PHEMA/SiO_2/n-HA凝胶膜的制备和生物相容性研究
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摘要
关节软骨损伤是骨科较为常见的一种疾病,软骨损伤后不能进行有效地修复,可导致关节软骨组织学环境、关节内力学环境和生物学环境的紊乱,使受损面积扩大,最终导致骨关节炎等严重疾患,寻觅理想的骨创伤修复材料成为治疗该类疾患的关键。
用于软骨组织工程研究的支架材料很多,主要天然材料和人工合成的材料。天然生物材料,修复效果较好但生物力学性能差,不能提供移植后的即时应力支撑,造成移植组织的有害应力损害和周围软骨的异常应力集中,影响修复效果。合成材料具有良好的力学性能,但是由于它的疏水性,使水分子难以在材料中扩散,也不利于关节内营养物质的输运,仿生性能较差。
甲基丙烯酸羟乙酯聚合物(PHEMA)水凝胶比较柔软,力学强度较高,具有一定的透氧性、亲水性,利于关节内外营养物质和代谢产物的交换。但是,PHEMA水凝胶其生物力学性能及机械性能不理想,改造方法是制备PHEMA的复合材料,本研究的设计思路,从改进PHEMA材料的力学强度出发,添加无机成分——SiO_2和n-HA,掺杂入无机成分之后,将使制备的PHEMA无机复合材料在生物力学性能和机械性能上得到改善,n-HA的引入主要考虑使所制备的材料更接近于天然仿生材料,使骨细胞更易生长。本研究的主要创新点在于引入两种无机成分于PHEMA中,并在制备过程通过工艺改革得到具有双层骨架的PHEMA/ SiO_2/ n-HA无机复合凝胶膜,通过生物学实验证明了本研究制备的材料具有良好的生物相容性,适宜于作为骨创伤的仿生修复材料。
本论文主要做了以下几方面研究:
1、以蒸馏水为溶剂,以N',N'-亚甲基双丙烯酰胺(MBA)为交联剂,制备了PHEMA水凝胶。通过电子扫描电镜对PHEMA凝胶的显微形貌进行了观察;研究了MBA添加量对PHEMA凝胶在蒸馏水中的溶胀特性、收缩动力学的影响。实验结果表明添加7.5wt%MBA的PHEMA水凝胶膜表面褶皱和沟痕更少,整体面光滑、表面形态理想,接近于软骨结构。既增加了材料的强度和减少了摩擦又不明显降低吸水量等重要指标。在生理温度下是稳定的,也更接近于人的关节软骨。
2、在前期单纯PHEMA的基础上,加入SiO_2拟制备出含无机盐SiO_2的PHEMA无机复合凝胶膜,以及PHEMA与n-HA复合材料膜,发现两种无机分子都能与HEMA形成良好的共溶混合物,基于生物软骨材料中软骨下骨富含钙,综合考虑,在制备的仿生生物软骨膜中引入两种无机成分,更能贴近原生材料,因此,通过混合HEMA与纳米级SiO_2和n-HA通过引发剂进行聚合,最终制备既含有SiO_2又含有n-HA的双层支架软骨凝胶膜,并对他们的理化性能、膨胀性能、机械性能作了探索。
PHEMA/SiO_2/n-HA无机复合凝胶膜中SiO_2为0.5 wt% ,n-HA的比为1 wt%时所制备的材料为双层支架式材料,它的吸水溶胀性能要比单纯PHEMA、PHEMA/SiO_2、PHEMA/n-HA无机复合凝胶膜低,保水性能良好。在滑动和固定磨料磨损条件下具有很高的耐磨性,PHEMA/SiO_2/n-HA无机复合凝胶膜具有良好的摩擦学性能,能满足受损关节软骨的替换修复后的正常摩擦耐受考验。蛋白吸附实验表明该双层框架结构的无机复合凝胶膜具有良好的吸附蛋白的作用,且在生物蛋白溶液中,不使蛋白质发生变性,揭示了所制备的PHEMA/SiO_2/n-HA无机复合凝胶膜具有良好的生物相容性,将会给生物细胞的培植生长提供一个良好的生存环境。
3、观察人工合成材料PHEMA对兔软骨细胞增殖影响及体内移植骨关节修复情况与炎症反应,采用胶原酶消化法培养兔股关节软骨细胞,免疫细胞化学法鉴定兔股关节软骨细胞;MTT法测定软骨细胞的增殖;流式细胞仪测软骨细胞的生长周期;比色法检测软骨细胞碱性磷酸酶活性; ELISA法测关节液IL-1β、TGF-β、TNF-α;HE染色观察关节修复情况;体内试验取健康成年新西兰大白兔10只,制备双侧直径为1.5cm膝关节缺损模型,采用同体对照方法实验侧(左侧)移植人工合成材料,对照侧自体移植缺损部份骨组织。结果表明PHEMA材料加入0.5% SiO_2不改变软骨细胞增殖活性;软骨细胞活性与PHEMA/0.5%SiO_2/ n-HA无机复合凝胶膜中n-HA含量有关;PHEMA/0.5%SiO_2/n-HA无机复合凝胶膜中含1%n-HA最适合软骨细胞生长;PHEMA/0.5%SiO_2/n-HA无机复合凝胶膜中含1%n-HA可促进软骨损伤缺损修复。
Articular cartilage injury is a common disease in orthopaedics. Articular cartilage injury repaired ineffectively would lead to the disorder of histological environment of articular cartilage, mechanical environment and biological environment, then expand the lesion area, and eventually result into osteoarthritis and other serious diseases. It is a key to find the ideal materials for bone trauma in the treatment of such diseases.
