Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839

Aims. This study aimed to determine the expression and clinical significance of a cartilage protein, cartilage oligomeric matrix protein (COMP), in knee osteoarthritis (OA) patients. Methods. A total of 270 knee OA patients and 93 healthy controls were recruited. COMP messenger RNA (mRNA) and protein levels in serum, synovial fluid, synovial tissue, and fibroblast-like synoviocytes (FLSs) of knee OA patients were determined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunohistochemistry. Results. COMP protein levels were significantly elevated in serum and synovial fluid of knee OA patients, especially those in the advanced stages of the disease. Serum COMP was significantly correlated with radiological severity as well as measures of body composition, physical performance, knee pain, and disability. Receiver operating characteristic curve analysis unveiled a diagnostic value of serum COMP as a biomarker of knee OA (41.64 ng/ml, area under the curve (AUC) = 1.00), with a sensitivity of 99.6% and a specificity of 100.0%. Further analysis uncovered that COMP mRNA expression was markedly upregulated in the inflamed synovium of knee OA, consistent with immunohistochemical staining revealing localization of COMP protein in the lining and sub-lining layers of knee OA inflamed synovium. Most notably, relative COMP mRNA expression in knee OA synovium was positively associated with its protein levels in serum and synovial fluid of knee OA patients. In human knee OA FLSs activated with tumour necrosis factor-alpha, COMP mRNA expression was considerably up-regulated in a time-dependent manner. Conclusion. All results indicate that COMP might serve as a supportive diagnostic marker for knee OA in conjunction with the standard diagnostic methods. Cite this article: Bone Joint Res 2024;13(6):261–271

Background. The anterior approach for total hip arthroplasty has recently been hypothesized to result in less muscle damage. While clinical outcome studies are essential, they are subject to patient and surgeon bias. We prospectively analyzed biochemical markers of muscle damage and inflammation in patients receiving anterior and posterior minimally-invasive total hip arthroplasty to provide objective evidence of the surgical insult. Methods. 29 patients receiving an anterior and 28 patients receiving a posterior total hip arthroplasty were analyzed. Peri-operative and radiographic data were collected to ensure similar cohorts. Creatine kinase, C-reactive protein, Interleukin-6, Interleukin-1beta, and Tumor necrosis factor-alpha were collected pre-operatively, post-operatively, and on post-operative days 1 and 2. Comparisons between the groups were made using the Student's t-test and Fisher's Exact test. Independent predictors of elevation in markers of inflammation and muscle damage were determined using multivariate logistic regression analysis. Results. Markers of inflammation were slightly decreased in direct anterior group (mean differences in C-reactive protein 27.5 [95% confidence interval −24.7–79.6] mg/dL, Interleukin-6 13.5 [95% confidence interval −11.5–38.4] pg/ml, Interleukin-1beta 42.6 [95% confidence interval −10.4–95.6], and Tumor necrosis factor-alpha 148.6 [95% confidence interval −69.3–366.6] pg/ml). The rise in creatine kinase was 5.5 times higher in the post anesthesia care unit (mean difference 150.3 [95% confidence interval 70.4–230.2] units/L, p < 0.05) and nearly twice as high cumulatively in the miniposterior approach group (305.0 [95% confidence interval −46.7–656.8] units/L, p < 0.05). Conclusion. Anterior total hip arthroplasty caused significantly less muscle damage compared to traditional posterior surgery as indicated by creatine kinase levels. The clinical importance of this rise needs to be delineated by further clinical studies. The overall physiologic burden as measured by markers of inflammation, however, appears to be similar. Objective measurement of muscle damage and inflammation provides an unbiased way of determining the immediate effects of surgical intervention in total hip arthroplasty patients

Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development

Introduction Bone morphogenetic protein-7 (BMP-7) is known to stimulate both cellular proliferation and extracellular matrix synthesis in the intervertebral disc but its protective role in apoptosis is unknown. The aim of this study was to determine whether BMP-7 protect cultured intervertebral disc cells following stimulation of apoptosis. Methods Nucleus pulposus tissues were obtained from consent individuals under surgical procedures and digested with collagenase prior to culturing. Cellular apoptosis was achieved by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide (H2O2) incubation. BMP-7 (Stryker) was used at 100ng/ml, 5 hours prior to the addition of apoptotic stimulation. Cellular apoptosis was detected by TUNEL assay, caspase-3 activity and caspase-3 protein expression. Cellular proliferation and viability was assayed by H3-thymidine incorporation and MTS assay respectively. Collagen II and aggrecan protein levels were measured using western blots and immunostaining. Proteoglycan synthesis was determined by (35)S-sulfate incorporation method. Nitric oxide and alkaline phosphatase activity were measured. Results Both extrinsic and intrinsic apoptotic pathways were induced by TNF-β or hydrogen peroxide with increased proteolytic activity of caspase-3 as well as cellular shrinkage and nuclear condensation. Addition of BMP-7 prior to stimulation of apoptosis resulted in complete block of the apoptotic effects of both inducers as well as the cellular nitric oxide induced by TNF-β and BMP-7 increases cellular viability, proliferation and extracellular matrix production in an apoptotic environment with no osteoblastic activity induction of discal cells. Discussion BMP-7 prevents apoptosis of cultured human disc cells induced by either tumor necrosis factor-alpha (TNF-β) or hydrogen peroxide. Induction of apoptosis led to down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3, however addition of BMP-7 alone prevented the effects observed. One possible mechanism of the anti-apoptotic effects of BMP-7 was shown by its retardation of the elevated levels of TNF-β induced nitric oxide

Abstract. BACKGROUND. Cell culture on tissue culture plastic (TCP) is widely used across biomedical research to understand the in vivo environment of a targeted biological system. However, growing evidence indicates that the characteristics of cells investigated in this way differ substantially from their characteristics in the human body. The limitations of TCP monolayer cell cultures are especially relevant for chondrocytes, the cell population responsible for producing cartilage matrix, because their zonal organization in hyaline cartilage is not preserved in a flattened monolayer assay. OBJECTIVE. Here, we contrast the response of primary human chondrocytes to inflammatory cytokines, tumor necrosis factor-alpha and interferon-gamma, via transcriptional, translational, and histological profiling, when grown either on TCP or within a 3D cell pellet (scaffold-less). We focus on anti-apoptotic (Bcl2), pro-apoptotic (Bax, Mff, Fis1), and senescent (MMP13, MMP1, PCNA, p16, p21) markers. RESULTS. We find that the 3D environment of the chondrocyte has a profound effect on the behavior and fate of the cell; in TCP monolayer cultures, chondrocytes become anti-apoptotic and undergo senescence in response to inflammatory cytokines, whereas in 3D cell pellet cultures, they exhibit a pro-apoptotic response. CONCLUSION. Our findings demonstrate that chondrocyte culture environment plays a pivotal role in cell behavior, which has important implications for the clinical applicability of in vitro research of cartilage repair. Although there are practical advantages to 2D cell cultures, our data suggest researchers should be cautious when drawing conclusions if they intend to extrapolate findings to in vivo phenomena. Our data demonstrates opposing chondrocyte responses in relation to apoptosis and senescence, which appear to be solely reliant on the environment of the culture system. This biological observation highlights that proper experimental design is crucial to increase the clinical utility of cartilage repair experiments and streamline their translation to therapy development. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Aims. Rheumatoid arthritis (RA), which mainly results from fibroblast-like synoviocyte (FLS) dysfunction, is related to oxidative stress. Advanced oxidation protein products (AOPPs), which are proinflammatory mediators and a novel biomarker of oxidative stress, have been observed to accumulate significantly in the serum of RA patients. Here, we present the first investigation of the effects of AOPPs on RA-FLSs and the signalling pathway involved in AOPP-induced inflammatory responses and invasive behaviour. Methods. We used different concentrations of AOPPs (50 to 200 µg/ml) to treat RA-FLSs. Cell migration and invasion and the expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), and MMP-13 were investigated. Western blot and immunofluorescence were used to analyze nuclear factor-κB (NF-κB) activation. Results. AOPPs promoted RA-FLS migration and invasion in vitro and significantly induced the messenger RNA (mRNA) and protein expression of TNF-α, IL-6, MMP-3, and MMP-13 in dose- and time-dependent manners. Moreover, AOPPs markedly activated the phosphorylation of nuclear factor-κB (NF-κB) p65 protein, which triggered inhibitory kappa B-alpha (IκBα) degradation, NF-κB p65 protein phosphorylation, and NF-κB p65 translocation into the nucleus. Furthermore, treatment with a neutralizing antibody specific to receptor for advanced glycation end products (RAGE) significantly suppressed aggressive behaviour and inflammation, decreased TNF-α, IL-6, MMP-3, and MMP-13 expression, and blocked AOPP-induced NF-κB pathway activation. Conclusion. The results indicate that AOPPs can enhance aggressive behaviour and the inflammatory response in RA-FLSs via the RAGE–NF-κB pathway. These results present AOPPs as a new class of potentially important mediators of progressive disease in RA patients. Cite this article: Bone Joint Res 2021;10(4):259–268

