Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and flow cytometry assays. Cell biofunction was detected by Western blotting. The expression profiles of messenger RNAs (mRNAs) in the EP and HDAC4 transfection groups were assessed using whole-transcriptome sequencing (RNA-seq). Volcano plot, Gene Ontology, and pathway analyses were performed to identify differentially expressed genes (DEGs). For verification of the results, the A289E/S246/467/632 A sites of HDAC4 were mutated to enhance the function of HDAC4 by increasing HDAC4 expression in the nucleus. RNA-seq was performed to identify the molecular mechanism of HDAC4 in chondrocytes. Finally, the top ten DEGs associated with ribosomes were verified by
Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time
Aims. MicroRNA-183 (miR-183) is known to play important roles in osteoarthritis (OA) pain. The aims of this study were to explore the specific functions of miR-183 in OA pain and to investigate the underlying mechanisms. Methods. Clinical samples were collected from patients with OA, and a mouse model of OA pain was constructed by surgically induced destabilization of the medial meniscus (DMM). Reverse transcription
The aims of this study were
to develop the Roche lightcycler Staphylococcal and Enterococcal PCR kits to facilitate diagnosis of hip and knee prosthetic infections To analyse results together with bacteriological and histological findings.
29 patients had non-inflammatory arthritis. 14/18 (77.8%) with positive cultures had staphylococci +/or enterococci isolated and 10 PCR results correlated. The other 11 patients had negative cultures. 9 patients had inflammatory arthritis. Six were culture negative and of the other three, 2 were positive for staphylococci on culture with 1 positive by PCR.
Enterococcal PCR confirmed culture positivity in 2/3 patients. An additional 5 positive PCR’s were obtained from patients’ culture negative for enterococci. It is not clear if these are false positives or more sensitive detection of enterococcal isolation.
Rheumatoid arthritis (RA) is a common chronic immune disease. Berberine, as its main active ingredient, was also contained in a variety of medicinal plants such as Berberaceae, Buttercup, and Rutaceae, which are widely used in digestive system diseases in traditional Chinese medicine with anti-inflammatory and antibacterial effects. The aims of this article were to explore the therapeutic effect and mechanism of berberine on rheumatoid arthritis. Cell Counting Kit-8 was used to evaluate the effect of berberine on the proliferation of RA fibroblast-like synoviocyte (RA-FLS) cells. The effect of berberine on matrix metalloproteinase (MMP)-1, MMP-3, receptor activator of nuclear factor kappa-Β ligand (RANKL), tumour necrosis factor alpha (TNF-α), and other factors was determined by enzyme-linked immunoassay (ELISA) kit. Transcriptome technology was used to screen related pathways and the potential targets after berberine treatment, which were verified by reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot (WB) technology.Aims
Methods
Aims. The present study investigated receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) gene expressions in giant cell tumour of bone (GCTB) patients in relationship with tumour recurrence. We also aimed to investigate the influence of CpG methylation on the transcriptional levels of RANKL and OPG. Methods. A total of 32 GCTB tissue samples were analyzed, and the expression of RANKL, OPG, and RUNX2 was evaluated by
Aims. To verify whether secretory leucocyte protease inhibitor (SLPI) can promote early tendon-to-bone healing after anterior cruciate ligament (ACL) reconstruction. Methods. In vitro: the mobility of the rat bone mesenchymal stem cells (BMSCs) treated with SLPI was evaluated by scratch assay. Then the expression levels of osteogenic differentiation-related genes were analyzed by real-time
Aims. Mechanical stimulation is a key factor in the development and healing of tendon-bone insertion. Treadmill training is an important rehabilitation treatment. This study aims to investigate the benefits of treadmill training initiated on postoperative day 7 for tendon-bone insertion healing. Methods. A tendon-bone insertion injury healing model was established in 92 C57BL/6 male mice. All mice were divided into control and training groups by random digital table method. The control group mice had full free activity in the cage, and the training group mice started the treadmill training on postoperative day 7. The quality of tendon-bone insertion healing was evaluated by histology, immunohistochemistry, reverse transcription
Tendinopathy is a tendon pathology often resulting from a failed healing response to tendon injury. Activated protein C (APC) is a natural anti-coagulant with anti-inflammatory and wound healing promoting functions, which are mainly mediated by its receptors, endothelial protein C receptor (EPCR) and protease activated receptors (PARs). This study aimed to determine whether APC stimulates tenocyte healing and if so, to assess the involvement of the receptors. Mouse-tail tenocytes were isolated from 3-week-old wild type (WT), PAR- 1 knockout (KO) and PAR-2 KO mice. The expression of EPCR, PAR-1 and −2 and the effect of APC on tenocytes tendon healing and the underlying mechanisms were investigated by Reverse transcription
Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels. 2. to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay,
Aims. Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. Methods. Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU),
Aims. Options for the treatment of intra-articular ligament injuries are limited, and insufficient ligament reconstruction can cause painful joint instability, loss of function, and progressive development of degenerative arthritis. This study aimed to assess the capability of a biologically enhanced matrix material for ligament reconstruction to withstand tensile forces within the joint and enhance ligament regeneration needed to regain joint function. Materials and Methods. A total of 18 New Zealand rabbits underwent bilateral anterior cruciate ligament reconstruction by autograft, FiberTape, or FiberTape-augmented autograft. Primary outcomes were biomechanical assessment (n = 17), microCT (µCT) assessment (n = 12), histological evaluation (n = 12), and
Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based
Introduction and Objective. Total joint replacement (TJR) is indicated for patients with end-stage osteoarthritis (OA) where conservative treatment has failed. Approximately 1.3 million primary hip replacement surgeries have been recorded in the United Kingdom since 2003 and this number is set to rise due to an increase in obesity as well as an ageing population. Total hip replacement (THR) has a survival rate of 85% at 20 years; the most common reason for failure is aseptic loosening which often occurs secondary to osteolysis caused by immune-mediated inflammation responses to wear debris generated from the materials used in the THR implant. Therefore, by understanding the biological steps by which biomaterials cause immune-mediated reactions it should be possible to prevent them in the future thereby reducing the number of costly revision surgeries required. Materials and Methods. The human osteoblast-like cell line (MG-63) was seeded at a density of 100,000 cell per well of a 6-well plate and treated with and increasing doses (0.5, 5, and 50mm. 3. per cell) of cobalt-chromium (CoCr) particles generated on a six-station pin-on-plate wear generator or commercially available ceramic oxide nanopowders (Al. 2. O. 3. and ZrO. 2. ) for 24 hours. TNF-alpha was used as a positive control and untreated cells as a negative control. Cells were then analysed by transmission electron microscopy (TEM) to determine whether the osteoblasts were capable of phagocytosing these biomaterials. MG-63 cells were used in conjunction with trypan blue and the XTT Cell Proliferation II Kit to assess cytotoxicity of the biomaterials investigated. Cells supernatants were also collected and analysed by enzyme-linked immunosorbant assay (ELISA) to investigate changes in pro-inflammatory protein secretion. Protein extracted from lysed cells was used for western blotting analysis to investigate RANKL protein expression to determine changes to osteolytic activation. Lysed cells were also used for RNA extraction and subsequent cDNA synthesis for real-time
Objectives. The aim of this study was to investigate the role of miR-126 in the development of osteoarthritis, as well as the potential molecular mechanisms involved, in order to provide a theoretical basis for osteoarthritis treatment and a novel perspective for clinical therapy. Methods. Human chondrocyte cell line CHON-001 was administrated by different doses of interleukin (IL)-1β to simulate inflammation. Cell viability, migration, apoptosis, IL-6, IL-8, and tumour necrosis factor (TNF)-α expression, as well as expression of apoptosis-related factors, were measured to assess inflammation. miR-126 expression was measured by
In a healthy joint, mechanical loading increases matrix synthesis and maintains cell phenotype, while reducing catabolic activities. It activates several pathways, most of them yet largely unknown, with integrins, TGF-β, canonical (Erk 1/2) and stress-activated (JNK) MAPK playing a key role. Degenerative joint diseases are characterized by Wnt upregulation and by the presence of proteolytic fibronectin fragments (FB-fs). Despite they are known to impair some of the aforementioned pathways, little is known on their modulatory effect on cartilage mechanoresponsiveness. This study aims at investigating the effect of mechanical loading in healthy and in vitro diseased cartilage models using pro-hypertrophic Wnt agonist CHIR99021 and the pro-catabolic FB-fs 30 kDa. Human primary chondrocytes from OA patients have been grown in alginate hydrogels for one week, prior to be incubated for 4 days with 3μM CHIR99021 or 1 μM FB-fs. Human cartilage explants isolated from OA patients have incubated 4 days with 3 μM CHIR99021 or 1 μM FB-fs. Both groups have then been mechanically stimulated (unconfined compression, 10% displacement, 1.5 hours, 1 Hz), using a BioDynamic bioreactor 5270 from TA Instruments. Expression of collagen type I, II and X, aggrecan, ALK-1, ALK-5, αV, α5 and β1 integrins, TGF-β1 have been assessed by
Objectives. Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. Methods. We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by
Tenomodulin (Tnmd) is the best known mature tendon factor for tendon and ligament tissues with reported important regulatory roles1. In addition, Tnmd C-terminal cysteine-rich domain has been descibed to exert anti-angiogenic functions in in vitro angiogenic assays as well as in vivo models of tendon injury and age-associated cardiac valve diseases1, 2. Interestingly, Tnmd expresson in the intervertebral disc (IVD), which is normally avascular tissue, has been also suggested3. Hence, the purpose of this study was first, to map the exact expression pattern of Tnmd during IVD development and aging and second, by implementing Tnmd-knockout mouse model, to examine if Tnmd plays a role in IVD homeostasis. Histological analyses (hematoxylin/eosin, Safranin O, CD31 for endothelium, TUNEL for apoptosis and type X collagen and Runx2 for hypetrophy) were performed on Tnmd −/−, Tnmd −/− and chondromodulin I Chmd 1 −/− (Tnmd only homolog) double knockout and wild type mice WT (n = three to five) to examine IVD degeneration.
Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by
Objectives. This study looked to analyse the expression levels of microRNA-140-3p and microRNA-140-5p in synovial fluid, and their correlations to the severity of disease regarding knee osteoarthritis (OA). Methods. Knee joint synovial fluid samples were collected from 45 patients with OA of the knee (15 mild, 15 moderate and 15 severe), ten healthy volunteers, ten patients with gouty arthritis, and ten with rheumatoid arthritis. The Kellgren–Lawrence grading (KLG) was used to assess the radiological severity of knee OA, and the patients were stratified into mild (KLG < 2), moderate (KLG = 2), and severe (KLG > 2). The expression of miR-140-3p and miR-140-5p of individual samples was measured by SYBR Green
The inflammatory cascade associated with prosthetic implant wear debris, in addition to diseases such as rheumatoid arthritis and periodontitis, it is shown to drastically influence bone turnover in the local environment. Ultimately, this leads to enhanced osteoclastic resorption and the suppression of bone formation by osteoblasts causing implant failure, joint failure, and tooth loosening in the respective conditions if untreated. Regulation of this pathogenic bone metabolism can enhance bone integrity and the treatment bone loss. The current study used novel compounds that target a group of enzymes involved with the epigenetic regulation of gene expression and protein function, histone deacetylases (HDAC), to reduce the catabolism and improve the anabolism of bone material in vitro. Human osteoclasts were differentiated from peripheral blood monocytes and cultured over a 17 day period. In separate experiments, human osteoblasts were differentiated from human mesenchymal stem cells isolated from bone chips collected during bone marrow donations, and cultured over 21 days. In these assays, cells were exposed to the key inflammatory cytokine involved with the cascade of the abovementioned conditions, tumour necrosis factor-α (TNFα), to represent an inflammatory environment in vitro. Cells were then treated with HDAC inhibitors (HDACi) that target the individual isoforms previously shown to be altered in pathological bone loss conditions, HDAC-1, −2, −5 and −7. Analysis of bone turnover through dentine resorptive measurements and bone mineral deposition analyses were used to quantify the activity of bone cells. Immunohistochemistry of tartrate resistant acid phosphatase (TRAP), WST-assay and automated cell counting was used to assess cell formation, viability and proliferation rates. Real-time
The detailed biomechanical mechanism of annulus fibrosus under abnormal loading is still ambiguous, especially at the micro and nano scales. This study aims to characterize the alterations of modulus at the nano scale of individual collagen fibrils in annulus fibrosus after in-situ immobilization, and the corresponding micro-biomechanics of annulus fibrosus. An immobilization model was used on the rat tail with an external fixation device. Twenty one fully grown 12-week-old male Sprague-Dawley rats were used in this study. The rats were assigned to one of three groups randomly. One group was selected to be the baseline control group with intact intervertebral discs (n=7). In the other two groups, the vertebrae were immobilized with an external fixation device that fixed four caudal vertebrae (C7-C10) for 4 and 8 weeks, respectively. Four K-wires were fixed in parallel using two aluminum alloy cuboids which do not compress or stretch the target discs. The immobilized discs were harvested and then stained with hematoxylin/eosin, scanned using atomic force microscopy to obtain the modulus at both nano and micro scales, and analyzed the gene expression with real-time
Background: The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established a 3D collagen gel system and studied the remodeling capacity of two different TSPC lines: young, Y-TSPC and aged/degenerative, A-TSPC. We specifically investigated the involvement of integrin receptors in the remodeling process. Methods: Y- and A-TSPC were derived from human Achilles tendon. RT-PCR was used to assess the expression of collagen-binding integrins. Integrins a1 and a11 were silenced by lentiviral delivery of shRNA in the Y-TSPC. Control-shRNA, a1-shRNA and a11-shRNA virus was given for 24h and then cells were selected with zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by
Pellino1 (Peli1) has been reported to regulate various inflammatory diseases. This study aims to explore the role of Peli1 in the occurrence and development of osteoarthritis (OA), so as to find new targets for the treatment of OA. After inhibiting Peli1 expression in chondrocytes with small interfering RNA (siRNA), interleukin (IL)-1β was used to simulate inflammation, and OA-related indicators such as synthesis, decomposition, inflammation, and apoptosis were detected. Toll-like receptor (TLR) and nuclear factor-kappa B (NF-κB) signalling pathway were detected. After inhibiting the expression of Peli1 in macrophages Raw 264.7 with siRNA and intervening with lipopolysaccharide (LPS), the polarization index of macrophages was detected, and the supernatant of macrophage medium was extracted as conditioned medium to act on chondrocytes and detect the apoptosis index. The OA model of mice was established by destabilized medial meniscus (DMM) surgery, and adenovirus was injected into the knee cavity to reduce the expression of Peli1. The degree of cartilage destruction and synovitis were evaluated by haematoxylin and eosin (H&E) staining, Safranin O/Fast Green staining, and immunohistochemistry.Aims
Methods
This study aimed to explore the role of small colony variants (SCVs) of A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR).Aims
Methods
Our aim was to investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Gonarthrosis (AMG). Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H&
E staining) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content.
Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders. We examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1PrαTACE’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation.Aims
Methods
Purpose: The introduction of supplementary cells into a region of diseased or damaged tissue is becoming a viable treatment strategy in many areas of medicine. Mesenchymal stem cells (MSCs) are attractive for this purpose because they represent an autologous, multipotent cell source. However, it has been recognized that populations of MSCs represent a heterogenous group of cells with each cell subpopulation possessing unique terminal differential capacity. The CD44 cell surface receptor has previously been identified on some of the cells within the MSC population. It is also present on chondrocytes and is thought to play a critical role in cartilage matrix generation and homeostasis. We hypothesized that a CD44+ purified subpopulation of MSCs will possess enhanced chondrogenic potential and be more suitable for articular cartilage regeneration. Method: Bone marrow aspirates were collected from orthopaedic patients undergoing iliac crest bone grafting. Human MSCs were isolated and cultured using standard techniques. Flow cytometry was utilized to identify the cell surface antigens characteristic of the MSC population. FACS was utilized to isolate the CD44 positive cells based on antigenic recognition, generating a CD44 positive population and a CD44 negative population. To confirm the multilineage potential of the isolates, defined media and culture conditions were utilized to differentiate both groups into osteocytes, adipocytes and chondrocytes.
Aim: To investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Osteoarthritis of the Knee (AMOA). Methods: Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H&
E staining) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content.
The aim of this study was to investigate the molecular features of progressive severities of cartilage damage, within the phenotype of Anteromedial Osteoarthritis of the Knee (AMOA). Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O and H&
E staining) and immunohistochemical analysis (Type I and II Collagen). Immunoassays were undertaken for Collagens I and II and GAG content.
Anteromedial Osteoarthritis of the Knee (AMOA) is a distinct phenotype of OA. Within this pattern of disease, the anterior third of the medial tibial plateau exhibits full thickness cartilage loss. The middle third has damaged partial thickness cartilage, and the posterior third has retained cartilage, which is seen on macroscopic visual assessment to be normal. This study investigates the molecular features of progressive severities of cartilage damage within this phenotype. Ten medial tibial plateau specimens were collected from patients undergoing unicompartmental knee replacements. The cartilage within the area of macroscopic damage was divided into equal thirds: T1(most damaged), to T3 (least damaged). The area of macroscopically undamaged cartilage was taken as a 4th sample, N. The specimens were prepared for histological (Safranin-O) and immunohistochemical analysis (Type I and II Collagen, proliferation and apoptosis). Immunoassays were undertaken for Collagens I and II and GAG content.
