HC/ACBM联合负压吸引修复恒河猴骨缺损的实验研究
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摘要
研究背景
临床上，面对因各种原因所致骨缺损、尤其是大块骨缺损，一直以来没有很理想的修复材料可供选择，而当前因车祸、肿瘤所导致的骨缺损发生率却呈上升趋势。骨缺损修复目前常用的支架材料主要有自体骨、同种异体骨、骨水泥、陶瓷及一些相关的衍生材料等，这些支架材料均存在不同程度的缺点。组织工程骨的兴起为骨缺损修复支架材料指明了新的发展方向，组织工程自1987年正式提出和确定后，在种子细胞、支架材料和组织构建技术等方面取得了明显进展，但是仍有一些问题尚待解决和完善，组织工程骨的血管化就是其中之一。
骨缺损植入修复材料后血管新生需要一个较长的过程，早期的氧和营养仅能靠受体的组织液渗透供应，并且植入的材料由于机体的凝血反应会被凝血块包裹，从而使渗透更加困难，因此其大小严重受到局限，这显然远远不能满足临床应用的要求。促进组织工程骨血管化的方法常见的有：用胶原、促血管生成的生长因子等修饰支架材料；或将成骨种子细胞与血管内皮细胞、成血管前体细胞共培养等，但是效果欠佳。考虑到移植的组织工程骨主要靠外周血管芽生的方式血管化，一些研究小组利用外周血管芽生的策略，提出了组织工程骨支架材料的预血管化技术：将材料和细胞复合物包埋于皮下、大网膜、筋膜或肌袋中待组织血管化，然后将血管化的组织移植到受区，或者将构建的组织工程骨内植入动静脉也能明显促进材料细胞复合物的血管化及成骨能力，但过程比较复杂，并且即便采用了这些方法，组织工程骨植入受区后，尤其是早期的血液供应仍然靠渗透进行，没有完全解决血管化的难题。
本课题组前期所研发的异种脱细胞骨基质(Acellular bone matrix，ACBM)支架材料较好地保留了细胞外骨基质的主要成分和完整结构，并且采用层层自组装技术，将肝素、壳聚糖复合在其表面，实验证明缓释、抗凝效果可靠；然后在此基础上联合封闭负压吸引技术(Vacuum Assisted closure,VAC)，使血液在早期即可在支架材料内部实现持续“灌注”，为构建的组织工程骨即时、持续提供氧和营养物质。以兔和猪骨缺损为模型的修复研究结果证实，HC/ACBM (Heparin-Chotison coated ACBM)材料结合封闭负压技术使骨缺损修复支架材料植入后即刻可以获得血液灌注，与非负压侧比较，血容量、血流量及新生血管数等均显著增多，并且能促进新骨形成。
但是，兔和猪和人差别较大，对于这种异种来源的支架材料应用于人还有待进一步研究。而恒河猴的解剖结构、生理学等方面与人高度相似，是一种较为理想的实验动物，为进一步验证其安全性和有效性，本实验首先按照ISO10993的标准检测了这种新材料的安全性和组织相容性的系列指标，并且在体外研究了模拟负压吸引灌注条件下培养对种子细胞与支架材料复合的影响，建立了恒河猴胫骨缺损的动物模型，初步探讨了HC/ACBM支架材料联合负压吸引对骨缺损修复过程中血流灌注和成骨的影响，为下一步的临床研究提供理论和实验依据。
材料与方法
1. HC/ACBM的生物安全性试验
将本课题组研制的HC/ACBM材料按照ISO10993的标准，制作了20%的HC/ACBM的浸提液进行了急性、亚急性毒理试验、细胞毒性试验、皮肤刺激性试验，以及HC/ACBM的肌肉埋植试验。
对骨髓来源间充质干细胞(mensenchymal stem cell，MSC)进行分离、培养，然后将HC/ACBM与细胞进行共培养，对表面改良后的材料与MSC的粘附性进行了研究。
2.负压吸引灌注培养方式对细胞与材料复合的影响
制作了体外模拟负压吸引进行细胞与材料共培养的灌注装置，以自制的这种装置将MSC和HC/ACBM共培养，对比研究了负压吸引灌注培养与静态培养对MSC的粘附、分布、生存、增殖等方面的影响。
3. HC/ACBM结合负压吸引修复恒河猴胫骨缺损
建立恒河猴胫骨缺损的模型，以双侧自体作为对照，分别观察了ACBM支架材料抗凝与不抗凝的比较，以及HC/ACBM联合负压吸引与不联合负压吸引的比较。利用CT及CT灌注观察了支架材料在修复后不同时间点的血流灌注和成骨情况。
主要结果和结论：
1.扫描电镜显示的超微结构表明，表面改良后的HC/ACBM仍保留了天然骨的多孔三维结构，并且HC/ACBM与MSC仍能紧密的粘附，证实HC/ACBM具有很好的粘附性。
2.各项安全性检测均证实HC/ACBM无明显的毒性，具有良好的安全性和组织相容性。
3.负压吸引灌注培养组与静态培养组的比较研究表明，负压吸引灌注培养条件下，细胞在材料表面和中心分布的更加均匀，TUNEL法检测没有发现粘附在材料上的细胞有明显的凋亡发生，Brdu法检测结果表明细胞与材料共培养时细胞可以正常进行增殖。
4. CT灌注成像显示，HC/ACBM时间密度曲线呈血液灌注的表现，而ACBM材料呈血液渗透曲线。血容量、血流量图象显示体内植入HC/ACBM这种抗凝材料修复骨缺损，不仅材料周边部分有血液灌注，材料中心出现血液的时间也明显早于非抗凝材料；
5.利用填充床式生物反应器的机理，在HC/ACBM抗凝支架材料植入后采用周期性低负压吸引，实验结果证实，构建的动物模型在HC/ACBM植入后早期就可以获得血液灌注，尤其是在材料的中心部分，与非负压侧比较，血容量、血流量等均显著增多，其新骨形成的质和量也显著改善。
Background
