应用富血小板血浆和骨髓基质细胞构建细胞-膜片及其异位成骨的实验研究

目的：探讨应用富血小板血浆（platelet rich plasma,PRP）和骨髓基质细胞（marrow stromal cells,MSCs）构建细胞-膜片的方法并评价膜片包裹珊瑚支架后的异位成骨能力。方法：将体外培养扩增的兔MSCs悬液与PRP混合,滴加到6孔培养板上,再滴加牛凝血酶溶液,形成凝胶样MSCs／PRP混合物,置于孵箱内培养。第1周用DMEM完全培养液,后2周改用DMEM诱导培养液。用得到的细胞-膜片包裹珊瑚圆片后,植入裸鼠背部皮下,以单纯珊瑚植入作对照。术后4周和8周取材,通过组织学观察,评价其异位成骨情况。结果：细胞-膜片具有一定的韧性和可操作性。扫描电镜观察显示：膜片由大量的纺锤样成骨细胞有规律地密集在一起而形成。组织学观察显示：术后4周和8周,膜-支架复合体珊瑚支架的表面及其孔洞内有大量的软骨或骨形成,以软骨内成骨的方式成骨;单纯珊瑚植入组,珊瑚表面及其孔洞内有大量纤维结缔组织长入,未见软骨或骨形成。结论：用PRP和MSCs构建细胞-膜片的方法简便,膜片包裹珊瑚支架形成的膜一支架复合体具有良好异位成骨能力。     

富血小板血浆对珊瑚支架上骨髓基质细胞增殖和成骨分化的影响

目的：评价富血小板血浆（plateletrichplasma，PRP）对珊瑚支架上骨髓基质细胞（marrowstromalcells，MSCs）增殖和成骨分化的影响。方法：将体外培养、扩增和诱导的兔MSCs悬液与同一供体来源的PRP混合，滴加到珊瑚圆片上，再滴加牛凝血酶溶液，形成珊瑚／MSCs／PRP复合物，继续在体外培养。以珊瑚／MSCs复合物和珊瑚／PRP复合物作对照。8d和14d，通过MTT法检测MSCs增殖情况；通过组织化学方法检测培养液中碱性磷酸酶活性和骨钙素含量；通过扫描电镜观察MSCs在珊瑚支架上附着、生长和增殖情况。结果：珊瑚／MSCs／PRP组的光密度值明显大于珊瑚／MSCs组（P〈0．05）；珊瑚／MSCs／PRP组培养液中的ALP活性和OC含量明显大于珊瑚／MSCs组（P〈0．05）；扫描电镜显示：珊瑚／MSCs／PRP组，大量MSCs附着于珊瑚表面和孔洞壁，可见孔洞内有血小板和纤维网格样结构存在；珊瑚／MSCs组，珊瑚表面和孔洞壁有少量MSCs附着；珊瑚／PRP组，珊瑚表面及其孔洞内有大量的血小板和纤维网格样结构存在。结论：PRP促进了珊瑚支架上MSCs的增殖、成骨分化和粘附。     

应用珊瑚／骨髓基质细胞／富血小板血浆组织工程骨修复兔颅骨缺损

目的：评价珊瑚／骨髓基质细胞（MSCs）／富血小板血浆（PRP）组织工程骨修复骨缺损的效果。方法：将兔MSCs悬液与同一供体来源的PRP混合后滴加到珊瑚团片上,形成珊瑚／MSCs／PRP复合物,用其修复MSCs和PRP来源兔颅骨极限缺损。自体颅骨或单纯珊瑚植入作为对照。术后6周和12周,通过组织学观察和组织形态测量分析．评价其骨修复效果。实验数据采用SPSS11．0软件包处理,两组之间差异采用t检验。结果：组织学观察显示,珊瑚／MSCs／PRP组,术后第6周,在整个缺损区珊瑚表面及其孔洞内有大量新骨形成;第12周,缺损区珊瑚完全降解吸收,被成熟的板层骨取代,缺损表现为完全的骨修复.图像分析显示：术后第6周和第12周,珊瑚／MSCs／PRP组的成骨量显著多于珊瑚组（P〈0．01）;术后第12周,珊瑚／MSCs／PRP组成骨量与自体骨组接近（P〉0．05）。结论：应用珊瑚作为支架材料、MSCs作为种子细胞、PRP作为内源性生长因子构建的组织工程骨修复骨缺损效果良好,与自体骨移植相近．是较理想的骨移植替代材料．     

