新型多孔自固化人工骨治疗骨质疏松骨缺损的实验研究
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摘要
一、研究背景
人口老龄化是目前困扰全球的问题,特别在我国,随着人口基数的增加和老龄化速度的加快,骨质疏松患者已达到9000余万,临床医生将面对大量骨质疏松伴骨缺损的病例。在骨质疏松环境下,破骨细胞活性增强,骨吸收大于骨形成,新骨生成能力下降,骨缺损愈合明显延迟,故治疗较单纯骨缺损更为棘手。传统的自体及异体骨移植仍是首选,但各自均存在难以克服的缺点,载体+因子的生物治疗模式取得了较好疗效,但载体材料有待进一步改进。虽然CPC携载骨生长因子治疗单纯骨缺损成果显著,但由于CPC的因子释放能力有限,而且骨质疏松骨缺损的愈合需要较高浓度的因子水平,故其在骨质疏松环璄下的应用受限。可降解微球携载因子后复合CPC,不仅使因子释放量大大增加,而且微球可降解成孔,明显加速材料的降解(解决CPC降解缓慢的临床应用障碍)。目前,主要以PLGA为材料制备微球,PLGA作为无机高分子物质,存在以下缺点:1、缓慢的降解速率阻碍新生骨组织的长入;2、降解产物呈酸性,累积后可损伤周围组织及细胞。明胶微球生物相容性好,常用作药物载体,微球经交联后降解可调,但常用的交联剂如GA细胞毒性较强,限制了明胶微球的应用,新型生物交联剂GP的出现有望解决这一问题,使明胶微球代替PLGA作为CPC的良好改性材料。
二、研究目的
1.以GP作为交联剂制备明胶微球,与GA交联的微球进行比较,探讨GP交联微球的性能优缺点,为多孔CPC的制备提供致孔材料。
2.通过对孔径率、生物力学强度及X线衍射等指标进行分析,确定复合型多孔CPC中明胶与骨水泥的最佳配比;并进行体外细胞实验及体内动物实验,综合评估多孔CPC的生物相容性。
3.明胶微球携载rhBMP-2后复合CPC制备新型多孔人工骨,测定其体外因子缓释曲线,通过细胞实验及体内异位成骨实验评估其成骨能力。
4.在绝经后骨质疏松模型体内,采用“二维组织形态-三维CT成像-生物力学特性”的综合手段,评估新型人工骨对促进骨质疏松骨缺损愈合、改善骨小梁微观结构及提高椎体生物力学强度的作用。
三、研究内容和方法
1.以改进的双相乳化冷凝聚合法制备明胶微球,分别以GP及GA进行交联。取60%交联度的GP及GA明胶微球,分散于PBS中,比较其粒径外观、溶胀及降解性能;两种微球分别携载rhBMP-2,测定载药量及包封率,观察10天内的药物缓释性能;收集GP及GA明胶微球浸提液,倍比稀释成100%、50%及25%的浓度,分别与小鼠成骨细胞共培养2天,以DMEM组为空白对照,MTT法检测细胞增殖,确定GP及GA微球的细胞毒级。
2.GP交联明胶微球以不同比例与CPC复合(0 wt%,2.5 wt%,5 wt%),制备多孔CPC,浸泡于PBS中1、3及5周后,测定材料孔径率、抗压强度及XRD情况,筛选出最佳比例。消化法培养成骨细胞接种于常规及多孔CPC支架上,SEM观察细胞形态;不同材料浸提液(0 wt %,2.5 wt% GM/CPC及聚苯乙烯)分别与成骨细胞共培养,钙黄绿素-AM及碘化苯啶标记后观察细胞生长状况,MTT法测定细胞增殖率,试剂盒测定ALP水平。CPC及GM/CPC分别植入山羊椎体骨缺损处,术后6周处死山羊,收集椎体标本,部分椎体制作不脱钙切片,丽春红三色染色后观察骨组织生长及材料降解情况,其余标体脱钙后制作石蜡切片,HE染色后观察组织炎症反应、骨组织生长及材料降解情况。
3.载rhBMP-2的明胶微球与CPC混合制备载因子多孔人工骨作为实验组(A),rhBMP-2/CPC作为对照组(B)。两者分别浸泡于5ml生理盐水,在1、4、7、14、21、28天时收集全部浸提液,ELISA法测定rhBMP-2的浓度,计算出各个时间点的累积释药量;两种材料浸提液分别与成骨细胞共培养,测定细胞增殖、ALP水平及钙化结节情况;材料植入小鼠大腿肌袋,术后3周处死小鼠,切取材料及周围组织,HE染色后进行组织学观察,同时测定材料周围组织中ALP及钙含量。
4.以卵巢切除法对15只成年山羊去势,低钙膳食6个月后建立骨质疏松模型。手术去除每只动物L2、L4椎体部分侧方骨质,造成约5 mm×10 mm×10 mm大小骨缺损,随机选取3只山羊不作任何处理,作为空白对照组观察骨缺损自愈情况。其余动物随机分成两组:实验组(A)植入rhBMP-2/GM/CPC;对照组(B)植入rhBMP-2/CPC。术后1周、7周及25周CT扫描后三维成像观察骨缺损愈合情况;术后7周及25周分两批处死动物,采用双能X线吸收骨密度仪测定手术椎体骨密度,组织病理学观察骨小梁微观结构及材料降解成骨情况,生物力学实验评估椎体骨质的结构力学强度。
四、研究结果
1. GP交联明胶微球需3天达到稳定的交联度,60%交联度的GP及GA微球在溶液中均呈规则圆形,粒径分别为78±18μm及74±10μm,载药量、包封率未见明显差异,但GP微球分散性更佳,溶胀率(89.0±4.8%)及10天内释药总量(78.8±4.96%)明显低于GA微球(118.0±7.6%、90.5±5.12%),抗降解能力(28天)强于GA微球(21天),100%、50%及25%浓度的GP浸提液细胞毒级均为Ⅰ级,而相应GA微球的细胞毒级分别为Ⅲ、Ⅲ及Ⅱ级。
2.GM/CPC的总孔径率、大孔率均随浸泡时间的延长而增加,但抗压强度逐渐下降,其中2.5 wt% GM/CPC最终具有相对较高的孔径率(65.5±1.12%)及较好的生物力学强度(6.3±0.6Mpa)。成骨细胞在多孔CPC支架上生长良好,细胞增殖及ALP水平均明显高于单纯CPC组,与聚苯乙烯组未见明显差异。两组材料植入体内3个月后,均未见不良炎症反应,多孔CPC可见材料边缘明胶微球降解,新生骨组织长入,有多核细胞出现降解材料,而单纯CPC无明显降解,材料内部无骨组织长入。
3.两种CPC的因子缓释曲线均由两部分组成:1天内的爆发式突释及随后的缓慢平稳释放;除第1天外,其余时间点多孔CPC的因子累积释放量均高于单纯CPC;28天后多孔CPC的累积释药量约为37.8±2.3%,明显高于单纯CPC的14.7±1.9%。A组成骨细胞增殖及ALP水平明显高于B组,且细胞形成的钙结节数量多形状大。体内实验发现两组均可见软骨形成,但A组软骨量更多,材料周围组织ALP及钙含量测定A组也明显高于B组。
