生物反应器内应用富血小板血浆构建血管化复合体修复骨坏死的研究
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摘要
股骨头缺血性坏死是由于各种病因破坏了股骨头的血液供应,造成的以股骨头内骨小梁和骨髓坏死为特征的临床常见病,治疗不及时可导致终生残疾,是骨科医生面临的一大难题。骨坏死区的根本问题是成骨祖细胞和血管缺乏,坏死区域骨血管的形成是骨组织修复的前提,各国学者将研究点集中于如何促进骨坏死区骨小梁再生、新生血管形成。富血小板血浆(Platelet-rich plasma,PRP)是新鲜全血经离心后获取的自体血小板的浓缩体,含有多种高浓度的生长因子,包括：血小板源性生长因子(platelet derived growth factor, PDGF)、转移生长因子(transforming growth factor, TGF-β)、血管内皮生长因子(vascular endothelial growth factor, VEGF)、类胰岛素生长因子(insulin-like growthfactor,IGF)、表皮生长因子(epidermal growth factor,EGF)等。本项目拟将骨髓基质细胞(bone marrow stromal cells, BMSCs)与p-磷酸三钙(p-TCP)在灌注式生物反应器(bioreatctor, BO)内复合三维培养,利用PRP的促血管生成、成骨作用,构建高质量的血管化复合体,在MRI实时导引下利用氩氦冷冻消融建立兔股骨头坏死动物模型,同时在MRI导引下通过氩氦刀探针钉道将构建的血管化复合体精确置入到股骨头骨坏死区,通过大体观察、X线检测、组织学评价观察其促进骨坏死修复的作用,期望为临床治疗早期股骨头缺血性坏死提供新的治疗方法。
1MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的实验研究
目的探讨MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的可行性和技术方法
方法取2-3月龄的健康的新西兰大白兔48只,静脉麻醉成功后置于开放式0.23TMRI系统中,MRI导引下在大转子下方1cm向股骨头负重区打入2mm导引针,位置满意后置入氩氦刀探针,左侧实验组股骨头氩氦冷冻消融2个循环20分钟,右侧股骨头对照组氩氦冷冻消融1个循环10分钟。分别于术后4周、8周、12周三个时间点行X线检查、大体观察和组织学观察,评估股骨头组织的坏死和修复情况。
结果术后4、8、12周股骨头组织学评估,实验组1组股骨头陷窝细胞分别是49.75士3.17,62.06±4.12,48.25±2.76,高于对照组2组39.13±4.48,50.69±3.84,37.50±3.86。术后8周实验组陷窝细胞率为62.06%。术后12周实验组股骨头塌陷率43.7%,对照组股骨头塌陷率12.5%。
结论MRI实时导引下利用氩氦冷冻消融成功的建立了兔股骨头缺血性坏死模型,术后4周、8周、12周实验组陷窝细胞率均明显高于对照组。
2灌注式生物反应器应用富血小板血浆构建细胞-支架复合体体内血管化评估目的灌注式生物反应器内应用富血小板血浆构建BMSCs-β-TCP复合体并评估其体内血管化情况。
方法从兔耳缘静脉抽取新鲜血液,采用二次离心法制备富血小板血浆,与低糖DMEM培养液制备全培养基。体外将兔骨髓间充质细胞与β-磷酸三钙陶瓷复合培养后植入到灌注式生物反应器内,定期更换全培养基,3周后取出复合体,进行扫描电镜检测。将各组不同方式培养的复合体植入到兔皮下组织内,术后12周,取出复合体,免疫组织化学法检测血管标志物血小板-内皮细胞粘附分子(Cluster of Diffrentiation31,CD31).第Ⅷ因子关连抗原(von willebrand factor,vWF)的表达.
结果实验组电镜扫描显示β—磷酸三钙(p-TCP)上可见有细胞粘附,细胞伸出伪足和p一磷酸三钙(β-TCP)相连接,骨髓间充质干细胞细胞在β-TCP多孔陶瓷支架上的黏附、伸展和增殖良好。实验组免疫组化结果显示血管标志物CD31.第Ⅷ因子关连抗原(VWF)呈强阳性表现,其他组CD31.vWF的表达较少。结论富血小板血浆富含多种细胞因子,灌注式生物反应器内应用富血小板血浆构建细胞-支架复合体可以明显促进复合体血管化。
3灌注式生物反应器内构建血管化BMSCs-β-TCP复合体修复骨坏死的实验研究
目的探讨灌注式生物反应内构建血管化BMSCs-β-TCP复合体促进骨坏死修复的作用。
方法分别用实验1、2描述的方法建立新西兰兔股骨头缺血性坏死模型和构建血管化复合体。30只兔随机分为六组,A组钉道中不植人任何材料,B组植入β-TCP支架,C组MSCs-β-TCP, D组MSCs-β-TCP+bioreatctor, E组MSCs-β-TCP+PRP,F组MSCs-β-TCP+bioreactor+PRP。将不同方式构建的BMSCs-β-TCP复合体沿股骨头建模制作的钉道植入到股骨头坏死区,术后12周利用大体观察、X线检查和组织学观察评估其促进骨坏死修复的作用。
结果术后12周,各组X线检查示：A组、B组均有部分股骨头骨皮质不完整,局部骨密度高；C、D、E组未见有股骨头明显塌陷；F组未见有股骨头塌陷,坏死区骨密度与宿主区骨密度相近。组织学结果显示：A组、B组见较多纤维组织,骨形成少；C、D、E组少量骨形成,骨小梁排列不整齐；F组骨形成量较多,骨小梁排列良好,与正常骨小梁结构相近。
结论生物反应器内构建血管化组织工程骨可以有效修复骨坏死,从而为临床治疗股骨头缺血性坏死提供了可行的方法。
Avascular necrosis of the femoral head is a major problem for orthopaedic surgeons, there is no way to cure. The basic problem of bone necrotic zone is the deficiency of osteoblast progenitor cells and vascular. Vascularization in necrotic area can increase the rate of bone tissue repair. Platelet-rich plasma(PRP) is autologous platelet concentrates after centrifugating the fresh whole blood, which contains growth factors and bioactive proteins,including platelet derived growth factor(PDGF),transforming growth factor(TGF-β),insulin-like growth factor(IGF),epidermal growth factor(EGF)and vascular endothelial growth factor(VEGF). PRP can enhance the tissue repair, vascularization and bone formation. This project aims to building high-quality vascularized tissue-engineered bone complex, cocultured three-dimensional dynamically bone marrow stromal cells (BMSCs) and beta three calcium of phosphoric acid ((3-TCP) in perfusion bioreactor, using the effect of angiogenesis and bone repair of PRP. Under the guidance of MRI, copying the rabbit model of necrosis of femoral head using cryoablation, the vascularized tissue-engineered bone complex is precisely plugged into the necrotic area of the femoral head through the argon helium cryoablatio probe tract. Gross observation, bone density measurement, MRI test, histological evaluation is used to observe bone repair in necrotic area, expecting to find a new breakthrough for the clinical treatment of early avascular necrosis of femoral head.1A novel animal model of osteonecrosis of the femoral head induced using a magnetic resonance imaging-guided argon-helium cryotherapy system
