Tsunemitsu Matsunaga

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.

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.

Effects of cerivastatin on vascularized allogenic bone transplantation in rats treated with cyclosporine A

The aim of the present study was to investigate the effects of oral cerivastatin (0.1 mg/kg/day) on vascularized allogenic transplanted bone that is treated with cyclosporine A (CsA) (10 mg/kg/day) and on vascularized isogenic transplanted bone that is not treated with CsA. Allogenic transplantation was performed on 12-week-old male DA rats with the major histocompatibility antigen (MHC) RT1a (as the donor) and age-matched male Lewis rats with MHC RT1l (as the recipient), and isogenic transplantation was performed on 12-week-old male Lewis rats. After transplantations, 20 rats (10 rats in each transplantation) were randomized into four groups to receive the following treatment for 16 weeks: (1) CsA plus cerivastatin vehicle or (2) CsA plus cerivastatin in the allogenic transplanted rats, and (3) CsA vehicle plus cerivastatin vehicle or (4) CsA vehicle plus cerivastatin in the isogenic transplanted rats. Bone biochemical markers, mineral density, and strength were measured at the end of the study period. Serum levels of osteocalcin (OC) and parathyroid hormone (PTH) and urinary deoxypyridinoline (DPD) level were higher in the allogenic transplanted rats than in the isogenic transplanted rats. In the allogenic transplanted rats, the cerivastatin treatment decreased urinary DPD levels, but not serum OC nor PTH levels. Furthermore, the cerivastatin treatment improved bone mineral density of the allogenic transplanted bones and bone strength of the allogenic reconstructed bones. In contrast, no effect of the cerivastatin treatment was observed in the isogenic transplanted rats. These results suggest that the cerivastatin treatment improves CsA-induced high-turnover osteopenia mainly through the inhibition of bone resorption.

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.