摘要
目前临床骨缺损常采用自体骨或同种异体骨等移植修复。自体骨移植存在着骨量有限和供区并发症等缺陷,同种异体骨移植有传播疾病和引发免疫排斥反应等不足。应用骨组织工程技术可避免自体骨或同种异体骨移植中存在的缺陷或不足,为骨缺损修复提供了一种新的方法。因此,自组织工程概念提出以来,骨组织工程就成为研究的热点并得到了迅速发展。
骨组织工程的研究包括三个方面:支架材料、种子细胞和生长因子。支架材料应具有多孔结构,为种子细胞提供足够的粘附和迁移空间,也为营养物质和代谢产物的运输提供通道。珊瑚是珊瑚虫死亡后遗留的外骨骼所形成的物质,呈三维多孔状结构,孔洞之间相互交通,主要成分是碳酸钙,类似无机骨,具有良好的生物相容性和可降解性,是骨组织工程较理想的支架材料之一。
富含血小板血浆(platelet rich plasma,PRP)是全血经过浓集、分离而得到的血液制品。其特点是PRP的血小板浓度比全血的血小板浓度升高4-5倍以上,含有高浓度的促进骨组织和软组织再生修复的生长因子,可作为内源性的生长因子而应用于骨组织工程。
骨髓基质细胞(marrow stromal cells, MSCs)具有多分化潜能,在特定条件下能向成骨细胞、软骨细胞和脂肪细胞等分化,增殖能力强,来源广泛,取材方便,供区损伤小,是骨组织工程较理想的种子细胞之一。
本研究将珊瑚作为支架、MSCs作为种子细胞、PRP作为内源性生长因子构建组织工程骨,通过体外培养、异位成骨和骨缺损修复等系列研究,来评价PRP对珊瑚支架上MSCs的增殖和成骨的影响;用PRP和MSCs在体外构建组织工程膜片并用其包裹珊瑚支架,通过异位成骨等研究,来评价其骨修复能力。
实验一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PRP对珊瑚支架上MSCs的增殖和成骨分化的影响。
方法:从兔髂骨骨髓中分离出MSCs,体外培养、扩增和诱导。取50μl细胞浓度为1.0×107/ml的MSCs悬液与同一供体来源的50μl PRP混合,滴加到直径8 mm、厚2 mm的珊瑚圆片上,再滴加10μl的牛凝血酶溶液,形成珊瑚/MSCs/PRP复合物,在饱和湿度、37oC、5%CO2条件下培养。以同样数量的MSCs或PRP制备的珊瑚/MSCs复合物和珊瑚/PRP复合物在体外培养作对照。8天和14天后,通过MTT法检测PRP对珊瑚支架上MSCs增殖的影响;通过对培养液中碱性磷酸酶(alkaline phosphatase, ALP)活性和骨钙素(osteocalcin, OC)含量检测,评价PRP对珊瑚支架上MSCs成骨分化的影响;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,体外培养8天,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;14天,珊瑚表面和孔洞有大量MSCs及其分泌的骨基质。珊瑚/MSCs组,体外培养8天,珊瑚表面和孔洞壁有少量MSCs附着;14天,珊瑚表面及其孔洞壁附着的MSCs周围出现分泌的骨基质。珊瑚/PRP组,体外培养8天和14天,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,未见MSCs和骨基质形成。
结论:PRP促进了珊瑚支架上MSCs的增殖和成骨分化。
实验二富血小板血浆对珊瑚支架上骨髓基质细胞异位成骨的影响
目的:评价PRP对珊瑚支架上骨髓基质细胞异位成骨的影响。
方法:从兔髂骨骨髓中分离出MSCs,体外培养、扩增和诱导。取50μl细胞浓度为1.0×108/ml的MSCs悬液与同一供体来源的50μl PRP混合,滴加到直径8 mm、厚2 mm的珊瑚圆片上,再滴加10μl的牛凝血酶溶液,形成珊瑚/MSCs/PRP复合物。以同样数量的MSCs或PRP制备珊瑚/MSCs复合物和珊瑚/PRP复合物。将三种复合物植入同一裸鼠的背部皮下,术后4周和8周取材。通过大体观察、组织学观察和组织形态测量分析来评价其异位成骨情况。
结果:珊瑚/MSCs/PRP植入后4W,在珊瑚表面及其孔洞内有大量的软骨形成;术后8W,珊瑚表面及其孔洞内有大量的板层骨形成,部分区域仍有软骨存在,珊瑚占据的区域明显缩小。珊瑚/MSCs植入后4W,珊瑚表面及其孔洞内也有软骨形成,并有纤维结缔组织长入;术后8W,珊瑚表面及其孔洞内有板层骨和纤维结缔组织充填。珊瑚/PRP植入后4W和8W,未见骨或软骨形成,珊瑚周围及其孔洞内有大量纤维组织充填,随着时间的推移,珊瑚占位逐渐缩小。珊瑚/MSCs/PRP组形成的软骨或骨质的量明显多于珊瑚/MSCs组(p<0.05)。
结论:PRP促进了珊瑚支架上MSCs异位成骨。
实验三珊瑚/骨髓基质细胞/富血小板血浆修复兔颅骨缺损
目的:评价珊瑚/MSCs/PRP修复骨缺损的效果。
