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The authors investigated the claims that silicone implants may be involved in autoimmune connective tissue diseases. Their experimental model in mice showed production of connective tissue antigen autoantibodies and susceptibility to autoimmune disease.

Staphylococcal enterotoxin B (SEB) treatment may not halt or lower arthritic response to collagen type II (CII) in mice and may increase the severity of collagen type II arthritis in arthritic mice. In a study to examine the changes of V-beta-8 T cell subsets during the induction of arthritis by CII immunization, genetically identical mice were injected with SEB and then with CII to induce an arthritic response, with saline alone, and with CII only. Mice with arthritis induced from CII injection were treated therapeutically with SEB as well. Mouse tissue was analysed for V-beta-8 and V-beta-6 activity and expression of V-beta T cell receptors. Mice were also physically monitored for arthritic symptoms. Mice treated with initially with SEB, compared with CII-only controls, showed a decrease in V-beta-8 T cells only and no change in CII antibody response. Mice with CII immunization showed no T cell compliment differences. Therefore, mice treated with SEB before onset of arthritis achieved no benefit. Mice treated with SEB after onset of arthritis suffered a hastening of the illness and more serious symptoms.

Wear particles produced from prosthetic joints may play critical roles in periprosthetic inflammatory reactions and osteolysis. The objective of this study was to quantify and compare the response to wear debris from different biomaterials at the bone–implant interface in a rat knee model.

This study investigated the therapeutic influence and potential mechanism of IL-10 in ameliorating orthopedic debris particle-induced inflammation and osteolysis. A murine air pouch with bone implantation and polyethylene particles was also used to evaluate the therapeutic effects of IL-10. The data suggested that the particle challenges significantly promoted macrophage activation and osteoclastogenesis, with dramatically increased macrophage infiltration into the pouch membranes and elevated tartrate-resistant acid phosphatase–positive cell deposition. Immunohistochemical stains revealed a significantly higher ratio of induced nitric oxide synthase–expressing cells in the particle-challenged group; treatment with IL-10 resulted in marked switching to CD163+cells. Also, IL-10 effectively reduced tartrate-resistant acid phosphatase–positive stained cells in the pouch membranes, and minimized the bone mineral density loss compared with untreated samples. Real-time PCR and Western blot examination indicated that IL-10 treatment significantly diminished the particle-induced IL-1β expression but promoted expression of CD163, transforming growth factor-β1, and CCR2. Furthermore, IL-10 significantly inhibited the ultra-high-molecular-weight polyethylene particle-elevated phospho-STAT1 and phospho–NF-κB p65 productions, and promoted phospho-STAT3 expression. Overall, the data indicate the pivotal effects of IL-10 on macrophage polarization. The effects of IL-10 in ameliorating local inflammation and osteolysis may be associated with macrophage polarization through the up-regulation of the Janus activating kinase/STAT3 signaling pathway, and the down-regulation of NF-κB and Janus activating kinase/STAT1 expression.

Fractured bones are often stabilised with orthopaedic fracture plates and screws until healed. If the plates and screws are removed, the vacant screw holes introduce a potential site for re-fracture. This study is aimed at simulating a laboratory torsional fracture test of a composite analogue tibia with vacant screw holes using a finite element (FE) model. This FE model is set up the same as the experimental torsion test, with a section from the distal portion of the tibia. The FE model contains over 35k second-order brick elements and nearly 165k nodes. It utilises an isotropic linear elastic material law with material properties obtained from the analogue tibia manufacturer. Comparisons between the experimental model and the FE model consider the fracture torque, fracture angle, and specific torsional stiffness. Stress contours of the FE model are compared to the fracture path of the experimental model. The FE model predicts the fracture location and a fracture torque within the standard deviation of that determined experimentally.

The objective of this work was to use a functional gait analysis technique to evaluate sciatic nerve repair through tissue-engineered nerve guides in a rodent animal model. The nerve guides were fabricated by blending collagen with chitosan material and evaluated over a 12-week period for motor and sensory nerve recovery assessed by gait analysis and behavioral testing. Gastrocnemius muscle weight measurements were obtained at the end of each experimental time point and correlated to motor nerve recovery. Functional gait analysis studied both the stance and swing phase angle formations during a normal gait cycle. During the stance phase, functional results revealed that blended nerve guides promoted increased motor nerve recovery than unblended chitosan nerve guides. Similar results were obtained from behavioral tests, indicating that blended nerve guides created increased sensitivity to applied stimulus compared to unblended nerve guides. Muscle strength also correlated with functional recovery and was significantly higher when compared to the unblended nerve guides. From this study, we conclude that collagen-blended chitosan nerve guides enhanced motor and sensory nerve recovery assayed through gait and behavioral testing compared to unblended nerve guides.