Abstract The aim of the present study was to establish a novel animal model of osteonecrosis of the femoral head (ONFH) using a magnetic resonance imaging (MRI)-guided argon-helium cryotherapy system. A total of48rabbits were used to generate the ONFH models. In group Ⅰ, the left femoral head of the rabbits received two cycles of argon-helium freezing-thawing under MRI guidance, while in group Ⅱ, the right femoral head of each rabbit received only one cycle of argon-helium freezing-thawing. X-ray, roentgenographic and histological examinations were performed. The percentages of lacunae in the femoral heads of group Ⅰ at weeks4,8and12following surgery (49.75±3.17,62.06±4.12and48.25±2.76%, respectively) were higher than those in group Ⅱ (39.13±4.48,50.69±3.84and37.50±3.86%, respectively). In addition, the percentage of empty lacunae in group Ⅰ was62.06%at week8following surgery. Therefore, an animal model of ONFH was successfully established using an argon-helium cryotherapy system. The percentage of empty lacunae in group Ⅰ was higher than that in group Ⅱ at weeks4,8and12after surgery.
2Construction of tissue engineered bone by using bioreactor and platelet-rich plasma
Abstract This study is to construct tissue engineered bone by using bioreactor and platelet-rich plasma (PRP). Bone marrow mesenchymal stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) were cultured in perfusion bioreactor and in PRP containing medium for21days to form BMSC-TCP composite. Rabbits were implanted with BMSC-TCP composite. Morphology of implanted BMSC-TCP composite was observed by scanning electron microscope and HE staining. Expression of CD31and VWF in implanted BMSC-TCP composite was detected by immunohistochemistry. After culturing in perfusion bioreactor and PRP, BMSCs were adhered on the β-TCP scaffold and the secretion of extracellular matrix was also seen. The stretching and proliferation of cells was good on the scaffold. Vascular endothelial cell markers of CD31and VEF were positively expressed. Tissue engineered bone can be constructed by using bioreactor and PRP. PRP, which contains multiple growth factors, may promote vascularization of tissue engineered bone.
3Repair of bone necrosis with vascularized tissue-engineered bone coculured in bioreactor using platelet-rich plasma
Abstract The aim of this study is to explore the effect of repair of bone necrosis with vascularized tissue-engineered bone coculured in bioreactor using platelet-rich plasma. A total of30New Zealand adult rabbits (3.0±0.3Kg) were used to generate the ONFH models using this way described in Experiment1.Both femoral heads of these rabbits received two circles of two cycles of argon-helium freezing-thawing under MRI guidance. Vascularize tissue-engineered bone cocultured in bioreactor using platelet-rich plasma using the way described in Experiment2. Thirty rabbits were randomly divided into groups A, B, C, D,E and F (5rabbits for each group). The defects were then repaired with various materials in the different groups:Group A with nothing, Group B withβ-TCP scaffold, Group C with MSCs-β-TCP composites, Group D with MSCs-β-TCP composites cocultured in bioreatctor, Group E with MSCs-β-TCP composites cocultured using PRP, and Group F with MSCs-β-TCP composites cocultured in bioreactor using PRP. All the rabbits were raised separately postoperatively. The rabbits were sacrificed and all femoral heads were evaluated with X-ray at twelve weeks postoperatively. The new bone formation area was evaluated from three slides of each specimen through the HPIAS-1000image analysis system and light microscopy. The percentage of new bone formation was calculated by the following formula:area of new bone÷area of observation×100%.In Group F, The ability of tissue engineered bone to repair the osteonecrosis was close to that of cancellous bone autograft. And there was no femoral head collapse. The new animal model of ONFH could be induced by argon-helium cryotherapy system, and the tissue engineering technique will provide an effective treatment.
1MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的实验研究
目的探讨MRI实时导引下氩氦冷冻消融建立新西兰兔股骨头缺血性坏死模型的可行性和技术方法
方法取2-3月龄的健康的新西兰大白兔48只,静脉麻醉成功后置于开放式0.23TMRI系统中,MRI导引下在大转子下方1cm向股骨头负重区打入2mm导引针,位置满意后置入氩氦刀探针,左侧实验组股骨头氩氦冷冻消融2个循环20分钟,右侧股骨头对照组氩氦冷冻消融1个循环10分钟。分别于术后4周、8周、12周三个时间点行X线检查、大体观察和组织学观察,评估股骨头组织的坏死和修复情况。
结果术后4、8、12周股骨头组织学评估,实验组1组股骨头陷窝细胞分别是49.75士3.17,62.06±4.12,48.25±2.76,高于对照组2组39.13±4.48,50.69±3.84,37.50±3.86。术后8周实验组陷窝细胞率为62.06%。术后12周实验组股骨头塌陷率43.7%,对照组股骨头塌陷率12.5%。
结论MRI实时导引下利用氩氦冷冻消融成功的建立了兔股骨头缺血性坏死模型,术后4周、8周、12周实验组陷窝细胞率均明显高于对照组。
2灌注式生物反应器应用富血小板血浆构建细胞-支架复合体体内血管化评估目的灌注式生物反应器内应用富血小板血浆构建BMSCs-β-TCP复合体并评估其体内血管化情况。
方法从兔耳缘静脉抽取新鲜血液,采用二次离心法制备富血小板血浆,与低糖DMEM培养液制备全培养基。体外将兔骨髓间充质细胞与β-磷酸三钙陶瓷复合培养后植入到灌注式生物反应器内,定期更换全培养基,3周后取出复合体,进行扫描电镜检测。将各组不同方式培养的复合体植入到兔皮下组织内,术后12周,取出复合体,免疫组织化学法检测血管标志物血小板-内皮细胞粘附分子(Cluster of Diffrentiation31,CD31).第Ⅷ因子关连抗原(von willebrand factor,vWF)的表达.
结果实验组电镜扫描显示β—磷酸三钙(p-TCP)上可见有细胞粘附,细胞伸出伪足和p一磷酸三钙(β-TCP)相连接,骨髓间充质干细胞细胞在β-TCP多孔陶瓷支架上的黏附、伸展和增殖良好。实验组免疫组化结果显示血管标志物CD31.第Ⅷ因子关连抗原(VWF)呈强阳性表现,其他组CD31.vWF的表达较少。结论富血小板血浆富含多种细胞因子,灌注式生物反应器内应用富血小板血浆构建细胞-支架复合体可以明显促进复合体血管化。
3灌注式生物反应器内构建血管化BMSCs-β-TCP复合体修复骨坏死的实验研究
目的探讨灌注式生物反应内构建血管化BMSCs-β-TCP复合体促进骨坏死修复的作用。
方法分别用实验1、2描述的方法建立新西兰兔股骨头缺血性坏死模型和构建血管化复合体。30只兔随机分为六组,A组钉道中不植人任何材料,B组植入β-TCP支架,C组MSCs-β-TCP, D组MSCs-β-TCP+bioreatctor, E组MSCs-β-TCP+PRP,F组MSCs-β-TCP+bioreactor+PRP。将不同方式构建的BMSCs-β-TCP复合体沿股骨头建模制作的钉道植入到股骨头坏死区,术后12周利用大体观察、X线检查和组织学观察评估其促进骨坏死修复的作用。
结果术后12周,各组X线检查示：A组、B组均有部分股骨头骨皮质不完整,局部骨密度高；C、D、E组未见有股骨头明显塌陷；F组未见有股骨头塌陷,坏死区骨密度与宿主区骨密度相近。组织学结果显示：A组、B组见较多纤维组织,骨形成少；C、D、E组少量骨形成,骨小梁排列不整齐；F组骨形成量较多,骨小梁排列良好,与正常骨小梁结构相近。
结论生物反应器内构建血管化组织工程骨可以有效修复骨坏死,从而为临床治疗股骨头缺血性坏死提供了可行的方法。
Avascular necrosis of the femoral head is a major problem for orthopaedic surgeons, there is no way to cure. The basic problem of bone necrotic zone is the deficiency of osteoblast progenitor cells and vascular. Vascularization in necrotic area can increase the rate of bone tissue repair. Platelet-rich plasma(PRP) is autologous platelet concentrates after centrifugating the fresh whole blood, which contains growth factors and bioactive proteins,including platelet derived growth factor(PDGF),transforming growth factor(TGF-β),insulin-like growth factor(IGF),epidermal growth factor(EGF)and vascular endothelial growth factor(VEGF). PRP can enhance the tissue repair, vascularization and bone formation. This project aims to building high-quality vascularized tissue-engineered bone complex, cocultured three-dimensional dynamically bone marrow stromal cells (BMSCs) and beta three calcium of phosphoric acid ((3-TCP) in perfusion bioreactor, using the effect of angiogenesis and bone repair of PRP. Under the guidance of MRI, copying the rabbit model of necrosis of femoral head using cryoablation, the vascularized tissue-engineered bone complex is precisely plugged into the necrotic area of the femoral head through the argon helium cryoablatio probe tract. Gross observation, bone density measurement, MRI test, histological evaluation is used to observe bone repair in necrotic area, expecting to find a new breakthrough for the clinical treatment of early avascular necrosis of femoral head.1A novel animal model of osteonecrosis of the femoral head induced using a magnetic resonance imaging-guided argon-helium cryotherapy system
Abstract The aim of the present study was to establish a novel animal model of osteonecrosis of the femoral head (ONFH) using a magnetic resonance imaging (MRI)-guided argon-helium cryotherapy system. A total of48rabbits were used to generate the ONFH models. In group Ⅰ, the left femoral head of the rabbits received two cycles of argon-helium freezing-thawing under MRI guidance, while in group Ⅱ, the right femoral head of each rabbit received only one cycle of argon-helium freezing-thawing. X-ray, roentgenographic and histological examinations were performed. The percentages of lacunae in the femoral heads of group Ⅰ at weeks4,8and12following surgery (49.75±3.17,62.06±4.12and48.25±2.76%, respectively) were higher than those in group Ⅱ (39.13±4.48,50.69±3.84and37.50±3.86%, respectively). In addition, the percentage of empty lacunae in group Ⅰ was62.06%at week8following surgery. Therefore, an animal model of ONFH was successfully established using an argon-helium cryotherapy system. The percentage of empty lacunae in group Ⅰ was higher than that in group Ⅱ at weeks4,8and12after surgery.