4.三维CT成像显示:空白对照组术后25周在椎体上仍可见较大骨缺损存在;A组骨缺损修复显著快于B组,且材料降解较快,剩余材料较B组少。组织病理学结果表明:术后7周A组的平均骨矿化率高于B组,但术后25周未见明显差异;A组的骨小梁三维结构(平均骨小梁厚度及密度、结点末端比)明显优于B组,而平均骨小梁间距低于B组;A组材料降解较快,25周后剩余材料约为50%,且可见大量新生骨长入,B组几乎未见明显降解,无新生骨长入。骨密度检测显示术后6周两组未见明显差异,但25周后A组明显高于B组。生物力学试验结果表明,术后7周及25周A组分别为4.2±1.2 MPa及7.8±1.4MPa,明显高于B组的2.4±0.9 MPa及4.6±0.8MPa。
五.结论
1.GP交联明胶微球所需时间稍长,60%交联度的明胶微球降解时间约28天左右,适合作为CPC的成孔材料,而且其缓释性能及生物相容性更佳,可广泛应用于骨组织工程领域。
2.利用明胶微球可成功制备多孔CPC,微球在2.5%比重时,材料孔径率高,强度较以前同等孔径率的多孔材料有一定改善。复合材料具有极好的生物相容性,微球在体内可缓慢降解成孔,使新生骨组织长入,从而更加速复合材料的降解,是良好的骨组织工程支架材料。
3.多孔CPC复合rhBMP-2与单纯rhBMP-2/CPC相比,因子的释放能力及骨诱导性能均有所提高。这种新型人工骨可加速骨质疏松骨缺损的愈合,显著改善局部骨微结构,增强局部骨生物力学性能,适合作为骨质疏松骨缺损生物治疗的理想材料。
Background
Osteoporosis is now becoming much more prevelent than ever, especially in our country, and the number of the people who suffers osteoporosis is exceeding 90 million, therefore, doctors are facing more patients with osteoporotic bone defects. Under circumstance of osteoporosis, osteoclasts are more active, and the ability of bone resorption is stronger than that of bone formation. So healing of this kind of bone defect is impaired, and cure of the bone defect is more diffucult. A bone-grafting procedure is a successful care to treat the bone defects, but there are many limitations with the materials used in the operation. Although improved effect on bone defects has been obtained by using carriers with growth factors, properties of the carriers still need to be upgraded. Normal bone defects can heal fast with rhBMP-2/CPC, but limit factor-release ability of CPC is not enough for osteoporotic bone defects because high concentrations of the growth factors are needed for osteoporotic bone defects. When degradable microspheres loaded with growth factors are mixed wih CPC to construct a macroporous composite, not only the release ability of the composite can be enhanced, but also the degradation rate of it is speeded up. But PLGA that is the most used material for microspheres has some limitations: 1. Slow degradatrion rate hampers new bone ingrowth. 2. Degradation products are harmful to cells and tissues around the materials. GMs, which are widely used as drug carriers, are biocompatible and controlled-degradable after crosslinking, but cytotoxic of the crosslinkers (such as GA) used for parepairing GMs is still an obstacle. The problem will be solved by a new bio-crosslinker—genipin, therefore, GMs may replace PLGA microspheres as a good additive to improve the properties of CPC.
Objective
1. To prepare GMs by using GP as a crosslinker and to evaluate the characteristics of the GMs for construction of macroporous CPC.
