Hideji Nishida

Reconstruction using an autograft containing tumour treated by liquid nitrogen

We describe a method of reconstruction using tumour-bearing autograft treated by liquid nitrogen in 28 patients. The operative technique consisted of en bloc excision of the tumour, removal of soft tissue, curettage of the tumour, drilling and preparation for internal fixation or prosthetic replacement before incubation for 20 minutes in liquid nitrogen, thawing at room temperature for 15 minutes, thawing in distilled water for ten minutes, and internal fixation with an intramedullary nail, plate or composite use of prosthetic replacement. Bone graft or cement was used to augment bone strength when necessary. The limb function was rated as excellent in 20 patients (71.4%), good in three (10.7%), fair in three (10.7%), and poor in two (7.1%). At the final follow-up six patients had died at a mean of 19.8 months after the operation, while 21 remained free from disease with a mean follow-up of 28.1 months (10 to 54). One patient is alive with disease. Bony union was seen at a mean of 6.7 months after the operation in 26 patients. Complications were encountered in seven patients, including three deep infections, two fractures, and two local recurrences. All were managed successfully. Our results suggest that this is a simple and effective method of biological reconstruction.

Pedicle frozen autograft reconstruction in malignant bone tumors

BackgroundStandardizing limb salvage surgery for malignant bone tumors should result in improved limb function after tumor excision and reconstruction. Recently, we developed and clinically applied a method of biological reconstruction using tumor-bearing autografts treated with liquid nitrogen. We report this newly modified technique using pedicle frozen autografts to save the continuity of anatomical structures.MethodsWe treated 33 malignant bone tumor patients. Diagnoses of the tumors were 17 osteosarcomas, 11 metastatic tumors, 2 Ewing’s sarcomas, 2 chondrosarcomas, and 1 undifferentiated pleomorphic sarcoma. The sites of the tumors were 23 femurs, 5 tibias, 4 humeri, and 1 calcaneus. Operative procedures consisted of exposing the tumor, performing one-site osteotomy or joint dislocation, rotating and freezing the tumor lesion in liquid nitrogen for 20 min, and reconstruction using intramedullary nailing, plates, or composite arthroplasty.ResultsPostoperative function was excellent in 25 patients (75.7%), good in 5 patients (15.1%), and fair in 3 patients (9.0%). At the final follow-up, 8 patients had died at a mean of 17 months postoperatively, and 18 patients remained disease-free for a mean follow-up period of 30 months (range 7–69 months). Seven patients were alive but with disease. Complications were encountered in 12 patients, including 4 deep infections, 3 fractures, 3 local recurrences from surrounding soft tissue, 2 nonunions, and 1 collapse. All were managed successfully.ConclusionsThe pedicle frozen autograft, which was newly developed to solve drawbacks of previously reported free frozen autografts, achieved success for reconstruction of malignant bone tumors. This is a new, simple, effective surgical technique for biological reconstruction that is still investigated but has potential for development.

Re-implantation of tumour tissue treated by cryotreatment with liquid nitrogen induces anti-tumour activity against murine osteosarcoma

We evaluated the possible induction of a systemic immune response to increase anti-tumour activity by the re-implantation of destructive tumour tissue treated by liquid nitrogen in a murine osteosarcoma (LM8) model. The tumours were randomised to treatment by excision alone or by cryotreatment after excision. Tissue from the tumour was frozen in liquid nitrogen, thawed in distilled water and then re-implanted in the same animal. In addition, some mice received an immunological response modifier of OK-432 after treatment. We measured the levels of interferon-gamma and interleukin-12 cytokines and the cytotoxicity activity of splenocytes against murine LM8 osteosarcoma cells. The number of lung and the size of abdominal metastases were also measured. Re-implantation of tumour tissue after cryotreatment activated immune responses and inhibited metastatic tumour growth. OK-432 synergistically enhanced the anti-tumour effect. Our results suggest that the treatment of malignant bone tumours by reconstruction using autografts containing tumours which have been treated by liquid nitrogen may be of clinical value.

Cryoimmunology for malignant bone and soft-tissue tumors

Several new methods have recently been developed for the treatment of malignant bone and soft-tissue tumors, and many of these targeted therapies have yielded promising initial results in clinical settings. As more sarcomas become amenable to effective molecular-targeting therapy, the need to evaluate the synergistic effects of combination therapies with anticancer drugs will grow. Other immunologic therapies have also been reported, such as exogenous cytokines, dendritic cell (DC) therapy and peptide vaccines. Cryoimmunology has shown promising results in some malignant tumors after cryosurgery and is expected to influence the next generation of tumor immunotherapy. In this report, we describe the induction of a systemic antitumor immune response following liquid nitrogen cryotreatment of a destructive murine osteosarcoma. Combining tumor cryotreatment with DCs to promote tumor-specific immune responses enhanced systemic immune responses and inhibited metastatic tumor growth. We also describe the induction of a systemic antitumor immune response following reconstruction for malignant bone tumors using frozen autografts treated with liquid nitrogen.