It has been previously shown that Low-Magnitude High-Frequency Vibration (LMHFV) is able to enhance ovariectomy-induced osteoporotic fracture healing in rats. Fracture healing begins with the inflammatory stage, and all subsequent stages are regulated by the infiltration of immune cells such as macrophages and the release of inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10). Therefore, the aim of this study was to investigate the effect of LMFHV treatment on the inflammatory response in osteoporotic fracture healing. In this study, ovariectomy-induced osteoporotic and sham-operated closed-femoral fracture SD-rats were randomized into three groups: sham control (SHAM), ovariectomized control (OVX-C) or ovariectomized vibration (OVX-V) (n=36, n=6 per group per time point). LMHFV (35Hz, 0.3g) was given 20 min/day and 5 days/week to OVX-V group. SHAM operation and ovariectomy were performed at 6-month and closed femoral fracture was performed at 9-month. Callus morphometry was determined by callus width from weekly radiography. Local expressions of inducible nitric oxide synthase (iNOS) (macrophage M1 marker), CD206 (macrophage M2 marker), TNF-α, IL-6 and IL-10 were detected by immunohistochemistry and quantified by colour threshold in ImageJ, assessed at weeks 1 and 2 post-fracture. Significant difference between groups was considered at p≤0.05 by one-way ANOVA. Callus formation was higher in OVX-V than that of OVX-C as shown by callus width at weeks 1 and 2 (p=0.054 and 0.028, respectively). Immunohistochemistry results showed that CD206 positive signal and the M2/M1 ratio which indicates the progression of macrophage polarization were significantly higher in OVX-V rats (p=0.053 and 0.049, respectively) when compared to OVX-C at week 1. Area fraction of TNF-α positive signal was significantly higher in SHAM and OVX-V rats at week 1 (p=0.01 and 0.033, respectively). IL-6 signal was also significantly higher in SHAM and OVX-V groups at week 1 (p=0.004 and 0.029, respectively). IL-10 expression was significantly lower in SHAM and OVX-V groups at week 1 (p=0.013 and 0.05, respectively). Here we have shown that LMHFV treatment promoted the shift from pro-inflammatory stage towards anti-inflammatory stage earlier. It has been reported that the polarization of pro-inflammatory macrophages M1 to anti-inflammatory macrophages M2 was indicative of the endochondral ossification process in the long bone fracture model. Besides, we found that LMHFV treatment enhanced pro-inflammatory markers of TNF-α and IL-6 and suppressed anti-inflammatory marker of IL-10 at week 1, showing that inflammatory response was enhanced at week 1 post-fracture. These inflammatory cytokines involved in fracture healing were shown to coordinate different fracture healing processes such as mesenchymal stem cell recruitment and angiogenesis. Our previous study has demonstrated that ovariectomized rats exhibit lower levels of inflammatory response after fracture creation. Therefore, we report that LMHFV treatment can modulate macrophage polarization from M1 to M2 at an earlier time-point and partly restore the impaired inflammatory response in OVX bones at the early stage of fracture healing that may lead to accelerated healing of osteoporotic fracture as shown by promoted callus formation

