| Osteosarcoma (OS) is a primary highly malignant tumor of the musculoskeletal system, with an incidence ranking second in primary bone tumors, a peak age of onset ranging from 15 to 25 years old, and high-incidence areas in the distal femur, the proximal humerus, the proximal tibia and the metaphysis. The clinical manifestations of this disease include limb pain, swelling, limping, pathological fractures, etc. Partial patients are accompanied by fever, anemia and weight loss. Moreover, distant metastasis may occur, which causes serious damage to the health of young people, leading to high mortality and disability rate. Before 1970, amputation was the standard treatment of OS.With the extensive application of neoadjuvant chemotherapy and the development of joint reconstruction technique, limb-salvage therapy has become the main trend in the surgical treatment of OS. The main content of this idea is to keep patients' own joint structure as far as possible, without increasing the risk of OS, to promote the functional recovery of the affected limb and overcome long-term complications such as limb shortening and prosthesis loosening caused by mechanical reconstruction, and thereby realizing the biological reconstruction of limb bone defects in malignant bone tumors. It is worth emphasizing that the limb-salvage therapy for bone tumors should first reach safe surgical margins in order to reduce local recurrence rate, and functional reconstruction is based on safe surgical margins. However, no research data regarding that how far resection is away from tumor margins can control local recurrence rate have been reported yet. The present study established an Mg-63 human OS xenograft model in nude mice and compared different therapeutic methods, to evaluate the correlation of surgical margins with local recurrence.In addition, among patients with OS, massive bone allograft is always used for reconstruction; however, nonunion of bone is increasingly becoming a challenge. BMP-2 can promote bone healing, and is also traditionally considered to play a role in the improvement of the local recurrence rate of OS. Therefore, BMP-2 is not recommended all the time. Our model also evaluated the correlation between the application of BMP-2 and local recurrence.During postoperative chemotherapy in patients with OS, multi-drug resistance may occur in partial patients, which seriously affects the efficacy of chemotherapy and always leads to the failure of chemotherapy, influencing prognosis. Through establishing an animal model of drug-resistant human OS cell line MG63/ADM, this study applied different chemotherapy regimens to evaluate the correlation of surgical margins with local recurrence. Arsenic trioxide (As2O3), which is the main component of arsenic, has good efficacy in malignant tumors of the hematopoietic system, the digestive system and the reproductive system; however, As2O3 has been rarely studied in OS. Our study also evaluated the effects of As2O3.Part I Evaluation of correlation between surgical margins and local recurrence by comparing different therapeutic methods[Objective] In surgery for OS, definition of an appropriate surgical margin, which not only can improve the effect of reconstruction and functional prognosis, but also can control local recurrence, is an issue that draws increasing attention. Through the establishment of an animal model of human OS cell line MG-63, different therapeutic methods were adopted to evaluate the correlation between surgical margins and local recurrence.[Methods] Establishment of human OS cell line Mg-63 in-situ xenograft model in nude mice Human OS cell line Mg-63 was routinely cultured and passaged in RPMI-1640 medium with 100 U/ml penicillin,10% fetal bovine serum, 100?g/ml streptomycin in a 5% CO2 incubator at 37?. Mg-63 cells during the logarithmic growth phase were collected and suspended with RPMI-1640 medium after digestion by trypsin. Cells were counted and adjusted to 2×107/ml. Under sterile conditions,25?l Mg-63 suspension was slowly injected into the proximal tibial medullary cavity of nude mice.1. Different therapeutic methods for nude mouse modelThe nude mice were randomly divided into 4 groups, with 30 mice per group: operation group, operation and single-drug chemotherapy (cisplatin or adriamycin) group, and operation and double-drug chemotherapy (cisplatin and adriamycin) group, respectively.2. Evaluation for different experimental groupsThe operation+single-drug chemotherapy group and the operation+double-drug chemotherapy group was compared with the operation group, respectively, to evaluate the correlation of surgical margins with local recurrence among different therapeutic methods.[Results] In the xenograft mouse model, the growth and development of OS followed a characteristic natural course, which simulated the growth process of human OS. For the operation group, no local recurrence occurred in patients with the range of surgical resection exceeding more than 994?m away the boundaries of OS lesions. For the operation+single-drug chemotherapy group, no local recurrence occurred in patients with the range of surgical resection exceeding more than 37?m away the boundaries of OS lesions. Comparison in surgical margins between the operation+single-drug chemotherapy group and the operation group showed significant differences (cisplatin, P<0.05; adriamycin, P<0.05). For the operation+double-drug chemotherapy group, no local recurrence occurred in patients with the range of surgical resection exceeding more than 12?m away the boundaries of OS lesions. Comparison in surgical margins between the operation+double-drug chemotherapy group and the operation group presented significant differences (P<0.05).