2Construction of tissue engineered bone by using bioreactor and platelet-rich plasma
Abstract This study is to construct tissue engineered bone by using bioreactor and platelet-rich plasma (PRP). Bone marrow mesenchymal stem cells (BMSCs) and β-tricalcium phosphate (β-TCP) were cultured in perfusion bioreactor and in PRP containing medium for21days to form BMSC-TCP composite. Rabbits were implanted with BMSC-TCP composite. Morphology of implanted BMSC-TCP composite was observed by scanning electron microscope and HE staining. Expression of CD31and VWF in implanted BMSC-TCP composite was detected by immunohistochemistry. After culturing in perfusion bioreactor and PRP, BMSCs were adhered on the β-TCP scaffold and the secretion of extracellular matrix was also seen. The stretching and proliferation of cells was good on the scaffold. Vascular endothelial cell markers of CD31and VEF were positively expressed. Tissue engineered bone can be constructed by using bioreactor and PRP. PRP, which contains multiple growth factors, may promote vascularization of tissue engineered bone.
3Repair of bone necrosis with vascularized tissue-engineered bone coculured in bioreactor using platelet-rich plasma
Abstract The aim of this study is to explore the effect of repair of bone necrosis with vascularized tissue-engineered bone coculured in bioreactor using platelet-rich plasma. A total of30New Zealand adult rabbits (3.0±0.3Kg) were used to generate the ONFH models using this way described in Experiment1.Both femoral heads of these rabbits received two circles of two cycles of argon-helium freezing-thawing under MRI guidance. Vascularize tissue-engineered bone cocultured in bioreactor using platelet-rich plasma using the way described in Experiment2. Thirty rabbits were randomly divided into groups A, B, C, D,E and F (5rabbits for each group). The defects were then repaired with various materials in the different groups:Group A with nothing, Group B withβ-TCP scaffold, Group C with MSCs-β-TCP composites, Group D with MSCs-β-TCP composites cocultured in bioreatctor, Group E with MSCs-β-TCP composites cocultured using PRP, and Group F with MSCs-β-TCP composites cocultured in bioreactor using PRP. All the rabbits were raised separately postoperatively. The rabbits were sacrificed and all femoral heads were evaluated with X-ray at twelve weeks postoperatively. The new bone formation area was evaluated from three slides of each specimen through the HPIAS-1000image analysis system and light microscopy. The percentage of new bone formation was calculated by the following formula:area of new bone÷area of observation×100%.In Group F, The ability of tissue engineered bone to repair the osteonecrosis was close to that of cancellous bone autograft. And there was no femoral head collapse. The new animal model of ONFH could be induced by argon-helium cryotherapy system, and the tissue engineering technique will provide an effective treatment.
引文
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[13]Hauzeur JPH, Pasteels JL. Bone biopsy as diagnostic criteria for aseptic necrosis of the femoral head [M] //Schoutens A, Arlet J, Gardeniers JWM, et al. Bone circulation and avascularization in normal and pathological conditions. New york:Plenum Pless,1993:277.
[14]Malizos KN, Quarles LD, Seaber AV, Rizk WS, Urbaniak JR. An experimental canine model of osteonecrosis:characterization of the repair process. J Orthop Res.1993 May;11(3):350-7.
[15]Nishino M, Matsumoto T, Nakamura T, Tomita K.Pathological and hemodynamic study in a new model of femoral head necrosis following traumatic dislocation.Arch Orthop Trauma Surg.1997;116(5):259-62.
