Takahiro Hashimoto

Skeletal reconstruction by vascularized allogenic bone transplantation: effects of statin in rats.

Background. Some statins have been reported to suppress the immune system and increase the expression of bone morphogenetic protein-2 gene that plays a pivotal role in bone regeneration. Methods. The effects of cerivastatin on skeletal reconstruction by vascularized bone allograft were investigated in a rat tibia-fibula graft model. After transplantation, the recipient rats were treated with vehicle, low-dose cerivastatin, high-dose cerivastatin, or cyclosporine A. Results. Transplanted bones treated with low-dose cerivastatin and vehicle failed to unite with the recipient bones. In contrast, high-dose cerivastatin induced the bone union as effectively as cyclosporine A. Histologically, high-dose cerivastatin-treated transplanted bones were nonvital, but new bone formation occurred at the outer layer of the nonvital cortex. Conclusion. These results indicate that statins could promote fracture healing. Because transplant recipients have the increased risks of osteoporotic fracture and hypercholesterolemia, statins may be a good choice in the treatment of these patients.

Transfer of latissimus dorsi muscle for the functional reconstruction of quadriceps femoris muscle following oncological resection of sarcoma in the thigh

Wide resection of tumours in the anterior compartment of the thigh frequently requires en bloc removal of the quadriceps femoris muscle. Such resection can result in significantly decreased muscle power of knee extension. Functional muscle transfer is a beneficial tool for the reconstruction process. Until now, however, there have been few reports on the outcome of quadriceps muscle reconstruction using free innervated muscle transfer. We reviewed 14 patients (seven women and seven men, mean age 53 years). The extent of tumour invasion required resection of the entire quadriceps in four cases, of three heads in six cases and of two heads in the remaining four cases. Local recurrence occurred in one patient only, and there were no major complications. In the four patients with entire resection of the quadriceps, the postoperative muscle manual test (MMT) result was 0-1 (mean, 0.5), but this recovered to between 2 and 3 (mean 2.3) after a mean follow-up of 70 months. In the six cases with resection of three heads, the MMT test result was 2 and active knee extension recovered almost fully. Transfer of free, functional latissimus dorsi muscle is best indicated for cases in which the entire quadriceps or three heads are resected. Active knee extension can be expected to improve to an MMT score of 2 after reinnervation of the transferred muscle. Microsurgical reconstruction following resection of soft-tissue sarcoma provides adequate functional and coverage reconstruction, together with better local control by allowing wider surgical margins.

Chimerism studies as an approach for the induction of tolerance to extremity allografts

SUMMARY Recent advances in the field of transplant immunology and reconstructive surgery have resulted in an increased interest in extremity allograft. Until now, more than 20 hand transplants have been performed in humans. Rejection is well controlled by currently available immunosuppressive drugs. The hand transplant, however, is not a life-supporting organ transplant and these drugs are unlikely to represent the final solution for hand transplantation due to serious adverse effects. The ultimate goal of extremity allograft is the induction of donor-specific immunotolerance. The major strategies for tolerance induction are: (1) T-cell costimulation blockade, (2) induction of mixed chimerism, (3) T-cell depletion, and (4) tolerance mediated by regulatory T cells. Amongst these, the establishment of a high level of chimerism may be the most stable strategy for donor-specific tolerance, and our laboratory has been investigating the induction of macrochimerism following extremity allotransplantation. Recently, some studies demonstrated that macrochimerism induces immunotolerance for extremity allograft in the rodent model. We made a new protocol using cyclophosphamide (CYP) and granulocyte colony-stimulation factor (G-CSF) to induce high-level chimerism following rat whole-limb allotransplantation. Limb allografting could function as a vascularised carrier for bone marrow transplantation, providing a continuous source of donor cells and contributing to a high level of chimerism in the recipient. Pretransplant CYP followed by G-CSF and FK506 treatment significantly prolong the survival of limb allografts, but frequently cause chronic graft-versus-host disease in the recipients. In this review, recent experimental chimerism studies are presented for tolerance induction and we review the prospect of clinical applicability in extremity allograft.