应用兔骨髓基质细胞膜片与珊瑚支架构建组织工程骨

目的：探讨应用富血小板血浆与骨髓基质细胞（BMSCs）构建骨髓基质细胞膜片，以及应用骨髓基质细胞膜片与珊瑚支架构建组织工程骨的可行性。方法：将取自同一供体兔的BMSCs与富血小板血浆复合后，共同在体外培养10天，构建出BMSCs膜片，用其包裹珊瑚支架后植入裸鼠背部皮下，另以单纯BMSCs种植到珊瑚支架及以单一珊瑚植入作为对照。术后4周和8周取材，通过组织学观察和组织形态测量分析，评价其成骨情况。采用SPSS11．0软件，对所得数据进行统计学分析。结果：BMSCs与富血小板血浆共同在体外培养10d，可构建出厚约2mm的BMSCs膜片。用其包裹珊瑚支架后植入裸鼠背部皮下4周和8周，在珊瑚表面及其孔洞内有大量软骨和骨质形成．其成骨量明显大于单纯BMSCs种植组。单一珊瑚植入组未见骨或软骨形成。结论：将富血小板血浆与BMSCs复合后共同在体外培养，可构建出具有一定厚度的BMSCs膜片，将此膜片包裹珊瑚支架所形成的膜片一支架复合体具有良好的成骨活性.     

细胞-膜片复合珊瑚支架修复兔颅骨极限缺损的实验研究

目的:评价富血小板血浆(platelet rich plasma,PRP)和骨髓基质细胞(marrow stromal cells,MSCs)构建的细胞-膜片包裹珊瑚支架后修复兔颅骨极限缺损的能力。方法:将体外培养扩增的兔MSCs悬液与同一供体来源的PRP混合,滴加到六孔培养板上,再滴加牛凝血酶溶液,形成凝胶样MSCs/PRP混合物,置于孵箱内培养。第1周用DMEM完全培养液,第2周用DMEM诱导培养液培养,形成细胞-膜片。将细胞-膜片取下后包裹珊瑚支架,植入供体兔颅骨直径15 mm的圆形缺损内,用自体颅骨和单纯珊瑚植入作对照。术后8周和16周取材,通过大体观察、组织学观察和组织形态测量分析等,评价其骨修复效果。结果:培养的细胞-膜片厚约2 mm,呈半透明胶冻样,有较好的韧性和可操作性。细胞-膜片/珊瑚组修复兔颅骨缺损效果明显优于单纯珊瑚修复组,在16周时得到完全的骨修复,与自体骨类似。结论:用MSCs和PRP在体外培养构建的细胞-膜片具有较强的成骨活性,包裹珊瑚支架后具有良好的骨修复能力。     

鸵鸟骨转化多相钙磷陶瓷支架负载骨髓基质细胞异位成骨性能研究

目的：观察鸵鸟骨转化多相钙磷陶瓷复合骨髓基质细胞后植入裸鼠皮下的成骨性能。方法：获取兔髂骨松质骨骨髓基质细胞，体外分离、扩增、诱导后接种于多相钙磷陶瓷支架。支架／细胞复合物植入裸鼠背部皮下，单纯支架材料植入作为对照。植入后4周、8周取材，通过大体、组织学观察评价成骨活性。结果：支架／细胞复合物植入后4周，新骨主要以软骨为主，部分区域见少量成熟骨，可见软骨内成骨方式。植入后8周，材料表面和孔隙内有更多的成熟骨形成，部分区域有血管和多核巨细胞分布。结论：支架／细胞复合物显示良好的成骨活性，鸵鸟骨转化多相钙磷陶瓷可以用于骨组织工程支架材料。     

重组人骨形成蛋白-2/珊瑚复合人工骨的研制及其诱导成骨的实验研究

目的：制备珊瑚（ｃｏｒａｌ）与重组人骨形成蛋白－２（ｒｈＢＭＰ－２）复合人工骨并测试其骨诱导活性。方法：将ｒｈＢＭＰ－２与珊瑚复合后植入鼠肌袋内。６６只小鼠随机分为３组：第１组为复合骨（１∶２０ｗ／ｗ）；第２组为复合骨（１∶４０ｗ／ｗ）；第３组为单独珊瑚。术后１、３、６周处死动物，光镜下观察，并用图象分析法作成骨定量测定，结果：术后１周，可见复合材料表面及孔洞内有软骨形成。３周出现编织骨。６周形成含骨髓的板层骨。珊瑚被部分吸收。诱导成骨的量有时间依赖性和ｒｈＢＭＰ－２剂量依赖性。结论：ｒｈＢＭＰ－２／ｃｏｒａｌ复合人工骨具有良好的诱导成骨能力；珊瑚可能是目前能使用的ｒｈＢＭＰ－２最合适的缓释载体。     