[16]Be jar J, Peled E, Boss JH. Vasculature deprivation--induced osteonecrosis of the rat femoral head as a model for therapeutic trials. Theor Biol Med Model.2005 Jul 5;2:24. Review.
[17]Hofstaetter JG, Wang J, Yan J, et al. The efects of alendronate in the treatment of experimental osteonecrosis of the hip in aduh rabbits[J]. Osteoarthritis Cartilage, 2009,17(3):362.370.
[18]Swiontkowski MF, Tepic S, Rahn BA, et al. The efect of fracture on femoral head blood flow. Osteonecrosis and re-vascularization studied in miniature swine[J]. Acta Orthop Scand,1993,64(2):196-202.
[19]Seiler JG, Kregor PJ, Conrad EU, et al. Posttraumatic oteonecrosis in a swine model Correlation of blood cell flux, MRI and histology [J]. Acta Onhop Scand, 1996,67(3):249-254.
[20]Kim HK, Su PH, Qiu YS. Histopathologic changes in growth-plate cartilage following ischemic necrosis of the capital femoral epiphysis. An experimental investigation in immature pigs. J Bone Joint Surg Am.2001 May;83-A(5):688-97.
[21]Koob TJ,Pringle D,Gedbaw E, Meredith J,Berrios R, Kim HK. Biomechanical properties of bone and cartilage in growing femoral head following ischemic osteonecrosis. J Orthop Res.2007 Jun;25(6):750-7.
[22]Hofstaetter JG, Roschger P, Klaushofer K, et al. Increased matrix mineralization in the immature femoral head following ischemic osteonecrosis[J]. Bone,2010, 46(2):379.385.
[23]Manggold J, Sergi C, Becker K, Lukoschek M, Simank HG. A new animal model of femoral head necrosis induced by intraosseous injection of et hanol. Lab Anim.2002 Apr;36(2):173-80.
[24]Zhu ZH, Gao YS, Luo SH, Zeng BF, Zhang CQ. An animal model of femoral head osteonecrosis induced by a single injection of absolute alcohol:an experimental study. Med Sci Monit.2011 Apr;17(4):BR97-102.
[25]Gao YS, Wang HF, Ding H, Zhang CQ. A novel rat model of osteonecrosis of the femoral head induced by periarticular injection of vascular endothelial growth factor receptor 2 antibody. J Surg Res.2013 Jul;183(1):e1-5. doi: 10.1016/j.jss.2013.01.046. Epub 2013 Feb 11.
[26]Conzemius MG, Brown TD, Zhang Y, et al. A new animal model of femoral head osteonecrosis:one that progresses to human like mechanical failure[J]. J Orthop Res,2002,20(2):303-309.
[27]Reed KL, Brown TD, Conzemius MG. Focal cryogen insults for inducing segmental osteonecrosis:computational and experimental assessments of thermal fields[J]. J Biomech,2003,36(9):1317-1326.
[28]范猛,汪爱媛,王玉,等.局部冷热交替损伤建立鸸鹋股骨头坏死塌陷模型.中国医学科学院学报.2011(04)：375-81
[29]Fan M, Peng J, Wang A, Zhang L, Liu B, Ren Z, Xu W, Sun J, Xu L, Xiao D, Qin L, Lu S, Wang Y, Guo QY. Emu model of full-range femoral head osteonecrosis induced focally by an alternating freezing and heating insult. J Int Med Res.2011;39(1):187-98.
[30]Li Y, Han R, Geng C, Wang Y, Wei L. A new osteonecrosis animal model of the femoral head induced by microwave heating and repaired with tissueengineered b one. Int Orthop.2009 Apr;33(2):573-80. doi:10.1007/s00264-008-0672-2. Epub 2008 Oct 28.
[31]Miyanishi K, Yamamoto T, Irisa T, Motomura G, Jingushi S, Sueishi K, Iwamoto Y. Effects of different corticosteroids on the development of osteonecrosis in rabbits. Rheumatology (Oxford).2005 Mar;44(3):332-6. Epub 2004 Dec 14. [32]Motomura G, Yamamoto T, Irisa T, Miyanishi K, Nishida K, Iwamoto Y. Dose effects of corticosteroids on the development of osteonecrosis in rabbits. J Rheumatol.2008 Dec;35(12):2395-9. Epub 2008 Oct 1.
[33]Yang L, BoydK, Kaste SC, et al. Amousemodelfor glucocorticoid-induced ostconeerosis:efect ofa steroid holiday[J]. JOrthopRes,2009,27(2):169-175
[34]程田,李月白,王义生.短期应用大剂量激素导致鸡股骨头坏死[J].郑州大学学报医学版,2009,44(2)：285—287。
[35]Mont M A, Jones L C, Hungerford D S. Nontraumatic osteonecrosis of the femoral head ten years later[J]. Bone Joint Surg Am,2006,88(5):1117-1132.
[36]Takano-Murakami R, Tokunaga K, Kondo N, Ito T, Kitahara H, Ito M, Endo N. Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats. Tohoku J Exp Med.2009 Jan; 217(1):51-8.
[37]王泳,高春锦,杨晋才,等.兔脊髓性股骨头坏死模型的建立[J].中国病理生理杂志,2009,25(11)：2233-2234、2239。
[38]Yamamoto T, Hirano K, Tsutsui H, et al. Corticosteroid enhances the experimental induction of osteonecrosis in rabbits witll Shwartzman reaction. Clin Orthop Relat Res,1995,316:235-243.
