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 polymerase chain reaction (PCR) is a quick test (4–5 hours) for detecting infections. The purpose of this study was to evaluate the negative predictive value of an automated multiplex PCR for the detection of microorganisms in synovial fluid obtained intraoperatively in unsuspected knee and hip revisions. The NPV of the multiplex PCR U-ITI system of synovial fluid compared to tissue cultures of knee and hip revisions was 95.7% and 92.5%, respectively. Cultures required several days for growth whereas the automated mPCR U-ITI system provided results within five hours. The multiplex PCR U-ITI system is a quick and reliable test in ruling out infection in presumed aseptic knee and hip revisions. With this test the number of unsuspected infected revisions can be lowered and antibiotic overtreatment as well as undertreatment after one-stage revision arthroplasty can be avoided. This directly results in a reduction in length of hospital stay, hospital costs and possible antibiotic resistance development

Abstract. Background. Progressive muscle ischaemia results in reduced aerobic respiration and increased anaerobic respiration, as cells attempt to survive in a hypoxic environment. Acute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency resulting in the production of Lactic acid by cells through anaerobic respiration. Our previous research has shown that it is possible to measure H+ ions concentration (pH) as a measure of progressive muscle ischaemia (in vivo) and hypoxia (in vitro). Our aim was to correlate intramuscular pH readings and cell viability techniques with the intramuscular concentration of key metabolic biomarkers [adenosine triphosphate (ATP), Phosphocreatine (PCr), lactate and pyruvate], to assess overall cell health in a hypoxic tissue model. Methods. Nine euthanised Wistar rats were used in a non-circulatory model. A pH catheter was used to measure real-time pH levels from one of the exposed gluteus medius muscles, while muscle biopsies were taken from the contralateral gluteus medius at the start of the experiment and subsequently at every 0.1 of a pH unit decline. The metabolic biomarkers were extracted from the snap frozen muscle biopsies and analyzed with standard fluorimetric method. Another set of biopsies were stained with Hoechst 33342, Ethidium homodimer-1 and Calcein am and imaged with a Zeiss LSM880 confocal microscope. Results. Our study shows that the direct pH electrode readings decrease with time and took an average of 69 minutes to drop to a pH of 6.0. The concentrations of ATP, pyruvate and PCr declined over time, and the concentration of lactate increased over time. At pH 6.0, both ATP and PCr concentrations had decreased by 20% and pyruvate has decreased by 50%, whereas lactate had increased 6-fold. The majority of cells were still viable at a pH of 6.0, suggesting that skeletal muscle cells are remarkably robust to hypoxic insult, although this was a hypoxic model where reperfusion was not possible. Conclusions. Our research suggests that histologically, skeletal muscle cells are remarkably robust to hypoxic insult despite the reduction in the total adenine nucleotide pool, but this may not reflect the full extent of cell injury and quite possibly irreversible injury. The timely restoration of blood flow in theory should halt the hypoxic insult, but late reperfusion results in cellular dysfunction and cell death due to localised free radical formation. Further research investigating the effects of reperfusion in vivo are warranted, as this may identify an optimal time for using pharmacological agents to limit reperfusion injury, around the time of fasciotomy to treat acute compartment syndrome. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Previous studies showed that telo-peptides degraded from type II collagen, a type of collagen fragments, could induce cartilage damage in bovine stifle joints. We aim to investigate the role of integrins (ITGs) and matrix metalloproteinases (MMPs) in collagen fragment-induced human cartilage damage that is usually observed in osteoarthritis (OA). We hypothesized that N-telopeptide (NT) derived from type II collagen could up-regulate the expression of β1 integrin (ITGB1) and then MMPs that may lead to osteoarthritic cartilage damage. Human chondrocytes were isolated from femoral head or tibial plateau of patients receiving arthroplasty (N = 24). Primary chondrocyte cultures were either treated with 30 µM NT, or 30 µM scrambled NT (SN), or PBS, or left untreated for 24 hrs. Total proteins and RNAs were extracted for examination of expression of ITGB1 and MMPs-3&13 with Western blotting and quantitative real-time PCR. Compared to untreated or PBS treated chondrocytes, NT-treated chondrocytes expressed significantly higher levels of ITGB1 and MMPs-3&-13. However, SN also up-regulated expression of ITGB1 and MMP-13. ITGB1 and MMPs-3&-13 might mediate the catalytic effect of NT, a type of collagen fragments, on human cartilage damage that is a hallmark of OA

