Tanghong Jia

The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure

Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone-implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.

Titanium particle-challenged osteoblasts promote osteoclastogenesis and osteolysis in a murine model of periprosthestic osteolysis.

The current study investigates the interactive behavior of titanium alloy particle-challenged osteoblastic bone marrow stromal cells (BMSCs) and macrophage lineage cells in a murine knee-prosthesis failure model. BMSCs were isolated from male BALB/c mice femurs and induced in osteogenic medium. At 24h after isolation, BMSCs in complete induction medium were challenged with 1, 3 or 5mgml(-1) titanium particles for 7days. Culture media were collected at 2, 4 and 6days and cells were harvested at 7days for alkaline phosphatase (ALP) assay/stains. Cell proliferation in the presence of Ti particles was periodically evaluated by MTT assay. Mice implanted with titanium-pin tibial implants were given an intra-articular injection of 50μl medium containing 5×10(5) Ti particles-challenged bone-marrow-derived osteoblastic cells, followed by a repeat injection at 2weeks post-operation. Control mice with titanium-pin implants received a naïve osteoblastic cell transfusion. After sacrifice at 4weeks, the implanted knee joint of each group was collected for biomechanical pin-pullout testing, histological evaluation and reverse transcriptase polymerase chain reaction analysis of mRNA extracted from the joint tissues. Ti particles significantly stimulated the proliferation of BMSC-derived osteoblastic cells at both high and low particle concentrations (p<0.05), with no marked differences between the particle doses. ALP expression was diminished following Ti particle interactions, especially in the high-dose particle group (p<0.05). In addition, the culture media collected from short-term challenged (48h) osteoblasts significantly increased the numbers of TRAP+ cells when added to mouse peripheral blood monocytes cultures, in comparison with the monocytes cells receiving naïve osteoblasts media (p<0.05). Intra-articular introduction of the osteoblastic cells to the mouse pin-implant failure model resulted in reduced implant interfacial shear strength and thicker peri-implant soft-tissue formation, suggesting that titanium particles-challenged osteoblasts contributed to periprosthetic osteolysis. Comparison of the gene expression profiles among the peri-implant tissue samples following osteoblast injection did not find significant difference in RunX2 or Osterix/Sp7 between the groups. However, MMP-2, IL-1, TNF-α, RANKL, and TRAP gene expressions were elevated in the challenged-osteoblast group (p<0.05). In conclusion, titanium alloy particles were shown to interfere with the growth, maturation, and functions of the bone marrow osteoblast progenitor cells. Particle-challenged osteoblasts appear to express mediators that regulate osteoclastogenesis and peri-prosthetic osteolysis.

Cell-based osteoprotegerin therapy for debris-induced aseptic prosthetic loosening on a murine model

Exogenous osteoprotegerin (OPG) gene modification appears a therapeutic strategy for osteolytic aseptic loosening. The feasibility and efficacy of a cell-based OPG gene delivery approach were investigated using a murine model of knee prosthesis failure. A titanium pin was implanted into mouse proximal tibia to mimic a weight-bearing knee arthroplasty, followed by titanium particles challenge to induce periprosthetic osteolysis. Mouse fibroblast-like synoviocytes were transduced in vitro with either AAV-OPG or AAV-LacZ before transfused into the osteolytic prosthetic joint 3 weeks post surgery. Successful transgene expression at the local site was confirmed 4 weeks later after killing. Biomechanical pullout test indicated a significant restoration of implant stability after the cell-based OPG gene therapy. Histology revealed that inflammatory pseudo-membranes existed ubiquitously at bone–implant interface in control groups, whereas only observed sporadically in OPG gene-modified groups. Tartrate-resistant acid phosphatase+osteoclasts and tumor necrosis factor α, interleukin-1β, CD68+ expressing cells were significantly reduced in periprosthetic tissues of OPG gene-modified mice. No transgene dissemination or tumorigenesis was detected in remote organs and tissues. Data suggest that cell-based ex vivo OPG gene therapy was comparable in efficacy with in vivo local gene transfer technique to deliver functional therapeutic OPG activities, effectively halted the debris-induced osteolysis and regained the implant stability in this model.

Combination gene therapy targeting on interleukin-1β and RANKL for wear debris-induced aseptic loosening