2. To investigate the best proportion of GMs and CPC powers in the composite macroporous CPC by analyzing porosity, biomechabical property and the result of XRD. To evaluate the biocompatibility of the composite in vitro and in vivo.
3. To study the controlled-release characteristic of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC. To evaluate the bone-induction ability of the both composite in vitro and in vivo.
4. To evaluate the effects of the new artificial bone on bone mineral density, biomechanical property, micro-architecture of osteoporotic goat vertebral and healing of ostoporotic bone defect in vivo.
Materials and methods
1. GMs, prepared by the improved emulsified cold-condensation method, were crosslinked by GP and GA, respectively. After being dispersed in PBS, two kinds of microspheres with the 60% degree of crosslinking were compared in terms of morphology, swelling and degrading properties;BMP-2 were loaded into the GP and GA microspheres, and encapsulation rate、drug loading and releasing capacity were measured; 100%、50% and 25% leaching liquid of GP and GA microspheres were cultured with rat osteoblast(DMEM group as the control), respectively, and cell proliferation were measured by MTT method to grade the cell cytotoxicity.
2. Macroporous CPC was developed using genipin-crosslinked GMs with three weight ratios (0 wt%, 2.5 wt% and 5 wt%). After the composites were soaked in PBS for 1, 3 and 5 weeks, the proper weight ratio of GM was conformed by analyzing porosity, compressive strength and XRD. The morphologies of osteoblasts were examined with SEM after the cells were seeded on the CPC and GM/CPC for two days. After the cells were cultured with different leaching liquor of three kind of materials (0 wt%, 2.5 wt% GM/CPC and polystyrene) for days, cell viability and ALP concentration were measured. And 6 weeks after implantation into the bone defect of goats, GM/CPC and CPC were obtained and histopathological method was applied to investigate the bone ingrowth and the material degradation.
3. The leaching liquor of rhBMP-2/CM/CPC or rhBMP-2/CPC was collected after both composites had been soaked in the Sodium Chloride for 1、4、7、14、21 and 28 days, and the concentration of rhBMP-2 was measured by ELISA method. Osteoblasts were cultured with both leaching liquor, and cell proliferation, ALP concentration and calcium nodule were investigated. Three weeks after implation in the muscle bags of the rats, the composites with the muscles around were collected and stained with hematoxylin and eosin; Ca and ALP concentrations of the muscles were also measured.