Fracture healing is an issue that has not yet been fully elucidated. It is generally accepted in the literature that head trauma accelerates fracture healing and causes higher volume callus tissue. Recent studies have examined the relationship between head trauma and fracture healing more molecularly. Based on this research; the aim of this study is to show the effect of head trauma on fracture healing radiologically and histologically and to investigate the relationship between serum β-Catenin level and fracture healing with the experiment we performed on rats. A total of 36 Wistar Albino female rats with a mean age of 24 weeks were included in the study with the permission of Mersin University Animal Experiments Local Ethics Committee. Six rats in the first group were not traumatized and their blood samples were collected on the day of the experiment started, end of the third week and end of the sixth week. In the second group, only head trauma was performed and blood samples were collected at the end of the third and sixth weeks. In the third group, only open femoral fracture model was applied, blood samples were collected at the third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. In the fourth group, both head trauma and open femur fracture model were applied, blood samples were collected at the end of third and sixth weeks and AP and Lateral radiographs of the fractured femurs were taken. After sacrification, femurs were dissected from the surrounding soft tissues and subjected to histological examination. The expression level of β-Catenin was measured by PCR from all blood samples. Direct radiographs of the third and fourth groups at 3 and 6 weeks were evaluated by two orthopedists according to Rust and Lane & Sandhu scoring system. The histomorphometric examination was performed by evaluating the Huo scoring and the ratio of fracture callus components (cartilage callus, bone callus, fibrous callus) to areas. According to PCR analysis, the change of expression of β-Catenin by weeks was not statistically significant in the first and second groups. However, a statistically significant decrease was observed in the 0–6 week interval in the third and fourth groups (p = 0.002, p <0.0001, respectively). In the radiological examination, the union scores of the rats with head trauma + femoral fracture were higher than the isolated femoral fractures at 3 weeks and 6 weeks. In histomorphometric examination, no statistically significant difference was found between head trauma + femur fracture group and isolated femur fracture group. In addition, there was no correlation between the groups in the correlation studies between radiological findings, histomorphmetric findings and PCR findings. Considering that each molecule involved in fracture healing processes has a time interval and concentration; We concluded that the expression levels of β-catenin can be repeated in smaller time periods including the early stages of fracture healing

Osteosarcoma is common in children and adolescents with high mortality due to rapid progression. Therapeutic approaches for osteosarcoma are limited and may cause side effects. Cannabinoid ligands exert antiproliferative, apoptotic effect in cancer cells via CB1/2 or TRPV1 receptors. In this study, we hypothesized that synthetic specific CB2R agonist CB65 might have an antiproliferative and apoptotic effect on osteosarcoma cell lines in vitro. If so, this agent might be a chemotherapeutic candidate for osteosarcoma, with prolonged release, increased stability and bioavailability when loaded into a liposomal system. We first determined CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT- PCR and FCM. CB65 reduced proliferation in osteosarcoma cells by WST-1 and RTCA. IC50 for MG63 and Saos-2 cells were calculated as 1.11×10-11 and 4.95×10-11 M, respectively. The antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. IC50 of CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively by FCM. CB65 was loaded into the liposomal system by thin film hydration method and particle size, polydispersity index, and zeta potentials were 141.7±0.6 nm, 0.451±0.026, and -10.9±0.3 mV, respectively. The CB65-loaded liposomal formulation reduced MG63 and Saos-2 cell proliferation by RTCA. IC50 of CB65 and CB65-loaded liposomal formulation induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 hours, respectively, by FCM. Scratch width was higher in CB65 and CB65-loaded liposome-treated cells compared to control. In this study, the real-time antiproliferative and apoptotic effect of synthetic specific CB2 agonist CB65 in osteosarcoma cell lines was demonstrated for the first time, and the real time therapeutic window was determined. The CB65-loaded liposomal formulation presents a potential treatment option that can be translated to clinic following its validation within animal models and production under GMP conditions

Introduction and Objective. Osteoarthritis (OA) represents one of the leading cause of disability all over the world. Cell therapies, mainly based on mesenchymal stem cells (MSCs), have shown to modulate the pathogenesis of OA in basic, preclinical and clinical studies. Adipose tissue (AT) have emerged as a rich and promising source of MSCs called adipose derived stem cells (ASCs). Different systems are available for processing lipoaspirate to purify the samples from oily and haemorrhagic fractions, minimizing the risk of complications and maximizing the biological yield for subsequent grafting. However, few studies compared the efficacy of the different processing devices already used in clinical practice. This study aims to characterize the products obtained by the use of two different systems such as micro-fragmentation or nano-fragmentation comparing them with the starting material (AT) and the collagenase isolated ASCs. Materials and Methods. AT from 12 donors arrived without selection to the laboratories: 4 lipoaspirated (LA), 4 micro-fragmented (mF) and 4 nano-fragmented (nF). The samples were divided into three aliquots for paraffin embedding, RNA extraction and digestion with collagenase for ASCs isolation. Paraffin embedded tissue sections were stained with hematoxylin-eosin to analyze morphology. RNA was extracted, retro-transcribed and analyzed with real-time PCR to analyze the expression of pluripotency genes (SOX2, NANOG and POU5F1) and inflammatory genes (IL-1beta and iNOS). Data were analyzed using Graphpad Prism 8.0 and expressed as mean ± SD. One-way ANOVA followed by Tukey test was used to compare the different groups. Results. The LA comprised small lobules, with intact cell membranes and structurally integer adipocytes. mF samples showed the presence of integer adipocytes, small lobules and higher amount of cell clusters. nF samples showed the almost completely absence of adipocytes, a high amount of cells without lipid content and a high amount of stromal matrix. Real-time PCR results showed the lowest expression levels of pluripotency genes in LA samples that were assumed equal to 1.0 and used to calculate the expression levels of the other samples. mF showed expression levels of pluripotency genes similar to AT. nF showed expression levels of pluripotency genes higher than AT and mF, but without statistically significant differences. ASCs showed statistically significant higher expression levels of these genes compared to LA and mF (p ≤ 0.001). Likewise, the expression of inflammatory genes resulted to be lowest in LA samples (assumed equal to 1.0), higher in mF samples and in nF samples without statistical significance. As expected, the highest values were found in ASCs isolated cells compared to all the other samples (p ≤ 0.0001). Conclusions. These results confirmed that micro-fragmentation (mF) and nano-fragmentation (nF) permitted to separate a cell mixture enriched in ASCs from a lipoaspirate sample without activating the inflammatory pathways. Both processing methods gave a minimally manipulated product suitable for OA cell therapy application. Further studies are needed to elucidate possible different activities of the ASCs enriched AT-derivatives