[39]Guan XY, Han D. Role of hypercoagulability in steroid-induced femoral head necrosis in rabbits. J Orthop Sci.2010 May; 15(3):365-70. doi: 10.1007/s00776-010-1452-6. Epub 2010 Jun 18.
[40]Matsui M, Saito S, Ohzono K, Sugano N, Saito M, Takaoka K, Ono K. Experimental steroid-induced osteonecrosis in adult rabbits with hypersensitivity vasculitis. Clin Orthop Relat Res.1992 Apr;(277):61-72.
[41]陈彦华,殷慧芬,王式鲁.马血清加激素诱导股骨头缺血性坏死动物模型的实验研究[J],山东中医药大学学报,2003(3)：222-223。
[42]Takaoka K, Yoshioka T, Hosoya T, et al:The repair process in experimentally induced avascular necrosis of the femoral head in dogs. Arch Orthop Trauma Surg 99: 109-115,1981.
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[44]Bonutti PM, Seyler TM, Delanois RE, et al:Osteonecrosis of the knee after laser or radiofrequency-assisted arthroscopy. treatment with minimally invasive knee arthroplasty. J Bone Joint Surg Am 88 (Suppl 3):69-75,2006.
[45]Robinson D, Halperin N, Nevo Z. Two freezing cycles ensure interface sterilization by cryosurgery during bone tumor resection. Cryobiology.2001 Aug;43(1):4-10.
[46]Lim CT,Tan LB, Nathan SS. Prospective evaluation of argon gas probe delivery for cryotherapy of bone tumours. Ann Acad Med Singapore.2012 Aug;41(8):347-53.
[47]Gage AA, Baust JG.Cryosurgery for tumors. J Am Coll Surg.2007 Aug; 205(2):342-56. Epub 2007 Jun 18.
[48]Yanagawa B, Holmes SD, Henry L, Hunt S and Ad N:Outcome of concomitant Cox-maze Ⅲ procedure using an argon-based cryosurgical system:a single-center experience with 250 patients. Ann Thorac Surg 95:1633-1639,2013.
[49]Albage A, Peterffy M and Kallner G:Learning what works in surgical cryoablation of atrial fibrillation:results of different application techniques and benefits of prospective follow-up. Interact Cardiovasc Thorac Surg 13:480-484, 2011.
[50]Ad N, Henry L and Hunt S:The concomitant cryosurgical Cox-maze procedure using argon based cryoprobes:12 month results. J Cardiovasc Surg (Torino) 52: 593-599,2011.
[51]Wu B, Xiao YY, Zhang X, Zhao L and Carrino JA:CT-guided percutaneous cryoablation of osteoid osteoma in children:an initial study. Skeletal Radiol 40: 1303-1310,2011.
[52]Tacke J, Speetzen R, Adam G, Sellhaus B, Glowinski A, Heschel I, Schaffter T, Schorn R, Grosskortenhaus S, Rau G, Gunther RW. Experimental MR imaging-guided interstitial cryotherapy of the brain. AJNR Am J Neuroradiol.2001 Mar;22(3):431-40.
[53]van den Bosch MA, Josan S, Bouley DM, Chen J, Gill H, Rieke V, Butts-Pauly K, Daniel BL. MR imaging-guided percutaneous cryoablation of the prostate in an animal model:in vivo imaging ofcryoablation-induced tissue necrosis with immedi ate histopathologic correlation. J Vase Interv Radiol.2009 Feb;20(2):252-8. doi: 10.1016/j.jvir.2008.10.030. Epub 2008 Dec.
[54]Tacke J1, Adam G,Haage P, Sellhaus B, Grosskortenhaus S, Gunther RW. MR-guided percutaneous cryotherapy of the liver: in vivo evaluation with histologic correlation in an animalmodel. J Magn Reson Imaging.2001 Jan;13(1):50-6.
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[56]OwanI,Burr DB,Turner CH,et al.Mechanotransduction in bone:Osteoblasts are more responsive to fluid forces than mechanicalstrain[J].Am Jphysiol,1997,273: C810-815.
[57]Xie Y, Hardouin P, Zhu Z, Tang T, Dai K, Lu J. Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size beta-tricalcium phosphate scaffold. Tissue Eng.2006 Dec;12(12):3535-43.
[58]Botchwey EA, Pollack SR, Levine EM, Laurencin CT. Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system. J Biomed Mater Res.2001 May;55(2):242-53.
[59]李德强,戴尅戎,汤亭亭,等[J].利用灌注式生物反应器体外构建大段组织工程化骨折的实验研究.中华创伤骨科杂志,2009(5)：455-459.
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[12]Sun S, Ren Q, Wang D, Zhang L, Wu S, Sun XT. Repairing cartilage defects using chondrocyte and osteoblast composites developed using a bioreactor, Chin Med J (Engl),2011 Mar;124(5):758-63
[13]Hauzeur JPH, Pasteels JL. Bone biopsy as diagnostic criteria for aseptic necrosis of the femoral head [M] //Schoutens A, Arlet J, Gardeniers JWM, et al. Bone circulation and avascularization in normal and pathological conditions. New york:Plenum Pless,1993:277.
