SCR技术即刻构建高成骨活性骨移植材料的实验研究
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摘要
背景:因高能量创伤、大段骨肿瘤切除、严重骨感染等因素所致的大段骨缺损十分常见,人工假体置换、脊柱融合等手术对骨移植材料的需求量也非常巨大。自体骨移植至今仍是评价骨移植效果的金标准,但自体骨移植增加了手术程序并引发许多新的问题如术区慢性疼痛、取骨量有限等。另外,自体骨移植也不适用于老年人、未成年人等。自体红骨髓局部注射在治疗骨不连、修复骨缺损等方面已取得了一定的成功,但所需骨髓量较大,局部有效成骨成分易流失,现在临床工作中应用较少。通过自体细胞体外扩增技术常规构建的组织工程骨是一种较为理想的骨修复材料,既能达到要求的细胞量,又能规避伦理道德限制。其缺点在于此方法往往需要两次手术:自体细胞的采集和体外扩增后的回植。体外构建获取时间较长,约需两周左右,且治疗费用较大、操作技术要求较高,因此目前在临床工作中广泛开展还存在一定的困难。选择性滞留(selective cell retention,SCR)技术是近年来国外学者报道的骨髓富集技术之一,主要是通过富集材料良好的黏附性能及适当的网孔结构,在负压作用下,使骨髓流经时骨髓中的有效成骨成分如干细胞及促成骨因子等可选择性地滞留于材料,然后将构建好的富集材料即刻回植体内。SCR技术在国外的动物试验及临床试验中已获得了较理想的效果。SCR技术的显著优势在于构建的移植材料成骨活性高,操作过程简单,安全性高,不涉及伦理问题等,为临床骨缺损修复带来了新的希望。
目的:(1)制备新型的骨髓富集材料,检测其性能;(2)探讨在SCR技术下富集材料对骨髓中有核细胞及促成骨生长因子的富集效果;(3)SCR技术下观察富集材料移植裸鼠肌袋内的成骨效果。
方法:(1)用自组装肽RADA16-I修饰青山羊脱钙骨基质(DBM),制备一种复合的骨髓富集材料,三维视频显微镜观察立体结构及检测孔径大小,扫描电镜观察其微观结构,X线光电子能谱分析仪(XPS)检测复合材料的结合情况,紫外分光光度计检测复合材料的稳定性能。(2)从青山羊髂骨抽取骨髓,置入富集器富集皿中,观察在SCR技术下富集系统对骨髓中有核细胞及生长因子的富集效果。(3)采用SCR技术即刻构建骨移植材料,在裸鼠模型上观察其成骨能力。将24只裸鼠随机分成三组,每组8只。骨移植材料分为如下三组(A组:SCR组,RADA16-I/DBM+SCR技术B组:骨髓浸泡组,RADA16-I/DBM+骨髓浸泡;C组:空白组,RADA16-I/DBM)。分别将A、B、C三组材料随机植入三组裸鼠臀肌内,做肌袋缝合。分别于术后4、8周分批处死裸鼠(每批每组处死4只),行X线片、CT值检测及三维CT、组织学观察,观察其成骨能力。
结果:(1)空白DBM具有天然骨的三维网孔结构,大孔隙内部虽有大量小孔隙相互连通,但孔径仍相对较大;复合材料RADA16-I/DBM可见无色透明样的胶状物黏附于三维网孔支架内部及DBM骨片表面。三维视频显微镜图像处理系统测得DBM孔径大小为(414.32±175.00)μm(n=60),RADA16-I/DBM复合材料孔径大小为(220.55±158.54)μm (n=60),p<0.05,认为RADA16-I/DBM组与DBM组孔径的总体均数差异显著。稳定性方面,RADA16-I/DBM浸泡液的紫外光谱与PBS液相比大致相同,光谱均不完整,说明对紫外敏感成分较少或无;0.5%RADA16-I肽溶液的紫外光谱表现出较强的紫外吸收特性,在222nm处有明显氨基酸吸收峰,且吸光度峰值大于2.0,说明有较多氨基酸成分存在于测试液中。DBM和RADA16-I多肽的主要成分是碳元素(C)、氮元素(N)、氧元素(O),与空白DBM组相比,RADA16-I/DBM中N的含量显著提高。(2)将复合材料RADA16-I/DBM和空白DBM的富集倍数进行统计分析,结果显示,RADA16-I/DBM复合材料对有核细胞的富集倍数为(6.05±0.96)(n=12),明显高于空白DBM对照组对有核细胞的富集倍数(2.27±0.41)(n=12),两者总体均数相比差异显著(p<0.05)。HE染色结果示:相同的富集条件下(骨髓量、支架材料量、富集时间、富集速度、富集次数均相同),RADA16-I/DBM复合材料中沿骨基质方向有成簇的有核细胞被募集,骨基质周围也有较多的有核细胞;对照组空白DBM中骨基质周围被募集的有核细胞较少。RADA16-I/DBM复合材料对骨髓中成骨相关的生长因子骨形态发生蛋白-2(BMP-2)、血小板源性生长因子(PDGF)、胰岛素样生长因子(IGF-I)的富集倍数分别为(23.03±2.68)倍、(19.68±4.02)倍、(31.02±2.09)倍,而DBM的相应富集倍数分别为(6.63±1.42)倍、(5.66±0.74)倍、(5.61±1.59)倍,二者总体均数相比有显著性差异(p<0.05),认为RADA16-I /DBM材料对成骨相关生长因子的富集倍数高于DBM材料。RADA16-I/DBM复合材料对BMP-2、PDGF、IGF-I的黏附率分别为(58%±7%)、(49%±10%)、(60%±9%),相比DBM的黏附率(33%±7%)、(28%±4%)、(28%±8%),二者总体均数差异性显著(p<0.05),认为RADA16-I/DBM材料对成骨相关生长因子的黏附率明显高于DBM材料。(3)裸鼠髂骨旁臀肌肌袋成骨实验观察,术后4周三组动物均没有明显的较高密度显影,8周SCR组较其它两组呈现高密度的钙化影像,提示有新骨生成;RADA16-I/DBM+骨髓浸泡组可见模糊密度影,提示有部分骨组织生成,但钙化程度不高;RADA16-I/DBM组不显影提示移植材料基本被吸收。CT结果显示:SCR组、RADA16-I/DBM+骨髓浸泡组、RADA16-I/DBM组骨移植区组织的CT值分别为(547.71±62.01)HU、(331.58±19.28)HU、(77.08±8.75)HU(n=8),三者总体均数有显著性差异(p<0.05),SCR组的CT值明显高于RADA16-I/DBM+骨髓浸泡组及RADA16-I/DBM组,即SCR技术构建的骨移植材料具有较高的成骨活性。组织学观察提示:SCR组移植材料有较高的成骨能力,RADA16-I/DBM+骨髓浸泡组移植材料骨生成能力不强,RADA16-I/DBM组材料基本被吸收,没有类骨组织形成。
