Background: Polymethylmethacrylate (PMMA) is a potent stimulant of inflammatory response. This study investigated the role of Prostaglandin E2 (PGE2), Platelet activating factor (PAF) and histamine and their specific antagonists in bone changes. Materials: 120 white-male-wistar rats were divided into ten groups. Using sterile technique, a 2mm drill hole was made in the tibia 1cm distal to the knee joint bilaterally. The left tibia was filled with Simplex particulate cement polymer (PMMA) and the right tibia was used as control. The first nine groups respectively received terfenadine 1mg/kg, 10mg/kg and 25mg/kg, alprazolam 0.08mg/kg, 0.32mg/kg and 0.64mg/kg, and naproxen 1mg/kg, 5mg/kg and 25mg/kg; however, the tenth group received no drug and served as control. The animals were killed after 16 weeks and implant areas were harvested aseptically and studied by one pathologist. Results: Our study revealed that the cellular reaction in the left side was statistically more than the right one in all cases (p< 0.05). Also, a significant decrease in histiocytes and giant cells was seen just in those groups that had received 10mg/kg and 25mg/kg of terfenadine, 0.32mg/kg and 0.64mg/kg of alprazolam and 5mg/kg and 25mg/kg of naproxen (P< 0.05) while administration of 1mg/kg naproxen resulted in significant decrease only in giant cells (P< 0.05) but not in histiocytes. Discussion: Previous studies have suggested that particulate debris, PGE2 production and inflammatory response are associated with arthroplasty loosening. This experiment has demonstrated that the increased cellular reaction by the membrane surrounding particulate cement polymer can be suppressed by administration of PGE2, PAF and histamine specific inhibitors. The use of these agents may be indicated in retarding the bone loss associated with early prosthetic loosening

The signaling molecule prostaglandin E2 (PGE2), synthesized by cyclooxygenase-2 (COX-2), is immunoregulatory and reported to be essential for skeletal stem cell function. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in osteoarthritis (OA) analgesia, but cohort studies suggested that long-term use may accelerate pathology. Interestingly, OA chondrocytes secrete high amounts of PGE2. Mesenchymal stromal cell (MSC) chondrogenesis is an in vitro OA model that phenocopies PGE2 secretion along with a hypertrophic OA-like cell morphology. Our aim was to investigate cause and effects of PGE2 secretion in MSC-based cartilage neogenesis and hypertrophy and identify molecular mechanisms responsible for adverse effects in OA analgesia. Human bone marrow-derived MSCs were cultured in chondrogenic medium with TGFβ (10ng/mL) and treated with PGE2 (1µM), celecoxib (COX-2 inhibitor; 0.5µM), AH23848/AH6809 (PGE2 receptor antagonists; 10µM), or DMSO as a control (n=3–4). Assessment criteria were proteoglycan deposition (histology), chondrocyte/hypertrophy marker expression (qPCR), and ALP activity. PGE2 secretion was measured (ELISA) after TGFβ withdrawal (from day 21, n=2) or WNT inhibition (2µM IWP-2 from day 14; n=3). Strong decrease in PGE2 secretion upon TGFβ deprivation or WNT inhibition identified both pathways as PGE2 drivers. Homogeneous proteoglycan deposition and COL2A1 expression analysis showed that MSC chondrogenesis was not compromised by any treatment. Importantly, hypertrophy markers (COL10A1, ALPL, SPP1, IBSP) were significantly reduced by PGE2 treatment, but increased by all inhibitors. Additionally, PGE2 significantly decreased ALP activity (2.9-fold), whereas the inhibitors caused a significant increase (1.3-fold, 1.7-fold, 1.8-fold). This identified PGE2 as an important inhibitor of chondrocyte hypertrophy. Although TGFβ and WNT are known pro-arthritic signaling pathways, they appear to induce a PGE2-mediated antihypertrophic effect that can counteract pathological cell changes in chondrocytes. Hampering this rescue mechanism via COX inhibition using NSAIDs thus risks acceleration of OA progression, indicating the need of OA analgesia adjustment

Aim: To examine the in vitro effects of several non-steroidal anti-inflammatory drugs (NSAIDs) on pro-inflammatory cytokines and PGE. 2. production by interface membrane from loose endoprosthesis of hip or knee arthroplasty. Since these factors are strongly implicated in the bone resorption process and aseptic prosthesis failure we hypothesize that the probable inhibition of their production by prophylactic administration of NSAIDs, will retard these processes. Materials and Methods: Interface membranes were harvested from ten patients who were subjected to revision surgery for aseptic total hip or knee replacement loosening and cultured for 72h in the absence or presence of therapeutic dosages of each, of aceclofenac, piroxicam, tenoxicam and indomethacin. Paracetamol was used as neutral control. In conditioned media the levels of IL-6, IL-1 (3, TNF-a and PGE2 were determined by ELISA and the data were analyzed by the Student’s t-test (significance level p< 0.05). Results: All the tested NSAIDs caused a statistically significant decrease on IL-6 and TNF-a levels, with aceclofenac and tenoxicam to be more effective (caused decrease in 7 out of 10 samples), while they had low or controversial effect on IL-1β production, except aceclofenac that seemed to augment the IL-1β levels (statistically significant increase in 5 out of 9 samples). Finally all the tested drugs, except paracetamol, caused a marked reduction (80–99%) of PGE2 levels. Conclusions: The stimulatory effect of IL-6 and TNF-α in the osteoclastic bone resorption process is well established. Considering the above results, the tested IMSAIDs (especially aceclofenac and tenoxicam) reduce the in vitro production of these mediators by interface membranes. Hence, it is reasonable to propose that the prophylactic treatment with these drugs could delay the process of the aseptic loosening. However, in order to support this hypothesis, more experiments are required by which the effects of them on other factors implicated in the loosening process, such as metalloproteinases, will be examined

Aseptic loosening of orthopaedic implants is usually attributed to the action of wear debris from the prosthesis. Recent studies, however, have also implicated physical pressures in the joint as a further cause of loosening. We have examined the role of both wear debris and pressure on the secretion of two chemokines, MIP-1α and MCP-1, together with M-CSF and PGE2, by human macrophages in vitro. The results show that pressure alone stimulated the secretion of more M-CSF and PGE. 2. when compared with control cultures. Particles alone stimulated the secretion of M-CSF and PGE. 2. , when compared with unstimulated control cultures, but did not stimulate the secretion of the two chemokines. Exposure of macrophages to both stimuli simultaneously had no synergistic effect on the secretion of the chemokines, but both M-CSF and PGE. 2. were increased in a synergistic manner. Our findings suggest that pressure may be an initiating factor for the recruitment of cells into the periprosthetic tissue

Objective. Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. Method. Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. Results. The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1β and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. Conclusion. These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280–8