方法:从兔髂骨骨髓中分离出MSCs,体外培养、扩增和诱导。取300μl细胞浓度为1.0×108/ml的MSCs悬液与同一供体来源的300μl PRP混合,滴加到直径15 mm、厚2 mm的珊瑚圆片上,再滴加100μl的牛凝血酶溶液,形成珊瑚/MSCs/PRP复合物。用其修复MSCs和PRP来源兔的直径15 mm颅骨缺损。自体颅骨或单纯珊瑚植入作对照。术后6周和12周,通过大体观察、X线片观察、组织学观察和组织形态测量分析,评价其骨修复效果。
结果:大体观察:珊瑚/MSCs/PRP组,术后6周,缺损修复区珊瑚孔洞内有淡红色组织充填,钳夹质地较韧,植入物周边与骨床之间无明显动度;术后12周,缺损区珊瑚支架材料消失,由硬度与周围骨床类似的组织替代,植入物与骨床完全融合。自体骨组,术后6周,缺损区再植的颅骨质地较硬,与周边骨床基本融合,无明显动度;术后12周,再植的颅骨与周边骨床完全融为一体,边界不清。单纯珊瑚组,术后6周,缺损区珊瑚支架材料清晰可见,钳夹质地较脆,植入物边界清楚,与周边骨床有轻微动度;术后12周,缺损区珊瑚支架材料消失,缺损区大部为质地较软的纤维组织充填,仅周边部质地稍硬,与周围骨床边界清楚。X线片观察:珊瑚/MSCs/PRP组,术后6周,缺损区有大块片状阻射影像,12周,缺损区呈现高密度阻射影,与周围骨床密度相近、边界欠清。自体骨组,术后6周,缺损区呈现高密度阻射影,术后12周,缺损区呈现高密度阻射影,与周围骨床密度一致,边界不清。单纯珊瑚组,术后6周,缺损区呈低密度的阻射影;12周,缺损区呈现透光影,仅周边部有少量阻射影。组织学观察:珊瑚/MSCs/PRP组,术后6周,在整个缺损区珊瑚表面及其孔洞内有大量的新骨形成;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。自体骨修复组,术后6周,再植的颅骨仍维持其原有形态和结构;12周,植入区结构与周围骨床一致,边界不清。单纯珊瑚组,术后6周,在骨床附近的珊瑚孔洞内有新骨形长入,缺损中心部珊瑚孔洞内仅有纤维组织充填;12周,缺损区珊瑚完全降解吸收,骨床附近的缺损有新骨修复,缺损中心的部分区域有充纤维组织充填,表现为不完全的骨修复。图像分析显示:术后6周和12周,珊瑚/MSCs/PRP组的成骨量明显多于珊瑚组(P<0.01);术后12周,珊瑚/MSCs/PRP组成骨量与自体骨组相近(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。
实验四应用富血小板血浆和骨髓基质细胞构建组织工程细胞膜片及其异位成骨的实验研究
目的:探讨应用PRP和MSCs构建组织工程细胞膜片的方法及评价膜片包裹珊瑚支架后的异位成骨能力。
方法:从兔髂骨骨髓中分离出MSCs,体外培养和扩增后,取500μl细胞浓度为1.0×107/ml的MSCs悬液与500μl来自同一供体的PRP混合,滴加到六孔培养板上,再滴加150μl的牛凝血酶溶液,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,后两周改用DMEM诱导培养液。3周后,培养板底部形成半透明胶冻样的厚约2mm的膜片。通过扫描电镜观察,了解膜片的结构情况。用膜片包裹直径8mm、厚2 mm珊瑚圆片,形成膜-支架复合体,将其植入裸鼠的背部皮下,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:大量的纺锤样成骨细胞有规律地密集在一起,在细胞的表面有散在的颗粒样骨基质形成。组织学观察显示:术后4周和8周,膜-支架复合体组,在珊瑚支架的表面及其孔洞内有大量的软骨和骨形成,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构建组织工程细胞膜片的方法简便,膜片包裹珊瑚支架形成的膜-支架复合体具有良好异位成骨能力。
Bone tissue engineering offers a promising new approach for bone repair. Compared to traditional autograft and allograft procedures, bone tissue engineering techniques based on autogenous cell/tissue transplantation would eliminate problems of donor scarcity, supply limitation, and pathogen transfer and immune rejection. Therefore, it has become a rapidly expanding research area since the emergence of the concept of tissue engineering.
