載骨髓間充質干細胞磷酸鈣骨水泥的制備
發(fā)布時間：2019-07-02 16:00
【摘要】：一、研究背景 隨著現代科學的進步，我們的生活步入了現代化，高速鐵路、飛機、汽車遍布周圍，高能量的損傷越來越多，組織缺損及功能障礙越來越常見。與此同時，人們的需求也在與時俱進，并不單純的講究保全生命，而是追求更高的生活品質，追求功能的恢復及重建。隨著現代醫(yī)學的發(fā)展，從上世紀80年代中期開始，組織工程——一個新興的概念開始興起。根據美國國家科學基金委員會的定義，組織工程是指應用細胞生物學、生物材料和工程學的原理，研究開發(fā)用于修復或改善人體病損組織或器官的結構、功能的生物活性替代物的一門科學。對于創(chuàng)傷而言，各種原因導致的骨缺損都嚴重影響患者生活質量，傳統(tǒng)的自體骨移植不但犧牲了自體健康的組織，且由于供區(qū)體積限制，很多情況下并不能完全修復骨質缺損。而骨組織工程的出現，為解決這個困擾骨科界數十年的問題展現了曙光。骨組織工程是指應用骨髓間充質干細胞、成骨細胞等相關合適的種子細胞，在各種可吸收的支架材料上種植，輔以具有相關生物活性的細胞因子，如骨形態(tài)發(fā)生蛋白（Bone morphogeneticprotein,BMP）、轉化生長因子（Transforming Growth Factor,TGF）等，修復缺損的骨組織，實現安全、有效、無副作用的醫(yī)療境界。在本課題中，我們試圖對傳統(tǒng)的磷酸鈣骨水泥加以改進，使之具有骨誘導性、速代謝性，高強度性，從而研制一種新型的骨替代材料，為大范圍骨質缺損的修復提供一種新的選擇。 二、研究目的 通過實驗將包含大鼠骨髓間充質干細胞（MSCs）的明膠微球載入到磷酸鈣骨水泥（CPC）中并使之存活，利用骨髓間充質干細胞的多向分化潛能促進磷酸鈣骨水泥的降解及骨誘導活性，從而研制一種新的具有生物活性的骨替代材料。 三、研究內容和方法 1.分離、培養(yǎng)大鼠骨髓間充質干細胞 我們選用大鼠的骨髓間充質干細胞作為種子細胞。通過傳統(tǒng)的貼壁培養(yǎng)法培養(yǎng)骨髓間充質干細胞，并對其進行傳代、鑒定，確保最終實驗所用的為分裂穩(wěn)定，具有多向分化潛能的干細胞。 2.制備載骨髓間充質干細胞的明膠微球 取1皿第三代骨髓間充質干細胞用胰酶消化備用，將一定量的明膠顆粒溶于DMEM培養(yǎng)基制備成13%的明膠溶液，取5ml溶液緩慢加入1ml細胞懸液，混勻后與橄欖油25ml均預熱至50℃。橄欖油中加入0.15mL Span-80后緩慢加入明膠溶液，于磁力攪拌儀上以900r／min攪拌15min后加入0.01g京尼平于室溫下繼續(xù)攪拌。為測試不同交聯度對明膠微球穩(wěn)定性的影響，分別交聯12h、24h、48h。后混合物驟冷至4℃，與含5g/L Tween-80的PBS液以1:1比例置于分液漏斗，清洗3次以去除橄欖油獲得明膠微球。置于DMEM培養(yǎng)基中于細胞培養(yǎng)箱保存?zhèn)溆谩?3.制備載骨髓間充質干細胞的磷酸鈣骨水泥 為確定合適的構成比，將載有MSCs的明膠微球與骨水泥分別按質量比2.5%（B）、5%（C）、10%（D）（w/w）充分混合后與磷酸鹽緩沖液按固液相2:1的比例混合為半流質糊狀物后置入預先備好的圓柱體模具中于37℃孵箱中固化4h后脫模后于DMEM培養(yǎng)基中保存?zhèn)溆谩Ａ碇苽鋯渭僀PC（A）作為空白對照。 4.測定載干細胞明膠微球的理化性質觀察明膠微球的外形、分散度，計算其細胞包封率、平均粒徑及溶脹率，用茚三酮法測定明膠微球的交聯度，觀察微球的降解時間。 5.熒光染色觀察微球內的骨髓間充質干細胞將明膠微球浸入DMEM中，用鈣黃綠素AM和碘化丙啶(PI)染色后分別于2h、12h、24h于熒光顯微鏡下觀察，其中綠色熒光表明細胞存活，紅色熒光表明細胞死亡。 6.測定新型骨水泥的理化性質表征新型磷酸鈣骨水泥的初凝時間、抗壓強度，計算其孔隙率及代謝時間，分析其代謝產物，觀察骨髓間充質干細胞在孔隙內的粘附及生長情況。 7.統(tǒng)計學處理數據統(tǒng)計用SPSS13.0進行統(tǒng)計分析，數據以均數±標準差(x±s)表示。采用AVONA方法，SNK-q檢驗進行兩兩比較，P0.05為差異有統(tǒng)計學意義。 四、研究結果 1.我們所培養(yǎng)的骨髓間充質干細胞已穩(wěn)定傳至第三代，經流式細胞儀檢測，本實驗分離、培養(yǎng)的MSCs符合國際標準。MSCs CD90表達為陽性，陽性率達到95.24%，而CD45則表達為陰性。 2.明膠微球的理化性質觀察交聯時間控制在24h內各組明膠微球的粒徑變化不大，，分別為(75±15)μm、(81.58±24.68)μm和(108.64±37.44)μm，24h后微球粒徑明顯減小為(27.88±7.46) μm，差異具有顯著性(P0.05)；交聯過的明膠微球降解時間明顯延長，較未交聯組差異差異具有顯著性(P0.05)。 3.磷酸鈣骨水泥的理化性質觀察在各組CPC中MSCs均生長良好，且與空白對照組比較，隨著明膠微球比例的增大，各組CPC的抗壓強度逐步降低，初凝時間延長，孔隙率增大，大孔率增高(P0.05)。 五、研究結論 1、本實驗分離、培養(yǎng)的骨髓間充質干細胞復合國際標準。 2、通過對明膠微球的理化性質測定，京尼平交聯24h后的明膠微球粒徑大小適中，降解時間以及載細胞率符合實驗要求，微球內攜載的MSCs存活良好。 3、通過對磷酸鈣骨水泥理化性質的測定，載MSCs的明膠微球與CPC的質量比為5%時，CPC的理化性質最為理想，細胞生長良好。 4、本實驗研究成果可為下一步體內實驗奠定堅實的基礎。
[Abstract]:I. Background of the study With the progress of modern science, our lives have entered the modern, high-speed railway, the plane, the automobile is spread around, the high-energy damage is more and more, the tissue defect and the dysfunction are more and more often See. At the same time, people's needs are advancing with the times, not only for the preservation of life, but for the higher quality of life, the recovery and the weight of the pursuit function. With the development of modern medicine, from the mid-1980s, the organization project _ an emerging concept has started. The tissue engineering, as defined by the National Commission of National Development, refers to the application of cellular biology, biological materials and engineering principles to study the development of a branch of a biological active substitute for the repair or improvement of the structure and function of a body or organ of a human body. In the case of trauma, the bone defect caused by various