原始骨血肿的骨再生潜能的实验研究

目的 探索骨折后冷藏原始骨血肿的骨再生潜能和对骨折愈合的影响。方法 在新西兰兔股骨、胫骨上制造骨缺损模型，3d后取出骨血肿并4℃冷藏．3d后冷藏的原始骨血肿回置到实验组骨缺损区，定期摄X线片和取出骨痂行组织学观察。结果 实验组从2、4、6周均见明显骨痂形成和骨膜反应，组织学观察可见有成骨样细胞形成。结论 冷藏的原始骨血肿仍有很好的骨再生潜能，可促进骨折、骨缺损区骨愈合。骨折行手术治疗尤其是闭合性骨折行手术治疗时，应考虑到原始骨血肿丢失对骨折愈合的影响，术中注意保存部分原始骨血肿可能有利于骨折、骨缺损的愈合。     

原始骨血肿的骨再生潜能的实验研究

目的　探索骨折后冷藏原始骨血肿的骨再生潜能和对骨折愈合的影响。方法　在新西兰兔股骨、胫骨上制造骨缺损模型 ,3d后取出骨血肿并 4℃冷藏 ,3d后冷藏的原始骨血肿回置到实验组骨缺损区 ,定期摄X线片和取出骨痂行组织学观察。结果　实验组从 2、 4、 6周均见明显骨痂形成和骨膜反应 ,组织学观察可见有成骨样细胞形成。结论　冷藏的原始骨血肿仍有很好的骨再生潜能 ,可促进骨折、骨缺损区骨愈合。骨折行手术治疗尤其是闭合性骨折行手术治疗时 ,应考虑到原始骨血肿丢失对骨折愈合的影响 ,术中注意保存部分原始骨血肿可能有利于骨折、骨缺损的愈合     

应用原始骨血肿对闭合性长骨骨折手术治疗的体会

目的探索新鲜原始骨血肿对骨折愈合的影响。方法随机选取新鲜闭合性长骨骨折病例，术中切开显露骨折断端时，留取了3～10mL原始骨血肿及血肿块，在内固定完成后回置骨折断端，定期拍摄CR片，观察骨折愈合情况。结果经观察证实实验组自4周开始即有明显骨痂生成。结论新鲜的原始骨血肿有很好的骨再生潜能，可促进骨折、骨缺损区骨愈合，因而在进行闭合的骨折切开复位内固定时，要考虑原始骨血肿丢失，应尽量保留骨折断端原始骨血肿，血凝块，在手术结束时回放，以期达到促进骨折愈合的目的。     

骨髓基质细胞促进引导性骨再生的研究

目的观察骨髓基质细胞增强引导性骨再生（GBR）修复骨缺损的能力。方法30只兔造成双桡骨干15mm骨缺损，以硅胶管桥接骨断端，实验组在硅胶管内注射自体骨髓基质细胞（MSC）1ml；对照组注射等量生理盐水。在不同时间内作X线片、大体、组织学观察及生化捡测。结果实验组成骨活跃，10周骨缺损完全修复，对照组各时间点骨修复均较实验组差，10周时仍无1只兔骨性愈合。术后4周实验组钙及碱性磷酸酶含量明显高于对照组（P〈0．01），术后8及10周实验组骨缺损区新生骨骨痂密度与相邻尺骨密度比值亦明显高于对照组（P〈0．01）。结论自体骨髓基质细胞可明显增强GBR修复骨缺损的能力。     

移植骨复合骨髓基质干细胞修复兔股骨缺损

目的观察同种异体骨复合骨髓基质干细胞对兔股骨大段缺损修复情况,探讨其修复骨缺损的可行性。方法将24只大耳兔随机分为2组,造成股骨大段缺损,对照组植入打孔同种异体骨,实验组植入打孔同种异体骨＋明胶海绵＋骨髓基质干细胞。术后2个月行X线片观察、病理组织学检查及骨密度测试。结果①放射学检查：对照组在异体骨结合部可见骨痂通过,实验组整个异体骨段均可见明显骨痂形成。②病理组织学检查：对照组可见少量内骨痂及外骨痂且被大量纤维结缔组织所分隔,实验组异体骨有坏死后弧形吸收窝,可见内外骨痂生长,髓腔内充满大量骨母细胞。③平均骨密度值测定：实验组高于对照组及正常股骨,差异有显著性（P〈0．05）。结论同种异体骨复合骨髓基质干细胞用于修复兔股骨大段缺损,较单纯异体骨成骨量大、迅速,能够对骨缺损进行有效的修复。     