Introduction. Osteoarthritis (OA) of the knee, a prevalently degenerative joint disorder provoked by articular cartilage loss, accounts for the leading cause of total knee arthroplasty. Autophagy is an indispensable intracellular event that maintains chondrocyte survival and metabolism. MicroRNAs are non-coding small RNAs participating in tissue morphogenesis, remodeling, and homeostasis. This study was undertaken to investigate the effect of microRNA-128 (miR-128) knockdown on the development of OA knees. Materials/Methods. Knee joints in rats were subjected to anterior cruciate ligament transection (ACLT) for inducing OA. Articular cartilage, synovium, and subchondral bone microarchitecture were assessed by OARSI scoring system, histomorphometry, and μCT imaging. Chondrocyte autophagy in terms of the expression of autophagic markers Atg4, Atg12, microtubule-associated protein 1 light chain 3 (LC3), and autophagosome formation was verified. Expression of microRNA, mRNA and signaling transduction were quantified with in situ hybridization, RT-
Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression.Aims
Methods
Introduction. The prevalence of symptomatic osteoarthritis (OA) in the knee is 11–11% compared to 3.4–4.4% in the ankle. In addition to this, 70% of ankle arthritis is post-traumatic while the vast majority of knee arthritis is primary OA. Several reports have previously implicated biochemical differences in extracellular matrix composition between these joint cartilages; however, it is unknown whether there is an inherent difference in their transcriptome and how this might affect their respective functionality under load, inflammatory environment etc. Therefore, we have analysed the transcriptome of ankle and knee cartilage chondrocytes to determine whether this could account for the lower prevalence and altered aetiology of ankle OA. Methods. Human full-depth articular cartilage was taken from the talar domes (n=5) and the femoral condyles (n=5) following surgical amputation. RNA was extracted and next generation sequencing (NGS) performed using the NextSeq®500 system. Statistical analysis was performed to identify differentially regulated genes (p adj < 0.05). Data was analysed using Integrated Pathway Analysis software and genes of interest validated by
Aim: Collagen meniscus implant (CMI) is a tissue engineering technique for the management of irreparable meniscal lesions. In this study we evaluate morphological and biochemical changes occurring in CMI after implantation, in order to better define tissue ingrowth inside the scaffold. Gene expression technique was also adopted to characterize the phenotype of the invading cells. Methods and materials: Morphological analysis was performed by light microscopy, immunohistochemistry (type I and II collagen), SEM and TEM on 5 biopsy specimens, harvested from 5 different patients (range, 6 to 16 months after surgery). Biochemical evaluation was carried out using Flurophore Assisted Carbohydrate Electrophoresis (FACE): this assay allowed to measure glycosaminoglycans (GAG) production in extracellular matrix of 2 biopsy specimens, harvested respectively 6 and 16 months after implantation.
Introduction. Stem cells are widely known in the state of the art of cell-based therapies. Recently, ADSCs are becoming a popular resource of adult stem cells across different fields, and latest publications show its wide application for the treatment of soft tissue injuries like tendon injuries, which represent a high percentage of the consultations in orthopaedic practitioners. Molecular-based therapies and local deliveries are necessary for an effective treatment of chronic tendon injuries. In this study, human ADSCs were selected to investigate its differentiation potential into the tendon phenotype. Customised cell culture media was used as the differentiation factor. Materials and Methods. In the present study, ADSCs were used in passage 3 to ensure pluripotency in vitro. Using the customised cell culture media, its time, concentration and frequency of refreshment effects were investigated. On the selected time points different techniques were performed: 1,) cells were harvested, and messenger RNA (mRNA) was examined by
Background. Adverse reactions to metal debris are implicated in the failure of metal-on-metal hip arthroplasty. The peri-implant tissues are often infiltrated by leukocytes which may cause observed immunological effects, including soft tissue necrosis and osteolysis. Cobalt ions from orthopaedic implants aberrantly activate the innate immune receptor human toll-like receptor-4 (TLR4), leading to inflammatory cytokine release including interleukin-8 (IL-8). IL-8 has been shown to increase expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These factors are essential for leukocyte adhesion to endothelium, which is required for leukocyte migration into tissues. This study investigates cobalt's effect on gene and protein changes in IL-8, ICAM-1 and VCAM-1 to determine their potential role in immune cell infiltration of peri-implant tissues. Methods. TLR4-expressing human dermal microvascular endothelial cells (HMEC-1) were treated with a range of clinically relevant cobalt ion concentrations. IL-8 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based
Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been reported to be a promising cellular therapeutic approach for various human diseases. The current study aimed to investigate the mechanism of BMSC-derived exosomes carrying microRNA (miR)-136-5p in fracture healing. A mouse fracture model was initially established by surgical means. Exosomes were isolated from BMSCs from mice. The endocytosis of the mouse osteoblast MC3T3-E1 cell line was analyzed. CCK-8 and disodium phenyl phosphate microplate methods were employed to detect cell proliferation and alkaline phosphatase (ALP) activity, respectively. The binding of miR-136-5p to low-density lipoprotein receptor related protein 4 (LRP4) was analyzed by dual luciferase reporter gene assay. HE staining, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemistry were performed to evaluate the healing of the bone tissue ends, the positive number of osteoclasts, and the positive expression of β-catenin protein, respectively.Aims
Methods
The increased incidence of type 2 Diabetes Mellitus is associated with an impaired skeletal structure and a higher prevalence of bone fractures. Sclerostin is a negative regulator of bone formation produced by osteocytes and there is recent evidence that its expression in serum is elevated in diabetic patients compared to control subjects. In this study, we test whether hyperglycemia affects serum and bone sclerostin levels in a rat model of type 2 Diabetes as well as sclerostin production by osteoblasts in culture. We used Zucker diabetic fatty (ZDF) male rats (n=6) that spontaneously develop obesity and frank diabetes around 8–9 weeks of age and Zucker lean rats as controls (n=6) to examine sclerostin expression in serum at 9, 11 and 13 weeks using a specific ELISA. Sclerostin expression in bone tibiae was examined at 12 weeks using immunocytochemistry. Rat osteoblast-like cells UMR-106 were cultured in the presence of increasing concentrations of glucose (5, 11, 22 and 44 mM) during 48 hours and sclerostin mRNA expression and release in the supernatant determined by
The role of N,N-dimethylformamide (DMF) in diabetes-induced osteoporosis (DM-OS) progression remains unclear. Here, we aimed to explore the effect of DMF on DM-OS development. Diabetic models of mice, RAW 264.7 cells, and bone marrow macrophages (BMMs) were established by streptozotocin stimulation, high glucose treatment, and receptor activator of nuclear factor-κB ligand (RANKL) treatment, respectively. The effects of DMF on DM-OS development in these models were examined by micro-CT analysis, haematoxylin and eosin (H&E) staining, osteoclast differentiation of RAW 264.7 cells and BMMs, H&E and tartrate-resistant acid phosphatase (TRAP) staining, enzyme-linked immunosorbent assay (ELISA) of TRAP5b and c-terminal telopeptides of type 1 (CTX1) analyses, reactive oxygen species (ROS) analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Cell Counting Kit-8 (CCK-8) assay, and Western blot.Aims
Methods
The October 2023 Shoulder & Elbow Roundup360 looks at: Arthroscopic capsular shift surgery in patients with atraumatic shoulder joint instability: a randomized, placebo-controlled trial; Superior capsular reconstruction partially restores native glenohumeral loads in a dynamic model; Gene expression in glenoid articular cartilage varies in acute instability, chronic instability, and osteoarthritis; Intra-articular injection versus interscalene brachial plexus block for acute-phase postoperative pain management after arthroscopic shoulder surgery; Level of pain catastrophizing rehab in subacromial impingement: secondary analyses from a pragmatic randomized controlled trial (the SExSI Trial); Anterosuperior versus deltopectoral approach for primary reverse total shoulder arthroplasty: a study of 3,902 cases from the Dutch National Arthroplasty Registry with a minimum follow-up of five years; Assessment of progression and clinical relevance of stress-shielding around press-fit radial head arthroplasty: a comparative study of two implants; A number of modifiable and non-modifiable factors increase the risk for elbow medial ulnar collateral ligament injury in baseball players: a systematic review.
Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs.Aims
Methods
This study was performed to explore the effect of melatonin on pyroptosis in nucleus pulposus cells (NPCs) and the underlying mechanism of that effect. This experiment included three patients diagnosed with lumbar disc herniation who failed conservative treatment. Nucleus pulposus tissue was isolated from these patients when they underwent surgical intervention, and primary NPCs were isolated and cultured. Western blotting, reverse transcription polymerase chain reaction, fluorescence staining, and other methods were used to detect changes in related signalling pathways and the ability of cells to resist pyroptosis.Aims
Methods
One of the most common bacteria in orthopaedic prosthetic infections is Staphylococcus Aureus. Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion on nanofabricated materials. We hypothesise that surface nanotopography impacts the differential ability of staphylococci species to adhere via altered metabolomics and may reduce orthopaedic implant infection rate. Bacteria were grown and growth conditions optimised. Polystyrene and titanium (Ti) nanosurfaces were studied. The polystyrene surfaces had different nanopit arrays, while the Ti surfaces expressed different nanowire structures. Adhesion analysis was performed using fluorescence imaging,
Osteoporosis (OP) is a metabolic bone disease, characterized by a decrease in bone mineral density (BMD). However, the research of regulatory variants has been limited for BMD. In this study, we aimed to explore novel regulatory genetic variants associated with BMD. We conducted an integrative analysis of BMD genome-wide association study (GWAS) and regulatory single nucleotide polymorphism (rSNP) annotation information. Firstly, the discovery GWAS dataset and replication GWAS dataset were integrated with rSNP annotation database to obtain BMD associated SNP regulatory elements and SNP regulatory element-target gene (E-G) pairs, respectively. Then, the common genes were further subjected to HumanNet v2 to explore the biological effects.Aims
Methods
To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed.Aims
Methods
This study explored the shared genetic traits and molecular interactions between postmenopausal osteoporosis (POMP) and sarcopenia, both of which substantially degrade elderly health and quality of life. We hypothesized that these motor system diseases overlap in pathophysiology and regulatory mechanisms. We analyzed microarray data from the Gene Expression Omnibus (GEO) database using weighted gene co-expression network analysis (WGCNA), machine learning, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify common genetic factors between POMP and sarcopenia. Further validation was done via differential gene expression in a new cohort. Single-cell analysis identified high expression cell subsets, with mononuclear macrophages in osteoporosis and muscle stem cells in sarcopenia, among others. A competitive endogenous RNA network suggested regulatory elements for these genes.Aims
Methods
Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA). Mesenchymal stem/stromal cells (MSCs) were isolated from rat bone marrow (BM) and used to evaluate the impact of 29-mer on chondrogenic differentiation of BM-MSCs in culture. Knee OA was induced in rats by a single intra-articular injection of monosodium iodoacetate (MIA) in the right knees (set to day 0). The 29-mer dissolved in 5% hyaluronic acid (HA) was intra-articularly injected into right knees at day 8 and 12 after MIA injection. Subsequently, the therapeutic effect of the 29-mer/HA on OA was evaluated by the Osteoarthritis Research Society International (OARSI) histopathological scoring system and changes in hind paw weight distribution, respectively. The regeneration of chondrocytes in damaged AC was detected by dual-immunostaining of 5-bromo-2'-deoxyuridine (BrdU) and chondrogenic markers.Aims
Methods
The involvement of cyclin D1 in the proliferation of microglia, and the generation and maintenance of bone cancer pain (BCP), have not yet been clarified. We investigated the expression of microglia and cyclin D1, and the influences of cyclin D1 on pain threshold. Female Sprague Dawley (SD) rats were used to establish a rat model of BCP, and the messenger RNA (mRNA) and protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and cyclin D1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. The proliferation of spinal microglia was detected by immunohistochemistry. The pain behaviour test was assessed by quantification of spontaneous flinches, limb use, and guarding during forced ambulation, mechanical paw withdrawal threshold, and thermal paw withdrawal latency.Aims
Methods
In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD.Aims
Methods
Rotator cuff (RC) injuries are characterized by tendon rupture, muscle atrophy, retraction, and fatty infiltration, which increase injury severity and jeopardize adequate tendon repair. Epigenetic drugs, such as histone deacetylase inhibitors (HDACis), possess the capacity to redefine the molecular signature of cells, and they may have the potential to inhibit the transformation of the fibro-adipogenic progenitors (FAPs) within the skeletal muscle into adipocyte-like cells, concurrently enhancing the myogenic potential of the satellite cells. HDACis were added to FAPs and satellite cell cultures isolated from mice. The HDACi vorinostat was additionally administered into a RC injury animal model. Histological analysis was carried out on the isolated supra- and infraspinatus muscles to assess vorinostat anti-muscle degeneration potential.Aims
Methods
The exact pathways of collagen remodeling in tendon tissue are not well understood. Therefore, we have established an ex vivo 3D collagen gel-based system and we studied the remodeling capacity of two different TSPC lines from young, Y-TSPC and aged/degenerative, A-TSPC donors. Here, we specifically focused on investigating the involvement of integrin receptors in the remodeling process. Integrins are transmembrane receptors consisting of alpha (a) and beta (b) subunits, which form cell-to-matrix bonds, activate various pathways and thereby control cell proliferation, differentiation and survival. Y- and A-TSPC were derived from human Achilles tendons and are fully described in Kohler et al. 2013. RT-PCR was used to assess the expression of collagen-binding integrins in the TSPC cultivated in collagen gels. Next, a1 and a11 integrins were silenced by stable lentiviral delivery of target-specific shRNA in the Y-TSPC. Control (con-shRNA), integrin (a1-shRNA) and integrin a11 (a11-shRNA) virus-containing supernatant was given for 24h and then cells were selected with 50 microg./ml zeocin for 10 days. The integrin knockdown (KD) efficiency was assessed by
Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67phox was involved in suramin-enhanced chondrocyte phenotype maintenance.Aims
Methods
Exosomes (exo) are involved in the progression of osteoarthritis (OA). This study aimed to investigate the function of dysfunctional chondrocyte-derived exo (DC-exo) on OA in rats and rat macrophages. Rat-derived chondrocytes were isolated, and DCs induced with interleukin (IL)-1β were used for exo isolation. Rats with OA (n = 36) or macrophages were treated with DC-exo or phosphate-buffered saline (PBS). Macrophage polarization and autophagy, and degradation and chondrocyte activity of cartilage tissues, were examined. RNA sequencing was used to detect genes differentially expressed in DC-exo, followed by RNA pull-down and ribonucleoprotein immunoprecipitation (RIP). Long non-coding RNA osteoarthritis non-coding transcript (OANCT) and phosphoinositide-3-kinase regulatory subunit 5 (PIK3R5) were depleted in DC-exo-treated macrophages and OA rats, in order to observe macrophage polarization and cartilage degradation. The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway activity in cells and tissues was measured using western blot.Aims
Methods
There is an increasing concern of osteoporotic fractures in the ageing population. Low-magnitude high-frequency vibration (LMHFV) was shown to significantly enhance osteoporotic fracture healing through alteration of osteocyte lacuno-canalicular network (LCN). Dentin matrix protein 1 (DMP1) in osteocytes is known to be responsible for maintaining the LCN and mineralization. This study aimed to investigate the role of osteocyte-specific DMP1 during osteoporotic fracture healing augmented by LMHFV. A metaphyseal fracture was created in the distal femur of ovariectomy-induced osteoporotic Sprague Dawley rats. Rats were randomized to five different groups: 1) DMP1 knockdown (KD), 2) DMP1 KD + vibration (VT), 3) Scramble + VT, 4) VT, and 5) control (CT), where KD was performed by injection of short hairpin RNA (shRNA) into marrow cavity; vibration treatment was conducted at 35 Hz, 0.3 g; 20 minutes/day, five days/week). Assessments included radiography, micro-CT, dynamic histomorphometry and immunohistochemistry on DMP1, sclerostin, E11, and fibroblast growth factor 23 (FGF23). In vitro, murine long bone osteocyte-Y4 (MLO-Y4) osteocyte-like cells were randomized as in vivo groupings. DMP1 KD was performed by transfecting cells with shRNA plasmid. Assessments included immunocytochemistry on osteocyte-specific markers as above, and mineralized nodule staining.Aims