OBJECTIVE: ASEPTIC LOOSENING IS A COMMON COMPLICATION OF TOTAL JOINT REPLACEMENT IN HUMANS: Our study examined the hypothesis that wear debris may influence vascular endothelial grow factor (VEGF) expression, and that blocking VEGF bioactivity might improve wear debris-induced inflammatory osteolysis in a mouse model. METHODS: Ultra high molecular weight polyethylene (UHMWPE) particles were introduced into established air pouches on BALB/c mice, followed by implantation of calvaria bone from syngeneic littermates. Mice were treated with recombinant VEGF, or VEGF inhibitor (VEGF R2/Fc chimera) or vehicle control, and mice without UHMWPE stimulation were also included. Pouch tissues were harvested 2 weeks after bone implantation for molecular and histological analyses. RESULTS: Exposure of UHMWPE particles increased VEGF expression at both mRNA and protein levels in pouch tissues. Immunostaining revealed intense VEGF staining predominantly in UHMWPE deposit foci surrounded by inflammatory cells. VEGF inhibitor treatment strongly attenuated tissue inflammation (cellular infiltration, membrane proliferation, and expression of interleukin 1ß and tumor necrosis factor-a in UHMWPE-stimulated pouch tissues). Further, VEGF inhibitor treatment caused a significant reduction in the number of TRAP+ cells, and effectively prevented UHMWPE particle-induced bone resorption of implanted calvaria (assessed by extent of collagen depletion and frequency of bone erosions). CONCLUSION: The observation that VEGF inhibitor treatment prevented UHMWPE particle-induced inflammatory osteolysis opens new possibilities for treatment of aseptic loosening, especially at an early stage.

Abstract: Introduction: The association between high bone mass and increased breast cancer risk has been established. Identification of polymorphisms and the resultant variant receptors suggests the possibility of differential effects on hormone responsive genes when complexed with the hormones. Both estrogen receptor-α (ER) and vitamin D receptor (VDR) polymorphisms have been associated with bone density. Thus, we examined these polymorphisms for association with increased breast cancer risk among US African-American and white women.Methods: A case–control study was conducted to measure ER and VDR polymorphisms and radial bone mineral density (BMD) in African-American and white women, and to examine the association between polymorphisms, bone density and breast cancer risk. Genotypes and bone density were obtained from 412 women (220 cases and 192 controls, with equal distribution between the two ethnic groups).Results: We found no evidence for an association between either the ER or VDR genotypes and breast cancer risk. Also, there was no difference in the risk of breast cancer by genotypes after adjusting for ethnicity. The addition of age, sex and ethnicity-specific BMD (Z-scores) did not significantly change the odds ratio for breast cancer.Conclusions: Our data suggest that the polymorphisms investigated had no effect on risk of breast cancer in this population. Thus, we found no evidence to support our hypothesis that breast cancer cases and controls would have a different distribution of ER and VDR genotypes. Furthermore, the polymorphisms were not associated with differences in bone mass and its relationship with breast cancer risk.

Quantitative analysis of peripheral nerve regeneration using nerve guides is commonly evaluated through histomorphometry and walking track analysis. We conducted a unique assessment of functional sciatic nerve recovery treated with chitosan nerve guides. We used video-gait analysis to evaluate the extent of functional nerve recovery by measuring the ankle angle at different gait cycle phases. We also correlated the gastrocnemius muscle weight measurements and histological analysis to functional nerve recovery. The chitosan group showed increased functional improvement compared to the control groups at the end of a 12-week period ( p < 0.05). Although both control and chitosan angle measurements were lower than those recorded for presurgery animals, the angle measurements significantly improved over the 12-week period. Stance phase duration of the gait cycle was also recorded, which showed a significant increase over the 12-week time period. The muscle weight parameter indicated a significant decrease in muscle atrophy and restoration of functional strength. Histological analysis revealed that the chitosan nerve guide provided significantly increased axonal growth. The functional results indicated that chitosan nerve guides enhanced functional improvement over no repair processes.

