Gasdermin-E Mediated Pyroptosis-A Novel Mechanism Regulating Migration, Invasion and Release of Inflammatory Cytokines in Rheumatoid Arthritis Fibroblast-like Synoviocytes.

Synovium fibroblast-like synoviocytes (FLSs) are important participants in the pathogenesis of synovitis and joint destruction in rheumatoid arthritis (RA). Pyroptosis is a pro-inflammatory and cell lytic programmed cell death mechanism mediated by gasdermin (GSDM) family proteins. In this study, we demonstrated the increased expression of GSDME and increased levels of GSDME-mediated pyroptosis in RA synovial tissues. In vitro, stimulation with TNF-α plus hypoxia mimicking the inflammatory and hypoxic environment in RA synovium induced GSDME-mediated pyroptosis in RA-FLSs in combination with the promotion of migration and invasion abilities and the release of inflammatory cytokines (IL-6, IL-8). Moreover, knockdown of GSDME significantly inhibited the proliferation rate, migration/invasion effects and cytokines released through the reduction of GSDME-mediated pyroptosis. The immunohistochemistry results showed that RA patients with high GSDME N-terminal (GSDME-NT) expression, which is the active form of GSDME, showed higher IL-6 expression in both lining and sublining layer of synovium than that in patients with low GSDME-NT expression, osteoarthritis and non-inflammatory orthopedic arthropathies. Our findings revealed a novel mechanism regulating cell proliferation, migration, invasion and inflammatory cytokines release during the process of GSDME mediated pyroptosis in RA.

Application of Rapid Prototyping Pelvic Model for Patients with DDH to Facilitate Arthroplasty Planning: A Pilot Study.

Total hip arthroplasty (THA) is challenging in cases of osteoarthritis secondary to developmental dysplasia of the hip (DDH). Acetabular deficiency makes the positioning of the acetabular component difficult. Computer tomography based, patient-individual three dimensional (3-D) rapid prototype technology (RPT)-models were used to plan the placement of acetabular cup so that a surgeon was able to identify pelvic structures, assess the ideal extent of reaming and determine the size of cup after a reconstructive procedure. Intraclass correlation coefficients (ICCs) were used to analyze the agreement between the sizes of chosen components on the basis of preoperative planning and the actual sizes used in the operation. The use of the 3-D RPT-model facilitates the surgical procedures due to better planning and improved orientation.

The clinical value of combined use of MR imaging and multi-slice spiral CT in limb salvage surgery for orthopaedic oncology patients: initial experience in nine patients.

BACKGROUND: The purpose of this prospective study was to evaluate the value of the combined use of MR imaging and multi-slice spiral CT for limb salvage surgery in orthopaedic oncology patients. PATIENTS AND METHODS: Nine consecutive patients with lower/upper limb malignant bone tumours (7 osteosarcomas and 2 chondrosarcomas) were treated with limb-salvaging procedures. Preoperative planning including determination of the osteotomy plane and diameters of the prosthesis was performed basing on the preoperative CT and MR images. The histopathology was performed as golden diagnostic criteria to evaluate the accuracy of CT and MR-based determination for tumour's boundary. RESULTS: The tumour extension measured on MRI was consistent with the actual extension (P>0.05, paired Student's t test), while the extension measured on CT imaging was less than the actual extension. The length, offset and alignment of the affected limb were reconstructed accurately after the operation. An excellent functional outcome was achieved in all patients. CONCLUSIONS: In the present study, MRI was found to be superior to CT for determining the tumour extension, combined use of MRI and CT measurement provided high precision for the fit of the prosthesis and excellent functional results.

Down-regulation of TNF-alpha by small interfering RNA inhibits particle-induced inflammation in vitro.