Introduction. The incidences of fragility fractures, often because of osteoporosis, are increasing. Research has moved towards bioresorbable scaffolds that provide temporary mechanical stability and promote osteogenesis. This research aims to fabricate a 3D printed composite Poly (l-lactic-co-glycolic acid)-strontium doped tricalcium phosphate (PLGA-SrTCP) scaffold and evaluate in an in vitro co culture study containing osteoporotic donor cells. Method. PLGA, PLGA TCP, and PLGA SrTCP scaffolds were produced using Fused Filament Fabrication (FFF). A four-group 35-day cell culture study was carried out using human bone marrow derived mesenchymal stem cells (hMSCs) from osteoporotic and control donors (monoculture) and hMSCs & human monocytes (hMCs) (Co culture). Outcome measures were biochemical assays, PCR, and cell imaging. Cells were cultured on scaffolds that had been pre-degraded for six weeks at 47°C prior to drying and gamma sterilisation. Result. 3D printed scaffolds were successfully produced by FFF. All groups in the study supported cell attachment onto the scaffolds, producing extracellular matrices as well as evidence of osteoclast cell structures. Osteoporotic cells increased CTSK activity and CAII activity and decreased ALP activity compared to controls. In control cultures, the addition of bTCP and bTCP/Sr to the PLGA reduced TRAP5b, CAII and ALP activity compared to PLGA alone. The addition of Sr did not show any differences between donors. Conclusion. This study details suitability of 3D printed polymer scaffolds for use in bone tissue applications. Both composite and pure polymer scaffolds promote osteogenesis in vitro. The introduction of ceramic filler and ion doping does not beneficially effect osteogenic potential and can reduce its ability compared to pure polymer. This study suggests the behaviour of control and osteoporotic cells are different and that osteoporotic cells are more prone to bone resorption. Therefore, it is important to design bone scaffolds that are specific to the patient as well as to the region of fracture

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 polymerase chain reaction (PCR) has certain role as potential diagnostic tools for such VNC infection. We have applied a real-time PCR system for the diagnosis of PJI, which is able to detect methicillin-resistant Staphylococcus (MRS) and distinguish gram-positive from gram-negative bacteria. The prominent advantage is that PCR is the singular way to identify MRS in such culture negative cases. Recent development of full-automatic PCR system may improve the time efficiency for routine application. In this presentation, we will show the overall sensitivity and specificity of our PCR system for diagnosing PJI and discuss the current problem and future prospect

Introduction. Cartilage comprises chondrocytes and extracellular matrix. The matrix contains different collagens, proteoglycans, and growth factors produced by chondroprogenitor cells that differentiate from proliferating to hypertrophic chondrocytes. In vitro chondrocyte growth is challenging due to differences in behaviour between 2D and 3D cultures. Our aim is to establish a murine 3D spheroid culture method using chondrocytes to study the complex interaction of cells on the chondro-osseous border during enchondral ossification. Method. Primary chondrocytes were isolated from the knee of WT new-born mice and used to form 10,000 cell number spheroids. We used the ATDC5-chondrocyte cell line as an alternative cell type. Spheroids were observed for 7, 14, and 21 days before embedding in paraffin for slicing. Alcian blue staining was performed to identify proteoglycan positive areas to prove the formation of extracellular matrix in spheroids. Collagen type 2, and Collagen type X expression were analyzed via quantitative real-time PCR and immunohistochemistry. Result. Alcian blue staining showed increasing matrix formation from day 7 to day 14 and proliferative chondrocytes at early time points. Both cell types showed increasing mRNA expression of Collagen type 2 from day 7 to day 21. Collagen type X positive staining starting from day 14 on confirmed the development of hypertrophic stage of chondrocytes. ATDC5 cells exhibited a slower progression in chondrogenic differentiation compared to primary chondrocytes. Conclusion. In chondrocyte spheroids, we observed proceeding differentiation of chondrocytes reaching hypertrophic phase. Primary chondrocytes showed faster development than ATDC5 cell line. Overall, spheroid culture of chondrocytes could be a good basis to study the interaction of different cells types of the chondro-osseous border by combination of chondrocytes with e.g., endothelial cells and osteoblasts within the spheroid. Those organoid cultures might also help to reduce animal experiments in the future, by mimicking complex regeneration procedures like bone growth or fracture healing. DFG(German Research Foundation)

