Celastrol increases osteosarcoma cell lysis by γδ T cells through up-regulation of death receptors.

γδ T cells has been shown to exhibit profound antitumor effects in a broad range of tumor entities, including OS. However, resistance to γδ T cells is a serious problem in the management of OS. This study investigates the impact of celastrol on the expression of death receptors 4/5 (DR4/5) on OS cell lines (HOS, U2OS) and cancer cell lysis by γδ T cells. The results showed that celastrol increased transcription of DR4/5 in HOS and U2OS, leading to increased cell surface, and total DR4/5 protein expression. Celastrol sensitizes OS cell lines or autologous OS cells to healthy donors-derived or OS patient-derived γδ T cell cytotoxicity in vitro. The induction of DR4/5 molecules increased lysis of HOS and U2OS by γδ T cells which was abolished by addition of a blocking TRAIL antibody. Importantly, the cytotoxic activity of γδ T cells was unaltered by small-dose celastrol. Taken together, our data show that celastrol up-regulated DR4/5 on OS cells to be responsible for intercellular TRAIL/APO-2L crosslink that confers increased cancer cell lysis by γδ T cells. These results suggest the clinical evaluation of celastrol in OS, especially in combination with immunotherapy approaches employing adoptive γδ T cell transfer.

Solitary haemangioma of the shaft of long bones: resection and reconstruction with autologous bone graft.

Bone haemangiomas are uncommon lesions, occurring in the skull or spine. A solitary haemangioma in the diaphysis of a long bone is rare. We retrospectively investigated six patients who presented with a solitary haemangioma in a long bone diaphysis. After segmental bone resection, the bone defect was replaced by a bone autograft. Patients were reviewed clinically and with radiographs. The mean follow-up was 6 years (range : 1-20 years). At the time of latest follow-up, no patient had a recurrence. Postoperative complications were one wound necrosis and one superficial wound infection. Union of the gap filling graft with the host bone was achieved in all patients at an average of 4 months (range: 3-8 months). The average Musculoskeletal Tumor Society functional score was 77% (range: 53%-90%) of normal at 6 months postoperatively, and 97% (range: 95%-99%) at the last follow-up evaluation. Segmental resection for solitary haemangioma and reconstruction with autologous bone graft can be considered as a suitable treatment option.

[Effect of zoledronate on the cytotoxicity of γδ T cells from PBMCs of osteosarcoma patients against osteosarcoma].

Objective To investigate the effect of zoledronate (Zol) on the cytotoxicity of γδ T cells from peripheral blood mononuclear cells (PBMCs) of osteosarcoma (OS) patients against OS. Methods γδ T cells, derived from PBMCs of patients with primary, recurrent and metastatic OS, were ex vivo expanded by Zol and IL-2. γδ T cell cytotoxicity against target cells was analyzed by a standard lactate dehydrogenase (LDH) release assay. IFN-γ secreted from γδ T cells was determined by ELISA kits. γδ T cells were incubated with different blocking Abs in order to investigate the mechanisms of recognition and killing of OS cells. Nude mice were inoculated with human OS cell line (HOS) by subcutaneous injection to create in vivo OS tumor models, and received normal saline, Zol, γδ T cells, or γδ T cells + Zol by tail vein injection, respectively. The inhibition of tumor growth by the different methods was observed and compared. Results γδ T cells from PBMCs of patients with OS were selectively expanded by Zol+IL-2 in vitro, and showed cytotoxicity against OS. In addition, γδ T cells showed significantly higher cytotoxicity against OS cells after treatment with Zol, and the mechanism of cytotoxicity was largely dependent on TCR pathway and perforin/plasmin pathway, partly on TRAIL pathway and NKG2D pathway. Combination treatment of γδ T cells and Zol had stronger and longer-lasting inhibition on tumor growth in vivo, compared with either drug alone. Conclusion Zol can promote γδ T cells' proliferation from PBMCs of OS patients and enhance γδ T cells' cytotoxicity against OS.

[Interferon-γ enhances human γδ T cell-mediated osteosarcoma cell killing in vitro].