Natural materials and synthetic materials are the main kinds of scaffolds used for research in cartilage tissue engineering. Natural materials have good repairing effect but poor biomechanical property, so they can not provide timely stress support after transplantation, which results in harmful stress damage of transplanted tissue and abnormal stress concentration around the cartilage and affects the recovery effect. Synthetic materials have good biomechanical property but poor hydrophilicity, so water molecules are difficult to diffuse in the synthetic materials and the transport of nutrition is restrained due to their hydrophobicity.
Poly-Hydroxyethyl methacrylate(PHEMA) hydrogel films has good pliability, mechanical strength, oxygen permeability and hydrophilicity, which are beneficial to exchange nutrition and metabolites between internal and external of joint. The biomechanical and mechanical properties of PHEMA are not ideal, so it is necessary to synthesize PHEMA composite to improve its properties. In this paper, SiO_2 and n-HA are introduced to PHEMA in order to improve the biomechanical and mechanical properties. Addition n-HA into PHEMA makes the synthetic material closer to the natural biomimetic materials for osteocyte growth. A double frame of PHEMA/SiO_2/n-HA inorganic composite hydrogel films is prepared by introducing two kinds of inorganic components (SiO_2 and n-HA ) into the PHEMA. Biological study has proved that the material has good biocompatibility and is suitable to be used as bionic repairing material for bone trauma.
The main work of this paper:
1.PHEMA hydrogel films are prepared by using H2O as solvent and MBA as crosslinking agent. The micro appearance of PHEMA was characterized by SEM. Effect of the addition amount of MBA on the swelling property and shrinking dynamics of PHEMA were studied. The results showed that the surface of PHEMA hydrogel films with 7.5wt%MBA is smooth and has less folds and grooves, close to the structure of cartilage. The addition of 7.5wt%MBA increases the strength and reduces the friction without significantly reducing the water absorption. This material is stable under physiological temperature and closer to human articular cartilage.
2. PHEMA/SiO_2 and PHEMA/n-HA inorganic composite hydrogel films were prepared respectively. Two kinds of inorganic ingredients can be mixed very well with HEMA. Subchondral bone is rich in calcium, so two kinds of inorganic ingredients were introduced into bionic perichondrium to make the synthetic PHEMA composites closer to original material. PHEMA/SiO_2/n-HA inorganic composite hydrogel films were prepared by polymerization HEMA with nano-SiO_2 and n-HA through the initiator. The physical and chemical properties, swelling properties and mechanical properties of PHEMA/SiO_2/n-HA inorganic composite hydrogel films were discussed.
