Effects of vitamin D analog, 22-oxa-1,25-dihydroxyvitamin D(3), on bone reconstruction by vascularized bone allograft.

We previously reported that vascularized bone allograft using immunosuppressants, such as cyclosporine A (CsA), is one approach for reconstruction of large bone defects in both experimental animals (Microsurgery 15:663; 1994) and clinically in humans (Lancet 347:970, 1996). Because immunosuppressive agents such as CsA induce significant side effects, including bone loss, other therapeutic agents supporting successful vascularized bone allografts have been sought after. We investigated the effects of 22-oxa-1,25-dihydroxyvitamin D(3) (OCT) on vascularized bone allograft, and compared its effects with CsA. Twelve-week-old DA rats with the major histocompatibility antigen (MHC) RT-1(a) were used as donors and age-matched Lewis rats with MHC RT-1(l) used as recipients. Allografted bones in rats treated with vehicle were rejected completely. Soft X-ray examination demonstrated that administration of OCT (0.5 microg/kg per day) for 12 weeks after bone graft induced bone union as effective as treatment for 12 weeks with CsA (10 mg/kg per day). Transplanted bones in OCT-treated rats showed higher bone mineral density than that in CsA-treated rats. Histologically, transplanted bones in OCT-treated rats at 12 weeks were nonvital, but these bones united with recipient vital bones. After cessation of 12 week treatment with OCT, new bone formation occurred around the grafted nonvital bones during a 9 month period. Transplanted bones in CsA-treated rats were vital and formed union with recipient bones, whereas cortical bones became thin when compared with nonvital bones in OCT-treated rats. Urinary deoxypyridinoline levels in rats treated with CsA were significantly higher than levels in rats treated with OCT, suggesting accelerated bone resorption in CsA-treated rats. These results suggest that OCT exerts an anabolic action on bone reconstruction by allogeneic bone transplantation.

Nerve regeneration and origin of Schwann cells in peripheral nerve allografts in immunologically pretreated rats.

For peripheral nerve allografts, the conditions for successful nerve regeneration and the possibility of transplanting Schwann cells were examined using a model of pretreated rats. Incomplete immunosuppression was achieved in recipient rats with donor red blood cell infusions given before allogeneic nerve grafting (RBC group). The origin of Schwann cells in the grafts was assessed by immunohistochemical staining from 1 week to 12 weeks after transplantation. In the RBC group, the replacement of donor Schwann cells by recipient cells was detected at 3-8 weeks, with the graft being filled with many of these cells at all times, and successful nerve regeneration was seen after 12 weeks on morphometric and electrophysiologic evaluations. In peripheral nerve allografts, pretreatment with donor-specific blood transfusion did not induce significant immunosuppression compared with allotransplantations of some tissues and organs. Clinically, if the state of immunosuppression can be controlled by RBC transfusion, it is possible that donor tissues may be replaced by recipient tissues and that nerves will regenerate successfully.

Successful nerve regeneration and persistence of donor cells after a limited course of immunosuppression in rat peripheral nerve allografts.

BACKGROUND: The origin of Schwann cells and effect of a limited course of immunosuppression using cyclosporine (CsA) were examined in rat peripheral nerve allotransplants. METHODS: Phenotypes of Schwann cells in groups without, with continuing, and with limited (12 weeks) CsA treatment were examined immunohistochemically in allogeneically and syngeneically transplanted animals from 4 to 36 weeks after transplantation. RESULTS: In the group receiving no CsA, little nerve regeneration was obtained; donor Schwann cells were rejected and replaced by recipient cells. In continuing and limited-course CsA groups, successful nerve regeneration was achieved at postoperative week 36, as was also observed in the syngeneic group. Schwann cells in the continuing CsA group remained donor-derived. In the limited-course CsA group, graft rejection and loss of function occurred after the withdrawal of CsA, and donor Schwann cells were replaced by recipient cells in the part of the graft where rejection had been complete. However, many donor Schwann cells remained at week 36, when the rejection response subsided. CONCLUSION: Possible clinical use of a limited course of immunosuppression was supported by this demonstration of long term persistence of donor Schwann cells.