Synovitis has been shown to play a role in pathophysiology of OA promoting cartilage destruction and pain. Synovium is mainly composed of synovial fibroblast (SF) and macrophage (SM) that guide synovial inflammation. Adipose stromal cells (ASC) promising candidate for cell therapy in OA are able to counteract inflammation. Two different subsets of macrophages have been described showing a pro-inflammatory (M1) and an anti-inflammatory (M2) phenotype. Macrophage markers: CD68, CD80 (M1-like) and CD206 (M2-like) were evaluated in osteoarthritic synovial tissue. GMP-clinical grade ASC were isolated from subcutaneous adipose tissue and M1-macrophages were differentiated from CD14+ obtained from peripheral blood of healthy donors. ASC were co-cultured in direct and indirect contact with activated (GM-CSF+IFNγ)-M1 macrophages for 48h. At the end of this co-culture we analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassay. PGE2 blocking experiments were performed. In moderate grade OA synovium we found similar percentages of CD80 and CD206. M1-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both co-culture conditions. Moreover, ASC induced the typical M2 macrophage markers IL10, CD163 and CD206. Blocking experiments showed that TNFα, IL6, IL10, CD163 and CD206 were significantly modulated by PGE2. We confirmed the involvement of PGE2/COX2 also in CD14+ OA synovial macrophages. In conclusion we demonstrated that ASC are responsible for the switching of activated-M1-like to a M2-like anti-inflammatory phenotype, mainly through PGE2. This suggested a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

Introduction: Selective COX-2 (Cyclooxygenase-2) inhibitors have been found to impede fracture healing. The effect of selective COX-2 inhibitors on tendon healing in a bone tunnel, however, is unknown. Methods: The authors performed bilateral anterior cruciate ligament reconstructions in 32 rabbits and used peripheral quantitative computed tomography (pQCT) to compare tendon-to-bone healing between tunnel aperture and midtunnel regarding bone mineral density (BMD) and ingrowth of new bone. Each animal was assigned to one of four groups. Two groups received selective COX-2 inhibitors orally for 3 weeks (Cele-coxib; 10 mg/kg/d), the two other groups received no COX-2 inhibitors (controls). The animals were sacrificed 3 and 6 weeks after surgery. In biomechanical testing maximum load to failure and stiffness of the tendon grafts were calculated from the load displacement curve and failure modes were recorded. To assess indirectly the effect on local COX-2 activity the synovial content of Prostaglandin E2 (PGE2), the major metabolite of arachnidonic acid metabolism and catalyzed by COX-2, was measured by Enzyme-linked Immunosorbent Assay (ELISA). Results: Animals treated with selective COX-2 inhibitors had significantly lower BMD at the tunnel aperture (P=.02). In all groups the BMD at the tunnel aperture was significantly higher in comparison with the midtunnel (P< .05). In the controls ingrowth of new bone was greater at the tunnel aperture at 3 weeks (P=.028). After 3 weeks of COX-2 inhibitor administration synovial fluid concentrations of PGE2 were significantly lowered (P=.018) and increased more than threefold by 6 weeks after surgery and 3 weeks after last drug administration (P=.022), while in the controls there was a decrease in PGE2 between week 3 and 6. At 6 weeks the controls exhibited a twofold increase in maximum load to failure (3 weeeks: 28.2±20.9 N; 6 weeks: 59.6±53.6 N; P=.394), whereas the COX-2 inhibitor treated specimens decreased 1.9fold (3 weeks: 69.3±50.5 N; 6 weeks: 37.4±16.8 N; P=.24). Maximum load to failure values correlated with PGE2 changes, but not statistically significant (r. 2. = −0,502; p=0,056). Failure modes at 3 and 6 weeks were rupture and degloving, respectively, of the tendon graft. Discussion: This study revealed decreased bone mineral density at the tunnel aperture at 3 weeks, an increase of the inflammatory mediator PGE2 and decreased graft stability with time after treatment with selective COX-2 inhibitors. Untreated controls appeared to have a more physiological healing course with a continuous decrease in PGE2 and an increase in graft stability. Our results suggest, that selective COX-2 inhibitors may delay tendon healing in a bone tunnel

Aims: The aim of this study was to investigate the ability of control and degenerate human nucleus pulposis to respond to an exogenous proinßammatory stimulus. Methods: Disc material from patients undergoing surgery for scoliosis, sciatica and low back pain was cultured under basal and lipopolysaccharride (LPS) stimulated conditions using a serumless technique. Levels of IL-1β, TNFα, LTB4, GM-CSF, IL-6, IL-8, MCP-1, PGE2, bFGF and TGFβ-1 in the media were estimated using commercially available enzyme linked immunoabsorbent assay kits. Results: Neither basal nor LPS stimulated control nucleus pulposis (NP) produced detectable levels of IL-1β, TNFα, LTB4 or GM-CSF. LPS induced a significant increase in scoliotic disc IL-8 production, p< .02. LPS induced signiþcant increases in degenerate disc IL-6, IL-8 and PGE2 production, p< .01, p< .001 and p< .005 respectively. LPS signiþcantly increased degenerate disc IL-6, IL-8 and PGE2 production compared to LPS stimulated scoliotic disc, p< .05, p< .02 and p< .003 respectively. Conclusions: Human nucleus pulposus can react to a pro-inßammatory stimulus by secreting IL-6, IL-8 and PGE2, suggesting that the NP may actively participate in the genesis of chemical radiculitis and dis-cogenic back pain

Heterotopic ossi?cation is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossi?cations are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossi?cation on a molecular level, therefore we aimed to determine whether indomethacin might influence heterotopic ossi?cation via impairing the chondrogenic phase of endochondral ossification. ATDC5, human bone marrow stem cells (hBMSCs) and rabbit periosteal agarose cultures were employed as progenitor cell models; SW1353, human articular chondrocytes and differentiated ATDC5 cells were used as matured chondrocyte cell models. All cells were cultured in the presence of (increasing) concentrations of indomethacin. The action of indomethacin was confirmed by decreased PGE2 levels in all experiments, and was determined by specific PGE2 ELISA. Gene- and protein expression analyses were employed to determine chondrogenic outcome. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a dose-dependent decrease in gene- and protein expression of chondrogenic and hypertrophic markers as well as decreased glycosaminoglycan content was observed. Even when hypertrophic differentiation was provoked the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre-differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onwards, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rationale behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications

Introduction and Objective. Anterior cruciate ligament reconstruction (ACLR) with tendon autografts is the “gold standard” technique for surgical treatment of ACL injuries. Common tendon graft choices include patellar tendon (PT), semitendinosus/gracilis “hamstring” tendon (HT), or quadriceps tendon (QT). Healing of the graft after ACLR may be affected by graft type since the tissue is subjected to mechanical stresses during post-operative rehabilitation that play important roles in graft integration, remodeling and maturation. Abnormal mechanical loading can result in high inflammatory and degradative processes and altered extracellular matrix (ECM) synthesis and remodeling, potentially modifying tissue structure, composition, and function. Because of the importance of load and ligamentization for tendon autografts, this study was designed to compare the differential inflammatory and degradative metabolic responses to loading by three tendon types commonly used for autograft ACL reconstruction. Materials and Methods. With IRB approval (IRB # 2009879) and informed patient consent, portions of 9 QT, 7 PT and 6 HT were recovered at the time of standard of care ACLR surgeries. Tissues were minced and digested in 0.2 mg/ml collagenase solution for two hours and were then cultured in 10% FBS at 5% CO. 2. , 37°C, and 95% humidity. Once confluent, cells were plated in Collagen Type I-coated BioFlex® plates (1 × 10. 5. cells/well) and cultured for 2 days prior to the application of strain. Then, media was changed to supplemented DMEM with 2% FBS for the application of strain. Fibroblasts were subjected to continuous mechanical stimulation (2-s strain and 10-s relaxation at a 0.5 Hz frequency) at three different elongation strains (mechanical stress deprivation-0%, physiologic strain-4%, and supraphysiological strain-10%). 9. for 6 days using the Flexcell FX-4000T strain system. Media was tested for inflammatory biomarkers (PGE2, IL-8, Gro-α, and MCP-1) and degradation biomarkers (GAG content, MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2). Significant (p<0.05) difference between graft sources were assessed with Kruskal-Wallis test and post-hoc analysis. Results are reported as median± interquartile range (IQR). Results. Differences in Inflammation-Related Biomarker Production (Figure 1): The production of PGE2 was significantly lower by HT fibroblasts compared to both QT and PT fibroblasts at all timepoints and strain levels. The production of Gro-α was significantly lower by HT fibroblasts compared to QT at all time points and strain levels, and significantly lower than PT on day 3 at 0% strain, and all strain levels on day 6. The production of IL-8 by PT fibroblasts was significantly lower than QT and HT fibroblast on day 3 at 10% strain. Differences in Degradation-Related Biomarker Production (Figure 2): The production of GAG by HT fibroblasts was significantly higher compared to both QT and PT fibroblasts on day 6 at 0% strain. The production of MMP-1 by the QT fibroblasts was significantly higher compared to HT fibroblasts on day 3 of culture at all strain levels, and in the 0% and 10% strain levels on day 6 of culture. The production of MMP-1 by the QT fibroblasts was significantly higher compared to PT fibroblasts at in the 0% and 4% strain groups on day 3 of culture. The production of TIMP-1 by the HT fibroblasts was significantly lower compared to PT fibroblasts on day 3 of culture. Conclusions. The results of this study identify potentially clinically relevant difference in the metabolic responses of tendon graft fibroblasts to strain, suggesting a lower inflammatory response by hamstring tendon fibroblasts and higher degradative response by quadriceps tendon fibroblasts. These responses may influence ACL autograft healing as well as inflammatory mediators of pain in the knee after reconstruction, which may have implications regarding graft choice and design of postoperative rehabilitation protocols for optimizing outcomes for patients undergoing ACL reconstruction. For any figures or tables, please contact the authors directly

Recently there has been considerable interest in the role of inflammatory mediator production by herniated degenerate discs. Modic has described MR endplate changes which have an inflammatory appearance and have been linked with discogenic back pain. To date there has been no biomechanical investigation of discs with associated Modic changes. The aim of this study is to determine if degenerate discs with associated Modic changes have higher levels of pro-inflammatory mediator production than those without Modic changes. Intervertebral disc tissue was obtained from 52 patients undergoing spinal surgery for sciatica [40] and discogram proven discogenic low back pain [12]. The tissue was cultured and the medium analysed for interleukin-6, interleukin-8 and prostaglandin E2 using an enzyme linked immunoabsorbetn assay method. Preoperative MR images of the patients were examined by a double blinded radiologist to determine the Modic status of the cultured disc level. Forty percent of patients undergoing surgery for discogenic low back pain had a Modic 1 change compared to only 12.5% of patients undergoing surgery for sciatica [p< .05] There was a statistically significant difference between levels of IL-6, IL-8 and PGE2 production by both the Modic1 [M1] and Modic2 [M2] groups compared to the Modic negative [NEG] group. IL-6:NEGvM1 p< .001, NEG v M2 p< .05, IL-8: NEG v M1 p< .01, NEG v M2 p> .05, PGE2: NEG v M1 p< 01, NEG v M2 p< .05. Modic changes have been associated with positive provocative discography by a number of authors. Pain generation requires the presence of nerves and hyperalgsia inducing mediators. Both IL-8 and PGE2 are known to induce hyperalgesia. The fact that Modic changes are associated with high levels of production of these mediators supports their role as an objective marker of discogenic low back pain

The role of nucleus pulposus (NP) biology in the genesis of sciatica is being increasingly investigated. The aim of this study was to examine the ability of control and degenerate human nucleus pulposus to respond to an exogenous pro-inflammatory stimulus. Control disc material was obtained from surgical procedures for scoliosis and degenerate disc tissue from surgical procedures for sciatica and low back pain. Disc specimens were cultured using a serumless technique under basal and lipopolysaccharride (LPS) stimulated conditions and the media harvested, aliquoted and stored at –80°C for subsequent analysis. Levels of IL-1β,TNFα, LTB4, GM-CSF, IL-6, IL-8, MCP-1, PGE2, bFGF and TGFβ-1 in the media were estimated using commercially available enzyme linked immunoabsorbent assay kits. Neither basal nor LPS stimulated control or degenerate NP produced detectable levels of IL-1β, TNFα, LTB4 or GM-CSF. Control disc IL-8 secretion increased significantly with LPS stimulation, p< .018. Degenerate disc IL-6, IL-8 and PGE2 production increased significantly with LPS stimulation, p< .01, p< .001 and p< .005 respectively. LPS stimulated degenerate NP secreted significantly more IL-6, IL-8 and PGE2 than LPS stimulated control NP, p < 0.05, 0.02 and 0.003 respectively. LPS induces an increase in both control and degenerate NP mediator production demonstrating the ability of human NP to react to a noxious stimulus by producing pro-inflammatory mediators. The difference in levels of basal and LPS stimulated mediator production between control and degenerate discs show that as a disc degenerates it increases both its level of inflammatory mediator production and its ability to react to a pro-inflammatory stimulus. The increased sensitivity of degenerating human NP to noxious stimuli and increased ability to respond with inflammatory mediator production support the role of NP as an active participant in the genesis of lumbar radiculopathy and discogenic back pain

Summary. Hyaluronan suppressed lipopolysaccharide-stimulated prostaglandin E. 2. production via intercellular adhesion molecule-1 through down-regulation of nuclear factor-κB. Administration of hyaluronan into rheumatoid joints may decrease prostaglandin E. 2. production by activated macrophages, which could result in improvement of arthritic pain. Introduction. Prostaglandin E. 2. (PGE. 2. ) is one of the key mediators of inflammation in rheumatoid arthritis (RA) joints. Intra-articular injection of high molecular weight hyaluronan (HA) into RA knee joints relieves arthritic pain. Although HA has been shown to inhibit PGE. 2. production in cytokine-stimulated synovial fibroblasts, it remains unclear how HA suppresses PGE. 2. production in catabolically activated cells. Furthermore, HA effect on macrophages has rarely been investigated in spite of their contribution to RA joint pathology. Objectives. This study was aimed to investigate the inhibitory mechanism of HA on lipopolysaccharide (LPS)-stimulated PGE. 2. in U937 human macrophage culture system. Methods. With or without pretreatment with one of HA, NS-398, and BAY11-7085, differentiated U937 macrophages were stimulated with LPS. In another set of experiments, the cells were incubated with anti-ICAM-1 antibody or non-specific IgG before pretreatment with HA. PGE. 2. concentrations of the cell-free supernatants were determined using an enzyme-linked immunosorbent assay. The cell lysates and nuclear extracts were prepared for immunoblot analysis. HA binding to ICAM-1 was evaluated by fluorescence microscopic analysis. Results. Stimulation of U937 macrophages with LPS enhanced PGE. 2. production in association with increased protein levels of cyclooxygenase-2 (COX-2). Pretreatment with HA of 2,700 kDa resulted in suppression of LPS-induced COX-2, leading to a decrease in PGE. 2. production. While LPS activated NF-κB pathway, inhibition studies using BAY11-7085 revealed the requirement of NF-κB for LPS-stimulated PGE. 2. production. HA down-regulated the phosphorylation and nuclear translocation of NF-κB by LPS. Fluorescence cytochemistry demonstrated that HA bound to ICAM-1 on U937 macrophages. Anti-ICAM-1 antibody reversed the inhibitory effects of HA on LPS-activated PGE. 2. , COX-2, and NF-κB. Conclusion. These results clearly demonstrated that HA suppressed LPS-stimulated PGE2 production via ICAM-1 through down-regulation of NF-κB. Clinical administration of high molecular weight HA into RA joints may decrease PGE2 production by activated macrophages, which could result in improvement of arthritic pain