PHEMA inorganic composite hydrogel films containing 0.5 wt%SiO_2/ and 1 wt%n-HA is a double-frame structure. It has better swelling property and water retention property than PHEMA, PHEMA/ SiO_2 and PHEMA/ n-HA inorganic composite hydrogel films respectively. PHEMA/SiO_2/n-HA inorganic composite hydrogel films have better tribological properties and can meet the request of normal friction after replacement of damaged articular cartilage. Protein adsorption experiments showed that the double-frame structure material has good adsorption of protein, and can not occur protein denaturation in the biological protein solution. The result indicated the prepared inorganic composite hydrogel films—PHEMA/SiO_2/ n-HA have good biocompatibility and will provide a good living environment to the growth of biological cells.
3.The effect of PHEMA inorganic composite hydrogel films on proliferation of the rabbit chondrocyte and on the recovery and inflammatory reaction of transplanted joint in vivo were observed. Rabbit hip chondrocytes is cultured by collagenase digestion. Immunity cell chemical method is used to identify the chondrocytes of the rabbit joint. Chondrocyte proliferation was determinded by MTT. Chondrocyte growth cycle was measured by flow cytometer. The basic phosphatase activity of the rabbit chondrocyte was detected by chromatometry. Joint fluid IL-1β, TGF-β, TNF-αare measured by ELISA. Joint recovery was observed by using HE staining. In vivo test, 10 rats (derived from healthy adult New Zealand white rabbits) were prepared with bilateral knee defect model 1.5cm in diameter, using the same body control methods with the experimental side (left) transplant synthetic material and the control side autotransplant bone tissue of defect part. The result indicated that PHEMA /0.5% SiO_2 inorganic composite hydrogel films can not change the proliferation activity of the rabbit chondrocyte. The proliferation reactivity of the chondrocyte is related to the content of n-HA in PHEMA/0.5%SiO_2/n-HA inorganic composite hydrogel films. PHEMA/0.5%SiO_2/n-HA inorganic composite hydrogel films containing 1%n-HA is best for the growth of the chondrocyte and promote the defect repair of the cartilage injury.
用于软骨组织工程研究的支架材料很多,主要天然材料和人工合成的材料。天然生物材料,修复效果较好但生物力学性能差,不能提供移植后的即时应力支撑,造成移植组织的有害应力损害和周围软骨的异常应力集中,影响修复效果。合成材料具有良好的力学性能,但是由于它的疏水性,使水分子难以在材料中扩散,也不利于关节内营养物质的输运,仿生性能较差。