云南白药促进骨缺损修复及引导性骨再生的实验研究

目的：阐明云南白药促进骨缺损修复及引导性骨再生的愈合机制，为临床上治疗骨折提供理论依据。方法：36只新西兰兔制作桡骨缺损不愈合模型和引导性骨再生模型，随机均分用药组及对照组，手术后3d、1、3、5、10、12周取材，观察骨痂愈合程度的差异及骨痂组织学变化，结果：实验组有明显的促进骨缺损修复作用，并在引导性骨再生中的膜内成骨作用强于对照组，结论：云南白药对骨缺损修复及引导性骨再生有明显的促进作用。     

低强度超声波对骨折愈合中胶原代谢影响的实验研究

目的：探讨低强度超声波对骨折愈合中Ⅰ,Ⅱ型胶原代谢的影响。方法：建立大鼠双侧胫骨近侧皮质骨缺损的动物模型,随机分为2组,实验组每日接受低强度超声波刺激,而对照组给与假刺激,于术后10,20,30天处死动物取标本（骨痂）进行组织学及免疫组化染色（SABC法）,结果：组织学检查显示,实验组的血肿机化,吸收,软骨性骨痂和软骨内化骨明显早于对照组;免疫组化检查显示;实验组在20天时Ⅱ型胶原的表达明显高于对照组,在30天时I型胶原的表达也略高于对照组,结论：低强度超声波通过增加骨痂内I、Ⅱ型胶原的合成来促进骨折的愈合,其中以Ⅱ型胶原的作用尤其明显。     

骨形态发生蛋白在骨折愈合中的作用

骨折愈合是一个复杂的组织学变化过程,涉及一系列不同的细胞活动,自1965年Urist提出骨诱导学说,并相继提纯骨形态发生蛋白(BMP)以来,对骨的再生修复均有了进一步的认识,并已开始将BMP初步应用于临床实践治疗骨不连接及骨缺损。对内源性BMP在骨折修复中的作用机制尚不清楚。本文应用骨形态发生蛋白单克隆抗体检测骨折愈合过程中外骨痂内BMP的分布及细胞定位,探讨BMP在闭合性骨折愈合外骨痂形成中的作用,以期对闭合性骨折之愈合获得进一步的认识。     

低强度脉冲超声波影响兔骨髓基质干细胞与β-磷酸三钙复合修复骨缺损的实验研究

[目的]探讨低强度脉冲超声波(LIPUS)影响β-磷酸三钙(β-TCP)与兔骨髓基质干细胞(BMSCs)复合体修复兔桡骨骨缺损的效果。[方法]从兔股骨粗隆部抽取骨髓,体外培养扩增,取第3代BMSCs接种于β-TCP培养1周,分为两组,一组予以LIPUS作用,另一组作为对照。以20只成年新西兰兔为研究对象,制作双侧桡骨远端骨缺损模型。一侧桡骨骨缺损处植入LIPUS作用过的细胞支架复合体做为实验组,另一侧植入LIPUS未作用过的细胞支架复合体做为对照组。分别于术后4周、8周处死新西兰兔,运用计算机图像分析X线片上骨痂的灰度密度,行组织学切片检查骨痂生长情况。[结果]X线片骨痂灰度密度测量提示实验组骨痂较对照组生长明显增多。组织学切片显示实验组血肿机化、吸收、骨小梁和骨基质形成早于对照组。[结论]在骨缺损修复早期,LIPUS对BM-SCs与β-TCP复合体修复兔骨缺损有明显的促进作用;而在骨缺损修复晚期,LIPUS的作用相应减弱。     