Osteoarthritis (OA) is a highly prevalent degenerative joint disorder characterized by joint pain and physical disability. Aberrant subchondral bone induces pathological changes and is a major source of pain in OA. In the subchondral bone, which is highly innervated, nerves have dual roles in pain sensation and bone homeostasis regulation. The interaction between peripheral nerves and target cells in the subchondral bone, and the interplay between the sensory and sympathetic nervous systems, allow peripheral nerves to regulate subchondral bone homeostasis. Alterations in peripheral innervation and local transmitters are closely related to changes in nociception and subchondral bone homeostasis, and affect the progression of OA. Recent literature has substantially expanded our understanding of the physiological and pathological distribution and function of specific subtypes of neurones in bone. This review summarizes the types and distribution of nerves detected in the tibial subchondral bone, their cellular and molecular interactions with bone cells that regulate subchondral bone homeostasis, and their role in OA pain. A comprehensive understanding and further investigation of the functions of peripheral innervation in the subchondral bone will help to develop novel therapeutic approaches to effectively prevent OA, and alleviate OA pain.

Cite this article: Bone Joint Res 2022;11(7):439–452.

Wear debris is a key factor in the pathophysiology of aseptic loosening of orthopaedic endoprostheses. Cobalt-chromium-molybdenum (Co-CrMo) alloys are used for metal-metal hip implants due to their enhanced wear resistance profiles. Whilst these alloys have widespread clinical application, little is known about their direct effect on osteoblast biology. To address this issue, in this study we have investigated particle-mediated inflammation, as a putative mechanism of aseptic loosening. The effects of Co2+ ions on the bone cellular milieu were assessed in vitro by profiling of classical inflammatory mediators. The inflammatory driver PGE2 was quantified and found to be increased, following osteoblast stimulation with metal ions, suggesting the initiation of a local inflammatory response to metal particle exposure. To determine the biological import of this molecular event, the role of metal ions in recruiting inflammatory cells by chemokine production was assessed. These data demonstrated significant induction of the chemokines, IL-8 and MCP-1 following both 12 and 24 hour exposure to 10ppm of Co2+. In this study, we demonstrate that Co2+ particles can rapidly induce chemotactic cytokines, IL-8 and MCP-1 early stress-responsive chemokines that function in activation and chemotaxis of monocytes, and PGE2, which stimulates bone resorption. We have shown that this induction occurs at a transcriptional level with significantly increased mRNA levels. These data lend further weight to the hypothesis that wear mediated osteolysis, is due, at least in part, to underlying chronic inflammation

Purpose: Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic patients rapidly express type X collagen (COL10A1), a marker of late-stage chondrocyte hypertrophy associated with endochondral ossification. We recently discovered that a novel atmospheric-pressure plasma-polymerized thin film substrate, named “nitrogen-rich plasma-polymerized ethylene” (PPE:N), is able to inhibit COL10A1 expression in committed MSCs. However, the cellular mechanisms implicated in the inhibition of COL10A1 expression by PPE:N surfaces are unknown. Method: Human mesenchymal stem cells (MSCs) were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for osteoarthritis. Bone marrow aspirates were processed and MSCs were cultured on commercial polystyrene (PS control) and on PPE:N surfaces in the presence of different kinases and cyclooxygenase inhibitors for 3 days. Total RNA was extracted with TRIzol reagent (Invitrogen, Burlington, ON) and the expression of COL10A1, cyclooxygenase-1 (COX-1), and 5-lipoxygenase (5-LOX) genes was measured by real-time quantitative RT-PCR. Results: Results showed that a non-specific inhibitor of cyclooxygenases reduced the expression of COL10A1. In contrast, inhibitors of protein kinases stimulated the expression of COL10A1. Furthermore, potent and selective inhibitors of COX-1 and 5-LOX also reduced the expression of COL10A1. However, COX-2 and 12-LOX inhibitors had no significant effect on the expression of COL10A1. COX-1 gene expression was also decreased when MSCs were incubated on “S5” PPE:N surfaces. Interestingly, MSCs did not express 5-LOX. Conclusion: PPE:N surfaces suppress COL10A1 expression through the inhibition of COX-1 which is directly implicated in the synthesis of prostaglandins. The decreased expression of COX-1 and COL10A1 in human MSCs cultured on PPE:N is therefore in agreement with the induction of the osteogenic capacity of rat bone marrow and bone formation by systemic or local injection of PGE2 in rats. However, PGE2 and other prostaglandins inhibited COL10A1 expression in chick growth plate chondrocytes. This suggests that the effect of prostaglandins on COL10A1 expression may be cell-specific or may be dependent on pre-existing patho-physiological conditions

The pathophysiology of discogenic low back pain is poorly understood. The morphological changes occurring in disc degeneration are well documented but unhelpful in determining if a particular degenerate disc will be painful or not. Herniated intervertebral disc tisssue has been shown to produce a number of pro-inflammatory mediators and cytokines. No similar studies have to date been done utilising disc material from patients with discogenic low back pain. The aim of this study was to compare levels of production of interleukin-6 (IL-6), interleukin-8 (IL-8) and Prostaglandin E2 (PGE2) in disc tissue from patients undergoing discectomy for sciatica with that from patients undergoing fusion for discogenic low back pain. Tissue from 50 patients undergoing discectomy for sciatica and 20 patients undergoing fusion for discogenic low back pain was cultured and the medium harvested for subsequent analysis using an enzyme linked immunoabsorbent assay method. Statistical analysis of the results was performed using the Mann-Whitney test. Disc specimens from both experimental groups produced measurable levels of all three mediators. Mean production of IL-6, IL-8 and PGE2 in the sciatica group was 26.2±75.7, 247±573 and 2255±3974 respectively. Mean production of IL-6, IL-8 and PGE2 in the low back pain group was 92±154, 776±987 and 3221±3350 respectively (data = mean production pg/ml ± 1 standard deviation). There was a statistically significant difference between the levels of IL-6 and IL-8 production in the sciatica and low back pain groups (p< 0.006 and p< 0.003 respectively). The high levels of pro-inflammatory mediator production found in disc tissue from patients undergoing fusion for discogenic LBP may indicate that nucleus pulposis pro-inflammatory mediator production is a major factor in the genesis of a painful lumbar disc. This could explain why some degenerate discs cause LBP while other morphologically similar discs do not

