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Structural features of the reprolysin atrolysin C and tissue inhibitors of metalloproteinases (TIMPs) interaction.
Atrolysin C is a P-I snake venom metalloproteinase (SVMP) from Crotalus atrox venom, which efficiently degrades capillary basement membranes, extracellular matrix, and cell surface proteins to produce hemorrhage. The tissue inhibitors of metalloproteinases (TIMPs) are effective inhibitors of matrix metalloproteinases which share some structural similarity with the SVMPs. In this work, we evaluated the inhibitory profile of TIMP-1, TIMP-2, and the N-terminal domain of TIMP-3 (N-TIMP-3) on the proteolytic activity of atrolysin C and analyzed the structural requirements and molecular basis of inhibitor-enzyme interaction using molecular modeling. While TIMP-1 and TIMP-2 had no inhibitory activity upon atrolysin C, the N-terminal domain of TIMP-3 (N-TIMP-3) was a potent inhibitor with a K(i) value of approximately 150nM. The predicted docking structures of atrolysin C and TIMPs were submitted to molecular dynamics simulations and the complex atrolysin C/N-TIMP-3 was the only one that maintained the inhibitory conformation. This study is the first to shed light on the structural determinants required for the interaction between a SVMP and a TIMP, and suggests a structural basis for TIMP-3 inhibitory action and related proteins such as the ADAMs.
Identification of a novel 82 kDa proMMP-9 species associated with the surface of leukaemic cells: (auto-)catalytic activation and resistance to inhibition by TIMP-1.
MMP-9 (matrix metalloproteinase 9) plays a critical role in tumour progression. Although the biochemical properties of the secreted form of proMMP-9 are well characterized, little is known about the function and activity of cell surface-associated proMMP-9. We purified a novel 82 kDa species of proMMP-9 from the plasma membrane of THP-1 leukaemic cells, which has substantial differences from the secreted 94 kDa proMMP-9. The 82 kDa form was not detected in the medium even upon stimulation with a phorbol ester. It is truncated by nine amino acid residues at its N-terminus, lacks O-linked oligosaccharides present in the 94 kDa proMMP-9, but retains N-linked carbohydrates. Incubation of 94 kDa proMMP-9 with MMP-3 generated the well-known 82 kDa active form, but the 82 kDa proMMP-9 was converted into an active species of 35 kDa, which was also produced by autocatalytic processing in the absence of activating enzymes. The activated 35 kDa MMP-9 efficiently degraded gelatins, native collagen type IV and fibronectin. The enzyme was less sensitive to TIMP-1 (tissue inhibitor of metalloproteinase 1) inhibition with IC50 values of 82 nM compared with 1 nM for the 82 kDa active MMP-9. The synthetic MMP inhibitor GM6001 blocked the activity of both enzymes, with similar IC50 values below 1 nM. The 82 kDa proMMP-9 is also produced in HL-60 and NB4 leukaemic cell lines as well as ex vivo leukaemic blast cells. It is, however, absent from neutrophils and mononuclear cells isolated from peripheral blood of healthy individuals. Thus, the 82 kDa proMMP-9 expressed on the surface of malignant cells may escape inhibition by natural TIMP-1, thereby facilitating cellular invasion in vivo.
Carcinomas contain a matrix metalloproteinase-resistant isoform of type I collagen exerting selective support to invasion.
Collagen fibers affect metastasis in two opposing ways, by supporting invasive cells but also by generating a barrier to invasion. We hypothesized that these functions might be performed by different isoforms of type I collagen. Carcinomas are reported to contain alpha1(I)(3) homotrimers, a type I collagen isoform normally not present in healthy tissues, but the role of the homotrimers in cancer pathophysiology is unclear. In this study, we found that these homotrimers were resistant to all collagenolytic matrix metalloproteinases (MMP). MMPs are massively produced and used by cancer cells and cancer-associated fibroblasts for degrading stromal collagen at the leading edge of tumor invasion. The MMP-resistant homotrimers were produced by all invasive cancer cell lines tested, both in culture and in tumor xenografts, but they were not produced by cancer-associated fibroblasts, thereby comprising a specialized fraction of tumor collagen. We observed the homotrimer fibers to be resistant to pericellular degradation, even upon stimulation of the cells with proinflammatory cytokines. Furthermore, we confirmed an enhanced proliferation and migration of invasive cancer cells on the surface of homotrimeric versus normal (heterotrimeric) type I collagen fibers. In summary, our findings suggest that invasive cancer cells may use homotrimers for building MMP-resistant invasion paths, supporting local proliferation and directed migration of the cells whereas surrounding normal stromal collagens are cleaved. Because the homotrimers are universally secreted by cancer cells and deposited as insoluble, MMP-resistant fibers, they offer an appealing target for cancer diagnostics and therapy.