Aseptic joint loosening is a key factor that reduces the life span of arthroplasty. There are currently few effective treatments for joint loosening except surgical revision. We explored the inhibitory effects of tumor necrosis factor (TNF)-alpha-targeted small interfering RNA (siRNA) on particle-induced inflammatory cytokine expression in the murine macrophage cell line, RAW264.7. siRNA targeting TNF-alpha was chemically synthesized and transfected into RAW264.7 cells by cationic liposomes. Ceramic, titanium, and polyethylene particles of different sizes (0.2-1.2 µm and 1.2-10 µm) were prepared to stimulate cells 24 h after siRNA transfection. The down-regulation of TNF-alpha mRNA and protein levels was quantitatively determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively, at different time intervals. Fluorescence microscopy showed that the siRNA transfection efficiency was 85.2% ± 3.5%. Real-time PCR showed that the levels of TNF-alpha mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups 3 h after stimulation (P < 0.05). Similarly, ELISA essays showed that the protein levels of TNF-alpha in the RNAi-treated groups were significantly decreased 24 h after transfection (P < 0.05). There were no significant differences in the inhibitory effect among the groups stimulated by different particle types or particle sizes (P > 0.05). Our results indicated that siRNA targeting TNF-alpha can inhibit TNF-alpha release from RAW264.7 cells when stimulated by ceramic, titanium, and polyethylene particles of different sizes and that the inhibitory effects were not related to the type or size of particle.

[Treatment of flatfoot deformity with concurrent spinal disease in children].

OBJECTIVE: To study the curative effect of operation to flatfoot deformity with concurrent spinal disease in children. METHODS: A retrospective survey was performed in 8 flatfoot patients with concurrent spinal disease from January 2004 to January 2008. Every case received special operative treatment. After casting, achilles tendon prolongation, reefing or tendon transfer, rehabilitation therapy was performed sequentially. Radiographs and clinical outcomes were evaluated during the follow-up. RESULTS: Six patients were followed up. There were 2 feet in 1 female and 7 feet in 5 males. The complications included tethered cord syndrome (n = 1), T12-S1 vertebral canal arachnoid cyst (n = 1), cerebral palsy and scoliosis (n = 1) and scoliosis and spinal bifida occult (n = 3). The average age was 8.4 (4 - 14) years old and the average follow-up time 22 (14 - 64) months. All cases were satisfied with the appearance and function according to Maryland foot score. Five were excellent, 2 good and 2 fair postoperatively versus 2 fair and 7 failed preoperatively. The total excellent and good rate was 77.8%. In the early stage after tendon transfer in 4 patients, the lateral view of height arch, talus-first metatarsal angle, calcaneus-navicular-first metatarsal angle and talonavicular coverage angle improved. Yet it lost the improved angle and reverted to the pre-operative state. No degenerative change was detected in tarsus joints during the long follow-up. CONCLUSION: The special and sequential surgical procedure may be an effective regimen in the treatment of flatfoot deformity with concurrent spinal disease in children.

IL-1beta sensitizes rat intervertebral disc cells to Fas ligand mediated apoptosis in vitro.

AIM: To determine the apoptotic effect of recombinant rat Fas Ligand on rat intervertebral disc cells pre-treated with IL-1beta in vitro, and the expression of Fas in cultured rat intervertebral disc cells. METHODS: Cells were isolated from the inner annulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and divided in 5 treatment groups. IL-1beta (10 ng/mL), FasL (5, 20 ng/mL) with/without IL-1beta (10 ng/mL) pre-treatment was respectively added in Dulbeccoos modified Eagleos medium and Hamos F-12 medium with 1% fetal bovine serum. After 32 h, the cells were stained with annexin V-FITC and propidium iodide to evaluate apoptosis using flow cytometry and to analysis transcription of Fas using RT-PCR. RESULTS: Compared with control group, FasL (20 ng/mL), IL-1beta (10 ng/mL)+FasL (5 ng/mL), and IL-1beta (10 ng/mL)+FasL (20 ng/mL) induced significant apoptosis of the disc cells (P<0.01). Apoptosis was also induced by FasL 5 ng/mL (P<0.05); whereas, apoptosis was not induced by IL-1beta (10 ng/mL) (P>0.05). IL-1beta (10 ng/mL) enhanced the apoptosis-inducing effects of FasL (5 ng/mL) and FasL (20 ng/mL) in disc cells. Fas gene transcription in all groups and Fas expression in the 5 treatment groups were approximately 1.2-2.1-fold greater than control group (respectively, P<0.05). Additionally, Fas expression in FasL with IL-1beta pre-treatment groups were significantly up-regulated than in FasL groups (P<0.01). CONCLUSION: The results of this study showed disc cells pre-treated with IL-1beta increased apoptotic rate in response to FasL in vitro and provided insights to understand Fas/FasL system-mediated apoptosis in disc cells which would be enhanced due to inflammation factor in degenerative disc.