结论:(1)制备的复合富集材料RADA16-I/DBM不但具有三维空间结构,适当的孔径,而且稳定性好,是一种较理想的骨髓富集材料。(2)应用SCR技术能使复合材料RADA16-I/DBM富集较高浓度的骨髓有核细胞,同时能富集较高浓度的促成骨生长因子。(3)SCR技术即刻构建的骨移植材料具有较高的成骨活性,在裸鼠肌袋成骨模型中有较强的成骨能力。
Background: Bone defects, which are caused by some factors such as high energy trauma, large bone tumor resection, and severe bone infection are common, and the demands for bone graft material about artificial prosthetic replacement and spinal fusion are also very great. Autologous bone graft is still the gold standard of bone transplantation, but it increases the surgical procedure and causes many new problems such as the chronic pain, limited bone etc. In addition, autogenous bone graft does not apply to the elderly, minors and so on. Local injection of autologous red bone marrow in the treatment of nonunion and bone defect have made some success. But it requires a large amount of bone marrow, and leads to the loss of effective ingredients of bone. Therefore, now it is seldom used in clinical work. Tissue engineering bone to be built with Autologous cells in vitro by conventional amplification is an ideal bone repair materials. It is not only meeting the requirements of the cell mass, but also avoiding the ethical and moral limits. The disadvantage is that amplification of autologous cells in vitro often requires twice operations: the collection of autologous cells in vitro and returning to the areas. It spends about two weeks in vitro for amplification and more cost for the treatment. There are still some difficulties to apply it for clinical works for its high level of technique. Selective cell retention technology(SCR) is reported as one of enriching technology of bone marrow by scholars of home and aboard in recent years. SCR technology may enrich effective ingredients such as stem cells and bone factor by the good adhesion and the appropriate mesh structure of enriched material by negative pressure of device when the bone marrow through them, thus it is easy to build a appreciate enriching material immediately. SCR technology has obtained ideal results in the foreign animal experiments and clinical trials. The significant advantages of SCR technology lies in the osteogenic activity, high safty in operation, and avoiding ethical issues. Thus SCR has brought new hope for clinical bone repair.