causes seriously affects the quality of life of the patient, and the traditional autogenous bone grafting not only sacrifices the self-healthy tissue, but also can not completely repair the bone defect due to the volume limitation of the donor area. The appearance of bone tissue engineering has shown a great deal of problems to solve the problem that has plagued the orthopedic industry for decades. Bone tissue engineering refers to the use of appropriate seed cells, such as bone marrow mesenchymal stem cells, osteoblasts and the like, to be grown on various absorbable scaffold materials, supplemented by cytokines with related biological activity, such as bone morphogenetic protein (BMP), transformation growth factor (BMP), and transformation growth factor (BMP). r, TGF) and the like, the bone tissue of the defect is repaired, the safe, effective and side-effect medical condition is realized, In this subject, we try to improve the traditional calcium phosphate cement to have bone-induced, rapid metabolic and high-strength, so as to develop a new kind of bone substitute material, which provides a new choice for the repair of large-scale bone defect. alternative. two, The purpose of this study was to load the gelatin microspheres containing the rat bone marrow mesenchymal stem cells (MSCs) into the calcium phosphate bone cement (CPC) and to make it survive, and to promote the degradation of the calcium phosphate bone cement by the multi-directional differentiation potential of the bone marrow mesenchymal stem cells. osteoinductive activity, thereby developing a new biological activity bone substitute material Study content and method 1. Separation, culture Rat Bone Marrow Mesenchymal Stem Cells: The Rat Bone Marrow Mesenchymal Stem Cells The mesenchymal stem cells are used as seed cells, bone marrow mesenchymal stem cells are cultured by a traditional adherent culture method, A stem cell with multi-directional differentiation potential. the gelatin microspheres carrying the bone marrow mesenchymal stem cells are used for digesting the third generation bone marrow mesenchymal stem cells with the pancreatin for standby, and a certain amount of the gelatin particles are dissolved in the DMEM medium to be prepared into 13 percent of the gelatin solution, and the gelatin microspheres 1 ml of cell suspension is slowly added to the solution, and then the solution is uniformly mixed. preheating to 50 & deg; C with 25 ml of olive oil, adding 0.15 mL of Span-80 into olive oil, slowly adding gelatin solution, stirring at 900 r/ min on a magnetic stirrer for 15min, and adding 0 in order to test the effect of different cross-linking degree on the stability of the gelatin microspheres, The mixture was quenched to 4 & deg; C for 12 h,24 h, and 48 h, respectively, and the PBS solution containing 5 g/ L Tween-80 was placed in a separatory funnel at a ratio of 1:1, 3 times to remove olive oil to obtain gelatin microspheres. The reserve was stored in the cell incubator in M medium. Preparation of Bone Marrow Mesenchymal Stem The calcium phosphate bone cement of the cells is used to determine the appropriate component ratio, and the gelatin microspheres loaded with MSCs and the bone cement are respectively mixed with the bone cement according to the mass ratio of 2.5% (B),5% (C),10% (D) (w/ w) and then mixed with the phosphate buffer solution according to the solid-liquid phase 2:1 to