Methods
Recent evidence suggests that the microbial community, its spatial distribution and activity play an important role in the prolongation of treatment and healing of chronic infections. Standard bacterial cultures often underestimate the microbial diversity present in chronic infections. This lack of growth is often due to a combination of inadequate growth conditions, prior usage of antibiotics and presence of slow-growing, fastidious, anaerobic or unculturable bacteria living in biofilms. Thus, diagnosis of chronic infections is challenged by lack of appropriate sampling strategies and by limitations in microbiological testing methods. The purpose of this study was to improve sampling and diagnosis of prosthetic joint infections (PJI) and chronic wounds, especially considering the biofilm issue. Systematic sampling, sonication of prosthesis and extended culture were applied on patients with chronic wounds and patients with suspected PJIs. Optimized DNA extraction,
Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of The human osteoblast-like Saos-2 cells infected with Aims
Methods
The most common bacteria in orthopaedic prosthetic infections are Staphylococcus, namely Staphylococcus Epidermidis (SE) and Staphylococcus Aureus (SA). Infection causes implant failure due to biofilm production. Biofilms are produced by bacteria once they have adhered to a surface. Nanotopography has major effects on cell behaviour. Our research focuses on bacterial adhesion and biofilm formation on nanofabricated materials. Bacteria studied were clinically relevant from an orthopaedic perspective, SA and SE. We hypothesise that that nanosurfaces can modulate bacterial adherence and biofilm formation and may reduce orthopaedic implant infection rate. Isolated bacteria were grown and growth conditions optimised. Bacterial concentrations were calculated by using qPCR. Statistical analysis allowed identification of optimal biofilm growth conditions. These were refined on standard, non-nanopatterned surfaces, and then control and nanopatterned polystyrene (nanopits) and titanium plates (nanowires). Adhesion analysis was performed using fluorescence imaging and
Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice. Chondrocytes were isolated from articular cartilage of mice and treated with 5 ng/ml lipopolysaccharide (LPS) or 10 ng/ml interleukin 1 beta (IL-1β) for 24 hours, and then treated with different concentrations of tBHQ (10, 20, and 40 μM) for 12 hours. The expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured. The expression levels of interleukin 6 (IL-6), IL-1β, and tumour necrosis factor alpha (TNF-α) leptin in plasma were measured using enzyme-linked immunoabsorbent assay (ELISA) kits. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathway proteins, and macrophage repolarization-related markers, were detected by western blot.Aims
Methods
Nanoscale topography increases the bioactivity of a material and stimulates specific responses (third generation biomaterial properties) at the molecular level upon first generation (bioinert) or second generation (bioresorbable or bioactive) biomaterials. We developed a technique (based upon the effects of nanoscale topography) that facilitated the in vitro expansion of bone graft for subsequent implantation and investigated the optimal conditions for growing new mineralised bone in vitro. Two topographies (nanopits and nanoislands) were embossed into the bioresorbable polymer Polycaprolactone (PCL). Three dimensional cell culture was performed using double-sided embossing of substrates, seeding of both sides, and vertical positioning of substrates. The effect of Hydroxyapatite, and chemical stimulation were also examined. Human bone marrow was harvested from hip arthroplasty patients, the mesenchymal stem cells culture expanded and used for cellular analysis of substrate bioactivity. The cell line specificity and osteogenic behaviour was demonstrated through immunohistochemistry, confirmed by real-time PCR and
Cell-free DNA (cfDNA) and circulating tumour DNA (ctDNA) are used for prognostication and monitoring in patients with carcinomas, but their utility is unclear in sarcomas. The objectives of this pilot study were to explore the prognostic significance of cfDNA and investigate whether tumour-specific alterations can be detected in the circulation of sarcoma patients. Matched tumour and blood were collected from 64 sarcoma patients (n = 70 samples) prior to resection of the primary tumour (n = 57) or disease recurrence (n = 7). DNA was isolated from plasma, quantified, and analyzed for cfDNA. A subset of cases (n = 6) underwent whole exome sequencing to identify tumour-specific alterations used to detect ctDNA using digital droplet polymerase chain reaction (ddPCR).Aims
Methods
Bone metastasis ultimately occurs due to a complex multistep process, during which the interactions between cancer cells and bone microenvironment play important roles. Prior to colonization of the bone, cancer cells must succeed through a series of steps that will allow them to gain migratory and invasive properties; epithelial-to-mesenchymal transition (EMT) is known to be integral here. The aim of this study was to determine the effects of G protein subunit alpha Q (GNAQ) on the mechanisms underlying bone metastasis through EMT pathway. A total of 80 tissue samples from patients who were surgically treated during January 2012 to December 2014 were used in the present study. Comparative gene analysis revealed that the GNAQ was more frequently altered in metastatic bone lesions than in primary tumour sites in lung cancer patients. We investigated the effects of GNAQ on cell proliferation, migration, EMT, and stem cell transformation using lung cancer cells with GNAQ-knockdown. A xenograft mouse model tested the effect of GNAQ using micro-CT analyses and histological analyses.Aims
Methods
In the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1β, and to explore its mechanism. SMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1β stimulation, and IL-1β stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated.Aims
Methods
Summary Statement. In articular cartilage defects, chemokines are upregulated and potentially induce the migration of bone marrow cells to accelerate the healing processes. Introduction. The treatment of damaged articular cartilages is one of the most challenging issues in sports medicine and in aging societies. In the microfracture technique for the treatment of articular cartilage defects, bone marrow cells are assumed to migrate from the bone marrow. Bone marrow cells are well-known for playing crucial roles in the healing processes, but how they can migrate from underlying bone marrow remains to be investigated. We have previously shown that SDF-1, one of chemokines, play crucial roles in the recruitment of mesenchymal stem cells in bone healing processes, and the induction of SDF-1 can induce a successful bone repair. If the migration can be stimulated by any means in the cartilage defects, a better result can be expected. The aim of this study was to elucidate the mechanisms of the migration of bone marrow cells and which factors contribute to the processes. Materials & Methods. Articular cartilage defects of 2 mm of diameter were created by drilling the cartilage with a wire to just the subchondral bone in 5-week-old SD rats. The width and depth of the created defects were confirmed by HE staining in histology. The healing tissues were harvested at days 2, 6, and 14 after the operation, and total RNAs were entracted. PCR array was conducted according to the manufacturer's instruction.
Non-coding microRNA (miRNA) in extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) may promote neuronal repair after spinal cord injury (SCI). In this paper we report on the effects of MSC-EV-microRNA-381 (miR-381) in a rodent model of SCI. In the current study, the luciferase assay confirmed a binding site of bromodomain-containing protein 4 (BRD4) and Wnt family member 5A (WNT5A). Then we detected expression of miR-381, BRD4, and WNT5A in dorsal root ganglia (DRG) cells treated with MSC-isolated EVs and measured neuron apoptosis in culture by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. A rat model of SCI was established to detect the in vivo effect of miR-381 and MSC-EVs on SCI.Aims
Methods
Objectives. Lower limb muscle power is thought to influence outcome following
total knee replacement (TKR). Post-operative deficits in muscle
strength are commonly reported, although not explained. We hypothesised
that post-operative recovery of lower limb muscle power would be
influenced by the number of satellite cells in the quadriceps muscle at
time of surgery. . Methods. Biopsies were obtained from 29 patients undergoing TKR. Power
output was assessed pre-operatively and at six and 26 weeks post-operatively
with a Leg Extensor Power Rig and data were scaled for body weight.