Clinically, the treatment of bone defects for various reasons, especially large bonedefect, is always the orthopedic surgeon’s problems. And the number of people required toundergo bone graft repair is rising because of car accidents, cancer and so on. The repairmaterials of basically has autologous bone, allograft bone, non-degradable bone cement,ceramics, related derivative materials and so on, and these materials are unsatisfied. Bonetissue engineering is the new technology for regeneration and repair, which is the new trendof development of bone defect repair. Since the1987officially proposed and determined,bone tissue engineering have achieved many progress in seed cells, scaffolds and thetechnology of structure. But, some problems need to be resolved. Revascularization is oneof the most important problems of them. As everybody knows, the angiogenesis requiresseveral days after bone defects implanted. The early nutrition supply of the cells can onlyrely on the body tissue fluid penetration. So, the size of scaffold material is severely limited.Only as skin and cartilage that can rely on diffusion to maintain a low metabolic untilcompletion of revascularization, which is obviously far from being able to meet therequirements of clinical applications. Meanwhile, the implant material will start thecoagulation reaction, thus make the blood penetration more difficult.
The common approachs to promote vascularization of bone tissue engineering,including collagen or vascular growth factors-modified scaffold, or oseogenesis seed cellsculture with the vascular endothelial cells or vessel precursor cells etc, but ineffective.Taking into account the way of vascularization of bone tissue engineering mainly rely onperipheral vascular buds born after transplantation, studies have used peripheral vascularshoot raw strategy, and puted forward the bone tissue engineering the pre-vascularizedtechnology of scaffolds: scaffolds and cells composite embeded in subcutaneous, omental,fascia or muscle bags, and then the pre-vascularization of tissue will transplant to therecipient or tissue engineered bone implant arteriovenous also can obviously promote the vascularization and osteogenic of the compound bone scaffolds. But the process is morecomplex, and even with these methods, tissue engineering bone after implantation torecipient, the blood supply, especially early blood supply remain dependent on thepenetration, not completely solve the problem of the early revascularization.
