摘要
寻找理想的生物填充材料来治疗和修复大段骨缺损，一直是骨科临床医生和材料学者共同努力的目标。理想的生物填充材料应该具备以下特点：1具有良好的生物相容性，该材料在体内没有细胞毒性和明显的炎性反应。2可吸收、可降解，且材料的降解速度与骨再生的速度相适应。3有良好的机械强度。4常温下易加工。5有合适的孔径和孔隙率，使得细胞能够长入等。自固化磷酸钙骨水泥（CPC）由于其良好的生物相容性、良好的骨传导和骨诱导作用而被材料学者所关注，然而，CPC因其脆性较强，无法应用于受力部位的骨缺损的修复和治疗。因此，改良和提高CPC的生物力学性状同时又保持其良好的促进骨再生的能力，也成为广大学者研究的热点。在CPC中引入不同的物质在某种程度上解决了CPC力学性状差的现象，生物蛋白胶（FS）是临床上应用广泛的一种生物粘合剂，对创伤止血、组织粘连以及促进组织再生方面有良好的作用。生物蛋白胶的引入是否能改良CPC的脆性，同时和周围骨组织又具有良好的生物相容性，且能刺激周围骨组织再生，仍需要进一步的研究探索。本研究旨在探寻复合材料CPC/FG的成骨效应以及寻求两者的最佳配比浓度。
1、不同配比浓度的复合材料CPC/FG材料性状的研究以及对成骨细胞生物学行为的影响
本实验拟通过构建不同配比的纳微米复合材料CPC/FG，探索不同配比复合材料的材料学特性，继而通过细胞学等技术和方法，研究CPC/FG诱导成骨的细胞学依据。
本实验拟通过对配比浓度为1:1、3:1、5:1（ml/g）的CPC/FG进行初凝和终凝时间的测定、电镜扫描以及体外生物力学测试，单纯的CPC作为实验对照组，来判断不同配比浓度的CPC/FG在材料学性能上的差异。以及通过研究成骨细胞在不同配比浓度的材料表面的黏附、增殖以及功能表达，评价不同材料在骨与材料界面的体外模型中促进成骨能力上的差异。初凝和终凝时间的测定结果（min）：1:1、3:1、5:1组的初凝时间分别为20.41±1.21、15.37±1.1、8.33±0.62；终凝时间分别为24.33±1.36、17.26±1.04、9.63±0.79，均能满足临床需要。从电镜扫描结果可以发现，CPC/FG的表面较单纯的CPC组致密而光滑，孔径也随着FG浓度的增加而降低，单纯的CPC表面颗粒直径悬殊较大，且有较大的孔洞存在。体外生物力学测试发现，任意配比浓度的CPC/FG，其压缩强度和弹性模量均较单纯的CPC组有显著的提高(p<0.05)，有效的提高和改善了CPC的机械性能。在体外细胞学实验中，成骨细胞贴附1h、2h后，四组细胞贴附数量无统计学差异，但细胞均完全展开且细胞骨架清晰可见，且均较完全空白对照组高(p<0.05)。细胞增殖1d、3d、7d后，四组材料增殖能力以及ALP（碱性磷酸酶活性）无明显差异，但均较完全空白对照组高(p<0.05)。由此可见，CPC中引入FG，提高了CPC的材料学性能，同时在体外细胞培养中也表现出良好的相容性。
2、不同配比浓度的复合材料CPC/FG在兔子体内促进骨组织再生的对比研究
为了研究CPC/FG在体内的成骨效应以及在体内促进骨组织再生的最佳配比浓度，该实验将配比浓度为1:1、3:1、5:1（ml/g）的CPC/FG模块分别植入新西兰大白兔的股骨髁部缺损中，单纯的CPC作为实验对照组，在术后4周、8周和12周进行micro-CT扫描、体内生物力学检测和组织学染色来判断刺激骨组织再生的最佳配比浓度。Micro-CT扫描结果显示，CPC/FG配比浓度为1:1时在体内代谢速度最快，成骨效果最佳。RMVF在配比浓度为1:1时，其数值为四组中最低(p<0.05)，术后4周为87.01±1.96%，8周为76.90±2.34%，12周为63.21±2.89%。BV/TV在术后4周、8周以及12周分别达到1.45±0.42%,7.35±1.45%,和29.10±1.67%，均为四组中最高(p<0.05)。体内生物力学测试中，任意配比浓度的CPC/FG，其压缩强度和弹性模量均较单纯的CPC组有显著的提高(p<0.05)，且CPC/FG配比浓度为1:1时，其机械性能最接近体内松质骨。组织学V-G染色在形态学上更直接的反应出CPC/FG配比浓度为1:1时在体内新生骨的成骨量最佳。术后12周，1:1组可见材料周边和中心部位被大量的骨组织所替代，且为成熟的骨小梁结构。由此可见，CPC/FG在配比浓度为1:1（ml/g）时，在体内促进骨组织再生方面，无论是机械性能还是生物学性能效果最佳。