Caffeine activates tumor suppressor PTEN in sarcoma cells

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Akt activation exerts a strong anti-apoptotic effect and inhibits key pro-apoptotic proteins. We investigated the effect of caffeine in the prevention of tumor cell proliferation and induction of cell death. We found that caffeine induced increased intracellular cAMP levels, PTEN activation and Akt inactivation, which together prevented proliferation of human osteosarcoma cells (MG63) and fibrosarcoma cells (HT1080). PTEN knockdown by siRNA reduced the effects of caffeine on Akt inactivation in osteosarcoma cells. These results indicate that the tumor suppressor PTEN signaling pathway contributes to the growth-inhibitory effect of caffeine on sarcoma cells. Our data suggest that caffeine and other drugs that act on this pathway could have promising therapeutic effects in the treatment of sarcoma patients.

The long-term outcome following the use of frozen autograft treated with liquid nitrogen in the management of bone and soft-tissue sarcomas

In 1999, we developed a technique for biological reconstruction after excision of a bone tumour, which involved using autografts of the bone containing the tumour treated with liquid nitrogen. We have previously reported the use of this technique in 28 patients at a mean follow up of 27 months (10 to 54). In this study, we included 72 patients who underwent reconstruction using this technique. A total of 33 patients died and three were lost to follow-up, at a mean of 23 months (2 to 56) post-operatively, leaving 36 patients available for a assessment at a mean of 101 months 16 to 163) post-operatively. The methods of reconstruction included an osteo-articular graft in 16, an intercalary in 13 and, a composite graft with prosthesis in seven. Post-operative function was excellent in 26 patients (72.2%), good in seven (19.4%), and fair in three (8.3%) according to the functional evaluation system of Enneking. No recurrent tumour occurred within the grafts. The autografts survived in 29 patients (80.6%), and the rates of survival at five and ten years were 86.1% and 80.6 %, respectively. Seven of 16 osteo-articular grafts (44%) failed because of fracture or infection, but all the composite and intercalary grafts survived. The long-term outcomes of frozen autografting, particularly using composite and intercalary grafts, are satisfactory and thus represent a good method of treatment for patients with a sarcoma of bone or soft tissue.

Activity of bone morphogenetic protein-7 after treatment at various temperatures: Freezing vs. pasteurization vs. allograft

Insufficient bone union is the occasional complication of biomechanical reconstruction after malignant bone tumor resection using temperature treated tumor bearing bone; freezing, pasteurization, and autoclaving. Since bone morphogenetic protein (BMP) plays an important role in bone formation, we assessed the amount and activity of BMP preserved after several temperature treatments, including -196 and -73°C for 20 min, 60 and 100°C for 30 min, 60°C for 10h following -80°C for 12h as an allograft model, and 4°C as the control. The material extracted from the human femoral bone was treated, and the amount of BMP-7 was analyzed using an enzyme-linked immunosorbent assay. Then, the activity of recombinant human BMP-7 after the treatment was assessed using a bioassay with NIH3T3 cells and immunoblotting analysis to measure the amount of phospho-Smad, one of the signaling substrates that reflect the intracellular reaction of BMPs. Both experiments revealed that BMP-7 was significantly better preserved in the hypothermia groups. The percentages of the amount of BMP-7 in which the control group was set at 100% were 114%, 108%, 70%, 49%, and 53% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. The percentages of the amount of phospho-Smad were 89%, 87%, 24%, 4.9%, and 14% in the -196, -73, 60, 100°C, and the allograft-model group, respectively. These results suggested that freezing possibly preserves osteoinductive ability than hyperthermia treatment.