Low back pain is more common in women than men, yet most studies of intervertebral disc (IVD) degeneration do not address sex differences. In humans, there are sex differences in spinal anatomy and degenerative changes in biomechanics, and animal models of chronic pain have demonstrated sex differences in pain transduction. However, there are few studies investigating sex differences in annular puncture IVD degeneration models. IVD puncture is known to result in progressive biomechanical alterations, but whether these IVD changes correlate with pain is unknown. This study used a rat IVD injury model to determine if sex differences exist in mechanical allodynia, biomechanics, and the relationship between them, six weeks after IVD injury. Procedures were IACUC approved. 24 male & 24 female four-month-old Sprague-Dawley rats underwent a sham or annular puncture injury surgery (n=12 male, 12 female). In injury groups, three lumbar IVDs were each punctured three times with a needle, and injected with tumor necrosis factor-alpha. Mechanical allodynia was tested biweekly using von Frey filaments. Six weeks after IVD injury, rats were euthanized and motion segments were dissected for non-destructive axial tension-compression and torsional rotation biomechanical testing. Two-way ANOVA with Bonferroni corrections identified statistically significant differences (p < 0 .05) and correlations used Pearson's coefficient. Annular puncture injury induced a significant increase in mechanical allodynia compared to sham in male but not female rats up to six weeks after injury. There was a significant sex effect on both torque range and torsional stiffness, with males exhibiting greater stiffness and torque range than females. Tensile stiffness, compressive stiffness, and axial range of motion showed no sex difference. Males and females showed similar patterns of correlation between variables when sham and injury groups were analyzed together, but correlations were stronger in males. Most correlations were clustered within testing approach: axial biomechanics negatively correlated, torsional biomechanics positively correlated, and von Frey thresholds positively correlated. Surprisingly, mechanical allodynia did not correlate with any biomechanics after injury, and the axial and torsional biomechanics showed little correlation. This study demonstrates that males and females respond to IVD injury differently. Given the absence of correlation between pain and biomechanics, pain cannot be attributed completely to biomechanical changes. This may explain why spinal fusion surgery, an intervention limited to the spine, has produced inconsistent results and is controversial for patients with low back pain. Thus, in addressing low back pain, we must consider both spinal tissues and the nervous system. Further, the limited correlation between axial and torsional biomechanics indicates that IVD injury may have distinct effects on nucleus pulposus and annulus fibrosus. Biomechanics did not differ between sham and injury at week six, suggesting healing after injury. It remains possible that acute biomechanical changes may initiate chronic pain pathogenesis. We conclude that the observed sex differences demonstrate the need for inclusion of both males and females in IVD injury and pain studies, and suggest that males and females may require different treatments for conditions that appear similar