Satellite cell content was assessed in two separate analyses, the
first cohort (n = 18) using immunohistochemistry and the second
(n = 11) by a new
The Wnt/b-catenin signaling pathway participates in normal adult bone and cartilage biology and seems to be involved in cartilage degeneration and subsequent OA progression. The aim of this study was to investigate the activation of Wnt/b-catenin pathway in osteoarthritis and the role of LRP5, a coreceptor of Wnt/b-catenin pathway, in human osteoarhritic chondrocytes. Human cartilage was obtained from 11 patients with primary osteoarthritis (OA) undergoing total knee and hip replacement surgery. Normal cartilage was obtained from 5 healthy individuals. b-catenin and LRP5 mRNA and protein levels were investigated using
Summary Statement. Survivin is a member of the inhibitor of apoptosis family, which may contribute to the progression of human MFH via inhibiting the mitochondrial apoptosis, and may be considered as a potent therapeutic target for the treatment of human MFH. Introduction. Survivin is a member of the inhibitor of apoptosis (IAP) family, which usually expresses in the embryonic lung and fetal organs in the developmental stages, but is undetectable in normal adult tissues other than thymus, placenta, CD34. +. stem cells, and basal colonic epitherial cells. However, several studies reported that survivin is highly expressed in various human malignancies, including sarcomas, and increased expression of survivin is an unfavorable prognostic marker correlating with decreased overall survival in cancer patients. We have previously reported that survivin was strongly expressed in human malignant fibrous histiocyoma (MFH), however, the roles of survivin in human MFH have not been studied. The aim of this study was to evaluate the effect of survivin inhibition on apoptotic activity in human MFH cells. Methods. Nara-H, a human MFH cell line which expresses the high levels of survivin, was used in this study. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO. 2. To evaluate the effect of survivin inhibition on MFH cell apoptosis, cells were transfected with either a survivin specific siRNA (survivin-siRNA) or a non-specific control siRNA (control-siRNA) by lipofection method. After siRNA transfection, the efficiency of siRNA knockdown of survivin was assessed by
Summary. Reciprocal metabolic reprogramming of MSCs and osteosarcoma cells influences tumor-stroma cross talk. Drugs targeting Warburg metabolism may define innovative therapeutic approaches in osteosarcoma. Introduction. Osteosarcoma (OS) is a malignant primary bone tumour of mesenchymal origin, in which cells with stem-like characteristics (CSCs) have been described. Recent studies have demonstrated a mutual interaction between stroma and tumor cells in exploiting a role in the pathogenesis and progression of cancer, and also in the enhancing stemness phenotype. Here we take in consideration the complex juxtacrine and paracrine intercellular cross talk played by mesenchymal stromal cells (MSCs) with adherent osteosarcoma cells and OS cells with stem-like characteristics (CSCs). Methods. MSCs were isolated from human adipose tissue and expanded. To evaluate the interaction between the stroma and the cancer cell compartment, we used two different osteosarcoma cancer cell lines (Saos-2 and HOS) and co-cultured them with MSCs. The different cell populations were sorted to study the reciprocal interaction including metabolic reprogramming. CSCs were obtained from SAOS-2 and HOS cell lines using the sphere formation assay and characterised for their self-renewal, mesenchymal stem cell properties and expression of pluripotency markers. CSCs sensitivity to paracrine factors produced by human MSCs was analysed in a model of co-culture system. Mitochondrial activity in the co-culture systems was also evaluated. Results. Our results revealed that upon intercellular contact, MSCs undergo Warburg metabolism and mitochondrial oxidative stress. In particular, the cell contact activated the stromal component, triggering autophagy and increased expression of monocarboxylate transporter-4 (MCT4) responsible for the extrusion of lactate. Conversely, osteosarcoma cancer cells, upon contact with MSCs, increased their aerobic metabolism and their development. In addition, using a co-culture method without cell contact, we observed that MSCs grown in serum-starvation conditions promoted proliferation of the CSCs obtained from OS cells, probably by secreting one or several soluble factors. By
Introduction. Low back pain is a major public health problem in our society. Degeneration of intervertebral disc (IVD) appears to be the leading cause of chronic low-back pain [1]. Mechanical stimulations including compressive and tensional forces are directly implicated in IVD degeneration. Several studies have implicated the cytoskeleton in mechanotransduction [2, 3], which is important for communication and transport between the cells and extracellular matrix (ECM). However, the potential roles of the cytoskeletal elements in the mechanotransduction pathways in IVD are largely unknown. Methods. Outer annulus fibrosus (OAF) and nucleus pulposus (NP) cells from skeletally mature bovine IVD were either seeded onto Flexcell¯ type I collagen coated plates or seeded in 3% agarose gels, respectively. OAF cells were subjected to cyclic tensile strain (10%, 1Hz) and NP cells to cyclic compressive strain (10%, 1Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy and RNA extracted for
We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by
Introduction: We studied the extracellular matrix (ECM) of 19 ruptured human Achilles tendons, comparing the tissue composition of specimens taken from area close to the rupture with specimens harvested from an apparently healthy area in the same tendon. The hypothesis was that the metabolism of these molecules is altered in patients with Achilles tendon rupture. Materials and Methods: We compared the gene expression and the protein localization of the main ECM molecules (collagen type I, decorin and versican) including enzymes involved in their metabolism as matrix metallo-proteases (MMP2 and 9) and tissue inhibitory of metal-loproteinase (TIMP 1 and 2) using a
Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty, the potential toxicity of metal ions in the periprosthetic area remains a cause for concern. Studies to date have assessed the acute effect of cobalt ions on osteoblasts over 48 hours. The aim of our study was to determine the response of osteoblasts to cobalt ions over a prolonged period of exposure. Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. ELISA were used to assess chemokine (IL-8, MCP-1 and TNF-α) secretion. Osteoblast gene expression was assessed using microarray analysis and
Introduction: In this study infrapatellar fat pad (IPFP) derived stem cells were expanded with and without Fibroblast Growth Factor-2 (FGF-2) supplementation and were compared with regards to their ability to proliferate and differentiate into chondrocytes. Materials and Methods: Cells were isolated from the IPFP tissue and expanded in monolayer culture with and without rhFGF-2 supplementation (final concentration 10ng/ ml). Cell aggregates were placed in chondrogenic media for two weeks. Gene expression studies were carried out using
Purpose. Vitamin D is a key regulator of bone homeostasis. The enzyme CYP24A1 is responsible for transforming vitamin D into 24,25(OH)2vitD. The putative biological activity of 24,25(OH)2vitD remains unclear. Previous studies showed an increase in the circulating levels of this metabolite following a fracture in chicks. Our laboratory has engineered a mouse model deficient for the Cyp24a1 gene for studying the role of 24,25(OH)2vitD. We set out to study the role of 24,25(OH)2vitD in endochondral and intramembranous bone formation in fracture repair in this mouse model based on the results of the chick fracture repair study. Method. Wild-type and mutant Cyp24a1 gene deficient mice were subjected to two different surgical procedures to simulate bone development and fracture repair. To mimic endochondral ossification, we devised a modified technique to perform intramedullary nailing of a mouse tibia followed by an induced fracture. To evaluate intramembranous ossification, we applied distraction osteogenesis to a mouse tibia using a mini Ilizarov external fixator apparatus. Histomorphometric parameters and gene expression differences in fracture repair between the mutant mice and the wild-type controls were measured using micro computed tomography, histology and reverse-transcription
Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny,
Despite the development of skeletal or mesenchymal stem cell (MSC) constructs aimed at creating viable cartilage and bone, few studies have examined the effects of cytokines present in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissues, or inhibition of these, on such constructs. This work addresses these issues using both in vitro and in vivo approaches and examines potential ways of overcoming the effects of cytokines on the integrity of cartilage and bone constructs. Synovial samples were obtained from RA or OA (n=10) patients undergoing elective hip or knee arthroplasty at Southampton General Hospital. Full ethical approval was obtained. Control bone marrow-derived stromal cells were obtained from patients undergoing emergency fractured neck of femur repair, cultured in basal, osteogenic (ascorbate and dexamethasone) and chondrogenic (transforming growth factor beta (TGFbeta3)) conditions. Differentiation towards bone and cartilage was assessed using alkaline phosphatase (ALP) staining, ALP and DNA biochemical assays and analysis of osteogenic/chondrogenic gene expression using
The giant cell tumor of bone (GCT) is a locally aggressive intraosseous neoplasm, with an uncertain biological behavior, constituted of giant multinuclear cells spread over tumoral tissue with a nucleus presenting the same features of the ovoid and fusiform cells forming its stroma. The local recurrence of GCT is often observed, mainly in the first three years after treatment, giving a rate of recurrence ranging in 20 to 50% of cases. Further aggravating the recurrence is the fact that after the relapse, the patient often also presents metastases in other organs. The aim of this study was to identify and to characterize differentially expressed genes that can be used in the prognostic, treatment and understanding of this physiopathology. To identify novel genes differentially expressed in GCT, we have applied rapid subtractive hybridization (RaSH). Samples of GCT and normal tissues were obtained at Tumor Bank of Barretos Cancer Hospital. After RNA extraction and cDNA synthesis the samples were submitted to Rapid hybridization Subtraction (RaSH) methodology for subtractive libraries elaboration. The RaSH subtractive libraries reveals the presence of 619 different clones including both normal and tumor tissues were identified. Of these, 450 in tumor sample and 169 in control tissue. Four biomarkers candidates were selected for validation: ZAK, KTN1, NEB, and ROCK1 genes, whose functions are, related to cell cycle checkpoint, transport of organelles, cytoskeletal matrix and cell adhesions. The validation of selected differentially expressed genes was performed using
