模拟微重力下胶原/壳聚糖/β-TCP修复体复合兔成骨细胞和软骨细胞体外培养的实验研究
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摘要
关节软骨缺损是临床的难题之一,自体关节软骨移植存在来源有限、创伤大、费用高等缺点,异体关节软骨移植难以排除免疫源性的问题。随着组织工程的发展,利用人工再生组织来修复关节软骨缺损,为临床解决这一难题提供了新的思路和方法。虽然关节软骨组织工程的研究已经取得了很大进展,但仍有大量的问题等待解决。尤其是软骨和软骨下骨的分层、断裂问题,和宿主的整合问题,关节软骨修复的长期效果等。
研究目的
将胶原、壳聚糖与β-磷酸三钙(Tricalcium Phosphate,TCP)有机复合,模仿体内关节软骨的分层结构,增加支架材料的力学强度,构建胶原/壳聚糖/β-TCP层状梯度支架材料。分别将扩增的经成骨诱导的兔骨髓间充质干细胞(BoneMesenchymal Stem Cells,BMSCs)和软骨细胞植入其中体外微重力下培养,对照组采用普通培养板培养,观察成骨细胞和软骨细胞在胶原/壳聚糖/β-TCP层状梯度修复体中的黏附、增殖及生物学性状变化,以评价该复合材料作为关节软骨组织工程支架的可行性,并观察模拟微重力对细胞及构建组织的效用。
研究方法
以细胞和支架材料为实验对象,以普通培养和微重力培养分两组对照。MTT实验分组:5个时间组:1天、3天、5天、7天、9天。5个平行孔/组,两培养组共50孔。
1.成骨细胞和材料复合:用全骨髓贴壁筛选法培养新西兰兔原代BMSCs,体外扩增后进行成骨诱导,3周后检测细胞碱性磷酸酶活性和钙结节生成情况,诱导成功后和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对细胞的影响;培养1周后行扫描电镜检查观察成骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物脱钙、石蜡包埋、切片,行苏木精一伊红染色及Ⅰ型胶原免疫组织化学染色,观察成骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
2.软骨细胞和材料复合:取新西兰幼兔的关节软骨消化培养软骨细胞体外培养扩增,取2、3代细胞行s—100细胞免疫化学染色,收集前4代软骨细胞作为种子细胞和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对软骨细胞的影响;培养7天后行扫描电镜检查观察软骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物进行脱钙、石蜡包埋、切片,行苏木精—伊红染色及Ⅱ型胶原免疫组织化学染色,观察软骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
统计学分析:MTT实验中,相同时间点两组之间的比较用两独立样本t检验;同组不同时间点比较采用完全随机设计单因素方差分析(One-way ANOVA);两组整体之间比较采用重复测量数据的方差分析。α=0.05为统计学检验水准。
研究结果
1.细胞形态:初接种时BMSCs呈球形,悬浮在培养液中,24h后开始贴壁,细胞呈长梭形,少量多角形,约1周左右细胞铺满培养瓶。加入成骨诱导培养液后细胞生长相对缓慢,其形态发生了改变,细胞逐渐增大,进而出现重叠生长。2周后细胞间可见圆形或卵圆形的钙化结节,结节周围细胞呈放射状分布。碱性磷酸酶染色阳性,Von Kossa法检测出钙化结节。体外单层培养的软骨细胞呈球形,悬浮在培养液中,5h后开始贴壁,细胞呈扁平状,大多数三角形,少量多角形,约1周细胞铺满培养瓶,呈“铺路石”样。第2、3代细胞S—100免疫细胞化学染色胞浆内呈阳性反应。
2.细胞/支架光镜下观察:细胞悬液滴加于经预湿处理的复合支架材料上,支架材料迅速膨胀,证明细胞扩散到支架内。接种24h后,倒置显微镜下见材料不透明,无法观察其内细胞生长情况,但可见大量成骨细胞或软骨细胞贴附在材料旁培养板内。
3.MTT检测:成骨细胞和材料复合后,除第1天两组之间无显著性差异外(P=0.706),其余时间点两组之间有显著性差异(P＜0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对成骨细胞在修复体内的增殖比普通培养起到了更好的促进作用。软骨细胞和材料复合后,不同时间点两组之间有显著性差异(P＜0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对软骨细胞在修复体内增殖比普通培养起到了更好的促进作用。
4.扫描电镜:培养7d后,细胞成团地吸附于修复体表面及孔隙侧壁,并可见细胞间通过突起相互连接,或伸出伪足贴附于支架孔隙壁上;从复合支架材料中间切开,普通培养组见其内部有少量细胞贴附于孔隙侧壁,模拟微重力组则可见到大量细胞,细胞数量比普通培养组明显增多。
5.HE染色和免疫组化结果:成骨细胞和材料:4周后,材料表面可见大量细胞,内部可见部分细胞聚集成团;细胞呈长梭形,细胞团中有基质分泌,沉积于细胞周围;Ⅰ型胶原免疫组织化学染色示胞浆内呈阳性反应。软骨细胞和材料:4周后,材料表面可见大量细胞黏附成层状,内部亦可见细胞聚集成团;细胞呈圆形或椭圆形,细胞团中有基质分泌,沉积于细胞周围,Ⅱ型胶原免疫组织化学染色示胞浆内呈阳性反应。普通培养组的细胞多聚集在修复体表面,细胞数量明显多于修复体内部,而模拟微重力组材料表面和材料内部细胞数量相差不大,内部细胞数量明显比普通培养组多。
结论
1.成功培养出兔原代软骨细胞和BMSCs,并成功诱导BMSCs为成骨细胞,为进一步的细胞和材料的接种提供了数量充足和生物学性能良好的种子细胞。
2.胶原/壳聚糖/β-TCP层状梯度复合体模拟了正常关节软骨分层结构,细胞相容性好,能提供细胞生长的三维环境。
3.转壁式旋转培养仪产生的模拟微重力可以使软骨细胞和成骨细胞在修复体内高密度生长,能更好的促进细胞增殖和细胞外基质分泌,提高了组织工程化关节软骨体外培养的质量,为关节软骨组织工程的研究开辟了新的道路。
Articular cartilage defects is one of the orthopedics clinical problems, articular cartilage autotransplantation limited by its shortcomings such as sources, trauma, the high cost. Articular cartilage allograft transplantation difficult to rule out the possibility of endogenous immune problems. Along with the development of tissue engineering, using artificial regeneration organizations to repair articular cartilage defects, providing new ideas and methods to solve the problem . Although the articular cartilage tissue engineering research has made great progress, there is still a large number of issues to be resolved. Particularly cartilage and subchondral bone tiered, fracture, the host integration, and the long-term effects of articular cartilage repairing.
