个体化组织工程骨修复长骨缺损实验与临床研究
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摘要
背景和目的
日本骨科学会(JOA)自1985年起每5年就开展一次全国性的有关骨移植材料使用情况的调查,发现随着现代医疗条件的改善,骨移植材料的使用每年都在增加,2007年最新一次发布的结果显示2000年至2004年5年间的163,564例组织移植病例中82.4%为骨移植,其中植骨材料的选择自体骨依然是首选,占56.4%;各种合成骨移植替代物占40%;骨库同种异体骨仅占3.6%,但与前五年同期比较三者各自所占移植总量的比值却表现为自体骨移植所占比例不断下降,人工骨移植比例持续上升,骨库同种异体骨增速最快,三者平均每年较前年递增倍数分别为1.32, 1.68,和1.72倍1,故而部分专家建议对于小儿骨缺损修复则不再首选自体骨移植。其原因主要是考虑:自体骨移植局部效果虽好,但仍属于“拆东墙补西墙”,且儿童青少年往往骨量有限,无法满足大量植骨的需要,造成术中困难,对较大骨囊肿造成囊腔内残留死腔而致术后复发,而且取自体骨增加手术时间、费用和感染几率,造成取骨区疼痛,影响儿童骨骼发育2。而亲属骨作为同种异体骨也并非临床植骨的最优选择,且难以保证其确切的疗效3。各国的研究者们曾经尝试采用多种人工替代材料修复这些骨缺损4,如各种人工合成材料、同种异体骨、人同种异体的脱钙骨基质等,虽然这些材料分别从某些特定的方面一定程度上弥补了自体骨植骨的缺点,但其固有缺陷决定了这些材料在骨需求量大,患者自身情况差时均不能较好地满足临床的需要及患者的要求。鉴于以组织工程骨修复大动物长骨缺损的实验研究已取得了突破性的进展5,有必要在此工作基础上谨慎地进行一部分临床试用性研究为今后组织工程骨正式走上临床,形成对长骨缺损的正规化、规模化地治疗模式完成临床观察数据的初步积累,进一步完善以组织工程骨修复长骨缺损治疗及随访的标准化操作规程6。然而目前我们所研制的组织工程骨构建需要花费较长的时间7且保存条件苛刻,不能达到“随取随用”的最终要求8。早期的研究发现,部分蛋白制品通过冷冻干燥技术处理后的真空包装可以达到在室温下保存2年而不发生变质的良好效果9,从最早应用于处理生物学材料直到今天Philip J.等使用冻干同种异体骨成功修复牙周骨缺损10,冻干技术在骨组织保存方面的研究走过了近六十年的发展历程,且还有研究表明,长期深低温保存对于骨组织组织学以及力学性能的影响可以忽略不计11,这无疑为组织工程骨这种对成骨效能和力学强度同时均有较高要求的组织工程产品的保存乃至产业化指出了今后的发展方向。
此项研究的临床试用是在获得了第三军医大学西南医院伦理委员会批准后进行的,实施本项试用的西南医院骨科已成为全军首家获得总后勤部卫生部批文的单位。
方法
1.本课题按照组织工程骨修复长骨缺损临床试用标准化操作规程(SOP)为特定的患者于术前3-4周开始构建个体化的组织工程骨,并在此过程中按规程中预定的检验项目对构建过程中的每个阶段的目标细胞或组织进行严格的筛选和测定,始终信守首先保障受试者人身安全的承诺。
2 .组织工程骨构建成功后由指定医师按照SOP中预定的临床路径展开对受试者围手术期的各项诊疗、随访观察和与受试者之间进行对其康复进程的长期动态交流,以便及时发现可能出现的问题,给予受试者有益的建议和帮助。临床疗效评价主要通过对受试者影像学及血液学各项指标的持续监测进行。
3.将当天手术预留的组织工程骨同期植入6周龄BALB/C裸鼠皮下,通过对其定期行X线检查、CT扫描以及取植入骨组织行切片、组织化学染色的方法评价植入骨的成骨情况,同时向裸鼠皮下注射与植入骨内等量的种子细胞混悬液,通过观察其生活习性有无改变及定期称重、取其重要脏器行切片、HE染色观察的方法初步评价种子细胞的致瘤性。
4.将个体化的组织工程骨按标准程序冻干后与未冻干的组织工程骨一并提取其中的成骨相关蛋白后通过western-blotting技术对其中BMP-2、TGF-β1以及IGF-1的表达进行测定,初步对个体化组织工程骨成骨机制和冻干对组织工程骨成骨能力的影响做一探索性研究。
结果
1.按照组织工程骨临床试用标准化操作规程构建个体化工程骨的过程中,种子细胞增殖迅速,诱导分化效率高,构建的骨组织内细胞基质分泌旺盛,几乎充满支架材料的每个孔隙。
2.严格按照组织工程骨SOP成功完成了30例试用者所需组织工程骨的构建,所有试用者手术过程顺利并获得了较完备的临床随访资料。资料显示腔隙性骨缺损植骨区于术后平均3月左右均可见较高密度的钙化影,术后12月左右病灶获得基本修复,CT检查发现髓腔样结构。术后随访1.5年的病例可发现完全的髓腔再通,抽血检查结果显示各项指标基本正常,符合术前对组织工程骨成骨效能较高且安全性良好的预测。
3.裸鼠皮下异位成骨实验中,术后1、2、3月复查X线片可观察到植骨区逐渐出现高密度影的同时对照侧DBM始终未显影;术后3月组织学HE及Masson染色切片可观察到组织块内部及外部均有编织骨形成,并有较多的成骨细胞及小血管长入;皮下注射种子细胞混悬液实验过程中,实验裸鼠未见明显生活习性改变,切取其心、肺、肝、肾做HE染色观察未见组织破坏或肿瘤样组织形成。
4.个体化组织工程骨内BMP-2、TGF-β1以及IGF-1三种抗体均呈阳性表达,细胞上架后电泳灰度值经统计学分析均与上架前有显著差异(P<0.01),表示细胞分泌蛋白量较未上架时有显著意义的增加,同一样本不同时间点电泳灰度值比较显示上架第5、7、9天与上架3、12、15天相比较有显著差异(P<0.05)表示三种蛋白的表达高峰时段为上架后5-9天。
结论
1.构建的个体化组织工程骨用于临床修复骨缺损未见明显的排斥反应,提示个体化的组织工程骨具有良好的组织相容性好和较为广泛的临床应用前景。动物实验和临床应用均观察到体外构建的组织工程骨具有良好的诱导成骨和促进骨愈合作用。
2.通过对组织工程骨临床试用者较长期的血清学持续监测和放射学动态观察,初步证实了组织工程骨用于修复各类长骨缺损有较强的生物安全性。
3.个体化的组织工程骨修复小儿长骨缺损显示出了可喜的临床效果,这为治疗各种病因造成的小儿骨缺损的这一临床难题提供了新的思路和方法。
4.根据我科制定并已提交SFDA讨论审定的组织工程骨构建标准操作规程所制备的个体化组织工程骨可分泌较多的BMP-2、TGF-β1以及IGF-1,且冻干后的个体化组织工程骨依然存在较高的成骨活性,为组织工程骨产品的较长期保存提供了可行性技术支持。
Background and objective