Engineering bone typically uses an artificial extracellular matrix (or scaffold), osteoblasts or cells that can become osteoblasts, and growth factors that promote cell recruitment, growth differentiation and mineralized bone tissue formation. The scaffold for cell seeding should be porous so that it provides a large internal surface for adhesion and migration of cells and makes it easy for the exchange of nutrients and metabolic waste. Natural coral, which is biocompatible and osteoconductive, has pore sizes and architecture similar to those of human bone. It is an excellent material for supporting marrow stromal cells (MSCs) or osteoblasts attachment, proliferation, and differentiation while gradually being degraded and finally replaced by new bone.
Platelet-rich plasma (PRP) is an autologous source of various growth factors that is obtained by sequestering and concentrating freshly drawn venous blood. When activated with thrombin and calcium chloride, the platelets in the PRP delivered a high concentration of growth factors into the recipient bed, which are all involved in reparative processes such as bone healing.
MSCs have a high proliferative capacity and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes. MSCs can be loaded onto biomaterials to create a bioactive composite. Adding expanded MSCs to biomaterials in vivo could significantly improve bone formation. However,the influence of PRP on expanded MSCs for osteogenesis and bone formation remains to be elucidated.
Our first hypothesis was that the combination of expanded MSCs with PRP in resorbable natural coral could promote osteogenesis and enhance bone formation. We further hypothesized that cell-sheet could be growed by combined MSCs and PRP and bone could be engineered in vivo by using the cell-sheet in combination with coral scaffold.
Experiment 1 Influence of platelet-rich plasma on proliferation and differentiation of MSCs in coral scaffold in vitro
Purpose: To investigate the Influence of PRP on proliferation and differentiation of MSCs in coral scaffold in vitro.
Method: A suspension of 5×106 MSCs in 50μl medium were dissolved in 50μl of PRP. The mixture of MSCs and PRP was spread on a coral disc with diameter of 8 mm and thickness of 2 mm, so coral/MSCs/PRP composite was obtained. A suspension of MSCs in 50μl medium was spread on a coral disc to obtain coral/MSCs composite. 50μl of PRP was added on a coral disc to obtain coral/PRP composite. Coral/MSCs/PRP composite was cultured in a humidified incubator for 8 and 14 days. The medium was changed every other day. Coral/MSCs composite and coral/PRP composite used as control. Then three types of composites were processed for to evaluate ALP activity, osteocalcin and proliferation of MSCs in the constructs. The composites were processed also for to evaluate adhesion, growth and proliferation of MSCs on coral scaffold by scanning electron microscope.
Results: The ALP activity and content of osteocalcin were significantly higher in the samples of coral/MSCs/PRP group compared to that of coral/MSC group at 8 and 14 days in vitro (p<0.05). A comparison of the results of MSCs proliferation showed that cell growth increases as a function of PRP in coral scaffolds. Scanning electron micrographs show the coral scaffold had interconnected porous networks, more cells adhered to the scaffolds of coral/MSCs/PRP than to that of coral/MSCs, and the MSCs were homogeneously distributed throughout the scaffolds. Some asteroid MSCs are bound by the fibrin network, and these cells are believed to be in an active proliferating phase. A lot of floccular extracellular matrix was evident, implying the presence of active secreting MSCs. SEM analysis confirmed the extensive growth of MSCs in the scaffolds of coral/MSCs/PRP over the 14 days.
Conclusion: These in vitro study results suggest that MSCs may be encouraged to proliferate and osteogenically differentiate by PRP in the coral scaffold, leading to increased osteogenesis in vitro.