Objective: Collagen, chitosan andβ-tricalcium phosphate are compounded organically to building layered gradient composite, in order to imitate the hierarchical structure of articular cartilage and increase the mechanical strength of scaffold. The osteoblasts were induced from rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a stimulated microgravity environment respectly, The effects of the composite scaffold on the cell adhesivity, proliferation and morphological changes were observed, to evaluate the feasibility of composite material as articular cartilage tissue engineering scaffolds and observe the effectiveness of simulated microgravity on cells and constructs.
Methods: Put the cells and scaffold as experimental object, The experiment were given to two groups: the general culture and microgravity culture, the general culture as a control. MTT assay subgroups: five time groups: first day, third day, fifth day, seventh days,ninth day. Five parallel hole / group, 50 holes totally.
1.BMSCs composite materials: the rabbit primary BMSCs were cultured by whole bone marrow adherent screening method and induced to osteoblasts,the result improved by detecting the activity of alkaline phosphatase and formation of calcium nodule after three weeks culture,then the BMSCs were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type I immunohistochemical staining after four weeks.2. Chondrocytes composite materials: the New Zealand immature rabbit primary chondrocytes were cultured in vitro amplification, the second and third generation cells were improved by S-100 immunocytochemistry staining ,then the first four generation chondrocytes were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type II immunohistochemical staining after four weeks.
Statistical analysis: MTT assay, the same time point of comparison between the two groups using two independent samples t-test .the same group at different time points compared using the completely randomized design of single-factor analysis of variance (One-way ANOVA); two overall comparison between the two groups using analysis of variance of repeated measures data. a= 0.05 is the level for the statistical test.
Results: 1. Cell morphology: BMSCs were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 24 h with its fusiform shape in majority and polygonal in minority. The cells covered culture flask about one week . After be induced, the cells grew slowly and changed its form into more large shape ,then overlapped one another. There were a mount of round or oval calcified nodules among cells two weeks later and distributed in radiat, the cells' Alkaline Phosphatase staining was positive and Von Kossa method staining of calcified nodules was positive. The chondrocytes were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 5h with its triangle shape in majority and polygonal in minority. The cells covered culture flask about one week with "paving stones" appearance. The S-100 immunocytochemical staining of chondrocytes ware positive.2. Cell / scaffold microscopy: After dropping cells suspension in the wet pretreatment scaffold, the scaffold expansion rapidly revealed that the cells spread in it. The result of inverted microscope revealed that the scaffolds were opaque and a large number of osteoblast and chondrocytes adhered the adjacent material. 3. MTT assay: After osteoblasts were seeded onto the scaffolds , There was a significant difference between two groups at different time points (P <0.05) except the first day (P = 0.706), and group-factors was also statistically significant (P = 0.000).
