Objectives. The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI). Methods. The 16s rDNA test combines a
A retrospective series of 45 cases of chronic osteomyelitis collected over a period of 14 years was histologically classified into tuberculous osteomyelitis (25) and chronic non-granulomatous osteomyelitis (20). The tuberculous osteomyelitis group was divided into three subgroups: a) typical granulomas (13 cases); b) ill-defined granulomas (seven cases), and c) suspected granulomas (five cases). An in-house
Objectives. Studies which consider the molecular mechanisms of degeneration and regeneration of cartilaginous tissues are seriously hampered by problematic ribonucleic acid (RNA) isolations due to low cell density and the dense, proteoglycan-rich extracellular matrix of cartilage. Proteoglycans tend to co-purify with RNA, they can absorb the full spectrum of UV light and they are potent inhibitors of
Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time
There is still no consensus on which concentration of mesenchymal stem cells (MSCs) to use for promoting fracture healing in a rat model of long bone fracture. To assess the optimal concentration of MSCs for promoting fracture healing in a rat model. Wistar rats were divided into four groups according to MSC concentrations: Normal saline (C), 2.5 × 106 (L), 5.0 × 106 (M), and 10.0 × 106 (H) groups. The MSCs were injected directly into the fracture site. The rats were sacrificed at 2 and 6 자 post-fracture. New bone formation [bone volume (BV) and percentage BV (PBV)] was evaluated using micro-computed tomography (CT). Histological analysis was performed to evaluate fracture healing score. The protein expression of factors related to MSC migration [stromal cell-derived factor 1 (SDF-1), transforming growth factor-beta 1 (TGF-β1)] and angiogenesis [vascular endothelial growth factor (VEGF)] was evaluated using western blot analysis. The expression of cytokines associated with osteogenesis [bone morphogenetic protein-2 (BMP-2), TGF-β1 and VEGF] was evaluated using real-time
Objectives. This study aimed to examine the effects of SRT1720, a potent SIRT1 activator, on osteoarthritis (OA) progression using an experimental OA model. Methods. Osteoarthritis was surgically induced by destabilization of the medial meniscus in eight-week-old C57BL/6 male mice. SRT1720 was administered intraperitoneally twice a week after surgery. Osteoarthritis progression was evaluated histologically using the Osteoarthritis Research Society International (OARSI) score at four, eight, 12 and 16 weeks. The expression of SIRT1, matrix metalloproteinase 13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5), cleaved caspase-3, PARP p85, and acetylated nuclear factor (NF)-κB p65 in cartilage was examined by immunohistochemistry. Synovitis was also evaluated histologically. Primary mouse epiphyseal chondrocytes were treated with SRT1720 in the presence or absence of interleukin 1 beta (IL-1β), and gene expression changes were examined by real-time
Ruling out an infection in one-stage knee and hip revisions for presumed aseptic failure by conventional tissue cultures takes up to 14 days. Multiplex
Intervertebral disc degeneration (IDD) affects more than 80% of the population all over the world. Current strategies for the treatment of IDD are based on conservative or surgical procedures with the aim of relieving pain. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy in recent decades, but studies showed that the particularly hostile microenvironment in the intervertebral disc (IVD) can compromise cells survival rate. The use of exosomes, extracellular vesicles released by various cell types, possess considerable economic advantages including low immunogenicity and toxicity. Exosomes allow intercellular communication by conveying functional proteins, RNA, miRNA and lipids between cells. The purpose of this study is to assess the therapeutic effects of exosomes derived from Wharton Jelly mesenchymal stromal cells (WJ-MSC) on human nucleuspulposus cells (hNPC) in an in vitro 3D culture model. Exosomes (exos) were isolated by tangential flow filtration of WJ-MSC conditioned media and characterized by: quantification with BCA test; morphological observation with TEM, surface marker expression by WB and size evaluation by NTA. Confocal microscopy has been used to identify exosomes marked with PKH26 and monitor fusion and/or incorporation in hNPC. hNPC were isolated from waste surgical material from patients undergoing discectomy (n = 5), expanded, encapsulated in alginate beads and treated with: culture medium (control group); WJ-MSC exos (WJ-exos) at different concentrations (10 μg/ml, 50 μg/ml and 100 μg/ml). They were then analysed for: cell proliferation (Trypan Blu); viability (Live/Dead Assay); quantification of nitrites (Griess) and glycosaminoglycans, GAG (DMBB). The hNPC in alginate beads treated for 7 days were included in paraffin and histologically analysed to determine the presence of extracellular matrix (ECM) components. Finally, the expression levels of catabolic and anabolic genes were evaluated through real-time