同侧腓骨移植结合外固定器治疗胫腓骨粉碎性骨折骨缺损

目的 评价同侧腓骨移植外固定支架固定治疗胫腓骨粉碎性骨折骨缺损的疗效。方法 38例均为新鲜骨折。其中，开放性骨折29例，闭合性骨折9例，均采用同侧腓骨移植，长约8～13cm，两端插入胫骨骨折两端骨髓腔内。单臂外固定支架分别在上、中、下三处，在不同平面和方向对骨折和植骨进行固定。同时将骨折粉碎骨块拼排在移植骨周围，术后早期活动。结果 38例术后随访6～24个月，骨痂形成时间6～8周。植骨与主骨愈合时间为6个月，无骨不愈合、骨髓炎感染发现。结论 胫腓骨粉碎性骨折、骨缺损，采用同侧排骨移植，既有移植骨功能，又可以直到支持和恢复肢体长度作用。骨折愈合快，临床疗效满意。     

激素性股骨头坏死患者骨髓基质细胞骨保护素/核因子kappa B受体活化因子配基蛋白表达的研究

目的观察激素性股骨头坏死患者骨髓基质细胞（bone marrow stroma cells,BMSCs）骨保护素（osteoprotegerin,OPG）/核因子kappa B受体活化因子配基（receptor activator of nuclear factor kappa B ligand,RANKL）蛋白表达情况,探讨长期应用激素导致股骨头坏死的另一病理机制。方法 2007年3月至2008年3月,取激素性股骨头坏死患者骨髓及股骨头骨组织35例（实验组）,股骨颈骨折患者骨髓及股骨头骨组织21例（对照组）。两组男女比例均为4：3;年龄41～70岁,实验组平均55.34岁,对照组平均55.33岁;实验组最近2年内接受过皮质激素治疗超过3周或超过1周的大剂量冲击治疗,对照组从未接受过超过1周的激素治疗。骨组织标本行多聚甲醛固定后石蜡包埋,HE染色。所取骨髓采用贴壁法分离培养骨髓基质细胞,采用蛋白免疫印迹（Western blot）技术检测骨髓基质细胞OPG和RANKL蛋白表达水平,并得出OPG/RANKL比值。结果 HE染色：实验组未见完整的骨小梁和骨单位,可见不连续的骨碎片,碎片骨陷窝内骨细胞大部分消失,周围大量炎性肉芽组织。对照组可见完整骨单位由板层骨构成,板层骨连续完整,围绕血管呈同心圆排列,小梁骨陷窝内可见骨细胞。骨髓基质细胞Western blot检测：实验组和对照组OPG/RANKL蛋白表达比分别为1.13±0.65和2.54±0.35,实验组明显低于对照组,有统计学差异（P〈0.05）。结论长期应用糖皮质激素致股骨头坏死可能与其调控骨髓基质细胞OPG和RANKL表达有关。     

Transplanted bone marrow cells localize to fracture callus in a mouse model.

Bone marrow contains many cellular elements that may contribute to fracture repair. We used a pluripotential stromal cell in a mouse model to demonstrate the presence of transplanted cells in fracture hematoma and subsequently in maturing fracture callus. Cells were transduced with traceable genes (lac Z and neomycin resistance) and traced in vivo after intravenous injection into syngeneic mice. These transduced cells home to bone marrow, suggesting that they might be detected in fracture callus. Cells were injected intravenously into mice and stabilized femoral shaft fractures were induced. Control mice received intravenous lactated-Ringer's solution prior to fracture. Callus tissue and marrow were examined histologically from I to 10 weeks after fracture to detect transplanted cells. Transplanted cells were detected in fracture callus in areas, and at times, of most active bone formation. Control specimens showed minimal staining of the callus tissue. Levels of the traceable gene in fracture callus increased, reached a peak between 3 and 4 weeks after fracture, then diminished and disappeared by 10 weeks post-fracture as woven bone at the fracture site was replaced by lamellar bone with cells from the host mouse. The results show that pluripotent bone marrow cells home to the marrow after systemic injection and localize in fracture callus.     

Generation of donor hematolymphoid cells after rat-limb composite grafting

BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.     

Immunosuppressive effect of tacrolimus (FK-506): Bone xenografts in rabbits

We examined the immunomodulatory effect of the macrolide antibiotic FK-506 (tacrolimus) in bone xenograft transplantation. Full-thickness pieces of iliac bone from mongrel dogs were transplanted into the iliac bone of Japanese white rabbits. FK-506 at a dose of 1.6 mg/kg/day was injected into the rabbits for 10 days after transplantation. in the animals treated with FK-506, inflammatory cell infiltration was remarkably reduced and revascularization accompanied by new bone formation occurred in the grafts. At 4 months after the transplantation, the formation of new bone and of mature new bone marrow were observed. in a control group, inflammatory cell infiltration was marked around the graft from 2 weeks after the transplantation. Revascularization from the recipient site to the graft in the control group was poor and only a small amount of new bone had formed at 4 months. Our findings suggest that short-term administration of FK-506 has a beneficial effect on experimental xenograft bone transplantation.     