The Japanese Orthopaedic Association Committeeon Tissue Transplantation and Regenerative Medicinehas conducted a nationwide survey of the status of bone graftingin Japan every 5 years from 1985. In the most recently research in 2007 they found that with the improvement of the modern medical conditions, the employment of the bone grafts kept increasing by each year, a total of 163,564 tissue transplantations were performed, and 134 782 (82.4%) of them were bone grafts. Of the bone grafts, 76,015 (56.4%) were autografts, 53,735 (40%) used a synthetic bone substitute, and 4886 (3.6%) were banked bone allografts. The proportion of synthetic bone substitutes increased, and the proportion of autografts decreased compared to the past 5 years, with the number of autografts, synthetic bone substitutes, and banked bone allografts increased 1.32, 1.68, and 1.72 times, respectively, during this period. Most significantly, the results suggest that Japanese orthopedic surgeons are tending to use synthetic bone substitutes largely as a replacement for autografts especially in the bone transplantations on children. In spite of the predominant properties of osteoinduction and osteoconduction autografts may have, there are limitations to the volume, shape, and size of autografts that can be excised. Excision of autogenous bone is also an invasive procedure. Furthermore, the bone mass of the autografts used to be deficient in children, in addition, this kind of operations may cause the pain in the donor site, the increase of the operation time, cost and probability of infections, even lead to the impediment of the adolescents’bone development. Same to some allografts, bones from the patients’relatives are not the optimum selection due to the indeterminateness of their curative effects. Scientists all over the world have tried many artificial substitutes to repair the bone defects such as the artificial synthetic materials, allografts, demineralized bone matrices from allografts, but none of them could fix the problems when the amount of the bones required are too large or the conditions of the patients are not sound. As we have gained so many breakthroughs on the research of repairing large bone defect of big animals using tissue-engineered bone(TEB), it is necessary to carry out some clinical experiments restrainedly for the sake of the TEB's formal clinical applications and the accumulation of rudiment data in order to form an orthonormal therapy mode for long bone defect. But the application of TEB is limited for the harsh demands of its conservation and transportation. To meet the final desire of the ideal tissue-engineered bone grafts“reach on demand”, literatures were reviewed by us and we found that in early research of William H’s group, the tissue they froze-dried and sealed in vacuum may be stored at room temperature for periods of at least two years without detectable change. In 2007 Philip J. declared they got successful result on the treatment of periodontal intrabony defects using freeze-dried bone. In addition to the conclusion Moshe Salai drew in 2001 that prolonged cryopreservation on the biomechanical properties of bone allografts came out insignificant, all the information give us a hint that the preservation even the full scale operation of our TEB should go well in this way.