3、模块型CPC/FG和注射型CPC/FG在兔子体内促进骨组织再生能力的对比研究
生物材料的制备方法不同，往往会致其有不同的超微结构，从而影响其机械性能和生物学性能。本实验拟通过对配比浓度为1:1（ml/g）的模块型CPC/FG和注射型CPC/FG在新西兰大白兔的股骨髁部缺损模型中刺激骨再生能力的对比，来寻找较优的材料制备方法。该实验同样用micro-CT扫描、体内生物力学检测和组织学染色的方法在术后6周和12周取材检测。Micro-CT扫描结果显示，注射型CPC/FG组体内降解速度较快，成骨能力较强。改组RMVF在术后6周和12周都低于模块型CPC/FG(p<0.05)，分别为70.63±2.38%和58.39±4.09%。改组BV/TV则较高(p<0.05)，分别为10.56±0.83%和39.73±4.47%。体内生物力学结果表明注射型CPC/FG的压缩强度和弹性模量在术后都较稳定，于周围松质骨形成较平衡的力学组合。该实验的组织学V-G染色支持micro-CT的数据，可见注射型CPC/FG在术后12周，材料被大量的网状骨小梁结构所替代，材料大量降解，残存的少量材料存在于骨组织网状空隙中间，材料和骨的生物相容性较佳。由此可见，相比模块型CPC/FG，注射型CPC/FG不仅实现了微创条件下的操作，同时在体内还表现出较优良的机械性能和生物学性能，有巨大的应用潜能。
本研究通过以上三部分的实验支持CPC/FG在促进成骨方面效果显著，克服了单纯CPC生物力学性能差的缺点，同时又保留了其良好的骨传导和骨诱导作用，且在注射型CPC/FG在配比浓度为1:1（ml/g）时，刺激成骨效果最佳。
The material researchers and the doctors in clinic focused on the research to find theideal biomaterial for the bone defects. The ideal material should include:(1) the goodbiocompatibility;(2) degradable and absorbable;(3) the good biomechanical properties;(4)preparation on the room temperature;(5) optimal pore size. Nowadays, CPC is the goodosteoinductive and osteoconductive material known by material scientists and is widelyused in bone tissue engineering. However, poor mechanical property of CPC has limitedits application into the non-load bearing condition. In order to improve the mechanicalproperties of CPC, other injectable materials such as chitosan, gelatin and collagen havebeen introduced into this material. Fibrin Glue (FG) is a kind of elastomeric,biocompatible material with sufficient adhesive strength, which has been approved by theChinese State Food and Drug Administration. It is unclear that the CPC/FG compositewhether could stimulate the bone regeneration. And it is also unclear that whether different ratio of CPC and FG has different properties for the bone reconstruction.
