Bilateral vascularized femoral bone transplant: a new model of vascularized bone marrow transplantation in rats, part I

We present a new model of vascularized bone marrow transplantation-bilateral vascularized femoral bone (BVFB) isograft transplant based on abdominal aorta and inferior vena cava. A total of 7 BVFB isograft transplants were performed between Lewis (RT1) rats. In the donor, both femoral bones were harvested based on the abdominal aorta and inferior vena cava. In the recipient, the harvested isograft transplants were transferred into the inguinal region (in 3 animals) and into the abdominal cavity (in 4 animals). The mean operation time was 3 hours and 35 minutes. The mean warm ischemic time was 35 minutes. The vascular pedicles of the transplants that were transferred into the inguinal region were thrombosed at day 7 posttransplantation. The vascular pedicles of transplants into the abdominal cavity were patent and the bones were viable during the follow-up period of 63 days posttransplant. We have confirmed the feasibility of BVFB transplantation based on abdominal aorta and inferior vena cava.     

Composite vascularized skin/bone graft model: a viable source for vascularized bone marrow transplantation

In this study, we introduce a new model for vascularized skin and bone marrow transplantation. Twenty-five Lewis (RT1(1)) rats were studied. Anatomic dissection studies were performed in 5 animals. In the experimental group, 10 isograft transplantations were performed between Lewis rats. Combined groin skin and femoral bone flaps were transplanted based on the femoral artery and vein. Transplants were evaluated on a daily basis. All flaps survived without problems over 100 days posttransplant. The skin component remained pink and pliable, and grew new hair. Histological examination of the femoral bone (except the femoral head) revealed active hematopoiesis with a viable compact and cancellous bone components on day 100 posttransplant. This model can be applied to tolerance induction studies across the major Histocompatibility (MHC) barrier, where bone will serve as donor of stem and progenitor cells, and the skin flap will serve as a monitor of graft rejection.     

Heterotopic sternum transplant in rats: A new model of a vascularized bone marrow transplantation

We introduced the heterotopic vascularized sternum transplant as a more simple and pure alternative to allogeneic hind limb transplantation for the study of bone marrow transplantation. We report the clinical and histopathological manifestations after transplantation of syngeneic and allogeneic sternal grafts with and without immunosuppression with FK-506. Syngeneic grafts maintained normal histology, whereas allografts showed rejection, which was prevented by FK-506. FK-506-treated allografts developed chimerism that was present throughout the observation period. Transplantation of the sternum may be a valuable model to study vascularized bone marrow transplantation and its effects on repopulation of bone marrow of the host, chimerism, and tolerance.     

Physical properties of autoclaved bone: Torsion test of rabbit diaphyseal bone

Structural properties of autoclaved diaphyseal bone in the rabbit were investigated by torsional test. Heat propagation into the bone was studied by means of thermocouples. The torsional test included 54 pairs of diaphyseal bones. Autoclaving was performed to the same degree of sterilization, although with variations of time and temperature. Standard autoclaving at 121°C for 20 min was found to cause a moderate decrease (23 per cent) in torsional strength. The decrease was more pronounced (35 per cent) for bones autoclaved at 110°C for 255 min and less (9 per cent) for those autoclaved at 131°C for 2 min. Heat propagation into bone during autoclaving proved to be rapid at both 121°C and 131°C., indicating that complete, uniform sterilization of diaphyseal bone may be performed to an accurate, predetermined degree. Diaphyseal bone subjected to standard autoclaving remains mechanically adequate for skeletal substitution. Reimplantation of autoclaved tumorous bone might provide a simple combined means for tumor devitalization and subsequent reconstruction.     

Physical properties of autoclaved bone. Torsion test of rabbit diaphyseal bone

Structural properties of autoclaved diaphyseal bone in the rabbit were investigated by torsional test. Heat propagation into the bone was studied by means of thermocouples. The torsional test included 54 pairs of diaphyseal bones. Autoclaving was performed to the same degree of sterilization, although with variations of time and temperature. Standard autoclaving at 121 degrees C for 20 min was found to cause a moderate decrease (23 per cent) in torsional strength. The decrease was more pronounced (35 per cent) for bones autoclaved at 110 degrees C for 255 min and less (9 per cent) for those autoclaved at 131 degrees C for 2 min. Heat propagation into bone during autoclaving proved to be rapid at both 121 degrees C and 131 degrees C, indicating that complete, uniform sterilization of diaphyseal bone may be performed to an accurate, predetermined degree. Diaphyseal bone subjected to standard autoclaving remains mechanically adequate for skeletal substitution. Reimplantation of autoclaved tumorous bone might provide a simple combined means for tumor devitalization and subsequent reconstruction.     

