異種骨支架與人骨髓間充質干細胞相容性的實驗研究
發(fā)布時間：2018-07-17 06:00
【摘要】：目的：(1)研究脫脂、脫蛋白后豬松質骨支架材料的生物學性能。(2)研究脊柱術中出血中的hBMSCs的細胞學特性。(3)探討不同方法改性的豬松質骨支架材料的細胞相容性情況。 方法：(1)通過超聲聯(lián)合化學方法對豬松質骨支架材料進行脫脂、脫蛋白去抗原處理。(2)大體觀察骨支架材料的形貌特征,組織切片HE染色觀察材料的組織學結構,比重法檢測支架材料的孔隙率,檢測支架材料的pH值及微生物生長情況。(3)通過密度梯度離心法分離脊柱手術出血中的hBMSCs;倒置顯微鏡觀察細胞生長情況,流式細胞儀分析第三代hBMSCs表面分子表達情況。對第三代hBMSCs細胞進行成骨誘導培養(yǎng),在誘導后第3、7、14、21天檢測細胞內堿性磷酸酶活性；在誘導培養(yǎng)第21天對礦化結節(jié)行茜素紅染色。(4)采用不同材料包被支架材料對支架材料改性,分為A、B、C三組,A組為胎牛血清包被,B組為I型膠原蛋白包被,C組為對照組。每塊材料上加入濃度為1×109/ml的細胞懸液250μ1。分別在第6、12、24、48小時采用胰蛋白酶消化細胞計數,計算三組的細胞粘附率；在第3、6、9、12、14天采用MTT法觀察三組的細胞增殖情況。 結果：(1)經過處理的骨支架材料外觀呈白色,其表面具有多孔結構微孔壁光滑,HE染色鏡下觀察到網狀骨小梁,骨小梁結構完整,無破壞,骨髓腔中血細胞、脂肪細胞去除,亦未見基質細胞殘留,橢圓形骨陷窩空虛,未見骨細胞核及其它結構。骨支架材料浸泡液pH值第1-7天平均7.356±0.034,呈中性。比重法測得骨支架材料的孔隙率高達81.34%士4.31%。在24、48、72小時三個檢測時間點,浸泡骨支架材料的完全培養(yǎng)基色澤清亮,無混濁及懸浮物,鏡下觀察,亦未發(fā)現細菌及真菌生長。(2)脊柱外科術中出血中分離出原代hBMSCs貼壁生長,呈梭形、多角形。流式細胞儀分析第三代hBMSCs高表達CD73、CD90、CD105,低表達CD14、CD19、CD34、CD45。 hBMSCs經成骨誘導分化后,隨著時間的延長,堿性磷酸酶活性增高。誘導分化培養(yǎng)21天后茜素紅染色顯示成骨細胞體外礦化特性。(3)采用胰蛋白酶消化法計計算細胞粘附率,hBMSCs粘附率在不同方法處理的支架上細胞粘附時相變化的趨勢不同,比較結果細胞粘附率B組A組C組。采用MTT法檢測hBMSCs增殖情況,C組細胞增值情況較A、B兩組增值情況比較均具有統(tǒng)計學差異(P0.05)；而第3、6、9、12、14天A、B兩組之間細胞增殖情況比較無統(tǒng)計學差異(P0.05)。 結論：(1)經脊柱術中出血分離的hBMSCs細胞符合間充質細胞特征,可在體外有效培養(yǎng)擴增。(2)經脫脂脫蛋白處理的異種骨支架其孔隙率、pH值均適合細胞生長,經過工型膠原蛋白及胎牛血清包被支架材料,I型膠原蛋白包被支架較胎牛血清更適于hBMSCs粘附,兩者均可促進hBMSCs在支架材料上生長。
[Abstract]:Objective: (1) to study the biological properties of porcine cancellous bone scaffolds after degreasing and deproteinizing. (2) to study the cytological characteristics of hBMSCs in intraoperative spinal hemorrhage. (3) to investigate the cytocompatibility of different modified porcine cancellous bone scaffolds. Methods: (1) the porcine cancellous bone scaffold was treated by ultrasonic and chemical methods. (2) the morphology of the scaffold was observed and the histological structure of the scaffold was observed by HE staining. Specific gravity method was used to detect the porosity of scaffolds, pH value of scaffold materials and microbial growth. (3) hBMSCs were separated from spinal bleeding by density gradient centrifugation, and cell growth was observed by inverted microscope. The surface molecular expression of the third generation hBMSCs was analyzed by flow cytometry. The third generation of hBMSCs cells were cultured by osteoblast induction, the activity of alkaline phosphatase was detected on the 3rd day after induction, and the mineralized nodules were stained with alizarin red on the 21st day after induction. (4) the scaffold materials were modified with different materials. Group A was divided into three groups: group A: fetal bovine serum capsule, group B, type I collagen coating, group C, control group. A cell suspension of 1 脳 109/ml was added to each material. The cell adhesion rate of the three groups was calculated by trypsin digestion at 48 hours and the cell proliferation of the three groups was observed by MTT assay on the 3rd day. Results: (1) the appearance of the treated bone scaffold was white, and the surface of the scaffold had a porous structure with a smooth wall. The meshwork was observed under HE staining. The bone trabeculae were intact, without destruction, blood cells and fat cells were removed from the medullary cavity. No stromal cells remained, oval bone lacunae were empty, bone nuclei and other structures were not found. The mean pH value of immersion solution of bone scaffold was 7.356 鹵0.034 on day 1-7, which was neutral. The porosity of bone scaffold material measured by specific gravity method was 81.34% 鹵4.31%. At three detection time points of 24: 48 ~ 72 hours, the complete culture medium for immersing bone scaffold materials was clear in color, free of turbidity and suspensions, and no bacteria or fungi were found under microscope. (2) Primary hBMSCs were isolated from spinal surgery bleeding to grow on the wall. It is fusiform and polygonal. Flow cytometry was used to analyze the high expression of CD73, CD90, CD105, and the low expression of CD14, CD19, CD34, CD45, CD45. after osteogenic induction, the activity of alkaline phosphatase increased with the prolongation of time. After 21 days of differentiation and culture, alizarin red staining showed the mineralized characteristics of osteoblasts in vitro. (3) the cell adhesion rate of hBMSCs was calculated by trypsin digestion method. Results the cell adhesion rate was compared in group B, group A, group C. MTT assay was used to detect the proliferation of hBMSCs. The proliferation of hBMSCs in group C was significantly higher than that in group A (P0.05), but there was no significant difference between group C and group A (P0.05). Conclusion: (1) the hBMSCs cells isolated by intraoperative spinal hemorrhage accord with the characteristics of mesenchymal cells and can be effectively cultured and amplified in vitro. (2) the porosity and pH value of the xenograft scaffolds treated with degreasing and deproteinization are suitable for cell growth. Type I collagen coated scaffold was more suitable for adhesion of hBMSCs than fetal bovine serum, both of which could promote the growth of hBMSCs on scaffold materials.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R318.08
