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Objective:To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs. Methods:MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains, and the 50% inhibitory concentration (half maximal inhibitory concentration, IC 50) of drug-resistant and sensitive strains was detected by MTT, and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity. RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells. Exosomes were identified by particle size analysis and Western Bolt detection. The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed, and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay. The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay. Tumor growth, serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group, drug-resistant+normal exosome group, drug-resistant+drug-resistant+drug-resistant exosome group were observed. MDR1 expression level in tumor tissue was detected by RT-PCR, Western Blot and immunohistochemistry. Results:The IC 50 of two adriamycin resistant strains were 2.21 vs. 11.81 μg/ml and 0.93 vs. 11.81 μg/ml, respectively, and the fluorescence intensity decreased faster than that of normal strains. The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16, 1.02±0.11 and drug-resistant 2.15±0.10, 2.127±0.12, respectively. The relative protein expression of P-gp was normal 0.92±0.11, 0.73±0.10 and drug-resistant 0.46±0.03, 0.30±0.04, the differences were statistically significant ( P<0.05). Drug-resistant exosomes can enter osteosarcoma cells through endocytosis and concentrate in the cytoplasm when co-cultured with normal strains. Osteosarcoma cells were co-cultured with drug-resistant exosomes at 2, 4, 6, and 8 μg/ml adriamycin, respectively. Compared with normal group, the proliferation level in drug-resistant group was significantly increased. Compared with the normal cell group 35.95±3.92, 6.72±3.55 and the normal exosome group 51.22±5.55, 19.31±1.93, the drug-resistant cell group 54.20±9.32, 19.24±2.88 and drug-resistant exosome group 76.40±5.41, 30.26±4.87, all had significantly higher cell mobility, the difference was statistically significant ( P<0.05). Exosome sequencing and biogenic analysis of 10 highly upregated miRNAs to validate mRNA expression differences between normal and drug-resistant strains by RT-PCR, showing a significant increase in miR-21-5p expression level of drug-resistant strains (5.89±0.26 vs. 0.99±0.06; 1.05±0.07 vs. 8.80±0.93, P<0.05), the difference was statistically significant ( P<0.05). In MG63 and U2OS, the normal cell group and drug-resistant cell group, and the normal exosome group and drug-resistant exosome group were compared, the tumor volume and the terminal tumor weight of nude mice were increased to varying degrees. MRNA relative expression levels of miR-21-5p in serum exosomes of nude mice after drug intervention were 0.86±0.07 and 0.86±0.05 in normal cell group, respectively. The values were 1.13±0.12, 1.14±0.12 in drug-resistant cell group, 0.71±0.05, 0.75±0.03 in normal exosome group, and 0.90±0.07, 0.93±0.04 in drug-resistant exosome group. Compared with normal and drug-resistant strains, the expression levels of normal and drug-resistant exosome groups were increased, with statistical significance ( P<0.05). Conclusion:The exosomes of drug-resistant cells in osteosarcoma could enhance the proliferation level and migration ability of cells through intercellular transfer of MDR1 and miRNAs. The expression of MDR1 and miR-21-5p in drug-resistant cells and tumor-forming nude mouse serum and tumor tissues were up-regulated which suggested that it might be involved in regulating the drug resistance process of osteosarcoma.
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Denosumab is a fully human monoclonal antibody on receptor activator of NF-κB ligand that has shown adjuvant treatment for giant cell tumour of bone (GCT).Clinical trials show that over 85％ of patients have significantly improved their clinical symptoms,imaging and histology.Currently mainly used for central axis bone such as the sacrum and spine of difficult surgical excision or limb recurrence or refractory GCT patients.Case reports have demonstrated complete response or tumor stabilization with denosumab,allowing for surgical procedures in simplify.However,the duration of the medication and the optimal therapeutic dose and long-term effects are not yet known.The local high recurrence rate after discontinuation of the drug remains a problem with the accumulation of clinical research,and the follow-up time is prolonged.More noteworthy is the possibility of pseudosarcoma,even malignant transformation after Denosumab treatment.This sarcoma transformation requires further controlled studies and long-term of follow-up to reach a definitive conclusion.In this paper,we retrospective analysis of the application status,imaging and histology related research of Denosumab in the treatment of GCT.Correct understanding of the value and clinical significance of this drug in GCT treatment requires a multi-center study and a long-term follow-up to evaluate the clinical value.
