骨髓间充质干细胞复合异种骨在兔体内的再血管化和成骨研究
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摘要
目的:
近年来组织工程学的迅速发展使得利用其原理和方法再生组织或器官的特点和优势得到广泛认同。作为组织工程研究的重要领域,骨组织工程研究在多方面均取得了令人振奋的研究成果,并已在临床得到初步应用,被认为是组织工程中最具有前途和可行性的领域之一。本课题根据骨组织工程学的基本原理,设计将骨髓间充质干细胞与带部分松质骨的小牛皮质骨板复合,植入新西兰大白兔髂骨内观察其成骨及再血管化情况,为构建理想的初步组织工程骨工程化产品提供理论依据。
方法:
1.兔骨髓间充质干细胞的分离、培养和鉴定
取新西兰大白兔骨髓,采用密度梯度离心法并结合传代获取、纯化细胞,观察细胞形态和表面标志物的表达,从而对其鉴定。MTT法测定第3、6代细胞的增殖情况,并绘制出生长曲线。
2.兔骨髓间充质干细胞的诱导成骨研究
取生长良好的传三代细胞,用成骨诱导培养基进行诱导,观察细胞形态,并分别于培养的第2天、第4天、第6天、第8天、第10天、第12天、第14天收集细胞,进行细胞内碱性磷酸酶含量测定;于诱导7天时行Ⅰ型胶原免疫细胞化学染色,于诱导14天行骨钙素免疫细胞化学染色,于诱导21天时行钙化结节染色;取传第三代,诱导7天,诱导14天,诱导21天后的的细胞行RT—PCR检测BMP—2,VEGF的mRNA含量和采用Western-blot检测BMP—2,VEGF的蛋白含量。
3.兔骨髓间充质干细胞与小牛骨的生物相容性研究
取第三代细胞接种在带部分松质骨的小牛皮质骨板上,接种后第4、8、12、24小时取样测定细胞的粘附率;将不同浓度的小牛骨浸提液与兔BMSCs体外培养2d,4d,7d,观察细胞的形态变化并用MTT检测,计算细胞的相对增殖度,用六级毒性分类进行分级;BMSCs与小牛骨复合培养2周后,取材行扫描电镜观察细胞在材料表面的生长增殖和基质分泌情况。
4.兔骨髓间充质干细胞复合小牛骨植入兔体内的实验研究
(1)分别将兔BMSCs成骨诱导后复合的异种骨(Ⅰ组),单纯异种骨(Ⅱ组),自体髂骨(Ⅲ组)随机植入兔髂骨。观察各组植入物的表面变化及周围组织反应。
(2)术后4周,8周,12周,24周行X线片检查,观察X线片变化情况。
(3)术后4周,8周,12周,24周取材行HE染色及VEGF,BMP-2免疫组织化学染色观察表达情况。
(4)RT-PCR检测体内各组术后4周,8周,12周,24周时VEGF,BMP-2的mRNA的表达情况。
结果:
1.经密度梯度离心结合传代所得到的细胞形态均一,呈长梭形,接近融合状态时可呈现旋涡样生长,传第10代细胞的增殖能力减弱,出现宽大梭形,排列紊乱。所分离的细胞表达CD44、CD29,仅约5%表达CD34。生长曲线显示第3、6代细胞均有较强的增殖能力。
2.兔骨髓间充质干细胞成骨诱导后,细胞形态由长梭形逐渐变为短梭形,多角形,最后成集落层叠生长;在诱导过程中,ALP含量逐渐增高,与对照组比较有显著性差异(P＜0.05);于诱导7天时Ⅰ型胶原免疫细胞化学染色,诱导14天骨钙素免疫细胞化学染色,诱导21天时钙化结节染色阳性;RT-PCR和Western-Blot结果显示诱导组和对照组均有BMP-2和VEGF表达,诱导组于诱导后第7天,两者表达最强。诱导组于诱导后第7天、第14天和第21天与对照组比较VEGF和BMP2表达有显著差异(P＜0.05)。
3.细胞接种4h,8h,12h,24h,细胞粘附率分别为47±4%,65±2%,77±3%,86±2%。不同浓度的小牛骨浸提液与兔BMSCs体外培养,细胞贴壁生长,形态良好,随着培养天数的增加细胞大量增殖,毒级为0-1级。BMSCs与小牛骨复合培养2周后行扫描电镜观察,细胞均在小牛骨表面生长,并可延入材料孔隙之中,有基质分泌。
4.各组材料复合体植入兔髂骨后,伤口生长良好,没有全身和局部的炎症和毒性反应,伤口一期愈合。X线显示髂骨组在术后12周基本达到骨质愈合,复合异种骨组在术后24周时与宿主骨结合部基本愈合,而单纯异种骨组术后24周与宿主骨愈合不满意。术后4周,8周,12周,24周取材行HE染色可观察到软骨化骨过程,各时间点VEGF,BMP-2免疫组织化学染色阳性,阳性程度Ⅲ组＞Ⅰ组＞Ⅱ组。术后4、8、12、24周各组均可见BMP2、VEGF的mRNA表达,BMP2在Ⅰ组、Ⅱ组、Ⅲ组之间相比,各组相比P＜0.05,有统计学意义,即成骨能力:Ⅲ组＞Ⅰ组＞Ⅱ组。VEGF在Ⅱ组与Ⅰ组、Ⅱ组与Ⅲ组之间相比,在各时间点P＜0.05,有统计学意义。Ⅰ组与Ⅲ组在术后8、12、24周时比较P＞0.05,无统计学意义。
结论:
1.兔BMSCs分离、提取操作简便易行,且有较强的生长增殖能力,可诱导分化为成骨细胞,符合组织工程种子细胞的基本条件。其在成骨诱导过程中,BMP—2和VEGF表达均先增强后减弱,VEGF表达可能对血管生成有促进作用。