Introduction:. Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation. METHODS:. The hNPCs, isolated from discectomy surgical waste material (n = 5), were expanded and encapsulated in alginate beads. The hNPCs were treated with: i) only growth medium (control); ii) medium with recombinant irisin (r-IR) at different concentrations (5, 10 and 25 ng / mL); iii) medium with Interleukin-1β (IL1β); iv) medium with IL1β for 24 h and then with IL1β and r-IR; v) medium with r-IR for 24 h and then with r-IR and IL1 β. We evaluated proliferation (trypan blue and PicoGreen), metabolic activity (MTT), nitrite concentration (Griess), and expression levels of catabolic and anabolic genes via real-time
Objectives. To determine the pattern of mutations of the WISP3 gene in clinically identified progressive pseudorheumatoid dysplasia (PPD) in an Indian population. Patients and Methods. A total of 15 patients with clinical features of PPD were enrolled in this study. Genomic DNA was isolated and
In the last decade, skeletal muscle has been recognized as an endocrine organ able to release molecules that may act as paracrine or endocrine factors, namely myokines. Among these, irisin is secreted upon muscle contraction after physical exercise (PE) and has been demonstrated to yield anabolic effects on different cell types. Recently, irisin has been shown to improve cortical bone mass, geometry and strength, hence resembling the effect of PE. It has also been reported that irisin levels in the serum and synovial fluid of patients with knee osteoarthritis (OA) were negatively correlated with OA severity. Therefore, we hypothesized that irisin may improve cartilage metabolism and blunt the osteoarthritic process. Human osteoarthritic chondrocytes (hOAC) were isolated from osteochondral specimens of patients undergoing total knee joint replacement. After in vitro expansion, hOAC were put in a three-dimensional culture system (alginate beads) and treated with either phosphate-buffered saline (control) or irisin (25 ng/mL). After 1 week, the amount of glycosaminoglycans (GAG) was evaluated using dimethylmethylene blue (DMMB) and PicoGreen assays. Quantitative real-time
Objectives. Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Materials and Methods. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time
Hypoxic Inducible Factor and Hypoxic mimicking agents (HMA) trigger the initiation and promotion of angiogenic-osteogenic cascade events. However, there has been paucity of studies investigating how HIF could be over expressed under chronic hypoxic conditions akin to that seen in sickle cell disease patients to help form a template for tackling the matter of macrocellular avascular necrosis. Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration, and the hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process studies have shown. There are still no established experimental models showing how this knowledge can be used for the evaluation of bone implant integration and suggest ways of improving osseointegration in sickle cell disease patients with hip arthroplasty and thereby prevent increased implant loosening. The aim of this study is to help develop an in vitro experimental model which would mimic the in vivo pathologic state in the bone marrow of sickle cell disease patients. It also seeks to establish if the hypoxic inducible factor (HIF) could be over expressed in vitro and thus enhancing osseointegration. MG63 osteoblastic cells were cultured under normoxia and hypoxic conditions (20%; and 1% oxygen saturation) for 48 and 72 hours. Cobalt chloride was introduced to the samples in order to mimic true hypoxia. Cells cultured under normoxic conditions and without cobalt chloride was used as the control in this study. The expression of the hypoxic inducible factor was assessed using the reverse transcriptase qualitative