4. Fifteen mature goats underwent ovariectomy and were placed on low cation relative to anion diet. After six months, bone defect of 5mm×10mm×10mm were made in L2、L4 of the goats, and three animals were not treated as blank control group. Others were randomly assigned to two groups and treated with rhBMP-2/GM/CPC (Group A) or rhBMP-2/CPC (Group B). After 1, 7 and 25 weeks, the defects were detected by CT scanning. Seven and twenty five weeks later, Animals were killed in batch, and BMD, compressive strength and micro-architecture of the vertebra were studied by dual energy X-ray absorption meter, biomechanical test and bone histomorphometry, respectively.
Results
1. GP and GA microspheres were both spherical with particle diameter of 78±18μm and 74±10μm, and there were no difference between both microspheres in drug loading and encapsulation rate; At the same crossling degree of 60%, GP microspheres, with longer degradation period (28 days) compared to GA microspheres (21 days), had better dispersibility, and swelling rate(89.0±4.8%)、percentage of cumulative drug releasing in 10 days(78.8±4.96%)were both lower than GA microsphere(118.0±7.6%、90.5±5.12%); Cell cytotoxicity of 100%、50% and 25% leaching liquid of GP microspheres were all at theⅠlevel, but leaching liquid of GA microspheres with corresponding concentration were at the level ofⅢ、Ⅲ、Ⅱ.
2. Porosity and macroporosity of the GM/CPC increased with the GMs increasing, but compression strength decreased after 1, 3 and 5 weeks of soaking, and the 2.5 wt% GM/CPC was the most favourable composite with high porosity and relative strong compressive strength. Osteoblasts showed normal morphology with both composites, but proliferation and differentiation of the cells were enhanced with the GM/CPC compared to the CPC. Both composites showed no adverse inflammatory reaction after weeks of implatation. New bones grew into the pores of the GM/CPC which resulted from GMs degradation, and coenocytes were present into the composite to degradate the material, but no new born could been seen inside of CPC.
3. The release characteristics of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC both comprised two phases: (a) an initial burst occurring during the first 24 h, (b) a linear slow steady release phase linear release phase for the rest of the time course (days 2–28). Except for the similar initial burst effect, rhBMP-2 release rate of rhBMP-2/GM/CPC was faster than that of rhBMP-2/CPC in all the other evaluated time periods. In 28 days, 37.8±2.3% of the loaded protein in GMs was released, while in the control group the release rate was 14.7±1.9%. Cell experiment showed that Cell proliferation and ALP concentration of group A were significantly higher than those of group B, and calcium nodus of group A was bigger. In vivo studies also showed that quantity of cartilage formation, ALP and calcium concentration were higher in the group A.
4. CT reconstrction analysis showd that bone defect healing was accelerated in the group A, and remain-volume of rhBMP-2/GM/CPC was smaller than that of rhBMP-2/CPC; in the untreated control group, a large defect could still be found in the vertebra after 25 weeks, although the bone defect became a little smaller. The results of biomechanical test showed that compressive strength of vertebra in the group A were 4.2±1.2 MPa for 7 weeks and 7.8±1.4Mpa for 25 weeks,which were higher than 2.4±0.9 Mpa and 4.6±0.8Mpa of the group B. Histomorphologically, trabecular thickness, relative trabecular volume and node/terminatio rate in the group A were higher than those in the group B, and trabecular separation was lower; more bone ingrowth was observed deeper into the rhBMP-2/GM/CPC composite, but no tissue formation was present inside the rhBMP-2/CPC composite; and almost half part of rhBMP-2/GM/CPC had degradated, but little resorption of rhBMP-2 /CPC could be detected. Mineralizition rate and bone density showed no difference between two groups after 7 weeks, but after 25 weeks the values were higher in the group A.
Conclusion
1.GMs crosslinked by GP have better biocompatibility and cotrolled-release ability than that crosslinked by GA, and GMs with crosslinking degree of 60% can be used as additive to construct macroporous CPC.
2. Macroporous CPC can be developed using GP-crosslinked GMs, and 2.5 wt% GM/CPC is the most favourable with high macroporosity and strong compressive strength. The composite is a good bone substitute with improved degradability and biocompatibility.
3.Controlled-release of rhBMP-2 and bone induction are both enhanced with rhBMP-2/GM/CPC compared to rhBMP-2/CPC. The new graft can accelerate healing of osteoporotic bone defect and improve bone quality significantly, therefore, it can be used as a promising bone substitute for bone defects of osteoporosis under non-loaded circumstances.
