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

Excessive apoptosis has been found in torn supraspinatus tendon1 and mechanically loaded tendon cells2. Following oxidative and other forms of stress, one family of proteins that is often unregulated are Heat Shock Proteins (HSPs). The purpose of this study was to determine if HSPs were unregulated in human and rat models of tendinopathy and to determine if this was associated with increased expression of regulators of apoptosis (cFLIP, Caspases 3& 8).

A running rat supraspinatus tendinopathy overuse model 3 was used with custom microarrays consisting of 5760 rat oligonucleotides in duplicate. Seventeen torn supraspinatus tendon and matched intact subscapularis tendon samples were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from ten patients undergoing arthroscopic stabilisation surgery and evaluated using semiquantative RT-PCR and immunohistochemistry.

Rat Microarray: Upregulation of HSP 27 (×3.4) & 70 (×2.5) and cFLIP (×2.2) receptor was noted in degenerative rat supraspinatus tendon subjected to daily treadmill running for 14 days compared to tendons of animals subject to cage activity only. Histological analysis: All torn human supraspinatus tendons exhibited changes consistent with marked tendinopathy. Matched subscapularis tendon showed appearances of moderate-advanced degenerative change. Apoptosis mRNA expression: The expression levels of caspase 3 & 8 and HSPs 27 & 70 were significantly higher in the torn edges of supraspinatus when compared to matched subscapularis tendon and control tendon (p< 0.01). cFLIP showed significantly greater (p< 0.001) expression in matched subscapularis compared to supraspinatus and control tendon. Immunohistochemical analysis: cFLIP, Caspase 3 & 8 and HSP 27 and 70 was confirmed in all samples of torn supraspinatus tendon. Significantly increased immunoactivity of Caspase 3& 8 and HSP 27 & 70 were found in torn supraspinatus (p< 0.001) compared to matched and normal subscapularis. The proteins were localized to tendon cells.

The finding of significantly increased levels of Heat Shock Proteins in human and rat models of tendinopathy with the co-expression of other regulators of apoptosis suggests that Heat Shock Proteins play a role in the cascade of stress activated-programmed cell death and degeneration in tendinopathy.

Aims. The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789

We undertook a study of the anti-tumour effects of hyperthermia, delivered via magnetite cationic liposomes (MCLs), on local tumours and lung metastases in a mouse model of osteosarcoma. MCLs were injected into subcutaneous osteosarcomas (LM8) and subjected to an alternating magnetic field which induced a heating effect in MCLs. A control group of mice with tumours received MCLs but were not exposed to an AMF. A further group of mice with tumours were exposed to an AMF but had not been treated with MCLs. The distribution of MCLs and local and lung metastases was evaluated histologically. The weight and volume of local tumours and the number of lung metastases were determined. Expression of heat shock protein 70 was evaluated immunohistologically. Hyperthermia using MCLs effectively heated the targeted tumour to 45°C. The mean weight of the local tumour was significantly suppressed in the hyperthermia group (p = 0.013). The mice subjected to hyperthermia had significantly fewer lung metastases than the control mice (p = 0.005). Heat shock protein 70 was expressed in tumours treated with hyperthermia, but was not found in those tumours not exposed to hyperthermia. The results demonstrate a significant effect of hyperthermia on local tumours and reduces their potential to metastasise to the lung