Study of microcirculation of rat femoral nerve and development of a new vascularized nerve graft model

This study was designed to investigate the microcirculation of the rat femoral nerve and to develop a vascularized nerve graft (VNG) model which is reliable, consistent, and simple. The rat was chosen for considerations of cost and simplicity of the model. One and 2 cm long femoral nerve segments were transected proximally and distally and left attached to the femoral artery and vein in all animals. Experimental groups included the following procedures: 1) the femoral artery and vein were ligated distally and an arteriovenous (AV) fistula just proximal to the ligature was constructed; 2) distal ligation only of the femoral artery and vein was done; 3) ligation of the femoral artery and vein was done both distally and proximally; 4) control: no ligation or construction of an AV fistula were done. Neurovascular segments in all groups were wrapped with a thin silicone sheath to prevent neurovascularization from the surrounding tissues. At seven days postoperatively, viability of the segments was assessed by fluorescein dye and microangiography studies, followed by histologic evaluation. Results showed that a 1- or 2-cm long femoral nerve segment, when left attached to distally ligated femoral vessels and an intact neurovascular sheath, is a simple design to reproduce and can serve reliably as a VNG model in the rat.     

带血管蒂骨瓣转移治疗双侧股骨头缺血性坏死的疗效分析

背景：非创伤性股骨头缺血性坏死（ONFH）常双侧发病,治疗更加困难,采用保留股骨头的治疗方法较为理想。目前,采用带血管蒂骨瓣转移治疗双侧ONFH疗效的报道甚少。 目的：探讨应用带血管蒂髂骨瓣转移治疗双侧ONFH的早中期临床疗效。 方法：2009年1月至2010年12月共收治双侧ONFH患者22例44髋,男10例,女12例;年龄22~41岁,平均32.6岁;体重指数（BMI）16.5~30,平均23.9。按ARCO分期标准分为：Ⅱb期9髋,Ⅱc期14髋,Ⅲa期8髋,Ⅲb期5髋,Ⅲc期8髋。术中所取血管蒂均为旋股外侧血管升支髂棘支骨瓣转移术。双髋分两次进行手术,手术间隔12~16个月,平均14个月。 结果：随访时间为36~47个月,平均40.3个月,单髋术中失血量200~500 ml,平均358 ml。双侧髋关节术后6个月、12个月的Harris髋关节评分（HHS）均较各自术前有明显提高;双侧髋关节术后相同时间点的HHS评分比较无统计学差异。初次手术侧1髋术后出现切口脂肪液化经换药痊愈,其余均无围手术期并发症。术后根据ARCO分期标准2髋由Ⅲb期病变进展至Ⅲc期;1髋由Ⅲc期进展至Ⅳ期,并于术后14个月进行人工关节置换手术。 结论：应用带血管蒂髂骨瓣转移分两次手术治疗双侧ONFH,合适的手术间隔对初次手术侧功能恢复影响较小,双侧髋关节术后早期临床功能评价较高,是治疗双侧ARCOⅡ~Ⅲ期ONFH的有效方法。     

Effectiveness of bone marrow transplantation for revitalizing a severely necrotic small bone: experimental rabbit model

Background

Although treating Kienböck disease is controversial, we previously applied a new method that was less invasive and comprised drilling, bone marrow (BM) transplantation, external fixation, and radiating low-intensity pulsed ultrasound. We reported good clinical results obtained by this new method, which were comparable to those obtained using other, rather invasive methods. Here, we investigated the effect of drilling holes and transplanting BM into necrotic bone in an animal model to further understand the effect of these methods on the revitalization of necrotic bone.

Methods

We used rabbit fourth tarsal bones, whose surfaces consist of cartilage and cortical bone, mimicking human lunate bone. We soaked the retrieved bones in liquid nitrogen to induce necrosis. After thawing, we inserted them separately into bilateral subcutaneous pouches in the backs of rabbits. A total of 60 rabbits were divided into four groups of 15 rabbits each: BM transplantation (BM group); peripheral blood transplantation (PB group); drilling (D group); control (C group). We sacrificed three rabbits to obtain six specimens in each group at 2, 4, 8, 12, and 20 weeks after operation and evaluated the specimens histomorphologically.

Results

In the BM group, significantly larger mineralizing surfaces, osteoblast surfaces, and osteoclast numbers were observed at 4, 8, and 12 weeks compared with those in the other groups. No significant differences were observed at 2 and 20 weeks in the groups except the mineralizing surface of the 20-week-BM group, which was significantly greater.