Millions of patients each year suffer from challenging non-healing bone defects secondary to trauma or disease (e.g. cancer, osteoporosis or osteomyelitis). Tissue engineering approach to non-healing bone defects has been investigated over the past few decades in a search for a novel solution for critical size bone defects. The success of the tissue engineering approach relies on three main pillars, the right type of cells; and appropriate scaffold; and a biologically relevant biochemical/ biophysical stimuli. When it comes to cells the mesodermal origin of mesenchymal stem cells and its well demonstrated multipotentiality makes it an ideal option to be used in musculoskeletal regeneration. For the presented set of experimental assays, fully characterised (passage 3 to 5)ovine adipose-derived mesenchymal stems cells (Ad-MSC) were cultured either in growth medium (GM) consisting of Dulbecco's Modification of Eagle's Medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% penicillin-streptomycin as a control or in osteogenic differentiation medium (DM), consisting of GM further supplemented with L- ascorbic acid (50 μg/ml), β-glycerophosphate (10 mM) and dexamethasone (100nM). Osteogenic differentiation was assessed biochemically by quantifying alkaline phosphatase (ALP) enzyme activity and alizarin red staining after 3, 7, 14 and 21 days in culture (where 1×105 cells/well were seeded in 24 well-plate, n=6/media type/ time point). Temporal patterns in osteogenic gene expression were quantified using real-time PCR for Runx-2, osteocalcin (OC), osteonectin (ON) and type 1 collagen (Col 1) at days 7, 15 and 21 (where 1×105 cells were seeded in T25 cell culture flasks for RNA extraction, n= 4 / gene/ media type/time point). The morphology of osteogenic cells was additionally evaluated by scanning electron microscopy (SEM) of cells seeded at low-density (1×102 cells) on glass coverslips for 2 weeks in GM or DM. The level of ALP activity of cells grown in osteogenic DM was significantly higher than the control growing in the standard growth medium (p ≤ 0.05) at days 3, 7 and 14. At 21 days there was a sharp drop in ALP values in the differentiating cells. Mineralisation, as evidenced by alizarin red staining, increased significantly by day 14 and then peaked at day 21. Quantitative real-time PCR confirmed early increases in Runx-2, Col 1 and osteonectin, peaking in the second week of culture, while osteocalcin peaked at 21 days of culture. Taken as a whole, these data indicate that ovine-MSCs exhibit a tightly defined pathway of initial proliferation and matrix maturation (up to 14 days), followed by terminal differentiation and mineralisation (days 14 to 21). SEM analysis confirmed the flattened, roughened appearance of these cells and abandoned extracellular matrix which resembled mature osteoblasts. Given the ready availability of adipose tissues, the use of Ad-MSCs as progenitors for bone tissue engineering applications is both feasible and reasonable. The data from this study indicate that Ad-MSCs follow a predictable pathway of differentiation that can be tracked using validated molecular and biochemical assays. Additional work is needed to confirm that these cells are osteogenic in vivo, and to identifying the best combination of scaffold materials and cell culture techniques (e.g. static versus dynamic) to accelerate or stimulate osteogenic differentiation for bone tissue engineering applications

Introduction and Objective. Intervertebral disc (IVD) degeneration accompanying with low back pain is a serious worldwide problem. Even though, surgical treatments are available for pain relief, there is an urgent need to establish enduring cell-based remedies. Notochordal (NC) cells as the ancestor of nucleus pulposus (NP) cells in human IVD are a promising therapeutic target. It has been reported that the loss of NC cells after childhood could promote the onset of disc degeneration. Thus, we firstly, aimed to optimise the culture of NC cells in vitro without using the FCS in alginate (3D) culture systems, secondly, investigate their behaviour in healthy and degenerate niche and lastly, co-culture these cells with degenerated NP cells to assess their regeneration potentials. Materials and Methods. Porcine NC cells were extracted using pronase treatment followed by overnight digestion in 0.01% collagenase II. After extraction, cells were culture in 1.2% alginate beads (gold standard 3D culture) in either low glucose DMEM or αMEM medium. Cells were harvested after 24 hours, 1 week and 2 weeks for gene expression analysis and formalin fixed paraffin embedding. Quantitative Real-Time PCR and Immuno-staining were performed for analysis of NC markers (KRT18, FOXA2 and T) and COL I as a negative marker. Next, NC cells were cultured in healthy and degenerate medium to assess their viability and behaviour. Results. A mixed phenotype of NC and NP cells was observed in alginate bead cultures. NC phenotype was observed within all culture conditions with production of GAGs and maintenance of vacuolated phenotype. Gene expression analysis showed no significant difference between the culture of NC cells in low glucose DMEM and αMEM medium. Interestingly, NC cell viability was maintained in both healthy and degenerate media, despite observing more dead cells in degenerate conditions. Current investigations are comparing the behaviour of NC cells in healthy and degenerate niche. Conclusions. Investigating the preservation of NC phenotype in alginate culture and studying their behaviour between healthy and degenerate conditions would lead us to better understand their characteristics in different niches and how we can further use them in therapeutic purposes for disc degeneration