Study Design: The effects of heat on porcine intertvertebral disc were studied experimentally. Objective: To assess the effects of in-vitro heating of porcine nucleus pulposus on expression of inducible heat shock protein 70 and subsequent modification of biochemical responses to an inflammatory insult in the heated intervertebral disc tissue. Subjects: Lumbar spines were harvested from six pigs. The nucleus pulposus was dissected from each intervertebral disc, divided into control (37°C) and heat shocked (42°C) groups then cultured in medium for one hour. All samples were then cultured at 37 C for a further two hours. After three hours tissue and supernatant were harvested from one third of the samples and the expression of inducible heat shock protein 70 (HSP70) was quantified via Western immunoblotting and enzyme linked immuno-sorbent assay (ELISA). The remaining samples were cultured either in normal medium or altered (pro-inflammatory) medium containing 5ug/ml bacterial lipopolysaccharide (LPS). At 24 hours the supernatant from these samples was analysed for both interleukin-8 (IL-8) and prostaglandin E2 (PGE2) secretion using ELISA. Outcome Measures: Western immunoblotting and enzyme linked immuno-sorbent assay (ELISA) for heat shock protein 70. ELISA for interleukin-8 (IL-8) and prostaglandin E2 (PGE2). Results: HSP70 expression was significantly increased in the heat shocked specimens. IL-8 and PGE2 secretion were significantly increased in nucleus pulposus exposed to LPS at both temperatures. The concentrations of IL-8 and PGE2 secreted in the heat shocked samples were significantly less than controls, particularly after exposure to LPS (p< 0.05, paired students t test). Conclusions: In vitro heating of porcine nucleus pulposus causes overexpression of HSP70. This heat shock effect can alter aspects of the biochemical response of the intervertebral disc tissue to an inflammatory insult. Intradiscal electrothermal therapy (IDET) may, in theory, reduce discogenic pain at temperatures as low as 42°C by generating similar heat-induced changes in the nuclear biochemistry of degenerate intervertebral discs

The expression of the mechanosensor, integrin αvβ3, is reduced in osteoporotic bone cells compared to controls. MLO-Y4 osteocytes experience altered mechanotransduction under estrogen deficiency and it is unknown whether this is associated with defective αvβ3 expression or signalling. The objectives of this study are to (1) investigate αvβ3 expression and spatial organisation in osteocytes during estrogen deficiency, and (2) establish whether altered responses of osteocytes under estrogen deficiency correlate to defective αvβ3 expression and functionality. MLO-Y4 cells were cultured as follows: Ctrl (no added estradiol), E+ (10nM 17β-estradiol for 5 days), and Ew (10nM 17β-estradiol for 3 days and withdrawal for 2 days). Cells were cultured with/without 0.5µM IntegriSense750 (αvβ3 antagonist). Laminar oscillatory fluid flow of 1Pa at 0.5Hz was applied for 1hr. αvβ3 content was quantified using an ELISA. The location and quantity of αvβ3 and focal-adhesions was determined by immunocytochemistry. Estrogen withdrawal under static conditions led to lower cell and focal-adhesion area (p<0.05), compared to E+ cells. Fluid flow led to higher αvβ3 content (p<0.05) in all groups, compared to static counterparts, with αvβ3 blocking altering this response. Fluid flow on Ew cells had the highest αvβ3 levels (p<0.05), but αvβ3 did not localise at focal-adhesions sites. Cell morphologies were similar after treatment with the αvβ3 antagonist to the Ew group. These results suggest there are fewer functional focal-adhesion sites at which αvβ3 integrins localise to facilitate mechanotransduction. To further understand these results, we are analysing osteocyte mechanotransduction by quantifying PGE2 and gene expression (COX-2, RANKL, OPG, SOST)

The authors wished to determine if macrophage activation and the release of osteolytic cytokines in response to orthopaedic wear debris could be suppressed pharmacologically with the use of anti-inflammatory and anti-oxidant agents. The current long-term results of total joint arthroplasty are limited by mechanical wear of the implants with an associated immune mediated bone lysis with subsequent loosening and eventual failure. It has been demonstrated that the osteolysis seen in cases of aseptic loosening is mediated by the immune system both directly and indirectly by activated macrophages. Macrophages indirectly cause osteolysis through release of the osteoclast activating cytokines TNFα, IL-1 and PGE2. They also directly resorb bone in small amounts when activated by wear particles. We utilised established cell culture models of both peripherally derived monocyte/macrophages and lymphocyte enriched co-cultures and examined the effects of polymethylmethacrylate particles alone on the cells in culture. The effect of anti-inflammatory and anti-oxidant agents (dexamethasone, diclofenac and n-acetyl cysteine) in varying concentrations was then examined using ELISA of cytokine release and electron microscopy to examine ultra structural responses. Cell viability was also measured in cultures over 24 hour periods (at 6, 12 and 24 hours) using Trypan blue exclusion and Coulter counter, while cell type and morphology were determined cytologically, including-naphthyl acetate esterase cytochemical identification and electron microscopy. The use of N-acetyl cysteine was associated with very significant suppression of TNF, IL-1 and PGE2 in both macrophage and lymphocyte enriched co-culture with no effect on cell viability. While diclofenac was also associated with significant decreases in cytokine expression, it was associated with a decrease in cell viability that approached significance. Dexamethasone did not have a reliable effect on these cytokines. Ultra-structural electron microscopic examination of the cells also demonstrated signs of definite down-regulation of cytoplasmic and nuclear activation. Novel anti-oxidant therapies and possibly other immune modulating drugs can eliminate the activation of macrophages in response to periprosthetic wear particles without any associated decrease in cell viability and thus may provide a means of reducing the incidence of loosening and failure of total joint arthroplasty

The current long term results of total joint arthroplasty are limited by mechanical wear of the implants with an associated immune mediated bone lysis with subsequent loosening and eventual failure. It has been demonstrated that the osteolysis seen in cases of aseptic loosening is mediated by the immune system, particularly, both directly and indirectly, by activated macrophages. Macrophages indirectly cause osteolysis through release of the osteoclast activating cytokines: TNFα, IL-1 and PGE2 and also directly resorb bone in small amounts when activated by wear particles. We wished to determine if macrophage activation and the release of osteolytic cytokines in response to orthopaedic wear debris could be suppressed pharmacologically, with the use of anti-inflammatory and anti oxidant agents. We utilised established cell culture models of both peripherally derived monocyte/macrophages and lymphocyte enriched co-cultures and examined the effects of polymethylmethacrylate particles alone on the cells in culture. The effects of anti-inflammatory and anti-oxidant agents (dexamethasone, diclofenac and n-acetyl cysteine) in varying concentrations were then examined using ELISA of cytokine release and electron microscopy to examine ultra structural responses. Cell viability was also measured in cultures over 24 hour periods (at 6, 12 and 24 hours) using Trypan blue exclusion and Coulter counter, while cell type and morphology were determined cytologically, including α-naphthyl acetate esterase cytochemical identification and electron microscopy. The use of N-acetyl cysteine was associated with very significant suppression of TNFα, IL-1β and PGE2 in both macrophage and lymphocyte enriched co-culture with no effect on cell viability. While diclofenac was also associated with significant decreases in cytokine expression it was associated with a decrease in cell viability that approached significance. Dexamethasone did not have a reliable effect on these cytokines. Ultra-structural electron microscopic examination of the cells also demonstrated signs of definite down-regulation of cytoplasmic and nuclear activation. We have demonstrated, therefore, that novel anti-oxidant therapies and possibly other immune modulating drugs can eliminate the activation of macrophages in response to peri-prosthetic wear particles without any associated decrease in cell viability and thus may provide a means of reducing the incidence of loosening and failure of total joint arthroplasty

