硫酸钙对骨修复的影响及硫酸钙富血小板血浆活性支架材料的研制
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摘要
因为创伤,特别是高能量创伤、关节置换翻修术、骨肿瘤切除术等原因造成大块骨缺损,需要进行骨移植的患者不断增多,临床上对骨移植材料的需求越来越大。国外商品化的硫酸钙(calcium sulfate,CS)在体内能完全降解、生物相容性好,其降解速度与新骨形成速度趋于一致,经临床应用证明是一种非常好的骨移植替代材料。目前我国尚无SFDA批准使用的国产外科级硫酸钙。我们通过条件筛选和技术改进,以柠檬酸+X为转晶剂制备出纯度高、晶形均一、具有良好生物相容性的柠檬酸化半水硫酸钙。通过系列实验证明该材料水化后形成的二水硫酸钙生物相容性好、体内能够降解吸收。在此基础上,我们进行了一系列的实验研究,通过对二水硫酸钙制备过程中的加压技术提高了硫酸钙的抗压强度;用包衣技术来控制硫酸钙的降解时间和释放速度,起到恒释、缓释的作用;通过复合BMP提高了硫酸钙人工骨的骨诱导活性;复合抗生素研制抗生素控缓释制剂,在硫酸钙的应用研究上取得了一定的进展,从理论上拓展了硫酸钙人工骨的应用前景。尽管硫酸钙作为一种骨移植替代材料,具有良好的生物相容性、可降解性、有一定的抗压强度,在临床上得到了广泛应用,但是,目前对硫酸钙修复骨缺损的具体机制研究并不多,特别是对硫酸钙是否具有骨诱导活性争议最大。我们的研究表明,复合BMP后,硫酸钙具有明确的骨传导、骨诱导双重活性。但是无论提纯还是重组BMP都存在潜在的安全性问题,目前仍然价格昂贵,本实验旨在研究硫酸钙对骨修复的影响,并在此基础上进一步研制具有骨传导、骨诱导双重活性的新型支架材料。
实验目的
研究硫酸钙对骨修复的影响,并在此基础上进一步研制具有骨传导、骨诱导双重活性的新型支架材料。
材料与方法
培养骨髓基质干细胞,用Transwell技术研究硫酸钙对上述细胞的趋化作用;用第二代功能基因芯片技术研究硫酸钙在骨髓基质干细胞向成骨细胞转化过程中对其基因表达的影响;研制硫酸钙富血小板血浆复合支架材料,并对其理化性质、生物相容性、骨诱导活性进行初步研究,观察其修复兔桡骨临界性骨缺损的情况。
结果
硫酸钙对人骨髓基质干细胞具有趋化作用;在人骨髓基质干细胞向成骨细胞转化的过程中,硫酸钙显著改变了成骨基因的表达,定量功能基因芯片可以同时检测目前已知的所有与成骨相关的基因,在本实验中共检测到89种基因,其中有23种基因表达改变显著(＞2倍)。基因表达上调超过2倍的基因包括:AMELY:2.11;BMP2:3.35;COL4A3:3.64;COMP:6.70;EGF:2.70;FLT1:3.10;IGF1:5.79;ITGA2:2.18;MMP10:13.12;MMP2:2.11;TGFB2:2.52;TGFBR1:2.82;VDR:2.63;VEGFA:2.61。表达下调超过2倍的有:COL2A1:-2.23;COL15A1:-2.47;COL1A1:-8.37;COL1A2:-2.17;COL5A1:-2.09;CSF2:-3.6;FGF1:-2.86;ITGA3:-5.41;MMP8:-5.05。硫酸钙/富血小板血浆支架材料晶形稳定,结构致密,抗压强度较高,可达38.5MPa,同松质骨相近;体外完全降解约需41天,硫酸钙复合富血小板血浆后能够诱导骨髓基质干细胞向成骨细胞转化,在裸鼠体内能够诱导异位骨化。复合富血小板血浆后,硫酸钙生物活性大大提高,能够修复兔桡骨节段性骨缺损,是一种较理想的具有骨诱导、骨传导双重活性的支架材料。
结论
硫酸钙对骨髓基质干细胞具有趋化作用;硫酸钙能够显著影响骨髓基质干细胞向成骨细胞转化过程中成骨基因的表达,具有潜在的骨诱导活性;硫酸钙富血小板血浆复合支架材料抗压强度高,生物相容性好,具有骨传导、骨诱导双重活性。
Because of trauma, especially high-energy trauma, arthroplasty revision surgery, removal of bone tumor causes massive bone defect, and the patients need for bone substitute has been increasing greatly. Commercial calcium sulfate is favored for its biocompatibility and completely degradation in body. The speed of degradation rate is accord with that of new bone formation. At present, China's no SFDA approved surgical calcium sulfate. We prepared surgical-grade a-calcium sulfate with citric acid which has good biocompatibility and can be absorbed along with its degradation. Compression technique was used to increase the compressive strength of the calcium sulfate and coating technique was used to control the degradation speed of calcium sulfate. We prepare delayed release materials by conbined calcium sulphate with BMP and antibiotics. Although calcium sulfate is a sort of biocompatible, biodegradable material which has good compressive strength, as a scaffold, we know little about its mechanim of reconstruction of bone defects. Calcium sulphate conbined with BMP has both bone conduction and bone induction ability, but expensive. The study was designed to find the effect of calcium sulphate on bone reconstruction and preparation of calcium sulfate/platelet-rich plasma bioactive scaffold for bone regeneration.