Introduction and Objective. Senescent bone cell overburden accelerates osteoporosis. Epigenetic alteration, including microRNA signalling and DND methylation, is one of prominent features of cellular senescence. This study aimed to investigate what role microRNA-29a signalling may play in the development of senile osteoporosis. Materials and Methods. Bone biopsy and serum were harvested from 13 young patients and 15 senior patients who required spine surgery. Bone mass, microstructure, and biomechanics of miR-29a knockout mice (miR-29aKO) and miR-29a transgenic mice (miR-29aTg) were probed using mCT imaging and three-point bending material test. Senescent cells were probed using senescence-associated b-galactosidase (SA-b-gal) staining. Transcriptomic landscapes of osteoblasts were characterized using whole genome microarray and KEGG bioinformatics. miR-29a and senescence markers p16. INK4a. , p21. Waf/cipl. and inflammatory cytokines were quantified using RT-PCR. DNA methylome was probed using methylation-specific PCR and 5-methylcytosine immunoblotting. Results. Senescent osteoblast overburden, DNA hypermethylation and oxidative damage together with significant decreases in serum miR-29a levels were present in bone specimens of aged patients. miR-29aKO mice showed a phenotype of skeletal underdevelopment, low bone mineral density and weak biomechanics. miR-29a knockout worsened age-induced bone mass and microstructure deterioration. Of note, aged miR-29aTg mice showed less bone loss and fatty marrow than aged wild-type mice. Transgenic overexpression of miR-29s compromised age-dysregulated osteogenic differentiation capacity of bone-marrow mesenchymal cells. In vitro, miR-29a promoted transcriptomic landscapes of antioxidant proteins in osteoblasts. The microRNA interrupted DNA methyltransferase (Dnmt3b)-mediated DNA methylation, inhibiting reactive oxygen radicals burst, IL-6 and RANKL production, and a plethora of senescent activity, including increased p16. INK4a. , p21. Waf/cipl. signalling and SA-b-gal activity. Conclusions. miR-29a loss is correlated with human age-mediated osteoporosis. miR-29a signalling is indispensable in bone mase homeostasis and microstructure integrity. Gain of miR-29a function is advantageous to delay age-induced bone loss through promoting antioxidant proteins to inhibit DNA hypermethylation-mediated osteoblast senescence. Collective investigations shine light onto the anabolic effects miR-29a signalling to bone integrity and highlight a new epigenetic protection strategy through controlling microRNA signalling to delay osteoblast senescence and senile osteoporosis development

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

Introduction and Objective. Achilles tendon defect is difficult problem for orthopedic surgeon, and therefore the development of new treatments is desirable. Platelet-rich fibrin (PRF), dense fibrin scaffold composed of a fibrin matrix containing many growth factors, is recently used as regenerative medicine preparation. However, few data are available on the usefulness of PRF on Achilles tendon healing after injury. The objective of this study is to examine whether PRF promotes the healing of Achilles tendon defect in vivo and evaluated the effects of PRF on tenocytes in vitro. Materials and Methods. PRF were prepared from rats according to international guidelines on the literature. To create rat model for Achilles tendon defect, a 4-mm portion of the right Achilles tendon was completely resected, and PRF was placed into the gap in PRF group before sewing the gap with nylon sutures. To assess the histological healing of Achilles tendon defect, Bonar score was calculated using HE, Alcian-blue, and Picosirius-red staining section. Basso, Beattie, Bresnahan (BBB) score was used for the evaluation of motor functional recovery. Biomechanical properties including failure tensile load, ultimate tensile stress, breaking elongation, and elastic modulus were measured. We examined the effects of PRF on tenocytes isolated from rat Achilles tendon in vitro. The number of viable cells were measured by MTS assay, and immunostaining of ki-67 was used for detection of proliferative cells. Migration of tenocytes was evaluated by wound closure assay. Protein or gene expression level of extracellular matrix protein, such as collagen, were evaluated by immunoblotting, immunofluorescence, or PCR. Phosphorylation level of AKT, FGF receptor, or SMAD3 was determined by western blotting. Inhibitory experiments were performed using MK-2206 (AKT inhibitor), FIIN-2 (FGFR inhibitor), SB-431542 (TGF-B receptor inhibitor), or SIS3 (SMAD3 inhibitor). All p values presented are two-sided and p values < 0.05 were considered statistically significant. Results. In rat Achilles tendon defects, Bonar score was significantly improved in PRF group compared to control group. Collagen deposition at the site of Achilles tendon defect was observed earlier in PRF group. Consistent with the histological findings, BBB score was significantly improved in PRF group. PRF also significantly improved the biomechanical properties of injured Achilles tendon. Furthermore, proliferating tenocytes, labelled by ki-67 were significantly increased in PRF group. These data suggested PRF prompted the healing of Achilles tendon defect. Thus, we further examined the effects of PRF on tenocytes in vitro. PRF significantly increased the number of viable cells, the proliferative cells labelled by ki-67, and migratory ability. Furthermore, PRF significantly increased the protein expression levels of collagen-I, collagen-III, α-SMA, and tenascin-C in tenocytes. Next, we examined the signalling pathway associated with PRF-induced proliferation of tenocytes. PRF increased the phosphorylation level and induced nuclear translocation of AKT, known as key regulator of cell survival. PRF also induced the phosphorylation of FGF receptor. Inhibition of AKT or FGF-receptor completely suppressed the positive effects of PRF on tenocytes. Furthermore, we found that inhibition of FGF receptor partially suppressed the phosphorylation of AKT by PRF. Thus, PRF induced the proliferation of tenocytes via FGFR/AKT axis. We further evaluated the signalling pathway associated with PRF-induced expression of extracellular matrix. PRF increased the phosphorylation levels of SMAD3 and induced nuclear translocation of SMAD3. Furthermore, inhibition of TGF-B receptor or SMAD3 suppressed increased expression level of extracellular matrix by PRF. Thus, PRF increased expression level of extracellular matrix protein via TGF-BR/SMAD3 axis. Conclusions. PRF promotes tendon healing of the Achilles tendon defect and recovery of exercise performance and biomechanical properties. PRF increases the proliferation ability or protein expression level of extracellular matrix protein in tenocytes via FGFR/AKT or TGF-βR/SMAD3 axis, respectively