Purpose: The osteogenic effects of BMPs on mesenchymal stem cells (MSCs) are less profound in human as compared to rodent. The mechanism for this phenomenon is unclear. This study evaluated the effects of macrophages on proliferation and BMP-2 induced osteogenic differentiation of human MSCs. Method: MSCs were isolated from human bone marrow. Human monocytes THP-1 (human acute monocytic leukemia cell line) were induced into macrophages by phorbol myristate acetate. The conditioned media (CM) from monocytes and macrophages were collected separately. After treated with CM from monocytes or macrophages for 5 and 7 days, the proliferation rate of human MSCs was determined by WST-8 assay. A group without CM served as control. Pretreated human MSCs were then induced towards osteogenic differentiation by osteoinductive medium supplemented with 0.1ug/ml BMP-2. Expression levels of osteogenic markers were determined by real-time
Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo. We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using
Osteoarthritis (OA) is a disabling joint disorder and mechanical loading is an important pathogenesis. This study aims to investigate the benefits of less mechanical loading created by intermittent tail suspension for knee OA. A post-traumatic OA model was established in 20 rats (12 weeks old, male). Ten rats were treated with less mechanical loading through intermittent tail suspension, while another ten rats were treated with normal mechanical loading. Cartilage damage was determined by gross appearance, Safranin O/Fast Green staining, and immunohistochemistry examinations. Subchondral bone changes were analyzed by micro-CT and tartrate-resistant acid phosphatase (TRAP) staining, and serum inflammatory cytokines were evaluated by enzyme-linked immunosorbent assay (ELISA).Aims
Methods
An ultra-high molecular weight polyethylene (UHMWPE) is widely used as bearing material in artificial joints, however, UHMWPE wear particles are considered to be a major factor in long-term osteolysis and loosening of implants. The wear particles activate macrophages, which release cytokines, stimulating osteoclasts, which results in bone resorption. The biological activity of the wear debris is dependent on the volume and size of the particles produced. Many researchers reported that the volume and size of particles were critical factors in macrophage activation, which particles in the size range of 0.1–1 mm being the most biological active. To minimize the amount of wear of UHMWPE and to enlarge the size of UHMWPE wear particle, a nano-level surface textured on Co-Cr-Mo alloy as a counterface material was invented (Figure 1). Although the generally-used surface for a conventional artificial joint has 10 nm roughness (G-1), the nano-level surface has a superfine surface of 1 nm with groove and dimples against the bearing area. The existence probability of groove or dimples, and their surface waviness were adjusted (P-1, 2, 3, 4 and W-1, 2). Pin-on-disc wear tester capable of multidirectional motions was used to verify that the nano-textured surface is the most appropriate for artificial joint. UHMWPE pin with an average molecular weight of 6.0 million was placed in contact with the disc and the contact pressure was 6.0 MPa. The disc and pin were lubricated by a water-based liquid containing the principal constituents of natural synovial fluid. Sliding speed of 12.12 mm/s had been applied for total sliding distance of 15 km. The nano-textured surfaces reduced the amount of UHMWPE wear, this would ensure the long-term durability of artificial joint (Figure 2). The wear particles isolated from lubricating liquid were divided broadly into two categories; one is “simple type” and the other is “complicated type”. The lengths in a longitudinal direction (Ll) and its orthogonal direction (Ls) for each particles (>150) were measured, and the each aspect ratio (= Ll/Ls) was calculated. No significant difference was found in the ratio between simple type and complicated type, and in the distributions of aspect ratios. However, the distributions of Ll, which means the size of UHMWPE wear particle, were dramatically changed by using the nano-textured surface (Figure 3). These results suggest that the nano-textured surface does not change the morphological aspect of UHMWPE particle but enlarges the size of UHMWPE particle. Cells (RAW264.7, blood, Mouse) were cultured with the particles in supplemented Dulbecco's modified Eagle's medium for 24 h in an atmosphere of 5% CO. 2. in air at 37 degrees C, and the
Purpose: Biomaterial-related infections continue to hamper the success of reconstructive and arthroplasty procedures in orthopaedic surgery. Staphylococci are the most common etiologic agents, with biofilm formation representing a major virulence factor. Environmental stress factors and sub-inhibitory concentration of some antibiotics have been identified to trigger staphylococcal biofilm formation through increased icaADBC expression. In staphylococci, production of polysaccharide intercellular adhesin (PIA) by the enzyme products of the icaADBC operon is the best understood mechanism of biofilm development, making the ica genes a potential target for biofilm inhibitors. Aims of the current study were. Determine the minimum inhibitory concentration (MIC) of Povidone-iodine. Investigate the effect of Povidone-iodine on icaADBC operon encoded staphylococcal biofilm formation. Investigate wether any observed changes on biofilm by Povidone-iodine is mediated through a change in icaADBC operon. Method: MIC of povidone – iodine for both reference strains and strains isolated from infected orthopaedic implants was determined. Biofilm assay was performed at different Povidone-iodine concentrations using 96-well polystyrene plates. Total RNA for cDNA synthesis was isolated from bacteria at different twofold dilutions of Povidone-iodine concentrations.
Despite the recent progress, non-metastatic pediatric osteosarcomas have now a 5-year overall survival (OS) around 75% and the metastatic forms are decreasing to 20–30%. To increase these survival rates, new molecular approaches are on development to understand and highlight new candidates for targeted therapies. Tyrosine kinase receptors (TKR) are one of this target class, where new drugs were especially developped, screening now a large spectrum of TKR. After the demonstration among cancers of TKR’s clinical utility as surrogate markers to guide the selection of patients susceptible to respond to these treatments, this success was recently tempered in part because of cancers developping resistance mechanisms to these drugs. A study was conducted to evaluate the interest of these molecular targets among pediatric osteosarcomas. Materiel and methods: 91 pediatric patients treated homogeneously with the French OS94 protocol were included in this analysis. Allelotyping, real-time
Dysregulation of differentiation genes involved in developmental signaling pathways seems to be a decisive event taking part in the multistep oncogenesis. As high grade osteosarcomas are histologically defined by the presence of malignant osteoblasts producing an osteoid component, we focused in a pediatric cohort, homogeneously treated with the French OS94 protocol, on the genomic status at diagnosis on tumor biopsies of several genes involved in flat and long bone formation. Material and methods: In 91 pediatric osteosarcomas, allelotyping analysis of FGFRs, TWIST, DERMO1, APC, MET, HGF, and SDC2 was done. After DNA extraction of paired blood and tumor samples, each locus was analysed by microsatellites bordering closely on each side the targeted genes. Complementary real-time
To investigate whether idiopathic osteonecrosis of the femoral head (ONFH) is related to impaired osteoblast activities. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head and the intertrochanteric region of patients with idiopathic ONFH, or from the intertrochanteric region of patients with osteoarthritis (OA), and compared their viability, mineralization capacity, and secretion of paracrine factors.Aims
Methods
The effect of the gut microbiota (GM) and its metabolite on bone health is termed the gut-bone axis. Multiple studies have elucidated the mechanisms but findings vary greatly. A systematic review was performed to analyze current animal models and explore the effect of GM on bone. Literature search was performed on PubMed and Embase databases. Information on the types and strains of animals, induction of osteoporosis, intervention strategies, determination of GM, assessment on bone mineral density (BMD) and bone quality, and key findings were extracted.Aims
Methods
Purpose: Current techniques for articular cartilage repair remain suboptimal. The best technique involves the introduction of cultured chondrocytes into the injury site. Experimental results of current chondrocyte culture and expansion techniques (passaging) have shown phenotypic alteration resulting in fibroblast-like cells. Therefore, treatment methods that propose the transplantation of cultured chondrocytes might be transplanting fibroblast-like cells instead of chondrocytes. This experiment explored the difference in genetic expression of chondrocytes left at confluence compared to chondrocytes that were passaged as performed in current culture techniques. It was hypothesized that chondrocytes left at confluence would maintain their collagen I and collagen II gene expression over time. Method: Fresh normal human articular cartilage was collected from deceased donor patients. The matrix was digested and the chondrocytes were plated in monolayer to create two groups. The first group was cultured and passaged 2? at confluence seven times. The second group was cultured at confluence and left for seven weeks, with medium changes every 3–4 days without passaging. At weekly intervals RNA was extracted from cells in both groups and analyzed with
Bone morphogenetic proteins (BMPs) are able to induce osteogenic differentiation in many cells, including muscle cells. However, the actual contribution of muscle cells to bone formation and repair is unclear. Our objective was to examine the capacity of myogenic cells to contribute to BMP-induced ectopic bone formation and fracture repair. Osteogenic gene expression was measured by
Purpose of study: To investigate the contribution of 11p15 defects to hemi-hypertrophy (HH) and clarify the potential association with Wilms’ tumour. Methods: Clinical data and blood/DNA samples were collected from patients with hemi- hypertrophy and from their parents. Where normal/abnormal tissue samples were available, fibroblast DNA was also analysed. Recruitment criteria included any patient with growth asymmetry. All patients were analysed for uni-parental disomy of 11p15 by