[Effect of interleukin-6 on the chondrocytes in the cartilage endplate of rabbits in vitro].

OBJECTIVE: To evaluate the effect of interleukin-6 (IL-6) on the biological behaviors of the chondrocytes in the cartilage endplate of rabbits. METHODS: Chondrocytes isolated from the cartilage endplate of New Zealand rabbits, verified for their biological characteristics by such means as toluidine blue staining for type II collagen, were treated with IL-6 at different concentrations. The proliferation of the chondrocytes was evaluated by MTT assay at different time points following the treatment, the cell cycle changes were determined by flow cytometry and the changes of aggrecan and type II collagen mRNAs detected by RT-PCR. RESULTS: At the concentrations of 10, 50 and 100 ng/ml, IL-6 did not obviously affect the rate of chondrocyte proliferation. IL-6 at 50 ng/ml resulted in no obvious changes of the cell cycle of the chondrocytes, but significantly decreased the expression of collagen IIa mRNA. CONCLUSION: IL-6 has no effect on the proliferation and cell cycle of the chondrocytes, but at higher concentrations, it inhibits matrix synthesis of the chondrocytes to promote intervertebral disc degeneration.

[Association of single nucleotide polymorphisms of IL-1b with lumbar disc disease].

This study was to explore the relationships between (-511)T>C and (+3954)C>T single nucleotide polymorphisms(SNP) in IL-1beta gene with lumbar intervertebral disc disease. We analyzed (-511)T>C and (+3954)C>T SNP in IL-1beta gene by the polymerase chain reaction-restriction fragment length polymorphism and electrophoresis methods respectively in 81 cases with lumbar disc disease and 101 healthy controls. The relationship between (-511)T>C and (+3954)C>T SNP in IL-1beta gene and lumbar disc disease in two groups was measured, so does the relationship between (-511)T>C and (+3954)C>T SNP in IL-1beta gene and intervertebral disc degeneration in those younger than 45-year-old. The results showed there were (-511)T>C and (+3954)C>T SNP in IL-1beta gene. There was a significant difference in the distribution of TT, TC and CC genotype or T, C genotype of (-511)T>C of IL-1beta in two groups. And there was no significant difference in the distribution of (+3954)C>T SNP in IL-1beta gene in two groups. There was no significant difference between the distribution of (-511)T>C and (+3954)C>T SNP in IL-1beta gene and intervertebral disc degeneration in those younger than 45-year-old. It suggested (-511)T>C SNP in IL-1beta gene be one of the susceptible alleles for Lumbar disc disease.

[The risk factors and preventive strategies of heterotopic ossification after artificial disc replacement in lumbar spine].