Objectives. Osteophytes are products of active endochondral and intramembranous ossification, and therefore could theoretically provide significant efficacy as bone grafts. In this study, we compared the bone mineralisation effectiveness of osteophytes and cancellous bone, including their effects on secretion of growth factors and anabolic effects on osteoblasts. Methods. Osteophytes and cancellous bone obtained from human patients were transplanted onto the calvaria of severe combined immunodeficient mice, with Calcein administered intra-peritoneally for fluorescent labelling of bone mineralisation. Conditioned media were prepared using osteophytes and cancellous bone, and growth factor concentration and effects of each graft on proliferation, differentiation and migration of osteoblastic cells were assessed using enzyme-linked immunosorbent assays, MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assays, quantitative real-time
Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold. To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm. 3. ) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time
While stable long-term clinical results have been achieved in total joint arthroplasty, periprosthetic joint infection (PJI) has been actualized as difficult issue in this decade. For accurate diagnosis, it is important to establish standard criteria such as MSIS criteria, and it is prevailing now. As an issue involving PJI, however, the existence of viable, but non-culturable (VNC) bacteria must be noticed. It is difficult to identify the VNC state infection, because microbiologic culture result shows negative and other markers tend to be negative. Here, molecular diagnosis based on
Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines/chemokines e.g. CCL3 and CCL4. The aim of this study was to evaluate whether TLR4-specific neutralising antibodies can prevent cobalt-mediated activation of TLR4. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with two different TLR4-specific monoclonal antibodies followed by 0.75mM of cobalt chloride (CoCl2). Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess CCL3/CCL4 protein secretion and real time-
Background. Rotator cuff disease (RCD) is the most common cause of shoulder pain and limitation of activities in sports and in repetitive work. The aetiology of RCD is not well established. A number of gene pathways are altered in RCD. Polymorphisms in Col1A1, Col5A1 (encoding collagen) and GDF5 (TGF-beta superfamily) can be associated with RCD susceptibility. Materials and Methods. Single-nucleotide polymorphisms (SNPs) in Col1A1, GDF5 and Col5A1 were genotyped in a case-control study with 103 RCD patients and 104 controls in Caucasian and African populations who suffered from injuries in any other anatomical location. All patients provided signed informed consent. Sampling was carried out with a puncture of the pad of a finger using a sterile, single-use lancet. NSPs were determined by real-time
Background. Despite the known multifactorial nature of scaphoid wrist fracture non-union, a possible genetic predisposition for the development of this complication remains unknown. This pilot study aimed to address this issue by performing Single Nucleotide Polymorphisms (SNPs) analysis of specific genes known to regulate fracture healing. Materials and Methods. We reviewed 120 patients in a retrospective case-control study from the Hand Surgery Department of Asepeyo Hospital. The case group comprised 60 patients with confirmed scaphoid wrist non-union, diagnosed by Magnetic Resonance Imaging (MRI) and Computed Tomography (CT). The control group comprised 60 patients with scaphoid fracture and complete bone consolidation. Sampling was carried out with a puncture of a finger pad using a sterile, single-use lancet. SNPs were determined by real-time
We have developed precision-engineered strontium eluting nanopatterned surfaces. Nanotopography has been shown to increase osteoblast differentiation, and strontium is an element similar to calcium, which has been proven to increase new bone formation and mineralization. This combination has great potential merit in fusion surgery and arthroplasty, as well as potential to reduce osteoporosis. However, osteoclast mediated osteolysis is responsible for the aseptic failure of implanted biomaterials, and there is a paucity of literature regarding osteoclast response to nanoscale surfaces. Furthermore, imbalance in osteoclast/osteoblast resorption is responsible for osteoporosis, a major healthcare burden. We aimed to assess the affect of strontium elution nanopatterned surfaces on osteoblast and osteoclast differentiation. We developed a novel human osteoblast/osteoclast co-culture system without extraneous supplementation to closely represent the in vivo environment. We assessed the surfaces using electron microscopy (SEM), protein expression using immunofluorescence and histochemical staining and gene expression using