OBJECTIVE: To investigate the cytotoxic effect of γδ T cells from osteosarcoma patients against interferon-γ (IFN-γ)-treated osteosarcoma cells in vitro. METHODS: Human γδ T cells were amplified by zoledronate from peripheral blood cells of osteosarcoma patients. The expression of Fas on the osteosarcoma cells were measured by flow cytometry and quantitative real-time PCR analysis before and after IFN-γ treatment. The cytotoxicity of γδ T cells against osteosarcoma cells was evaluated with LDH assay. RESULTS: IFN-γ significantly enhanced the susceptibility of the osteosarcoma cell lines HOS and U2OS to the cytotoxicity of γDelta; T cells from osteosarcoma patients (P<0.01). IFN-γ obviously up-regulated the expression of Fas in HOS and U2OS cells (P<0.01). Anti-FasL mAb failed to inhibit the cytotoxicity of γδ T cells in untreated osteosarcoma targets (P>0.05), but significantly impaired γδ T cell cytotoxicity in IFN-γ pre-treated osteosarcoma targets (P<0.01). CONCLUSION: IFN-γ can enhance the cytotoxic effect of human γδ T cells from osteosarcoma patients against osteosarcoma cells in vitro.

[Transplantation of collagen-chitosan nerve conduits filled with glial cell line-derived neurotrophic factor gene-modified schwann cells for the repair of sciatic nerve defect].

OBJECTIVE: To explore the effects of the transplantation of collagen-chitosan nerve scaffold containing glial cell line-derived neurotrophic factor(GDNF)gene modified schwann cells on the recovery of long-distance sciatic nerve defect. METHODS: The rat models of 8 mm long-distance sciatic nerve defect were established and divided into three groups, with 6 rats in each group. In GDNF-Sch group, the defect was repaired by GDNF modified Schwann cells combined with collagen-chitosan nerve scaffold. In Sch group, the defect was repaired by Schwann cells combined with collagen-chitosan nerve scaffold. In the control group, the defect was repaired by autologous nerve graft. Sciatic function index(SFI)was detected 3, 6, and 12 weeks after surgery. After 12 weeks, the tibialis anterior muscle wet weight, electrophysiology, and regenerated nerve morphology were detected. RESULTS: The SFI in the operated side significantly differed among these three groups after 6 and 12 weeks(P<0.05). Along with prolonged treatment, the GDNF-Sch group had similar SFI recovery with the control group but significantly better SFI recovery than Sch group. After 12 weeks, the sensory nerve conduction velocity in the GDNF-Sch and Sch group was not significantly different(P>0.05)but was significantly lower than that in the control group(P<0.05). Both the GDNF-Sch group and Sch group had significantly lower sensory nerve amplitude comparing with the control group(P<0.05), whereas that in the GDNF-Sch group was significantly higher than that in the Sch group(P<0.05). GDNF-Sch group and the control group had significantly higher motor nerve conduction velocity and amplitude than Sch group(P<0.05), while no such statistically significant difference was seen between the two groups(P >0.05). After 12 weeks, the wet weight of the bridging side of the tibial muscle in the control group, Sch group, and GDNF-Sch group was(0.360±0.020), (0.250±0.018), and(0.310±0.025)g, which were significantly lower than the control side [(0.440±0.031), (0.420±0.024), and(0.430±0.027)g, respectively(P<0.05)]. Muscle wet weight in bridge side of GDNF-Sch group and the control group were significantly higher than in Sch group(P<0.05), but it was not significantly different between the GDNF-Sch group and the control group(P>0.05). CONCLUSION: Transplantation of collagen-chitosan nerve scaffold containing GDNF gene modified Schwann cells can remarkably facilitate sciatic nerve defect recovery, with a milimar effectiveness as autologous nerve grafting.

Comparing the chondrogenic potential in vivo of autogeneic mesenchymal stem cells derived from different tissues.

