改良复合磷酸钙骨水泥诱导成骨作用的体外研究
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摘要
背景:植骨是治疗骨缺损和骨不连最常用的方法,由于自体骨和同种异体骨来源有限且无可塑性等缺点使人工植骨材料成为当前研究的热点。最早在临床使用的可塑形较好的植入材料为聚甲基丙烯酸甲酯(PMMA),但是,由于PMMA固化时产生热量,可灼伤周围正常骨组织,又不能被机体吸收和重塑,阻碍骨折愈合,不适合作为创伤性骨缺损和骨不连的植骨材料。其后研究的可吸收的骨移植替代物主要有硫酸钙、磷酸钙羟基磷灰石等。硫酸钙类在体内降解较快,与新骨长入速度不一致,且质地较脆、机械强度有限,一般不用于治疗骨干部骨折的缺损。
磷酸钙骨水泥具有优良的生物学和力学性能,但是普通的磷酸钙骨水泥的固化产物羟基磷灰石虽然含有微孔结构,其孔径大小多在微米以下至数微米之间,孔径不能允许细胞及血管的透过和生长,这类骨水泥在人体中非常稳定,吸收和降解通常仅发生在骨水泥的表面且吸收速率非常缓慢,且一般不具有诱导成骨活性,骨爬行替代时间较长,一定程度上限制了其临床应用价值。因此,有必要对其改进配方,使其更符合临床需要。
我们通过优化普通磷酸钙骨水泥的配方,并与同种异体骨粉混合,以同种异体骨粉作为新骨长入的微孔结构研制了改良复合可吸收磷酸钙骨水泥(BCPC),动物实验显示该BCPC作为植骨材料可以诱导成骨。但一种新的植骨材料应用于临床之前,必须阐明其作用机制。本实验拟通过体外实验进一步研究该材料对兔骨髓间充质干细胞(Bonemesenchymalstemcells,BMSCs)的生长和诱导分化成骨作用,探索其临床应用价值。
目的:通过体外实验观察改良复合磷酸钙骨水泥(BCPC)生物相容性,对兔BMSCs的生长和诱导成骨分化作用,以及其负载促骨形成因子的能力。
材料和方法:实验分4组:①对照组;②普通磷酸钙骨水泥组(CPC组);③改良复合磷酸钙骨水泥组(BCPC组);④负载BMP组(加BMP的BCPC组)。将各组材料与传代至第三代的兔骨髓间充质干细胞在24孔板上复合培养,每日倒置相差显微镜下观察细胞生长情况,细胞计数并绘制细胞增殖曲线,碱性磷酸酶测试盒检测细胞合成碱性磷酸酶活性变化,扫描电镜观察细胞在材料表面的生长及长入情况。复合培养第10天各组取相同数目细胞,real-timePCR检测成骨及成软骨相关基因ALP、OSX、CollgenⅠ、CollagenⅡ、AggrecanmRNA表达情况,2-△△CT法比较这些基因mRNA表达差别。
结果:与普通骨水泥比较,配制的BCPC具有更疏松的结构,细胞计数结果显示复合培养后各组细胞增殖无统计学差别,倒置显微镜观察细胞与载体边缘接触良好。复合培养第7天后,BCPC组及加BMP组表达更高的碱性磷酸酶活性,扫描电镜观察细胞在BCPC表面生长增殖良好,逐渐伸出伪足并长入材料孔隙,而CPC组细胞只在材料表面生长。复合培养第10天,BCPC组及加BMP组与CPC组及对照组比较,其成骨或成软骨相关基因ALP、OSX、CollagenⅠ、CollagenⅡ、AggrecanmRNA表达有统计学差别。结论:配制的改良复合磷酸钙骨水泥具有良好的生物相容性和一定的成骨诱导能力,其疏松的结构及更大的孔径结构适宜细胞长入,并可作为BMP因子的良好释放载体而增强其对骨髓间充质干细胞的诱导分化成骨作用。
Background: Bone grafting is the most commonly used method in treatment of bone defectsand nonunion. Since autogenous or allogeneic bone is limited in sourse and has poor quality ofplasticity, artificial bone graft material is becoming the research hotspot currently. The earliestapplicable artificial graft material in clinic with good plasticity was poly(methyl methacrylate)(PMMA), but PMMA could generate heat during curing which may burn the surrounding normalbone tissue, and it can not be absorbed and reshaped in the body which would impede fracturehealing. Therefore PMMA was not suitable as a bone graft material for traumatic bone defectsand bone nonunion. Subsequent arisen bone graft substitutes mainly included calcium sulfate,various calcium phosphates and so on. Calcium sulfate degradated fast in the body and wasinconsistent with the time for new bone grow. Its brittle texture and poor mechanical madecalcium sulfate generally not suitable for the treatment of defect in bone shaft.
Calcium phosphate bone cement is of excellent biological and mechanical properties and hasbe applicated widely in clinic. Although the curing product hydroxyapatite of ordinary calciumphosphate bone cement contains microporous structure, the pore size, however, usually variedfrom below micron to several microns and can not allow cells and blood vessels to grow into itsdeep surface. Furthermore this type of bone cement is very stable in the body.The absorption anddegradation usually only occurs on its surface and the absorption rate is very slow, it is belived tohas no osteoinductive activity and the“bone creeping substitution”process usually needs a longtime. These deficiency limits their clinical application to a certain extent.Therefore there is aneed to improve its formulation so as to make it more adaptive in clinic.
We developed a modified absorbable calcium phosphate bone cement composite (Bone &Calcium phosphate cement, BCPC) through optimizating the formulation of common calciumphosphate cement and mixing it with allogeneic bone meal. Animal experiment had showed thatthis BCPC could induce bone formation when as a bone graft material. In this study, we planedto further study its proliferation and osteoblast differentiation effect on rabbit bone mesenchymalstem cells(BMSCs) in vitro so as to explore its clinical value.
Objective To investigate in vitro the biocompatibility of modified calcium phosphate bone cement(BCPC) and its effect on the growth state and osteogenic differentiation of rabbit BMSCs,as well as its capacity for loading relative factors to promote bone formation.
Methods The experiment included four different groups:①control group;②ordinary calciumphosphate bone cement group(CPC group);③modified composite calcium phosphate bonecement group(BCPC group);④BCPC added exogenous BMP factor group(BMP group).BMSCs of rabbit were co-cultured with vectors respectively in the four groups. Cells growth wasobserved by an inverted phase microscope everyday and through drawing the cell growth curve.The activity changement of alkaline phosphatase (ALP) was detected by an alkaline phosphatasetest kit and the growth state of BMSCs on the surface of vectors was observed by a scanningelectron microscpe. Real-time PCR was used to measure the expression of relative genemRNA(ALP、OSX、CollagenⅠ、CollagenⅡ、Aggrecan) for osteogenesis and chondroblast.
Results There was no statistical difference of cell proliferation amone the four groups. TheBMSCs contacted well with the edges of vectors and had a good growth on the surface of thecarriers. Investigation through scanning electron microscpe showed the pseudopodias wasextended from the cells into the pores of BCPC,while in CPC group, cells growed only on thesurface of vector. Higher activity of ALP was detected in BMP group and BCPC group than theother two groups 7 days after co-culturing with vectors. In the 10th day after co-culturing, theexpression of relative gene mRNA(ALP、OSX、CollagenⅠ、CollagenⅡ、Aggrecan) forosteogenesis and chondroblast was significant higher in BCPC group and BMP group than thecontrol group.
Conclusion The BCPC has good biocompatibility and can induce osteoblast to a certain degree.Its loose structure and the larger aperture enable cells to grow into its deep layers as well as afavorble delivery system of BMP factor to strengthen the effect of osteoblast differentiation ofthe BMSCs.
磷酸钙骨水泥具有优良的生物学和力学性能,但是普通的磷酸钙骨水泥的固化产物羟基磷灰石虽然含有微孔结构,其孔径大小多在微米以下至数微米之间,孔径不能允许细胞及血管的透过和生长,这类骨水泥在人体中非常稳定,吸收和降解通常仅发生在骨水泥的表面且吸收速率非常缓慢,且一般不具有诱导成骨活性,骨爬行替代时间较长,一定程度上限制了其临床应用价值。因此,有必要对其改进配方,使其更符合临床需要。
我们通过优化普通磷酸钙骨水泥的配方,并与同种异体骨粉混合,以同种异体骨粉作为新骨长入的微孔结构研制了改良复合可吸收磷酸钙骨水泥(BCPC),动物实验显示该BCPC作为植骨材料可以诱导成骨。但一种新的植骨材料应用于临床之前,必须阐明其作用机制。本实验拟通过体外实验进一步研究该材料对兔骨髓间充质干细胞(Bonemesenchymalstemcells,BMSCs)的生长和诱导分化成骨作用,探索其临床应用价值。
目的:通过体外实验观察改良复合磷酸钙骨水泥(BCPC)生物相容性,对兔BMSCs的生长和诱导成骨分化作用,以及其负载促骨形成因子的能力。
材料和方法:实验分4组:①对照组;②普通磷酸钙骨水泥组(CPC组);③改良复合磷酸钙骨水泥组(BCPC组);④负载BMP组(加BMP的BCPC组)。将各组材料与传代至第三代的兔骨髓间充质干细胞在24孔板上复合培养,每日倒置相差显微镜下观察细胞生长情况,细胞计数并绘制细胞增殖曲线,碱性磷酸酶测试盒检测细胞合成碱性磷酸酶活性变化,扫描电镜观察细胞在材料表面的生长及长入情况。复合培养第10天各组取相同数目细胞,real-timePCR检测成骨及成软骨相关基因ALP、OSX、CollgenⅠ、CollagenⅡ、AggrecanmRNA表达情况,2-△△CT法比较这些基因mRNA表达差别。
结果:与普通骨水泥比较,配制的BCPC具有更疏松的结构,细胞计数结果显示复合培养后各组细胞增殖无统计学差别,倒置显微镜观察细胞与载体边缘接触良好。复合培养第7天后,BCPC组及加BMP组表达更高的碱性磷酸酶活性,扫描电镜观察细胞在BCPC表面生长增殖良好,逐渐伸出伪足并长入材料孔隙,而CPC组细胞只在材料表面生长。复合培养第10天,BCPC组及加BMP组与CPC组及对照组比较,其成骨或成软骨相关基因ALP、OSX、CollagenⅠ、CollagenⅡ、AggrecanmRNA表达有统计学差别。结论:配制的改良复合磷酸钙骨水泥具有良好的生物相容性和一定的成骨诱导能力,其疏松的结构及更大的孔径结构适宜细胞长入,并可作为BMP因子的良好释放载体而增强其对骨髓间充质干细胞的诱导分化成骨作用。
Background: Bone grafting is the most commonly used method in treatment of bone defectsand nonunion. Since autogenous or allogeneic bone is limited in sourse and has poor quality ofplasticity, artificial bone graft material is becoming the research hotspot currently. The earliestapplicable artificial graft material in clinic with good plasticity was poly(methyl methacrylate)(PMMA), but PMMA could generate heat during curing which may burn the surrounding normalbone tissue, and it can not be absorbed and reshaped in the body which would impede fracturehealing. Therefore PMMA was not suitable as a bone graft material for traumatic bone defectsand bone nonunion. Subsequent arisen bone graft substitutes mainly included calcium sulfate,various calcium phosphates and so on. Calcium sulfate degradated fast in the body and wasinconsistent with the time for new bone grow. Its brittle texture and poor mechanical madecalcium sulfate generally not suitable for the treatment of defect in bone shaft.
Calcium phosphate bone cement is of excellent biological and mechanical properties and hasbe applicated widely in clinic. Although the curing product hydroxyapatite of ordinary calciumphosphate bone cement contains microporous structure, the pore size, however, usually variedfrom below micron to several microns and can not allow cells and blood vessels to grow into itsdeep surface. Furthermore this type of bone cement is very stable in the body.The absorption anddegradation usually only occurs on its surface and the absorption rate is very slow, it is belived tohas no osteoinductive activity and the“bone creeping substitution”process usually needs a longtime. These deficiency limits their clinical application to a certain extent.Therefore there is aneed to improve its formulation so as to make it more adaptive in clinic.
We developed a modified absorbable calcium phosphate bone cement composite (Bone &Calcium phosphate cement, BCPC) through optimizating the formulation of common calciumphosphate cement and mixing it with allogeneic bone meal. Animal experiment had showed thatthis BCPC could induce bone formation when as a bone graft material. In this study, we planedto further study its proliferation and osteoblast differentiation effect on rabbit bone mesenchymalstem cells(BMSCs) in vitro so as to explore its clinical value.
Objective To investigate in vitro the biocompatibility of modified calcium phosphate bone cement(BCPC) and its effect on the growth state and osteogenic differentiation of rabbit BMSCs,as well as its capacity for loading relative factors to promote bone formation.
Methods The experiment included four different groups:①control group;②ordinary calciumphosphate bone cement group(CPC group);③modified composite calcium phosphate bonecement group(BCPC group);④BCPC added exogenous BMP factor group(BMP group).BMSCs of rabbit were co-cultured with vectors respectively in the four groups. Cells growth wasobserved by an inverted phase microscope everyday and through drawing the cell growth curve.The activity changement of alkaline phosphatase (ALP) was detected by an alkaline phosphatasetest kit and the growth state of BMSCs on the surface of vectors was observed by a scanningelectron microscpe. Real-time PCR was used to measure the expression of relative genemRNA(ALP、OSX、CollagenⅠ、CollagenⅡ、Aggrecan) for osteogenesis and chondroblast.
Results There was no statistical difference of cell proliferation amone the four groups. TheBMSCs contacted well with the edges of vectors and had a good growth on the surface of thecarriers. Investigation through scanning electron microscpe showed the pseudopodias wasextended from the cells into the pores of BCPC,while in CPC group, cells growed only on thesurface of vector. Higher activity of ALP was detected in BMP group and BCPC group than theother two groups 7 days after co-culturing with vectors. In the 10th day after co-culturing, theexpression of relative gene mRNA(ALP、OSX、CollagenⅠ、CollagenⅡ、Aggrecan) forosteogenesis and chondroblast was significant higher in BCPC group and BMP group than thecontrol group.
Conclusion The BCPC has good biocompatibility and can induce osteoblast to a certain degree.Its loose structure and the larger aperture enable cells to grow into its deep layers as well as afavorble delivery system of BMP factor to strengthen the effect of osteoblast differentiation ofthe BMSCs.
引文
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