This study investigated the efficacy of a combination gene therapy to repress interleukin-1 (IL-1) and receptor activator of nuclear factor NF-kappa B ligand (RANKL) for the treatment of particulate debris-induced aseptic loosening, and tried to explore the molecular mechanism of the exogenous gene modifications on osteoclastogenesis. RAW cells activated by titanium particles were transduced with DFG-IL-1Ra (retroviral vector encoding IL-1 receptor antagonist) and AAV-OPG (adeno-associated viral vectors—osteoprotegerin) individually or in combination for 4 weeks. Pro-inflammatory cytokines in culture media were determined by enzyme-linked immunosorbent assay, and gene expressions of RANK, IL-1β, c-Fos, TRAF6, JNK1 and CPK were examined using real-time PCR. An established knee-implant-failure mouse model was employed to evaluate the efficacy of the in vivo double-gene therapy. The surgical implantation of a titanium alloy pin into the proximal tibia was followed by monthly challenge with titanium debris. Peri-implant gene transfers of IL-1Ra and OPG (respectively or in combination) were given 3 weeks after surgery. The combination of OPG and IL-1Ra gene transfer exhibited strong synergetic effects in blockage of inflammation and osteoclastogenesis at 8 weeks after gene modification. The combination therapy reversed peri-implant bone resorption and restored implant stability when compared with either single gene transduction. Real-time PCR data indicated that the action of IL-1Ra gene therapy may be mediated via the JNK1 pathway, while the reduction of osteoclastogenesis by OPG gene modification may be regulated by c-Fos expression. In addition, both gene modifications resulted in significant diminishment of TRAF6 expression.

Effects of Ti, PMMA, UHMWPE, and Co–Cr wear particles on differentiation and functions of bone marrow stromal cells

This study investigates the roles of orthopedic biomaterial particles [Ti-alloy, poly(methyl methacrylate) (PMMA), ultrahigh-molecular-weight polyethylene (UHMWPE), Co-Cr alloy] on the differentiation and functions of bone marrow stromal cells (BMSCs). Cells were isolated from femurs of BALB/c mice and cultured in complete osteoblast-induction medium in presence of micron-sized biomaterial particles at various doses. 3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase assay were performed for cell proliferation and cytotoxicity. Differentiation and function of osteoblasts were evaluated by alkaline phosphatase (ALP), osteocalcin, RANKL, OSX, and Runx2 expressions. Murine interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α in culture media were determined by enzyme-linked immunosorbent assay. Challenge with low doses of Ti, UHMWPE, or Co-Cr particles markedly promoted the bone marrow cell proliferation while high dose of Co-Cr significantly inhibited cell growth (p < 0.05). Cells challenged with low dose of PMMA or UHMWPE particles (0.63 mg/mL) exhibited strong ALP activity, whereas Ti and Co-Cr groups showed minimal effects (p < 0.05). UHMWPE and Ti particles also promoted higher expression of proinflammatory cytokines. Real-time polymerase chain reaction data suggested that cells treated with low dose (0.5 mg/mL) particles resulted in distinctly diminished RANKL expression compared to those exposed to high concentrated (3 mg/mL) particles. In conclusion, various types of wear debris particles behaved differently in the differentiation, maturation, and functions of osteogenic cells; and the particulate debris-interacted BMSCs may play an important role in the pathogenesis and process of the debris-associated aseptic prosthetic loosening.

Recombinant human erythropoietin prevents motor neuron apoptosis in a rat model of cervical sub-acute spinal cord compression.

The objective of the study was to investigate the effects of recombinant human erythropoietin (rhEPO) in a rat model of cervical sub-acute spinal cord compression. 80 Wistar rats were randomly divided into 4 groups. Rats in the sham group (Group A, n=5) underwent surgical procedures without cervical spinal cord compression; while rats in other groups were subjected to the spinal compression process. In the control group (Group B, n=25), rats received an i.v. injection of 1 mL saline at day 7 post-surgery. Rats in the low-dose group (Group C, n=25) and the high-dose group (Group D, n=25) were treated with rhEPO at 500 units/kg body-weight and 5000 units/kg, respectively, via intravenous injection at day 7 post surgery. Limb motor function was scored by Basso-Beattie-Bresnahan (BBB) standards at 3, 7, 14, 21 and 28 days post-surgery. The distribution and quantities of EPO and its receptor (EPO-R) in the compressed segment of the spinal cord were detected by immunohistochemistry. Motor neuron apoptosis in the spinal cord was evaluated using TUNEL staining and flow cytometry at the indicated time points. Finally, IL-8, TNF-α, IL-6, and IL-1β levels in the compressed cervical spinal cord were determined by ELISA within the lesion epicenter at each time point post-surgery. The data suggest that expression of EPO-R was significantly increased following sub-acute cervical spinal cord compression; Groups C and D exhibited better BBB scores at all observed time points compared with the control group (p<0.01). Using TUNEL staining and FCM, we observed that rhEPO profoundly inhibited motor neuron apoptosis in the spinal cord at day 21 (p<0.01). Additionally, treatment with rhEPO halted the elevation of inflammatory cytokines. rhEPO administration decreased motor neuron apoptosis in the cervical spinal cord, improved motor functions and reduced the inflammatory response in a sub-acute cervical spinal cord compression model. Moreover, sustained treatment with low doses of rhEPO revealed a positive therapeutic effect.