重组人骨形成蛋白—2／珊瑚复合人工骨的研制及其诱导成骨的？…

目的：制备珊瑚（ｃｏｒａｌ）与重组人骨形成蛋白－２（ｒｈＢＭＰ－２）复合人工骨并测试其骨诱导活性，方法：将ｒｈＢＭＰ－２与珊瑚复合后植入鼠肌袋内，６６只小鼠随机分为３组，第１组为复合骨（１：２０ｗ／ｗ）；第２组为复合骨（１：４０ｗ／ｗ）；第３组为单独珊瑚，术后１，３，６周处死动物。光镜下观察，并用图象分析法作成骨定量测定。结果：术后１周，可见复合材料表面及孔洞内有软骨形成。３周出现编织骨。６周形成     

珊瑚复合人骨髓基质细胞构建组织工程骨

目的：观察人骨髓基质细胞在角孔珊瑚上立体培养后的生长情况及其在体内的成骨能力。方法：穿刺抽吸人髂骨区骨髓基质细胞，体外培养扩增、诱导后，接种于角孔珊瑚中立体培养5d，观察细胞在角孔珊瑚表面的贴附及伸展情况；将上述细胞/角孔珊瑚复合物植入体内，观察植入物在体内的成骨情况。结果：细胞可在角孔珊瑚中立体培养成活。体内植入后4周，复合物中有少量新骨形成；体内植入后8周，复合物中出现大量较成熟骨组织；而对照组4、8周均无骨组织形成。结论：穿刺抽吸的人骨髓基质细胞可作为骨组织工程的种子细胞，角孔珊瑚可作为骨组织工程的支架材料。     

磷酸钙钠/β-磷酸三钙陶瓷支架接种骨髓基质细胞成骨性能的研究

目的：观察磷酸钙钠／β-磷酸三钙(NaCaPO2/β-TCP)支架接种骨髓基质细胞后植入裸鼠皮下的成骨性能。方法：通过理化方法,将煅烧牛松质骨转化为NaCaPO2/β-TCP双相钙磷陶瓷。获取兔髂骨松质骨骨髓基质细胞,体外分离、扩增、诱导后,接种于NaCaPO2/β-TCP支架。将支架／细胞复合物植入裸鼠背部皮下,NaCaPO2/β-TCP陶瓷单纯植入作为对照。植入后4、8周取材,通过大体、组织学观察,评价成骨活性。结果：支架／细胞复合物植入后4周,材料表面见相对较成熟的骨组织,内部主要为软骨;植入后8周,大量骨小梁形成。可见骨髓腔、骨髓细胞及脂肪细胞,在支架材料和骨组织的邻接区域见成骨细胞及破骨细胞。可见软骨内成骨方式。结论：NaCaPO2/β-TCP支架接种骨髓基质细胞后显示良好的成骨活性,能够促进未成熟骨矿化,可以作为骨组织工程支架材料。     

Influence of platelet-rich plasma on ectopic bone formation of bone marrow stromal cells in porous coral

This study evaluated the effect of platelet-rich plasma (PRP) on the bone formation of marrow stromal cells (MSCs) in porous coral. MSCs in 50 μl of PRP were seeded into natural coral disks (diameter 8.0 mm; thickness 2.0 mm). The composites were clotted and cultured in vitro or implanted subcutaneously into nude mice. Coral scaffolds loading MSCs or PRP alone acted as control. Alkaline phosphatase (ALP) activity of the specimens cultured in vitro for 7 and 14 days was measured, and the level of ectopic bone formation was investigated 4 and 8 weeks after operation. The samples from the coral/PRP/MSC group exhibited significantly higher ALP activity, compared with that from the coral/MSC group or the coral/PRP group (p < 0.05). New bone and/or cartilage formation could be observed in specimens from both coral/PRP/MSC and coral/MSC groups in ectopic sites, and osteogenesis followed the pattern of endochondral bone formation. Histomorphometric analyses showed enhanced cartilage and/or bone formation in the coral/PRP/MSC group, 4 and 8 weeks after implantation. No bone or cartilage formation could be observed in the coral/PRP group. The authors concluded that PRP could improve the ALP activity of MSCs on coral and increase ectopic bone formation.     