The xenogeneic acellular hone matrix (ACBM) scaffold which developed by ourresearch group retains the major components and complete structure of the extracellularmatrix of bone. With layer-by-layer of self-packaging technology, heparin (HEP) andchitosan (HC) compound were coated on ACBM’s surface. The experiment showed thatslow-release and reliable anticoagulation effects were satisfied. And then combined vacuumassisted closure (VAC), allowed blood to continuous infusion at an early stage, provided thescaffolds of tissue engineered bone real-time, continuous oxygen and nutrients. Repairdefects in rabbit and pig results confirmed that the HC/ACBM (Heparin-Chotison coatedACBM) scaffold with the vacuum assisted closure make repair materials can be obtainedimmediately blood infusion after implantation. And compared with the non-negativepressure side, the blood volume, blood flow and neovascularization number weresignificantly increased and the quality and quantity of new bone formation alsosignificantly improved.
In order to further promote application of HC/ACBM scaffold materials, this study inaccordance with ISO10993standard to detect related safety series indicators of the newmaterial. In vitro, we studied the influence of simulation of the negative pressure drainageto seed cells compound scaffold materials. And we selected the rhesus monkeys which wasvery close to human as experimental animals, established the animal models of rhesus tibialdefect. Then we study how the HC/ACBM scaffold materials with VAC impact the bloodperfusion and bone formation during the process of bone defect repair, provided to thetheory and experiment basis for the next step of the clinical research.
Materials and methods
1. The HC/ACBM biological safety test
According to the standard of ISO10993, we study on the biological safety ofHC/ACBM materials. We prepared20%liquid extracts of HC/ACBM for acute or subacutesystemic toxicological tests, in vitro cytotoxicity tests, skin irritation tests and local effects after implantation tests.
The research has isolated and cultured bone marrow derived mesenchymal stem cells.And then we studied the adhesion to MSC to the material modified surface by HC/ACBMand the stem cells co-cultured.
2. The influence of the negative pressure drainage infusion to the cells compositematerials
The research has developed a cell culture device with perfusion simulation of suctiondrainage in vitro. With this homemade device, the MSCs and HC/ACBM scaffold materialco-cultured. Compared to the static culture, we investigate the impact of the negativepressure drainage infusion how to impact the adhesion to HC/ACBM, distribution, survival,the proliferation of MSCs.
3. HC/ACBM combined with vacuum assisted closure repair rhesus monkeys tibialdefect
Established rhesus tibial defect model, bilateral self control, we compared the ACBMwith or without anticoagulation, and HC/ACBM combination of vacuum assisted closure ordo not use. Using CT and CT perfusion studied the blood perfusion and osteogenesis atdifferent time points after repair rhesus monkey tibia defect with the HC/ACBM.
The main results and conclusions:
1. The scanning electron microscopy (SEM) shows that the ultrastructure ofHC/ACBM retained the natural bone nets holes and porosity. Results of MSC andHC/ACBM co-culture suggested that MSC can close adhesion to the ACBM with surfacemodified, and scaffold materials have the characteristic of well adhesion.
2. In accordance with the methods and procedures developed by the ISO10993standard, acute or subacute systemic toxicological tests, in vitro cytotoxicity tests, skinirritation tests and local effects after implantation tests both confirmed the HC/ACBM nosignificant toxicity, has the good biological safety and compatibility.
3. The cells culture in device with perfusion simulation of suction drainage shows thatthe cells adhere to surface and center of the material, and with a more uniform distribution.TUNEL assay found the cells adhesion to the HC/ACBM’s were no obvious apoptosis.Brdu assay results also showed that cell proliferation of cells adhesion to the HC/ACBM’s could normal proliferate. In contrast, the static cultured cells were mainly distributed in theouter surface of the HC/ACBM, while the central parts were very few.
4. CT perfusion imaging shows that time density curve of HC/ACBM group is bloodperfusion performance, and ACBM group is blood penetration curve. Blood volume, bloodflow in the images show the implant HC/ACBM with anticoagulation to repair bone defect,scaffold materials have a blood perfusion not only surrounding peripheral parts, materialscenter appearance of blood in the early time also obvious in this anticoagulant scaffoldmaterials;
5. In this study, using the mechanism of the packed bed bioreactor, and imposedperiodic low negative pressure in HC/ACBM with anticoagulation scaffold afterimplantation. The experimental results demonstrate that, animals after implantation inHC/ACBM with negative pressure can get blood perfusion earlier, especially in the centralparts of scaffold materials. Compared with the non-negative pressure side, the negativepressure side had more blood volume, blood flow significantly increased, and the qualityand quantity of new bone formation also significantly improved.