This clinical research was approved by the Ethics Committee of Southwest Hospital, which made the department of orthopedics of southwest hospital come to be the first unit in PLA authorized to carry out such clinical applications of tissue-engineered bone by the ministry of health, PLA General Logistics Department.
Methods
1. The individualized TEB(iTEB) was constructed according to the SOP for the clinical application in the treatment of long bone defects 3-4 weeks before the admission. In this procedure, strict screening and determination based on SOP were used on the target cells or tissues in different phases of the construction. Commitments of the subject’s physical security are sticked to from the beginning to the end in this research.
2. The patients were informed to hospitalization after the construction of TEB was accomplished, all the perioperative check-ups, follow-up visits and the long-term developmental communications between the subjects and the clinicians during the restoration were executed according to the clinical path for these patients only. An identical resident is appointed to accomplish the above work, which is responsible to find out possible problems promptly and provide helpful recommendations for the subjects. The evaluation of the curative effect was mainly through the persistent monitoring of the radiology and hemotolgy results.
3. Nude mice of 6 weeks old (male:female=1:1) were chosen to be the host of the implantation subcutaneously and were evaluated the osteogenesis or oncogenicity by periodic check through X-ray, CT scan and pathological slice and histochemical stain of the graft and the vital organs respectively.
4. Key proteins like BMP-2, TGF-β1 and IGF-1 which should be produced in tissue-engineered bone and freeze-dried tissue-engineered bone were detected by western-blot method in order to explore the mechanism of the individualized tissue-engineered grafts’bone formation and the possible influence of freeze-drying on our TEB.
Results
1. Rapid proliferation, efficient induction and differentiation of the seedling cells for the construction of TEB was documented according to the SOP, productive secretion of the cell matrix was found in almost every pore of DBM under the inverted phase contrast microscope. Strict screening and determination of the cells and tissues all through the procedure were carried out and the physical security of the subjects is confirmed at least.