Membrane antigen expression of syngeneically but heterotopically transplanted hepatocytes in rats.

The expression of membrane antigens on rat hepatocytes transplanted syngeneically and heterotopically was analyzed immunohistochemically using monoclonal antibodies against rat hepatocytes. Isolated adult and fetal hepatocytes were able to survive in the spleen, salivary gland, thymus, or subcapsular region of the kidney for various periods after transplantation. Fairly clear expression of HAM2, 4, and 8 antigens was observed on hepatocytes transplanted into syngeneic spleen, suggesting that the cells might be functionally equivalent to hepatocytes in situ. HAM4 antigen was localized specifically on the newly formed bile-canalicular faces of hepatocytes. The expression of HAM2 (MHC class I) antigen on the transplanted hepatocytes appeared much stronger on the side facing lymphoid tissues, than on the other faces, suggesting that some immunological reactions may take place between hepatocytes and lymphoid tissue. HAM8 antigen, which is localized on gap junctions between neighboring hepatocytes in rat liver, was also recognized between transplanted hepatocytes. In salivary glands where hepatocytes were transplanted, bile-canaliculus-like structures were observed not only between neighboring hepatocytes but also between hepatocytes and salivary acinar cells, suggesting good interaction between the two different epithelial cell types. Hepatocytes transplanted into thymus appeared viable, but most showed fatty degeneration. Some healthy hepatocytes survived in the interlobular connective tissue and the thymic cortical tissue. When fetal hepatocytes were transplanted heterotopically, they formed a mass consisting of hepatocytes and bile duct-like structures 7 wk after transplantation. The inoculated hepatocytes possessed HAM4 antigen, which was not recognized on fetal hepatocytes at day 14 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

Tensor fasciae latae flap: alternative donor as a functioning muscle transplantation.

The functioning tensor fasciae late muscle was used for a patient with both a complete defect of the deltoid muscle and a defect of overlying skin. The configuration of the tensor fasciae latae including the length of the muscle belly as well as the muscle fiber arrangement was similar to that of the deltoid. In addition, the spatial relationship between the muscle, donor vessels, and motor nerve fulfilled the requirements for the recipient site of deltoid reconstruction. The transferred muscle successfully replaced the function of the deltoid and provided sufficient strength for elevation of the arm. Simultaneous skin coverage was also satisfactory. The case report here clearly indicates that the tensor fasciae latae muscle is a promising candidate for functioning muscle transplantation, and can also be applied for other disorders. However, several points such as limited motor nerve length should be considered when tensor fasciae latae is used as a functioning muscle.

Revascularized intercalary bone allografts with short-term immunosuppression with cyclosporine in the canine.

To study the healing process of vascularized intercalary bone allograft after withdrawal of immunosuppressive drugs, allotransplantation of the tibia diaphysis with a vascular pedicle was performed in eight adult mongrel dogs (group 2) and assessments were made both during administration and after discontinuation of cyclosporin A. As controls, similar grafts with the vascular pedicles were removed and reimplanted back to the same animals (five dogs, group 1). Allotransplantation of frozen stored bone without a vascular pedicle (10 dogs, groups 3A and 3B) were also compared. No union occurred in most cases of frozen stored bone allotransplant because the transplanted bone was resorbed, leading to loosening and subsequent failure of osteosynthesis with the plate and screws used. Under cyclosporin A immunosuppression, bony union (i.e., when trabeculae were seen crossing the graft-recipient junction with obliteration of the junction line) occurred at almost similar time intervals in all dogs of group 2 (bone allotransplant with a vascular pedicle) by 3 months postoperatively, which was similar to those of group 1. No systemic side effects of cyclosporin A were observed. Cyclosporin A was discontinued 3 months following graft implantation. The bone graft became avascular within a week following withdrawal of cyclosporin A. However, bone union was maintained, and the transplanted bone never showed bone resorption, sclerosis, or fracture on serial radiographs up to the time the animals were sacrificed, between 5 and 14 months later. Histology at sacrifice showed that the transplanted allografts were being replaced at both ends by fresh bone derived from the transplantation bed. We conclude on the basis of the results of this study that solid bony union can be obtained in allotransplanted bone with a vascular pedicle if cyclosporin A is given for a brief period. After cyclosporin A is withdrawn, although the bone becomes nonviable secondary to rejection occurring in the blood vessels, its skeletal structure remains intact, enabling it to maintain its structural support while awaiting replacement by bony ingrowth from both ends of the graft.