人口老龄化是目前困扰全球的问题,特别在我国,随着人口基数的增加和老龄化速度的加快,骨质疏松患者已达到9000余万,临床医生将面对大量骨质疏松伴骨缺损的病例。在骨质疏松环境下,破骨细胞活性增强,骨吸收大于骨形成,新骨生成能力下降,骨缺损愈合明显延迟,故治疗较单纯骨缺损更为棘手。传统的自体及异体骨移植仍是首选,但各自均存在难以克服的缺点,载体+因子的生物治疗模式取得了较好疗效,但载体材料有待进一步改进。虽然CPC携载骨生长因子治疗单纯骨缺损成果显著,但由于CPC的因子释放能力有限,而且骨质疏松骨缺损的愈合需要较高浓度的因子水平,故其在骨质疏松环璄下的应用受限。可降解微球携载因子后复合CPC,不仅使因子释放量大大增加,而且微球可降解成孔,明显加速材料的降解(解决CPC降解缓慢的临床应用障碍)。目前,主要以PLGA为材料制备微球,PLGA作为无机高分子物质,存在以下缺点:1、缓慢的降解速率阻碍新生骨组织的长入;2、降解产物呈酸性,累积后可损伤周围组织及细胞。明胶微球生物相容性好,常用作药物载体,微球经交联后降解可调,但常用的交联剂如GA细胞毒性较强,限制了明胶微球的应用,新型生物交联剂GP的出现有望解决这一问题,使明胶微球代替PLGA作为CPC的良好改性材料。
二、研究目的
1.以GP作为交联剂制备明胶微球,与GA交联的微球进行比较,探讨GP交联微球的性能优缺点,为多孔CPC的制备提供致孔材料。
2.通过对孔径率、生物力学强度及X线衍射等指标进行分析,确定复合型多孔CPC中明胶与骨水泥的最佳配比;并进行体外细胞实验及体内动物实验,综合评估多孔CPC的生物相容性。
3.明胶微球携载rhBMP-2后复合CPC制备新型多孔人工骨,测定其体外因子缓释曲线,通过细胞实验及体内异位成骨实验评估其成骨能力。
4.在绝经后骨质疏松模型体内,采用“二维组织形态-三维CT成像-生物力学特性”的综合手段,评估新型人工骨对促进骨质疏松骨缺损愈合、改善骨小梁微观结构及提高椎体生物力学强度的作用。
三、研究内容和方法
1.以改进的双相乳化冷凝聚合法制备明胶微球,分别以GP及GA进行交联。取60%交联度的GP及GA明胶微球,分散于PBS中,比较其粒径外观、溶胀及降解性能;两种微球分别携载rhBMP-2,测定载药量及包封率,观察10天内的药物缓释性能;收集GP及GA明胶微球浸提液,倍比稀释成100%、50%及25%的浓度,分别与小鼠成骨细胞共培养2天,以DMEM组为空白对照,MTT法检测细胞增殖,确定GP及GA微球的细胞毒级。
2.GP交联明胶微球以不同比例与CPC复合(0 wt%,2.5 wt%,5 wt%),制备多孔CPC,浸泡于PBS中1、3及5周后,测定材料孔径率、抗压强度及XRD情况,筛选出最佳比例。消化法培养成骨细胞接种于常规及多孔CPC支架上,SEM观察细胞形态;不同材料浸提液(0 wt %,2.5 wt% GM/CPC及聚苯乙烯)分别与成骨细胞共培养,钙黄绿素-AM及碘化苯啶标记后观察细胞生长状况,MTT法测定细胞增殖率,试剂盒测定ALP水平。CPC及GM/CPC分别植入山羊椎体骨缺损处,术后6周处死山羊,收集椎体标本,部分椎体制作不脱钙切片,丽春红三色染色后观察骨组织生长及材料降解情况,其余标体脱钙后制作石蜡切片,HE染色后观察组织炎症反应、骨组织生长及材料降解情况。
3.载rhBMP-2的明胶微球与CPC混合制备载因子多孔人工骨作为实验组(A),rhBMP-2/CPC作为对照组(B)。两者分别浸泡于5ml生理盐水,在1、4、7、14、21、28天时收集全部浸提液,ELISA法测定rhBMP-2的浓度,计算出各个时间点的累积释药量;两种材料浸提液分别与成骨细胞共培养,测定细胞增殖、ALP水平及钙化结节情况;材料植入小鼠大腿肌袋,术后3周处死小鼠,切取材料及周围组织,HE染色后进行组织学观察,同时测定材料周围组织中ALP及钙含量。
4.以卵巢切除法对15只成年山羊去势,低钙膳食6个月后建立骨质疏松模型。手术去除每只动物L2、L4椎体部分侧方骨质,造成约5 mm×10 mm×10 mm大小骨缺损,随机选取3只山羊不作任何处理,作为空白对照组观察骨缺损自愈情况。其余动物随机分成两组:实验组(A)植入rhBMP-2/GM/CPC;对照组(B)植入rhBMP-2/CPC。术后1周、7周及25周CT扫描后三维成像观察骨缺损愈合情况;术后7周及25周分两批处死动物,采用双能X线吸收骨密度仪测定手术椎体骨密度,组织病理学观察骨小梁微观结构及材料降解成骨情况,生物力学实验评估椎体骨质的结构力学强度。
四、研究结果
1. GP交联明胶微球需3天达到稳定的交联度,60%交联度的GP及GA微球在溶液中均呈规则圆形,粒径分别为78±18μm及74±10μm,载药量、包封率未见明显差异,但GP微球分散性更佳,溶胀率(89.0±4.8%)及10天内释药总量(78.8±4.96%)明显低于GA微球(118.0±7.6%、90.5±5.12%),抗降解能力(28天)强于GA微球(21天),100%、50%及25%浓度的GP浸提液细胞毒级均为Ⅰ级,而相应GA微球的细胞毒级分别为Ⅲ、Ⅲ及Ⅱ级。
2.GM/CPC的总孔径率、大孔率均随浸泡时间的延长而增加,但抗压强度逐渐下降,其中2.5 wt% GM/CPC最终具有相对较高的孔径率(65.5±1.12%)及较好的生物力学强度(6.3±0.6Mpa)。成骨细胞在多孔CPC支架上生长良好,细胞增殖及ALP水平均明显高于单纯CPC组,与聚苯乙烯组未见明显差异。两组材料植入体内3个月后,均未见不良炎症反应,多孔CPC可见材料边缘明胶微球降解,新生骨组织长入,有多核细胞出现降解材料,而单纯CPC无明显降解,材料内部无骨组织长入。
3.两种CPC的因子缓释曲线均由两部分组成:1天内的爆发式突释及随后的缓慢平稳释放;除第1天外,其余时间点多孔CPC的因子累积释放量均高于单纯CPC;28天后多孔CPC的累积释药量约为37.8±2.3%,明显高于单纯CPC的14.7±1.9%。A组成骨细胞增殖及ALP水平明显高于B组,且细胞形成的钙结节数量多形状大。体内实验发现两组均可见软骨形成,但A组软骨量更多,材料周围组织ALP及钙含量测定A组也明显高于B组。