临床上，面对因各种原因所致骨缺损、尤其是大块骨缺损，一直以来没有很理想的修复材料可供选择，而当前因车祸、肿瘤所导致的骨缺损发生率却呈上升趋势。骨缺损修复目前常用的支架材料主要有自体骨、同种异体骨、骨水泥、陶瓷及一些相关的衍生材料等，这些支架材料均存在不同程度的缺点。组织工程骨的兴起为骨缺损修复支架材料指明了新的发展方向，组织工程自1987年正式提出和确定后，在种子细胞、支架材料和组织构建技术等方面取得了明显进展，但是仍有一些问题尚待解决和完善，组织工程骨的血管化就是其中之一。
骨缺损植入修复材料后血管新生需要一个较长的过程，早期的氧和营养仅能靠受体的组织液渗透供应，并且植入的材料由于机体的凝血反应会被凝血块包裹，从而使渗透更加困难，因此其大小严重受到局限，这显然远远不能满足临床应用的要求。促进组织工程骨血管化的方法常见的有：用胶原、促血管生成的生长因子等修饰支架材料；或将成骨种子细胞与血管内皮细胞、成血管前体细胞共培养等，但是效果欠佳。考虑到移植的组织工程骨主要靠外周血管芽生的方式血管化，一些研究小组利用外周血管芽生的策略，提出了组织工程骨支架材料的预血管化技术：将材料和细胞复合物包埋于皮下、大网膜、筋膜或肌袋中待组织血管化，然后将血管化的组织移植到受区，或者将构建的组织工程骨内植入动静脉也能明显促进材料细胞复合物的血管化及成骨能力，但过程比较复杂，并且即便采用了这些方法，组织工程骨植入受区后，尤其是早期的血液供应仍然靠渗透进行，没有完全解决血管化的难题。
本课题组前期所研发的异种脱细胞骨基质(Acellular bone matrix，ACBM)支架材料较好地保留了细胞外骨基质的主要成分和完整结构，并且采用层层自组装技术，将肝素、壳聚糖复合在其表面，实验证明缓释、抗凝效果可靠；然后在此基础上联合封闭负压吸引技术(Vacuum Assisted closure,VAC)，使血液在早期即可在支架材料内部实现持续“灌注”，为构建的组织工程骨即时、持续提供氧和营养物质。以兔和猪骨缺损为模型的修复研究结果证实，HC/ACBM (Heparin-Chotison coated ACBM)材料结合封闭负压技术使骨缺损修复支架材料植入后即刻可以获得血液灌注，与非负压侧比较，血容量、血流量及新生血管数等均显著增多，并且能促进新骨形成。
但是，兔和猪和人差别较大，对于这种异种来源的支架材料应用于人还有待进一步研究。而恒河猴的解剖结构、生理学等方面与人高度相似，是一种较为理想的实验动物，为进一步验证其安全性和有效性，本实验首先按照ISO10993的标准检测了这种新材料的安全性和组织相容性的系列指标，并且在体外研究了模拟负压吸引灌注条件下培养对种子细胞与支架材料复合的影响，建立了恒河猴胫骨缺损的动物模型，初步探讨了HC/ACBM支架材料联合负压吸引对骨缺损修复过程中血流灌注和成骨的影响，为下一步的临床研究提供理论和实验依据。
材料与方法
1. HC/ACBM的生物安全性试验
将本课题组研制的HC/ACBM材料按照ISO10993的标准，制作了20%的HC/ACBM的浸提液进行了急性、亚急性毒理试验、细胞毒性试验、皮肤刺激性试验，以及HC/ACBM的肌肉埋植试验。
对骨髓来源间充质干细胞(mensenchymal stem cell，MSC)进行分离、培养，然后将HC/ACBM与细胞进行共培养，对表面改良后的材料与MSC的粘附性进行了研究。
2.负压吸引灌注培养方式对细胞与材料复合的影响
制作了体外模拟负压吸引进行细胞与材料共培养的灌注装置，以自制的这种装置将MSC和HC/ACBM共培养，对比研究了负压吸引灌注培养与静态培养对MSC的粘附、分布、生存、增殖等方面的影响。
3. HC/ACBM结合负压吸引修复恒河猴胫骨缺损
建立恒河猴胫骨缺损的模型，以双侧自体作为对照，分别观察了ACBM支架材料抗凝与不抗凝的比较，以及HC/ACBM联合负压吸引与不联合负压吸引的比较。利用CT及CT灌注观察了支架材料在修复后不同时间点的血流灌注和成骨情况。
主要结果和结论：
1.扫描电镜显示的超微结构表明，表面改良后的HC/ACBM仍保留了天然骨的多孔三维结构，并且HC/ACBM与MSC仍能紧密的粘附，证实HC/ACBM具有很好的粘附性。
2.各项安全性检测均证实HC/ACBM无明显的毒性，具有良好的安全性和组织相容性。
3.负压吸引灌注培养组与静态培养组的比较研究表明，负压吸引灌注培养条件下，细胞在材料表面和中心分布的更加均匀，TUNEL法检测没有发现粘附在材料上的细胞有明显的凋亡发生，Brdu法检测结果表明细胞与材料共培养时细胞可以正常进行增殖。
4. CT灌注成像显示，HC/ACBM时间密度曲线呈血液灌注的表现，而ACBM材料呈血液渗透曲线。血容量、血流量图象显示体内植入HC/ACBM这种抗凝材料修复骨缺损，不仅材料周边部分有血液灌注，材料中心出现血液的时间也明显早于非抗凝材料；
5.利用填充床式生物反应器的机理，在HC/ACBM抗凝支架材料植入后采用周期性低负压吸引，实验结果证实，构建的动物模型在HC/ACBM植入后早期就可以获得血液灌注，尤其是在材料的中心部分，与非负压侧比较，血容量、血流量等均显著增多，其新骨形成的质和量也显著改善。
Background