2. Thirty cases of clinical probation were accomplished according to the SOP of TEB strictly and self-contained follow-up materials were obtained simultaneously. According to the materials, comparatively high-density calcification shadows were observed 3 months after the implantation when capsular bone defect cases were analyzed, then foci almost disappeared at about 12 months after operation, furthermore, cavum pulpi were found completely recanalized in the 1.5 years case. Bone callous was found across the gap 6 months after the implantation and the recanalization of cavum pulpi was observed by CT scan throughout the cases.
3. Ectopia osteogenesis experiment on nude mice demonstrated gradually ossification along with the extension of the time post-operation. Both the internal and the external part of the tissue have formation signs of woven bone with major osteoblasts and small vessles infiltration. No obvious change of life habit was observed and no tumor-like tissue was discovered by HE staining of the slice of heart, lung, liver and kidney taken from the mice injected with cell suspensions.
4. The expressions of the three proteins are positive both in TEB and freeze-dried TEB, with significant difference to the pure DBM. The climax of their secretion lies in the period of 5-9 days after the cells were seeded into the scaffold.
Conclusion
1. No visible rejections were observed during the application of individualized TEB for the treatment of patients with long bone defects, which implies that the constructed TEB has good histocompatibility and extensive prospects for clinical application. Both the result of the clinical application and ectopia osteogenesis experiment in nude mice showed comparatively doughty bone induction and osteogenesis of TEB at the same time.
2. The biological security and curative effect of TEB in the treatment of different kinds of long bone defects were tentatively confirmed by monitoring the serology and imageology results of the patients in differents periods of rehabilitation dynamically.
3. Felicitous curative results were manifested in the treatments of children’s long bone defects with individualized TEB, which provided a novel path for the theraphy of juvenile bone defects casued by different etiological factors.
4. Much osteogenic key proteins such as BMP-2, TGF-β1 and IGF-1 in the individualized tissue-engineered bone constructed according to the standard operating procedure which we preferred were confirmed within the individualized tissue-engineered bone and freeze-dried TEB by WB method.