4.三维CT成像显示:空白对照组术后25周在椎体上仍可见较大骨缺损存在;A组骨缺损修复显著快于B组,且材料降解较快,剩余材料较B组少。组织病理学结果表明:术后7周A组的平均骨矿化率高于B组,但术后25周未见明显差异;A组的骨小梁三维结构(平均骨小梁厚度及密度、结点末端比)明显优于B组,而平均骨小梁间距低于B组;A组材料降解较快,25周后剩余材料约为50%,且可见大量新生骨长入,B组几乎未见明显降解,无新生骨长入。骨密度检测显示术后6周两组未见明显差异,但25周后A组明显高于B组。生物力学试验结果表明,术后7周及25周A组分别为4.2±1.2 MPa及7.8±1.4MPa,明显高于B组的2.4±0.9 MPa及4.6±0.8MPa。
五.结论
1.GP交联明胶微球所需时间稍长,60%交联度的明胶微球降解时间约28天左右,适合作为CPC的成孔材料,而且其缓释性能及生物相容性更佳,可广泛应用于骨组织工程领域。
2.利用明胶微球可成功制备多孔CPC,微球在2.5%比重时,材料孔径率高,强度较以前同等孔径率的多孔材料有一定改善。复合材料具有极好的生物相容性,微球在体内可缓慢降解成孔,使新生骨组织长入,从而更加速复合材料的降解,是良好的骨组织工程支架材料。
3.多孔CPC复合rhBMP-2与单纯rhBMP-2/CPC相比,因子的释放能力及骨诱导性能均有所提高。这种新型人工骨可加速骨质疏松骨缺损的愈合,显著改善局部骨微结构,增强局部骨生物力学性能,适合作为骨质疏松骨缺损生物治疗的理想材料。
Background
Osteoporosis is now becoming much more prevelent than ever, especially in our country, and the number of the people who suffers osteoporosis is exceeding 90 million, therefore, doctors are facing more patients with osteoporotic bone defects. Under circumstance of osteoporosis, osteoclasts are more active, and the ability of bone resorption is stronger than that of bone formation. So healing of this kind of bone defect is impaired, and cure of the bone defect is more diffucult. A bone-grafting procedure is a successful care to treat the bone defects, but there are many limitations with the materials used in the operation. Although improved effect on bone defects has been obtained by using carriers with growth factors, properties of the carriers still need to be upgraded. Normal bone defects can heal fast with rhBMP-2/CPC, but limit factor-release ability of CPC is not enough for osteoporotic bone defects because high concentrations of the growth factors are needed for osteoporotic bone defects. When degradable microspheres loaded with growth factors are mixed wih CPC to construct a macroporous composite, not only the release ability of the composite can be enhanced, but also the degradation rate of it is speeded up. But PLGA that is the most used material for microspheres has some limitations: 1. Slow degradatrion rate hampers new bone ingrowth. 2. Degradation products are harmful to cells and tissues around the materials. GMs, which are widely used as drug carriers, are biocompatible and controlled-degradable after crosslinking, but cytotoxic of the crosslinkers (such as GA) used for parepairing GMs is still an obstacle. The problem will be solved by a new bio-crosslinker—genipin, therefore, GMs may replace PLGA microspheres as a good additive to improve the properties of CPC.
Objective
1. To prepare GMs by using GP as a crosslinker and to evaluate the characteristics of the GMs for construction of macroporous CPC.