OBJECTIVE: to compare the chondrogenic ability of mesenchymal stem cells (MSCs) derived from different tissues in rabbits' full-thickness articular cartilage defects. METHODS: sixty New Zealand white rabbits of ordinary grade with a body weight of 2.5 approximately 3.5kg were selected for this study. Six were sacrificed for preparation of deminerized bone matrix (DBM) as scaffold. Fifty-four were used for cartilage defects model. Full-thickness cartilage defect of knee joint was created on trochlear groove at two sides of the femur with a diameter of 4 mm and thickness of 3 mm. All 54 rabbits were randomly divided into 6 groups and treated by autogeneic MSCs isolated from bone marrow, periosteum, synovium, adipose tissue and muscle, respectively. The 6th group was a control group with nothing plugged into the defects. Every three rabbits were killed at three time points, which were 4, 8, and 12 weeks after the operation in each group. The reparative tissue samples were evaluated grossly, histologically, immunohistochemically, and graded according to gross and histological scales 12 weeks postoperatively. We input the scores into SPSS 11.5 software and the analysis of variance (one-way-ANOVA) and student-newman-keuls (SNK-q) test were used to process statistical analysis and find out if the differences between each group had statistical significance. RESULTS: fifty-four rabbits are included in the final analysis. The defects are all repaired by hyaline-like tissue except the control group. The bone-marrow-MSCs produced much more cartilage matrix than that of other groups. Gross and histological grading scale indicates that the defects repaired by MSCs isolated from bone marrow are superior to that repaired by MSCs isolated from periosteum, synovium, adipose tissue, and muscle (p < 0.05). In adipose-MSCs and muscle-MSCs group, some defects are even repaired by fibrous tissue. CONCLUSION: bone-marrow-MSCs have greater in vivo chondrogenic potential than periosteum-, synovium-, adipose- and muscle-MSCs.

In vitro and in vivo biocompatibility investigation of diamond-like carbon coated nickel-titanium shape memory alloy.

OBJECTIVE: To investigate the biocompatibility of diamond-like carbon (DLC) coated nickel-titanium shape memory alloy (NiTi SMA) in vitro and in vivo. METHODS: The in vitro study was carried out by co-culturing the DLC coated and uncoated NiTi SMA with bone marrow mesenchymal stem cells (MSCs), respectively, and the in vivo study was carried out by fixing the rabbits' femoral fracture model by DLC coated and uncoated NiTi SMA embracing fixator for 4 weeks, respectively. The concentration of the cells, alkaline phosphatase (AKP), and nickel ion in culture media were detected, respectively, at the first to fifth day after co-culturing. The inorganic substance, osteocalcin, alkaline phosphatase (ALP), and tumor necrosis factor (TNF) in callus surrounding fracture and the Ni(+) in muscles surrounding fracture site, liver and brain were detected 4 weeks postoperatively. RESULTS: The in vitro study showed that the proliferation of MSCs and the expression of AKP in the DLC-coated group were higher than the uncoated group (P < 0.05), while the uncoated group released more Ni(2+) into the culture media than that in the coated group (P < 0.05). The in vivo study revealed that the inorganic substance and AKP, osteocalcin, and TNF expression were significantly higher in the DLC coated NiTi SMA embracing fixator than that in the uncoated group (P < 0.05). Ni(2+) in liver, brain, and muscles surrounding the fracture were significantly lower in the DLC coated groups than that in the uncoated group (P < 0.05). CONCLUSION: Nickel-titanium shape memory alloy coated by diamond-like carbon appears to have better biocompatibility in vitro and in vivo compared to the uncoated one.

Nervus cutaneus femoris posterior pedicle flap for repairing large soft tissue defects at the heel or inferior segment of the shank.

OBJECTIVE: To investigate the clinical effect of the nervus cutaneus femoris posterior pedicle flap on repairing large soft tissue defects at the heel or inferior segment of the shank. METHODS: Totally 14 cases were followed up for 8-22 months (mean 15.5 months) to observe the clinical effects of nervus cutaneus femoris posterior pedicle flap on repairing large soft tissue defects of the heel or inferior segment of the shank. Among them, there were 3 patients afflicted with infection and cutaneous defects in the middle and inferior segment of the shank after internal fixation of open fracture, 4 patients with soft tissue defects of the ankle and uncovered tendo calcaneus, and 7 patients with soft tissue defects of the heel and exposed calcaneus. RESULTS: The flaps survived well in 13 cases and partial necrosis occurred in 1 case that was thereafter cured with changing dressing. Various extents of pain and stiffness of the knee joints were present in all cases and disappeared through 1-8 weeks' (mean 3.2 weeks) functional exercises. The last follow-up showed that all the flaps kept good texture and satisfactory appearance. CONCLUSIONS: The nervus cutaneus femoris posterior pedicle flap, having the advantages of simple surgical procedures, anastomosing the nerves and restoring the sensation of recipient site, can be used for recovering large soft tissue defects of the shank and ankle.