甲基丙烯酸羟乙酯聚合物(PHEMA)水凝胶比较柔软,力学强度较高,具有一定的透氧性、亲水性,利于关节内外营养物质和代谢产物的交换。但是,PHEMA水凝胶其生物力学性能及机械性能不理想,改造方法是制备PHEMA的复合材料,本研究的设计思路,从改进PHEMA材料的力学强度出发,添加无机成分——SiO_2和n-HA,掺杂入无机成分之后,将使制备的PHEMA无机复合材料在生物力学性能和机械性能上得到改善,n-HA的引入主要考虑使所制备的材料更接近于天然仿生材料,使骨细胞更易生长。本研究的主要创新点在于引入两种无机成分于PHEMA中,并在制备过程通过工艺改革得到具有双层骨架的PHEMA/ SiO_2/ n-HA无机复合凝胶膜,通过生物学实验证明了本研究制备的材料具有良好的生物相容性,适宜于作为骨创伤的仿生修复材料。
本论文主要做了以下几方面研究:
1、以蒸馏水为溶剂,以N',N'-亚甲基双丙烯酰胺(MBA)为交联剂,制备了PHEMA水凝胶。通过电子扫描电镜对PHEMA凝胶的显微形貌进行了观察;研究了MBA添加量对PHEMA凝胶在蒸馏水中的溶胀特性、收缩动力学的影响。实验结果表明添加7.5wt%MBA的PHEMA水凝胶膜表面褶皱和沟痕更少,整体面光滑、表面形态理想,接近于软骨结构。既增加了材料的强度和减少了摩擦又不明显降低吸水量等重要指标。在生理温度下是稳定的,也更接近于人的关节软骨。
2、在前期单纯PHEMA的基础上,加入SiO_2拟制备出含无机盐SiO_2的PHEMA无机复合凝胶膜,以及PHEMA与n-HA复合材料膜,发现两种无机分子都能与HEMA形成良好的共溶混合物,基于生物软骨材料中软骨下骨富含钙,综合考虑,在制备的仿生生物软骨膜中引入两种无机成分,更能贴近原生材料,因此,通过混合HEMA与纳米级SiO_2和n-HA通过引发剂进行聚合,最终制备既含有SiO_2又含有n-HA的双层支架软骨凝胶膜,并对他们的理化性能、膨胀性能、机械性能作了探索。
PHEMA/SiO_2/n-HA无机复合凝胶膜中SiO_2为0.5 wt% ,n-HA的比为1 wt%时所制备的材料为双层支架式材料,它的吸水溶胀性能要比单纯PHEMA、PHEMA/SiO_2、PHEMA/n-HA无机复合凝胶膜低,保水性能良好。在滑动和固定磨料磨损条件下具有很高的耐磨性,PHEMA/SiO_2/n-HA无机复合凝胶膜具有良好的摩擦学性能,能满足受损关节软骨的替换修复后的正常摩擦耐受考验。蛋白吸附实验表明该双层框架结构的无机复合凝胶膜具有良好的吸附蛋白的作用,且在生物蛋白溶液中,不使蛋白质发生变性,揭示了所制备的PHEMA/SiO_2/n-HA无机复合凝胶膜具有良好的生物相容性,将会给生物细胞的培植生长提供一个良好的生存环境。
3、观察人工合成材料PHEMA对兔软骨细胞增殖影响及体内移植骨关节修复情况与炎症反应,采用胶原酶消化法培养兔股关节软骨细胞,免疫细胞化学法鉴定兔股关节软骨细胞;MTT法测定软骨细胞的增殖;流式细胞仪测软骨细胞的生长周期;比色法检测软骨细胞碱性磷酸酶活性; ELISA法测关节液IL-1β、TGF-β、TNF-α;HE染色观察关节修复情况;体内试验取健康成年新西兰大白兔10只,制备双侧直径为1.5cm膝关节缺损模型,采用同体对照方法实验侧(左侧)移植人工合成材料,对照侧自体移植缺损部份骨组织。结果表明PHEMA材料加入0.5% SiO_2不改变软骨细胞增殖活性;软骨细胞活性与PHEMA/0.5%SiO_2/ n-HA无机复合凝胶膜中n-HA含量有关;PHEMA/0.5%SiO_2/n-HA无机复合凝胶膜中含1%n-HA最适合软骨细胞生长;PHEMA/0.5%SiO_2/n-HA无机复合凝胶膜中含1%n-HA可促进软骨损伤缺损修复。
Articular cartilage injury is a common disease in orthopaedics. Articular cartilage injury repaired ineffectively would lead to the disorder of histological environment of articular cartilage, mechanical environment and biological environment, then expand the lesion area, and eventually result into osteoarthritis and other serious diseases. It is a key to find the ideal materials for bone trauma in the treatment of such diseases.
Natural materials and synthetic materials are the main kinds of scaffolds used for research in cartilage tissue engineering. Natural materials have good repairing effect but poor biomechanical property, so they can not provide timely stress support after transplantation, which results in harmful stress damage of transplanted tissue and abnormal stress concentration around the cartilage and affects the recovery effect. Synthetic materials have good biomechanical property but poor hydrophilicity, so water molecules are difficult to diffuse in the synthetic materials and the transport of nutrition is restrained due to their hydrophobicity.