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Objective To analyze clinical efficacy of artificial prosthesis in giant cell tumor in distal femur,and to investigate risk factors affecting prosthesis failure and functional outcomes.Methods 42 patients with giant cell tumor of bone in distal femur,who had undergo prosthesis replacement from January 2002 to May 2015,were enrolled in this study.There were 24 males and 18 females,with an average age of 38.53± 12.87 years.There were 28 primary patients and 14 relapsed patients,including 11 cases of recurrence once and 3 cases of twice.Three-dimensional finite element model was used to analyze the effect of different angles of deviation of the spinal needle on the deformation of the bone wall.The correlations between the factors such as age,sex,occupation,prosthesis type,and other factors on prosthesis loosening were compared.Biomechanical effect of lower limbs caused by prosthesis offset angle was analyzed through gait analysis.Analyzed the effects of primary tumor or recurrence,prosthesis service status,and length of surgical osteotomy on joint function.Results A total of 42 patients were followed up by 20-158 months,with an average of 68.7 months.The 3 year survival rate of prosthesis was 83.33％ for 3 years and 57.14％ for 5 years.The major reason of prosthesis failure was loose (18/42,42.8％).X-ray films showed 19 cases of prosthetic intramedullary nail and sagittal bias of medullary force line angle ＞ 3° in the first follow-up.Osteotomy length (OR=0.132,P=0.0027) and offset angle of needle (OR=25.000,P=0.000) were significantly correlated to prosthesis loose.A length more than 12 cm and angle more than 3° were easier to result in prosthesis loose.There were no significant correlation between prosthesis failure and patients age,gender,occupation and prosthesis type.Gait analysis shows that the unsuitable bias angle of the prosthesis can significantly change the joint force of the prosthesis.The average score of MSTS 93 function evaluation was 25.43±4.256,excellent in 33 cases,good in 7 cases and poor in 2 cases.Function of patients with primary GCT were better than that of recurrent ones.Patients with one 1 times recurrence were better than that of recurrence twice (P=0.003).Patients without prosthesis loosening and revision were better than that with loosening (P=0.001).Patients with an osteotomy length less than 12 cm had a poorer function than that with more than 12 cm (P=0.002).Conclusion The main factors affecting distal femoral prosthesis replacement therapy of GCT is loosening,which was caused by broach and medullary cavity mismatch,osteotomy length,prosthesis rotation,prosthesis position.The function of the prosthesis is mainly affected by operation times,prosthesis status,osteotomy length and low patella.
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Objective To retrospectively analyze clinical features,treatment methods and efficacy of giant cell tumor of bone in proximal tibia,and to investigate risk factors affecting tumor recurrence and functional outcomes.Methods A total of 250 patients with giant cell tumor of bone in proximal tibia confirmed by pathology,who had undergone surgical treatment from March 2000 to July 2014,were enrolled in this study.There were 132 males and 118 females,with an average age of (34.59±12.86) years.A total of 140 patients who were followed up for more than 3 years were included in this study,and there were 72 males and 68 females,with an average age of (34.46± 11.96) years.There were 11 cases of Campanacci grade Ⅰ,58 cases of grade Ⅱ,71 cases of grade Ⅲ and pathological fracture of 47 cases.According to surgical methods,they were divided into bone grafting group (49 cases),bone cement filling group (34 cases),prosthesis group (46 cases) and others group (11 cases).The epidemiology,clinical and radiographic features and risk factors affecting tumor recurrence and functional outcomes were analyzed.Results A total of 140 patients were followed up,the follow-up period was 36-324 months,with an average of 95.4 months,and the median follow-up time was 88 months.Recurrence was found in 26 cases,and recurrence rate was 18.57％,with an average recurrence interval of 25.85 months.Recurrence was found in 17 cases in the first 2 years.The 5-year free survival rate was 77.60％.The recurrence rates were 18.37％ in bone grafting group,20.59％ in bone cement filling group,15.22％ in prosthesis group and 27.27％ in the others group,no statistically difference was found on recurrence rate and free survival rate (P=0.805,P=0.558).Recurrence was not related to all kinds of factors.A variety of related factors affecting postoperative recurrence were analyzed,sex,the first diagnosis of the original recurrence,left and right side,whether the eccentricity,fracture,cortical bone destruction,soft tissue mass,surgical methods,high-speed grinding,auxiliary application,and there was no significant correlation between recurrence and these factors.The MSTS 93 score was 25.26±4.31.Function of the primary patients was better than that of recurrence (P=0.044).Function of the patients treated with curettage with or without internal fixation was better than that with segmental resection (P=0.011).Function of the patients treated with grafting or bone cement filling was better than that with prosthesis or allograft-prosthesis reconstruction (P=0.004).There were no significant correlation between MSTS function score and gender,left and right side,whether the eccentricity,whether fractures,cortical bone destruction (Campanacci grade),whether there is soft tissue mass,whether the use of assisted inactivation,whether the use of grinding or internal fixation.Conclusion Various surgical methods had no significant effect on the recurrence of proximal tibial GCT,as for GCT in proximal tibia,there is no relation between recucrrence and related factors.Whether primary tumor and surgical methods are two important factors affecting limb function.