The trap hypothesis: α2M and protease inhibition
In the 1970s, the Strangeways Laboratory in Cambridge consisted of a small number of groups collectively focused on the mechanisms of pathological connective-tissue damage. One of these groups, headed by Alan Barrett, was breaking ground on the destruction of the protein components of the matrix and was therefore heavily involved in identifying and categorizing newly emerging types of tissue-degrading enzymes. These enzymes, which Alan Barrett urges scientists to call peptidases, are also commonly called proteases or proteinases*. In the early 1970s, there were about 100 described human peptidases, a reasonable sampling of the 500-600 now known in humans in the post-genomic age. Approximately 2% of the human genome encodes peptidases, and roughly 1% encodes proteins with the ability to inhibit these enzymes. As the peptidases developed different catalytic mechanisms to solve the problem of cleaving the notoriously stable peptide bond, so the families of protease inhibitors acquired distinct strategies to regulate peptidase action.The strategies are usually directed towards blocking the peptidase active site directly or, less commonly, by allosteric mechanisms. But perhaps the most bizarre mechanism is that performed by members of the protein clan exemplified by the human protein α2-macroglobulin (α2M). © 2006 Biochemical Society.
Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate.
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.
Activation of membrane-type matrix metalloproteinase 3 zymogen by the proprotein convertase furin in the trans-Golgi network.
Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases implicated in tumor invasion and metastasis, must undergo zymogen activation prior to expressing any proteolytic activity. Although the cysteine-switch model predicts the well-established autoactivation process, approximately 40% of the known MMPs possess a conserved RXKR motif between their pro- and catalytic domains and, thus, could be activated directly by members of the proprotein convertase family. To further understand this process, we analyzed the activation of proMT3-MMP as a model system. We demonstrated that the conversion of MT3-MMP zymogen into active form is dependent on both the furin-type convertase activity and the R(116)RKR motif. Consistently, MT3-MMP was colocalized with furin in the trans-Golgi network by confocal microscopy. However, neither furin activity nor its recognition site in MT3-MMP is required for the observed colocalization. In fact, the colocalization pattern remains intact, even in the presence of brefeldin A, an agent known to block endoplasmic reticulum to Golgi trafficking. Yet, brefeldin A completely blocked the activation of MT3-MMP. A23187, a calcium ionophore known to block furin maturation, also blocked proMT3-MMP activation but had minimal effect on the colocalization between MT3-MMP and furin. Thus, furin processes MT3-MMP zymogen in the trans-Golgi network, where they colocalize independently of their apparent enzyme-substrate relationship.
Efficacy of the MMP inhibitor MMI270 against lung metastasis following removal of orthotopically transplanted human colon cancer in rat.
We have investigated the antitumor effects of synthetic MMP inhibitor MMI270 against postoperative lung metastasis from colon cancer in nude rat. The KM12SM human colon cancer cells were injected into the cecal wall, and at 5 weeks after the injection, the cecum was removed including the tumor. Then, 30 mg/kg of MMI270 was administered perorally twice per day for 2 or 4 weeks, either immediately after removal or after week 2 after the removal. At week 7 after the removal, lung metastasis was significantly inhibited by the early administration of MMI270 immediately after the tumor removal but not by the late administration. The survival rates were significantly higher in the rats treated by early administration of MMI270 compared to the survival rate in untreated rats. Moreover, no lung metastasis was detected in some rats with 24-weeks' survival treated by early administration. Lower microvessel density, lower PCNA Index and higher Apoptotic Index in the lung metastases of the rats treated with MMI270 were found compared to those in untreated rats. A beneficial effect of by early administration of MMI270 against postoperative lung metastases may be expected through inhibiting neovascularization of metastases in nude rat.