日本骨科学会(JOA)自1985年起每5年就开展一次全国性的有关骨移植材料使用情况的调查,发现随着现代医疗条件的改善,骨移植材料的使用每年都在增加,2007年最新一次发布的结果显示2000年至2004年5年间的163,564例组织移植病例中82.4%为骨移植,其中植骨材料的选择自体骨依然是首选,占56.4%;各种合成骨移植替代物占40%;骨库同种异体骨仅占3.6%,但与前五年同期比较三者各自所占移植总量的比值却表现为自体骨移植所占比例不断下降,人工骨移植比例持续上升,骨库同种异体骨增速最快,三者平均每年较前年递增倍数分别为1.32, 1.68,和1.72倍1,故而部分专家建议对于小儿骨缺损修复则不再首选自体骨移植。其原因主要是考虑:自体骨移植局部效果虽好,但仍属于“拆东墙补西墙”,且儿童青少年往往骨量有限,无法满足大量植骨的需要,造成术中困难,对较大骨囊肿造成囊腔内残留死腔而致术后复发,而且取自体骨增加手术时间、费用和感染几率,造成取骨区疼痛,影响儿童骨骼发育2。而亲属骨作为同种异体骨也并非临床植骨的最优选择,且难以保证其确切的疗效3。各国的研究者们曾经尝试采用多种人工替代材料修复这些骨缺损4,如各种人工合成材料、同种异体骨、人同种异体的脱钙骨基质等,虽然这些材料分别从某些特定的方面一定程度上弥补了自体骨植骨的缺点,但其固有缺陷决定了这些材料在骨需求量大,患者自身情况差时均不能较好地满足临床的需要及患者的要求。鉴于以组织工程骨修复大动物长骨缺损的实验研究已取得了突破性的进展5,有必要在此工作基础上谨慎地进行一部分临床试用性研究为今后组织工程骨正式走上临床,形成对长骨缺损的正规化、规模化地治疗模式完成临床观察数据的初步积累,进一步完善以组织工程骨修复长骨缺损治疗及随访的标准化操作规程6。然而目前我们所研制的组织工程骨构建需要花费较长的时间7且保存条件苛刻,不能达到“随取随用”的最终要求8。早期的研究发现,部分蛋白制品通过冷冻干燥技术处理后的真空包装可以达到在室温下保存2年而不发生变质的良好效果9,从最早应用于处理生物学材料直到今天Philip J.等使用冻干同种异体骨成功修复牙周骨缺损10,冻干技术在骨组织保存方面的研究走过了近六十年的发展历程,且还有研究表明,长期深低温保存对于骨组织组织学以及力学性能的影响可以忽略不计11,这无疑为组织工程骨这种对成骨效能和力学强度同时均有较高要求的组织工程产品的保存乃至产业化指出了今后的发展方向。
此项研究的临床试用是在获得了第三军医大学西南医院伦理委员会批准后进行的,实施本项试用的西南医院骨科已成为全军首家获得总后勤部卫生部批文的单位。
方法
1.本课题按照组织工程骨修复长骨缺损临床试用标准化操作规程(SOP)为特定的患者于术前3-4周开始构建个体化的组织工程骨,并在此过程中按规程中预定的检验项目对构建过程中的每个阶段的目标细胞或组织进行严格的筛选和测定,始终信守首先保障受试者人身安全的承诺。
2 .组织工程骨构建成功后由指定医师按照SOP中预定的临床路径展开对受试者围手术期的各项诊疗、随访观察和与受试者之间进行对其康复进程的长期动态交流,以便及时发现可能出现的问题,给予受试者有益的建议和帮助。临床疗效评价主要通过对受试者影像学及血液学各项指标的持续监测进行。
3.将当天手术预留的组织工程骨同期植入6周龄BALB/C裸鼠皮下,通过对其定期行X线检查、CT扫描以及取植入骨组织行切片、组织化学染色的方法评价植入骨的成骨情况,同时向裸鼠皮下注射与植入骨内等量的种子细胞混悬液,通过观察其生活习性有无改变及定期称重、取其重要脏器行切片、HE染色观察的方法初步评价种子细胞的致瘤性。
4.将个体化的组织工程骨按标准程序冻干后与未冻干的组织工程骨一并提取其中的成骨相关蛋白后通过western-blotting技术对其中BMP-2、TGF-β1以及IGF-1的表达进行测定,初步对个体化组织工程骨成骨机制和冻干对组织工程骨成骨能力的影响做一探索性研究。
结果
1.按照组织工程骨临床试用标准化操作规程构建个体化工程骨的过程中,种子细胞增殖迅速,诱导分化效率高,构建的骨组织内细胞基质分泌旺盛,几乎充满支架材料的每个孔隙。
2.严格按照组织工程骨SOP成功完成了30例试用者所需组织工程骨的构建,所有试用者手术过程顺利并获得了较完备的临床随访资料。资料显示腔隙性骨缺损植骨区于术后平均3月左右均可见较高密度的钙化影,术后12月左右病灶获得基本修复,CT检查发现髓腔样结构。术后随访1.5年的病例可发现完全的髓腔再通,抽血检查结果显示各项指标基本正常,符合术前对组织工程骨成骨效能较高且安全性良好的预测。
3.裸鼠皮下异位成骨实验中,术后1、2、3月复查X线片可观察到植骨区逐渐出现高密度影的同时对照侧DBM始终未显影;术后3月组织学HE及Masson染色切片可观察到组织块内部及外部均有编织骨形成,并有较多的成骨细胞及小血管长入;皮下注射种子细胞混悬液实验过程中,实验裸鼠未见明显生活习性改变,切取其心、肺、肝、肾做HE染色观察未见组织破坏或肿瘤样组织形成。
4.个体化组织工程骨内BMP-2、TGF-β1以及IGF-1三种抗体均呈阳性表达,细胞上架后电泳灰度值经统计学分析均与上架前有显著差异(P<0.01),表示细胞分泌蛋白量较未上架时有显著意义的增加,同一样本不同时间点电泳灰度值比较显示上架第5、7、9天与上架3、12、15天相比较有显著差异(P<0.05)表示三种蛋白的表达高峰时段为上架后5-9天。
结论
1.构建的个体化组织工程骨用于临床修复骨缺损未见明显的排斥反应,提示个体化的组织工程骨具有良好的组织相容性好和较为广泛的临床应用前景。动物实验和临床应用均观察到体外构建的组织工程骨具有良好的诱导成骨和促进骨愈合作用。
2.通过对组织工程骨临床试用者较长期的血清学持续监测和放射学动态观察,初步证实了组织工程骨用于修复各类长骨缺损有较强的生物安全性。
3.个体化的组织工程骨修复小儿长骨缺损显示出了可喜的临床效果,这为治疗各种病因造成的小儿骨缺损的这一临床难题提供了新的思路和方法。
4.根据我科制定并已提交SFDA讨论审定的组织工程骨构建标准操作规程所制备的个体化组织工程骨可分泌较多的BMP-2、TGF-β1以及IGF-1,且冻干后的个体化组织工程骨依然存在较高的成骨活性,为组织工程骨产品的较长期保存提供了可行性技术支持。
Background and objective
The Japanese Orthopaedic Association Committeeon Tissue Transplantation and Regenerative Medicinehas conducted a nationwide survey of the status of bone graftingin Japan every 5 years from 1985. In the most recently research in 2007 they found that with the improvement of the modern medical conditions, the employment of the bone grafts kept increasing by each year, a total of 163,564 tissue transplantations were performed, and 134 782 (82.4%) of them were bone grafts. Of