HSV-sr39TK-GCv/ACV系统抑制小鼠异基因骨髓移植后移植物抗宿主病

目的 研究突变型单纯疱疹病毒胸苷激酶一更昔洛韦/阿昔洛韦(HSV-sr39TK-GCV/ACV)系统对小鼠异基因骨髓移植后移植物抗宿主病(GVHD)的影响.方法 采用改良的磷酸钙沉淀法,以携带HSv-sr39TK基因的慢病毒感染C57BL/6小鼠的脾淋巴细胞.制得sr39TK+T淋巴细胞.以C57BL/6小鼠为供者,Balb/c小鼠为受者进行骨髓移植,受者移植前接受60>Coγ射线照射.实验分6组进行:(1)GCV组共30只小鼠,均于骨髓移植的同时输注sr39TK+T淋巴细胞.其中10只于骨髓移植当天至第6天腹腔注射GCV 0.5 mg/d,10只于骨髓移植后第7～13天腹腔注射GCV0.5 mg/d,10只于骨髓移植后第12～18天腹腔注射GCV 0.5 nag/d;(2)ACV组共30只小鼠,骨髓移植与sr9TK+T淋巴细胞输注同GCV组,其中10只于骨髓移植当天至第6天腹腔注射ACV 0.5mg/d,10只于骨髓移植后第7～13天腹腔注射ACV 0.5 mg/d,10只于骨髓移植后第12～18天腹腔注射ACV 0.5 mg/d;(3)移植对照组仅行骨髓移植;(4)脾细胞对照组行骨髓移植和脾淋巴细胞输注;(5)GCV对照组在脾细胞对照组的基础上于骨髓移植后第7～13天腹腔注射GCV 0.5 mg/d.(6)sr39TK对照组行骨髓移植和sr9TK+T淋巴细胞输注.观察各组受者的存活时间、GVHD的发生情况及程度.结果 GCV对照组、sr9TK对照组、睥细胞对照组和移植对照组小鼠均于骨髓移植后19 d内死亡.GCV组移植当天用药者、第7天用药者和第12天用药者的存活时间分别为(36.70±5.20)d、(40.30±4.69)d和(27.10±4.85)d.ACV组移植当天用药者、第7天用药者和第12天用药者的存活时间分别为(36.50±5.26)d、(46.20±3.61)d和(30.90±5.21)d.GCV组和ACV组受者的存活时间均长于4个对照组(P<0.01),GCV组和ACV组中第7天用药者的存活时间和5(1 d存活率优于其它各时间用药者,差异有统计学意义(P<0.05),而ACV组第7天用药者又明显优于GCV组第7天用药者(P<0.05).4个对照组小鼠移植后10～12 d均开始出现Ⅲ～Ⅳ级GVHD.GCV组和ACV组死亡小鼠可见Ⅱ～Ⅳ级GVHD.而该两组中长期存活受者仅有Ⅰ～Ⅱ级GVHD.结论 HSV-sr9TK-GCV/ACV系统对小鼠异基因骨髓移植后的GVHD有一定的抑制作用;ACV的效果优于GCV;移植后7 d时应用ACV的效果较佳.     

Vascularized bone marrow transplantation: A new surgical approach using isolated femoral bone/bone marrow