2.带部分松质骨的小牛皮质骨可作为骨组织工程中BMSCs的细胞载体,无毒,生物相容性好,基本满足骨组织工程的需要。体内实验表明其是一种较为理想的骨组织工程支架材料,在组织相容性、力学性能及成骨效果等方面均令人满意。兔BMSCs复合带部分松质骨的小牛皮质骨支架材料经体外诱导后具有在新西兰兔体内构建组织工程骨的能力。
Objective:
In recent years,the characteristics and advantages of tissue engineering which could renew tissues or organs have been widely recognized.As an important area of tissue engineering,bone tissue engineering which has achieved many exciting research results and preliminary clinical application,is considered to be one of the most promising and feasibility areas.The subject is according to the basic principles of bone tissue engineering.BMSCs and calf bone composites implanted into rabbit ilium,and then observe the osteogenesis and revascularization during the process of bone healing.The ideal is to provide a theoretical basis for building the initial tissue-engineered bone products.
Methods:
1.The isolation,cultivation and identification about the rabbit marrow mesenchymal stem cell
Cells were isolated from the New Zealand white rabbits' bone marrow via density gradient centrifugation.Light and electron microscope was used to observed the cell morphology and Flow cytometry was used to examine the expression of cell surface antigen.The cell culture growth curve was obtained based on observation of the proliferation status of 3~(rd),6~(th) cell generation by MTT method.
2.Study of osteoblastic differentiation potentiality of the rabbit bone marrow mesenchymal stem cell in vitro
The well 3~(rd) generation cells were utilized for osteoblasts differentiation in osteogenic inductive conditioned medium,which was changed every three days.Light and electron microscope was used to observed the cell morphology.Cells were collected for determination of intracellular alkaline phosphatase respectively in the first 2,4,6,8,10,12, 14 days culture.TypeⅠcollagen and osteocalcin(OCN) assayed with immunocytochemical stain and calcium node detected by staining after several days' culture BMSCs under osteogenic inductive condition.Using RT-PCR detection of BMP-2,VEGF mRNA content and using Western-blot detection of BMP-2,VEGF protein content after 0 days,7 days,14 days,21 days osteogenic induction.