Caffeine-potentiated chemotherapy for metastatic osteosarcoma

BackgroundThe prognosis for patients with metastatic osteosarcoma is still poor despite the development of effective adjuvant and neoadjuvant chemotherapy regimens. We have developed caffeine-potentiated chemotherapy for treatment of high-grade bone and soft tissue sarcomas based on the ability of caffeine to enhance the cytocidal effects of anticancer drugs. We report results of caffeine-potentiated chemotherapy for patients with osteosarcoma with pulmonary metastases.MethodsWe analyzed retrospectively overall survival and some prognostic factors for 41 patients with osteosarcoma/pulmonary metastases who were treated with caffeine-potentiated chemotherapy between 1990 and 2006.ResultsThe mean follow-up of all patients was 32.7 months. At the time of the final follow-up, 11 patients were alive and 30 had died of disease. Overall survival rates at 2 and 5 years were 38% and 28%, respectively. We identified the primary tumor site, the histological response to preoperative chemotherapy, the number of pulmonary nodules at initial identification, the timing of pulmonary metastasis identification, and the existence of extrapulmonary metastasis as prognostic factors. Especially, the number of pulmonary nodules at initial identification and the timing of pulmonary metastasis identification were independent, strong prognostic factors. Patients with solitary pulmonary metastasis had good prognoses, and their overall 5-year survival rate was 60%; in contrast, survival was 28% in patients with two to five pulmonary nodules, and no patients with more than six nodules survived 5 years. Patients with pulmonary metastasis identified after completion of treatment had the best prognosis, whereas patients with pulmonary metastases identified during treatment had the worst prognosis.ConclusionsCaffeine-potentiated chemotherapy prolonged survival of patients who had osteosarcoma with pulmonary metastasis. Especially, patients with pulmonary metastasis identified after completion of treatment or with a solitary pulmonary nodule had good prognoses.

Antimicrobial megaprostheses supported with iodine

Deep infection associated with implants remains a serious complication of orthopedic surgery. We developed iodine coating for titanium implants. In this study, we performed a clinical trial of iodine-coated megaprostheses to evaluate its safety and antibacterial effects. Forty-seven patients with malignant bone tumor or pyogenic arthritis were treated using iodine-supported titanium megaprostheses between July 2008 and May 2013. The mean age was 53.6 years (range, 15–85 years). Twenty-six patients were males and 21 were females. The diagnoses included malignant bone tumor in 29 cases, infected total knee arthroplasty in 11 cases, chronic osteomyelitis due to pyogenic arthritis in six cases and loosening of total knee arthroplasty in one case. The iodine-supported implants used were 42 Kyocera Limb Salvage System and five KOBELCO K-MAX K-3. These megaprostheses were used to prevent infection in 21 patients, treat active infections in 26 patients. The mean follow-up period was 30.1 months (range, 8–50). Infection was prevented in 20 out of 21 patients. Only one patient had surgical site infection caused by Pseudomonas aeruginosa and was cured by intravenous administration of antibiotics alone without removal of the implant. In 26 treatment cases involving one- or two-stage revision surgery, infection subsided without any additional surgery. In all cases, there were no signs of infection at the time of the last follow-up. White blood cell and C-reactive protein levels returned to normal within four weeks after surgery. To confirm systemic effects of iodine, thyroid hormone levels in the blood were examined. Abnormalities of thyroid gland function were not detected. Loosening of the implants was not observed. Excellent bone ingrowth and ongrowth were found around iodine-supported megaprostheses. The iodine-supported titanium megaprostheses are highly effective and show promise for the prevention and treatment of infections in large bone defects. No cytotoxicity or adverse effects were detected with this treatment.

Cryoimmunologic Antitumor Effects Enhanced by Dendritic Cells in Osteosarcoma

BackgroundWe previously reported a limb-salvage technique by treating tumor-bearing bone with liquid nitrogen. We also reported systemic antitumor immunity was enhanced by cryotreatment in a murine osteosarcoma (LM8) model. We therefore combined the cryotreatment of tumor with dendritic cells to promote tumor-specific immune responses.Questions/purposesWe determined whether our technique could enhance systemic immune response and inhibit metastatic tumor growth in a murine osteosarcoma model.Materials and MethodsTo evaluate activation of the immune response, we prepared six groups of C3H mice (80 mice total): (1) excision only, (2) dendritic cells without reimplantation of the cryotreated primary tumor, (3) reimplantation of the cryotreated primary tumor alone, (4) dendritic cells combined with reimplantation of the cryotreated primary tumor, (5) dendritic cells exposed to cryotreated tumor lysates without reimplantation of the cryotreated primary tumor, and (6) dendritic cells exposed to cryotreated tumor lysates with reimplantation of the cryotreated primary tumor. We then compared and verified the activation state of each group’s antitumor immunity.ResultsMice that received dendritic cells exposed to cryotreated tumor lysates with reimplantation of the cryotreated primary tumor group had high serum interferon γ, reduced pulmonary metastases, and increased numbers of CD8(+) T lymphocytes in the metastatic areas.ConclusionsCombining tumor cryotreatment with dendritic cells enhanced systemic immune responses and inhibited metastatic tumor growth.Clinical RelevanceWe suggest immunotherapy could be developed further to improve the treatment of osteosarcoma.