2. To investigate the best proportion of GMs and CPC powers in the composite macroporous CPC by analyzing porosity, biomechabical property and the result of XRD. To evaluate the biocompatibility of the composite in vitro and in vivo.
3. To study the controlled-release characteristic of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC. To evaluate the bone-induction ability of the both composite in vitro and in vivo.
4. To evaluate the effects of the new artificial bone on bone mineral density, biomechanical property, micro-architecture of osteoporotic goat vertebral and healing of ostoporotic bone defect in vivo.
Materials and methods
1. GMs, prepared by the improved emulsified cold-condensation method, were crosslinked by GP and GA, respectively. After being dispersed in PBS, two kinds of microspheres with the 60% degree of crosslinking were compared in terms of morphology, swelling and degrading properties;BMP-2 were loaded into the GP and GA microspheres, and encapsulation rate、drug loading and releasing capacity were measured; 100%、50% and 25% leaching liquid of GP and GA microspheres were cultured with rat osteoblast(DMEM group as the control), respectively, and cell proliferation were measured by MTT method to grade the cell cytotoxicity.
2. Macroporous CPC was developed using genipin-crosslinked GMs with three weight ratios (0 wt%, 2.5 wt% and 5 wt%). After the composites were soaked in PBS for 1, 3 and 5 weeks, the proper weight ratio of GM was conformed by analyzing porosity, compressive strength and XRD. The morphologies of osteoblasts were examined with SEM after the cells were seeded on the CPC and GM/CPC for two days. After the cells were cultured with different leaching liquor of three kind of materials (0 wt%, 2.5 wt% GM/CPC and polystyrene) for days, cell viability and ALP concentration were measured. And 6 weeks after implantation into the bone defect of goats, GM/CPC and CPC were obtained and histopathological method was applied to investigate the bone ingrowth and the material degradation.
3. The leaching liquor of rhBMP-2/CM/CPC or rhBMP-2/CPC was collected after both composites had been soaked in the Sodium Chloride for 1、4、7、14、21 and 28 days, and the concentration of rhBMP-2 was measured by ELISA method. Osteoblasts were cultured with both leaching liquor, and cell proliferation, ALP concentration and calcium nodule were investigated. Three weeks after implation in the muscle bags of the rats, the composites with the muscles around were collected and stained with hematoxylin and eosin; Ca and ALP concentrations of the muscles were also measured.