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

INTRODUCTION. Endochondral ossification in the growth plate is directly responsible for skeletal growth and its de novo bone-generating activity. Growth plates are vulnerable to disturbances that may lead to abnormal skeletal development. Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used analgesics but have been reported to impair endochondral ossification-driven fracture healing. Despite the general awareness that NSAIDs affect endochondral ossification, the consequences of NSAIDs on skeletal development are unknown. We hypothesise that the NSAID celecoxib leads to impaired growth plate development and consequently impairs skeletal development. METHODS. Healthy skeletally immature (5 weeks old) C57BL/6 mice were treated for ten weeks with celecoxib (daily oral administration 10 mg/kg) or placebo (water) (institutional approval 2013–094) (n=12 per group). At 15 weeks postnatally, total growth plate thickness, the thickness of specific growth plate zones, (immuno)histological analysis of extracellular matrix composition in the growth plate, cell number and cell size, longitudinal bone growth and bone micro-architecture by micro-CT were analysed. Inhibition of COX-2 activity was confirmed by determining PGE2 levels in plasma using an ELISA. RESULTS. No significant difference in total growth plate thickness or thickness of the resting zone, proliferative or hypertrophic zone was found between groups. Staining of growth plate extracellular matrix components revealed, however, a significantly higher proteoglycan content and less collagen type II staining in the proliferative zone. In the hypertrophic zone of the growth plates of celecoxib treated mice collagen type X was hardly detectable as compared to placebo mice. In addition, a significantly decreased cell number was observed in the hypertrophic zone of the growth plate and cells were significantly smaller in the celecoxib group. Micro-CT analysis of the subchondral bone region directly beneath the growth plate showed significantly higher bone density, bone volume density and trabecular thickness following celecoxib treatment. Despite the detected differences in extracellular matrix composition of the growth plate, no difference was found in the length of the tibia in celecoxib treated mice. DISCUSSION. In summary, there are no measurable differences found in murine skeletal formation as a result of treatment with celecoxib in this study. However, there are notable phenotypic features found in the maturation of the growth plate (hypertrophic zone and subchondral bone) as a result from the celecoxib treatment, of which the potential consequences we do not yet understand. SIGNIFICANCE. When follow-up actions from the use of celecoxib on the growing individual are found this may warrant re-evaluation for the use of celecoxib in these individuals

Bone is one of the most highly adaptive tissues in the body, possessing the capability to alter its morphology and function in response to stimuli in its surrounding environment. The ability of bone to sense and convert external mechanical stimuli into a biochemical response, which ultimately alters the phenotype and function of the cell, is described as mechanotransduction. This review aims to describe the fundamental physiology and biomechanisms that occur to induce osteogenic adaptation of a cell following application of a physical stimulus. Considerable developments have been made in recent years in our understanding of how cells orchestrate this complex interplay of processes, and have become the focus of research in osteogenesis. We will discuss current areas of preclinical and clinical research exploring the harnessing of mechanotransductive properties of cells and applying them therapeutically, both in the context of fracture healing and de novo bone formation in situations such as nonunion.

Cite this article: Bone Joint Res 2019;9(1):1–14.

In both Enchondromatosis (EC) and Multiple Osteochondromas (MO), multiple benign cartilaginous tumours occur, which have a severely increased risk of malignant progression. Preventing new tumor formation and malignant progression would benefit the prognosis of these patients. A protective effect of selective Cox-2 inhibitor celecoxib, has been suggested against development and growth of colorectal cancer in familial syndromes. At last year’s EMSOS meeting we reported on expression of Cox-2 in 37% (central) – 46% (peripheral) of conventional chondrosarcomas. mRNA levels of EC related tumours were slightly higher than the solitary tumours. Celecoxib treatment of the chondrosarcoma cell lines resulted in a 3 fold decrease of PGE2 levels already at 5 μM. A significant decrease in proliferation was found at 10 μM in OUMS27 and at 25 μM in SW1353 and CH2879 compared to DMSO controls. For the present study we assessed the (prophylactic) effect of celecoxib on chondrosarcoma growth in vivo using a xenograft model of immunoincompetent nude mice which were injected with cell line CH2879 subcutaneously. Tumour volume was measured during 8 weeks. Celecoxib serum levels were determined by HPLC. Expression of proliferation marker Ki-67 and Cox-2 was assessed by IHC. Our in vivo results also showed a beneficial effect of high dose prophylactic celecoxib treatment. Tumour volumes were negatively correlated with celecoxib serum levels (r2=0.152). However, at the end of pubertal growth of the mice, a catch-up tumour growth was observed, resulting in the absence of differences in tumour volume between control and treatment groups. Accordingly, proliferation marker Ki67 was higher expressed in the treated groups at sacrifice. This suggests that there is no role for celecoxib in the treatment of adult chondrosarcoma patients. Celecoxib treatment of younger patients, especially to prevent formation of new tumours in EC and OC patients, might be beneficial, however more research is necessary

As our understanding of hip function and disease improves, it is evident that the acetabular fossa has received little attention, despite it comprising over half of the acetabulum’s surface area and showing the first signs of degeneration. The fossa’s function is expected to be more than augmenting static stability with the ligamentum teres and being a templating landmark in arthroplasty. Indeed, the fossa, which is almost mature at 16 weeks of intrauterine development, plays a key role in hip development, enabling its nutrition through vascularization and synovial fluid, as well as the influx of chondrogenic stem/progenitor cells that build articular cartilage. The pulvinar, a fibrofatty tissue in the fossa, has the same developmental origin as the synovium and articular cartilage and is a biologically active area. Its unique anatomy allows for homogeneous distribution of the axial loads into the joint. It is composed of intra-articular adipose tissue (IAAT), which has adipocytes, fibroblasts, leucocytes, and abundant mast cells, which participate in the inflammatory cascade after an insult to the joint. Hence, the fossa and pulvinar should be considered in decision-making and surgical outcomes in hip preservation surgery, not only for their size, shape, and extent, but also for their biological capacity as a source of cytokines, immune cells, and chondrogenic stem cells.

Cite this article: Bone Joint Res 2020;9(12):857–869.

Aseptic loosening is a growing problem for orthopaedic surgeons and the importance of elevated hydrostatic pressure in its development in vivo is now well documented, but the mechanisms by which pressure could enhance loosening are unclear. We have demonstrated that hydrostatic pressures increased MP synthesis of cytokines, chemokines, PGE2 and M-CSF in vitro, all of which are implicated in bone resorption. 1,25-dihydroxy vitamin D3 (1,25D3) has a pivotal role in bone resorption. It stimulates osteoclastic bone resorption and formation, causes fusion of committed osteoclast precursor cells and activates mature osteoclasts in vitro. Under the correct conditions, macrophages (MP) have the ability to differentiate into osteoclasts. Research has shown that MP can synthesise 1,25D3 and changes in this synthesis occur during MP differentiation. We therefore examined how the application of hydrostatic pressure to MP in vitro influenced their synthesis of 1,25D3. In this study, normal human peripheral blood MP (5x105/ml) were cultured for 7 days then exposed to physiological pressure (34.5x10-3MPa) and/or UHMWPE particles (8mg/ml) and the effect on synthesis of 1,25D3 by endogenous 1a-hydroxylase (1aOHase) was studied. MP were incubated with H3-25, hydroxy vitamin D and 1,25D3 synthesis was analysed by HPLC. 1,25D3 synthesis was increased in cells under pressure by an average of 17% compared to static controls. In situ hybridisation (ISH) was used to demonstrate expression of 1aOHase. Image analysis showed a small increase in 1aOHase mRNA in response to pressure and to particles, and a larger increase to the two stimuli simultaneously. Expressed as % of maximum +Pressure + Particles 100%;+ Particles 59%; +Pressure 37%; No Stimulus < 0.1%. These results suggest that 1,25D3 may be one of the factors which stimulates osteoclastic bone resorption in aseptic loosening. As both these stimuli are likely to be present in vivo, such synthesis could further exacerbate loosening