Objective: (1) Preparating a new type of bone marrow enriching materials and testing its performance. (2) Approching the enriching effects of SCR technology for the nuclear cells and growth factors with bone formation in the bone marrow. (3) Observing the bone formation results of transplanting with enriching material within muscle pouch of nude mice.
Methods: (1) Goat demineralized bone matrix (DBM) is combinated with self-assembling peptide RADA16-Ⅰto composite materials in PBS solution, followed by the analysis of three-dimensional structure and pore size though three-dimensional video microscope ,microscopic structure though scanning electron and relationship between RADA16-Ⅰwith DBM though X ray photoelectron spectroscopy analyzer (XPS),and also test the stability of composite materials though UV spectrophotometer. (2) Aspirating the iliac bone marrow from goats and inserting into the enriching dish to observe the enriching effect of RADA16-Ⅰ/DBM about nucleated cells and growth factors with bone formation in bone marrow by SCR. (3) Observing the osteogenesis of bone graft material in muscle pouch of nude mice by SCR technology instantly building bone graft material. 24 mice were randomly divided into three groups, n = 8. Bone graft materials were divided into the following three groups (A ,SCR group, RADA16-I/DBM +SCR materials; B, bone marrow immersion group,RADA16-I/DBM + bone marrow immersion materials; C, blank group, RADA16-I/DBM material).Group A, B, C were implanted into muscle pouch beside both of iliac of nude mice of three groups randomly and then suture muscle pouch. After 4 and 8 weeks of the surgery, respectively, nude mice were killed in batches (Four of mice were killed in each batch of each group), and detecting the osteogenic potential by X-ray,three dimensions CT and histological observation to observe the bone formation ability.
Results: (1) Blank DBM has three-dimensional mesh structure of natural bone. Although a large number of big pores within the small pores connected with each other, the aperture are still relatively large. However, the colorless jelly like adhesion in three-dimensional mesh scaffolds and the DBM bone surface is visible in composite materials RADA16-I/DBM. Measuring DBM pore size (414.32±175.00)μm (n = 60), RADA16-I/DBM pore size (220.55±158.54)μm (n = 60), p <0.05, through image processing system of three-dimensional video microscope. It is that overall mean diameter was significantly different between DBM group and RADA16-I/DBM group. Detecting about the stability of RADA16-I/DBM shows that its spectrum of immersion UV is not the full, which is roughly the same spectra to PBS solution, indicating that UV-sensitive components are less or no about them; UV spectra show a more strong UV absorption properties of amino acids in the 222nm absorption peak about 0.5% RADA16-I peptide in solution, and the absorbance peak is greater than 2.0, indicating that more amino acids present in the test solution. DBM and RADA16-I peptide, both of them are mainly consisted of carbon (C), nitrogen (N), oxygen (O). The N of RADA16-I /DBM is increasing significantly compared with the control group DBM; (2)The statistical calculations between RADA16-I/DBM and DBM enriching effects showed that enriching effect of nucleated cells was (6.05±0.96) (n = 12 )about RADA16-I/DBM composite materials ,which is significantly higher than the control group DBM which is(2.27±0.41) (n = 12), and the overall mean difference between them is significant (p <0.05). The resuits of HE staining shows that: under the same enriching conditions (amount of bone marrow,amount of materials, enriching time, enriching speed and the number