form a semi-fluid paste, and then the calcium phosphate bone cement is put into a pre-prepared cylindrical mould to be cured in a 37 DEG C cold box After 4 h, the preparation was stored in DMEM medium. and 4. determining the physical and chemical properties of the loaded stem cell gelatin microspheres to observe the appearance and the dispersion degree of the gelatin microspheres, and calculating the cell encapsulation rate, the average particle diameter and the swelling rate of the gelatin microspheres, The cross-linking degree of the gelatin microspheres was measured and the degradation time of the microspheres was observed. 6. The physical and chemical properties of the new bone cement were used to characterize the initial setting time and the compressive strength of the new type of calcium phosphate bone cement. The porosity and the metabolic time of the new bone cement were calculated, and its metabolism was analyzed. The adhesion and growth of bone marrow-derived mesenchymal stem cells in the pores were observed. 13.0 Statistical analysis is performed and data is expressed in the mean square standard deviation (x% s). The AVONA method, S SNK-q媯
本文編號：2509068
[Abstract]:I. Background of the study With the progress of modern science, our lives have entered the modern, high-speed railway, the plane, the automobile is spread around, the high-energy damage is more and more, the tissue defect and the dysfunction are more and more often See. At the same time, people's needs are advancing with the times, not only for the preservation of life, but for the higher quality of life, the recovery and the weight of the pursuit function. With the development of modern medicine, from the mid-1980s, the organization project _ an emerging concept has started. The tissue engineering, as defined by the National Commission of National Development, refers to the application of cellular biology, biological materials and engineering principles to study the development of a branch of a biological active substitute for the repair or improvement of the structure and function of a body or organ of a human body. In the case of trauma, the bone defect caused by various causes seriously affects the quality of life of the patient, and the traditional autogenous bone grafting not only sacrifices the self-healthy tissue, but also can not completely repair the bone defect due to the volume limitation of the donor area. The appearance of bone tissue engineering has shown a great deal of problems to solve the problem that has plagued the orthopedic industry for decades. Bone tissue engineering refers to the use of appropriate seed cells, such as bone marrow mesenchymal stem cells, osteoblasts and the like, to be grown on various absorbable scaffold materials, supplemented by cytokines with related biological activity, such as bone morphogenetic protein (BMP), transformation growth factor (BMP), and transformation growth factor (BMP). r, TGF) and the like, the bone tissue of the defect is repaired, the safe, effective and side-effect medical condition is realized, In this subject, we try to improve the traditional calcium phosphate cement to have bone-induced, rapid metabolic and high-strength, so as to develop a new kind of bone substitute material, which provides a new choice for the repair of large-scale bone defect. alternative. two, The purpose of this study was to load the gelatin