Conclusions

We examined the efficacy of drilling and of BM transplantation for regenerating necrotic bone in a rabbit model. Our experiments suggest that drilling with BM transplantation to the necrotic bone accelerates bone formation and remodeling.     

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.     

Comparison of internal bone transport and vascularized fibular grafting for femoral bone defects

OBJECTIVE: To compare results between vascularized fibular grafting and internal bone transport for large bone defects of the femur. DESIGN: Retrospective review of patients. SETTING: University teaching hospitals. PATIENTS: This study included 37 patients with femoral bone loss: 20 patients were treated with internal bone transport and seventeen patients were treated with vascularized fibular grafting. MAIN OUTCOME MEASUREMENTS: The outcomes investigated were external fixation time, external fixation index, bone results (union, infection, deformity, leg length discrepancy), and functional results. RESULTS: The mean amount of filled defect was 8.4 cm with internal bone transport and 8.9 cm with vascularized fibular grafting. The external fixation index was 1.4 months/cm with internal bone transport and 1 month/cm with vascularized fibular grafting. The bone results and functional results of the internal bone transport were excellent in 65% and 0%, good in 5% and 45%, fair in 5% and 40%, poor in 25% and 15%, respectively, whereas those of the vascularized fibular grafting were excellent in 35% and 0%, good in 25% and 47%, fair in 5% and 35%, and poor in 35% and 18%. CONCLUSION: With vascularized fibular grafting, careful monitoring of circulation and early intervention surgery is necessary to avoid vascular failure. With internal bone transport, repeated radical debridement until control of infection is achieved, bone grafting at the docking site for early union, and avoiding stress fracture are recommended to improve bone results.     

Avascular necrosis of the femoral head: role of vascularized bone grafts

This article presents the history and development of as well as the results from the various techniques of vascularized bone grafting for the treatment of osteonecrosis (ON) of the femoral head. The authors have treated more than 2600 patients who had femoral head ON, using a vascularized fibular graft by way of an intraosseous approach. The results from this vast experience are summarized and certain pearls and pitfalls regarding the treatment of femoral head ON using the free vascularized fibular graft are highlighted.     

Host-derived neoangiogenesis with short-term immunosuppression allows incorporation and remodeling of vascularized diaphyseal allogeneic rabbit femur transplants

The purpose of this study was to demonstrate that living bone allotransplants can incorporate, remodel, and maintain mechanical properties without long-term immunosuppression in a fashion comparable to living autotransplants. For this, viability is maintained by repair of nutrient vessels and neovascularization from implanted host-derived vasculature. Microsurgically revascularized femoral diaphysis allotransplants were transferred from young male New-Zealand-White (NZW) into 4 groups of male Dutch-Belted (DB) rabbits. Short-term immunosuppression by tacrolimus (IS, groups 4 and 5) and host-derived neovascularization (NV) from implanted fascial flaps was used to maintain viability (groups 3 and 5) as independent variables. Group 2 received neither IS nor NV. Vascularized pedicled autotransplants were orthotopically transplanted in group 1. After 16 weeks, transplants were evaluated using radiologic, histologic, biomechanical, and histomorphometric parameters. Vascularized bone allotransplants treated with both short-term IS and host-derived NV (group 5) healed in a fashion similar to pedicled autotransplants (group 1). Their radiographic scores were higher than other groups. Groups with patent fascial flaps (3 and 5) showed significantly greater neoangiogenesis than ligated controls (2 and 4). Tacrolimus administration did not affect neoangiogenesis. Elastic modulus and ultimate stress were significantly greater in autogenous bone than in allotransplanted femora. Biomechanical properties were not significantly different among allotransplants. Bone turnover was decreased with IS, but increased with NV by the implanted fascial flaps. Living allogeneic femoral allotransplants treated with short-term IS and host-derived neoangiogenesis can lead to stable transplant incorporation in this rabbit model. The combination of both factors optimizes bone healing. Transplant mineralization is improved with neoangiogenesis but diminished with IS. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 763–770, 2009     

Allogenic grafting of vascularized bone segments under immunosuppression. Clinical results in the transplantation of femoral diaphyses

Trauma surgery lack, substitute, for the reconstruction of large defects of the long bones. Encouraged by the promising results of bone allotransplantation in animal models, we successfully performed vascularized bone transplantation in humans. Vascularized femoral diaphyses were allogenically transplanted into three patients suffering from chondrosarcoma or post-traumatic osteomyelitis with postoperative immunosuppression. The bone segments were harvested from multi-organ donors and perfused with UW solution. After back-table preparation, the grafts were transplanted into the defect zone. Interlocking devices were used in these operations. Vascular anastomoses were performed in end-to-side technique. The early clinical course of the patients was not free of anatomical, technical, or immunological complications. However, all patients are currently free of malignancy and infection. They are also free of pain and full weight bearing. We conclude that allogenic grafting of vascularized bone segments has the potential to become an alternative for the replacement of large bone defects. Received: 21 July 1997 Received after revision: 17 December 1997 Accepted: 9 January 1998     

A comparison of vascularized and nonvascularized bone transfer in rabbits: a roentgenographic, scintigraphic, and histologic evaluation

Extensive bone defects caused by bone tumor resection, osteomyelitis, congenital pseudoarthrosis, post-traumatic bone loss, or femoral head necrosis, require large bone grafts. Such large defects usually are not amenable to conventional, nonvascularized cancellous grafts. By using vascularized bone grafts that do not undergo creeping substitution, that heal rapidly and are not depending on the surrounding tissue, better, safer, and faster results can be obtained. To compare recoveries after vascularized grafts with those after conventional, nonvascularized grafts, experiments were carried out in a rabbit model. They demonstrated good viability and better and faster healing of the microvascular grafts, using radiography, scintigraphy, light microscopy of bone osteocytes and vessels, and tetracycline double-labeling evaluation techniques.     

Osteogenic enhancement of diaphyseal reconstruction: Comparison of bone grafts in the rabbit

We investigated incorporation of autoclaved autografts in segmental defects of rabbit humeri for comparison with a previous study on similar grafts supplemented with demineralized allogeneic bone matrix (DABM). We also made similar reconstructions with frozen allografts—both DABM and nonsupplemented allografts.

Before the animals were killed at 8 months, they underwent scintigraphy, showing that all 28 humeral reconstructions were metabolically active. Faxitrone radiography showed nonunion in three of nine with autoclaved autografts and in two of eight with frozen allografts, whereas all 11 DABM-supplemented frozen allografts had incorporated. Taking into account only the 23 healed reconstructions, the mean torsional strength in relation to the contralateral nonoperated on humeri was 0.81 for all three groups. Histologically, new bone enveloping, partly replacing, the implants was more abundant in DABM-supplemented reconstructions.

Our study shows that osteogenic enhancement is more important than the type of nonviable bone chosen for diaphyseal repair. However, if healing is obtained, osteogenic enhancement per se does not increase the strength.     

Allogeneic vascularized transplantation in cases of bone and joint defects

This paper presents preliminary results of allogeneic vascularized transplantations of three femoral diaphyses and four total human knee joints. Grafts were harvested from multi-organ-donors and immediately transplanted. Osteosyntheses were performed employing intramedullary nails. Vascular pedicles of the grafts were anastomosed in end-to-side technique. Immunosuppression mainly based on Cyclosporine and Azathioprine. Grafts' perfusion was demonstrated by DSA and Duplex-sonograms, bone metabolism by SPECT-scintigraphy. Five months following transplantation osteotomies demonstrated consolidation in conventional X-rays. Biopsies of the grafted bone revealed intact osteocytes and arthroscopy demonstrated intact synovial, chondral and ligamentous structures. From the technical aspect vascularized transplantation of the femoral diaphyses and total knee joints is feasible. The main problems are of immunologic nature. Transplantations were performed respecting the ABO-compatibility but with a large HLA-mismatch. Acute and chronic rejection crises may damage the grafts. At least in synovial joints live-long immunosuppression of the recipients seems to be unavoidable.     

Allogeneic vascularized transplantation in cases of bone and joint defects

This paper presents preliminary results of allogeneic vascularized transplantations of three femoral diaphyses and four total human knee joints. Grafts were harvested from multi-organ-donors and immediately transplanted. Osteosyntheses were performed employing intramedullary nails. Vascular pedicles of the grafts were anastomosed in end-to-side technique. Immunosuppression mainly based on Cyclosporine and Azathioprine. Grafts' perfusion was demonstrated by DSA and Duplex-sonogramms, bone metabolism by SPECT-scintigraphy. Five months following transplantation osteotomies demonstrated consolidation in conventional X-rays. Biopsies of the grafted bone revealed intact osteocytes and arthroscopy demonstrated intact synovial, chondral and ligamentous structures. From the technical aspect vascularized transplantation of the femoral diaphyses and total knee joints is feasible. The main problems are of immunologic nature. Transplantations were performed respecting the ABO-compatibility but with a large HLA-mismatch. Acute and chronic rejection crises may damage the grafts. At least in synovial joints livelong immunosuppression of the recipients seems to be unavoidable.