1、The mechanical and biological properties of an injectable calcium phosphatecement-fibrin glue composite for bone regeneration in vitro
Calcium phosphate cement (CPC) that can be injected to form a scaffold in situ haspromise for the repair of bone defects. However, its low-strength limits the CPC to nonstress-bearing repairs. Fibrin glue (FG) with good sticking property and biocompatibilityis possible used to reinforce the CPC. The objective of this study was to investigate theeffects of FG on the mechanical and biological properties of CPC in an injectable CPC/FGcomposite. The initial setting time of this CPC/FG was delayed compared with the CPCcontrol at different powder/liquid (P/L) mass ratio (p>0.05). At a P/L of5, the strengthwas (38.4±4.32) MPa for the CPC/FG, much higher than (27.42±2.85) MPa for the CPCalone (p <0.05). Osteoblast cell adhesion and proliferation were examined by MTTmethod and alkaline phosphatase (ALP) was assessed using test kits. The data allsupported that CPC/FG at any ratio could stimulate the activity of the osteoblast cell. Theresults demonstrate that this stronger CPC/FG scaffold may be useful for osteoblastcelll-based bone regeneration in moderate load-bearing orthopedic applications.
2、The Mechanical and Biological Studies of Calcium Phosphate Cement-Fibrin Gluefor Bone Reconstruction of Rabbit femoral defects
In order to improve the mechanical and biological properties of calcium phosphatecement (CPC, nanometer-biomaterial) for bone reconstruction in the rabbit femoral defectmodel, fibrin glue (FG, the natural product, purified from the blood) was introduced atthree different ratios. The CPC powder and the FG solution were mixed respectively at thepower/liquid (P/L) ratios (g/ml) of1:1,3:1and5:1(g/ml), and pure CPC was used ascontrol. After being implanted into the femoral defect in rabbit, the healing process wasevaluated by micro-CT scan, biomechanical testing and histological examination. Bymicro-CT analysis the P/L ratio of1:1(g/ml) group indicated largest quantity of new boneformation at4w,8w, and12w after implantation respectively. BV/TV of1:1group was highest in the four groups, which was1.45±0.42%,7.35±1.45%, and29.10±1.67%at4w,8w, and12w after the operation respectively. In the biomechanical tests, thecompressive strength and the elastic modulus of three CPC/FG groups were much higherthan those of the pure CPC group at the determined time point (p<0.05). The histologicalevaluation also showed the best osseointegration in the1:1group at4w,8w, and12w afterthe operation respectively. In1:1group, the bone grew into the pore of cement in thelaminar arrangement and connected with the cement tightly at the12thweek after theoperation. This present study indicated that CPC/FG composite at the P/L ratio of1:1(g/ml)stimulated bone regeneration better than any other designed group, which suggested thatCPC/FG at the P/L ratio of1:1has significant potential as the bioactive material for thetreatment of bone defects.
3、The Comparison between the remolded CPC/FG and the injectable CPC/FG forthe Reconstruction of femoral defects in a rabbit model
The aim of this study was to compare the remolded CPC/FG composite with theinjectable CPC/FG composite at the P/L ratio of1:1(g/ml) for new bone formation.Calcium phosphate cement (CPC) is a family of injectable biomaterial made of calciumphosphate, and FG is the natural product purified from human blood. In this study, theremolded CPC/FG composite at the P/L ratio of1:1(g/ml) was defined as Group A, andthe injectable one was defined as Group B. After being implanted into the cavity defect offemur in rabbits, the healing process was evaluated by micro-CT scan, biomechanicaltesting and histological examination at6w and12w after the operation. In micro-CTanalysis, Group B indicated larger quantity of new bone formation at6week and12weekafter the operation. The compressive strength of Group B was lower than that of Group Aat12week (p<0.05), and the elastic modules of Group B was more stable after theoperation compared with Group A. The histological analysis showed the similar imagingas micro-CT analysis. This present study indicated that the injectable CPC/FG compositeat the P/L ratio of1:1(g/ml) stimulated bone regeneration better than the remolded one.
The study showed that the CPC/FG could stimulate the bone regeneration, and the injectabla CPC/FG at the P/L ratio of1:1(ml/g) has the best activity for new boneformation.
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