Experiment 2 Influence of platelet-rich plasma on ectopic bone formation of bone marrow stromal cells in porous coral
Purpose: To evaluate the effect of PRP on ectopic bone formation of MSCs in porous coral.
Methods: Natural coral disks with diameter of 8 mm and thickness of 2 mm were used in this study. A suspension of 5×10~6 MSCs in 50μl medium were dissolved in 50μl of PRP. The mixture of MSCs and PRP was spread on a coral disc. The constructs were implanted into the dorsal subcutaneous area of athymic mice. Coral scaffolds seeded with MSCs alone or added with PRP alone acted as control. The ectopic bone formation was investigated by gross examination, histological observation and Histomorphometric analyses 4 and 8 weeks after operation.
Results: Gross examination showed that three types of constructs retained their original shape. The tissue formed in the coral/MSCs/PRP or coral/MSCs constructs was pink and hard to the touch with surgical forceps, a feature absent in the coral/PRP constructs. There was a trend of progressively increased pink and hardness between coral/MSCs and coral/MSCs/PRP constructs. New bone and/or cartilage formation could be observed in specimens from both coral/MSCs/PRP group and coral/MSCs group in ectopic sites, and osteogenesis followed the pattern of endochondral bone formation. Histomorphometric analyses showed enhanced cartilage and/or bone formation in coral/MSCs/PRP group 4 and 8 weeks after implantation, compared with the coral/MSCs group. In contrast, no bone or cartilage formation could be observed in coral/PRP group.
Conclusion: Combination of MSCs with PRP in porous coral could improve increased ectopic bone formation.
Experiment 3 Treatment of rabbit calvaria defects with bone marrow stromal cells in combination with platelet-rich plasma in porous coral
Purpose: To evaluate local bone formation following surgical implantation of MSCs in combination with PRP in porous coral using a critical-size rabbit calvaria defect model.
Methods: Natural coral disks with diameter of 15 mm and thickness of 2 mm were used in this study. A suspension of 3.3×107 MSCs in 300μl medium were dissolved in 300μl of PRP. The mixture of MSCs and PRP was spread on a coral disc. The construct was implanted into a critical-size 15mm rabbit calvaria defect. Coral disc alone or autograft used as control. The bone formation was investigated by gross examination, X-ray, histological observation and Histomorphometric analyses 6 and 12 weeks after operation.
Results: Six weeks after grafting, new bone was distributed throughout the coral scaffolds in specimens from the test group, and new bone appeared only in the periphery region of the coral scaffold from the coral group. Histomorphometric data revealed a significantly higher bone area in test group than in the coral group (p<0.05). Twelve weeks after grafting, the bone defects of the test group were repaired fully with bone, when those of the coral group were repaired partly with bone and fibrous tissue was evident in the central region of defects. The test group had evident advantage over the coral group in term of bone regeneration (p<0.05). There was no statistically significant difference in bone formation between the test group and autograft group at 12 weeks post-surgery (p>0.05).
Conclusion: Combination of MSCs with PRP in porous coral could enhance the bone healing considerably.
Experiment 4 Cell-sheet constructed by combined marrow stromal cells and Platelet-rich plasma and its ectopic bone formation
Purpose: To investigate method of growing cell-sheet by combined MSCs and PRP and its ectopic bone formation.
Methods: A suspension of 5×10~6 MSCs in 500μl medium were dissolved in 500μl of PRP. The mixture of MSCs and PRP was plated in one well of 6-well plate and cultured in a humidified incubator for 3 weeks. The culture media was standard DMEM for first week and then was changed to osteogenic DMEM for next two weeks. The medium was changed every other day. MSCs sheet was formed and could detached intact from the substratum using a cell scraper. The sheet was processed for to evaluate its construction by scanning electron microscope. The sheet was then wrapped around the coral scaffold, and the sheet–scaffold construct was implanted into the dorsal subcutaneous area of athymic mice. Coral scaffolds acted as control. The ectopic bone formation was investigated by histological observation 4 and 8 weeks after operation.
Results: The sheet comprising multilayered spindle-like osteoblasts possessed good mechanical properties. New bone and/or cartilage formation could be observed both inside and outside the scaffold in specimens from the sheet–scaffold construct group in ectopic sites 4 and 8 weeks after implantation, and osteogenesis followed the pattern of endochondral bone formation. In contrast, no bone or cartilage formation could be observed in coral group.
Conclusion: The method of growing cell-sheet by combined MSCs and PRP was convenient and simple, and bone graft could be engineered through combination of the sheet and coral scaffold.
引文
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