Significance of elbow extension in reconstruction of prehension with reinnervated free-muscle transfer following complete brachial plexus avulsion.

Thirty-one patients with complete avulsion of the brachial plexus underwent reconstruction of elbow extension by intercostal nerve transfer following reconstruction of prehension with either a single or double free-muscle transfer. Long-term results of elbow extension were evaluated in 24 patients. Reinnervation of the triceps muscle took longer than that of the transferred muscle on serial electromyographic examinations, and the eventual strength of the triceps muscle was weak. None attained M5 grade, 2 achieved M4 grade, 4 achieved M3 grade, 8 achieved M2 grade, 5 achieved M1 grade, and another 5 achieved M0 grade. However, despite the weak recovery, 14 patients were able to obtain useful functional recovery of the triceps muscle, enabling it to stabilize the elbow joint against the transferred muscle, which acted as simultaneous elbow flexor and wrist or finger extensor. Elbow stability is imperative in order to obtain voluntary finger function following free-muscle transfer. Should the triceps muscle fail to recover following intercostal nerves neurotization, transferring the reinnervated infraspinatus to the triceps is an optional procedure to provide stabilization of the elbow.

Use of the AO hook-plate for treatment of unstable fractures of the distal clavicle.

INTRODUCTION: We used a new internal fixative implant, the AO clavicle hook-plate, for treatment of unstable fractures of the distal clavicle. This study describes the operative procedure and the clinical results obtained, as well as discussion of the advantages and problems encountered. PATIENTS AND METHODS: Fifteen consecutive patients with unstable fractures of the distal clavicle (Neer type II) were treated using AO clavicle hook-plates. The average age of patients was 47 years and there were 13 males and 2 females. The mean follow-up period was 15.5 months. Plain radiographs of clavicles were used to assess bony union. Functional recovery of the shoulder joint was assessed using the Constant-Murley scoring system. RESULTS: All fractures eventually achieved solid bony union within 4 months after surgery. Thirteen patients (87%) showed hook migration into the acromion. Clinical results were excellent with a mean Constant-Murley score of 89 points at final follow-up. CONCLUSION: AO clavicle hook-plates are useful fixative implants for unstable fractures of the distal clavicle. Static fixation was achieved and physiotherapy can be started immediately after surgery. Early removal of the implant is recommended however because hooks inserted under the acromion migrated into the bone in most cases.

Effects of bisphosphonate treatment on bone repair under immunosuppression using cyclosporine A in adult rats.