Abstract. Objective. SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between mesenchymal stromal cells (MSCs) and chondrocytes by comparing SOX gene expression in their co-culture and respective monocultures. Methods. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in flow cytometry and were allowed to undergo trilineage differentiation for subsequent histological investigation. SOX5, SOX6, and SOX9 expression of co-cultures and monoculture controls were quantified by TaqMan quantitative real-time PCR. Experiments were performed in triplicate. Results. AMSC phenotype was evidenced by the expression of CD105, CD73, CD90 & heterogeneous CD34 but not CD45, CD14, CD19 & HLA-DR in flow cytometry, and also differentiation into chondrogenic, osteogenic and adipogenic lineages with positive Alcian blue, Alizarin Red and Oil Red O staining. The expression of SOX5, SOX6, and SOX9 was greater in observed co-cultures than would be expected from an expression profile modelled from monocultures. Conclusions. These findings provide evidence for the upregulation of SOX family transcription factors expression during the co-culture of MSCs and chondrocytes, suggesting an active induction of chondrogenic differentiation and change of cell fate amidst a microenvironment that facilitates cell-contact and paracrine secretion. This provides insight into the chondrogenic potential and therapeutic effects of MSCs preconditioned by the chondrocyte secretome (or potentially chondrocytes reinvigorated by the MSC secretome), and ultimately, cartilage repair. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between and preconditioning of mesenchymal stem cells (MSCs) with chondrocytes through comparing SOX gene expression in their co-culture and respective monocultures. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Samples were handled according to the 2004 UK Human Tissue Act. Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in flow cytometry and were allowed to undergo trilineage differentiation for subsequent histological investigation. SOX5, SOX6, and SOX9 expression of co-cultures and monoculture controls were quantified by TaqMan quantitative real-time PCR. Experiments were performed in triplicate. AMSC phenotype was evidenced by the expression of CD105, CD73, CD90 & heterogenous CD34 but not CD45, CD14, CD19 & HLA-DR in flow cytometry, and also differentiation into chondrogenic, osteogenic and adipogenic lineages with positive Alcian blue, Alizarin Red and Oil Red O staining. The expression of SOX5, SOX6, and SOX9 were greater in observed co-cultures than would be expected from an expression profile modelled from monocultures. The findings provides evidence for the upregulation of SOX family transcription factors expression during the co-culture of MSCs and chondrocytes, suggesting an active induction of chondrogenic differentiation and change of cell fate amidst a microenvironment that facilitates cell-contact and paracrine secretion. This provides insight into the chondrogenic potential and therapeutic effects of MSCs preconditioned by the chondrocyte secretome (or potentially chondrocytes reinvigorated by the MSC secretome), and ultimately, cartilage repair