After chondrocytes were seeded onto the scaffolds , There was a significant difference between the two groups at different time points (P <0.05), and group-factors was also statistically significant (P = 0.000).4. Scaning Electron Microscope: After cultured 7 days, the cells agglomerate adsorbed on the surface and pore walls of scaffolds, linked with each other by cell processes or stretched out pseudo-foot attached to the pore wall. Discussion from the middle of scaffold, there were a small number of cells attached to pore wall.5. HE staining and immunohistochemical staining: Osteoblasts and scaffolds: After 4 weeks, there were a large number of cells with spherical shape at the surface and a small amount of cells overlapped in it , around which deposited much extra cell matrix .The typeⅠcollagen immunohistochemistry show positive response within the cytoplasm. Chondrocytes and scaffolds: After 4 weeks,there were a large number of cells with round shape at the surface and a small amount of cells overlapped in it, around which deposited much extra cell matrix . The typeⅡcollagen immunohistochemistry show positive response within the cytoplasm.
Conclusion: 1. We develope successfully New Zealand rabbit primary chondrocytes and BMSCs, and successfully induced BMSCs to osteoblasts, which provided sufficient quantity and better biological properties cells resource for further experiment. 2. Collagen / chitosan /β-TCP layered gradient composites simulate the hierarchical structure of normal articular cartilage, which can provide three-dimensional growth environment for cells with its better compatibility. 3. Simulated microgravity from rotating wall vessel bioreactor can promote osteoblasts and chondrocytes grow in high density in scaffold, and promote the secretion of extracellular matrix, which improve the quality of tissue-engineered articular cartilage in vitro and open a new way for the articular cartilage tissue engineering research.
研究目的
将胶原、壳聚糖与β-磷酸三钙(Tricalcium Phosphate,TCP)有机复合,模仿体内关节软骨的分层结构,增加支架材料的力学强度,构建胶原/壳聚糖/β-TCP层状梯度支架材料。分别将扩增的经成骨诱导的兔骨髓间充质干细胞(BoneMesenchymal Stem Cells,BMSCs)和软骨细胞植入其中体外微重力下培养,对照组采用普通培养板培养,观察成骨细胞和软骨细胞在胶原/壳聚糖/β-TCP层状梯度修复体中的黏附、增殖及生物学性状变化,以评价该复合材料作为关节软骨组织工程支架的可行性,并观察模拟微重力对细胞及构建组织的效用。
研究方法
以细胞和支架材料为实验对象,以普通培养和微重力培养分两组对照。MTT实验分组:5个时间组:1天、3天、5天、7天、9天。5个平行孔/组,两培养组共50孔。
1.成骨细胞和材料复合:用全骨髓贴壁筛选法培养新西兰兔原代BMSCs,体外扩增后进行成骨诱导,3周后检测细胞碱性磷酸酶活性和钙结节生成情况,诱导成功后和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对细胞的影响;培养1周后行扫描电镜检查观察成骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物脱钙、石蜡包埋、切片,行苏木精一伊红染色及Ⅰ型胶原免疫组织化学染色,观察成骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
2.软骨细胞和材料复合:取新西兰幼兔的关节软骨消化培养软骨细胞体外培养扩增,取2、3代细胞行s—100细胞免疫化学染色,收集前4代软骨细胞作为种子细胞和修复体复合体外普通培养和旋转培养仪模拟微重力培养,进行MTT检测比较两组细胞增殖情况并观察模拟微重力对软骨细胞的影响;培养7天后行扫描电镜检查观察软骨细胞在修复体材料内黏附生长情况;4周后,细胞和材料复合物进行脱钙、石蜡包埋、切片,行苏木精—伊红染色及Ⅱ型胶原免疫组织化学染色,观察软骨细胞的生物学性状变化及模拟微重力对复合物的力学影响。