Abstract. Introduction. Risk factors for osteoarthritis include raised BMI and female gender. Whether these two factors influenced synovial gene expression was investigated using a triangulation and modelling strategy which generated 12 datasets of gene expression in synovial tissue from three knee pathologies with matching BMI groups, obese and overweight, and gender distributions. Methodology. Intra-operative synovial biopsies were immersed in RNAlater at 4oC before storage at -80oC. Total RNA was extracted using RNAeasy with gDNA removal. Following RT- PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Linear models were prepared in limma with gender and BMI factors incorporated sequentially for each pathology comparison, generating 12 models of probes differentially expressed at FDR p<0.05 and Bayes number, B>0. Data analysis of differently expressed genes utilized Ingenuity Pathway Analysis and Cytoscape with Cluego and Cytohubba plug-ins. Results. Expression of 453 synovial genes was influenced by BMI and gender, 360 encode proteins such as HIF-1a, HSF1, HSPA4, HSPA5. Top canonical pathways include Unfolded protein response, Protein Ubiquiitation and Clathrin mediated endocytosis signalling linked by modulation of heat shock proteins, comparable to pathology dependent regulation. In addition BMI and gender modulate gene expression in the NRF2-mediated oxidative stress response pathway with down regulation of Glutathione-S-transferases potentially down regulating antioxidant defences. Conclusion. The enhanced risk of osteoarthritis induced by an elevated BMI and female gender maybe include differential expression of heat shock proteins and genes in the NRF2 pathway

Aim. The aim of our study was to analyze putative genes for virulence factors of Cutibacterium isolates obtained from implant-associated infections. Methods. We analyzed 64 isolates of Cutibacterium spp. (C. acnes (53/64), C. avidum (6/64), C. granulosum (4/64), C. namnetense (1/64)) using NextSeq 550 (Illumina, San Diego, CA, USA) and performed genomic analysis of 24 genes associated with virulence factors (VFs) of C. acnes previously reported in the literature. Most isolates were obtained from implant-associated infections (IAI) between 2012–2021 at the Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana. Additionally, we included the first C. namnetense isolated in our laboratory from surgical site infection. Results. C. acnes and C. namnetense have the highest number of VFs among those examined. The VFs gntK (shikimate kinase) and HYL-IB / II (hyaluronate lyase) are absent in phylotype IA. 1. (sequence types (ST) A, C, D according to the SLST scheme). Repressor gene of porphyrin synthesis, deoR is present in all Cutibacterium spp. isolates. The phylotypes II and IB show a similar distribution of VFs, with the presence of the VFs rcsB (compound for biofilm formation) and HYL-IA (hyaluronate lyase), which are absent in other C. acnes phylotypes and other Cutibacterium spp. In phylotypes IA. 1. and IB, the sequence of genes encoding VFs dsA1 and dsA2 does not have 100% genomic coverage, possibly indicating homologs between species. The isolates of C. acnes and C. namnetense possess all three CAMP (1,2,4) factors, which are not detected in other Cutibacterium spp. However, further analysis revealed species-specific CAMP factors in C. avidum and C. granulosum. Both species also have similar other genes for VFs, mainly encoding heat shock proteins and lipases, while VFs related to biofilm production are mostly absent (rcsB, ytpA). Conclusion. We found several differences in the distribution of VFs among Cutibacterium spp. isolated from IAI

Background: The cells of the intervertebral disc must synthesise and maintain their surrounding matrix for it to function normally, providing all its physiological and mechanical properties. However, disc cells survive in an environment that most cells would not tolerate, ie with a low pH and relatively little oxygen. Cells which experience such potentially damaging conditions, including excessive heat, elicit a stress response and synthesise a range of proteins, called heat shock proteins (Hsps); these facilitate repair and survival or removal of damaged cells. Methods and Results: We have studied Hsp production by disc cells, both in vitro and in vivo. We measured Hsps produced by bovine skin and disc cells grown in monolayer and heated up to 45°C and also immunostained human surgical discs for stress proteins, Hsp27 and Hsp72. Disc cells responded differently to dermal fibroblasts; when freshly isolated they had a reduced or attenuated stress response and produced much less Hsp 70 than freshly isolated skin cells. After culturing in monolayer (by passage 2) all cells produced more Hsps. Human surgical discs produced varying amounts of Hsp, with most being produced by cells in herniated discs, particularly those within clusters of cells. Conclusion: Our results suggest that intervertebral disc cells in vivo normally have a reduced stress response. Hsp production is considered to protect against damage, suggesting that the reduced response may contribute to disc degeneration and back pain. The prosurvival stress response of disc cells could provide a novel therapeutic target in patients with degenerative disc disease. Conflict of Interest: None. Source of Funding: Wolfson Charitable Trust