of enrichment are the same),to RADA16-I/DBM composites material, clusters of nucleated cells along the direction of bone matrix were raising and bone matrix is also surrounded by more nucleated cells; To control group DBM, nucleated cells are less around the bone matrix. Enriching effects about growth factors of bone formation of RADA16-I/DBM showed that bone morphogenetic protein -2 (BMP-2), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I) of the enrichment factor were (23.03±2.68) times, (19.68±4.02) times, (31.02±2.09) times respectively, while the corresponding enriching effects for the DBM were (6.63±1.42) times, (5.66±0.74) times, (5.61±1.59) Times, both of the overall mean was significantly different (p <0.05), that is enriching effects about bone growth factors of RADA16-I/DBM were higher than DBM. Adhesion rates of RADA16-I / DBM about BMP-2, PDGF, IGF-I were (58%±6.7%), (49%±10%), (60%±8.9%), compared to DBM's corresponding (33%±7.1%), (28%±3.7%), (28%±7.7%), both of the overall mean was significantly different (p <0.05), that is adhesion rate of RADA16-I/DBM for bone growth factors was significantly higher than DBM. (3) Observating from muscle pouch beside iliac bone of nude mice the bone graft area of animals in the 4 weeks does
not show significantly high-density screenage. SCR group shows high density of calcification than other two images in the 8 weeks, which suggests new bone formation at the bone graft area. Meanwhile, RADA16-I/DBM + bone marrow immersion group shows fuzzy density screenage, suggesting that new bone has been formatted partly, but the degree of calcification is low.However, blank group, RADA16-I/DBM does not show screenage, which suggests that the graft material is absorbed on the whole, and CT results shows that: CT values of bone graft of SCR group, RADA16-I/DBM + bone marrow immersion and RADA16-I/DBM were (547.71±62.01) HU, (331.58±19.28) HU, (77.08±8.75) HU (n = 8) respectively, and the overall number of those were significantly different (p <0.05), that is the CT value of the SCR group, was significantly higher than RADA16-I/DBM + bone marrow immersion and RADA16-I/DBM, the bone graft materials built by SCR have highly osteogenic activity. Histological observations suggest that: SCR group show higher graft osteogenesis and RADA16-I/DBM + bone marrow immersion materials show some capabilities of bone formation, but the ability of bone formation is not strong.But, RADA16-I/DBM group materials are basically absorbed, and without neoplastic bone formation.
Conclusions: (1) RADA16-I/DBM composite material which not only has the three-dimensional structure and the appropriate pore size, but also has good stability is an ideal bone marrow enriched material. (2) RADA16-I/DBM composite materials can enrich highly concentrative nucleus cells and highly concentrative bone growth factors from bone marrow by SCR technology.(3) Bone graft material built instantly by SCR technology has a high osteogenic activity for bone formation in muscle pouches of nude mice model.
目的:(1)制备新型的骨髓富集材料,检测其性能;(2)探讨在SCR技术下富集材料对骨髓中有核细胞及促成骨生长因子的富集效果;(3)SCR技术下观察富集材料移植裸鼠肌袋内的成骨效果。
方法:(1)用自组装肽RADA16-I修饰青山羊脱钙骨基质(DBM),制备一种复合的骨髓富集材料,三维视频显微镜观察立体结构及检测孔径大小,扫描电镜观察其微观结构,X线光电子能谱分析仪(XPS)检测复合材料的结合情况,紫外分光光度计检测复合材料的稳定性能。(2)从青山羊髂骨抽取骨髓,置入富集器富集皿中,观察在SCR技术下富集系统对骨髓中有核细胞及生长因子的富集效果。(3)采用SCR技术即刻构建骨移植材料,在裸鼠模型上观察其成骨能力。将24只裸鼠随机分成三组,每组8只。骨移植材料分为如下三组(A组:SCR组,RADA16-I/DBM+SCR技术B组:骨髓浸泡组,RADA16-I/DBM+骨髓浸泡;C组:空白组,RADA16-I/DBM)。分别将A、B、C三组材料随机植入三组裸鼠臀肌内,做肌袋缝合。分别于术后4、8周分批处死裸鼠(每批每组处死4只),行X线片、CT值检测及三维CT、组织学观察,观察其成骨能力。