[Effect factors analysis of knee function recovery after distal femoral fracture operation].

OBJECTIVE: To investigate the effect factors of knee function recovery after operation in distal femoral fractures. METHODS: From January 2001 to May 2007, 92 cases of distal femoral fracture were treated. There were 50 males and 42 females, aged 20-77 years old (average 46.7 years old). Fracture was caused by traffic accident in 48 cases, by falling from height in 26 cases, by bruise in 12 cases and by tumble in 6 cases. According to Müller's Fracture classification, there were 29 cases of type A, 12 cases of type B and 51 cases of type C. According to American Society of Anesthesiologists (ASA) classification, there were 21 cases of grade I, 39 cases of grade II, 24 cases of grade III, and 8 cases of grade IV. The time from injury to operation was 4 hours to 24 days with an average of 7 days. Anatomical plate was used in 43 cases, retrograde interlocking intramedullary nail in 37 cases, and bone screws, bolts and internal fixation with Kirschner pins in 12 cases. After operation, the HSS knee function score was used to evaluate efficacy. Ten related factors were applied for statistical analysis, to knee function recovery after operation in distal femoral fractures, such as age, sex, preoperative ASA classification, injury to surgery time, fracture type, treatment, reduction quality, functional exercise after operation, whether or not CPM functional training and postoperative complications. RESULTS: Wound healed by first intention in 88 cases, infection occurred in 4 cases. All patients followed up 16-32 months with an average of 23.1 months. Clinical union of fracture was achieved within 3-7 months after operation. Extensor device adhesions and the scope of activities of <80 degrees occurred in 29 cases, traumatic arthritis in 25 cases, postoperative fracture displacement in 6 cases, mild knee varus or valgus in 7 cases and implant loosening in 6 cases. According to HSS knee function score, the results were excellent in 52 cases, good in 15 cases, fair in 10 cases and poor in 15 cases with an excellent and good rate of 72.83%. Single factor analysis showed that age, preoperative ASA classification, fracture type, reduction quality, whether or not CPM functional exercise, and postoperative complications were significantly in knee function recovery (P < 0.05). logistic regression analysis showed that the fracture type, quality of reduction, whether or not CPM functional exercise, and age were major factors in the knee joint function recovery. CONCLUSION: Age, preoperative ASA classification, fracture type, reduction quality, and whether or not CPM functional training, postoperative complications factors may affect the knee joint function recovery. Adjustment to the patient's preoperative physical status, fractures anatomic reduction and firm fixation, early postoperative active and passive functional exercises, less postoperative complications can maximize the restoration of knee joint function.

[Strategy of the treatment for dislocation of cervical vertebra].

OBJECTIVE: To study the strategy of the treatment for dislocation of cervical vertebra. METHODS: The clinical data of 39 cases with dislocation of cervical vertebra were analyzed. Among them,29 were male and 10 were female. The average age was 40 years old (range from 6 to 74 years old). Segment of dislocation: 15 cases in C(1,2), 1 case in C(3,4), 9 cases in C(4,5), 9 cases in C(5,6), 5 cases in C(6,7). Spinal injury according to Frankel grade, 9 cases were A grade,8 were B, 5 were C, 8 were D, 8 were E, 1 case had radicular symptom. Thirty-two cases were early and rapidly treated with traction (progressive weight). Seventeen cases were treated with operation. RESULTS: Traction-reduction was successful in 90% of patients. According to Frankel grade, 32 cases averagely improved 0.63 grades. Six cases of severe spinal injury accompany with interlocking of zygopophysis died. CONCLUSION: Inspecting weight of traction is important in rapid traction-reduction for dislocation of cervical vertebra. The choice of surgical treatment depends on the degree of reduction, the result of MRI,the grade of spinal trauma and the status of patients.