Donor cell repopulation of whole-limb allografts in the rat: detection with green fluorescent protein.

Background: Although cell traffic between donor and recipient has previously been observed during allogeneic organ transplantation, little is known about cell traffic following whole-limb allografting. Whole-limb grafts are composed of composite tissues, and thus cell repopulations of recipients may be different for each component. This study was conducted using green fluorescent protein (GFP) transgenic rats to define cell repopulation of whole-limb allografts. Methods: Twenty-four hind-limb allotransplants were performed across GFP-positive (Wistar background) and GFP-negative (Lewis) rats. Eighteen recipient animals were treated with continuous FK506 immunosuppression at a dose of 0.5 mg/kg/day up to 6 months after transplantation and assessed until 18 months posttransplantation. The expression of the GFP gene was examined under 489-nm excitation light and semiquantitatively assessed by polymerase chain reaction. Results: Allografted limbs showed acute rejection in nontreated recipients, but no rejection episodes occurred in FK506-treated recipients until 18 months posttransplantation. Intense GFP expression was noted in allotransplanted GFP-negative limbs at 18 months posttransplant. GFP expression was especially marked at the interfollicular epidermis in the skin component and the endothelial cells. Polymerase chain reaction using GFP-specific primers confirmed the presence of the GFP gene in these tissues. Allotransplanted GFP-positive limbs retained marked GFP expression at the muscle fiber. Conclusions: The authors’ results demonstrate that recipient-derived cells gradually migrate into grafted skin, endothelial cells, muscle, and bone marrow cells. Recipient-derived stem cells may contribute to this cell renewal within the graft. Repopulation of antigenic skin components in the graft with recipient cells may also help in avoiding rejection.

Stimulation of neo-angiogenesis by combined use of irradiated and vascularized living bone graft for oncological reconstruction

Reconstruction for large bone and osteochondral defects following musculoskeletal tumor excision remains challenging. Mega-prosthesis is clearly a useful reconstructive tool. Because the survival time of tumor patients has been increasing due to better treatment options, the aim of our group is to achieve complete biological reconstruction without using any artificial materials. With this approach, durability would not be a limitation. In the present study, we reviewed the biological reconstructive procedures currently available for large bone defects after tumor excision. Devitalized bone autograft is particularly well suited in the region where allografts are not readily available. However, the complication rate, such as infection and spontaneous bone resorption, was unexpectedly high due to non-viable graft. In an attempt to reduce these complications, we have used irradiated bone autograft in combination with free vascularized viable bone graft. In an experimental study, we demonstrated a neo-vascularization effect of vascularized bone graft with devitalized bone autograft, i.e. to convert dead bone into living bone. Clinically, this technique is best indicated for reconstruction of intercalary bone defect, especially tibial shaft. Some degree of articular change occurs after irradiation and cannot be prevented, even with the combined use of vascularized bone graft. In our experience, secondary procedures such as surface replacement prosthesis are necessary to treat the osteoarthritis in such cases, even if the radiological finding is severe. The rationale for a combined vascularized and irradiated bone autograft is the cumulative advantage provided by the biological properties of the former with the mechanical endurance of the latter.

Effects of cessation of immunosuppression on skeleton reconstructed by vascularized bone allograft in rats

In the present study, we investigated the effects of cessation of immunosuppression on skeleton reconstructed by vascularized allogenic bone transplantation in a rat tibio–fibula graft model. Twelve‐week‐old male 25 Dark Agouti rats with the major histocompatibility antigen (MHC) RT1a were used as donors and age‐matched male 25 Lewis rats with MHC RT11 were used as recipients. Among them, 20 rats were randomly allocated to 8‐week cyclosporine A (CsA) followed by 8‐week CsA vehicle group or continuous 16‐week CsA group. The remaining 5 rats received CsA for 8 weeks followed by no further treatment for next 40 weeks (long‐term observation group). In the CsA followed by vehicle group as well as the continuous CsA group, the structure of the reconstructed bones was maintained, though the transplanted bones in former group were found to be partly non‐vital. The CsA followed by vehicle group had higher bone mineral density of the transplanted bones and stronger strength of the reconstructed bones than the continuous CsA group. In the long‐term observation group, the structure of the reconstructed bones was still maintained and the transplanted bones were almost vital. These results suggest that long‐term strong immunosuppression may not be necessary for successful reconstruction of large bone defect by vascularized bone allograft.

