水凝胶仿生关节软骨材料的制备与性能评价
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摘要
人工材料修复受损软骨,实现与周围机体组织的完好整合,阻止软骨进一步退化,被认为是很有发展潜力的一种软骨修复手段。本研究从材料仿生和功能仿生的角度出发,采用原位复合的方法在聚乙烯醇(PVA)水溶液中合成晶相纳米羟基磷灰石(n-HA)颗粒,然后经过冷冻解冻和辐照交联相结合的方法制备n-HA/PVA水凝胶仿生关节软骨材料,并对相关性能进行了评价,主要结果如下：
1.反复冷冻解冻和γ射线辐照交联相结合的方法制备的PVA水凝胶具有与天然软骨相似的三维多孔网络结构和含水量(约75%)；拉伸和压缩特征曲线都呈现出典型的非线性关系,力学强度随PVA初始浓度和冷冻解冻次数的增加而增大,随辐照剂量的增加先增大后减小,辐照剂量为100kGy时力学强度最高。
2.在PVA水溶液中原位合成的晶相HA陶瓷颗粒为纳米针状结构,PVA分子链在HA结晶过程中起晶核和矿化模板作用,HA相和PVA基体相之间存在一定的氢键结合；n-HA/PVA复合水凝胶材料具有三维多孔网络结构,辐照处理后结构更为致密。
3. n-HA/PVA复合水凝胶材料的拉伸和压缩力学响应分别符合多项式和指数变化规律；力学强度随辐照剂量和n-HA添加量的增大先上升后下降,辐照剂量为100kGy、n-HA含量为6wt%时机械力学性能最优。
4.采用两相有限元方法模拟压凹过程中液体相所承受的载荷情况,结果表明天然软骨和复合水凝胶材料均具有两相特性；Voight-Boltzmann串联模型能够准确地描述自然软骨和复合水凝胶的压凹蠕变行为,说明它们有着极为相似的粘弹性力学特征。
5.摩擦起始阶段,两相润滑机制起主导作用,摩擦系数小,随着载荷作用时间的延长,摩擦系数渐渐增大并达到平衡,润滑机制转为边界润滑；与软骨-软骨摩擦配副相比,水凝胶-软骨摩擦配副的摩擦系数相对较高,但比不锈钢-软骨摩擦配副的摩擦系数小且稳定；复合凝胶材料的摩擦系数随接触应力的增大而增大,而且在低载区摩擦系数的增加幅度较高载区大；分析纳米羟基磷灰石填充和辐照交联改性的协同效果表明,当n-HA添加量为5wt%,辐照剂量为100kGy时,获得的复合水凝胶材料的生物摩擦学性能最优；经过1h的摩擦实验,磨损轨迹表面形成了一个深度约60μm的凹坑,表面粗糙度较实验前有所降低,而且与之配副的软骨销表面粗糙度是天然软骨自配副软骨销表面粗糙度的3倍,但仅仅是与不锈钢配副的软骨表面粗糙度的60%。
6.溶血和MTT试验结果表明,n-HA/PVA复合水凝胶不具有急性溶血作用和细胞毒性；局部植入实验表明材料未引起大兔的肝、肾、脾等脏器以及肌肉和滑膜组织明显的毒性反应,说明制备的水凝胶仿生软骨材料具有良好的生物相容性。
Development of artificial materials that filling the damaged area and integrating with surrounding tissue, preventing further degeneration of articular cartilage, is considered as a promising approach to repair cartilage defect. By imitating the material and function characteristics of nature articular cartilage, in the present work, n-HA/PVA composite hydrogels were prepared by in-situ synthesis, freezing-thawing combined with y-ray irradiation methods. A series of experimental methods were used to investigate properties of n-HA/PVA hydrogel biomimetic articular cartilage, the results are as following:
1. PVA hydrogel prepared by repeated freezing-thawing method followed with y-ray irradiation had similar internal three-dimensional structure and water content (approximately 75%) as nature articular cartilage. Both uniaxial tensile and unconfined compressive properties of PVA hydrogel were found to exhibit nonlinear behavior. Mechanical strength increased as initial concentration of PVA and freezing-thawing cycles increased; the strength increased first with the increasing of irradiation dose and dropped when it reached 100kGy.