OBJECTIVE: Pristane induced arthritis (PIA) is a seropositive experimental murine model that closely resembles rheumatoid arthritis (RA). Immune reactivity to a broad spectrum of autoantigens has been recognized in this disease model. We investigated the specificity of the autoimmune response in PIA to determine whether reactivity to connective tissue antigens is associated with the development of arthritis. METHODS: DBA/1 mice were injected with pristane and evaluated for development of joint disease and autoimmunity. Lymph nodes, spleen, sera, and arthritic paws were investigated at 1, 2, 4, 6, 9, and 12 months postinjection. T cell responses to 16 different joint components were evaluated using proliferation assays, and sera were assayed by ELISA for antibodies to these joint antigens. Cytokine concentrations after antigenic stimulation were assessed by ELISA in cultured cell supernatants and by real-time polymerase chain reaction using mRNA from spleens and arthritic paws. RESULTS: ELISA revealed positive responses to glucose-6-phosphate isomerase, chondroitin sulfate B, collagen I, collagen II, aggrecan, and DNA between 4 and 12 months post-pristane injection. In vitro tumor necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin 6 (IL-6) responses were detected during reactions to most antigens tested, while IL-4 responses were absent. Cytokine analysis in arthritic joints revealed consistent expression of IL-1, IL-4, IL-6, TNF-a, and IFN-g mRNA. CONCLUSION: These results indicate that PIA animals develop both T cell and antibody responses to a broad spectrum of connective tissue antigens. Biglycan, aggrecan, and decorin may be relevant antigens in the pathogenesis of PIA, but no specific reaction pattern could be associated with the occurrence of disease. The data suggest that the development of pristane arthritis is not dependent upon reactivity against a single connective tissue antigen, but is a polyspecific autoimmune response to joint components elicited in pristane injected mice.

It is recognized that the chronic inflammation in periprosthetic tissue that contributes to implant failure frequently is provoked by the presence of wear debris. Some wear debris is inevitable because of the nature of the prosthesis, but not all patients develop severe inflammatory responses. The precise factors that mediate the severity of tissue inflammation to wear debris has yet to be fully defined. Because wear debris retrieved from periprosthetic tissue consists of a heterogeneous mixture of materials with various sizes and shapes, this study evaluated the influence of two major physical aspects of ultrahigh molecular weight polyethylene UHMWPE wear debris shape and surface texture using a model of tissue inflammation. UHMWPE debris particulates recovered from 50 periprosthetic tissue samples were examined by scanning electron microscopy and categorized into four groups based upon aspect ratio and surface texture of the material. The four groups were defined as: 1 smooth and globular, 2 smooth and fibular, 3 rough and globular, and 4 rough and fibular. Histological analysis and ELISA assays were conducted to evaluate variations in cellular responses and cytokine production between the groups. The strongest expression of tumor necrosis factor alpha and interleukin1 beta was found in tissues exposed to UHMWPE debris with both a rough surface texture and fibular shape, and this response was significantly elevated over debris particles with a smooth surface texture and globular shape. The data suggest that both shape and texture influence the severity of specific inflammatory responses and that rough debris surface texture exerts a marked effect on adverse tissue responses when combined with particles that have a sharp, elongated shape. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 64A: 457–464, 2003

Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)–mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris–induced bone loss in a murine model of osteolysis. Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or β-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses. Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ–transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor κB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss. OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene–induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris–associated osteolysis and aseptic loosening.

Chitosan scaffolds appear to be suitable for a variety of tissue engineering applications. This study addressed the biocompatibility of chitosan in a mouse implantation model. Porous chitosan scaffolds were implanted in mice, and animals were sacrificed after 1, 2, 4, 8, or 12 weeks. Macroscopic inspection of the implantation site revealed no pathological inflammatory responses. Histological assessment indicated marked neutrophil accumulation within the implant, which resolved with increasing implantation time. Gram staining and limulus assays revealed no evidence of infection or endotoxin. Collagen was observed within the chitosan pore spaces, indicating that connective tissue matrix was deposited within the implant. Angiogenic activity associated with the external implant surface was also observed. Cellular immune responses were determined by lymphocyte proliferation assays, and antibody responses were measured using ELISA techniques. These assays indicated a very low incidence of chitosanspecific reactions. Although there was a large migration of neutrophils into the implantation area, there were minimal signs of any inflammatory reaction to the material itself. This preliminary study demonstrates that chitosan has a high degree of biocompatibility in this animal model. Overall, the findings suggest that chitosan may be suitable for the development of implantable materials. © 2001 Wiley Periodicals, Inc. J Biomed Mater Res 59: 585–590, 2002

