摘要
背景:聚乳酸类高分子聚合物与羟基磷灰石复合材料的研究是目前骨修复材料的研究热点。聚L-乳酸/聚L-乳酸接枝的纳米羟基磷灰石(PLLA/PLLA-gHAP)为具有独立知识产权的新型骨修复材料,具有较好的力学强度,其与骨修复的功能细胞成骨细胞间的相互作用尚无相关报道。目的:观察新型骨修复材料PLLA/PLLA-gHAP对成骨细胞黏附、增殖功能的影响。设计、时间及地点:细胞学体外实验,于2006-03/2007-03在东北师范大学细胞与遗传研究所完成。材料:①将骨修复材料PLLA/PLLA-gHAP及PLLA制备成2cm×2cm×0.5mm的板,浸泡、冲洗、消毒灭菌后备用。②将PLLA/PLLA-gHAP、PLLA两种板剪成0.5cm×0.5cm×0.5mm的小块,加入DMEM培养液,配成10,100g/L两种质量浓度的浸提液,于37℃孵箱中浸提72h,4℃保存备用。方法:取孕19～20d的Wistar大鼠胎鼠的颅骨进行成骨细胞体外培养。①光镜下观察成骨细胞形态,应用碱性磷酸酶染色、钙结节染色对其进行鉴定。②将成骨细胞分别接种在PLLA/PLLA-gHAP和PLLA材料板上,24h后应用细胞技术器检测细胞黏附数量,扫描电镜观察细胞形态。③MTT法测定成骨细胞在不同质量浓度(10,100g/L)PLLA/PLLA-gHAP材料浸提液中的增殖情况,并与PLLA材料进行对比。主要观察指标:①成骨细胞在PLLA/PLLA-gHAP和PLLA材料上的黏附数量和形态。②成骨细胞在PLLA/PLLA-gHAP和PLLA材料浸提液中的增殖情况。结果:①在PLLA/PLLA-gHAP表面黏附的细胞数略多于PLLA材料,且黏附于PLLA/PLLA-gHAP材料表面的成骨细胞直径较大,伸出的伪足与材料间形成一种牢固的锚状结构。而在PLLA表面的细胞细胞直径较小,多为球形,很少伸出伪足。②10g/LPLLA/PLLA-gHAP浸提液组的细胞活性明显高于10g/LPLLA浸提液组和空白对照组;两种材料100g/L质量浓度组的细胞活性均低于空白对照组,但PLLA/PLLA-gHAP组的细胞活性仍高于PLLA组。结论:①成骨细胞在PLLA/PLLA-gHAP材料表面较PLLA材料表面显示更好的黏附能力。②成骨细胞在PLLA/PLLA-gHAP材料浸提液中较在PLLA材料浸提掖中显示更好的增殖能力。③新型PLLA/PLLA-gHAP材料具有较好的成骨细胞相容性,可能作为骨修复材料使用。
BACKGROUND： Studies regarding poly（L-lactide）/hydroxyapatite composite materials are the hot spot for bone repairing. The poly（L-lactide）/poly（L-lactide） surface grafted hydroxyapatite （PLLA/PLLA-gHA） is a newly repairing material with excellent mechanical strength. However, the interaction between PLLA/PLLA-gHA with osteoblasts is poorly understood. OBJECTIVE： To assess the effect of PLLA/PLLA-gHA on adhesion and proliferation of osteoblasts. DESIGN, TIME AND SETTING： The in v#ro cytology experiment was performed at the Institute of Genetics and Cytology, Northeast Normal University between March 2006 and March 2007. MATERIALS： （1）PLLA/PLLA-gHA and PLLA were prepared for plate with 2 cmx2 crux0.5 mm in size, and the plate were reserved following soak, washing and sterilization. （2）PLLA/PLLA-gHA and PLLA plates were cut into 0.5 cm×0.5 cm×0.5 mm pieces, cultured with DMEM to obtain leaching liquor with 10 g/L, 100 g/L, and then extracting at the 37℃ incubator for 72 hours followed by reserving at 4℃.
METHODS： The osteoblasts which were derived from the fetal rat cranium of the 19 20 days pregnant Wistar rats were cultured in vitro. （1）The osteoblasts were observed under the light microscope and identified by the alkaline phosphates （ALP） staining and alizarin red staining.（2）Rat osteoblasts were seeded on the PLLA/PLLA-gHA and PLLA plate, the adherent cell number and morphological changes of osteoblasts were observed by cell arithmometer and scanning electron microscopy after 24 hours of culture. （3）The osteoblasts were also cocultured with the leaching liquor of PLLA/PLLA-gHA and PLLA. The proliferation of the cells was determined by inverted microscope and MTT method. MAIN OUTCOME MEASURES： （1）The adherent cell number and morphological changes of osteoblasts on two materials. （2） The cell proliferation in PLLA/PLLA-gHA and PLLA leaching liquor. RESULTS： （1）The adherent number of osteoblasts on the surface of PLLA,＇PLLA-gHA was higher with larger diameter than that of PLLA, and the osteoblasts are more easily to adherent and spread on PLLA/PLLA-gHA. Cells on the PLLA surface had smaller diameter with globular shapes. （1）The cellular activity of osteoblasts in the 10 g/L leaching liquor of PLLA/PLLA-gHA was higher than that of PLLA and blank control group. The cellular activity of 100 g/L leaching liquor of PLLA/PLLA-gHA and PLLA group were lower than blank control group, however, the PLLA/PLLA-gHA group was still higher than PLLA group. CONCLUSION： The osteoblasts show better adhesion on the surface of the PLLA/PLLA-gHA than PLLA and higher proliferation rate in the leaching liquor of PLLA/PLLA-gHA than PLLA. The PLLA/PLLA-gHA exhibit excellent biocompatibility to rat osteoblasts, which can be used as bone repairing materials.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第34期6617-6621,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金面上项目(30670563)~~