结果:(1)空白DBM具有天然骨的三维网孔结构,大孔隙内部虽有大量小孔隙相互连通,但孔径仍相对较大;复合材料RADA16-I/DBM可见无色透明样的胶状物黏附于三维网孔支架内部及DBM骨片表面。三维视频显微镜图像处理系统测得DBM孔径大小为(414.32±175.00)μm(n=60),RADA16-I/DBM复合材料孔径大小为(220.55±158.54)μm (n=60),p<0.05,认为RADA16-I/DBM组与DBM组孔径的总体均数差异显著。稳定性方面,RADA16-I/DBM浸泡液的紫外光谱与PBS液相比大致相同,光谱均不完整,说明对紫外敏感成分较少或无;0.5%RADA16-I肽溶液的紫外光谱表现出较强的紫外吸收特性,在222nm处有明显氨基酸吸收峰,且吸光度峰值大于2.0,说明有较多氨基酸成分存在于测试液中。DBM和RADA16-I多肽的主要成分是碳元素(C)、氮元素(N)、氧元素(O),与空白DBM组相比,RADA16-I/DBM中N的含量显著提高。(2)将复合材料RADA16-I/DBM和空白DBM的富集倍数进行统计分析,结果显示,RADA16-I/DBM复合材料对有核细胞的富集倍数为(6.05±0.96)(n=12),明显高于空白DBM对照组对有核细胞的富集倍数(2.27±0.41)(n=12),两者总体均数相比差异显著(p<0.05)。HE染色结果示:相同的富集条件下(骨髓量、支架材料量、富集时间、富集速度、富集次数均相同),RADA16-I/DBM复合材料中沿骨基质方向有成簇的有核细胞被募集,骨基质周围也有较多的有核细胞;对照组空白DBM中骨基质周围被募集的有核细胞较少。RADA16-I/DBM复合材料对骨髓中成骨相关的生长因子骨形态发生蛋白-2(BMP-2)、血小板源性生长因子(PDGF)、胰岛素样生长因子(IGF-I)的富集倍数分别为(23.03±2.68)倍、(19.68±4.02)倍、(31.02±2.09)倍,而DBM的相应富集倍数分别为(6.63±1.42)倍、(5.66±0.74)倍、(5.61±1.59)倍,二者总体均数相比有显著性差异(p<0.05),认为RADA16-I /DBM材料对成骨相关生长因子的富集倍数高于DBM材料。RADA16-I/DBM复合材料对BMP-2、PDGF、IGF-I的黏附率分别为(58%±7%)、(49%±10%)、(60%±9%),相比DBM的黏附率(33%±7%)、(28%±4%)、(28%±8%),二者总体均数差异性显著(p<0.05),认为RADA16-I/DBM材料对成骨相关生长因子的黏附率明显高于DBM材料。(3)裸鼠髂骨旁臀肌肌袋成骨实验观察,术后4周三组动物均没有明显的较高密度显影,8周SCR组较其它两组呈现高密度的钙化影像,提示有新骨生成;RADA16-I/DBM+骨髓浸泡组可见模糊密度影,提示有部分骨组织生成,但钙化程度不高;RADA16-I/DBM组不显影提示移植材料基本被吸收。CT结果显示:SCR组、RADA16-I/DBM+骨髓浸泡组、RADA16-I/DBM组骨移植区组织的CT值分别为(547.71±62.01)HU、(331.58±19.28)HU、(77.08±8.75)HU(n=8),三者总体均数有显著性差异(p<0.05),SCR组的CT值明显高于RADA16-I/DBM+骨髓浸泡组及RADA16-I/DBM组,即SCR技术构建的骨移植材料具有较高的成骨活性。组织学观察提示:SCR组移植材料有较高的成骨能力,RADA16-I/DBM+骨髓浸泡组移植材料骨生成能力不强,RADA16-I/DBM组材料基本被吸收,没有类骨组织形成。
结论:(1)制备的复合富集材料RADA16-I/DBM不但具有三维空间结构,适当的孔径,而且稳定性好,是一种较理想的骨髓富集材料。(2)应用SCR技术能使复合材料RADA16-I/DBM富集较高浓度的骨髓有核细胞,同时能富集较高浓度的促成骨生长因子。(3)SCR技术即刻构建的骨移植材料具有较高的成骨活性,在裸鼠肌袋成骨模型中有较强的成骨能力。
Background: Bone defects, which are caused by some factors such as high energy trauma, large bone tumor resection, and severe bone infection are common, and the demands for bone graft material about artificial prosthetic replacement and spinal fusion are also very great. Autologous bone graft is still the gold standard of bone transplantation, but it increases the surgical procedure and causes many new problems such as the chronic pain, limited bone etc. In addition, autogenous bone graft does not apply to the elderly, minors and so on. Local injection of autologous red bone marrow in the treatment of nonunion and bone defect have made some success. But it requires a large amount of bone marrow, and leads to the loss of effective ingredients of bone. Therefore, now it is seldom used in clinical work. Tissue engineering bone to be built with Autologous cells in vitro by conventional amplification is an ideal bone repair materials. It is not only meeting the requirements of the cell mass, but also avoiding the ethical and moral limits. The disadvantage is that amplification of autologous cells in vitro often requires twice operations: the collection of autologous cells in vitro and returning to the areas. It spends about two weeks in vitro for amplification and more cost for the treatment. There are still some difficulties to apply it for clinical works for its high level of technique. Selective cell retention technology(SCR) is reported as one of enriching technology of bone marrow by scholars of home and aboard in recent years. SCR technology may enrich effective ingredients such as stem cells and bone factor by the good adhesion and the appropriate mesh structure of enriched material by negative pressure of device when the bone marrow through them, thus it is easy to build a appreciate enriching material immediately. SCR technology has obtained ideal results in the foreign animal experiments and clinical trials. The significant advantages of SCR technology lies in the osteogenic activity, high safty in operation, and avoiding ethical issues. Thus SCR has brought new hope for clinical bone repair.
