和模拟体液(SBF)中的吸水率通过重量分析法进行检测。MTT实验表明支架对兔角膜成纤维细胞无毒性;兔角膜成纤维细胞培养72小时后仍有强烈增殖的趋势。将支架浸入SBF中8周后,其重量发生明显变化,大量的类骨磷灰石颗粒在支架孔壁上沉积,表明其具有好的生物活性。这些反映了PVA/Gel支架作为一种人工角膜的周边支架具有很大的应用潜力。
Damaged cartilage has a limited ability to repair itself due to the absence of vascularization and nerve endings in the tissue. Thus the treatment of cartilage lesion is one of the most important problems in orthopedic surgery. Reconstruction of articular cartilage defects using adult osteochondral allografts is lack of lateral integration of the grafts to the surrounding tissue, and sophisticated autologous chondrocytes transplantation can not provide implants with mechanical strength early.
Hydrogel can be used as artificial cartilages due to their good lubrication and elasticity. Among various synthetic hydrophilic polymers used in the preparation of hydrogel, polyvinyl alcohol (PVA) hydrogel has definitely gained biomedical applications because of its inherent non-toxicity, good biocompatibility and desirable physical properties such as elasticity and high swelling property in aqueous solutions. Nano-hydroxyapatite (n-HA) has been proved to have good biocompatibility and osteoconductivity, and it is expected to induce calcification and biological bond between implant and subchondral bone. Gelatin (Gel), a connective tissue protein, is well known for its non-toxic, good biocompatibility and biodegradability. The incorporation of Gel in the composite is expected to enhance the biocompatibility.
In this paper, an artificial cartilage material and a potential scaffold for cartilage tissue engineering based on PVA, n-HA and Gel were prepared, respectively. The contents included the following.
1. A novel composite, composed of n-HA, PVA and Gel, was prepared by blending them in solution. Water content and burning test were used to analyze the homogeneity of the composites. The materials were also characterized by Scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDAX), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and the water absorption rate of the hydrogels with different ratio of n-HA/PVA/Gel were tested. The results showed that the prepared hydrogels had good homogeneity and suitable water absorption rate. The n-HA/PVA/Gel hydrogel could be used as an artificial cartilage material.
2. The n-HA/PVA/Gel composite was characterized by Fourier transform infrared absorption spectroscopy (FT-IR), X-ray diffraction (XRD) and the possible interior chemical bonds were also discussed. Hydrogen bonds and coordination bonds may exist between n-HA, PVA and Gel. Mechanical strength and water absorption of the prepared composite were investigated, respectively. The results show that water absorption of the samples is enhanced from 77% to 150% with the increase of PVA, and that n-HA/PVA/Gel composite has similar mechanical properties to natural cartilage. The composite has excellent biocompatibility in vivo.
3. A novel porous composite material composed of n-HA, PVA and Gel was fabricated by emulsification. SEM showed that the material had a well-interconnected porous structure including a lot of micropores (less than 20μm) on the walls of macropores (100~500μm). The composite had a porosity of 78% and showed high water absorption rate up to 312.7% indicating a good water-swellable behavior. When immersed in water, the weight of the scaffold continuously decreased. However, after immersion in simulated body fluid (SBF), the weight continuously increased because Ca2+ and PO43- ions deposited on the surface and the internal surfaces of the material pores. The deposits were proved to be carbonated hydroxyapatite by thin-film X-ray diffraction (TF-XRD), FT-IR and EDAX. The composite was proved to be non-cytotoxicity by MTT assay. The n-HA/PVA/Gel material was also implanted subcutaneously in the dorsal region of adult female rats. After 12 weeks of implantation, the porous material adhered tightly with the surrounding tissue, and the ingrowth of fibrous tissue as well as the material’s partial degradation was observed.
4. PVA and Gel were used to fabricate a gelatinous composite. The PVA/Gel composite was characterized by IR and XRD, which indicated that PVA and Gel was cross-linked via hydrogen bondings. PVA/Gel scaffold was prepared by emulsification and its microstructure was observed with SEM. The scaffold had a high porosity up to 76% and the pore size is fit for tissue ingrowth. Water absorption of the scaffolds in deionized water and in SBF was monitored by gravimetric procedure. The scaffold was proved to be non-toxic for rabbit corneal fibroblasts by MTT assay. We observed that rabbit corneal fibroblasts proliferated intensively in fluorescence photos after 72 hours of cell culture. After 8 weeks immersion in water and SBF, the scaffold’s weight varied obviously and a lot of apatite deposited along the internal surfaces of the porous structure, which demonstrated that the scaffold had good bioactivity and degradation property. The data herein reflect the promising utility of PVA/Gel scaffold as fringe scaffold of keratoprosthesis.
引文
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