Sulfated glycosaminoglycans control the extracellular trafficking and the activity of the metalloprotease inhibitor TIMP-3.
Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.
Clinical effectiveness and cost-effectiveness of open and arthroscopic rotator cuff repair [the UK Rotator Cuff Surgery (UKUFF) randomised trial].
BACKGROUND: Uncertainty exists regarding the best management of patients with degenerative tears of the rotator cuff. OBJECTIVE: To evaluate the clinical effectiveness and cost-effectiveness of arthroscopic and open rotator cuff repair in patients aged ≥ 50 years with degenerative rotator cuff tendon tears. DESIGN: Two parallel-group randomised controlled trial. SETTING: Nineteen teaching and district general hospitals in the UK. PARTICIPANTS: Patients (n = 273) aged ≥ 50 years with degenerative rotator cuff tendon tears. INTERVENTIONS: Arthroscopic surgery and open rotator cuff repair, with surgeons using their usual and preferred method of arthroscopic or open repair. Follow-up was by telephone questionnaire at 2 and 8 weeks after surgery and by postal questionnaire at 8, 12 and 24 months after randomisation. MAIN OUTCOME MEASURES: The Oxford Shoulder Score (OSS) at 24 months was the primary outcome measure. Magnetic resonance imaging evaluation of the shoulder was made at 12 months after surgery to assess the integrity of the repair. RESULTS: The mean OSS improved from 26.3 [standard deviation (SD) 8.2] at baseline to 41.7 (SD 7.9) at 24 months for arthroscopic surgery and from 25.0 (SD 8.0) at baseline to 41.5 (SD 7.9) at 24 months for open surgery. When effect sizes are shown for the intervention, a negative sign indicates that an open procedure is favoured. For the intention-to-treat analysis, there was no statistical difference between the groups, the difference in OSS score at 24 months was -0.76 [95% confidence interval (CI) -2.75 to 1.22; p = 0.452] and the CI excluded the predetermined clinically important difference in the OSS of 3 points. There was also no statistical difference when the groups were compared per protocol (difference in OSS score -0.46, 95% CI -5.30 to 4.39; p = 0.854). The questionnaire response rate was > 86%. At 8 months, 77% of participants reported that shoulder problems were much or slightly better, and at 24 months this increased to 85%. There were no significant differences in mean cost between the arthroscopic group and the open repair group for any of the component resource-use categories, nor for the total follow-up costs at 24 months. The overall treatment cost at 2 years was £2567 (SD £176) for arthroscopic surgery and £2699 (SD £149) for open surgery, according to intention-to-treat analysis. For the per-protocol analysis there was a significant difference in total initial procedure-related costs between the arthroscopic group and the open repair group, with arthroscopic repair being more costly by £371 (95% CI £135 to £607). Total quality-adjusted life-years accrued at 24 months averaged 1.34 (SD 0.05) in the arthroscopic repair group and 1.35 (SD 0.05) in the open repair group, a non-significant difference of 0.01 (95% CI -0.11 to 0.10). The rate of re-tear was not significantly different across the randomised groups (46.4% and 38.6% for arthroscopic and open surgery, respectively). The participants with tears that were impossible to repair had the lowest OSSs, the participants with re-tears had slightly higher OSSs and the participants with healed repairs had the most improved OSSs. These findings were the same when analysed per protocol. CONCLUSION: In patients aged > 50 years with a degenerative rotator cuff tear there is no difference in clinical effectiveness or cost-effectiveness between open repair and arthroscopic repair at 2 years for the primary outcome (OSS) and all other prespecified secondary outcomes. Future work should explore new methods to improve tendon healing and reduce the high rate of re-tears observed in this trial. TRIAL REGISTRATION: Current Controlled Trials ISRCTN97804283. FUNDING: This project was funded by the National Institute for Health Research Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 19, No. 80. See the NIHR Journals Library website for further project information.