Introduction. Long bone surgery and marrow instrumentation represent significant surgical insults, and may cause severe local and systemic sequelae following both planned and emergent surgery. Preconditioning is a highly conserved evolutionary endogenous protective mechanism, but finding a clinically safe and acceptable method of induction has proven difficult. Glutamine, a known inducer of the heat shock protein (HSP) response, offers pharmacological modulation of injury through clinically acceptable preconditioning. This effect has not been previously demonstrated in an orthopaedic model. Aims. The aim of the study was to test the hypothesis that glutamine preconditioning protects against the local and systemic effects of long bone trauma in a rodent model. Methods. Thirty two adult male Sprague-Dawley rats were randomised into four groups:. Control group which received trauma without preconditioning,. Normal Saline preconditioning 1 hour before trauma,. Glutamine preconditioning 1 hour before trauma, and. Glutamine preconditioning 24 hours prior to trauma. Trauma consisted of bilateral femoral fracture following intramedullary instrumentation. Blood samples were taken just prior to the insult, and at an interval four hours following this. The animals were then sacrificed, bronchioalveolar lavage (BAL) performed and skeletal muscle and lung harvested for evaluation. Results. Glutamine pretreated rats had lower CK levels at 4 hours than those treated with normal saline. Renal dysfunction was less in pre-treated animal, and there was a significant reduction in neutrophil infiltration into BAL fluid. Finally glutamine pre-treated rats showed less muscle and lung oedema. This effect was more pronounced for the group which received glutamine 24 hours before trauma than the group receiving glutamine one hour before trauma. Conclusion. This data suggests that preconditioning with a single bolus of intravenous glutamine prior to planned orthopaedic intervention may afford local and end-organ protection

Background. Intervertebral disc cells exist in a challenging physiological environment. Disc degeneration occurs early in life implying that disc cells may no longer be able to maintain a functional tissue. We hypothesise that disc cells have a stress response different from most other cells because of the disc environment. We have compared the stress response of freshly isolated and cultured bovine nucleus pulposus (NP) cells with bovine dermal fibroblasts, representative of cells from a vascularised tissue. Methods. Freshly isolated and passaged bovine NP cells and dermal fibroblasts were cultured for 3 days then subjected to either thermal stress at 45°C for 1h followed by recovery times of 6, 24 and 48h or nutrient stress involving culture without serum for 6, 24 and 48 h. At each time point, cell number and viability were assessed and heat shock protein 70 (Hsp70) measured in cell lysates by an enzyme-linked immunosorbent assay. Results. In response to thermal stress, both freshly isolated and passaged dermal fibroblasts and also passaged NP cells showed a rapid elevation of Hsp70. In contrast, freshly isolated NP cells exhibited an attenuated Hsp70 response. With nutrient stress, Hsp70 increased with time in all dermal fibroblasts and passaged NP cells after 24 h, but freshly isolated NP cells responded differently again, producing less Hsp70 than controls. Conclusion. Freshly isolated bovine NP cells have a reduced response to applied stresses. This pilot study suggests that NP disc cells may have adapted to their physiologically challenging in vivo environment by attenuating their response to environmental stress. No conflicts of interest. Sources of Funding: The Wolfson Charitable Trust and Genodisc (EC's 7. th. Framework Programme (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626). This abstract has not been previously published in whole or substantial part nor has it been presented previously at a national meeting