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 polymerase chain reaction (PCR). Therefore, the objective of the present study is to compare and optimise different homogenisation methods and RNA isolation kits for an array of cartilaginous tissues. Materials and Methods. Tissue samples such as the nucleus pulposus (NP), annulus fibrosus (AF), articular cartilage (AC) and meniscus, were collected from goats and homogenised by either the MagNA Lyser or Freezer Mill. RNA of duplicate samples was subsequently isolated by either TRIzol (benchmark), or the RNeasy Lipid Tissue, RNeasy Fibrous Tissue, or Aurum Total RNA Fatty and Fibrous Tissue kits. RNA yield, purity, and integrity were determined and gene expression levels of type II collagen and aggrecan were measured by real-time PCR. Results. No differences between the two homogenisation methods were found. RNA isolation using the RNeasy Fibrous and Lipid kits resulted in the purest RNA (A260/A280 ratio), whereas TRIzol isolations resulted in RNA that is not as pure, and show a larger difference in gene expression of duplicate samples compared with both RNeasy kits. The Aurum kit showed low reproducibility. Conclusion. For the extraction of high-quality RNA from cartilaginous structures, we suggest homogenisation of the samples by the MagNA Lyser. For AC, NP and AF we recommend the RNeasy Fibrous kit, whereas for the meniscus the RNeasy Lipid kit is advised. Cite this article: M. Peeters, C. L. Huang, L. A. Vonk, Z. F. Lu, R. A. Bank, M. N. Helder, B. Zandieh Doulabi. Optimisation of high-quality total ribonucleic acid isolation from cartilaginous tissues for real-time polymerase chain reaction analysis. Bone Joint Res 2016;5:560–568. DOI: 10.1302/2046-3758.511.BJR-2016-0033.R3

Tendon healing is a complex process that often results in compromised healing of the tendon tissue. It has recently been shown that temporal changes in the expression profile and the histological tissue quality of the tendons occur during the early healing process after acute Achilles tendon rupture. Whether these changes are accompanied by an altered healing process, is not yet known and was the aim of the present study. Tendon biopsies were obtained from 24 patients with acute Achilles tendon rupture at the time of surgery (2–9 days after rupture) and examined histologically as well as on RNA level. Histologically, the tendon architecture, the amount of aligned collagen, glycosaminoglycan and fat as well as the cellularity, vascularity and immune cell infiltration were determined. On RNA level the expression of markers for the modeling/remodeling (MMPs and TIMPs), collagens (1, 3, 5), tendon markers (scleraxis, tenomodulin), pro- and anti-inflammatory markers (IL-1beta, IL6, IL10, IL33, TNFa, TGF-beta1, COX2) and immune cell markers (CD3, CD68, CD80, CD206) were analyzed by Real-Time PCR. To determine the clinical outcome, the patients were followed up 12 months after the operation and the following scores were recorded: Subjective score, Tegner score, Visual Analog Scale (VAS) pain, VAS function, Matles Test, Achilles tendon total rupture score (ATRS), Therman 100-points score, Heel rise test. Statistics: Spearman correlation analysis. Correlation analysis shows that early post-rupture surgery is associated with better clinical outcome (ATRS Score: p=0.022). Histologically, a good functional healing outcome shows a positive correlation to the amount of aligned collagen (Heel Rise Test: p = 0.009) and glycosaminoglycans in the tendon (Heel Rise Test: p = 0.026, Matles difference: p = 0.029), as well as a negative correlation to the fat content (Thermann score: p = 0.018, subjective score: p = 0.027, VAS function: p = 0.031). On RNA level, a good healing outcome correlates with increased expression of MMP13, collagen 1, 3, 5 (Heel Rise Test: p = 0.019, p = 0.048, p = 0.030), and TIMP2 (Tegner Score: p = 0.040), TGF-beta1 (Thermann Score: p = 0.032) and CD80 (ATRS: p = 0.025, Thermann score:, p = 0.032). Whereas a limited healing outcome is associated with an increased expression of MMP2 (Heel Rise Test: p = 0.033), MMP3 (Matles Test: p=0.001, Heal Rise test p = 0.017), and IL33 (Tegner Score: p = 0.047). The results of the study show a clear relationship between the tendon biology at the time of the surgery and the clinical and functional healing outcome 12 months after the operation. Especially matrix formation and remodeling play a crucial role, while the examined immunological factors seem to influence the tendon healing to a lesser extent. The modulation of matrix formation could potentially lead to improved treatment options in the future

Summary Statement. Low-intensity pulsed ultrasound (LIPUS) enhanced osteogenic differentiation of osteoprogenitor cells derived from mouse induced pluripotent cells (iPSCs) without embryoid body formation. Our findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs. Introduction. iPSCs represent a promising cell source for regenerative medicine such as bone regeneration because of their unlimited self-renewal property and ability of differentiation into all somatic cell types. Recently, we developed an efficient protocol for generating a highly homogeneous population of osteoprogenitor cells from embryonic stem cells by using a direct-plating method without EB formation step. It is well-recognised that LIPUS accelerates the fracture healing. There have been several reports showing that LIPUS stimulates the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. To date, effect of LIPUS on iPSCs remains unknown. In this study, we investigated in vitro effect of LIPUS on osteogenic differentiation of osteoprogenitor cells derived from mouse iPS cells via a direct-plating method. Methods. Murine iPSC colonies were dissociated with trypsin-EDTA, and obtained single cells were cultured on gelatin-coated plates without feeders in MSC medium and FGF-2. Adherent fibroblastic cells obtained by this direct-plating technique were termed as direct-plated cells (DPCs). DPCs were evaluated for cell-surface protein expression using flow cytometry. Expression levels of Oct-3/4 mRNA in iPSCs and DPCs were analyzed by real-time PCR. For osteogenic differentiation, DPCs were divided into two groups: (1) control group: DPCs cultured in osteogenic medium (OM) without LIPUS, and (2) LIPUS group: DPCs cultured in OM with LIPUS treatment. LIPUS was given through the bottom of the culture plates for 20 minutes daily. After 14-day culture, osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity and Alizarin red S staining. Expression of osteoblast-related genes, Rnux2 and ALP was also analyzed by real-time PCR. Results. Flow cytometry analysis revealed DPCs had similar characteristics to MSCs. Expression level of Oct-3/4 in DPCs was robustly down-regulated compared to that in iPSCs, suggesting DPCs lost pluripotency. After 14-day osteogenic induction, ALP activity was shown to be higher in LIPUS group than control group on days 3 and 7. Real-time PCR analysis revealed that in LIPUS group, expression level of Runx2 on day 1 and that of ALP on days 3 and 5 were significantly up-regulated compared to control group. The quantity of calcium deposition measured by Alizarin red staining on day 14 was shown to be higher in LIPUS group than control group. Conclusion. The novel direct-plating method described here provides a significant technical advance over conventional methods of isolating iPSC-induced osteoprogenitor cells by avoiding the embryonic body formation that often leads to heterogeneous, variable, and unpredictable osteogenic differentiation. Our results demonstrated that osteogenic differentiation of osteoprogenitor cells from iPSCs was robustly increased by LIPUS treatment. LIPUS may be a promising enhancer of osteogenesis of iPSCs. These findings provide insights on the development of LIPUS as an effective technology for bone regeneration strategies using iPSCs

Summary Statement. The stable inhibition of miR-214 in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days, which will provide a potential bone anabolic strategy for treatment of osteoprosis. Introduction. MiR-214 has a crucial role in suppressing bone formation and miR-214 inhibition in osteogenic cells may be a potential anabolic strategy for ameliorating osteoporosis (Wang X, et al. 2013). An aged ovariectomised rat has been regarded as a golden model to test bone anabolic agents for reversing established osteoporosis in aged postmenopausal women (Li X, et al. 2009). However, there is still lack of evidence to demonstrate bone anabolic potential of therapeutic inhibition of miR-214 within osteogenic cells in the golden model. So, it should be necessary to establish RNAi-based administration protocol toward stable inhibition of miR-214 at a low level in the golden model. A targeted delivery system specifically facilitating Antagomir-214 approaching osteogenic cells, i.e. (Asp-Ser-Ser). 6. -liposome (Zhang G, et al 2012), was employed in this study. Objectives. This study was to investigate optimal dosage and duration for therapeutic inhibition of miR-214 within osteogenic cells in the aged osteoporotic rats induced by ovariectomy. Materials and Methods. Six-month-old female Sprague-Dawley rats were ovariectomised (OVX) and left untreated for 12 months to establish aged osteoporosis. To determine the optimal dosage for therapeutic inhibition of miR-214, the OVX rats were injected intravenously with the AntagomiR-214 at a dosage of 0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg, 6mg/kg and 8mg/kg (n=6 for each dosage group) delivered by (Asp-Ser-Ser). 6. -liposome, respectively. Thereafter, miR-214 expression level in osteogenic cells from bilateral femur was quantified at day 2 post injection by real-time PCR analysis in combination with laser captured dissection (LCM). To determine the optimal duration of miR-214, the OVX rats were intravenously injected with the AntagomiR-214 (AntagomiR-214 group) or non-sense AntagomiR-214 (NC group) delivered by (Asp-Ser-Ser). 6. -liposome at the optimal dosage or (Asp-Ser-Ser). 6. -liposome alone (Vehicle group). Then, the miR-214 level in osteogenic cells from bilateral femur was quantified at 1, 3, 5, 7, 9, 12, 14, 16, 21 day after the single dosing (n=6 for each time-point) by real-time PCR analysis in combination with LCM, respectively. To examine the long-term effect of the AntagomiR-214 after periodic pulsed dosing, the OVX rats were administrated with the AntagomiR-214 at the optimal dosage and duration for 5 repeated injections and then the miR-214 level in osteogenic cells from bilateral femur was quantified by real-time PCR analysis in combination with LCM. Results. The miR-214 level was efficiently decreased in a dose-dependent manner by the AntogomiR-214 and reached the level lower than 10% of the baseline at a dosage of 4 mg/kg at least in the aged osteoporotic rats. The effective duration for miR-214 at a level lower than 50% of the baseline lasted for 7 days in the osteoporotic rats after the single dosing. The miR-214 level was continuously lowered until 28 days and continuously maintained later at the level lower than 10% of the baseline by the 5 pulsed dosing of the AntagomiR-214 at an interval of 7 days and at a dosage of 4 mg/kg in the osteoporotic rats. Conclusions. The stable inhibition of miR-214 for bone anabolic strategy in the aged osteoporotic rats induced by OVX could be achieved by periodic administration of AntagomiR-214 at a dosage of 4 mg/kg and at an interval of 7 days