A layered electrospun and woven surgical scaffold to enhance endogenous tendon repair.
Surgical reattachments of tendon to bone in the rotator cuff are reported to fail in around 40% of cases. There are no adequate solutions to improve tendon healing currently available. Electrospun, sub-micron materials, have been extensively studied as scaffolds for tendon repair with promising results, but are too weak to be surgically implanted or to mechanically support the healing tendon. To address this, we developed a bonding technique that enables the processing of electrospun sheets into multi-layered, robust, implantable fabrics. Here, we show a first prototype scaffold created with this method, where an electrospun sheet was reinforced with a woven layer. The resulting scaffold presents a maximum suture pull out strength of 167N, closely matched with human rotator cuff tendons, and the desired nanofibre-mediated bioactivity in vitro and in vivo. This type of scaffold has potential for broader application for augmenting other soft tissues.
Mortality rates at 10 years after metal-on-metal hip resurfacing compared with total hip replacement in England.
STUDY QUESTION: How do 10 year mortality rates compare between patients undergoing metal-on-metal (MoM) hip resurfacing and those undergoing total hip replacement in England? SUMMARY ANSWER: Patients in England with hip osteoarthritis who underwent MoM hip resurfacing between 1999 and 2012 have reduced long term mortality compared with those who underwent cemented and uncemented THR.
Dickkopf-3 is upregulated in osteoarthritis and has a chondroprotective role.
OBJECTIVE: Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance. METHODS: Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1β (IL1β) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFβ and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays. RESULTS: Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1β and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFβ signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited. CONCLUSIONS: We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.
Feasibility of surgical randomised controlled trials with a placebo arm: a systematic review.
OBJECTIVES: To find evidence, either corroborating or refuting, for many persisting beliefs regarding the feasibility of carrying out surgical randomised controlled trials with a placebo arm, with emphasis on the challenges related to recruitment, funding, anaesthesia or blinding. DESIGN: Systematic review. DATA SOURCES AND STUDY SELECTION: The analysis involved studies published between 1959 and 2014 that were identified during an earlier systematic review of benefits and harms of placebo-controlled surgical trials published in 2014. RESULTS: 63 trials were included in the review. The main problem reported in many trials was a very slow recruitment rate, mainly due to the difficulty in finding eligible patients. Existing placebo trials were funded equally often from commercial and non-commercial sources. General anaesthesia or sedation was used in 41% of studies. Among the reviewed trials, 81% were double-blinded, and 19% were single-blinded. Across the reviewed trials, 96% (range 50-100%) of randomised patients completed the study. The withdrawal rate during the study was similar in the surgical and in the placebo groups. CONCLUSIONS: This review demonstrated that placebo-controlled surgical trials are feasible, at least for procedures with a lower level of invasiveness, but also that recruitment is difficult. Many of the presumed challenges to undertaking such trials, for example, funding, anaesthesia or blinding of patients and assessors, were not reported as obstacles to completion in any of the reviewed trials.