[14]Malizos KN, Quarles LD, Seaber AV, Rizk WS, Urbaniak JR. An experimental canine model of osteonecrosis:characterization of the repair process. J Orthop Res.1993 May;11(3):350-7.
[15]Nishino M, Matsumoto T, Nakamura T, Tomita K.Pathological and hemodynamic study in a new model of femoral head necrosis following traumatic dislocation.Arch Orthop Trauma Surg.1997;116(5):259-62.
[16]Be jar J, Peled E, Boss JH. Vasculature deprivation--induced osteonecrosis of the rat femoral head as a model for therapeutic trials. Theor Biol Med Model.2005 Jul 5;2:24. Review.
[17]Hofstaetter JG, Wang J, Yan J, et al. The efects of alendronate in the treatment of experimental osteonecrosis of the hip in aduh rabbits[J]. Osteoarthritis Cartilage, 2009,17(3):362.370.
[18]Swiontkowski MF, Tepic S, Rahn BA, et al. The efect of fracture on femoral head blood flow. Osteonecrosis and re-vascularization studied in miniature swine[J]. Acta Orthop Scand,1993,64(2):196-202.
[19]Seiler JG, Kregor PJ, Conrad EU, et al. Posttraumatic oteonecrosis in a swine model Correlation of blood cell flux, MRI and histology [J]. Acta Onhop Scand, 1996,67(3):249-254.
[20]Kim HK, Su PH, Qiu YS. Histopathologic changes in growth-plate cartilage following ischemic necrosis of the capital femoral epiphysis. An experimental investigation in immature pigs. J Bone Joint Surg Am.2001 May;83-A(5):688-97.
[21]Koob TJ,Pringle D,Gedbaw E, Meredith J,Berrios R, Kim HK. Biomechanical properties of bone and cartilage in growing femoral head following ischemic osteonecrosis. J Orthop Res.2007 Jun;25(6):750-7.
[22]Hofstaetter JG, Roschger P, Klaushofer K, et al. Increased matrix mineralization in the immature femoral head following ischemic osteonecrosis[J]. Bone,2010, 46(2):379.385.
[23]Manggold J, Sergi C, Becker K, Lukoschek M, Simank HG. A new animal model of femoral head necrosis induced by intraosseous injection of et hanol. Lab Anim.2002 Apr;36(2):173-80.
[24]Zhu ZH, Gao YS, Luo SH, Zeng BF, Zhang CQ. An animal model of femoral head osteonecrosis induced by a single injection of absolute alcohol:an experimental study. Med Sci Monit.2011 Apr;17(4):BR97-102.
[25]Gao YS, Wang HF, Ding H, Zhang CQ. A novel rat model of osteonecrosis of the femoral head induced by periarticular injection of vascular endothelial growth factor receptor 2 antibody. J Surg Res.2013 Jul;183(1):e1-5. doi: 10.1016/j.jss.2013.01.046. Epub 2013 Feb 11.
[26]Conzemius MG, Brown TD, Zhang Y, et al. A new animal model of femoral head osteonecrosis:one that progresses to human like mechanical failure[J]. J Orthop Res,2002,20(2):303-309.
[27]Reed KL, Brown TD, Conzemius MG. Focal cryogen insults for inducing segmental osteonecrosis:computational and experimental assessments of thermal fields[J]. J Biomech,2003,36(9):1317-1326.
[28]范猛,汪爱媛,王玉,等.局部冷热交替损伤建立鸸鹋股骨头坏死塌陷模型.中国医学科学院学报.2011(04)：375-81
[29]Fan M, Peng J, Wang A, Zhang L, Liu B, Ren Z, Xu W, Sun J, Xu L, Xiao D, Qin L, Lu S, Wang Y, Guo QY. Emu model of full-range femoral head osteonecrosis induced focally by an alternating freezing and heating insult. J Int Med Res.2011;39(1):187-98.
[30]Li Y, Han R, Geng C, Wang Y, Wei L. A new osteonecrosis animal model of the femoral head induced by microwave heating and repaired with tissueengineered b one. Int Orthop.2009 Apr;33(2):573-80. doi:10.1007/s00264-008-0672-2. Epub 2008 Oct 28.
[31]Miyanishi K, Yamamoto T, Irisa T, Motomura G, Jingushi S, Sueishi K, Iwamoto Y. Effects of different corticosteroids on the development of osteonecrosis in rabbits. Rheumatology (Oxford).2005 Mar;44(3):332-6. Epub 2004 Dec 14. [32]Motomura G, Yamamoto T, Irisa T, Miyanishi K, Nishida K, Iwamoto Y. Dose effects of corticosteroids on the development of osteonecrosis in rabbits. J Rheumatol.2008 Dec;35(12):2395-9. Epub 2008 Oct 1.
[33]Yang L, BoydK, Kaste SC, et al. Amousemodelfor glucocorticoid-induced ostconeerosis:efect ofa steroid holiday[J]. JOrthopRes,2009,27(2):169-175
[34]程田,李月白,王义生.短期应用大剂量激素导致鸡股骨头坏死[J].郑州大学学报医学版,2009,44(2)：285—287。
[35]Mont M A, Jones L C, Hungerford D S. Nontraumatic osteonecrosis of the femoral head ten years later[J]. Bone Joint Surg Am,2006,88(5):1117-1132.