Background. Osteoarthritis (OA), a common degenerative disorder of synovial joints, is characterized by disruption of the extracellular matrix (ECM) homeostasis with an overall misbalance towards cartilage catabolism. Integrins are alpha/beta heterodimeric transmembrane proteins transmitting chemical and biomechanical signals into the cells. There is a growing consensus that changes of ECM composition by proteolytic degradation of matrix constituents, or alteration of the biomechanical microenvironment of chondrocytes caused by chronic stress or injury significantly increase the risk of OA through the perturbation of integrin signaling. In order to further investigate the role of the b1 integrin subfamily in OA, we have challenged hip cartilage explants dissected for mice lacking beta1 integrins in chondrocytes by cytokines, ECM degradation products or mechanical stimulation. Methods. Femoral articular cartilages were avulsed from hip joints of 6 weeks old wild type (WT) and b1fl/fl-PrxCre mutant (MT) mice. For the chemically-induced OA-like stimulation, femoral caps were cultured for 3 days in serum-free DMEM/F12 with or without the supplementation of interleukin-1a (IL1a), 120kDa cell-binding fibronectin fragments (120FNf), or tumor necrosis factor-alpha (TNFa) + oncostatin M (OM). Sulphated glycosaminoglycan (sGAG) release of the explants were measured in the supernatants by the 1,9-dimethylmethlene blue (DMMB) assay. Proteoglycan loss was monitored by Safranin-O (SO) staining on cryo-sections of the explants. For the cartilage injury model, avulsed femoral caps were either directly snap-frozen or kept in serum-free DMEM/F12 for 4 hours before snap-freezing. Gene expression changes were analyzed by quantitative RT-PCR using a pre-determined set of genes regulated by injury. Results. Articular cartilages of MT mice were found to have consistently higher release of GAGs when exposed to cytokines or 120FNf. IL-1a exerted the highest catabolic stimulation. The ex vivo biochemical analysis was further verified by SO staining demonstrating more pronounced proteoglycan loss on MT sections compared to WT. Assessing the mRNA of articular cartilages subjected to the injury model, revealed expression changes in genes which have been previously implicated in OA: Il1a (interleukin 1, alpha) and Ptgs2 (prostaglandin-endoperoxide synthase 2) were upregulated in MT mice; whereas Il1rl1 (interleukin 1 receptor-like 1) and Nos2 (nitric oxide synthase 2) expression levels were significantly reduced in MT compared to WT. Conclusion. The data imply that b1 integrins play a protective role against cytokine- and fibronectin fragment-induced cartilage degradation. Our findings also suggest that b1 integrins modulate the expression of catabolic factors upon mechanical insults

Summary. RNAi targeting p110β reduces TNF-alpha production and osteolysis in response to wear particles. Introduction. Aseptic joint loosening is a key factor that reduces the life span of joint prosthesis. Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aims to explore the effect of p110β-targeted small interfering RNA (siRNA) and lentivirus on particle-induced inflammatory cytokine expression in murine macrophage. Methods. siRNA and lentivirus targeting p110β were transfected and infected prior to particle stimulation, respectively. Ceramic and titanium particles of different sizes were prepared to stimulate macrophages. Fluorescence microscopy showed that the siRNA transfection and lentivirus infection efficiency were 74.2 ± 4.2% and 92.3 ± 2.6%, respectively. Results. Real-time polymerase chain reaction (PCR) showed that the levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups (P<0.05), respectively. Similarly, enzyme-linked immunosorbent assay (ELISA) showed that protein levels of TNF-alpha in RNAi-treated groups were significantly decreased after transfection or infection (P<0.05), respectively. Western Blot showed that Phospho-Akt activation was significantly reduced by RNAi. As assessed by CT and micro-CT, particle implantation induced a significant osteolysis effect in mice calvaria, which was limited by p110β-lentivirus addition. Conclusions. p110β subtype of PI3K, followed by activation of phosphor-AKT (Ser473), may possibly participate in the regulation of activating macrophages by wear particles, ultimately resulting in the secretion of TNF-α and osteolysis

