Objective To investigate feasible immunotherapy strategy using tumor specific cell against osteosarcoma,and to analyze the therapeutic effect of adoptive cellular infusion therapy on osteosarcoma.Methods Decitabine (DAC) was employed as a hypomethylating agent for the treatment in osteosarcoma cell lines HOS and U2OS.After treatment,the expression of cancer-testis antigen (CTA) was evaluated by PCR and Western Blot.In animal studies,human osteosarcoma cell line HOS,which was transfected by luciferase and HLA-A0201 in previous,was inoculated into immune deficient NOD-SCID mice to establish osteosarcoma xenografts.Ex-vivo expanded CTA specific homo CD8+T-ells were labeled with DiR and injected into the mice via the tail vein.In vivo imaging system was utilized to detect the distribution of administrated CD8+ T-cells.In addition,the progression of tumor xenografts was monitored.Moreover,mouse K7M2 osteosarcoma cell line was used to establish animal models in immune competent BALB/c mice.Immune competent models were utilized to evaluate the effectiveness of hypomethylating treatment in regarding to spontaneous immune attack against tumors.Flow cytometry was used to analyze the proportion of intratumoral lymphocytes and the status of these effector antitumor immune cells,and to reveal the effect of hypomethylating treatment in facilitating lymphocyte infiltration and activation.Results The expression of all the evaluated cancer/testis antigens were elevated in HOS and U2OS osteosarcoma cell lines after hypomethylating treatment with DAC.The proliferation of in vitro cultured osteosarcoma cells can be significantly suppressed after at least 5 d treatment with DAC.Besides,DAC alone controlled osteosarcoma cell proliferation.In immune deficient mouse models,hypomethylating pre-treatment resulted in successful T-cell homing to tumor sites.Moreover,the combination treatment with DAC and CTA specific T-cell adoptive transfer significantly suppressed tumor proliferation.In immune competent mouse models,hypomethylating treatment with DAC improved autologous T-cell infiltration into the tumor,and strengthened the activity of intratumoral CD8+ T-cells,elevated the secretion of IFN-gamma,granzyme B and perforin by CD8+ T-cells.Conclusion Hypomethylating treatment is able to suppress osteosarcoma cell proliferation,improve the expression of CTA in osteosarcoma cells,and consequently provide optimal environment for CTA specific T-cell adoptive therapy.

Objective To investigate the safety and efficacy of curettage and excision for giant cell tumor of the cervical spine.Methods Retrospectively analyzed 6 cases of giant cell tumors patients who underwent surgical resection of cervical spine between April 2006 and July 2015.There were 2 males and 4 females with an average age of 31.3 years old (ranged from 18 to 42 years).Among them,4 cases were located in C2 vertebral body,1 case located in C3 vertebral body,1 case in C4 spinous pro cess and vertebral lamina.In this study,all the 6 patients underwent intralesional surgery,3 patients were Enneking grade ⅡA,which 1 case underwent curettage,2 cases underwent extended curettage;three cases were Enneking grade ⅢB,which 1 case underwent extended curettage,and 2 cases underwent subtotal spondylectomy.Three patients received local inactivation of the lesion.One patient received local radiotherapy after first posterior occipital-cervical fixation,and underwent posterior and anterior tumor resection,bone grafting and internal fixation 3 months later.One patient was treated with denosumab preoperatively,3 patients were treated with bisphosphonates postoperatively.The neurological status was evaluated by Frankel classification pre-and postoperatively,the pain scale was evaluated by Visual Analogue Scale (VAS) pre-and postoperatively.Imaging examination was used to assess the location and the extent of the tumor preoperatively,as well as the tumor recurrence postoperatively.Results The mean follow-up period was 61.1 months (ranged from 25 to 134 months).One case underwent posterior surgery,while other 5 cases underwent anterior and posterior surgery;3 patients underwent one-stage surgery,3 patients underwent multi-staged surgery.The mean intraoperative bleeding was 2 142 ml (ranged from 300 to 9100 ml).The mean preoperative VAS score was 3 (ranged from 2 to 5),the neck pain of the patients were all relieved postoperatively,and the mean VAS score was 0.The Frankel grade were E in all 6 patients of pre-and postoperation.One intracranial epidural hematoma occurred on the second day after operation,emergency evacuation of hematoma was then performed and the postoperative recovery was good.At the latest follow-up,no internal fixation loosening or failure occurred,and no tumor recurrence was observed in all patients.Conclusion Intralesional curettage and excision of the cervical spinal giant cell tumor is a safe and effective surgical method which can achieve satisfactory local control and prognosis.

Objective:To explore the curative effect of the "domino" sequential method with prosthesis preservation in the treatment of infection around the prosthesis after limb salvage surgery for bone tumors.Methods:A retrospective analysis of 11 patients with peripheral prosthetic infections after limb salvage surgery with prosthesis preserving "domino" from January 2016 to January 2020 was retrospectively analyzed, including 8 males and 3 females; age 21-74 years old, with an average of 51.8 years old. There were 6 cases of knee prosthesis for distal femoral tumor, 2 cases of proximal tibia knee prosthesis, 2 cases of pelvic prosthesis infection, and 1 case of middle femoral prosthesis. Before the operation, 8 cases had fever, and 2 cases had sinus. Sequential treatment failure is defined as: recurrence of infection. The treatment method is the debridement and lavage of the prosthesis and the systemic combined local application of drug-sensitive antibiotics to evaluate the patient's blood routine, C-reflective protein, erythrocyte sedimentation rate and other indicators, X-ray and CT of the surgical site, and the Musculoskeletal Tumor Society (MSTS) score.Results:The positive rate of microorganism culture was 72.7% (8/11), including 2 cases of Staphylococcus aureus, 1 case of Staphylococcus epidermidis, 1 case of methicillin-resistant Staphylococcus epidermidis, 1 case of methicillin-resistant Staphylococcus aureus, abalone There were 2 cases of Acinetobacter mannifolia and 1 case of Streptococcus degalactiae. The MSTS score before treatment and at the last follow-up after treatment increased from 10.91±2.31 points to 20.73±3.52 points, the difference was statistically significant ( t=7.162, P<0.05). A total of 3 cases of sequential treatment failed to control infection, and the operation success rate was 72.7% (8/11). One case was amputation, one case was long-term replacement of wound dressing, and one case was switched to antibiotic bone cement combined with intramedullary nail reverse double insertion technique to control infection. Conclusion:For bone tumors with clinical infection symptoms less than one month after limb salvage surgery, the use of prosthesis-preserving "domino" sequential method for treatment of prosthetic infections is desirable for early and mid-term clinical efficacy.

Objective:To investigate the alteration of immune microenvironment in giant cell tumor of bone (GCTB) after denosumab treatment from the aspect of immune cellsat single-cell level.Methods:During Nov 2018 and May 2020, fresh tumor excision tissues from GCTB cases were collected and received CyTOF analyses. CyTOF datasets were analyzed and visualized by t-distributed stochastic neighbor embedding (TSNE) method of reduction dimension. The compositions of immune cells in GCTB with or without denosumab treatment were compared. The supernatant of culture medium of ex vivo inoculated primary tumor tissues was harvested to clarify if the culturing supernatant could affect cell growth.Results:A total of 15 primary GCTB cases and three denosumab-treated samples were included in this study and were sent for CyTOF and multicolor FACS assay. GCTB was featured of T-cell and macrophage-like myeloid cell-dominant immune microenvironment. After denosumab treatment, the percentage of T-cells was significantly elevated, while the number of macrophage-like myeloid cells were reduced. Furthermore, the ratio of macrophage-like myeloid cells in total live cells was associated with the treatment period of denosumab. The multinuclear osteoclast like giant cells were characterized by the expression of γδTCR, while most of the intratumoral CD8+ T-cells were activated PD-1+CD69+T-cells. The culturing supernatant of denosumab treatment-free GCTB tissues reinforced cell proliferation in vitro, while this phenomenon was not seen when using denosumab treated tissues.Conclusion:Illustrated the immune cell atlas of GCTB, and preliminarily investigated the potential effects of immune cells on tumor progression in GCTB, providing some theoretical clues for prolonged use of denosumab in unresectable GCTB cases.

Objective:To explore the clinical characteristics of dislocation after resection and reconstruction of tumors involving pelvic area II, and to try to propose intraoperative and postoperative techniques to prevent its occurrence.Methods:From March 2011 to March 2021, 122 patients with resection and reconstruction involving pelvic area II were retrospectively analyzed. Among them, 17 had postoperative dislocation, 32 had pelvic area II, and 31 had pelvic area I+II. There were 40 cases in the pelvic area II+III, and 19 cases in the pelvic area I+II+III. There were 49 female patients and 73 male patients; the mean age was 47 years (9-73 years). The Musculoskeletal Tumor Society (MSTS) score of lower extremity patients after reduction was evaluated, and the clinical characteristics of dislocation, such as dislocation direction, dislocation time and reduction method, were counted and analyzed, and feasible prevention measures were analyzed based on the imaging characteristics after resection and reconstruction. dislocation method.Results:Among the 122 patients, there were 17 cases of dislocation, and the dislocation rate was 13.7%. Among them, 12 cases were anterior dislocation, accounting for 70.6% of anterior dislocation; 5 cases were posterior dislocation, and the proportion of posterior dislocation was 29.4%, difference (χ 2=4.52, P=0.033). There were 12 cases of dislocation within 3 months after operation, accounting for 70.6%; 1 case of dislocation occurred in 5 months after operation, and the other 4 cases of dislocation occurred for more than one year. The MSTS score of the dislocation patients after reduction was 56.1±15.6% (20%-80%). Combined with postoperative imaging examinations, the feasible methods for preventing dislocation include: The anteversion angle of the acetabular cup can be appropriately reduced (0°-10°); The acetabular cup can be appropriately shifted to the rear of the rotation center; Appropriately reducing the anteversion angle of the femoral neck prosthesis, the combination of the three can reduce the occurrence of femoral-acetabular prosthesis impingement; at the same time, the affected limb is controlled in a neutral position with strict nail shoes after surgery to avoid the occurrence of external rotation. Conclusion:The dislocation after tumor resection and reconstruction in pelvic area II is mostly anterior dislocation. Postoperative CT scan of the pelvis shows that the space between the neck of the femoral stem prosthesis and the lower edge of the acetabulum is small, which is prone to impingement. If the anteversion angle of the acetabular cup is appropriately reduced, the acetabular prosthesis is appropriately displaced posteriorly, and the anteversion angle of the femoral neck is reduced by 5°, the possibility of femoral-acetabular impingement can be reduced, thereby reducing the risk of postoperative dislocation.

Objective:To investigate the surgical method and clinical effect of O-arm navigation mini-open burring for osteoid osteoma.Methods:Eighteen patients with osteoid osteoma were treated with O-arm guided grinding drill from June 2021 to May 2022, including 15 males and 3 females, the age was (18.4 ±10.9) years (range 2 to 44 years), and the course of disease ranged from 1 week to 3 years (mean 14.2 months). The lesions sites included 6 cases of proximal femur, 3 cases of distal femur, 4 cases of proximal tibia, 1 case of distal tibia, 2 cases of proximal fibula and 1 case of distal and proximal humerus. During the operation, the O-arm navigation was used to determine the location of the focus, the muscle and soft tissue was peeled off to the bone surface through a 1-4 cm small incision, the channel retractor was placed, and the burr was registered as a navigation recognition device to gradually remove the bone on the surface of the tumor nest, and the tumor nest was scraped with a curette for pathological examination; according to the navigation image, the focus was enlarged removed with burr and the grinding range was confirmed by the O-arm X-ray machine before the end of the operation. The patients were followed up for 6 to 15 months (mean 9.5 months). CT scans were performed before and after surgery for imaging comparison in order to figure out whether it had residual lesions or recurrence. The visual analogue score (VAS) of pain was used as a parameter for evaluating the clinical efficacy.Results:The operation time of 18 cases was 40-175 min, with an average of 89.3 min. The time required to establish navigation image was 18.0 ±4.1 min (range 13 ~ 22 min). The length of the incision was 2.7±1.1 cm (range 1-4 cm). All patients achieved complete curettage of the lesions, and osteoid osteoma was confirmed by pathology after operation. All the patients could move to the ground 24 hours after operation, and the pain was significantly relieved from 3 to 7 d after operation, and the pain almost disappeared 3 months after operation. The VAS score of 18 cases was 5.33±1.24 before surgery, 2.79±1.32 on the 3rd day, 1.86±1.21 on the 7th day, 0.86±0.93 on the 1st month, 0.33±0.48 on the 3rd month, and 0.09±0.29 on the 6th month after operation, and the difference was statistically significant ( F=58.50, P<0.001). There were no serious complications during and after operation, and the success rate of treatment (no recurrence of symptoms, no residual recurrence of imaging lesions, no serious complications after operation) was 100%. Conclusion:Treatment of osteoid osteoma with mini-open excision using burrs under the navigation of O-arm is a simple, safe, minimally invasive and efficient technique. Intraoperative precise positioning and the use of burr with navigation to remove a larger area than the tumor nest are the keys to successful treatment.