Objective: (1) Preparating a new type of bone marrow enriching materials and testing its performance. (2) Approching the enriching effects of SCR technology for the nuclear cells and growth factors with bone formation in the bone marrow. (3) Observing the bone formation results of transplanting with enriching material within muscle pouch of nude mice.
Methods: (1) Goat demineralized bone matrix (DBM) is combinated with self-assembling peptide RADA16-Ⅰto composite materials in PBS solution, followed by the analysis of three-dimensional structure and pore size though three-dimensional video microscope ,microscopic structure though scanning electron and relationship between RADA16-Ⅰwith DBM though X ray photoelectron spectroscopy analyzer (XPS),and also test the stability of composite materials though UV spectrophotometer. (2) Aspirating the iliac bone marrow from goats and inserting into the enriching dish to observe the enriching effect of RADA16-Ⅰ/DBM about nucleated cells and growth factors with bone formation in bone marrow by SCR. (3) Observing the osteogenesis of bone graft material in muscle pouch of nude mice by SCR technology instantly building bone graft material. 24 mice were randomly divided into three groups, n = 8. Bone graft materials were divided into the following three groups (A ,SCR group, RADA16-I/DBM +SCR materials; B, bone marrow immersion group,RADA16-I/DBM + bone marrow immersion materials; C, blank group, RADA16-I/DBM material).Group A, B, C were implanted into muscle pouch beside both of iliac of nude mice of three groups randomly and then suture muscle pouch. After 4 and 8 weeks of the surgery, respectively, nude mice were killed in batches (Four of mice were killed in each batch of each group), and detecting the osteogenic potential by X-ray,three dimensions CT and histological observation to observe the bone formation ability.
Results: (1) Blank DBM has three-dimensional mesh structure of natural bone. Although a large number of big pores within the small pores connected with each other, the aperture are still relatively large. However, the colorless jelly like adhesion in three-dimensional mesh scaffolds and the DBM bone surface is visible in composite materials RADA16-I/DBM. Measuring DBM pore size (414.32±175.00)μm (n = 60), RADA16-I/DBM pore size (220.55±158.54)μm (n = 60), p <0.05, through image processing system of three-dimensional video microscope. It is that overall mean diameter was significantly different between DBM group and RADA16-I/DBM group. Detecting about the stability of RADA16-I/DBM shows that its spectrum of immersion UV is not the full, which is roughly the same spectra to PBS solution, indicating that UV-sensitive components are less or no about them; UV spectra show a more strong UV absorption properties of amino acids in the 222nm absorption peak about 0.5% RADA16-I peptide in solution, and the absorbance peak is greater than 2.0, indicating that more amino acids present in the test solution. DBM and RADA16-I peptide, both of them are mainly consisted of carbon (C), nitrogen (N), oxygen (O). The N of RADA16-I /DBM is increasing significantly compared with the control group DBM; (2)The statistical calculations between RADA16-I/DBM and DBM enriching effects showed that enriching effect of nucleated cells was (6.05±0.96) (n = 12 )about RADA16-I/DBM composite materials ,which is significantly higher than the control group DBM which is(2.27±0.41) (n = 12), and the overall mean difference between them is significant (p <0.05). The resuits of HE staining shows that: under the same enriching conditions (amount of bone marrow,amount of materials, enriching time, enriching speed and the number of enrichment are the same),to RADA16-I/DBM composites material, clusters of nucleated cells along the direction of bone matrix were raising and bone matrix is also surrounded by more nucleated cells; To control group DBM, nucleated cells are less around the bone matrix. Enriching effects about growth factors of bone formation of RADA16-I/DBM showed that bone morphogenetic protein -2 (BMP-2), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I) of the enrichment factor were (23.03±2.68) times, (19.68±4.02) times, (31.02±2.09) times respectively, while the corresponding enriching effects for the DBM were (6.63±1.42) times, (5.66±0.74) times, (5.61±1.59) Times, both of the overall mean was significantly different (p <0.05), that is enriching effects about bone growth factors of RADA16-I/DBM were higher than DBM. Adhesion rates of RADA16-I / DBM about BMP-2, PDGF, IGF-I were (58%±6.7%), (49%±10%), (60%±8.9%), compared to DBM's corresponding (33%±7.1%), (28%±3.7%), (28%±7.7%), both of the overall mean was significantly different (p <0.05), that is adhesion rate of RADA16-I/DBM for bone growth factors was significantly higher than DBM. (3) Observating from muscle pouch beside iliac bone of nude mice the bone graft area of animals in the 4 weeks does
