复合富血小板血浆的可注射藻酸盐组织工程骨的构建和体外增殖、矿化的实验研究
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摘要
目的:观察富血小板血浆(Platelet-Rich Plasma,PRP),作为一种生长因子,在可注射藻酸盐(Alginate,A)骨组织工程(Bone Tissue Engineering)中对成骨细胞(Osteoblasts,OBs)增殖及矿化功能的影响,探讨可注射藻酸盐骨组织工程复合富血小板血浆的可行性。
方法:①体外大鼠成骨细胞OBs的分离、培养及鉴定:取新生Wistar大鼠颅骨,采用多次酶消化法分离OBs,进行体外培养,Gomori钙钻法碱性磷酸酶染色和茜素红矿化结节染色鉴定OBs。②MTT法检测细胞的增殖及活力:取第三代Wistar大鼠成骨细胞与1.5%藻酸盐、富血小板血浆复合。实验分三组,1.实验组(成骨细胞/藻酸盐/20%PRP);2.对照组(成骨细胞/藻酸盐);3.成骨细胞贴壁生长组。体外连续培养14d,分别于培养的第2、3、4、5、6、7、10、12、14d,行MTT法检测实验组、对照组与贴壁生长细胞增殖能力,酶标仪测定光密度值(OD值),每个样本设三个复孔,求其平均值并绘制细胞生长曲线。③荧光四环素标记法行矿化结节测定:取第三代Wistar大鼠成骨细胞,与1.5%藻酸盐支架材料和富血小板血浆复合。实验分三组,1.实验组(成骨细胞/藻酸盐/20%PRP);2.对照组(成骨细胞/藻酸盐);3.成骨细胞贴壁生长组。在培养的第14d、24d、31d,对实验组、对照组与单层培养组,行荧光四环素法标记矿化结节,倒置荧光显微镜下观察各组矿化情况,分别统计各组不同时间点的矿化结节数目、矿化面积百分数。
结果:①成骨细胞鉴定:Gomori钙钻法碱性磷酸酶染色后细胞呈强阳性反应,胞浆内呈现灰黑色颗粒或块状沉淀;茜素红矿化结节染色后矿化结节呈现深红色,结节周围细胞胞浆也呈红染。②MTT法检测细胞的增殖及活力:与单层培养相比,成骨细胞藻酸盐三维培养未见明显的细胞毒性,在培养周期内细胞增殖一直呈上升趋势。含20%PRP藻酸盐三维培养组增殖能力明显高于无PRP的三维培养组,差异具有统计学意义(P＜0.05),尤其是培养的第3-6d。③荧光四环素标记法矿化结节测定:与单层培养相比,成骨细胞藻酸盐三维培养的荧光强度、数目、面积,在培养周期内随培养时间延长而一直呈增多增大的趋势。与对照组相比,含20%PRP实验组的矿化结节数目增多、矿化面积增大,组间各时间点的差异均有统计学意义(P＜0.05)。
结论:①成骨细胞的藻酸盐三维培养体系,细胞代谢活性及功能接近、甚至优于单层培养。
②成骨细胞/藻酸盐/PRP复合物,三者能很好的结合,均匀分布,三维培养下,能促进成骨细胞大量增殖,其构建可行。
③PRP作为藻酸盐组织工程骨的生长因子,能促进成骨细胞矿化功能。
Objective:To study the effects of Platelet-Rich Plasma in Alginate based Bone Tissue Engineering on osteoblasts viabilities and mineralization in vitro and confirm whether PRP can be used as the growth factor resource of injectable Alginate scaffold based Bone Tissue Engineering.
Material and methods:①The isolation,culture and identification of osteoblasts in vitro:New born rat calvarid osteoblasts were isolated by the enzyme digesting for several times and cultured in vitro.The cells were observed by inverted phase contrast microscope.Osteoblasts were identified by ALP dyeing and mineralization nodules dyeing.②The study of osteoblastic proliferation by MTT assay:Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5% Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 14 days.On the days 2,3,4,5,6,7,10,12,14d,the proliferation of OBs was measured by MTT assay respectively,then the growth curves were drawn according to OD values.③The study of osteoblastic mineralization by tetracycline hydrochloride staining: Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5%Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 31 days.On the days 14,24,31d, mineralization nodules of osteoblasts were observed by tetracycline hydrochloride staining under fluorescence inverted phase contrast microscope.Mineralization nodules numbers and mineralizing surface percentage were measured respectively per field of vision in every group at different time.
Results:①The isolation,culture and identification of osteoblasts in vitro:The cells possessed the characteristics of osteoblastic typical morphology.ALP dyeing and mineralization nodules dyeing were positive.The cells can form bone nodules in vitro.②The study of osteoblastic proliferation by MTT assay:Osteoblastic proliferation inclination in Alginate three-dimensionally cultured group remained climbing on,in contrast to the monolayer culture one,and Alginate three-dimension culture system's biocompatibility was no worse than that of the monolayer culture one.Moreover MTT assay results(OD values)indicated cytoactive of the experimental group was much higher than that of the controlled,which difference had statistical significance (P＜0.05).③The study of osteoblastic mineralization by tetracycline hydrochloride staining:The intensity,numbers and volume of fluorescence in Alginate three-dimensionally cultured group remained going on,in contrast to the monolayer culture group.And this inclination became obvious with culture lasting.Moreover, between the experimental group and the control one,there was significant difference in mineralization nodules number and mineralizing surface percentage on the culture days of 24th and 31st(P＜0.05).
Conclusions:Construction of an injectable tissue engineered bone with Alginate, osteoblasts and PRP does not require sophisticated techniques and ensures good biological property in vitro of the artificial bone that can be easily shaped and allows better growth,better function of the OBs,suggesting great potential of this technique for use.Platelet-Rich Plasma can be used as growth factor resource of alginate bone tissue engineering.
方法:①体外大鼠成骨细胞OBs的分离、培养及鉴定:取新生Wistar大鼠颅骨,采用多次酶消化法分离OBs,进行体外培养,Gomori钙钻法碱性磷酸酶染色和茜素红矿化结节染色鉴定OBs。②MTT法检测细胞的增殖及活力:取第三代Wistar大鼠成骨细胞与1.5%藻酸盐、富血小板血浆复合。实验分三组,1.实验组(成骨细胞/藻酸盐/20%PRP);2.对照组(成骨细胞/藻酸盐);3.成骨细胞贴壁生长组。体外连续培养14d,分别于培养的第2、3、4、5、6、7、10、12、14d,行MTT法检测实验组、对照组与贴壁生长细胞增殖能力,酶标仪测定光密度值(OD值),每个样本设三个复孔,求其平均值并绘制细胞生长曲线。③荧光四环素标记法行矿化结节测定:取第三代Wistar大鼠成骨细胞,与1.5%藻酸盐支架材料和富血小板血浆复合。实验分三组,1.实验组(成骨细胞/藻酸盐/20%PRP);2.对照组(成骨细胞/藻酸盐);3.成骨细胞贴壁生长组。在培养的第14d、24d、31d,对实验组、对照组与单层培养组,行荧光四环素法标记矿化结节,倒置荧光显微镜下观察各组矿化情况,分别统计各组不同时间点的矿化结节数目、矿化面积百分数。
结果:①成骨细胞鉴定:Gomori钙钻法碱性磷酸酶染色后细胞呈强阳性反应,胞浆内呈现灰黑色颗粒或块状沉淀;茜素红矿化结节染色后矿化结节呈现深红色,结节周围细胞胞浆也呈红染。②MTT法检测细胞的增殖及活力:与单层培养相比,成骨细胞藻酸盐三维培养未见明显的细胞毒性,在培养周期内细胞增殖一直呈上升趋势。含20%PRP藻酸盐三维培养组增殖能力明显高于无PRP的三维培养组,差异具有统计学意义(P＜0.05),尤其是培养的第3-6d。③荧光四环素标记法矿化结节测定:与单层培养相比,成骨细胞藻酸盐三维培养的荧光强度、数目、面积,在培养周期内随培养时间延长而一直呈增多增大的趋势。与对照组相比,含20%PRP实验组的矿化结节数目增多、矿化面积增大,组间各时间点的差异均有统计学意义(P＜0.05)。
结论:①成骨细胞的藻酸盐三维培养体系,细胞代谢活性及功能接近、甚至优于单层培养。
②成骨细胞/藻酸盐/PRP复合物,三者能很好的结合,均匀分布,三维培养下,能促进成骨细胞大量增殖,其构建可行。
③PRP作为藻酸盐组织工程骨的生长因子,能促进成骨细胞矿化功能。
Objective:To study the effects of Platelet-Rich Plasma in Alginate based Bone Tissue Engineering on osteoblasts viabilities and mineralization in vitro and confirm whether PRP can be used as the growth factor resource of injectable Alginate scaffold based Bone Tissue Engineering.
Material and methods:①The isolation,culture and identification of osteoblasts in vitro:New born rat calvarid osteoblasts were isolated by the enzyme digesting for several times and cultured in vitro.The cells were observed by inverted phase contrast microscope.Osteoblasts were identified by ALP dyeing and mineralization nodules dyeing.②The study of osteoblastic proliferation by MTT assay:Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5% Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 14 days.On the days 2,3,4,5,6,7,10,12,14d,the proliferation of OBs was measured by MTT assay respectively,then the growth curves were drawn according to OD values.③The study of osteoblastic mineralization by tetracycline hydrochloride staining: Osteoblastic suspension was prepared by combining the third passage of OBs with 1.5%Alginate.The cells were divided into three groups:1.the experimental group (1.5%Alginate/OBs/20%PRP)2.the control group(1.5%Alginate/OBs)3.the monolayer culture group,and cultured in vitro for 31 days.On the days 14,24,31d, mineralization nodules of osteoblasts were observed by tetracycline hydrochloride staining under fluorescence inverted phase contrast microscope.Mineralization nodules numbers and mineralizing surface percentage were measured respectively per field of vision in every group at different time.
Results:①The isolation,culture and identification of osteoblasts in vitro:The cells possessed the characteristics of osteoblastic typical morphology.ALP dyeing and mineralization nodules dyeing were positive.The cells can form bone nodules in vitro.②The study of osteoblastic proliferation by MTT assay:Osteoblastic proliferation inclination in Alginate three-dimensionally cultured group remained climbing on,in contrast to the monolayer culture one,and Alginate three-dimension culture system's biocompatibility was no worse than that of the monolayer culture one.Moreover MTT assay results(OD values)indicated cytoactive of the experimental group was much higher than that of the controlled,which difference had statistical significance (P＜0.05).③The study of osteoblastic mineralization by tetracycline hydrochloride staining:The intensity,numbers and volume of fluorescence in Alginate three-dimensionally cultured group remained going on,in contrast to the monolayer culture group.And this inclination became obvious with culture lasting.Moreover, between the experimental group and the control one,there was significant difference in mineralization nodules number and mineralizing surface percentage on the culture days of 24th and 31st(P＜0.05).
Conclusions:Construction of an injectable tissue engineered bone with Alginate, osteoblasts and PRP does not require sophisticated techniques and ensures good biological property in vitro of the artificial bone that can be easily shaped and allows better growth,better function of the OBs,suggesting great potential of this technique for use.Platelet-Rich Plasma can be used as growth factor resource of alginate bone tissue engineering.
引文
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2.Klein CP,van der Lubbe HB,de Groot K,et al.A plastic composite of alginate with calcium phosphate granulate as implant material:an in vivo study[J].Biomaterials,1987,8(4):308-310.
3.Suzuki Y,Tanihara M,Ohnishi K,et al.Cat peripheral nerve regeneration across 50mm gap repaired with a novel nerve guide composed of freeze-dried alginate gel[J].Neurosci Lett,1999,259(2):75-78.
4.Thorn as S.Alginate dressings in surgery and wound management-Part1[J].J Wound Care,2000,9(2):56-60.
5.Heald VA,Jay TR,Downing R.Assessment of the reproducibility of Alginate encapsulation of pancreatic islets using the MTT colorimetric assay[J].Cell Transplant,1994,3(4):333-337.
6.Ueng SWN,Lee SS,Lin SS,et al.Biodegradable alginate antibiotic Beads[J].Clin Orthop,2000,NOV(380):250-259.
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