Severe ulnar nerve palsy caused by synovial chondromatosis arising from the pisotriquetral joint: a case report and review of literature.

We report here the unique case of 60-year-old man with severe ulnar nerve palsy caused by synovial chondromatosis arising from the pisotriquetral joint. At operation, the tumor entrapped the ulnar nerve proximal to the Guyon canal so that it was severely paralyzed. The ulnar neurovascular bundle could be separated safely under the microscope. To our knowledge, this type of severe neuropathy has not been reported before. Although synovial chondromatosis associated with peripheral nerve neuropathy is extremely rare, we should be aware of the existence of this type of compression neuropathy in the upper limb.

Prolonged survival of rat whole-limb allografts treated with cyclophosphamide, granulocyte colony-stimulation factor and FK506.

We evaluated the efficacy of a new protocol using cyclophosphamide (CYP), granulocyte colony‐stimulation factor (G‐CSF) and FK506 to induce high level chimerism following rat whole‐limb allotransplantation. The present study investigated the dose requirement and toxicity of CYP monotherapy in inducing stable bone marrow chimerism. Fifty‐six whole‐limb allotransplants from LacZ transgenic rats to LEW rats were performed. CYP at a dose of 100 mg to 200 mg/kg was injected 2 days before transplantation and G‐CSF of 25 μg/kg/day was given for 4 days. FK506 was used for 28 days at 1 mg/kg/day. The level of chimerism was evaluated by semi‐quantitative polymerase chain reaction. The survival of limb allografts in recipients treated with CYP of 150 mg/kg was significantly prolonged to 107 days. The onset of rejection was more prolonged to 158 days in recipients with CYP of 200 mg/kg, with two of eight grafts surviving >1 year and three recipients (38%) showed chronic, nonlethal GVHD with a high level of bone marrow chimerism. Limb allografting could contribute to chimerism in the recipient. Pretreatment with CYP had the dose‐dependent effects of prolonging the survival of limb allografts. A CYP dose of 200 mg/kg appears to significantly prolong limb graft survival but frequently causes chronic nonlethal GVHD in the longer surviving recipients.

Frontalis sling procedure for ocular myasthenia gravis

A 39-year-old woman was diagnosed with myasthenia gravis when she was 8 years old. Although many treatments – such as cholinesterase inhibitors and steroids – had been given to the patient, her condition did not improve sufficiently. As she demonstrated bilateral 3 mm levator function without any eye movement disturbances, bilateral frontalis sling procedures were performed with an autologous fascia lata. One year after the operation, the operated upper eyelids showed symmetrically appropriate heights with good functional outcome. A sling procedure with an autologous fascia lata was suitable for correcting poor levator function of an ocular myasthenia gravis case.

Sarcoma in the forearm and hand: clinical outcomes and microsurgical reconstruction for limb salvage.

Sarcomas in the forearm and hand are very rare, and surgical outcomes have been unclear. The aim of this study was to examine oncologic outcome, microvascular reconstruction, and functional evaluation. A retrospective review was performed in 19 patients who were treated for soft tissue or osseous sarcoma. All 12 patients with sarcoma in the forearm were treated with limb salvage and needed microvascular reconstruction. Flap survival was excellent. Local recurrence occurred in 4 patients, 3 of which had previously undergone inadequate resection. Three patients had distant recurrence and 1 died of disease. The mean Enneking functional score was 83% at a mean follow-up period of 37 months. Although sarcomas often metastasize, the overall survival rate is excellent. Wide marginal resection during initial surgery is the most predictive factor for tumor control. Microvascular cutaneous, myocutaneous, or osteocutaneous flap reconstruction is essential for limb salvage and provides reliable, safe coverage with reasonable preservation of function.