the bone grafts, 76,015 (56.4%) were autografts, 53,735 (40%) used a synthetic bone substitute, and 4886 (3.6%) were banked bone allografts. The proportion of synthetic bone substitutes increased, and the proportion of autografts decreased compared to the past 5 years, with the number of autografts, synthetic bone substitutes, and banked bone allografts increased 1.32, 1.68, and 1.72 times, respectively, during this period. Most significantly, the results suggest that Japanese orthopedic surgeons are tending to use synthetic bone substitutes largely as a replacement for autografts especially in the bone transplantations on children. In spite of the predominant properties of osteoinduction and osteoconduction autografts may have, there are limitations to the volume, shape, and size of autografts that can be excised. Excision of autogenous bone is also an invasive procedure. Furthermore, the bone mass of the autografts used to be deficient in children, in addition, this kind of operations may cause the pain in the donor site, the increase of the operation time, cost and probability of infections, even lead to the impediment of the adolescents’bone development. Same to some allografts, bones from the patients’relatives are not the optimum selection due to the indeterminateness of their curative effects. Scientists all over the world have tried many artificial substitutes to repair the bone defects such as the artificial synthetic materials, allografts, demineralized bone matrices from allografts, but none of them could fix the problems when the amount of the bones required are too large or the conditions of the patients are not sound. As we have gained so many breakthroughs on the research of repairing large bone defect of big animals using tissue-engineered bone(TEB), it is necessary to carry out some clinical experiments restrainedly for the sake of the TEB's formal clinical applications and the accumulation of rudiment data in order to form an orthonormal therapy mode for long bone defect. But the application of TEB is limited for the harsh demands of its conservation and transportation. To meet the final desire of the ideal tissue-engineered bone grafts“reach on demand”, literatures were reviewed by us and we found that in early research of William H’s group, the tissue they froze-dried and sealed in vacuum may be stored at room temperature for periods of at least two years without detectable change. In 2007 Philip J. declared they got successful result on the treatment of periodontal intrabony defects using freeze-dried bone. In addition to the conclusion Moshe Salai drew in 2001 that prolonged cryopreservation on the biomechanical properties of bone allografts came out insignificant, all the information give us a hint that the preservation even the full scale operation of our TEB should go well in this way.