[36]Takano-Murakami R, Tokunaga K, Kondo N, Ito T, Kitahara H, Ito M, Endo N. Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats. Tohoku J Exp Med.2009 Jan; 217(1):51-8.
[37]王泳,高春锦,杨晋才,等.兔脊髓性股骨头坏死模型的建立[J].中国病理生理杂志,2009,25(11)：2233-2234、2239。
[38]Yamamoto T, Hirano K, Tsutsui H, et al. Corticosteroid enhances the experimental induction of osteonecrosis in rabbits witll Shwartzman reaction. Clin Orthop Relat Res,1995,316:235-243.
[39]Guan XY, Han D. Role of hypercoagulability in steroid-induced femoral head necrosis in rabbits. J Orthop Sci.2010 May; 15(3):365-70. doi: 10.1007/s00776-010-1452-6. Epub 2010 Jun 18.
[40]Matsui M, Saito S, Ohzono K, Sugano N, Saito M, Takaoka K, Ono K. Experimental steroid-induced osteonecrosis in adult rabbits with hypersensitivity vasculitis. Clin Orthop Relat Res.1992 Apr;(277):61-72.
[41]陈彦华,殷慧芬,王式鲁.马血清加激素诱导股骨头缺血性坏死动物模型的实验研究[J],山东中医药大学学报,2003(3)：222-223。
[42]Takaoka K, Yoshioka T, Hosoya T, et al:The repair process in experimentally induced avascular necrosis of the femoral head in dogs. Arch Orthop Trauma Surg 99: 109-115,1981.
[43]Cetik O, Cift H, Comert B and Cirpar M:Risk of osteonecrosis of the femoral condyle after arthroscopic chondroplasty using radiofrequency:a prospective clinical series. Knee Surg Sports Traumatol Arthrosc 17:24-29,2009.
[44]Bonutti PM, Seyler TM, Delanois RE, et al:Osteonecrosis of the knee after laser or radiofrequency-assisted arthroscopy. treatment with minimally invasive knee arthroplasty. J Bone Joint Surg Am 88 (Suppl 3):69-75,2006.
[45]Robinson D, Halperin N, Nevo Z. Two freezing cycles ensure interface sterilization by cryosurgery during bone tumor resection. Cryobiology.2001 Aug;43(1):4-10.
[46]Lim CT,Tan LB, Nathan SS. Prospective evaluation of argon gas probe delivery for cryotherapy of bone tumours. Ann Acad Med Singapore.2012 Aug;41(8):347-53.
[47]Gage AA, Baust JG.Cryosurgery for tumors. J Am Coll Surg.2007 Aug; 205(2):342-56. Epub 2007 Jun 18.
[48]Yanagawa B, Holmes SD, Henry L, Hunt S and Ad N:Outcome of concomitant Cox-maze Ⅲ procedure using an argon-based cryosurgical system:a single-center experience with 250 patients. Ann Thorac Surg 95:1633-1639,2013.
[49]Albage A, Peterffy M and Kallner G:Learning what works in surgical cryoablation of atrial fibrillation:results of different application techniques and benefits of prospective follow-up. Interact Cardiovasc Thorac Surg 13:480-484, 2011.
[50]Ad N, Henry L and Hunt S:The concomitant cryosurgical Cox-maze procedure using argon based cryoprobes:12 month results. J Cardiovasc Surg (Torino) 52: 593-599,2011.
[51]Wu B, Xiao YY, Zhang X, Zhao L and Carrino JA:CT-guided percutaneous cryoablation of osteoid osteoma in children:an initial study. Skeletal Radiol 40: 1303-1310,2011.
[52]Tacke J, Speetzen R, Adam G, Sellhaus B, Glowinski A, Heschel I, Schaffter T, Schorn R, Grosskortenhaus S, Rau G, Gunther RW. Experimental MR imaging-guided interstitial cryotherapy of the brain. AJNR Am J Neuroradiol.2001 Mar;22(3):431-40.
[53]van den Bosch MA, Josan S, Bouley DM, Chen J, Gill H, Rieke V, Butts-Pauly K, Daniel BL. MR imaging-guided percutaneous cryoablation of the prostate in an animal model:in vivo imaging ofcryoablation-induced tissue necrosis with immedi ate histopathologic correlation. J Vase Interv Radiol.2009 Feb;20(2):252-8. doi: 10.1016/j.jvir.2008.10.030. Epub 2008 Dec.
[54]Tacke J1, Adam G,Haage P, Sellhaus B, Grosskortenhaus S, Gunther RW. MR-guided percutaneous cryotherapy of the liver: in vivo evaluation with histologic correlation in an animalmodel. J Magn Reson Imaging.2001 Jan;13(1):50-6.
[55]Klein-Nulend J,van der PlasA,Semeins CM,et al.SenSitivity of osteocytes to biomechanical stress in vitro[J]. FasebJ,1995,9(5):441-445.
[56]OwanI,Burr DB,Turner CH,et al.Mechanotransduction in bone:Osteoblasts are more responsive to fluid forces than mechanicalstrain[J].Am Jphysiol,1997,273: C810-815.
[57]Xie Y, Hardouin P, Zhu Z, Tang T, Dai K, Lu J. Three-dimensional flow perfusion culture system for stem cell proliferation inside the critical-size beta-tricalcium phosphate scaffold. Tissue Eng.2006 Dec;12(12):3535-43.
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