Purpose
To find the effect of calcium sulphate on bone reconstruction and prepare calcium sulfate /platelet-rich plasma bioactive scaffold for bone regeneration.
Materials and methods
Culture bone marrow stromal stem cells and research the role of calcium sulphate in chematropism of BMSCs; investigate the effect of calcium sulphate on the gene expression of BMSCs by the second generation of gene chip technology during bone marrow stromal stem cells induced into osteoblast-lieke cells. Prepare platelet-rich plasma and calcium sulfate compound scaffold, and research its physico-chemical properties and biocompatibility. Restoring the segmental bone defect of rabbit with the scaffold and observing the osteoinduction role of the material.
Results
Calcium sulfate of bone marrow stem cells have the role of Chemokines in human bone marrow stromal stem cells. Calcium sulphate significantly change the gene exression during the process of the cells induced into osteoblasts. Quantitative functional gene chip can detect all currently known osteoblast-related genes. 89 genes were detected in this experiment. 23 kinds of them change significantly (> 2-fold). Compared with the contrast group, 14 genes of the experimental group increased more than two times: AMELY: 2.11; BMP2: 3.35; COL4A3: 3.64; COMP: 6.70; EGF: 2.70; FLT1: 3.10; IGF1: 5.79; ITGA2: 2.18; MMP10: 13.12; MMP2:2.11; TGFB2: 2.52; TGFBR1: 2.82; VDR: 2.63; VEGFA: 2.61. 9 genes reduced more than two-fold: COL2A1: -2.23; COL15A1: -2.47; COL1A1: -8.37; COL1A2: -2.17; COL5A1: -2.09; CSF2: -3.6; FGF1: -2.86; ITGA3: -5.41; MMP8: -5.05. Calcium Sulfate / platelet-rich plasma has stable craystle and high compressive strength, up to 38.5 MPa, which similar to cancellous bone. Completely degradation time in vitro is about 41 days. Calcium sulfate combined with platelet-rich plasma can induce bone marrow stromal stem cells to osteoblast-like cells and induce ectopic ossification in nude mice. Biological activity of calcium sulfate was greatly improved by combing with platelet-rich plasma. It can be used to restore critical bone defec of rabbit radial. It is an ideal bone substitute with bone induction and conduction.
Conclusion
Calcium sulfate of bone marrow stem cells have the role of Chemokines in human bone marrow stromal stem cells. Calcium sulphate significantly change the gene exression during the process of the cells induced into osteoblasts. Calcium sulphate has potential bone induction ability. Calcium Sulfate/platelet-rich plasma is an ideal bone substitute with bone induction and conduction.