[Conclusion] This model reliably simulates the clinical course, radiological imaging and therapeutic methods of human OS, and also successfully references the experience in chemotherapy for human OS. Compared with the operation group, the locally controlled distance from surgical margins to tumor margins decreased obviously in the operation+single-drug chemotherapy group, while reduced significantly in the operation+double-drug chemotherapy group.Part ? Correlation Between Application of Exogenous BMP-2 and Local Recurrence[Objective] The role of BMP-2 in the promotion of bone healing has been confirmed in a large number of preclinical experiments; in clinic, BMP-2 is also widely used in some orthopedic surgeries not for tumors. In surgery for OS, nonunion of bone is still a challenge among patients with reconstruction using allografts. Traditionally, BMP-2 is not suggested due to its role in the promotion of tumor recurrence. Through the establishment of an animal model of human OS, the correlation between application of exogenous BMP-2 and local recurrence was evaluated.[Methods] A human OS cell line Mg-63 in-situ xenograft model in nude mice was established. Then, the animal model was randomly divided into 2 groups, with 30 mice per group:operation+BMP-2 group and operation+double-drug chemotherapy+BMP-2 group, respectively. Afterwards, the operation+BMP-2 group was compared with the operation group, and the operation+double-drug chemotherapy+BMP-2 group was compared with the operation+double-drug chemotherapy group, respectively.[Results] For the operation+30?g BMP-2 group, no local recurrence occurred in patients with bone resection margins greater than 687?m and resection margins of soft tissue greater than 640?m; while local recurrence was found in patients with bone resection margins below 91 ?m and resection margins of soft tissue below 80?m. Compared with the operation group, no apparent differences were detected (P>0.05). For the operation+double-drug chemotherapy+30?g BMP-2 group, local recurrence occurred in patients with bone resection margins below 9?m and resection margins of soft tissue below 8?m; while no patients with bone resection margins greater than 11?m and resection margins of soft tissue greater than 9?m presented local recurrence. Compared with the operation+double-drug chemotherapy group, no evident differences were found (P>0.05).[Conclusion] In both operation group and operation combined with chemotherapy group, application of exogenous BMP-2 in operative site after tumor resection does not increase the risk of local recurrence.Part ? Evaluation of correlation between surgical margins and local recurrence by establishing an animal model of drug-resistant human OS cell line MG63/ADM[Objective] During chemotherapy for OS, multi-drug resistance may occur in partial patients, which leads to the failure of chemotherapy. The correlation between surgical margins and local recurrence was evaluated by establishing a model of drug-resistant human OS cell line MG63/ADM and treating with operation and different chemotherapy regimens.[Methods] 1. Drug-resistant cell line MG63/ADM was established.2. A drug-resistant human OS cell line MG63/ADM in-situ xenograft model in nude mice was established. 3. The animal model was randomly divided into 4 groups, with 30 mice per group: operation group,operation+adriamycin group, operation+As2O3 group, and operation+adriamycin+As2O3 group, respectively.4. Comparison was conducted among different experimental groups.[Results] For the operation group, no patients with bone resection margins greater than 936?m and resection margins of soft tissue greater than 748?m presented local recurrence;For the operation+adriamycin group, no patients with bone resection margins greater than 705?m and resection margins of soft tissue greater than 627?m presented local recurrence; compared with the operation group, no apparent differences were found (P>0.05). For the operation+As2O3 group, no local recurrence was detected in patients with bone resection margins greater than 582?m and resection margins of soft tissue greater than 494?m; comparison with the operation group demonstrated no significant differences in resection margins of soft tissue or bone resection margins (P>0.05). For the operation+adriamycin+As2O3 group, no local recurrence occurred in patients with bone resection margins greater than 52?m and resection margins of soft tissue greater than 44pm; compared with the operation+adriamycin group, there were significant differences in both resection margins of soft tissue and bone resection margins (P<0.05).[Conclusion] Wide surgical excision and adjuvant chemotherapy are two important measures for the treatment of OS. For OS with multi-drug resistance, the range of surgical resection should be appropriately expanded. As2O3 plays a certain role in reversing multi-drug resistance in OS. |
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