2. HA active ceramic particles formed in PVA solution by in-situ synthesis method showed nano-sized needlelike structure. PVA polymer chains acted as template during the formation of HA particles. A hydrogen-bonding was observed to exist between dispersed HA phase and PVA polymer matrix. Internal structure observation by SEM technique showed a three-dimensional network structure and the structure became denser after y-ray irradiation.
3. Uniaxial tensile and unconfined compressive mechanical properties of n-HA/PVA hydrogel conformed polynominal and exponential change rules, respectively. Mechanical strength of n-HA/PVA hydrogel increased first and then dropped with increasing of irradiation dose and n-HA content, it reached the maximum value when the irradiation dose was 100kGy and n-HA content was 6 wt%.
4. A biphasic finite element model was used to simulate the load-bearing condition of liquid phase during indentation creep test, and it was derived that both nature articular cartilage and hydrogel presenting biphasic characteristics. Voight-Boltzmann model was established to describe creep respose of articular cartilage and hydrogel accurately. The results showed hydrogel prepared in our study had similar viscoelastic performance as nature articular cartilage.
5. Friction tests were performed on a reciprocating motion pin-on-plate machine. Friction coefficient rose gradually with increasing loading time up to a constanct value at about 30mins. Initial low friction coefficient was attributed to an effective biphasic lubrication action, after it reached an equivalent state, boundary lubrication mechanism came into action. Friction coefficient of hydrogel-cartilage contact was larger than cartilage-cartilage contact, but much smaller and stabler than stainless steel-cartilage contact. An increase in contact stress was found to increase the friction levels between the articulating surfaces of hydrogel and cartilage, and the change at low load condition was more significant than at high load condition. Frictional properties of hydrogel sliding against articular cartilage were controlled both by irradiation dose and n-HA content, the results showed that friction behavior reached an optimal level when the irradiation dose was 100kGy and n-HA content was 5wt%. It was found a concave on the wear track with the depth of 60μm after 1h loading during friction test, and surface roughness of hydrogel after friction test was smaller than before. Surface roughness of cartilage in hydrogel-cartilage contact was 3 times of it in cartilage-cartilage contact, but 60% of it in stainless steel-cartilage contact.
6. Hemolysis and MTT tests showed that the hydrogel prepared in our study would not induce hemolytic effect to blood or cytotoxic effect to cells, and local implantation tests indicated that the hydrogel didn't bring toxicity to liver, kidney, spleen, muscle or capsule. The hydrogels showed to have excellent biocompatibility.
1.反复冷冻解冻和γ射线辐照交联相结合的方法制备的PVA水凝胶具有与天然软骨相似的三维多孔网络结构和含水量(约75%)；拉伸和压缩特征曲线都呈现出典型的非线性关系,力学强度随PVA初始浓度和冷冻解冻次数的增加而增大,随辐照剂量的增加先增大后减小,辐照剂量为100kGy时力学强度最高。
2.在PVA水溶液中原位合成的晶相HA陶瓷颗粒为纳米针状结构,PVA分子链在HA结晶过程中起晶核和矿化模板作用,HA相和PVA基体相之间存在一定的氢键结合；n-HA/PVA复合水凝胶材料具有三维多孔网络结构,辐照处理后结构更为致密。
3. n-HA/PVA复合水凝胶材料的拉伸和压缩力学响应分别符合多项式和指数变化规律；力学强度随辐照剂量和n-HA添加量的增大先上升后下降,辐照剂量为100kGy、n-HA含量为6wt%时机械力学性能最优。
4.采用两相有限元方法模拟压凹过程中液体相所承受的载荷情况,结果表明天然软骨和复合水凝胶材料均具有两相特性；Voight-Boltzmann串联模型能够准确地描述自然软骨和复合水凝胶的压凹蠕变行为,说明它们有着极为相似的粘弹性力学特征。
5.摩擦起始阶段,两相润滑机制起主导作用,摩擦系数小,随着载荷作用时间的延长,摩擦系数渐渐增大并达到平衡,润滑机制转为边界润滑；与软骨-软骨摩擦配副相比,水凝胶-软骨摩擦配副的摩擦系数相对较高,但比不锈钢-软骨摩擦配副的摩擦系数小且稳定；复合凝胶材料的摩擦系数随接触应力的增大而增大,而且在低载区摩擦系数的增加幅度较高载区大；分析纳米羟基磷灰石填充和辐照交联改性的协同效果表明,当n-HA添加量为5wt%,辐照剂量为100kGy时,获得的复合水凝胶材料的生物摩擦学性能最优；经过1h的摩擦实验,磨损轨迹表面形成了一个深度约60μm的凹坑,表面粗糙度较实验前有所降低,而且与之配副的软骨销表面粗糙度是天然软骨自配副软骨销表面粗糙度的3倍,但仅仅是与不锈钢配副的软骨表面粗糙度的60%。
6.溶血和MTT试验结果表明,n-HA/PVA复合水凝胶不具有急性溶血作用和细胞毒性；局部植入实验表明材料未引起大兔的肝、肾、脾等脏器以及肌肉和滑膜组织明显的毒性反应,说明制备的水凝胶仿生软骨材料具有良好的生物相容性。