实验目的
研究硫酸钙对骨修复的影响,并在此基础上进一步研制具有骨传导、骨诱导双重活性的新型支架材料。
材料与方法
培养骨髓基质干细胞,用Transwell技术研究硫酸钙对上述细胞的趋化作用;用第二代功能基因芯片技术研究硫酸钙在骨髓基质干细胞向成骨细胞转化过程中对其基因表达的影响;研制硫酸钙富血小板血浆复合支架材料,并对其理化性质、生物相容性、骨诱导活性进行初步研究,观察其修复兔桡骨临界性骨缺损的情况。
结果
硫酸钙对人骨髓基质干细胞具有趋化作用;在人骨髓基质干细胞向成骨细胞转化的过程中,硫酸钙显著改变了成骨基因的表达,定量功能基因芯片可以同时检测目前已知的所有与成骨相关的基因,在本实验中共检测到89种基因,其中有23种基因表达改变显著(＞2倍)。基因表达上调超过2倍的基因包括:AMELY:2.11;BMP2:3.35;COL4A3:3.64;COMP:6.70;EGF:2.70;FLT1:3.10;IGF1:5.79;ITGA2:2.18;MMP10:13.12;MMP2:2.11;TGFB2:2.52;TGFBR1:2.82;VDR:2.63;VEGFA:2.61。表达下调超过2倍的有:COL2A1:-2.23;COL15A1:-2.47;COL1A1:-8.37;COL1A2:-2.17;COL5A1:-2.09;CSF2:-3.6;FGF1:-2.86;ITGA3:-5.41;MMP8:-5.05。硫酸钙/富血小板血浆支架材料晶形稳定,结构致密,抗压强度较高,可达38.5MPa,同松质骨相近;体外完全降解约需41天,硫酸钙复合富血小板血浆后能够诱导骨髓基质干细胞向成骨细胞转化,在裸鼠体内能够诱导异位骨化。复合富血小板血浆后,硫酸钙生物活性大大提高,能够修复兔桡骨节段性骨缺损,是一种较理想的具有骨诱导、骨传导双重活性的支架材料。
结论
硫酸钙对骨髓基质干细胞具有趋化作用;硫酸钙能够显著影响骨髓基质干细胞向成骨细胞转化过程中成骨基因的表达,具有潜在的骨诱导活性;硫酸钙富血小板血浆复合支架材料抗压强度高,生物相容性好,具有骨传导、骨诱导双重活性。
Because of trauma, especially high-energy trauma, arthroplasty revision surgery, removal of bone tumor causes massive bone defect, and the patients need for bone substitute has been increasing greatly. Commercial calcium sulfate is favored for its biocompatibility and completely degradation in body. The speed of degradation rate is accord with that of new bone formation. At present, China's no SFDA approved surgical calcium sulfate. We prepared surgical-grade a-calcium sulfate with citric acid which has good biocompatibility and can be absorbed along with its degradation. Compression technique was used to increase the compressive strength of the calcium sulfate and coating technique was used to control the degradation speed of calcium sulfate. We prepare delayed release materials by conbined calcium sulphate with BMP and antibiotics. Although calcium sulfate is a sort of biocompatible, biodegradable material which has good compressive strength, as a scaffold, we know little about its mechanim of reconstruction of bone defects. Calcium sulphate conbined with BMP has both bone conduction and bone induction ability, but expensive. The study was designed to find the effect of calcium sulphate on bone reconstruction and preparation of calcium sulfate/platelet-rich plasma bioactive scaffold for bone regeneration.
Purpose
To find the effect of calcium sulphate on bone reconstruction and prepare calcium sulfate /platelet-rich plasma bioactive scaffold for bone regeneration.
Materials and methods
Culture bone marrow stromal stem cells and research the role of calcium sulphate in chematropism of BMSCs; investigate the effect of calcium sulphate on the gene expression of BMSCs by the second generation of gene chip technology during bone marrow stromal stem cells induced into osteoblast-lieke cells. Prepare platelet-rich plasma and calcium sulfate compound scaffold, and research its physico-chemical properties and biocompatibility. Restoring the segmental bone defect of rabbit with the scaffold and observing the osteoinduction role of the material.
Results
Calcium sulfate of bone marrow stem cells have the role of Chemokines in human bone marrow stromal stem cells. Calcium sulphate significantly change the gene exression during the process of the cells induced into osteoblasts. Quantitative functional gene chip can detect all currently known osteoblast-related genes. 89 genes were detected in this experiment. 23 kinds of them change significantly (> 2-fold). Compared with the contrast group, 14 genes of the experimental group increased more than two times: AMELY: 2.11; BMP2: 3.35; COL4A3: 3.64; COMP: 6.70; EGF: 2.70; FLT1: 3.10; IGF1: 5.79; ITGA2: 2.18; MMP10: 13.12; MMP2:2.11; TGFB2: 2.52; TGFBR1: 2.82; VDR: 2.63; VEGFA: 2.61. 9 genes reduced more than two-fold: COL2A1: -2.23; COL15A1: -2.47; COL1A1: -8.37; COL1A2: -2.17; COL5A1: -2.09; CSF2: -3.6; FGF1: -2.86; ITGA3: -5.41; MMP8: -5.05. Calcium Sulfate / platelet-rich plasma has stable craystle and high compressive strength, up to 38.5 MPa, which similar to cancellous bone. Completely degradation time in vitro is about 41 days. Calcium sulfate combined with platelet-rich plasma can induce bone marrow stromal stem cells to osteoblast-like cells and induce ectopic ossification in nude mice. Biological activity of calcium sulfate was greatly improved by combing with platelet-rich plasma. It can be used to restore critical bone defec of rabbit radial. It is an ideal bone substitute with bone induction and conduction.
Conclusion
Calcium sulfate of bone marrow stem cells have the role of Chemokines in human bone marrow stromal stem cells. Calcium sulphate significantly change the gene exression during the process of the cells induced into osteoblasts. Calcium sulphate has potential bone induction ability. Calcium Sulfate/platelet-rich plasma is an ideal bone substitute with bone induction and conduction.
引文
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