Summary. RNAi targeting TNF-alpha inhibits particle-induced inflammation and osteolysis. Introduction. Over 1000,000 joint prostheses are implanted every year in the world. Aseptic joint loosening is a key factor that reduces the longevity of joint prosthesis. Prosthetic wear particles are thought to play a central role in the initiation and development of periprosthetic osteolysis, leading to aseptic loosening of prostheses. This study aims to investigate the effect of RNA interference (RNAi) targeting tumor necrosis factor-alpha (TNF-α) gene on particle-induced inflammation and osteolysis in macrophages in vitro and in vivo. Methods. An in vitro-transcribed small interfering (siRNA) sequences targeting mouse TNF-alpha gene from four candidates was screened and identified and then a lentivirus vector expressing short hairpin RNA (shRNA) was constructed to allow an efficient expression of TNF-alpha-siRNA. Lentivirus-mediated shRNA was transduced into mouse macrophages cells line RAW 264.7. Ceramic particles and titanium particles were added to stimulate cells 24 h after lentivirus transduction. TNF-alpha expression at both mRNA and protein levels were detected quantitatively by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) at different time intervals. Lentivirus-mediated shRNA suspension was locally administered into the murine calvarial model following local particle injection. Multi-slice spiral CT scan was used to evaluate the osteolysis of calvaria by detecting the width of the cranial sutures. Results. Lentivirus-mediated shRNA was effectively transfected, and inhibited the expression of TNF-alpha both in mRNA and protein level in RAW 264.7. Multi-slice spiral CT scan showed that lentivirus-mediated shRNA significantly suppressed osteolysis of mouse calvaria. Conclusion. Our investigation demonstrated that lentivirus-mediated shRNA targeting TNF-alpha gene could inhibit particle-induced inflammation and osteolysis in vitro and in vivo. Therefore, lentivirus-mediated gene therapy has the potential to be developed into a novel therapeutic strategy in the treatment of aseptic joint loosening

Aims: Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the inflammatory response to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation and we have studied the effect of single nucleotide polymorphisms (SNPs) within the TNF gene. Methods: We performed a case control study of 9 SNPs (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THR. Cases consisted of 44 patients with early aseptic loosening and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that were clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening. DNA was extracted from venous blood and genotyped by Snapshot assay. Results: Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. A significant association was found for the -863 SNP and aseptic loosening (p< 0.05; OR=2.36; 95% CI: 0.976 – 5.71). A trend towards association was found between the -863A SNP and deep infection (p=0.80; OR=2.42; CI: 0.800 – 7.34). Conclusions: Genetic polymorphism of TNF-alpha may play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment

Endochondral ossification involves a well ordered sequence of cellular events. Chondrocytes change their morphology and functions and are ultimately removed by the process of apoptosis. A variety of apoptotic-related signals have been characterised. These include Fas receptor (FasR)/Fas ligand (FasL), p53 and Bcl family. However, there is little known regarding the activity of these signals in the process of fracture healing. The purpose of this study was to investigate mRNA expression of apoptotic signals using RNase protection assay (RPA) and immunohistochemistry in endochondral bone formation. BALB/C mice aged 8 to 10 weeks were used for this study. First, a transverse fracture was made in the right tibia. Mice were euthanised at 1, 2 and 3 weeks postfracture. The calluses were harvested and studied for the expression of caspase-8, a key enzyme of apoptosis, and apoptosis inducers: tumour necrosis factor-alpha (TNF-α) and its receptor p55, FasL and Fas receptor (FasR), and TNF-related apoptosis-inducing ligand (TRAIL). Four mice at each timepoint were used for immunostaining of fracture callus. Sections were incubated with primary antibody then labelled by avi-din-biotin complex method. Another four to ten tibiae were used for RPA. Fracture callus were harvested and snap frozen in liquid nitrogen. RNA was isolated by TRI reagent and BCP, and mRNAs expression of apoptotic signals were detected. At each timepoint, mRNA of caspase-8, TNF-α, p55, FasL,FasR and TRAIL were detected by RPA. Immunostainings clearly showed that those apoptotic-related proteins were expressed by callus chondrocytes. Cartilaginous callus is replaced by woven bone in endochondral ossification. In this process, chondrocytes should be removed by the process of apoptosis in which death factors are elaborated directly in both an autocrine and paracrine manner