Percutaneous fibrin glue therapy for meningeal cysts of the sacral spine with or without aspiration of the cerebrospinal fluid

OBJECT The authors assessed the efficacy of computed tomography (CT)-guided percutaneous injection of fibrin glue to treat meningeal cysts of the sacral spine in patients with back pain, and evaluated the necessity for cerebrospinal fluid (CSF) aspiration before glue injection. METHODS Of the 31 patients in this study, 15 underwent injection of fibrin glue under CT guidance after aspiration of more than 15 ml of CSF (Group A), and 16 patients were treated with the glue but without CSF aspiration (Group B). Clinical results were evaluated after an average of 23 months of follow-up, and changes on the imaging studies were also evaluated. The clinical outcome and postoperative complications were analyzed. RESULTS All 31 patients experienced resolution or marked improvement of symptoms for as long as 28 months after fibrin glue therapy. No patient experienced recurrence of symptoms during the follow-up interval. The postoperative pain relief was statistically significant (p < 0.001) according to evaluations in which a 100-mm visual analog pain scale was used. There were no statistical differences between the two groups (p > 0.05). CONCLUSIONS Percutaneous CT-guided fibrin glue therapy for sacral arachnoid cysts may be a definitive therapy. It is unnecessary to aspirate the CSF before injection of the fibrin glue.

Urodynamic Study of Bladder Function for Patients with Lumbar Spinal Stenosis Treated by Surgical Decompression

Lumbar spinal stenosis usually leads to different degrees of nerve damage, presenting with back and leg pain, and/or neurogenic bladder symptoms. To determine whether lumbar decompression improved urological function, bladder dysfunction was evaluated in this retrospective study of 26 patients with lumbar spinal stenosis who had undergone lumbar decompression surgery. Urodynamic study procedures were performed pre-operatively and 6 months post-operatively. The Japanese Orthopaedic Association score rating system and Oswestry Disability Index were employed for clinical evaluation. Following surgery, post-voiding residual urine, maximum cystometric capacity and maximum flow rate improved significantly. There was no statistically significant improvement in voided volume, bladder compliance, maximum detrusor pressure or upper urinary tract damage. Urodynamic study was important in the diagnosis of neurogenic bladder dysfunction, prevention of renal deterioration and assessment of post-operative effects after surgical decompression for patients with lumbar spinal stenosis.

Destructive pathological changes in the rat spinal cord due to chronic mechanical compression : Laboratory investigation

OBJECT Chronic mechanical compression of the spinal cord, which is commonly caused by degeneration of the spine, impairs motor and sensory functions insidiously and progressively. Yet the exact mechanisms of chronic spinal cord compression (SCC) remain to be elucidated. To study the pathophysiology of this condition, the authors developed a simple animal experimental model that reproduced the clinical course of mechanical compression of the spinal cord. METHODS A custom-designed compression device was implanted on the exposed spinal cord of female Wistar rats between the T-7 and T-9 vertebrae. A root canal screw attached to a plastic plate was tightened 1 complete turn (1 pitch) every 7 days for 6 weeks. The placement of the compression device and the degree of compression were validated every week using radiography. Furthermore, a motor sensory deficit index was also calculated every week. After 3, 6, 9, or 12 weeks, the compressed T7-9 spinal cords were harvested and examined histologically. RESULTS Lateral projection of the thoracic spine showed a progressively increasing rate of mean spinal cord narrowing in the compression group. Motor and sensory deficiencies were observed from Week 3 onward; paralysis was observed in 2 rats at Week 12. Motor deficiency appeared earlier than sensory deficiency. Obvious pathological changes were observed starting at Week 6. The number of neurons in the gray matter of rats with chronic compression of the spinal cord decreased progressively in the 6- and 9-week compression groups. In the white matter, myelin destruction and loss of axons and glia were noted. The number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive neurons increased in the ventral-to-dorsal direction. The number of TUNEL-positive cells increased from Week 6 onward and peaked at Week 9. CONCLUSIONS This practical model accurately reproduces characteristic features of clinical chronic SCC, including progressive motor and sensory disturbances after a latency and insidious neuronal loss.

Antigen-Specific Tolerogenic Dendritic Cells Ameliorate the Severity of Murine Collagen-Induced Arthritis

Dendritic cells (DCs) play important roles in initiation of the pathogenic processes of autoimmune disorders, such as rheumatoid arthritis (RA). Tolerogenic dendritic cells (tolDCs) are generated from naïve DCs and induce T cell tolerance; thus, they represent a promising strategy for specific cellular therapy for autoimmune diseases. In this study, we generated green fluorescent protein (GFP)-labeled tolDCs and confirmed their phenotypes and biological functions. We found that tolDCs suppressed the memory lymphocyte response and exhibited strong tolerogenic potential; thus, these cells show promise for the treatment of autoimmune diseases. Additionally, a collagen-induced arthritis (CIA) mouse model was used to test the role of tolDCs in vivo. The results of a further mechanistic experiment revealed that tolDCs suppressed inflammatory arthritis at least partially by up-regulating regulatory T (Treg) cells. Collectively, our data suggest that tolDCs may be used as a promising alternative therapy for inflammatory arthritis.