OBJECTIVE: To analyze the risk factors of heterotopic (HO) ossification after total lumbar disc replacement and probe the preventive strategies for it. METHODS: The radiographs and clinical data of 78 discs in 65 patients who received artificial lumbar disc replacement (ADR) from April 1998 to December 2003 were analyzed retrospectively by two radiologists and one orthopaedic surgeon and then postoperative HO were graded according to McAfee system. The bony formations in disc spaces, time of HO were found, and range of motion (ROM) of the operated levels were measured on radiographic films. In addition, the risk factors such as preoperative peri-annulus ossification, bony endplates injuries, and subsided or mal-position of the prostheses were also analyzed by Logistic regression analysis. RESULTS: Postoperative HO was found in 10 spaces of 9 cases. Class I of HO were occurred in 7 patients at means 2.1 years postoperatively with normal range of motion preserved. Three of them turned into class II or III with 10 degrees of mean ROM in the following 2.5 years. Another 2 (2/9) cases with preoperative peri-annulus ossification had bridging trabecular bone (class III) between the endplates and 9 degrees of ROM 2 years after surgery, then turned into class IV at 6 years with 0 degrees and 4 degrees of motion in the operated levels. As the risk factors of HO, preoperative annulus ossification (2 cases), bony endplates injuries (5 cases), mal-positioned prostheses (2 cases) and subsided prostheses (2 cases) were found simultaneity with significant positive relation to HO occurred (P < 0.05). CONCLUSIONS: Factors such as preoperative ossification of annulus, endplate injuries, prosthesis subsided and mal-position would have higher risks to have HO occurred after ADR, but ROM of most affected levels are preserved. Strict control indication and avoid all above risk factors can prevent HO occurring effectively.

[Design and experiment of the pedicle aiming device for the cervical spine].

OBJECTIVE: To probe the accuracy and safety of using an aiming device in the transpedicular fixation of cervical spine. METHODS: Eight cervical specimens were obtained. We used the computed tomography (CT) to scan C3 to C7, measured the medial angle of the pedicle, and determined the location of the pedicle projecting on the articular process. Then we took the oblique X-ray film, measured the cephalic/caudal angle of the pedicle, and determined the location of the pedicle projecting on the articular process. All the specimens were equally divided into two groups. Screws of 2.8 mm x 30 mm, were used. Specimens in one group were inserted with the transpedicular screw manually, while specimens in the other one inserted with the transpedicular screw using a self-designed aiming device that can be modulate at the three dimensions according to the angles of the pedicles. RESULTS: The first group totally had 40 screws from C3 to C7. There were 13 screws in the pedicle, 9 violated the walls of the pedicle but not involved the adjunct structure, and 18 injured the important structure such as spinal cord, verteberal artery, or nerve root. In the other group, only 4 screws violated the walls of the pedicle but not involved the adjunct structure, and the others all in the pedicles. The difference was of statistical significance (P < 0.01). CONCLUSION: In the cervical spine, transpedicular fixation using an aiming device can improve the accuracy and safety during operation.

[The role of artificial disc replacement in restoring lumbar segment motion and intervertebral height].

OBJECTIVE: To investigate the role of artificial disc replacement (ADR) in restoring lumbar segment motion and intervertebral height (IH). METHODS: Seventy-five patients with intervertebral disc diseases underwent artificial disc replacement and were divided into 3 groups to be followed up for 2 years (group A, n = 32), 3 years (group B, n = 27), and 5 years (group C, n = 19) respectively. Before the operation and during the follow-up the range of motion (ROM) of the involved lumbar segments and IH were assessed. RESULTS: The mean ROM and IH of the group A were 15 degrees and 14.1 mm respectively during the follow-up 2 years after operation. The mean ROM and IH of the group B were 14.2 degrees and 13.8 mm respectively during the follow-up 2 years after the operation and 13.7 degrees and 13.4 mm respectively during the follow-up 3 years after operation. The mean ROM and IH of the group C were 9.1 degrees and 11.5 mm respectively during the follow-up 2 years after the operation and 8.8 degrees and 11.0 mm respectively during the follow-up 3 years after operation, and 8.0 degrees and 10.3 mm respectively during the follow-up 5 years after operation. No significant differences in ROM and IH were found among different groups at any time point (all P > 0.05). Malposition was found in immediately after operation in 11 patients, all of the group C. During the follow-up, 3 cases of prostheses subsidence and 3 cases of annular ossification were found. No severe complications appeared. CONCLUSION: ADR is effective in restoring the ROM and IH of vertebrae suffering from intervertebral disc diseases.