新型骨修复材料的制备与体外研究
发布时间：2018-09-06 14:01
【摘要】：目的 本研究以PGA和β-TCP为基料制备复合支架材料,并观察复合支架材料的微观结构,测试其孔隙率、抗压强度。将富血小板血浆(Platelet-Rich-Plasma,简称PRP)作为内源性生长因子与制备出的复合支架材料复合研究其对成骨细胞的增殖、分化作用。为动物实验及临床应用提供实验依据。 方法 1.改良溶液浇铸-离子沥滤法制备质量比1：1(A组)与1：3(B组)的PGA/β-TCP复合支架材料,扫描电镜(SEM)观察两组支架材料的微结构,用万能材料试验机对制备的试样进行抗压缩强度的测试,用液体置换法测试PGA/β-TCP复合支架材料的孔隙率。 2.成骨细胞的原代培养：无菌条件下处死一日龄SD大鼠,超净台下剥离出颅骨片,Ⅰ型胶原酶消化成单个细胞后以10%胎牛血清的DMEM培养基培养、纯化。传至第三代,用倒置显微镜观察细胞形态,碱性磷酸酶染色法、茜素红钙结节染色法鉴定成骨细胞。 3.取SD大鼠全血,两步离心法制备PRP,将上步培养的三代成骨细胞接种于制备好的PGA/β-TCP复合支架材料上并与PRP复合。实验A组为成骨细胞复合5%PRP/PGA/β-TCP支架,B组为成骨细胞复合10%PRP/PGA/β-TCP支架。对照组为成骨细胞复合不加PRP的PGA/β-TCP材料。于培养的1d、2d、3d时做细胞增殖MTT检测,2d、4d、6d做碱性磷酸酶定量检测。 结果 1.SEM照片可见两组支架材料均为三维多孔结构,孔隙的连通性好。β-TCP均匀地分散于聚合物中,B组支架材料中p-TCP颗粒明显多于A组。B组支架材料的抗压缩强度大于A组,差异有统计学意义(P0.01)；A、B组支架材料孔隙率均大于85%,A组孔隙率大于B组,差异有统计学意义(P0.01)。 2.倒置显微镜下可见48h后细胞贴壁生长良好,细胞体积较大,呈三角形、多角形或不规则形。胞浆丰富并向外伸展,有突起。7d后细胞长满培养瓶,细胞间出现融合,细胞边界不清。第三代细胞碱性磷酸酶染色可见多数细胞细胞核、胞质内染色呈阳性。钙结节茜素红染色可见细胞之间有团块状的钙盐沉积,呈橘红色或深红色。矿化结节大小不一,有的多个矿化结节可以融合。 3.实验A、B组与对照组的细胞增殖随时间的延长而增加,实验A、B组较对照组各相同时点细胞增殖明显上升,同一时间点,实验B组测得的OD值最高,差异有统计学意义(P0.05)；3组在2d时检测出碱性磷酸酶的活性均较低, 4、6d时三组成骨细胞ALP的活性随时间的延长呈增加趋势,实验B组成骨细胞ALP的活性随时间变化最为明显,其6d时成骨细胞ALP含量显著高于2d时的ALP含量。对照组各相同时点ALP的活性虽也有升高,但都低于实验两组。差异有统计学意义(P0.05)。 结论 1.A、B组PGA/β-TCP复合支架材料孔隙率均在85%以上,B组复合支架抗压缩强度显著高于A组,B组(1：3质量比)PGA/β-TCP复合支架更能适应临床对支架材料的要求。 2.PRP可以促进PGA/β-TCP复合支架上成骨细胞的增殖及提高ALP的活性。其中10%浓度的PRP对PGA/β-TCP组复合支架材料上成骨细胞增殖及ALP活性作用要高于5%PRP浓度组。
[Abstract]:objective
In this study, PGA and beta-TCP were used to prepare composite scaffolds, and the microstructure of composite scaffolds was observed, the porosity and compressive strength were tested. It provides experimental evidence for animal experiment and clinical application.
Method
1. The PGA/beta-TCP composite scaffolds with mass ratio of 1:1 (group A) and 1:3 (group B) were prepared by improved solution casting-ion leaching method. The microstructure of the scaffolds was observed by scanning electron microscopy (SEM). The compressive strength of the prepared samples was tested by universal material testing machine. The porosity of PGA/beta-TCP composite scaffolds was measured by liquid displacement method.
2. Primary culture of osteoblasts: one-day-old SD rats were sacrificed under aseptic conditions, skull slices were stripped off under super-clean table, and the collagenase type I was digested into a single cell and cultured in DMEM medium with 10% fetal bovine serum, and then passed to the third generation. Cell morphology was observed by inverted microscope, alkaline phosphatase staining and alizarin red calcium nodule staining. Osteoblasts.
3. PRP was prepared from the whole blood of SD rats by two-step centrifugation method. Three generations of osteoblasts were inoculated into PGA/beta-TCP composite scaffolds and combined with PRP. The osteoblasts in group A were mixed with 5% PRP/PGA/beta-TCP scaffolds, while the osteoblasts in group B were combined with 10% PRP/PGA/beta-TCP scaffolds. Beta -TCP material. MTT was detected in cultured 1D, 2D and 3D, and 2D, 4D and 6D were used for quantitative detection of alkaline phosphatase.