rhBMP-2、胶原、珊瑚复合骨诱导骨形成生物活性的实验研究

目的：研究珊瑚、胶原、ｒｈＢＭＯＰ－２复合骨异位诱导新骨形成的活性和载体在体内降解情况。方法：选择珊瑚和１型胶 为ｒｈＢＭＰ－２载体,采用肌袋异位成骨的动物模型,通过大体观察、组织测量等手段,研究复合骨诱导新骨形成的特点、珊瑚降解的过程。结果：ｒｈＢＭＰ－２、珊瑚和胶原复合骨诱导骨组织形成,在一定范围内随作用时间延长新骨形成量持续增加,其中ｒｈＢＭＰ－２、胶原、珊瑚复合骨具有骨引导性和骨诱导性,珊     

Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits

In the present study, we compared bone regeneration ability in sinus floor elevation between a tissue engineering method using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP), and a promising new method using particulate cancellous bone and marrow (PCBM) and PRP. Bilateral sinus floor elevation procedures were performed in 18 adult Japanese white rabbits. MSCs/PRP or PCBM/PRP complexes were grafted to each maxillary sinus in the same rabbits. The MSCs were isolated from rabbit iliac crest marrow, and PRP was obtained from peripheral blood. PCBM were collected from the rabbit iliac crest and mixed with PRP. The animals were sacrificed at 2, 4, and 8 weeks after transplantation, and the bone formation ability of each implant was evaluated histologically and histometrically. According to the histological observations, both sites (MSCs/PRP and PCBM/PRP) showed well newly formed bone and neovascularization at 2 and 4 weeks. However, at 8 weeks, the lamellar bone was observed to be occupied by fatty marrow in large areas in both sites. There was no significant difference in bone volume or augmented height between MSCs/PRP and PCBM/PRP groups each week, but there were significant differences in bone volume and augmented height between 2 and 8 weeks in PCBM/PRP or MSCs/PRP groups and in bone volume between 4 and 8 weeks in the PCBM/PRP group (P<0.05). These results suggest that the MSCs/PRP complex may well be used for bone regeneration in sinus floor elevation, compared with the PCBM/PRP complex.     

The Influence of PRP on Early Bone Formation in Membrane Protected Defects. A Histological and Histomorphometric Study in the Rabbit Calvaria

Background: Platelet rich plasma (PRP) has been proposed to be a useful adjunct to bone grafting. Purpose: The aim of the present study was to assess new bone formation in bone regeneration procedures using platelet rich plasma (PRP) alone or in combination with autogenous bone. Materials and Methods: Four surgically created, monocortical defects 5 mm in diameter in the calvariae of 15 New Zealand rabbits were grafted with a coagulum‐filled control, PRP, particulated autogenous bone alone (A), or combined with PRP (A‐PRP). Results: Mean platelet concentration of 1,761,930 ± 680,200/µl was achieved (5.30 ± 2.63 × fold of baseline). Animals were sacrificed 1, 2, and 4 weeks later. Histomorphometric analysis showed no statistical difference for total new bone formation at any time point, however, a detailed analysis revealed a statistically significant higher percentage of lamellar bone than woven bone for the autogenous bone group at 2 weeks; all other groups demonstrated equal percentages of either bone type. At 4 weeks, all groups revealed a statistically greater component of lamellar bone over woven bone. Graft resorption rate was similar for both A and A‐PRP. PRP platelet concentration was significantly positively correlated with TGF‐beta1 but not with PDGF‐AB. Conclusions: Within the limits of the chosen animal model, this study demonstrated that PRP during early healing, whether alone or mixed with autogenous bone, did not lead to greater bone remodelling, as compared to coagulum. In contrast, autogenous bone alone demonstrated accelerated bone remodelling at 2 weeks.     

辛伐他汀与骨粉复合物填充大鼠胫骨骨缺损修复作用的实验研究

目的研究辛伐他汀作为激活物对大鼠胫骨骨缺损修复的促进作用。方法实验组用辛伐他汀与Bio-Oss骨粉复合物填入大鼠胫骨骨缺损模型,对照组骨缺损单纯填入Bio-Oss骨粉,空白组骨缺损部位不放入骨粉。术后4、8、12周分别处死大鼠,定性、定量分析,观测骨增量变化和骨缺损修复进程。结果空白组无法正常愈合;实验组在新骨生成和改建速度上明显好于对照组。结论辛伐他汀有促进新骨生成作用,加速了骨缺损修复进程。