UNLABELLED: The effect of cyclosporine A on bone turnover remains unclear. Using adult rats with vascularized bone transplantation, we show that long-term cyclosporine A administration increases bone turnover and zoledronic acid treatment enhances the reconstruction of cyclosporine A-administered skeleton. Bisphosphonates might be efficacious in human bone repair under immunosuppression using cyclosporine A. INTRODUCTION: Bisphosphonate treatment effectively prevents bone loss after transplantation. However, recent evidence from gain- and loss-of-function experiments has indicated that calcineurin inhibitors, such as cyclosporine A (CsA), reduce bone turnover, and severely suppressed bone turnover might delay the union of human fractured bone. The purpose of this study was to investigate the effects of bisphosphonate treatment on the repair of CsA-administered skeleton. METHODS: After skeletal reconstruction by vascularized tibial grafting, adult recipient rats were treated with intramuscular CsA (10 mg/kg/day) and low-dose (0.2 microg/kg/week) or high-dose (2 microg/kg/week) subcutaneous zoledronic acid alone or in combination for 8 weeks. Biochemical parameters were measured in blood and urine. The reconstructed skeleton was analyzed using soft X-ray, histology, dual energy X-ray absorptiometry, and three-point bending test. RESULTS: CsA induced mild renal dysfunction, hyperparathyroidism and high bone turnover. High-dose zoledronic acid delayed cortical bone union at the distal host-graft junction, but its combination with CsA did not cause such a delay. High-dose zoledronic acid prevented CsA-induced bone loss and bone fragility in the reconstructed skeleton. CONCLUSION: In this rat model, long-term CsA administration increases bone turnover, at least partly, through hyperparathyroidism and high-dose zoledronic acid treatment does not impair the union of CsA-administered bone.

Studies of allotransplantation of bone using immunohistochemistry and radioimmunoassay in rats.

Immunohistochemistry and radioimmunoassay were used to study the rejection mechanism of allografted bone tissue in rats through changes in major histocompatibility complex (MHC) antigen expression by osteocytes in the allograft and in alloantibody levels in the serum of the host rats. Some osteocytes in normal rat bone tissue expressed MHC Class I antigen, and the degree of expression varied among the osteocytes. The expression of MHC Class I antigens on osteocytes of allografted bone tissue increased with time after allograft transplantation. When DA strain bone tissue was allografted to F344 strain host tissue, a marked decrease in the number of osteocytes was observed, and almost all of the remaining osteocytes strongly expressed MHC Class I antigens in the allografted bone tissue three weeks after transplantation. F344 strain rat recipients with DA strain rat bone grafts showed high titers of anti-Class I alloantibody. These titers increased two weeks after transplantation. Major histocompatibility complex Class I antigens on osteocytes may play an important role in allograft rejection, as has been observed in other transplanted organs in the rat.

Femoral reconstruction by single, folded or double free vascularised fibular grafts.

We reviewed 17 patients for a mean of 25 months period after free vascularised fibular transfer to reconstruct massive bone defect or recalcitrant nonunion of the femur. There were 11 cases of posttraumatic nonunion and six patients had a large bony defect following resection of bone tumour. Ten patients underwent double or folded and seven patients underwent single vascularised fibula graft transfer. Mean bony defect of the femur was 6.5 cm and mean length of grafted fibula was 15 cm. Revision surgery due to postoperative vascular complications was required in five cases. Twenty-three out of 24 (96%) vascularised fibulas were transferred successfully. The resultant outcome was successful in 15 out of 16 (94%) patients with confirmed bone union. Stress fracture occurred in three inlay fibula grafts. Hypertrophic change of the fibula graft was significantly noted in inlay grafts as compared to onlay grafts. All patients could walk without brace at a mean of 11 months postoperatively. Donor-site morbidity was minimum. Vascularised fibula grafting is a reliable and safe reconstructive procedure for massive femur defects. Folded or double fibula grafts cannot prevent stress fractures and the key point is to rigidly stabilise the femur in an anatomically aligned position.

Experience with the pedicled latissimus dorsi flap for finger reconstruction.

The pedicled latissimus dorsi flap was used for the reconstruction of finger function in 12 patients with severely disabled hands. The fascial origin of the muscle was sutured directly to finger flexors in 6 patients, and to extensors in the other 6 patients in the forearm without severing the neurovascular pedicle. Voluntary contraction of the muscle was obtained in all patients. Useful tendon gliding was achieved in 6 patients, thereby providing total active motion of fingers of >100 degrees. In the remaining 6 patients, however, several problems such as stiff finger joints prevented appropriate tendon gliding, resulting in total active motion of <60 degrees. When these problems are overcome, the pedicled latissimus dorsi muscle can yield satisfactory excursion for extrinsic tendons of the fingers. In contrast to free muscle transplantation, early and predictable recovery of function can be obtained using this particular technique without any risk of denervation of the muscle.