BACKGROUND: Orthotopic composite tissue (limb) transplantation in rats is a unique model for vascularized bone marrow transplantation because bone marrow cells and bone marrow stroma are transplanted by microsurgical means, thus creating immediate bone marrow space and engraftment. However, it contains a skin component and other musculoskeletal tissues that complicate issues related to tolerance induction. MATERIALS AND METHODS: To study only aspects of vascularized bone marrow transplantation, we created a new isolated vascularized bone marrow transplant model in rats. The common iliac (or femoral) artery and vein were microsurgically anastomosed to the recipient abdominal aorta and inferior vena cava in an end-to-side fashion, respectively. Syngeneic male Lewis (RT1(1), n = 20) and allogeneic male BN (RT1(n), n = 10) donors were transplanted to female Lewis recipients. To establish rejection criteria, we examined histopathology and used the polymerase chain reaction (PCR) to assess microchimerism of donor male bone marrow cells in the peripheral blood of female recipients using rat Y chromosome (sex-determining region Y)-specific primers. RESULTS: All recipients were healthy and remained stable without major complications for up to 300 days posttransplant. Morphologically, syngeneic male Lewis bone marrow showed a near-normal appearance. Allogeneic male BN bone marrow was clearly rejected. Male bone marrow cells were detected by PCR in the peripheral blood of all syngeneic recipients, but not in allogeneic blood specimens. CONCLUSIONS: A new surgical approach to bone marrow transplantation was established. This consisted of the vascularized femoral bone/bone marrow transplant. Further analyses regarding the ability of vascularized femoral bone marrow transplants to induce systemic transplantation tolerance in adult rats will provide insights into not only various issues of immunology but also the potential clinical application of vascularized bone marrow transplantation.     

自体成骨细胞的诱导培养及其生物学特性

目的 诱导培养来自自体骨髓间充质干细胞的成骨细胞或成骨细胞的前体细胞，进行自体成骨细胞异体骨复合移植实验研究。方法 抽取动物骨髓、抗凝稀释，以适当条件培养诱导，分化为成骨细胞并分析细胞生物学特性。成骨细胞生长在同种异体骨制成的载体上，形成复合物，植入人工缺损处，观察骨修复情况。结果 可以诱导培养出大量的成骨细胞或成骨细胞的前体细胞。自体成骨细胞异体松质骨复合物修复骨缺损的效果明显优于对照组。结论 本实验为自体成骨细胞异体骨复合移植的临床应用提供一个简单有效的方法。     

Successful allogeneic leg transplantation in rats in conjunction with intra-bone marrow injection of donor bone marrow cells

BACKGROUND: We have recently established a new method for bone marrow transplantation (BMT) in mice: bone marrow cells are directly injected into the intra-bone marrow (IBM) cavity. IBM-BMT induces persistent donor-specific tolerance and enhances the rapid recovery or reconstitution of the hematolymphoid system of donor origin without any signs of graft-versus-host disease (GVHD) or graft failure. Furthermore, the prior injection of fludarabine can reduce the irradiation dose to the sublethal level (4.5 Gy x 2). Therefore, we hypothesize that IBM-BMT plus fludarabine is applicable to allogeneic leg transplantation in rats. METHODS: Brown Norway (BN; RT1An) rats were injected intravenously with 50 mg/kg of fludarabine phosphate, followed by sublethal fractionated irradiation (4.5 Gy x 2) 1 day before IBM-BMT. The hind limbs from Fischer 344 (F344; RT1Al) rats were transplanted on day 0, and bone marrow cells (3 x 10(7) cells/50 microL) obtained from the donor F344 rats were injected into the bone marrow cavity of the left tibias of the recipient BN rats. RESULTS: The hematolymphoid cells in the recipient BN rats were completely reconstituted by the cells of the donor F344 rats. The limbs transplanted from the donor F344 rats were accepted for >1 year without any clinical signs of rejection (10 of 10). The lymphocytes of the BN rats showed tolerance to both donor-type and recipient-type major histocompatibility complex determinants in mixed lymphocyte reaction, but showed a significant response to the third-party major histocompatibility complex determinants. CONCLUSIONS: Using a combination of the injection of fludarabine, low-dose irradiation, and IBM-BMT, we have succeeded in allogeneic limb transplantation without using any immunosuppressants after the operation. This strategy would be applicable to the transplantation of other vascularized organs in humans.     

骨髓间充质干细胞对异种胰岛移植排斥反应的影响

目的 探讨骨髓间充质干细胞(mesenchymal stem cell,MSC)对异种胰岛移植排斥反应的影响.方法 建立人-Wistar大鼠异种胰岛移植模型,选取造模成功的30只糖尿病大鼠,随机分为对照组和MSC组,每组15只.用Lewis大鼠股骨与胫骨培养制成的MSC进行干预治疗,MSC组植入人胰岛细胞和MSC,对照...