Retroviral vectors encoding the human IL-1 antagonist (IL-1Ra) gene and the human tumor necrosis factor soluble receptor (sTNF-R) gene were investigated using an in vivomodel of the inflammatory response to orthopedic wear debris. Air pouches established in BALB/c mice were injected with polymethylmethacrylate (PMMA) particles to provoke an inflammatory reaction, and infected with retroviral vectors expressing IL-1Ra, sTNF-R or a LacZmarker gene. Pouch membranes and fluids were harvested after 48 or 72 hours for analyses. Positive PCR reactions for Neogenes were observed specifically in DNA extracted from the membrane of retroviral-infected pouches. ELISA assays revealed the presence of human IL-1Ra in pouch fluid from DFG-IRAP-Neotransduced mice, but not control animals. Histological evaluation indicated that the IL-1Ra gene transfer was associated with markedly decreased inflammation in the model, with resolution of the edematous phase of the reaction, decreased pouch fluid accumulation, and lowered macrophage influx. The data suggest that the air pouch model represents a useful tool to evaluate gene therapy, and demonstrate that IL-1Ra gene therapy may be an appropriate therapeutic approach to inflammation.

This study investigated immunological responses to Staphylococcus aureusbone infection. Because considerable immunological information is available on the mouse, a murine model of acute hematogenous osteomyelitis was established. Osteomyelitis was created in the proximal tibia of C3H/HeJ mice by a tibial epiphyseal fracture followed by intravenous bacterial inoculation with Staphylococcus aureus(strain LS‐1). Swelling and warmth of the limb was found, and following limb exposure, abscess formation was evident in the proximal tibia. Histological examination revealed distortion primarily at the hypertrophic zone of the physis and polymorphonuclear leukocyte infiltration throughout the damaged area of the proximal tibia. Local infection was demonstrated at the fracture site, evidenced by the recovery of Staphylococcus aureusfollowing microbiological analysis of tissue specimens. Polymerase chain reaction was utilized to detect 16S ribosomal prokaryotic nucleic acid to demonstrate that the diagnosis of osteomyelitis could be established in the absence of conventional microbiological techniques. The infected mice had an increase of circulating large leukocytes (granulocytes) and an elevation of total serum immunoglobulin. Flow cytometry revealed significant increases in splenic B lymphocytes and in lymph‐node CD4+ T lymphocytes. These results indicate that an experimental model of acute hematogenous osteomyelitis that closely resembles the pathology of the disease in humans may be consistently induced in mice. Furthermore, marked immunological changes may be observed in response to the Staphylococcus aureusbone infection.

Pristane‐induced arthritis (PIA) is an experimental seropositive arthritis that is characterized by serologic and cellular immune abnormalities and is dependent on the presence of a competent CD4+ T cell population. We examined the regulation of PIA by genes of the major histocompatibility complex (MHC) and the Mls‐1loci to determine whether the selection of the T cells that infiltrate arthritic joints is a critical factor in disease susceptibility. Genetic regulation of PIA was investigated using F1hybrid and congenic strain analysis to determine the influence of MHC and Mls‐1genes. The T cell receptor Vβphenotypes of lymph node cells and T cells infiltrating arthritic joints were examined with 2‐color flow cytometry and reverse transcription‐polymerase chain reaction techniques. F1hybrid offspring from 2 major PIA‐susceptible strains (DBA/1 × BALB/c) were resistant to the induction of arthritis because of the interaction between genes of the MHC and the Mls‐1loci, which modified the T cell repertoire. This conclusion was supported by the observed resistance to PIA in BALB/c‐Mls‐1amice, where T cells expressing the Vβ8.1 and Vβ6 phenotypes were absent. The receptor phenotype of T cells infiltrating arthritic joints in DBA/1 mice was markedly skewed toward Vβ8.1 and Vβ6 compared with the population observed in lymph nodes from either PIA or normal control DBA/1 mice. The data support the hypothesis that PIA is a T cell‐mediated disease. While pristane causes a polyclonal T cell expansion that gives rise to lymphadenopathy, the development of arthritis in susceptible strains of mice occurs due to the preservation of specific T cell subsets with the capacity to infiltrate synovial joints.