Experimental study on vascularized bone allografts for reconstruction of massive bone defects.

To study the healing mechanism of vascularized bone allografts under short-term as well as long-term immunosuppression with cyclosporin A, experimental vascularized intercalary bone allograft transplantation was carried out between inbred rats using the tibiofibula graft model. Bone scintigram and radiographs were used as an indicator for early detection of rejection after transplantation and bone union. In vascularized bone allografts under long-term immunosuppression with cyclosporin A, early bone union and continuous incorporation were similar to that observed in vascularized bone autograft transplantation. When administration of cyclosporin A was discontinued before completion of bone union, the graft was rejected and bone union was delayed. Apparent swelling on the operated limb associated with a decrease in bone scintigram uptake suggested the occurrence of rejection of the allograft. Vascularized bone allograft transplantation is useful for reconstruction of massive bone defects only if immunosuppressants are used and maintained at least until bone union is obtained.

Vascularized bone allografts: review of current concepts.

Advances in the field of microsurgery over the past two decades, together with the discovery of a powerful immunosuppressive, cyclosporin A, have brought vascularized osteochondral allografts closer to reality. In this review, the authors describe the history of laboratory research on vascularized osteochondral allografts using immunosuppression, as well as their experimental and preliminary clinical experiences.

Results of vascularized joint allograft under immunosuppression with cyclosporine.

The effects of cyclosporine (CsA), a strong immunosuppressive drug, on vascularized allogeneic joint transplantations were examined. An orthotopical transplant model of a vascularized knee joint allograft was developed using inbred DA and Lewis rats to investigate the fate of grafts following withdrawal of short-term immunosuppression compared to continuous immunosuppression with CsA. Five isograft controls acquired solid bone union at both femur and tibia sites within 4 weeks, and joint function as skeletal support was maintained until 25 weeks. Without immunosuppression, ten allografts were severely rejected within the first week, and joint destruction occurred immediately. Twenty-five short-term immunosuppressed rats acquired solid union, but, after withdrawal of immunosuppression, grafted joints showed gradual rejection and were destroyed due to pathological fractures or joint instability, although partial revascularization from the recipient occurred. Ten allografts under continuous immunosuppression at a dose of 10 mg/kg/day showed no rejection and remained viable for 12 weeks postoperatively, but thereafter all rats died. Death was considered to be a side effect of CsA. Fifteen animals, under continuous immunosuppression at a dose of 5 mg/kg/day, showed no rejection except in the bone marrow; the grafted joint function was not effected until 25 weeks. Continuous treatment with low and nontoxic doses (5 mg/kg/day) of CsA was necessary to maintain the functions of the grafted joint.

Nerve-regenerating effect of 15-deoxyspergualin. Peripheral nerve allotransplants in the rat.

We studied the effect of two immunosuppressive agents, 15-deoxyspergualin and cyclosporine A, on various nerve allografts in inbred rats whose major histocompatibility complex was mismatched. As allografts, we used the sciatic nerve (20 mm) and the saphenous nerve (20 mm). We found that 1) fresh peripheral nerve allografts with a short course of 15-deoxyspergualin and cyclosporine A therapy induced more regenerated axons than autografts did, 2) a short course of 15-deoxyspergualin therapy provided better nerve regeneration than cyclosporine A therapy in all forms of nerve allografts and large caliber nerve allografts induced more regenerated axons.

A new immunosuppressant, FTY720, prolongs limb allograft survival in rats.