Characterisation of tendons at different length scales using atomic force microscopy and polarised light microscopy may provide insight into tendon disease
© 2014 by Nova Science Publishers, Inc. Chronic, non- inflammatory tendon disorders are widespread and debilitating. The aetiology of these disorders is unclear but is believed to be related to tissue degeneration associated with altered mechanical loading. For example, studies have reported biological and cellular adaptations in torn tendons indicative of increased amounts of compressive and shear loading. However, due to a lack of suitable characterisation techniques, the role of mechanical loading in tendon degeneration and tearing is largely unknown. Given the strong correlation between age and tendinopathy, and unprecedented population aging, these disorders will become increasingly prevalent. Improved understanding of noninflammatory tendon disorders is therefore urgently needed to aid clinician decisionmaking.As the fibrous tissues that connect muscles to bones, tendons must be strong and flexible whilst facilitating efficient energy storage and return. This is achieved through the use of a fibre-reinforced composite material with a hierarchical, rope-like structure consisting of collagen fibrils, fibres and fascicles embedded in a mucopolysaccharide matrix. Like all biological materials, tendons exhibit a dynamic relationship between loading environment and material properties; cells tailor the proportions and dimensions of the extra-fibrillar matrix and fibrous elements of tendons to better suit the mechanical environment they experience. Given this strong, dynamic relationship between loading and material properties, knowledge of the material adaptations of healthy and torn tendons could provide significant insight into the role of the mechanical environment in the aetiology and pathology of tendon disorders. To date, the material properties of healthy and torn tendons, and in particular the structural adaptations, have been largely unreported. This is largely caused by a lack of techniques suitable for characterising the material adaptations of small, surgically obtained, formalin-fixed tissue samples. Historically, structural investigations at small length scales (ultrastructure) have been restricted to transmission electron microscopy, a technique which requires glutaraldehyde fixed samples and complex sample preparation, and provides a limited amount of structural information and no direct mechanical information. Recently, however, it has been demonstrated that atomic force microscopy is a highly suitable technique for biological characterisation, requiring little, if any, sample preparation and providing a wealth of structural and mechanical information, even for formalin-fixed samples. In this chapter, atomic force microscopy, polarised light microscopy and instrumented indentation are used to characterise the adaptations of small, formalin-fixed animal tendon samples that have been exposed to different loading environments in vivo, to gain insight into the effect of mechanical loading on the structural properties of tendons. The results demonstrate that atomic force microscopy and polarised light microscopy are highly suitable techniques for characterising the structural properties of surgically obtained, formalin-fixed tendon samples; loading environment is demonstrated to significantly affect the structural adaptations of healthy tendons with regions exposed to compressive stresses exhibit thinner fibres, shorter crimp lengths and thinner, less aligned fibrils compared with regions exposed to tensile and shear stresses. However, comparison of mechanical data obtained using AFM and instrumented indentation reveal that a great deal of additional work is required in order to establish appropriate methodologies and data analysis algorithms before these techniques can be reliably used to characterise the micro-mechanical properties of tendon. The application of these techniques to human samples could provide information regarding the material adaptations of healthy, degenerative and torn tendons that could provide valuable information regarding the role of mechanical environment in the aetiology and pathology of tendon disorders.
MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1.
Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.
A survey on beliefs and attitudes of trainee surgeons towards placebo.
BACKGROUND: The aim of this study was to investigate the beliefs and attitudes of trainee surgeons regarding placebo interventions, in surgical practice and in research, and to compare them to those of senior orthopaedic surgeons. METHODS: An invitation to participate in an online survey was sent to all the email addresses in the members' database of the British Orthopaedic Trainees Association (BOTA). RESULTS: All 987 members of BOTA were invited to participate in the survey and 189 responded (19 %). The majority of trainees think that the placebo effect is real (88 %), has therapeutic benefits (88 %) and that placebo manipulations are permissible (98 %). Sixty per cent of respondents agree that placebo can be used outside of research, most commonly, to distinguish between organic and non-organic symptoms (36 %). Trainees are more likely than senior surgeons to use placebo for pain management (34 % vs. 12 %). They are mainly concerned about the risk of side effects associated with the use of placebo (80 %) and prefer placebo interventions with minimal invasiveness. Seventy-three per cent respondents would recruit patients into the proposed randomised controlled surgical trial. CONCLUSIONS: The views regarding efficacy, permissibility and indications for placebo among trainees are similar to those of orthopaedic consultants. Orthopaedic trainees regard placebo as permissible and show willingness to recruit into placebo-controlled trials. However, they seem to have limited understanding of mechanisms of placebo effect and underestimate its ubiquity.
A review of current surgical practice in the operative treatment of proximal humeral fractures: Does the PROFHER trial demonstrate a need for change?