3.Study of histocomapatibility of cortical strut with partial cancellous bone and its potentiality used as the carrier for BMSCs
The well 3~(rd) generation cells were seeded in calf cortical strut with partial cancellous bone,and then the adhesion ratio was detected in the first 4,8,12,24 hours.BMSCs were cultured in calf bone leach liquor with different concentration.Then the cells were observed by contrast phase microscope,RGR(relative growth rate) were measured by MTT and cytotoxicity was graded after 2 days,4 days,7 days culture.The growth, proliferation and matrix secretion of BMSCs in calf cortical strut were observed by scanning electron microscope after 2 weeks culture.
4.An Experimental study of rabbit BMSCs combined with calf bone implants into rabbits
BMSCs induced osteogenic composite calf bone(groupⅠ),Simple calf bone group(groupⅡ) or autogenous iliac implant(groupⅢ) were randomly implanted in rabbit ilium.The changes of implants surface and tissue reaction around the implant were observed.All of implants were performed X-ray examination and observed the changes after 4,8,12 and 24 weeks.All of implants were performed examinations of HE stain and immunohistochemistry staining of VEGF and BMP-2 to observe the expression after operation 4,8,12,24 weeks..The expression of VEGF and BMP-2 mRNA were detected by RT-PCR after operation 4,8,12,24 weeks.
Results:
1.BMSCs were very purified and all in the whirlpool shape.Flow cytometry showed that more than 90%cells expressed CD44 and CD29, while only 5%of the cells expressed CD34.Cell culture growth curve showed that cells of the 3~(rd) and 6~(th) generation have high proliferation while the 10~(th) generation have lower proliferation.
2.During rabbit BMSCs osteogenic induction,cell morphology gradually changed from a long fusiform to short fusiform,polygonal,and finally stacked into colony growth.In the induction process,ALP content was gradually increased,compared with the control group,there is a significant difference(P＜0.05.Immunocytochemical staining showed TypeⅠcollagen and OCN positive.Calcium node detected by staining was also positive.RT-PCR and Western-Blot showed that BMP-2 and VEGF were expressed in induced group and control group.The strongest expression of BMP-2 and VEGF was in the 7~(th) day after BMSCs osteogenic induction.There is a significant difference(P＜0.05) in the in the 7~(th) day, 14~(th) day,21~(th) day after BMSCs osteogenic induction compared to the control group.
3.The adhesion ratio were 47%±4%,65%±2%,77%±3%,86%±2%, respectively;after seeding 4,8,12,24 hours.The rabbit BMSCs showed normal morphology and proliferation increased with culture time.The toxicity gradation was zero to one.The BMSCs could be adhered the surface of calf cortical strut and extended in the cancellous bone,and extracellular matrices were found in it.
4.The wound was well,inflammation and toxicity reaction in body or local part were not observed in all groups.The X-ray showed that groupⅠhas reached bone healing after implant 12 weeks,groupⅡalmost got host bone junction healing after implant 24weeks,while groupⅢwere not satisfying healing.The process of ossifications from cartilages were observed in all groups by HE stain and BMP-2 and VEGF were positive by immunocytochemical stain.BMP2、VEGF mRNA were expressed in all groups.Compared between groupⅠ,Ⅱ,Ⅲ,there is statistical significance of BMP2 expression in each group(P＜0.05).Compared groupⅡwith groupⅠandⅢ,there is statistical significance of BMP2 expression(P＜0.05).Compared groupⅠwith groupⅢ,there is statistical significance of BMP2 expression(P＞0.05).
Conclision:
1.The method of isolation and culture of BMSCs is simple and feasible.BMSCs could be induced into obteoblasts with stably expression of osteoblastic phenotype in vitro.BMSCs may be accorded as a kind of seed cells for tissue engineering.During BMSCs osteogenic induction, BMP-2 and VEGF were first expressed increased and then decreased.The expression of VEGF may play a role in promoting angiogenesis.
2.The calf cortical strut with partial cancellous bone could be used for cells carrier in bone tissue engineering.It was an innocuity,better histocompatibility material and could be doubled as osteo-conductibility and osteo-inductivity for satisfying the demand of bone tissue engineering.The cortical strut with partial cancellous bone was an ideally material for bone tissue engineering which was satisfied in histocompatibiity,the speed of degradation,the efficiency of biomechanics and ossification.Rabbit BMSCs seeded in the cortical strut with partial cancellous bone could construct tissue engineering bone by osteoinductation in vivo.
