摘要
第一部分聚乳酸/纳米羟基磷灰石可吸收椎间融合器的制备及其生物力学测试
目的:制备出聚乳酸/纳米羟基磷灰石可吸收椎间融合器,并对其进行生物力学性能的测试。
方法:采用热致相分离法制备出不同纳米羟基磷灰石质量百分比(0、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%)的聚乳酸/纳米羟基磷灰石可吸收材料,通过电镜扫描进行表征,测量可吸收材料的压缩强度、抗弯强度、弯曲模量,通过比较得出聚乳酸与纳米羟基磷灰石的最佳质量配比,并由此制备出聚乳酸/纳米羟基磷灰石可吸收椎间融合器。以6具青年男性新鲜腰椎(L1～L5)尸体标本进行试验,分为三组:正常组、自体髂骨组、聚乳酸/纳米羟基磷灰石可吸收椎间融合器组即材料组,分别进行脊柱三维运动测试、最大拔出力实验、压缩毁损实验。
结果:当纳米羟基磷灰石的质量比20%时,其力学性能达到峰值,通过电镜扫描,所制备聚乳酸/纳米羟基磷灰石可吸收材料微观结构为纳米级,具有良好的孔隙率,因此,以聚乳酸与纳米羟基磷灰石质量比为80:20的配比,制备出聚乳酸/纳米羟基磷灰石可吸收椎间融合器。在前屈、左右旋转、左右侧弯的加载中,材料组中L3/4节段之间运动范围,均较髂骨组减少;在前屈、左右侧弯加载中,材料组均较正常组减少;在最大拔出力测试中,材料组的最大拔出阻力较大;在压缩毁损实验中,材料组对椎体有很好的保护作用,而且它承载能力明显好于自体髂骨组。
结论:所制备聚乳酸/纳米羟基磷灰石可吸收椎间融合器,其微观结构为纳米级,与天然纤维支架相似,预计具有良好的生物相容性,力学性能较好,植入椎间后,具有较好稳定性和抗压性能,为临床应用提供了力学基础。
第二部分聚乳酸/纳米羟基磷灰石/骨髓间充质干细胞复合椎间融合器的动物实验研究
目的:检测聚乳酸/纳米羟基磷灰石可吸收材料的细胞相容性,制备聚乳酸/纳米羟基磷灰石/骨髓间充质干细胞复合椎间融合器,观察其椎间融合效果以及材料的可吸收性。
方法:采集山羊骨髓间充质干细胞进行细胞培养,将上一步所制备聚乳酸/纳米羟基磷灰石可吸收材料与骨髓间充质干细胞混合培养,采用MTT法检测干细胞在可吸收材料孔隙中黏附和增殖情况,并进行电镜扫描。将聚乳酸/纳米羟基磷灰石可吸收椎间融合器与骨髓间充质干细胞混合培养制备出聚乳酸/纳米羟基磷灰石/骨髓间充质干细胞复合椎间融合器,将其与聚乳酸/纳米羟基磷灰石可吸收椎间融合器以及自体髂骨植入同一山羊椎体间,术后分期收集标本,进行CT扫描,生物力学测试、大体观察、光镜观察和电镜扫描。
结果:骨髓间充质干细胞与聚乳酸/纳米羟基磷灰石可吸收材料混合培养6小时后,骨髓间充质干细胞在聚乳酸/纳米羟基磷灰石复合支架材料上的黏附率为72.1%。材料组细胞增殖情况明显高于对照组,电镜扫描也显示干细胞在可吸收材料支架中具有较好的黏附和增殖情况,通过椎间融合器植入山羊体内术后融合效果的观察,术后短期内,自体髂骨具有较好的融合效果,随着时间延长,可吸收材料均能达到与自体髂骨相同的融合效果,而且,聚乳酸/纳米羟基磷灰石/骨髓间充质干细胞复合椎间融合器能较快的达到良好的椎间融合。
结论:本研究制备出的聚乳酸/纳米羟基磷灰石可吸收椎间融器具有良好的生物相容性,并且通过进一步复合,制备出具有生物活性的可吸收椎间融合器,证明能够满足椎间骨组织替代的要求,具有广阔的应用前景。
第三部分聚乳酸/纳米羟基磷灰石可吸收椎间融合器的生物安全性研究
目的:观察聚乳酸/纳米羟基磷灰石可吸收材料的生物安全性。
方法:将聚乳酸/纳米羟基磷灰石可吸收材料浸提液对SD大鼠进行腹腔内注射,观察大鼠的急性毒性反应;在SD大鼠脊柱两侧种植聚乳酸/纳米羟基磷灰石可吸收材料和单纯聚乳酸试样,于术后15、30、60、90天取材,观察试样周围组织的炎症反应和纤维囊壁情况以及心、肝、肾等脏器情况。
结果:所有实验动物在观察期间无一例死亡,各动物活动、进食、生长情况良好,未出现腹泻,震颤,呼吸困难,运动机能减退等情况;术后15天,聚乳酸/纳米羟基磷灰石可吸收材料组和聚乳酸组周围均可见中性粒细胞,炎症反应Ⅱ级,囊壁反应均Ⅳ级。术后30天,炎症反应Ⅰ级,囊壁反应Ⅲ级,术后60天,炎症反应Ⅰ级、囊壁反应均Ⅱ级,术后90天,炎症反应和囊壁反应均为Ⅰ级或以下;术后各时间点观察,心、肝、肾等脏器均正常。
结论:该自制聚乳酸/纳米羟基磷灰石可吸收材料植入局部反应良好,有较好的生物安全性。
Part 1 Preparation and Biomechanical Study of PLA/nano-hydroxyapatite Absorbable Interbody Fusion Cage
Object:To produce the PLA/nano-hydroxyapatite absorbable interbody fusion cage and analyze the biomechanical capability of the interbody fusion cage.