OBJECTIVES: The PROximal Fracture of the Humerus: Evaluation by Randomisation (PROFHER) trial has recently demonstrated that surgery is non-superior to non-operative treatment in the management of displaced proximal humeral fractures. The objective of this study was to assess current surgical practice in the context of the PROFHER trial in terms of patient demographics, injury characteristics and the nature of the surgical treatment. METHODS: A total of ten consecutive patients undergoing surgery for the treatment of a proximal humeral fracture from each of 11 United Kingdom hospitals were retrospectively identified over a 15 month period between January 2014 and March 2015. Data gathered for the 110 patients included patient demographics, injury characteristics, mode of surgical fixation, the grade of operating surgeon and the cost of the surgical implants. RESULTS: A majority of the patients were female (66%, 73 of 110). The mean patient age was 62 years (range 18 to 89). A majority of patients met the inclusion criteria for the PROFHER trial (75%, 83 of 110). Plate fixation was the most common mode of surgery (68%, 75 patients), followed by intramedullary fixation (12%, 13 patients), reverse shoulder arthroplasty (10%, 11 patients) and hemiarthroplasty (7%, eight patients). The consultant was either the primary operating surgeon or supervising the operating surgeon in a large majority of cases (91%, 100 patients). Implant costs for plate fixation were significantly less than both hemiarthroplasty (p < 0.05) and reverse shoulder arthroplasty (p < 0.0001). Implant costs for intramedullary fixation were significantly less than plate fixation (p < 0.01), hemiarthroplasty (p < 0.0001) and reverse shoulder arthroplasty (p < 0.0001). CONCLUSIONS: Our study has shown that the majority of a representative sample of patients currently undergoing surgical treatment for a proximal humeral fracture in these United Kingdom centres met the inclusion criteria for the PROFHER trial and that a proportion of these patients may, therefore, have been effectively managed non-operatively.Cite this article: Mr B. J. F. Dean. A review of current surgical practice in the operative treatment of proximal humeral fractures: Does the PROFHER trial demonstrate a need for change? Bone Joint Res 2016;5:178-184. DOI: 10.1302/2046-3758.55.2000596.
H3K27me3 demethylases regulate in vitro chondrogenesis and chondrocyte activity in osteoarthritis.
BACKGROUND: Epigenetic changes (i.e., chromatin modifications) occur during chondrogenesis and in osteoarthritis (OA). We investigated the effect of H3K27me3 demethylase inhibition on chondrogenesis and assessed its utility in cartilage tissue engineering and in understanding cartilage destruction in OA. METHODS: We used a high-content screen to assess the effect of epigenetic modifying compounds on collagen output during chondrogenesis of monolayer human mesenchymal stem cells (MSCs). The impact of GSK-J4 on gene expression, glycosaminoglycan output and collagen formation during differentiation of MSCs into cartilage discs was investigated. Expression of lysine (K)-specific demethylase 6A (UTX) and Jumonji domain-containing 3 (JMJD3), the HEK27Me3 demethylases targeted by GSK-J4, was measured in damaged and undamaged cartilage from patients with OA. The impact of GSK-J4 on ex vivo cartilage destruction and expression of OA-related genes in human articular chondrocytes (HACs) was assessed. H3K27Me3 demethylase regulation of transforming growth factor (TGF)-β-induced gene expression was measured in MSCs and HACs. RESULTS: Treatment of chondrogenic MSCs with the H3K27me3 demethylase inhibitor GSK-J4, which targets JMJD3 and UTX, inhibited collagen output; expression of chondrogenic genes, including SOX9 and COL2A1; and disrupted glycosaminoglycan and collagen synthesis. JMJD3 but not UTX expression was increased during chondrogenesis and in damaged OA cartilage, suggesting a predominant role of JMJD3 in chondrogenesis and OA. GSK-J4 prevented ex vivo cartilage destruction and expression of the OA-related genes MMP13 and PTGS2. TGF-β is a key regulator of chondrogenesis and articular cartilage homeostasis, and TGF-β-induced gene expression was inhibited by GSK-J4 treatment of both chondrogenic MSCs and HACs. CONCLUSIONS: Overall, we show that H3K27me3 demethylases modulate chondrogenesis and that enhancing this activity may improve production of tissue-engineered cartilage. In contrast, targeted inhibition of H3K27me3 demethylases could provide a novel approach in OA therapeutics.