近年来组织工程学的迅速发展使得利用其原理和方法再生组织或器官的特点和优势得到广泛认同。作为组织工程研究的重要领域,骨组织工程研究在多方面均取得了令人振奋的研究成果,并已在临床得到初步应用,被认为是组织工程中最具有前途和可行性的领域之一。本课题根据骨组织工程学的基本原理,设计将骨髓间充质干细胞与带部分松质骨的小牛皮质骨板复合,植入新西兰大白兔髂骨内观察其成骨及再血管化情况,为构建理想的初步组织工程骨工程化产品提供理论依据。
方法:
1.兔骨髓间充质干细胞的分离、培养和鉴定
取新西兰大白兔骨髓,采用密度梯度离心法并结合传代获取、纯化细胞,观察细胞形态和表面标志物的表达,从而对其鉴定。MTT法测定第3、6代细胞的增殖情况,并绘制出生长曲线。
2.兔骨髓间充质干细胞的诱导成骨研究
取生长良好的传三代细胞,用成骨诱导培养基进行诱导,观察细胞形态,并分别于培养的第2天、第4天、第6天、第8天、第10天、第12天、第14天收集细胞,进行细胞内碱性磷酸酶含量测定;于诱导7天时行Ⅰ型胶原免疫细胞化学染色,于诱导14天行骨钙素免疫细胞化学染色,于诱导21天时行钙化结节染色;取传第三代,诱导7天,诱导14天,诱导21天后的的细胞行RT—PCR检测BMP—2,VEGF的mRNA含量和采用Western-blot检测BMP—2,VEGF的蛋白含量。
3.兔骨髓间充质干细胞与小牛骨的生物相容性研究
取第三代细胞接种在带部分松质骨的小牛皮质骨板上,接种后第4、8、12、24小时取样测定细胞的粘附率;将不同浓度的小牛骨浸提液与兔BMSCs体外培养2d,4d,7d,观察细胞的形态变化并用MTT检测,计算细胞的相对增殖度,用六级毒性分类进行分级;BMSCs与小牛骨复合培养2周后,取材行扫描电镜观察细胞在材料表面的生长增殖和基质分泌情况。
4.兔骨髓间充质干细胞复合小牛骨植入兔体内的实验研究
(1)分别将兔BMSCs成骨诱导后复合的异种骨(Ⅰ组),单纯异种骨(Ⅱ组),自体髂骨(Ⅲ组)随机植入兔髂骨。观察各组植入物的表面变化及周围组织反应。
(2)术后4周,8周,12周,24周行X线片检查,观察X线片变化情况。
(3)术后4周,8周,12周,24周取材行HE染色及VEGF,BMP-2免疫组织化学染色观察表达情况。
(4)RT-PCR检测体内各组术后4周,8周,12周,24周时VEGF,BMP-2的mRNA的表达情况。
结果:
1.经密度梯度离心结合传代所得到的细胞形态均一,呈长梭形,接近融合状态时可呈现旋涡样生长,传第10代细胞的增殖能力减弱,出现宽大梭形,排列紊乱。所分离的细胞表达CD44、CD29,仅约5%表达CD34。生长曲线显示第3、6代细胞均有较强的增殖能力。
2.兔骨髓间充质干细胞成骨诱导后,细胞形态由长梭形逐渐变为短梭形,多角形,最后成集落层叠生长;在诱导过程中,ALP含量逐渐增高,与对照组比较有显著性差异(P＜0.05);于诱导7天时Ⅰ型胶原免疫细胞化学染色,诱导14天骨钙素免疫细胞化学染色,诱导21天时钙化结节染色阳性;RT-PCR和Western-Blot结果显示诱导组和对照组均有BMP-2和VEGF表达,诱导组于诱导后第7天,两者表达最强。诱导组于诱导后第7天、第14天和第21天与对照组比较VEGF和BMP2表达有显著差异(P＜0.05)。
3.细胞接种4h,8h,12h,24h,细胞粘附率分别为47±4%,65±2%,77±3%,86±2%。不同浓度的小牛骨浸提液与兔BMSCs体外培养,细胞贴壁生长,形态良好,随着培养天数的增加细胞大量增殖,毒级为0-1级。BMSCs与小牛骨复合培养2周后行扫描电镜观察,细胞均在小牛骨表面生长,并可延入材料孔隙之中,有基质分泌。
4.各组材料复合体植入兔髂骨后,伤口生长良好,没有全身和局部的炎症和毒性反应,伤口一期愈合。X线显示髂骨组在术后12周基本达到骨质愈合,复合异种骨组在术后24周时与宿主骨结合部基本愈合,而单纯异种骨组术后24周与宿主骨愈合不满意。术后4周,8周,12周,24周取材行HE染色可观察到软骨化骨过程,各时间点VEGF,BMP-2免疫组织化学染色阳性,阳性程度Ⅲ组＞Ⅰ组＞Ⅱ组。术后4、8、12、24周各组均可见BMP2、VEGF的mRNA表达,BMP2在Ⅰ组、Ⅱ组、Ⅲ组之间相比,各组相比P＜0.05,有统计学意义,即成骨能力:Ⅲ组＞Ⅰ组＞Ⅱ组。VEGF在Ⅱ组与Ⅰ组、Ⅱ组与Ⅲ组之间相比,在各时间点P＜0.05,有统计学意义。Ⅰ组与Ⅲ组在术后8、12、24周时比较P＞0.05,无统计学意义。
结论:
1.兔BMSCs分离、提取操作简便易行,且有较强的生长增殖能力,可诱导分化为成骨细胞,符合组织工程种子细胞的基本条件。其在成骨诱导过程中,BMP—2和VEGF表达均先增强后减弱,VEGF表达可能对血管生成有促进作用。
2.带部分松质骨的小牛皮质骨可作为骨组织工程中BMSCs的细胞载体,无毒,生物相容性好,基本满足骨组织工程的需要。体内实验表明其是一种较为理想的骨组织工程支架材料,在组织相容性、力学性能及成骨效果等方面均令人满意。兔BMSCs复合带部分松质骨的小牛皮质骨支架材料经体外诱导后具有在新西兰兔体内构建组织工程骨的能力。
Objective:
In recent years,the characteristics and advantages of tissue engineering which could renew tissues or organs have been widely recognized.As an important area of tissue engineering,bone tissue engineering which has achieved many exciting research results and preliminary clinical application,is considered to be one of the most promising and feasibility areas.The subject is according to the basic principles of bone tissue engineering.BMSCs and calf bone composites implanted into rabbit ilium,and then observe the osteogenesis and revascularization during the process of bone healing.The ideal is to provide a theoretical basis for building the initial tissue-engineered bone products.
Methods:
1.The isolation,cultivation and identification about the rabbit marrow mesenchymal stem cell
Cells were isolated from the New Zealand white rabbits' bone marrow via density gradient centrifugation.Light and electron microscope was used to observed the cell morphology and Flow cytometry was used to examine the expression of cell surface antigen.The cell culture growth curve was obtained based on observation of the proliferation status of 3~(rd),6~(th) cell generation by MTT method.
2.Study of osteoblastic differentiation potentiality of the rabbit bone marrow mesenchymal stem cell in vitro
The well 3~(rd) generation cells were utilized for osteoblasts differentiation in osteogenic inductive conditioned medium,which was changed every three days.Light and electron microscope was used to observed the cell morphology.Cells were collected for determination of intracellular alkaline phosphatase respectively in the first 2,4,6,8,10,12, 14 days culture.TypeⅠcollagen and osteocalcin(OCN) assayed with immunocytochemical stain and calcium node detected by staining after several days' culture BMSCs under osteogenic inductive condition.Using RT-PCR detection of BMP-2,VEGF mRNA content and using Western-blot detection of BMP-2,VEGF protein content after 0 days,7 days,14 days,21 days osteogenic induction.
3.Study of histocomapatibility of cortical strut with partial cancellous bone and its potentiality used as the carrier for BMSCs
The well 3~(rd) generation cells were seeded in calf cortical strut with partial cancellous bone,and then the adhesion ratio was detected in the first 4,8,12,24 hours.BMSCs were cultured in calf bone leach liquor with different concentration.Then the cells were observed by contrast phase microscope,RGR(relative growth rate) were measured by MTT and cytotoxicity was graded after 2 days,4 days,7 days culture.The growth, proliferation and matrix secretion of BMSCs in calf cortical strut were observed by scanning electron microscope after 2 weeks culture.