Despite improvements in techniques and materials, aseptic loosening of artificial hip joints remains as the most serious problem. This study investigated mechanical and biological effects of biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on prevention of aseptic loosening. To examine mechanical effects of MPC grafting, we performed hip simulator tests (3million cycles) using cross-linked polyethylene (CL-PE) liners with or without nano-grafting of MPC onto articulating surface (MPC liner/CL-PE liner) and PE liner against CoCrMo heads. To examine biological responses of macrophages and osteoblasts, we prepared MPC nanoparticles (500nm). Using in vitro/vivo murine particle-induced osteolysis model, we examined biological effects of MPC nanoparticles on osteoclastogenesis. The friction torque was about 90% lower in MPC liners than control liners. Total amounts of wear produced from MPC liner was about 1/5and 1/30 of those from CL-PE and PE liners, respectively. Three-dimensional analysis and SEM analysis of MPC liners revealed no or little wear. The effect of MPC nanografting was maintained even after the test, because XPS analysis confirmed the remainder of specific spectra of MPC on the liner surface. When nanoparticles were exposed to cultured mouse macrophages, MPC nano particles were hardly phagocytosed by macrophages and did not enhance the concentration of bone resorptive cytokines and PGE2. Furthermore, culture medium of macrophages exposed to MPC nanoparticles did not induce RANKL expression in osteoblasts and osteoclastogenesis from bone marrow cells. In vivo murine osteolysis model, particle-induced bone resorption was hardly observed in mice implanted MPC nanoparticles. Some medical devices grafted MPC onto itssurface have been already used under authorization of the FDA. This study demonstrated that MPC grafting markedly decreases wear production. In addition, even if wear particles are produced, they are biologically inert in respect to phagocytosis by macrophages and subsequent resorptive actions, suggesting an epochal improvement of artificial hip joints preventing aseptic loosening

Objectives: The replacement tissue used for anterior cruciate ligament reconstruction undergoes extensive biologic remodelling and incorporation after implantation. These changes, in which the tendon loses some of its characteristic features and adopts those typically associated with ligaments, has been referred to as ligamentization. The purpose of this study was to identify the proinflammatory response in the healing graft in the early phase. Methodes: Twenty New Zealand White Rabbits underwent ACL reconstruction with a semitendinosus tendon. Animals were sacrificed at 3 and 6 weeks. The harvested tissue including parts of remaining grafted tendon and genuine anterior cruciate ligament at time of the surgery as well as the tendon graft withdrawn at sacrification were prepared for immunohistochemical, histomorphometry and electromicroscopical analysis; synovia samples were taken at the sacrification as well. The tissues were immunostained for IL-1beta, TGF-beta, TNF-alpha (induction of inflammatory cascade), COX-2 (mediator of inflammatory response), Matrix Metalloproteinases (MMP-1, MMP-3, MMP-13, matrix destructive enzymes), TIMP-2 (Tissue Inhibitor of MMPs); the PGE2 (mediator of inflammatory response) content in the synovia was quantified by ELISA. Results: At 3 weeks after surgery the COX-2+ cells accounted for 70% of all cells present in the graft tissue, and decreased to 28% at 6 weeks. Similar, IL-1beta+ cells within the tendon decreased from week 3 to week 6. Controversly, there was an increase of COX-2, IL-1beta and MMP-1 in the intercellular tissue. The numbers of COX-2+ cells and IL-1beta+ cells at 3 weeks as well as the intercellular area stained positiv for COX-2, IL-1beta and MMP-1 at 6 weeks were significantly larger compared to the genuine ACL (p =< 0.05). At 3 weeks some cells stained positiv for MMP-3 and MMP-13, but not at 6 weeks. There was a slight pericellular staining for TIMP-2 at 3 weeks. TGF-beta+ cells and TNF-alpha+ cells were almost not detectable at every time point. Thus, proinflammatory cytokines and MMPs were synthesized in the early phase after ACL reconstruction by the tendon cells and accumulated at 6 weeks in the intercellular tissue. Conclusions: In the early phase of the graft healing after ACL reconstruction, there was a signifikant increase in proinflammatory cytokines and matrix destructive enzymes in the tendon graft. With the capability of synthesizing cytokines, tendon cells may play a critical role in tendon healing at early time points. Facing the widespread use the bias of cox-2 inhibitors on these immunologic processes has to be checked. Activating matrix destructive enzymes, cytokines appear to be crucial for connective tissue remodelling and graft stability after ACL reconstruction

Aims

The aim of this study was to systematically review the literature for evidence of the effect of a high-fat diet (HFD) on the onset or progression of osteoarthritis (OA) in mice.

Osteolysis and periprosthetic bone loss have been a concern since Charnley’s original reports of metal on Teflon. Willart and Semlitch were the first investigators to propose a biologic mechanism for osteolysis associated with particulate wear debris. Harris in 1976 and Goldring 1983 describe the presence of macrophages and giant cells in the synovial membrane at the bone cement interface in loose THR. Initially it was associated with cement and it was called cement disease. Reports of resorption around cementless implants led to the realization that PE alone was good enough to create bone loss. Aetiology: Submicron wear particles are phagocytosed by macrophages resulting in release of various cellular mediators from these activated cells. Cellular mediators playing significant role in osteolysis are IL-1, IL-6, TNF-a, PGE2. These mediators lead to stimulation and differentiation of osteoclasts and inhibition of osteoblasts. These factors together assist in the dissolution of bone at the interface allowing for micromotion of the prosthesis that leads to further generation of wear debris. On top of the above there is release of collagenase, stromelysin, gelatinase which further destroy the bone. Another active area of research involves roles at Rank, Rank and osteoprotegerin. Recently there is extensive work done as far as it concerns the role of endotoxin in osteolysis and periprosthetic bone loss. It still remains a controversial issue. Other researchers have studied the effects of elevated periarticular hydrostatic pressure and fluid access in the development of osteolysis (effective joint space). Particles bioreactivity: It has been shown that the major determinants of particle bioreactivity are particle size, composition, shape, and concentration. Particles of submicron size are more stimulatory and there is a dose dependent response. Concerning the composition it has been found that UHMWPE, CoCr and stainless steel particles induce more severe reactions than Titanium and alumina ceramic. It also has been found that Al2O3 particles were more easily phagocytosed than UHMWPE at the same size and concentration but TNF-a release was higher with than UHMWPE with Al2O3. Concerning the metal to metal particles it has been found that the volumetric wear is less than M/P with smaller particles and less intensive tissue reaction but Shanbhag reported that bioreactivity of metal wear debris is a function of the total surface area and not the volume of wear debris and casts doubts at the theory that metal to metal wear particles produce a less intense biological response. Concerning the highly crosslinked PE it has been found that wear debris from gamma crossed –linked remelted PE contains very few fibrils after a dose of 5 Mrads and virtually none after 9.5 Mrads. Clinical Manifestations: The majority of patients with osteolysis are asymptomatic. Pain is caused mainly from a fracture.Ultimately periprosthetic bone loss results in aseptic loosening. Furthermore if the component becomes loose bone loss often progress more rapidly resulting in large bone defects that can lead to catastrophic failure or fracture. Radiographic manifestations: Characteristic radiographic patterns of osteolysis have been described on both the femoral and acetabular side with cemented and cementless components. Recent studies have suggested that plain radiographs often underestimate the extent of osteolysis and CT or MRI may be necessary to assess the true extent of the bone loss