Development of artificial materials that filling the damaged area and integrating with surrounding tissue, preventing further degeneration of articular cartilage, is considered as a promising approach to repair cartilage defect. By imitating the material and function characteristics of nature articular cartilage, in the present work, n-HA/PVA composite hydrogels were prepared by in-situ synthesis, freezing-thawing combined with y-ray irradiation methods. A series of experimental methods were used to investigate properties of n-HA/PVA hydrogel biomimetic articular cartilage, the results are as following:
1. PVA hydrogel prepared by repeated freezing-thawing method followed with y-ray irradiation had similar internal three-dimensional structure and water content (approximately 75%) as nature articular cartilage. Both uniaxial tensile and unconfined compressive properties of PVA hydrogel were found to exhibit nonlinear behavior. Mechanical strength increased as initial concentration of PVA and freezing-thawing cycles increased; the strength increased first with the increasing of irradiation dose and dropped when it reached 100kGy.
2. HA active ceramic particles formed in PVA solution by in-situ synthesis method showed nano-sized needlelike structure. PVA polymer chains acted as template during the formation of HA particles. A hydrogen-bonding was observed to exist between dispersed HA phase and PVA polymer matrix. Internal structure observation by SEM technique showed a three-dimensional network structure and the structure became denser after y-ray irradiation.
3. Uniaxial tensile and unconfined compressive mechanical properties of n-HA/PVA hydrogel conformed polynominal and exponential change rules, respectively. Mechanical strength of n-HA/PVA hydrogel increased first and then dropped with increasing of irradiation dose and n-HA content, it reached the maximum value when the irradiation dose was 100kGy and n-HA content was 6 wt%.
4. A biphasic finite element model was used to simulate the load-bearing condition of liquid phase during indentation creep test, and it was derived that both nature articular cartilage and hydrogel presenting biphasic characteristics. Voight-Boltzmann model was established to describe creep respose of articular cartilage and hydrogel accurately. The results showed hydrogel prepared in our study had similar viscoelastic performance as nature articular cartilage.
5. Friction tests were performed on a reciprocating motion pin-on-plate machine. Friction coefficient rose gradually with increasing loading time up to a constanct value at about 30mins. Initial low friction coefficient was attributed to an effective biphasic lubrication action, after it reached an equivalent state, boundary lubrication mechanism came into action. Friction coefficient of hydrogel-cartilage contact was larger than cartilage-cartilage contact, but much smaller and stabler than stainless steel-cartilage contact. An increase in contact stress was found to increase the friction levels between the articulating surfaces of hydrogel and cartilage, and the change at low load condition was more significant than at high load condition. Frictional properties of hydrogel sliding against articular cartilage were controlled both by irradiation dose and n-HA content, the results showed that friction behavior reached an optimal level when the irradiation dose was 100kGy and n-HA content was 5wt%. It was found a concave on the wear track with the depth of 60μm after 1h loading during friction test, and surface roughness of hydrogel after friction test was smaller than before. Surface roughness of cartilage in hydrogel-cartilage contact was 3 times of it in cartilage-cartilage contact, but 60% of it in stainless steel-cartilage contact.
6. Hemolysis and MTT tests showed that the hydrogel prepared in our study would not induce hemolytic effect to blood or cytotoxic effect to cells, and local implantation tests indicated that the hydrogel didn't bring toxicity to liver, kidney, spleen, muscle or capsule. The hydrogels showed to have excellent biocompatibility.
引文
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