Pristane-induced arthritis was investigated in DBA/1, DBA/2, and BALB/c mice, and F1 hybrid mice generated from inter-crosses between these strains. The incidence of disease in FI hybrid mice was significantly lower than the susceptible parental strains (DBA/I and BALB/c). and resistance to arthritis was observed in both DBA/2 mice and the (DBA/2 × BALB/c) FI hybrid mice. Several cellular immune abnormalities were observed in pristane-injected DBA/1 mice. Con A mitogen responses were depressed following pristane injection, and a functional suppressor cell population was detected. Delayed type hypersensitivity responses to type II collagen were observed in pristane injected mice. The intraperitoneal injection of pristane appears to alter immune regulation and induce autoimmune responses to connective tissue components.

In vitro cellular immune responses to metallic and polymeric implant materials in particulate form were measured preoperatively in 185 patients. The patients were candidates for either primary total joint replacement (n = 65) or revision arthroplasty (n = 120). Proliferative cellular responses to polymethylmethacrylate‐ particles in patients with osteoarthritis at revision surgery for aseptic loosening were significantly higher than the responses of patients with osteoarthritis at either primary surgery or surgical revision for mechanical failure of the prosthesis or, sepsis. The responses to particles of cobalt‐chromium alloy at revision surgery were also higher than the responses at primary surgery. The responses were reevaluated in 32 patients after a minimum of 10 months following surgery to correlate individual changes in the biological responses with clinical progress. Reevaluation at early follow‐up of patients who had undergone primary surgery revealed significantly elevated proliferative responses and in vitro cytokine production in response to poly‐methylmethacrylate and cobalt‐chromium alloy particles compared with their preoperative responses. In contrast, the response at follow‐up to polymethylmethacrylate was significantly reduced in patients who had undergone revision surgery, and this reduction corresponded with a marked improvement in pain, joint motion, and function following revision surgery. These data suggest that specific cellular responses to polymethylmethacrylate or cobalt‐chromium alloy particles, or both, may be associated with loose or painful prostheses.

The T cell repertoire expressed by Tsk2 mice, a novel experimental model of systemic sclerosis, was examined to determine whether cells infiltrating the areas of involved skin exhibit a T cell receptor (TCR) bias. Reverse transcription-polymerase chain reactions (RT-PCR) were conducted using RNA extracted from lymph nodes and skin from Tsk2 mice and from normal mice, with an oligonucleotide primer library specific for the variable region of the TCR (β) chain, RT-PCR signals were observed in all lymph node cell (LNC) samples from both Tsk2 mice and control mice, with eighteen of the twenty-one Vβ types present. In contrast, cDNA extracted from areas of involved skin from Tsk2 mice exhibited a restricted pattern, with positive Vβ signals corresponding to eight T cell subtypes (Vβ1, 6, 8.1, 8.2, 10, 11, 16, and 18). Band strength analysis revealed that three Vβ subtypes dominated within this restricted pattern (Vβ8.1, 11, and 18). Moreover, this pattern of VB bias was consistent among the four skin samples from different Tsk2 mice. These data suggest that a restricted T cell population participates in the inflammatory cell infiltrate of Tsk.2 skin.

Using the curative model of adjuvant arthritis, adult male Sprague-Dawley rats were treated with vehicle or etodolac (1, 3, and 8 mg/kg/day, po) for 9 days. Rats were sacrificed after 1, 2, 4, or 9 daily doses, and paw volume, PGE2concentrations, and N-acetyl-β-D-glucosaminidase (NAG) activity were determined in the left adjuvant-injected hindpaws. All three doses of etodolac caused a significant decrease in PGE2concentrations after the first dose, and the decreases persisted for 2, 4, and 9 days of treatment, respectively. In rats given four daily doses of 3 and 8 mg/kg/day of etodolac, the paw volume was significantly decreased by about 50%, compared with that of the arthritic controls. A significant decrease in NAG activity was observed only after nine daily doses of 8 mg/kg/day etodolac. The sequence of anti-inflammatory events manifested following etodolac treatment would appear to be an initial inhibition of PGE2synthesis, followed by resorption of fluid, and then by a reduction in macrophage infiltration.

Patients with locally advanced or metastatic gastric adenocarcinoma received an i.v. bolus of 4′-epi-doxorubicin, 75/mg/m2/cycle, every 21 days. Partial responses were observed in 5 of 23 evaluable patients (21.7%). Treatment was generally well tolerated and toxicity was mild. The response rate to epirubicin appears to be very similar to that reported for doxorubicin. Larger doses of epirubicin could be safely used in future studies, and further evaluation of epirubicin in phase III trials is indicated.

Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-α (hTNF-α) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-α antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-α and local expression of various proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming a critical role of hTNF-α in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-α treatment was observed in young mice but not in aged mice.