Sepsis and multiple-organ failure are common sequelae of multiple trauma. Although sepsis is considered to result from bacteria translocating through the gut mucosa, evidence for that is lacking. In order to define the chronic involvement of bacterial translocation, fracture was induced after crushing of the right femor on its mid in 11 rabbits. Blood was collected at regular time intervals for quantitative culture and for estimation of endotoxins (LPS) by the QCL LAL-assay, tumor necrosis factor-alpha (TNFalpha) by a bioassay in L929 fibrosarcoma cell line and malondialdehyde (MDA) by HPLC. After death, segments of liver, lung and spleen were cut for quantitative culture. Mean +/−SE of the log10 of viable cells in blood were 2.48 +/− 0.43, 3.16 +/− 0.46, 2.77 +/− 0.69 and 2.12 +/− 0.43 at 2, 4, 24 and 48 hours after fracture. Respective values for LPS were 1.50 +/−0.29, 1.54 +/− 0.44, 1.17 +/− 0.17 and < 1.00; for MDA 3.57 +/− 0.55, 7.50 +/− 3.00, 15.77 +/− 12.26 and 5.07 +/− 2.18 μM; and for TNFalpha 11.8 +/− 1.2, 36.7 +/− 25.9, 40.7 +/− 24.0 and 56.8 +/− 45.3 pg/ml. Positive tissue cultures for Serratia marscecens and Pseudomonas aeruginosa were found for six rabbits. Median survival for animals drawn positive tissue cultures was 1.00 days and 7.00 days for animals with negative tissue cultures (p: 0.0092). It is concluded that bacterial translocation is a process occurring early in a significant percentage in the field of multiple trauma. Its occurrence is accompanied by rapid progression to death. Further research is mandatory to clarify the reasons favoring that process in certain hosts compared to others

Introduction: Tumor necrosis factor-alpha (TNF-a) has been shown to be a potent stimulator of bone resorption in vitro and in vivo, and has been identified as an important factor in aseptic loosening of total joint replacements. In order to investigate the effects of TNF-a at the bone-cement interface, we developed a rabbit model in which a slow-release pellet containing a known amount of TNF-a was inserted adjacent to a polymethylmethacryate (PMMA) implant in the distal femur. Methods: 25 male New Zealand white rabbits were used in this IACUC-approved study. After routine exposure of the distal femur, a 3 mm drill bit was used to drill through the intercondylar region into the medullary canal of the distal femur. A resorbable pellet containing 0, 420, 4200, 42 000 or 420 000 pg of TNF-a (n=5 animals per dose level) was inserted into the drill hole, immediately followed by a cylindrical PMMA implant (20 mm long). Animals were euthanized 42 days after surgery. The right femora were excised, radiographed, and processed for histology. Ground sections were prepared at the level of the proximal implant. Semi-automated image analysis was used to quantify cortical bone area, porosity and fractional surfaces (quiescent, osteoid and eroded). Data from control and treatment animals were compared with a one-way analysis of variance (ANOVA) using p< 0.05. Results: All of the animals recovered well after surgery. Radiographically, all of the implants appeared to be stable, with no evidence of linear or cystic osteolysis. Local delivery of TNF-a for 6 weeks had no effect on cortical bone area or porosity. However, TNF-a stimulated bone resorption and decreased new bone formation at the endosteal surface (p< 0.05); these effects were not dose-dependent but were seen in all of the TNF-a groups. Discussion: Our data provide direct evidence that local release of TNF-a is capable of inducing endosteal bone resorption in vivo. Additional studies are now needed to determine the effects of other proinflammatory cytokines in this animal model. However, based on these results, it appears that targeted blockade of TNF-a release or activity may provide a rational therapeutic approach to osteolysis and aseptic loosening