Poly-Hydroxyethyl methacrylate(PHEMA) hydrogel films has good pliability, mechanical strength, oxygen permeability and hydrophilicity, which are beneficial to exchange nutrition and metabolites between internal and external of joint. The biomechanical and mechanical properties of PHEMA are not ideal, so it is necessary to synthesize PHEMA composite to improve its properties. In this paper, SiO_2 and n-HA are introduced to PHEMA in order to improve the biomechanical and mechanical properties. Addition n-HA into PHEMA makes the synthetic material closer to the natural biomimetic materials for osteocyte growth. A double frame of PHEMA/SiO_2/n-HA inorganic composite hydrogel films is prepared by introducing two kinds of inorganic components (SiO_2 and n-HA ) into the PHEMA. Biological study has proved that the material has good biocompatibility and is suitable to be used as bionic repairing material for bone trauma.
The main work of this paper:
1.PHEMA hydrogel films are prepared by using H2O as solvent and MBA as crosslinking agent. The micro appearance of PHEMA was characterized by SEM. Effect of the addition amount of MBA on the swelling property and shrinking dynamics of PHEMA were studied. The results showed that the surface of PHEMA hydrogel films with 7.5wt%MBA is smooth and has less folds and grooves, close to the structure of cartilage. The addition of 7.5wt%MBA increases the strength and reduces the friction without significantly reducing the water absorption. This material is stable under physiological temperature and closer to human articular cartilage.
2. PHEMA/SiO_2 and PHEMA/n-HA inorganic composite hydrogel films were prepared respectively. Two kinds of inorganic ingredients can be mixed very well with HEMA. Subchondral bone is rich in calcium, so two kinds of inorganic ingredients were introduced into bionic perichondrium to make the synthetic PHEMA composites closer to original material. PHEMA/SiO_2/n-HA inorganic composite hydrogel films were prepared by polymerization HEMA with nano-SiO_2 and n-HA through the initiator. The physical and chemical properties, swelling properties and mechanical properties of PHEMA/SiO_2/n-HA inorganic composite hydrogel films were discussed.
PHEMA inorganic composite hydrogel films containing 0.5 wt%SiO_2/ and 1 wt%n-HA is a double-frame structure. It has better swelling property and water retention property than PHEMA, PHEMA/ SiO_2 and PHEMA/ n-HA inorganic composite hydrogel films respectively. PHEMA/SiO_2/n-HA inorganic composite hydrogel films have better tribological properties and can meet the request of normal friction after replacement of damaged articular cartilage. Protein adsorption experiments showed that the double-frame structure material has good adsorption of protein, and can not occur protein denaturation in the biological protein solution. The result indicated the prepared inorganic composite hydrogel films—PHEMA/SiO_2/ n-HA have good biocompatibility and will provide a good living environment to the growth of biological cells.
3.The effect of PHEMA inorganic composite hydrogel films on proliferation of the rabbit chondrocyte and on the recovery and inflammatory reaction of transplanted joint in vivo were observed. Rabbit hip chondrocytes is cultured by collagenase digestion. Immunity cell chemical method is used to identify the chondrocytes of the rabbit joint. Chondrocyte proliferation was determinded by MTT. Chondrocyte growth cycle was measured by flow cytometer. The basic phosphatase activity of the rabbit chondrocyte was detected by chromatometry. Joint fluid IL-1β, TGF-β, TNF-αare measured by ELISA. Joint recovery was observed by using HE staining. In vivo test, 10 rats (derived from healthy adult New Zealand white rabbits) were prepared with bilateral knee defect model 1.5cm in diameter, using the same body control methods with the experimental side (left) transplant synthetic material and the control side autotransplant bone tissue of defect part. The result indicated that PHEMA /0.5% SiO_2 inorganic composite hydrogel films can not change the proliferation activity of the rabbit chondrocyte. The proliferation reactivity of the chondrocyte is related to the content of n-HA in PHEMA/0.5%SiO_2/n-HA inorganic composite hydrogel films. PHEMA/0.5%SiO_2/n-HA inorganic composite hydrogel films containing 1%n-HA is best for the growth of the chondrocyte and promote the defect repair of the cartilage injury.
引文
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