4.An Experimental study of rabbit BMSCs combined with calf bone implants into rabbits
BMSCs induced osteogenic composite calf bone(groupⅠ),Simple calf bone group(groupⅡ) or autogenous iliac implant(groupⅢ) were randomly implanted in rabbit ilium.The changes of implants surface and tissue reaction around the implant were observed.All of implants were performed X-ray examination and observed the changes after 4,8,12 and 24 weeks.All of implants were performed examinations of HE stain and immunohistochemistry staining of VEGF and BMP-2 to observe the expression after operation 4,8,12,24 weeks..The expression of VEGF and BMP-2 mRNA were detected by RT-PCR after operation 4,8,12,24 weeks.
Results:
1.BMSCs were very purified and all in the whirlpool shape.Flow cytometry showed that more than 90%cells expressed CD44 and CD29, while only 5%of the cells expressed CD34.Cell culture growth curve showed that cells of the 3~(rd) and 6~(th) generation have high proliferation while the 10~(th) generation have lower proliferation.
2.During rabbit BMSCs osteogenic induction,cell morphology gradually changed from a long fusiform to short fusiform,polygonal,and finally stacked into colony growth.In the induction process,ALP content was gradually increased,compared with the control group,there is a significant difference(P＜0.05.Immunocytochemical staining showed TypeⅠcollagen and OCN positive.Calcium node detected by staining was also positive.RT-PCR and Western-Blot showed that BMP-2 and VEGF were expressed in induced group and control group.The strongest expression of BMP-2 and VEGF was in the 7~(th) day after BMSCs osteogenic induction.There is a significant difference(P＜0.05) in the in the 7~(th) day, 14~(th) day,21~(th) day after BMSCs osteogenic induction compared to the control group.
3.The adhesion ratio were 47%±4%,65%±2%,77%±3%,86%±2%, respectively;after seeding 4,8,12,24 hours.The rabbit BMSCs showed normal morphology and proliferation increased with culture time.The toxicity gradation was zero to one.The BMSCs could be adhered the surface of calf cortical strut and extended in the cancellous bone,and extracellular matrices were found in it.
4.The wound was well,inflammation and toxicity reaction in body or local part were not observed in all groups.The X-ray showed that groupⅠhas reached bone healing after implant 12 weeks,groupⅡalmost got host bone junction healing after implant 24weeks,while groupⅢwere not satisfying healing.The process of ossifications from cartilages were observed in all groups by HE stain and BMP-2 and VEGF were positive by immunocytochemical stain.BMP2、VEGF mRNA were expressed in all groups.Compared between groupⅠ,Ⅱ,Ⅲ,there is statistical significance of BMP2 expression in each group(P＜0.05).Compared groupⅡwith groupⅠandⅢ,there is statistical significance of BMP2 expression(P＜0.05).Compared groupⅠwith groupⅢ,there is statistical significance of BMP2 expression(P＞0.05).
Conclision:
1.The method of isolation and culture of BMSCs is simple and feasible.BMSCs could be induced into obteoblasts with stably expression of osteoblastic phenotype in vitro.BMSCs may be accorded as a kind of seed cells for tissue engineering.During BMSCs osteogenic induction, BMP-2 and VEGF were first expressed increased and then decreased.The expression of VEGF may play a role in promoting angiogenesis.
2.The calf cortical strut with partial cancellous bone could be used for cells carrier in bone tissue engineering.It was an innocuity,better histocompatibility material and could be doubled as osteo-conductibility and osteo-inductivity for satisfying the demand of bone tissue engineering.The cortical strut with partial cancellous bone was an ideally material for bone tissue engineering which was satisfied in histocompatibiity,the speed of degradation,the efficiency of biomechanics and ossification.Rabbit BMSCs seeded in the cortical strut with partial cancellous bone could construct tissue engineering bone by osteoinductation in vivo.
引文
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