The osteoclastogenesis is regulated by a complex signaling system between the pro-apoptotic factors (Bax-Cyclin E2-Cdk2) and the tumor necrosis factor family (RANKL-RANK-OPG). Extracorporeal Shock Waves Therapy (ESWT) have recently been used in orthopaedic treatments to induce bone repair, but their mechanisms of action are not sufficiently investigated. So we studied the effect of shock-waves on murine osteoblastic cells. Osteoblast cultures were subjected to a single shock-wave with combinations of low energy intensities (0.05mJ/mm2) and 500 number of shocks (impulses), whereas control cells received no treatment. We valued the cell viability quantifying the expressions of Bax and Opg by PCR. We found an immediate negative effect on cell viability, that occurs with an increase of Bax protein expression after 3 hours of treatment. After a longer time lapse a stimulatory effect on cell proliferation, as reflected by the increase of a G(1)-S phase marker, was observed. In fact, in the following 24, 48 and 72 hours after ESW treatment, we found a stronger association of Cyclin E2 and Cdk2, forming active cyclin E-Cdk2 kinase, compared to untreated cells at the same times. We further explored the molecular mechanism for the ESW induction of osteogenesis: by
Purpose. The data regarding the effects of noggin on bone morphogenetic protein (BMP)-induced osteogenesis of mesenchymal stem cells (MSCs) are controversial. Most studies performed in rodent cells/models indicated that noggin was a negative regulator of BMP-2-induced osteogenesis; however, one study conducted with human MSCs in culture showed that the addition of noggin induced osteogenesis in vitro. To clear the controversy, we designed this study to evaluate the effects of knocking down noggin gene expression on BMP-2-induced osteogenesis of human bone marrow-derived primary MSCs in vitro. Method. MSCs were isolated from human tibial bone marrow by density gradient centrifugation. Two noggin small interfering RNAs (siRNAs) were used in this study to knockdown noggin gene expression. There were four study groups: MSCs with no transfection of siRNA (named as NT group), MSCs transfected with non-targeting negative control siRNA (named as control group), MSCs transfected with noggin siRNA1 (named as NOGsi1 group), and MSCs transfected with noggin siRNA2 (named as NOGsi2 group). After transfection, MSCs were induced to undergo osteogenic differentiation by incubating in basal medium containing 0.1 μg/ml BMP-2 for 35 days. The expression levels of osteoblastic marker genes were measured by real-time
Aims: Extracorporeal Shock Waves Therapy (ESWT) has recently been used in orthopaedic treatments to induce bone repair, but their mechanisms of action are not sufficiently investigated. So we studied the effect of shockwaves on murine osteoblastic cells. Methods: Osteoblast cultures were subjected to a single shockwave with combinations of low energy intensities (0,05mJ/mm2) and 500 number of shocks (impulses), whereas control cells received no treatment. Cell division and apoptosis are interconnected and regulated by cyclins, kinases, Bax and Opg. We valued the cell viability quantifying by PCR the expressions of these molecules which regulate cell-cycle. Results: We found an immediate negative effect on cell viability, that occurs with an increase of Bax protein expression after 3 hours of treatment. After a longer time lapse a stimulatory effect on cell proliferation, as reflected by the increase of a G(1)-S phase marker, was observed. In fact, in the following 24, 48 and 72 hours after ESW treatment, we found a stronger association of Cyclin E2 and Cdk2, forming active cyclin E-Cdk2 kinase, compared to untreated cells at the same times. We further explored the molecular mechanism for the ESW induction of osteogenesis: by
In tissue engineering, scaffolds are vitalized by cells in vitro. Human mesenchymal stem cells (hMSC) are very interesting because of their ability to differentiate towards the osteogenic lineage and their self renewing capacity. Yet, it is important that implanted cells do not disseminate and exhibit unwanted cell growth outside the implantation site. Therefore the aim of this study was to detect migrated cells in organs of mice after implantation of a composite (cell-scaffold) substitute. HMSC (Cambrex, USA) were inoculated on a clinically approved 3D scaffold (Tutobone(TM), Tutogen, Germany). One composite and one scaffold without cells were implanted subcutanously, left and right paravertebrally in athymic nude mice (nu/nu). After 2, 4, 8 and 12 weeks constructs were explanted and organs (liver, spleen, lungs, kidney, heart, testicles, brain and blood) were harvested. The entire organs were homogenized and genomic DNA was isolated for qualitative and
Background: The success of the increasing number of arthroplasty, spinal instrumentation and other implanted orthopaedic devices is hampered by device-related infections. More than half of these infections are caused by staphylococcal biofilm mediated antibiotic resistance. The hope of preventing prosthetic joint infection by antibiotic loaded cement is threatened by emerging resistant organisms. No bacterial resistance to betadine has been reported. Current intervention strategy is focussed on prevention of initial device colonisation and inhibition of genes encoding biofilm formation. Aim:. Determine the minimum inhibitory concentration (MIC) of betadine. Investigate the effect of betadine on icaADBC operon encoded staphylococcal biofilm formation. Investigate wether betadine can prevent bacterial adherence and biofilm formation by inhibition of the encoding genes. Methods: MIC of betadine for both reference strains and strains isolated from infected orthopaedic implants was determined. Biofilm assay was performed at different betadine concentrations using 96-well polystyrene plates. Total RNA for cDNA synthesis was isolated from bacterial at different twofold dilutions of betadine concentrations.
The aim of our study is to investigate the effect induced by alternated mechanical loading on Notch-1 in mandibular condylar cartilage (MCC) of growing rabbits. A total of 64 ten-day-old rabbits were randomly divided into two groups according to dietary hardness: normal diet group (pellet) and soft diet group (powder). In each group, the rabbits were further divided into four subgroups by feeding time: two weeks, four weeks, six weeks, and eight weeks. Animals would be injected 5-bromo-2′-deoxyuridine (BrdU) every day for one week before sacrificing. Histomorphometric analysis of MCC thickness was performed through haematoxylin and eosin (HE) staining. Immunochemical analysis was done to test BrdU and Notch-1. The quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure expression of Notch-1, Jagged-1, and Delta-like 1 (Dll-1).Aims
Methods
Introduction: Chondral injuries of the knee are commonly seen at arthroscopy, yet there is no consensus on the most appropriate treatment method. However, untreated cartilage injury predisposes to osteoarthritis contributing to pain and disability. For cell-based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient cells for therapeutic intervention. Although recent reports demonstrated the central role of oxygen in the function and differentiation of chondrocytes, little is known of the effect of physiological low oxygen concentrations during the expansion of the cells and whether this alters their chondrogenic capacity. Methods: Articular mouse chondrocytes were prepared from the distal femoral condyles of adult mice and chondrocytes were liberated by collagenase type II treatment. Cells were cultured in RPMI 1640 media in monolayer under normoxic or hypoxic conditions (5% O2). Chondrogenic potential was subsequently assessed by plating the cells under micromass conditions and glycosaminoglycan deposition was determined by alcian blue staining. Having determined that oxygen tension infiuences murine chondrocyte expansion and differentiation, similar studies were conducted using adult human chondrocytes taken from knee arthroplasty off-cuts, and Aggrecan (ACAN) gene expression was analyzed using real-time
An ultra-high molecular weight polyethylene (UHMWPE) is widely used as bearing material in artificial joints, however, UHMWPE wear particles are considered to be a major factor in long-term osteolysis and loosening of implants. The wear particles activate macrophages, which release cytokines, stimulating osteoclasts, which results in bone resorption. The biological activity of the wear debris is dependent on the volume and size of the particles produced. Many researchers reported that the volume and size of particles were critical factors in macrophage activation, which particles in the size range of 0.1–1 mm being the most biological active. To minimize the amount of wear of UHMWPE and to enlarge the size of UHMWPE wear particle, a nano-level surface texturing on Co-Cr-Mo alloy as a counterface material was invented. Although the generally-used surface for a conventional artificial joint has 10 nm roughness (Surface A), the nano-level textured surface invented has a superfine surface of 1 nm with 3% of groove and dimples against the bearing area. The depths of groove and dimples are less than 50 nm (Surface F). Pin-on-disc wear tester capable of multidirectional motions was used to verify that the nano-textured surface is the most appropriate for artificial joint. UHMWPE pin with an average molecular weight of 6.0 million was placed in contact with the disc and the contact pressure was 6.0 MPa. The disc and pin were lubricated by a water-based liquid containing the principal constituents of natural synovial fluid. Sliding speed of 12.12 mm/s had been applied for total sliding distance of 15 km. The superfine surface with nano-level grooves and dimples (Surface F) reduced the amount of UHMWPE wear, this would ensure the long-term durability of artificial joint. The wear particles isolated from lubricating liquid were divided broadly into two categories; one is “simple type” and the other is “complicated type”. The lengths in a longitudinal direction (Ll) and its orthogonal direction (Ls) for each particles (>150) were measured, and the each aspect ratio (= Ll/Ls) was calculated. No significant difference was found in the ratio between simple type and complicated type, and in the distributions of aspect ratios. However, the distributions of Ll, which means the size of UHMWPE wear particle, were dramatically changed by using the nano-textured surface (Figure 2). These results suggest that the nano-textured surface does not change the morphological aspect of UHMWPE particle but enlarges the size of UHMWPE particle. Cells (RAW264.7, blood, Mouse) were cultured with the particles in supplemented Dulbecco's modified Eagle's medium for 24 h in an atmosphere of 5% CO. 2. in air at 37 degrees C, and the
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. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology.Aims
Methods