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

Introduction: Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. IPC has been most comprehensively studied in cardiothoracic surgery, to date there has been little use of this powerful phenomenon in orthopaedic surgery. In this study, we report on the first clinical trial of IPC on human skeletal muscle, and show the potential of IPC in orthopaedics using global gene expression analysis. Methods: After local ethics committee approval and informed consent, patients undergoing primary knee arthroplasty were randomly assigned into an IPC group and a control group. Diabetic patients or patients with an ankle/brachial index of less than 1 were excluded. The IPC consisted of three five-minute periods of tourniquet insufflation on the operative limb, interrupted by five minute periods of reperfusion. The tourniquet was again insufflated and the operation started. The control group simply had tourniquet insufflation as normal prior to the start of surgery. Muscle samples were taken from the operative knee of all patients at the immediate onset of surgery (t=0), and again, at one hour into the surgery (t=1). Total RNA was extracted from the muscle samples, and the gene expression profiles were determined using microarray technology. Results: Comparison of IPC and control samples identified 702 transcripts with differences of ≥1.5-fold in their expression. Of these, 137 were altered at t=0 while 565 were altered at t=1. Amongst these changes was an up-regulation in the expression of a number of heat shock proteins (HSPs) in the IPC group as compared to the control group. Notably, there was up-regulation of the well known cytoprotective/anti-apoptotic gene, HSP72, at one hour post IPC (1.5-fold, p=0.039). There was also up-regulation of important oxidative stress defense genes, such as glutathione-S-transferase (1.6-fold, p = 0.021) and superoxide dismutase 2 (3.6-fold, p= 0.048). Microarray analysis also revealed a down-regulation in the expression of genes involved in metabolism, down-regulation of pro-apoptotic genes and up-regulation of genes necessary for transformation to a hypoxia-tolerant state. Discussion: We present convincing evidence that IPC is beneficial to human skeletal muscle and for the first time show that IPC of human skeletal muscle works in the clinical setting. In this study, the protective effect of IPC involved a down-regulation in the expression of genes associated with metabolism, and an up-regulation in the expression of genes that provide protection from cell stress, oxidative stress and apoptosis. HSPs, and especially HSP72, have well documented roles in cell stress protection. Their presence has been cited by other studies as an indicator of cell adaptation to stress

Aims

Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH.

Objectives

Tranexamic acid (TXA) is an anti-fibrinolytic medication commonly used to reduce perioperative bleeding. Increasingly, topical administration as an intra-articular injection or perioperative wash is being administered during surgery. Adult soft tissues have a poor regenerative capacity and therefore damage to these tissues can be harmful to the patient. This study investigated the effects of TXA on human periarticular tissues and primary cell cultures using clinically relevant concentrations.

Introduction

The pathogenesis of rotator cuff disease (RCD) is complex and not fully understood. This systematic review set out to summarise the histological and molecular changes that occur throughout the spectrum of RCD.

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry.

We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008).

These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

Abnormal wear of cobalt-containing metal-on-metal joints is associated with inflammatory pseudotumours. Cobalt ions activate human toll-like receptor 4 (TLR4), which normally responds to bacterial lipopolysaccharide (LPS) in sepsis. Activation of TLR4 by LPS increases the expression of chemokines IL-8 and CXCL10, which recruit leukocytes and activated T-cells, respectively. This study was designed to determine whether cobalt induces a similar inflammatory response to LPS by promoting the expression of IL-8 and CXCL10. A human monocytic cell line, derived from acute monocytic leukaemia, was treated with cobalt ions and expression of IL-8 and CXCL10 measured at mRNA and protein levels. Cobalt-treated macrophages showed a 60-fold increase in IL-8 mRNA, and an eightfold increase in production of the mature chemokine (both p < 0.001); expression of the CXCL10 gene and protein was also significantly increased by cobalt (both p < 0.001). Experiments were also performed in the presence of CLI-095, a TLR4-specific antagonist which abrogated the cobalt-mediated increase in IL-8 and CXCL10 expression.

These findings suggest that cobalt ions induce inflammation similar to that observed during sepsis by the simultaneous activation of two TLR4-mediated signalling pathways. These pathways result in increased production of IL-8 and CXCL10, and may be implicated in pseudotumour formation following metal-on-metal replacement.

Cite this article: Bone Joint J 2014; 96-B:1172–7.

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.