A new immunosuppressant, FTY720, was applied to limb allotransplants and its effectiveness was investigated. Using inbred rats, for which the major histocompatibility complexes were completely mismatched, 31 limb transplantations were performed and FTY720 was administered at a dose of 1.5 or 3 mg per kilogram per day for 10 days postoperatively. Rejection was monitored by the appearance of the skin of the grafted hind limb, soft radiograph, microangiography, and histology. In animals receiving no immunosuppressive therapy, the mean onset of rejection was 4.2+/-1.0 days postoperatively, and the grafted limbs became acutely necrotic. The mean onset of rejection was 6.0+/-0.9 days in animals receiving FTY720 at a dose of 1.5 mg per kilogram per day and 7.9+/-1.5 days in animals receiving FTY720 at a dose of 3 mg per kilogram per day. Survival of the grafted limbs was significantly prolonged compared with that in animals without immunosuppression. The immunosuppressive effect of FTY720 appeared to be dose dependent; however, complete suppression of rejection could not be obtained with FTY720 therapy alone.

Longer survival of rat limb allograft. Combined immunosuppression of FK-506 and 15-deoxyspergualin.

We studied the individual and synergistic effect of 3 immunosuppressive drugs, FK-506 (1 mg/kg/day), 15-deoxyspergualin (2.5 mg/kg/day) and cyclosporine (15 mg/kg/day) in a DA/Lewis rat limb allotransplantation model. 74 right hindlimb transplantations were performed. The median time for onset of rejection was 4 days in animals without immunosuppression, 37 days in animals receiving cyclosporine immunosuppression for 30 days, 61 days in animals receiving FK-506 for 30 days, 36 days in animals receiving a 30-day course of cyclosporine and, in the first 15 days, a course of 15-deoxyspergualin, and 76 days in animals receiving a 30-day course of FK-506 and 15-deoxyspergualin in the first 15 days. The combination of cyclosporine with 15-deoxyspergualin did not prolong graft survival and no synergistic effect was evident. In contrast, survival time in rat limb allografts receiving FK-506 and 15-deoxyspergualin was longer than in those receiving single FK-506 therapy. Our findings suggest a positive synergistic immunosuppressive effect with FK-506 and 15-deoxyspergualin in limb allotransplantation.

Functioning muscle transplantation after wide excision of sarcomas in the extremity.

Free functioning muscle transplantation was performed after resection of 23 sarcomas in the extremity. There were 21 soft tissue sarcomas and two malignant bone tumors. The tumor resection was performed with a wide margin in all except two patients who had a marginal margin in a limited area. The consequent extensive soft tissue defect received free musculocutaneous flaps, the motor nerve of which was repaired in the recipient site. The most frequent procedure was latissimus dorsi transplantation to replace thigh muscles in 17 cases. The other donors included gracilis, tensor fascia lata, and rectus femoris, which were selected according to the site of defects. Patients were followed up for a mean of 60 months (range, 13-119 months). The grafted muscles showed reinnervation at a mean of 6 months postoperatively in all patients except for a 75-year-old patient. Obtained contraction of the muscles was powerful in 18 patients and fair in four patients. Performance of the salvaged limb significantly improved after recovery of the muscles. Although there were five distant recurrences, local recurrence was seen in one patient with systemic metastases. Because muscle loss could be compensated functionally for by the innervated free muscle transfer, the method encouraged surgeons to perform more radical tumor excisions and this may have contributed to the excellent local tumor control that was achieved. Thus, functioning muscle transplantation was extremely useful in limb salvage surgery from the functional and oncologic viewpoints.

Vascular complication in free tissue transfer to the leg.

The lower-extremity free flap has a high incidence of vascular complication. A retrospective study of 70 free flap transplants in 70 patients (1987-2000) was conducted to investigate factors leading to vascular complication and free flap failure. The overall success rate was 92% (64 of 70 flaps); the incidence of vascular complications was 22% (16 of 70 flaps). Among 16 complicated flaps, 7 were transferred in severely crushed legs, and 4 were in multioperated legs attributable to chronic osteomyelitis. Venous thrombosis occurred in 12 flaps (86%). The success rate of the patients operated on at Yamaguchi University Hospital was higher (96%) than at other hospitals tested. The key factors contributing to improved outcome in free tissue transplantation in the leg were careful preoperative planning for highly traumatized legs and proper selection of the recipient vein and of the hospital at which immediate reexploration can be performed for vascular complicated flaps.