4. Fifteen mature goats underwent ovariectomy and were placed on low cation relative to anion diet. After six months, bone defect of 5mm×10mm×10mm were made in L2、L4 of the goats, and three animals were not treated as blank control group. Others were randomly assigned to two groups and treated with rhBMP-2/GM/CPC (Group A) or rhBMP-2/CPC (Group B). After 1, 7 and 25 weeks, the defects were detected by CT scanning. Seven and twenty five weeks later, Animals were killed in batch, and BMD, compressive strength and micro-architecture of the vertebra were studied by dual energy X-ray absorption meter, biomechanical test and bone histomorphometry, respectively.
Results
1. GP and GA microspheres were both spherical with particle diameter of 78±18μm and 74±10μm, and there were no difference between both microspheres in drug loading and encapsulation rate; At the same crossling degree of 60%, GP microspheres, with longer degradation period (28 days) compared to GA microspheres (21 days), had better dispersibility, and swelling rate(89.0±4.8%)、percentage of cumulative drug releasing in 10 days(78.8±4.96%)were both lower than GA microsphere(118.0±7.6%、90.5±5.12%); Cell cytotoxicity of 100%、50% and 25% leaching liquid of GP microspheres were all at theⅠlevel, but leaching liquid of GA microspheres with corresponding concentration were at the level ofⅢ、Ⅲ、Ⅱ.
2. Porosity and macroporosity of the GM/CPC increased with the GMs increasing, but compression strength decreased after 1, 3 and 5 weeks of soaking, and the 2.5 wt% GM/CPC was the most favourable composite with high porosity and relative strong compressive strength. Osteoblasts showed normal morphology with both composites, but proliferation and differentiation of the cells were enhanced with the GM/CPC compared to the CPC. Both composites showed no adverse inflammatory reaction after weeks of implatation. New bones grew into the pores of the GM/CPC which resulted from GMs degradation, and coenocytes were present into the composite to degradate the material, but no new born could been seen inside of CPC.
3. The release characteristics of rhBMP-2 from rhBMP-2/GM/CPC and rhBMP-2/CPC both comprised two phases: (a) an initial burst occurring during the first 24 h, (b) a linear slow steady release phase linear release phase for the rest of the time course (days 2–28). Except for the similar initial burst effect, rhBMP-2 release rate of rhBMP-2/GM/CPC was faster than that of rhBMP-2/CPC in all the other evaluated time periods. In 28 days, 37.8±2.3% of the loaded protein in GMs was released, while in the control group the release rate was 14.7±1.9%. Cell experiment showed that Cell proliferation and ALP concentration of group A were significantly higher than those of group B, and calcium nodus of group A was bigger. In vivo studies also showed that quantity of cartilage formation, ALP and calcium concentration were higher in the group A.
4. CT reconstrction analysis showd that bone defect healing was accelerated in the group A, and remain-volume of rhBMP-2/GM/CPC was smaller than that of rhBMP-2/CPC; in the untreated control group, a large defect could still be found in the vertebra after 25 weeks, although the bone defect became a little smaller. The results of biomechanical test showed that compressive strength of vertebra in the group A were 4.2±1.2 MPa for 7 weeks and 7.8±1.4Mpa for 25 weeks,which were higher than 2.4±0.9 Mpa and 4.6±0.8Mpa of the group B. Histomorphologically, trabecular thickness, relative trabecular volume and node/terminatio rate in the group A were higher than those in the group B, and trabecular separation was lower; more bone ingrowth was observed deeper into the rhBMP-2/GM/CPC composite, but no tissue formation was present inside the rhBMP-2/CPC composite; and almost half part of rhBMP-2/GM/CPC had degradated, but little resorption of rhBMP-2 /CPC could be detected. Mineralizition rate and bone density showed no difference between two groups after 7 weeks, but after 25 weeks the values were higher in the group A.
Conclusion
1.GMs crosslinked by GP have better biocompatibility and cotrolled-release ability than that crosslinked by GA, and GMs with crosslinking degree of 60% can be used as additive to construct macroporous CPC.
2. Macroporous CPC can be developed using GP-crosslinked GMs, and 2.5 wt% GM/CPC is the most favourable with high macroporosity and strong compressive strength. The composite is a good bone substitute with improved degradability and biocompatibility.
3.Controlled-release of rhBMP-2 and bone induction are both enhanced with rhBMP-2/GM/CPC compared to rhBMP-2/CPC. The new graft can accelerate healing of osteoporotic bone defect and improve bone quality significantly, therefore, it can be used as a promising bone substitute for bone defects of osteoporosis under non-loaded circumstances.
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