Total joint arthroplasty is the most significant advance in the treatment of end-stage arthritic disease of major joints. Despite the clinical success of this surgical procedure, however, some total joint prostheses fail, and although a failed prosthesis can be replaced, the results of revision arthroplasty are not as good as the first time. Studying the failed prosthesis and the associated bone and soft tissues provides insight into the causes of failure. Most prosthetic failures are the result of structural limitations of the implant components. Although material failure may be sudden, a much more common cause is gradual aseptic loosening of the prostheses. Aseptic loosening is caused by both mechanical (gradual loss of material by wear) and biological (osteoclastic resorption of adjacent bone) factors. Wear particles induce a foreign body reaction characterized by a pseudomembrane composed of granulomatous tissues including macrophages, fibroblasts, giant cells, and osteoclasts in addition to debris particles. The extent of this response is driven by the number, size, composition, surface area, and types of particles present. Although there are differences in the relative local toxicity of each of these particles, the end result is the same. These mechanical and biological factors are unavoidable, and the success of a total joint prosthesis depends on the rate with which they occur. Polyethylene wear particles (1–200 ?) are the primary cause of loosening. They are strongly birefringent under polarized light microscopy. Smaller particles are phagocytized by histiocytes, whereas larger particles are surrounded by foreign body giant cells. Fragmentation of PMMA may also cause particulate debris. The presence of these particles (30–100 ?) may be deduced by empty spaces into the soft tissues, often bordered by foreign body giant cells, since PMMA is dissolved by xylene during routine histological techniques. Metal oxides form on the surface of chrome-cobalt or titanium alloys due to an electrolytic process, and stresses on the surface of the metal shear the oxides into the surrounding tissues, causing a black pigmentation of the tissues. Histologically, the black deposits of oxidized metals are seen extracellularly as well as in the cytoplasm of histiocytes. In addition to oxidation, metal undergoes corrosion and, as a result, metal ions enter the soft tissues and the bloodstream. A ceramic-on-ceramic coupling generates a significantly lower amount of debris as compared to the conventional metal-on polyethylene solution. When present, ceramic debris cause a mild histiocytic reaction without giant cells and virtually no osteoclastic bone resorption. There are various secretory proteins at the interfacial membrane that can affect bone turnover, including the cytokines IL-1, IL-6, Il-10, and TNF-a. Other factors involved with bone resorption include the enzymes responsible for catabolism of the organic component of bone, such as MMPs. Prostaglandins, in particular PGE2, are also known to be important intercellular messengers in the osteolytic cascade. More recently, several mediators known to be involved in stimulation or inhibition of osteoclast differentiation and maturation, such as RANKL and osteoprotegerin, have been suggested as key factors in the development and progression of osteolysis. Infection around a prosthesis also causes loosening in approximately 1–5% of cases. Total joint prostheses become infected by two mechanisms, wound contamination at the time of surgery by Staph. aureus or Staph.epidermidis, and late hematogenous spread of organisms (Staphylo- and Streptococci, E. Coli, Pseudomonas, and anaerobes). The following factors facilitate bacterial growth. First, reaming and sawing, as well as PMMA polymerization, cause necrosis of necrotize bone adjacent to the implant, and such nonvascularized area permits bacteria to grow, safe from circulating host defenses. Second, a highly hydrated matrix of extracellular polymeric substances (biofilm) is formed that defends bacteria from antibiotics and phagocytosis. Third, some metals, such as nickel or cobalt, may depress macrophage function. The distinguishing histologic features of an infected prosthesis is an acute inflammatory reaction: a finding of > 5 PMN or of > 50 lymphocytes/hp field are presumptive for infection. Because some low-grade infections fail to stimulate an acute inflammatory reaction, they go undiagnosed until postoperative period when microbacterial culture results are available. To date, no single routinely used clinical or laboratory test has been shown to achieve ideal sensitivity and specificity for the diagnosis of prosthetic joint infection, and in most cases the diagnosis depends on a combination of clinical features, radiographic findings, and laboratory results. Intra-operative tissue cultures may be falsely negative because of prior antimicrobial exposure, a low number of organisms, inappropriate culture media, or atypical organisms. Conversely, cultures may be falsely positive because of contamination in the operating room, during transport, or in the laboratory. If the implant is removed, the entire prosthesis can be cultured. Moreover, because prosthetic joint infection is a biofilm-mediated infection, techniques that sample bacteria in biofilm, such as sonication or enzymatic treatment, may improve the diagnosis of prosthetic joint infection. More recently, molecular techniques are being used to detect nucleic acid in samples from infected patients even when conventional techniques are negative because of unusual microbial growth requirements or failure to grow after antimicrobial exposure or due to unfavourable environmental conditions. A disadvantage of such approach is its extreme sensitivity, leading to the possibility of false positive results. The clinical presentation of prosthetic joint infection may be indistinguishable from that of aseptic implant failure. In many cases, culture of granulomatous tissue around failed prostheses, preoperatively diagnosed as aseptically loosened, reveals the presence of bacteria that may per se significantly contribute to the recruitment, maturation and activation of osteoclasts and that superimpose to the foreign body reaction to wear debris. The presence of a smouldering infection in case of “aseptic” failure observed in revision arthroplasties. A systematic investigation on all retrieved implants is mandatory to define the precise role of each potential factor contributing to the pathogenesis of failure, in order to further improve the quality of care of patients having total joint arthroplasty

Objectives

Osteoarthritis (OA) is the most common form of arthritis, affecting approximately 15% of the human population. Recently, increased concentration of nitric oxide in serum and synovial fluid in patients with OA has been observed. However, the exact role of nitric oxide in the initiation of OA has not been elucidated. The aim of the present study was to investigate the role of nitric oxide in innate immune regulation during OA initiation in rats.

Osteoarthritis (OA) is an important cause of pain, disability and economic loss in humans, and is similarly important in the horse. Recent knowledge on post-traumatic OA has suggested opportunities for early intervention, but it is difficult to identify the appropriate time of these interventions. The horse provides two useful mechanisms to answer these questions: 1) extensive experience with clinical OA in horses; and 2) use of a consistently predictable model of OA that can help study early pathobiological events, define targets for therapeutic intervention and then test these putative therapies. This paper summarises the syndromes of clinical OA in horses including pathogenesis, diagnosis and treatment, and details controlled studies of various treatment options using an equine model of clinical OA.

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis.

In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition.

Cite this article: Bone Joint J 2013;95-B:1021–5.

The incidence of acute and chronic conditions of the tendo Achillis appear to be increasing. Causation is multifactorial but the role of inherited genetic elements and the influence of environmental factors altering gene expression are increasingly being recognised. Certain individuals’ tendons carry specific variations of genetic sequence that may make them more susceptible to injury. Alterations in the structure or relative amounts of the components of tendon and fine control of activity within the extracellular matrix affect the response of the tendon to loading with failure in certain cases.

This review summarises present knowledge of the influence of genetic patterns on the pathology of the tendo Achillis, with a focus on the possible biological mechanisms by which genetic factors are involved in the aetiology of tendon pathology. Finally, we assess potential future developments with both the opportunities and risks that they may carry.

Cite this article: Bone Joint J 2013;95-B:305–13.