not show significantly high-density screenage. SCR group shows high density of calcification than other two images in the 8 weeks, which suggests new bone formation at the bone graft area. Meanwhile, RADA16-I/DBM + bone marrow immersion group shows fuzzy density screenage, suggesting that new bone has been formatted partly, but the degree of calcification is low.However, blank group, RADA16-I/DBM does not show screenage, which suggests that the graft material is absorbed on the whole, and CT results shows that: CT values of bone graft of SCR group, RADA16-I/DBM + bone marrow immersion and RADA16-I/DBM were (547.71±62.01) HU, (331.58±19.28) HU, (77.08±8.75) HU (n = 8) respectively, and the overall number of those were significantly different (p <0.05), that is the CT value of the SCR group, was significantly higher than RADA16-I/DBM + bone marrow immersion and RADA16-I/DBM, the bone graft materials built by SCR have highly osteogenic activity. Histological observations suggest that: SCR group show higher graft osteogenesis and RADA16-I/DBM + bone marrow immersion materials show some capabilities of bone formation, but the ability of bone formation is not strong.But, RADA16-I/DBM group materials are basically absorbed, and without neoplastic bone formation.
Conclusions: (1) RADA16-I/DBM composite material which not only has the three-dimensional structure and the appropriate pore size, but also has good stability is an ideal bone marrow enriched material. (2) RADA16-I/DBM composite materials can enrich highly concentrative nucleus cells and highly concentrative bone growth factors from bone marrow by SCR technology.(3) Bone graft material built instantly by SCR technology has a high osteogenic activity for bone formation in muscle pouches of nude mice model.
引文
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2 Connolly JF,Guse R,Tiedeman J,Dehne R.Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop 1991;266:259–270.
3 Connolly J,Guse R,Lippiello L,etal.Development of an osteogenic bone-marrow preparation.J Bone Joint Surg (Am),2005,71:684-691.
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5 Kon E, Murglia A, Corsi A, et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatie ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res, 2000, 49: 328–337.
6 Isogai N, Kusuhara H, Ikada Y, et al. Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue Eng, 2006 , 12(4): 691–703.
7 Daisuke T, Isador H, Lieberman, et al. Clinical and radiographic outcomes of lumbar interbody fusion using allograft enriched with bone marrow derived cells prepared by selective stem cell retention. The Spine Journal, 2004, 4:118–119.
8 Muschler GF, Matsukura Y, Nitto H, et al. Selective retention of bone marrow-derived cells to enhance spinal fusion. Clin Orthop Relat Res, 2005, ( 432): 242–251.
9 Brodke D, Pedrozo HA, Kaput TA, et al. Bone grafts prepared with selective cell retention technology heal canine segmental defects as effectively as autograft. J Orthop Res, 2006, 24(5): 857–866.
10 Gupta MC, Theerajun yaporn T, Maitra S, et al. Efficacy of mesenchymal stem cell enriched grafts in an ovine posterolateral lumbar spine model.Spine, 2007, 32(7): 720–727.
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2 Connolly JF,Guse R,Tiedeman J,Dehne R.Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop 1991;266:259–270.
3 Connolly J,Guse R,Lippiello L,etal.Development of an osteogenic bone-marrow preparation.J Bone Joint Surg (Am),2005,71:684-691.
4张蒲,干耀恺,李华等.富集骨髓干细胞结合β-磷酸三钙在脊柱融合的临床应用[J].临床骨科杂志,2006,9(1):1-4.
5 Kon E, Murglia A, Corsi A, et al. Autologous bone marrow stromal cells loaded onto porous hydroxyapatie ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res, 2000, 49: 328–337.
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