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BACKGROUND: Bone defects forming after resection of bone tumor and tumor-like lesion are often packed with autogeneic bone. But sample amount cannot completely meet the clinical demand and different degrees of complications are often left in the bone graft donor region. All these problems remarkably limit the application of autogeneic bone. Allogenic bone is increasingly widely used in the clinical practice due to its structure and biological characteristics similar to autogeneic bone, rich sources, long-term preservation, easy to use and other advantages. OBJECTIVE: To investigate the biocompatibility and clinical application effects of allogeneic bone in filling and repairing benign bone tumor and tumor-like lesion after resection and curettage.DESIGN: A retrospective analysis. SETTING: Department of Pelvis Surgery, Second Affiliated Hospital of Inner Mongolia Medical College. PARTICIPANTS: Totally 230 patients (156 males and 74 females, with age of 5-56 years) were admitted to Department of Bone Tumor, Second Affiliated Hospital of Inner Mongolia Medical College from December 1999 to December 2005 to undergo curettage and hyperthermia inactivation due to benign bone tumor and tumor-like lesion and to receive the treatment by filling and repairing bone defects with lyophilized small-segment allogeneic bone, and were recruited for this study. Written informed consents of treatment were obtained from all the patients. The protocol was approved by the Hospital's Ethics Committee. METHODS: Allogeneic bone grafts (Shanxi Aorui Biomedical Co.,Ltd /Shanxi Provincial Medical Tissue Banking) were used to fill and repair bone defects. Patients who had benign bone tumor, bone cyst or osteofibrous dysplasia underwent cyst curretage. Allogeneic bones were used to pack empty cavity. Therapeutic effects were assessed according to the scoring criteria of allogeneic bone transplantation from Mankin et al, consisting of satisfactory and unsatisfactory two levels. X-ray plain films of surgery sites were taken at postoperative 3, 6 and 12 months. The patients were followed up for 38 months on average in order to observe the therapeutic effects. MAIN OUTCOME MEASURES: Histocompatibility of allogeneic bone in filling and repairing bone tumor defects. RESULTS: All 230 patients participated in the final analysis. ① Biocompatibility of allogeneic bone: Postoperatively, minority of patients had mild immunological rejections. Such allogeneic bone grafts had a good biocompatibility. They could directly fuse with bone tissue in the implantation location of patients, but not inhibit the normal activity of osteocytes on the allogeneic bone grafts or interfere the natural substitution of autogeneic osteocytes, i.e. there were no or less immunological rejections. Bone union was obtained in all the patients at postoperative 6-18 months (6.5 months on average). Thirty-four patients presented exudation of light yellow liquid from incision. Incisions healed in 30 (14.8%) patients at postoperative 2 weeks and in 4 (1.7%) in later time. Altogether 196 (85.2%) patients obtained satisfactory therapeutic effects, but 34 (14.8%) obtained unsatisfactory therapeutic effects. CONCLUSION: Small-segment allogeneic bones have good histocompatibility and osteogenesis, and they are good bone grafts in the bone transplantation.