The biological response to implant-derived wear particles is recognized as one of the main factors involved in the development of periprosthetic osteolysis. Wear particles induce a foreign-body inflammatory response that results in the formation of a periprosthetic membrane and progresses over time to aseptic loosening and implant failure. Upon exposure to particles, macrophages and other cell types release inflammatory cytokines to the periprosthetic milieu such as inter-leukin-1 beta (IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) which contribute to bone resorption. Heat shock proteins (HSP) are intra-cellular proteins involved in the maintenance of cellular homeostasis. The stress inducible form of the Hsp70 family protein, Hsp72, has been detected in circulation, acting as a factor capable of regulating pro-inflammatory cytokines secretion and it has been demonstrated that induces the production of pro-inflammatory cytokines via the CD14 and Toll-like receptor-mediated signal transduction pathway. We hypothesized that Hsp72 could be involved in the inflammatory response to wear particles. To this aim, we investigated Hsp72 and its receptor, CD14, in interfacial membrane specimens obtained from patients undergoing revision surgery for aseptic loosening of uncemented acetabular cups (n=7). Distribution of both proteins was assessed by immunofluorescence and examined by confocal laser scanning microscopy. Hsp72 was detected in the periprostehetic membranes, colocalizing with CD14. Explants of membranes were cultured in vitro and levels of Hsp72 and IL-6 were determined by ELISA after 24, 48 and 72 h (n=9). Cultured membranes released IL-6 to culture medium in a time-dependent manner (p< 0.05), while Hsp72 levels decreased during same observation period (p< 0.05). These data suggest that, rather than being produced by the periprosthetic tissue, Hsp72 might be recruited by CD14+ cells from extracellular fluids. In this regard, preliminary data indicated that soluble Hsp72 levels in sera from patients undergoing revision surgery due to aseptic loosening were significantly lower than those from age-matched control subjects (n=6; p< 0.001). To investigate the involvement of Hsp72 in the inflammatory response to wear particles, we used a cell culture model of THP-1 cells driven to the monocyte/macrophage differentiation pathway. These cells were exposed to titanium particles of phagocytosable sizes, either in the presence or absence of exogenously added Hsp72. results obtained to date indicate that Hsp72 is able to modulate the titanium-induced TNF-alpha, IL-1 beta and IL-6 secretion (p< 0.05). Altogether, our data suggest that Hsp72 could be a novel mediator involved in wear particles-induced osteolysis and prosthetic failure

Tumour necrosis factor-alpha is a proinflammatory cytokine that has been implicated in the propagation of inflammatory responses to bacterial infection and wear debris particles around loosened total hip replacements (THR). Individual TNF responses to such stimuli may be dictated by genetic variation. Single nucleotide polymorphisms (SNPs) at several loci within the TNF gene are associated with disease severity and susceptibility in a number of inflammatory conditions, but only a few SNPs have been screened in any one study. 14 SNPs have been identified within the TNF gene. Our unit has previously demonstrated that 5 SNPs are monomorphic in a sample group of UK Caucasians. We performed a case control study of the remaining 9 polymorphic positions (−1031, −863, −857, −376, −308, −238, +489, +851 and +1304) for possible association with deep sepsis or aseptic loosening. All patients included in the study were Caucasian and had had a cemented Charnley THR and polyethylene cup. Cases consisted of 44 patients with early aseptic loosening (defined as that occurring within 6 years of implantation and findings at revision surgery or by the criteria of Hodgkinson et al for the acetabulum and Harris for the femoral stem) and 30 patients with microbiological evidence at surgery of deep infection. Controls consisted of 85 THRs that had remained clinically asymptomatic for over 10 years and demonstrated no radiographic features of aseptic loosening or ‘at risk’ signs as described by Wroblewski et al. DNA was extracted from venous blood and genotyped by Snapshot assay. Genotype and allele frequencies for all SNPs were in Hardy-Weinberg equilibrium between THR controls and a random sample of UK Caucasians. The most significant associations were between the −238A (p< 0.05) and −863T (p< 0.05) alleles and aseptic loosening. A trend towards association was found between the −863A SNP and deep infection (p=0.80). The −238 A/G and −863 G/T genotypes were associated with deep infection (p< 0.05). No other significant associations were found. Genetic polymorphism of TNF appears to play a significant role in THR aseptic loosening and possibly in deep infection. SNP markers may serve as predictors of implant survival and response to therapy such as anti-TNF treatment

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Aims

Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI).