富含血小板血浆在骨组织工程中的应用研究
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摘要
目的:1.传统的细胞支架复合方法是直接将细胞悬液滴加于支架上的静置接种法。但此方法接种效率低,不仅细胞流失量大而且不能均匀地分布在三维支架的内部。所以有学者用纤维蛋白原和胶原等水凝胶物质的双相性,将细胞输送并包埋于支架内,提高了细胞接种效率。本研究试将富含血小板血浆(platelet-rich plasmaPRP)替代水凝胶类物质作为双相接种媒介构建组织工程骨,以探索更好的细胞支架复合方法。因为PRP不仅具有类似水凝胶的双相性,重要的是其中的血小板可释放大量生长因子。2.目前在PRP制备中,抗凝剂的使用多样化,没有研究关注抗凝剂对PRP活性的影响,本研究欲明确抗凝剂在PRP制备和应用中的重要性,及用于PRP制备的最佳抗凝剂,从而为PRP的实际应用提供参考。
方法:1.分别用自体PRP、自体纤维蛋白原、细胞培养基为媒介构建细胞/支架复合物,通过显微镜和电镜观察、复合物中细胞数量检测、复合物碱性磷酸酶活性测定及成骨基因表达分析,比较三种接种方法的细胞接种效率及细胞在支架内的增殖、分化状态。将三种接种方法构建的细胞/支架复合物用于修复山羊颅骨缺损,通过影像学、组织学检查比较三种细胞/支架复合物体内成骨能力。2.用肝素钠、枸橼酸钠、抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁四种抗凝剂制备PRP,通过电镜观察血小板的微观结构,检测血浆中可溶性P-选择素浓度,测定PRP转移生长因子释放量,观察PRP对细胞增殖的影响。
结果:1.与传统静置接种法比较,PRP和自体纤维蛋白原双相接种法明显提高细胞接种效率。PRP较自体纤维蛋白原、细胞培养基显著促进细胞在支架中的增殖,同时不抑制细胞的分化。以PRP为媒介构建的细胞/支架复合物较其它两种复合物明显促进体内新骨形成。2.枸橼酸-茶碱-腺苷-潘生丁抗凝剂与其它抗凝剂相比,能长时间保持血小板结构完整,减少血小板在PRP制备过程中的激活。抗凝枸橼酸葡萄糖对血小板的保护作用较枸橼酸-茶碱-腺苷-潘生丁弱,但二者与肝素钠和枸橼酸钠相比,均能使PRP释放更多的生长因子,并有效地促进细胞增殖。
结论:1.以PRP为媒介的双相细胞接种法是一种简单、有效、经济、实用的细胞/支架复合物构建方法,其应用可以增强组织工程骨的临床应用性。2.抗凝剂是影响PRP质量的重要因素,抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁复合型抗凝剂的应用可以保证PRP的质量、提高PRP生物效力。
Objectives:1.Conventional static seeding method is not efficient in establishing 3D cell/scaffold composites,so fibrinogen and other hydrogels are used to increase seeding efficiency.Platelet-rich plasma(PRP) is derived from autologous whole blood and rich in fibrinogen and piatelets which release multiple active factors in response to thrombin. Therefore,we think using platelet-rich plasma(PRP) as seeding intermedium instead of fibrinogen and other hydrogels can improve current static seeding technology.2.In PRP clinical or research studies,the anticoagulant application does not draw enough attentions though anticoagulants might influence the quality and then biological effect of PRP.This study conducted a comparative study to investigate the effect of the anticoagulants on PRP quality wishing to clarify the appropriate anticoagulant used in PRP production.
Methods:To test our hypothesis,this study evaluated seeding efficiency,proliferation and differentiation of BMSCs seeded into PLGA porous scaffolds via PRP,autologous fibrinogen,and DMEM respectively.Additionally,BMSCs/PLGA constructs established by above three intermediums were implanted in goat calvarial defects to evaluate the capability of PRP in enhancing bone regeneration by radiological and histological assay. 2.The microstructure change of platelets collected via heparin,citrate,acid citrate dextrose(ACD) and citrate-theophylline-adenosine-dipyridamole(CTAD) was observed by TEM following time course.The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma.The TGF-β1 release amount of activated PRP of four groups was measured and the biological effect of PRP on cell proliferation was also studied.
Results:1.PRP not only could increase BMSCs seeding efficiency like fibrinogen,but also could significantly stimulate BMSCs proliferation without inhibition BMSCs differentiation in vitro and obviously enhance bone regeneration in vivo compared with DMEM and fibrinogen.2.CTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation.ACD slightly surpassed heparin and citrate in above two aspects.ACD-PRP and CTAD-PRP released significantly more TGF-β1 and significantly enhanced hBMSCs proliferation compared with heparin and citrate.
Conclusion:1.Therefore,PRP seeding strategy is both valuable and convenient for application in bone tissue engineering and this new seeding method will hopefully enhance the clinical utility of engineered bone.2.The PRP quality and biological effects were strongly associated with the type of Anticoagulants.ACD and CTAD are optimal anticoagulants in PRP production for they can maintain platelet viability at a high level.
方法:1.分别用自体PRP、自体纤维蛋白原、细胞培养基为媒介构建细胞/支架复合物,通过显微镜和电镜观察、复合物中细胞数量检测、复合物碱性磷酸酶活性测定及成骨基因表达分析,比较三种接种方法的细胞接种效率及细胞在支架内的增殖、分化状态。将三种接种方法构建的细胞/支架复合物用于修复山羊颅骨缺损,通过影像学、组织学检查比较三种细胞/支架复合物体内成骨能力。2.用肝素钠、枸橼酸钠、抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁四种抗凝剂制备PRP,通过电镜观察血小板的微观结构,检测血浆中可溶性P-选择素浓度,测定PRP转移生长因子释放量,观察PRP对细胞增殖的影响。
结果:1.与传统静置接种法比较,PRP和自体纤维蛋白原双相接种法明显提高细胞接种效率。PRP较自体纤维蛋白原、细胞培养基显著促进细胞在支架中的增殖,同时不抑制细胞的分化。以PRP为媒介构建的细胞/支架复合物较其它两种复合物明显促进体内新骨形成。2.枸橼酸-茶碱-腺苷-潘生丁抗凝剂与其它抗凝剂相比,能长时间保持血小板结构完整,减少血小板在PRP制备过程中的激活。抗凝枸橼酸葡萄糖对血小板的保护作用较枸橼酸-茶碱-腺苷-潘生丁弱,但二者与肝素钠和枸橼酸钠相比,均能使PRP释放更多的生长因子,并有效地促进细胞增殖。
结论:1.以PRP为媒介的双相细胞接种法是一种简单、有效、经济、实用的细胞/支架复合物构建方法,其应用可以增强组织工程骨的临床应用性。2.抗凝剂是影响PRP质量的重要因素,抗凝枸橼酸葡萄糖、枸橼酸-茶碱-腺苷-潘生丁复合型抗凝剂的应用可以保证PRP的质量、提高PRP生物效力。
Objectives:1.Conventional static seeding method is not efficient in establishing 3D cell/scaffold composites,so fibrinogen and other hydrogels are used to increase seeding efficiency.Platelet-rich plasma(PRP) is derived from autologous whole blood and rich in fibrinogen and piatelets which release multiple active factors in response to thrombin. Therefore,we think using platelet-rich plasma(PRP) as seeding intermedium instead of fibrinogen and other hydrogels can improve current static seeding technology.2.In PRP clinical or research studies,the anticoagulant application does not draw enough attentions though anticoagulants might influence the quality and then biological effect of PRP.This study conducted a comparative study to investigate the effect of the anticoagulants on PRP quality wishing to clarify the appropriate anticoagulant used in PRP production.
Methods:To test our hypothesis,this study evaluated seeding efficiency,proliferation and differentiation of BMSCs seeded into PLGA porous scaffolds via PRP,autologous fibrinogen,and DMEM respectively.Additionally,BMSCs/PLGA constructs established by above three intermediums were implanted in goat calvarial defects to evaluate the capability of PRP in enhancing bone regeneration by radiological and histological assay. 2.The microstructure change of platelets collected via heparin,citrate,acid citrate dextrose(ACD) and citrate-theophylline-adenosine-dipyridamole(CTAD) was observed by TEM following time course.The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma.The TGF-β1 release amount of activated PRP of four groups was measured and the biological effect of PRP on cell proliferation was also studied.
Results:1.PRP not only could increase BMSCs seeding efficiency like fibrinogen,but also could significantly stimulate BMSCs proliferation without inhibition BMSCs differentiation in vitro and obviously enhance bone regeneration in vivo compared with DMEM and fibrinogen.2.CTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation.ACD slightly surpassed heparin and citrate in above two aspects.ACD-PRP and CTAD-PRP released significantly more TGF-β1 and significantly enhanced hBMSCs proliferation compared with heparin and citrate.
Conclusion:1.Therefore,PRP seeding strategy is both valuable and convenient for application in bone tissue engineering and this new seeding method will hopefully enhance the clinical utility of engineered bone.2.The PRP quality and biological effects were strongly associated with the type of Anticoagulants.ACD and CTAD are optimal anticoagulants in PRP production for they can maintain platelet viability at a high level.
引文
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