Methods:PLA/nano-hydroxyapatite compound material of different n-HA mass fraction(0、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%) was produced by the thermal induced phase separation method.The absorbable material was characterized by the scanning electron microscope observation.Its mechanics performance(pressure strength、bending strength、curving module) were tested.PLA/nano-hydroxyapatite absorbable interbody fusion cage was produced according to the best n-HA mass fraction.6 fresh lumber spine specimens(L1-L5) from young male cadavers were used,The specimens were classified into 3 groups:the normal group,auto iliac bone group and PLA/n-HA interbody fusion cage group.The specimens were measured by the three dimensional motional stability test,the pullout test,and compression destoryed test. capability of PLA/nano-hydroxyapatite compound material reached the peak value.The absorbable material had the three dimensional porous micro-structure.The PLA/nano-hydroxyapatite absorbable interbody fusion cage was produced according to the 20%mass fraction of HA.The absorbable interbody fusion cage exhibited a significant increase in stability between L3 and L4 in flexion,left/right lateral bending and left/right axial rotation than iliac crest bone.The absorbable interbody fusion cage exhibited a significant increase in stability between L3 and L4 in flexion,left/right lateral bending than the normal group.The material group had the more pullout resistance than iliac crest bone group. The absorbable interbody fusion cage group can protect vertebrae,and it has very good load capacity better than iliac bone group.
Conclusion:The produced PLA/nano-hydroxyapatite absorbable interbody fusion cage has the three dimensional porous micro-structure. Its micro-structure is similar to extracellular matrix.It is producted that the material has the better biocompatibility.It has the better capability of mechanics,stability and anti-compression.
Object:To detect the cell compatibility of PLA/nano-hydroxyapatite absorbable material.Produce PLA/nano-hydroxyapatite/ Bone marrow derived mesenchymal stem cells(MSCs) Compound Interbody Fusion Cage.Observe the its effect of interbody fusion and absorbability.
Methods:Bone marrow derived mesenchymal stem cells were extracted from the bone marrow in goats and cultured.Bone marrow derived mesenchymal stem cells were cultured with PLA/nano-hydroxyapatite absorbable material.Tested the cell multiplication and adhesion under the influence of the material by MTT methods.PLA/nano-hydroxyapatite absorbable interbody fusion cage and Bone marrow derived mesenchymal stem cells were cocultured to produce PLA/nano-hydroxyapatite/ Bone marrow derived mesenchymal stem cells(MSCs) Compound Interbody Fusion Cage.It was imbedded the interbody of goat lumbar to evaluate the effect of interbody fusion.
Results:When Bone marrow derived mesenchymal stem cells were seeded into scaffolds,the cell attachment rate in PLA/nano-hydroxyapatite was 72.1%.The cell proliferation assay showed that the cell number in PLA/nano-hydroxyapatite was higher than that in control group.In a short term after operation,the fusion effect of iliac crest bone group was best.But the last all the group can reach the same fusion effect.
Conclusion:The PLA/nano-hydroxyapatite absorbable material has the better biocompatibility.It can be used to repair the bone defects in interbody fusion.
Objective:Conserve the Biosafety of PLA/nano-hydroxyapatite material by animal experiments,prepare for clinical application.
Methods:The leaching liquor of the material was injected into mice to test the acute toxicity.To explore the biocompatibility of PLA/nano-hydroxyapatite absorbable material,implant test were performed in SD rat.
Results:All animals were alive in the labor period.No significant difference in general situation,the food utilization rate and the body weight relative growth rate.There was only mild inflammatory reaction and thin fibrosis membrane in the early days after PLA/ PLA/nano-hydroxyapatite absorbable material plate implantation,the fibrosis membrane was nearly disappeared and most of the imflammatory reaction recovered 3 months later.The heart,liver and kidney were all normal.
Conclusion:The self-made PLA/nano-hydroxyapatite absorbable material has a good Biosafety,it has a great clinical application prospects.
引文
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