This clinical research was approved by the Ethics Committee of Southwest Hospital, which made the department of orthopedics of southwest hospital come to be the first unit in PLA authorized to carry out such clinical applications of tissue-engineered bone by the ministry of health, PLA General Logistics Department.
Methods
1. The individualized TEB(iTEB) was constructed according to the SOP for the clinical application in the treatment of long bone defects 3-4 weeks before the admission. In this procedure, strict screening and determination based on SOP were used on the target cells or tissues in different phases of the construction. Commitments of the subject’s physical security are sticked to from the beginning to the end in this research.
2. The patients were informed to hospitalization after the construction of TEB was accomplished, all the perioperative check-ups, follow-up visits and the long-term developmental communications between the subjects and the clinicians during the restoration were executed according to the clinical path for these patients only. An identical resident is appointed to accomplish the above work, which is responsible to find out possible problems promptly and provide helpful recommendations for the subjects. The evaluation of the curative effect was mainly through the persistent monitoring of the radiology and hemotolgy results.
3. Nude mice of 6 weeks old (male:female=1:1) were chosen to be the host of the implantation subcutaneously and were evaluated the osteogenesis or oncogenicity by periodic check through X-ray, CT scan and pathological slice and histochemical stain of the graft and the vital organs respectively.
4. Key proteins like BMP-2, TGF-β1 and IGF-1 which should be produced in tissue-engineered bone and freeze-dried tissue-engineered bone were detected by western-blot method in order to explore the mechanism of the individualized tissue-engineered grafts’bone formation and the possible influence of freeze-drying on our TEB.
Results
1. Rapid proliferation, efficient induction and differentiation of the seedling cells for the construction of TEB was documented according to the SOP, productive secretion of the cell matrix was found in almost every pore of DBM under the inverted phase contrast microscope. Strict screening and determination of the cells and tissues all through the procedure were carried out and the physical security of the subjects is confirmed at least.
2. Thirty cases of clinical probation were accomplished according to the SOP of TEB strictly and self-contained follow-up materials were obtained simultaneously. According to the materials, comparatively high-density calcification shadows were observed 3 months after the implantation when capsular bone defect cases were analyzed, then foci almost disappeared at about 12 months after operation, furthermore, cavum pulpi were found completely recanalized in the 1.5 years case. Bone callous was found across the gap 6 months after the implantation and the recanalization of cavum pulpi was observed by CT scan throughout the cases.
3. Ectopia osteogenesis experiment on nude mice demonstrated gradually ossification along with the extension of the time post-operation. Both the internal and the external part of the tissue have formation signs of woven bone with major osteoblasts and small vessles infiltration. No obvious change of life habit was observed and no tumor-like tissue was discovered by HE staining of the slice of heart, lung, liver and kidney taken from the mice injected with cell suspensions.
4. The expressions of the three proteins are positive both in TEB and freeze-dried TEB, with significant difference to the pure DBM. The climax of their secretion lies in the period of 5-9 days after the cells were seeded into the scaffold.
Conclusion
1. No visible rejections were observed during the application of individualized TEB for the treatment of patients with long bone defects, which implies that the constructed TEB has good histocompatibility and extensive prospects for clinical application. Both the result of the clinical application and ectopia osteogenesis experiment in nude mice showed comparatively doughty bone induction and osteogenesis of TEB at the same time.
2. The biological security and curative effect of TEB in the treatment of different kinds of long bone defects were tentatively confirmed by monitoring the serology and imageology results of the patients in differents periods of rehabilitation dynamically.
3. Felicitous curative results were manifested in the treatments of children’s long bone defects with individualized TEB, which provided a novel path for the theraphy of juvenile bone defects casued by different etiological factors.
4. Much osteogenic key proteins such as BMP-2, TGF-β1 and IGF-1 in the individualized tissue-engineered bone constructed according to the standard operating procedure which we preferred were confirmed within the individualized tissue-engineered bone and freeze-dried TEB by WB method.
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