Result
1. SEM images showed that the two groups of scaffolds were three-dimensional porous structure with good pore connectivity. Beta-TCP was dispersed evenly in the polymer. The p-TCP particles in group B were significantly more than those in group A. The compressive strength of group B was higher than that of group A, and the difference was statistically significant (P 0.01). Compared with group B, the difference was statistically significant (P0.01).
2. Under inverted microscope, the cells adhered to the wall and grew well 48 hours later. The cells were large, triangular, polygonal or irregular in size. The cytoplasm was abundant and extended outward with protuberances. After 7 days, the cells grew into culture flasks and fused with each other, and the cell boundary was unclear. Alizarin red staining of calcium nodules showed massive calcium deposits between cells, which were orange or dark red. The mineralized nodules varied in size and some of them could fuse.
3. The proliferation of cells in group A, B and control increased with time. The proliferation of cells in group A and B increased significantly at the same time point compared with the control group. At the same time point, the OD value of group B was the highest, and the difference was statistically significant (P 0.05); the activity of alkaline phosphatase was lower in the three groups at the 2nd day.
The activity of ALP in osteoblasts of experimental group B was the most obvious change with time. The content of ALP in osteoblasts of experimental group B was significantly higher than that of osteoblasts of 2 days at 6 days. The activity of ALP in control group was also increased at the same time, but was lower than that of experimental group (P 0.05). ).
conclusion
1.The porosity of PGA/beta-TCP composite scaffolds in group A and group B was above 85%. The compressive strength of PGA/beta-TCP composite scaffolds in group B was significantly higher than that in group A. The PGA/beta-TCP composite scaffolds in group B (1:3 mass ratio) were more suitable for the clinical requirements of scaffolds.
2. PRP can promote the proliferation of osteoblasts on PGA/beta-TCP composite scaffolds and increase the activity of ALP. The effect of 10% PRP on osteoblasts proliferation and ALP activity on PGA/beta-TCP composite scaffolds was higher than that on 5% PRP group.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
[Abstract]:objective
In this study, PGA and beta-TCP were used to prepare composite scaffolds, and the microstructure of composite scaffolds was observed, the porosity and compressive strength were tested. It provides experimental evidence for animal experiment and clinical application.
Method
1. The PGA/beta-TCP composite scaffolds with mass ratio of 1:1 (group A) and 1:3 (group B) were prepared by improved solution casting-ion leaching method. The microstructure of the scaffolds was observed by scanning electron microscopy (SEM). The compressive strength of the prepared samples was tested by universal material testing machine. The porosity of PGA/beta-TCP composite scaffolds was measured by liquid displacement method.
2. Primary culture of osteoblasts: one-day-old SD rats were sacrificed under aseptic conditions, skull slices were stripped off under super-clean table, and the collagenase type I was digested into a single cell and cultured in DMEM medium with 10% fetal bovine serum, and then passed to the third generation. Cell morphology was observed by inverted microscope, alkaline phosphatase staining and alizarin red calcium nodule staining. Osteoblasts.
3. PRP was prepared from the whole blood of SD rats by two-step centrifugation method. Three generations of osteoblasts were inoculated into PGA/beta-TCP composite scaffolds and combined with PRP. The osteoblasts in group A were mixed with 5% PRP/PGA/beta-TCP scaffolds, while the osteoblasts in group B were combined with 10% PRP/PGA/beta-TCP scaffolds. Beta -TCP material. MTT was detected in cultured 1D, 2D and 3D, and 2D, 4D and 6D were used for quantitative detection of alkaline phosphatase.
Result
1. SEM images showed that the two groups of scaffolds were three-dimensional porous structure with good pore connectivity. Beta-TCP was dispersed evenly in the polymer. The p-TCP particles in group B were significantly more than those in group A. The compressive strength of group B was higher than that of group A, and the difference was statistically significant (P 0.01). Compared with group B, the difference was statistically significant (P0.01).
2. Under inverted microscope, the cells adhered to the wall and grew well 48 hours later. The cells were large, triangular, polygonal or irregular in size. The cytoplasm was abundant and extended outward with protuberances. After 7 days, the cells grew into culture flasks and fused with each other, and the cell boundary was unclear. Alizarin red staining of calcium nodules showed massive calcium deposits between cells, which were orange or dark red. The mineralized nodules varied in size and some of them could fuse.
3. The proliferation of cells in group A, B and control increased with time. The proliferation of cells in group A and B increased significantly at the same time point compared with the control group. At the same time point, the OD value of group B was the highest, and the difference was statistically significant (P 0.05); the activity of alkaline phosphatase was lower in the three groups at the 2nd day.
The activity of ALP in osteoblasts of experimental group B was the most obvious change with time. The content of ALP in osteoblasts of experimental group B was significantly higher than that of osteoblasts of 2 days at 6 days. The activity of ALP in control group was also increased at the same time, but was lower than that of experimental group (P 0.05). ).
conclusion
1.The porosity of PGA/beta-TCP composite scaffolds in group A and group B was above 85%. The compressive strength of PGA/beta-TCP composite scaffolds in group B was significantly higher than that in group A. The PGA/beta-TCP composite scaffolds in group B (1:3 mass ratio) were more suitable for the clinical requirements of scaffolds.
2. PRP can promote the proliferation of osteoblasts on PGA/beta-TCP composite scaffolds and increase the activity of ALP. The effect of 10% PRP on osteoblasts proliferation and ALP activity on PGA/beta-TCP composite scaffolds was higher than that on 5% PRP group.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
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