microspheres containing the rat bone marrow mesenchymal stem cells (MSCs) into the calcium phosphate bone cement (CPC) and to make it survive, and to promote the degradation of the calcium phosphate bone cement by the multi-directional differentiation potential of the bone marrow mesenchymal stem cells. osteoinductive activity, thereby developing a new biological activity bone substitute material Study content and method 1. Separation, culture Rat Bone Marrow Mesenchymal Stem Cells: The Rat Bone Marrow Mesenchymal Stem Cells The mesenchymal stem cells are used as seed cells, bone marrow mesenchymal stem cells are cultured by a traditional adherent culture method, A stem cell with multi-directional differentiation potential. the gelatin microspheres carrying the bone marrow mesenchymal stem cells are used for digesting the third generation bone marrow mesenchymal stem cells with the pancreatin for standby, and a certain amount of the gelatin particles are dissolved in the DMEM medium to be prepared into 13 percent of the gelatin solution, and the gelatin microspheres 1 ml of cell suspension is slowly added to the solution, and then the solution is uniformly mixed. preheating to 50 & deg; C with 25 ml of olive oil, adding 0.15 mL of Span-80 into olive oil, slowly adding gelatin solution, stirring at 900 r/ min on a magnetic stirrer for 15min, and adding 0 in order to test the effect of different cross-linking degree on the stability of the gelatin microspheres, The mixture was quenched to 4 & deg; C for 12 h,24 h, and 48 h, respectively, and the PBS solution containing 5 g/ L Tween-80 was placed in a separatory funnel at a ratio of 1:1, 3 times to remove olive oil to obtain gelatin microspheres. The reserve was stored in the cell incubator in M medium. Preparation of Bone Marrow Mesenchymal Stem The calcium phosphate bone cement of the cells is used to determine the appropriate component ratio, and the gelatin microspheres loaded with MSCs and the bone cement are respectively mixed with the bone cement according to the mass ratio of 2.5% (B),5% (C),10% (D) (w/ w) and then mixed with the phosphate buffer solution according to the solid-liquid phase 2:1 to form a semi-fluid paste, and then the calcium phosphate bone cement is put into a pre-prepared cylindrical mould to be cured in a 37 DEG C cold box After 4 h, the preparation was stored in DMEM medium. and 4. determining the physical and chemical properties of the loaded stem cell gelatin microspheres to observe the appearance and the dispersion degree of the gelatin microspheres, and calculating the cell encapsulation rate, the average particle diameter and the swelling rate of the gelatin microspheres, The cross-linking degree of the gelatin microspheres was measured and the degradation time of the microspheres was observed. 6. The physical and chemical properties of the new bone cement were used to characterize the initial setting time and the compressive strength of the new type of calcium phosphate bone cement. The porosity and the metabolic time of the new bone cement were calculated, and its metabolism was analyzed. The adhesion and growth of bone marrow-derived mesenchymal stem cells in the pores were observed. 13.0 Statistical analysis is performed and data is expressed in the mean square standard deviation (x% s). The AVONA method, S SNK-q媯
本文編號：2509068
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