统计学分析:MTT实验中,相同时间点两组之间的比较用两独立样本t检验;同组不同时间点比较采用完全随机设计单因素方差分析(One-way ANOVA);两组整体之间比较采用重复测量数据的方差分析。α=0.05为统计学检验水准。
研究结果
1.细胞形态:初接种时BMSCs呈球形,悬浮在培养液中,24h后开始贴壁,细胞呈长梭形,少量多角形,约1周左右细胞铺满培养瓶。加入成骨诱导培养液后细胞生长相对缓慢,其形态发生了改变,细胞逐渐增大,进而出现重叠生长。2周后细胞间可见圆形或卵圆形的钙化结节,结节周围细胞呈放射状分布。碱性磷酸酶染色阳性,Von Kossa法检测出钙化结节。体外单层培养的软骨细胞呈球形,悬浮在培养液中,5h后开始贴壁,细胞呈扁平状,大多数三角形,少量多角形,约1周细胞铺满培养瓶,呈“铺路石”样。第2、3代细胞S—100免疫细胞化学染色胞浆内呈阳性反应。
2.细胞/支架光镜下观察:细胞悬液滴加于经预湿处理的复合支架材料上,支架材料迅速膨胀,证明细胞扩散到支架内。接种24h后,倒置显微镜下见材料不透明,无法观察其内细胞生长情况,但可见大量成骨细胞或软骨细胞贴附在材料旁培养板内。
3.MTT检测:成骨细胞和材料复合后,除第1天两组之间无显著性差异外(P=0.706),其余时间点两组之间有显著性差异(P＜0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对成骨细胞在修复体内的增殖比普通培养起到了更好的促进作用。软骨细胞和材料复合后,不同时间点两组之间有显著性差异(P＜0.05),模拟微重力组明显比培养板组细胞增殖力高。两组之间整体比较也有统计学意义(P=0.000),证明模拟微重力对软骨细胞在修复体内增殖比普通培养起到了更好的促进作用。
4.扫描电镜:培养7d后,细胞成团地吸附于修复体表面及孔隙侧壁,并可见细胞间通过突起相互连接,或伸出伪足贴附于支架孔隙壁上;从复合支架材料中间切开,普通培养组见其内部有少量细胞贴附于孔隙侧壁,模拟微重力组则可见到大量细胞,细胞数量比普通培养组明显增多。
5.HE染色和免疫组化结果:成骨细胞和材料:4周后,材料表面可见大量细胞,内部可见部分细胞聚集成团;细胞呈长梭形,细胞团中有基质分泌,沉积于细胞周围;Ⅰ型胶原免疫组织化学染色示胞浆内呈阳性反应。软骨细胞和材料:4周后,材料表面可见大量细胞黏附成层状,内部亦可见细胞聚集成团;细胞呈圆形或椭圆形,细胞团中有基质分泌,沉积于细胞周围,Ⅱ型胶原免疫组织化学染色示胞浆内呈阳性反应。普通培养组的细胞多聚集在修复体表面,细胞数量明显多于修复体内部,而模拟微重力组材料表面和材料内部细胞数量相差不大,内部细胞数量明显比普通培养组多。
结论
1.成功培养出兔原代软骨细胞和BMSCs,并成功诱导BMSCs为成骨细胞,为进一步的细胞和材料的接种提供了数量充足和生物学性能良好的种子细胞。
2.胶原/壳聚糖/β-TCP层状梯度复合体模拟了正常关节软骨分层结构,细胞相容性好,能提供细胞生长的三维环境。
3.转壁式旋转培养仪产生的模拟微重力可以使软骨细胞和成骨细胞在修复体内高密度生长,能更好的促进细胞增殖和细胞外基质分泌,提高了组织工程化关节软骨体外培养的质量,为关节软骨组织工程的研究开辟了新的道路。
Articular cartilage defects is one of the orthopedics clinical problems, articular cartilage autotransplantation limited by its shortcomings such as sources, trauma, the high cost. Articular cartilage allograft transplantation difficult to rule out the possibility of endogenous immune problems. Along with the development of tissue engineering, using artificial regeneration organizations to repair articular cartilage defects, providing new ideas and methods to solve the problem . Although the articular cartilage tissue engineering research has made great progress, there is still a large number of issues to be resolved. Particularly cartilage and subchondral bone tiered, fracture, the host integration, and the long-term effects of articular cartilage repairing.
Objective: Collagen, chitosan andβ-tricalcium phosphate are compounded organically to building layered gradient composite, in order to imitate the hierarchical structure of articular cartilage and increase the mechanical strength of scaffold. The osteoblasts were induced from rabbit bone marrow-derived mesenchymal stem cells and chondrocytes were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a stimulated microgravity environment respectly, The effects of the composite scaffold on the cell adhesivity, proliferation and morphological changes were observed, to evaluate the feasibility of composite material as articular cartilage tissue engineering scaffolds and observe the effectiveness of simulated microgravity on cells and constructs.