本文編號：2129322
[Abstract]:Objective: (1) to study the biological properties of porcine cancellous bone scaffolds after degreasing and deproteinizing. (2) to study the cytological characteristics of hBMSCs in intraoperative spinal hemorrhage. (3) to investigate the cytocompatibility of different modified porcine cancellous bone scaffolds. Methods: (1) the porcine cancellous bone scaffold was treated by ultrasonic and chemical methods. (2) the morphology of the scaffold was observed and the histological structure of the scaffold was observed by HE staining. Specific gravity method was used to detect the porosity of scaffolds, pH value of scaffold materials and microbial growth. (3) hBMSCs were separated from spinal bleeding by density gradient centrifugation, and cell growth was observed by inverted microscope. The surface molecular expression of the third generation hBMSCs was analyzed by flow cytometry. The third generation of hBMSCs cells were cultured by osteoblast induction, the activity of alkaline phosphatase was detected on the 3rd day after induction, and the mineralized nodules were stained with alizarin red on the 21st day after induction. (4) the scaffold materials were modified with different materials. Group A was divided into three groups: group A: fetal bovine serum capsule, group B, type I collagen coating, group C, control group. A cell suspension of 1 脳 109/ml was added to each material. The cell adhesion rate of the three groups was calculated by trypsin digestion at 48 hours and the cell proliferation of the three groups was observed by MTT assay on the 3rd day. Results: (1) the appearance of the treated bone scaffold was white, and the surface of the scaffold had a porous structure with a smooth wall. The meshwork was observed under HE staining. The bone trabeculae were intact, without destruction, blood cells and fat cells were removed from the medullary cavity. No stromal cells remained, oval bone lacunae were empty, bone nuclei and other structures were not found. The mean pH value of immersion solution of bone scaffold was 7.356 鹵0.034 on day 1-7, which was neutral. The porosity of bone scaffold material measured by specific gravity method was 81.34% 鹵4.31%. At three detection time points of 24: 48 ~ 72 hours, the complete culture medium for immersing bone scaffold materials was clear in color, free of turbidity and suspensions, and no bacteria or fungi were found under microscope. (2) Primary hBMSCs were isolated from spinal surgery bleeding to grow on the wall. It is fusiform and polygonal. Flow cytometry was used to analyze the high expression of CD73, CD90, CD105, and the low expression of CD14, CD19, CD34, CD45, CD45. after osteogenic induction, the activity of alkaline phosphatase increased with the prolongation of time. After 21 days of differentiation and culture, alizarin red staining showed the mineralized characteristics of osteoblasts in vitro. (3) the cell adhesion rate of hBMSCs was calculated by trypsin digestion method. Results the cell adhesion rate was compared in group B, group A, group C. MTT assay was used to detect the proliferation of hBMSCs. The proliferation of hBMSCs in group C was significantly higher than that in group A (P0.05), but there was no significant difference between group C and group A (P0.05). Conclusion: (1) the hBMSCs cells isolated by intraoperative spinal hemorrhage accord with the characteristics of mesenchymal cells and can be effectively cultured and amplified in vitro. (2) the porosity and pH value of the xenograft scaffolds treated with degreasing and deproteinization are suitable for cell growth. Type I collagen coated scaffold was more suitable for adhesion of hBMSCs than fetal bovine serum, both of which could promote the growth of hBMSCs on scaffold materials.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R318.08
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