Clinically, the treatment of bone defects for various reasons, especially large bonedefect, is always the orthopedic surgeon’s problems. And the number of people required toundergo bone graft repair is rising because of car accidents, cancer and so on. The repairmaterials of basically has autologous bone, allograft bone, non-degradable bone cement,ceramics, related derivative materials and so on, and these materials are unsatisfied. Bonetissue engineering is the new technology for regeneration and repair, which is the new trendof development of bone defect repair. Since the1987officially proposed and determined,bone tissue engineering have achieved many progress in seed cells, scaffolds and thetechnology of structure. But, some problems need to be resolved. Revascularization is oneof the most important problems of them. As everybody knows, the angiogenesis requiresseveral days after bone defects implanted. The early nutrition supply of the cells can onlyrely on the body tissue fluid penetration. So, the size of scaffold material is severely limited.Only as skin and cartilage that can rely on diffusion to maintain a low metabolic untilcompletion of revascularization, which is obviously far from being able to meet therequirements of clinical applications. Meanwhile, the implant material will start thecoagulation reaction, thus make the blood penetration more difficult.
The common approachs to promote vascularization of bone tissue engineering,including collagen or vascular growth factors-modified scaffold, or oseogenesis seed cellsculture with the vascular endothelial cells or vessel precursor cells etc, but ineffective.Taking into account the way of vascularization of bone tissue engineering mainly rely onperipheral vascular buds born after transplantation, studies have used peripheral vascularshoot raw strategy, and puted forward the bone tissue engineering the pre-vascularizedtechnology of scaffolds: scaffolds and cells composite embeded in subcutaneous, omental,fascia or muscle bags, and then the pre-vascularization of tissue will transplant to therecipient or tissue engineered bone implant arteriovenous also can obviously promote the vascularization and osteogenic of the compound bone scaffolds. But the process is morecomplex, and even with these methods, tissue engineering bone after implantation torecipient, the blood supply, especially early blood supply remain dependent on thepenetration, not completely solve the problem of the early revascularization.