Methods: Put the cells and scaffold as experimental object, The experiment were given to two groups: the general culture and microgravity culture, the general culture as a control. MTT assay subgroups: five time groups: first day, third day, fifth day, seventh days,ninth day. Five parallel hole / group, 50 holes totally.
1.BMSCs composite materials: the rabbit primary BMSCs were cultured by whole bone marrow adherent screening method and induced to osteoblasts,the result improved by detecting the activity of alkaline phosphatase and formation of calcium nodule after three weeks culture,then the BMSCs were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type I immunohistochemical staining after four weeks.2. Chondrocytes composite materials: the New Zealand immature rabbit primary chondrocytes were cultured in vitro amplification, the second and third generation cells were improved by S-100 immunocytochemistry staining ,then the first four generation chondrocytes were seeded onto the scaffolds and cultured in vitro, The effects of the composite scaffold between the two groups on the cell adhesivity, proliferation, morphological changes, and synthesis of the extracellular matrix were observed by MTT assay, scanning electron microscopy one week later, hematoxylin- eosin stain and collagen type II immunohistochemical staining after four weeks.
Statistical analysis: MTT assay, the same time point of comparison between the two groups using two independent samples t-test .the same group at different time points compared using the completely randomized design of single-factor analysis of variance (One-way ANOVA); two overall comparison between the two groups using analysis of variance of repeated measures data. a= 0.05 is the level for the statistical test.
Results: 1. Cell morphology: BMSCs were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 24 h with its fusiform shape in majority and polygonal in minority. The cells covered culture flask about one week . After be induced, the cells grew slowly and changed its form into more large shape ,then overlapped one another. There were a mount of round or oval calcified nodules among cells two weeks later and distributed in radiat, the cells' Alkaline Phosphatase staining was positive and Von Kossa method staining of calcified nodules was positive. The chondrocytes were suspended in culture medium with spherical shape at the beginning stage, then adhered culture flask and elongated after 5h with its triangle shape in majority and polygonal in minority. The cells covered culture flask about one week with "paving stones" appearance. The S-100 immunocytochemical staining of chondrocytes ware positive.2. Cell / scaffold microscopy: After dropping cells suspension in the wet pretreatment scaffold, the scaffold expansion rapidly revealed that the cells spread in it. The result of inverted microscope revealed that the scaffolds were opaque and a large number of osteoblast and chondrocytes adhered the adjacent material. 3. MTT assay: After osteoblasts were seeded onto the scaffolds , There was a significant difference between two groups at different time points (P <0.05) except the first day (P = 0.706), and group-factors was also statistically significant (P = 0.000).
After chondrocytes were seeded onto the scaffolds , There was a significant difference between the two groups at different time points (P <0.05), and group-factors was also statistically significant (P = 0.000).4. Scaning Electron Microscope: After cultured 7 days, the cells agglomerate adsorbed on the surface and pore walls of scaffolds, linked with each other by cell processes or stretched out pseudo-foot attached to the pore wall. Discussion from the middle of scaffold, there were a small number of cells attached to pore wall.5. HE staining and immunohistochemical staining: Osteoblasts and scaffolds: After 4 weeks, there were a large number of cells with spherical shape at the surface and a small amount of cells overlapped in it , around which deposited much extra cell matrix .The typeⅠcollagen immunohistochemistry show positive response within the cytoplasm. Chondrocytes and scaffolds: After 4 weeks,there were a large number of cells with round shape at the surface and a small amount of cells overlapped in it, around which deposited much extra cell matrix . The typeⅡcollagen immunohistochemistry show positive response within the cytoplasm.
Conclusion: 1. We develope successfully New Zealand rabbit primary chondrocytes and BMSCs, and successfully induced BMSCs to osteoblasts, which provided sufficient quantity and better biological properties cells resource for further experiment. 2. Collagen / chitosan /β-TCP layered gradient composites simulate the hierarchical structure of normal articular cartilage, which can provide three-dimensional growth environment for cells with its better compatibility. 3. Simulated microgravity from rotating wall vessel bioreactor can promote osteoblasts and chondrocytes grow in high density in scaffold, and promote the secretion of extracellular matrix, which improve the quality of tissue-engineered articular cartilage in vitro and open a new way for the articular cartilage tissue engineering research.
引文
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