The xenogeneic acellular hone matrix (ACBM) scaffold which developed by ourresearch group retains the major components and complete structure of the extracellularmatrix of bone. With layer-by-layer of self-packaging technology, heparin (HEP) andchitosan (HC) compound were coated on ACBM’s surface. The experiment showed thatslow-release and reliable anticoagulation effects were satisfied. And then combined vacuumassisted closure (VAC), allowed blood to continuous infusion at an early stage, provided thescaffolds of tissue engineered bone real-time, continuous oxygen and nutrients. Repairdefects in rabbit and pig results confirmed that the HC/ACBM (Heparin-Chotison coatedACBM) scaffold with the vacuum assisted closure make repair materials can be obtainedimmediately blood infusion after implantation. And compared with the non-negativepressure side, the blood volume, blood flow and neovascularization number weresignificantly increased and the quality and quantity of new bone formation alsosignificantly improved.
In order to further promote application of HC/ACBM scaffold materials, this study inaccordance with ISO10993standard to detect related safety series indicators of the newmaterial. In vitro, we studied the influence of simulation of the negative pressure drainageto seed cells compound scaffold materials. And we selected the rhesus monkeys which wasvery close to human as experimental animals, established the animal models of rhesus tibialdefect. Then we study how the HC/ACBM scaffold materials with VAC impact the bloodperfusion and bone formation during the process of bone defect repair, provided to thetheory and experiment basis for the next step of the clinical research.
Materials and methods
1. The HC/ACBM biological safety test
According to the standard of ISO10993, we study on the biological safety ofHC/ACBM materials. We prepared20%liquid extracts of HC/ACBM for acute or subacutesystemic toxicological tests, in vitro cytotoxicity tests, skin irritation tests and local effects after implantation tests.
The research has isolated and cultured bone marrow derived mesenchymal stem cells.And then we studied the adhesion to MSC to the material modified surface by HC/ACBMand the stem cells co-cultured.
2. The influence of the negative pressure drainage infusion to the cells compositematerials
The research has developed a cell culture device with perfusion simulation of suctiondrainage in vitro. With this homemade device, the MSCs and HC/ACBM scaffold materialco-cultured. Compared to the static culture, we investigate the impact of the negativepressure drainage infusion how to impact the adhesion to HC/ACBM, distribution, survival,the proliferation of MSCs.
3. HC/ACBM combined with vacuum assisted closure repair rhesus monkeys tibialdefect
Established rhesus tibial defect model, bilateral self control, we compared the ACBMwith or without anticoagulation, and HC/ACBM combination of vacuum assisted closure ordo not use. Using CT and CT perfusion studied the blood perfusion and osteogenesis atdifferent time points after repair rhesus monkey tibia defect with the HC/ACBM.
The main results and conclusions:
1. The scanning electron microscopy (SEM) shows that the ultrastructure ofHC/ACBM retained the natural bone nets holes and porosity. Results of MSC andHC/ACBM co-culture suggested that MSC can close adhesion to the ACBM with surfacemodified, and scaffold materials have the characteristic of well adhesion.
2. In accordance with the methods and procedures developed by the ISO10993standard, acute or subacute systemic toxicological tests, in vitro cytotoxicity tests, skinirritation tests and local effects after implantation tests both confirmed the HC/ACBM nosignificant toxicity, has the good biological safety and compatibility.
3. The cells culture in device with perfusion simulation of suction drainage shows thatthe cells adhere to surface and center of the material, and with a more uniform distribution.TUNEL assay found the cells adhesion to the HC/ACBM’s were no obvious apoptosis.Brdu assay results also showed that cell proliferation of cells adhesion to the HC/ACBM’s could normal proliferate. In contrast, the static cultured cells were mainly distributed in theouter surface of the HC/ACBM, while the central parts were very few.
4. CT perfusion imaging shows that time density curve of HC/ACBM group is bloodperfusion performance, and ACBM group is blood penetration curve. Blood volume, bloodflow in the images show the implant HC/ACBM with anticoagulation to repair bone defect,scaffold materials have a blood perfusion not only surrounding peripheral parts, materialscenter appearance of blood in the early time also obvious in this anticoagulant scaffoldmaterials;
5. In this study, using the mechanism of the packed bed bioreactor, and imposedperiodic low negative pressure in HC/ACBM with anticoagulation scaffold afterimplantation. The experimental results demonstrate that, animals after implantation inHC/ACBM with negative pressure can get blood perfusion earlier, especially in the centralparts of scaffold materials. Compared with the non-negative pressure side, the negativepressure side had more blood volume, blood flow significantly increased, and the qualityand quantity of new bone formation also significantly improved.
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