Periosteum is important for bone homoeostasis through the release of bone morphogenetic proteins (BMPs) and their effect on osteoprogenitor cells. Smoking has an adverse effect on fracture healing and bone regeneration. The aim of this study was to evaluate the effect of smoking on the expression of the BMPs of human periosteum. Real-time polymerase chain reaction was performed for BMP-2,-4,-6,-7 gene expression in periosteal samples obtained from 45 fractured bones (19 smokers, 26 non-smokers) and 60 non-fractured bones (21 smokers, 39 non-smokers). A hierarchical model of BMP gene expression (BMP-2 > BMP-6 > BMP-4 > BMP-7) was demonstrated in all samples. When smokers and non-smokers were compared, a remarkable reduction in the gene expression of BMP-2, -4 and -6 was noticed in smokers. The comparison of fracture and non-fracture groups demonstrated a higher gene expression of BMP-2, -4 and -7 in the non-fracture samples. Within the subgroups (fracture and non-fracture), BMP gene expression in smokers was either lower but without statistical significance in the majority of BMPs, or similar to that in non-smokers with regard to BMP-4 in fracture and BMP-7 in non-fracture samples. In smokers, BMP gene expression of human periosteum was reduced, demonstrating the effect of smoking at the molecular level by reduction of mRNA transcription of periosteal BMPs. Among the BMPs studied, BMP-2 gene expression was significantly higher, highlighting its role in bone homoeostasis.

Lubricin is a proteoglycan that is a boundary lubricant in synovial joints and both a surface and collagen inter-fascicular lubricant in ligaments. The purpose of this study was to characterise the mRNA levels for lubricin in the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medial collateral ligament (MCL), and lateral collateral ligament (LCL) in aging and surgically-induced menopausal rabbits. We hypothesised that lubricin mRNA levels would be increased in ligaments from aging and menopausal rabbits compared with ligaments from normal rabbits. All four knee ligaments (ACL, PCL, MCL, LCL) were isolated from normal (1-year-old rabbits, n=8), aging (3-year-old rabbits, n=6), and menopausal (1-year-old rabbits fourteen weeks after surgical ovariohysterectomy, n=8) female New Zealand White rabbits. RT-qPCR was used to evaluate the mRNA levels for lubricin normalised to the housekeeping gene 18S. After removing outliers, data for normal, aging, and menopausal rabbits for each knee ligament (ACL, PCL, MCL, LCL) were compared using ANOVA with linear contrasts or Kruskal-Wallis test with Conover post-hoc analysis. For ACLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.056). For PCLs, trends for increased lubricin mRNA levels were found when comparing menopausal and aging rabbits with normal rabbits (p<0.092). For MCLs, the mRNA levels for lubricin were increased in menopausal and aging rabbits compared with normal rabbits (p<0.050). For LCLs, no differences in lubricin mRNA levels were detected comparing the three groups. For all four knee ligaments (ACL, PCL, MCL, LCL), no differences in lubricin mRNA levels were detected comparing the ligaments from menopausal rabbits with those from aging rabbits. Lubricin plays a role in collagen fascicle lubrication in ligaments (1,2). Increased lubricin gene expression was associated with mechanical changes (including decreased modulus and increased failure strain) in the aging rabbit MCL (3). Detection of similar molecular changes in the ACL, and possibly the PCL, may indicate that their mechanical properties may also change as a result of increased lubricin gene expression, thereby potentially pre-disposing these ligaments to damage accumulation. Compared to aging ligaments, aging tendons exhibited decreased lubricin gene and protein expression, and increased stiffness (4). Although opposite changes than aging ligaments, these findings support the relationship between lubricin and modulus/stiffness. The similarities between ligaments in the aging and menopausal groups may suggest that surgically-induced menopause results in a form of accelerated aging in the rabbit ACL, MCL and possibly PCL

Purpose. Angiogenesis and osteogenesis are essential for bone growth, fracture repair, and bone remodeling. VEGF has an important role in bone repair by promoting angiogenesis and osteogenesis. In our previous study, endothelial progenitor cells (EPCs) promoted bone healing in a rat segmental bone defect as confirmed by radiological, histological and microCT evaluations (Atesok, Li, Schemitsch 2010); EPC treatment of fractures resulted in a significantly higher strength by biomechanical examination (Li, Schemitsch 2010). In addition, cell-based VEGF gene transfer has been effective in the treatment of segmental bone defects in a rabbit model (Li, Schemitsch et al 2009); Purpose of this study: Evaluation of VEGF gene expression after EPC local therapy for a rat segmental bone defect. Method. Rat bone marrow-derived EPCs were isolated from the rat bone marrow by the Ficoll-paque gradient centrifuge technique. The EPCs were cultured for 7 to 10 days in endothelial cell growth medium with supplements (EGM-2-MV-SingleQuots, Clonetics). and collected for treatment of the rat segmental bone defect. EPCs were identified by immunocytochemistry staining with primary antibodies for CD34, CD133, FLK-1, and vWF. A total of fifty six rats were studied. A five millimeter segmental bone defect was created in the middle 1/3 of each femur followed by mini plate fixation. The treatment group received 1×106 EPCs locally at the bone defect and control animals received saline only. Seven control and seven EPC treated rats were included in each group at 1, 2, 3 and 10 weeks. Animals were sacrificed at the end of the treatment period, and specimens from the fracture gap area were collected and immediately frozen. Rat VEGF mRNA was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantified by VisionWorksLS. All measurements were performed in triplicate. Results. Cultured EPCs at 1 week showed positive staining for CD34, CD133, Flk-1 and vWf markers. The EPC group had a greater VEGF expression than the control group at week 1, 2 and 3 but not at week 10. Three VEGF isoforms were detected in this rat model: VEGF120, VEGF164 and VEGF188. VEGF120 and VEGF164 levels peaked at two weeks, while VEGF188 levels peaked at three weeks. All three VEGF isoform levels were low at ten weeks. Conclusion. EPC-based therapy for a segmental bone defect results in increased VEGF expression during the early period of fracture repair. In addition, the specific VEGF isoform may be a key regulator of the bone healing process. These findings demonstrate that EPCs promote fracture healing by increasing VEGF levels and thus stimulating angiogenesis, a process that is essential for early callus formation and bone regeneration

Aim. Vertebral osteomyelitis (VO) is an infection of the spine mostly caused by bacterial pathogens. The pathogenesis leading to destruction of intervertebral discs (IVD) and adjacent vertebral bodies (VB) is poorly described. We aimed to investigate the connection between infection, bone- and disc-metabolism in VO patients. Method. Fourteen patients with VO (infection group) and 14 patients with incomplete burst fractures of the spine (fracture group as controls) were included prospectively. Demographic data, treatment details, laboratory infection markers, and patient-reported outcome were assessed. Tissue biopsies from affected IVDs and adjacent VBs were analyzed for mRNA-expression levels of 18 target genes including chemokines, adipokines and genes involved in bone-metabolism by RT-qPCR. Results. The Receptor activator of NF-κB/Osteoprotegerin (RANK/OPG) expression ratio was elevated in VB and IVD of the infection group (p<0.001 and p=0.028, respectively). The RANK-ligand (RANKL)/OPG expression ratio was elevated in VB of the infection group (p<0.01). Expressions of the chemokines IL8 and CCL20 were higher in VB samples of the infection group. The expression of leptin was higher in IVD tissue, the mRNA expression of omentin and resistin was lower in VBs of the infection group. OPG mRNA expression was lower in infected VB and in IVD tissue compared to the fracture group. Conclusions. We identified similar expression patterns of pro-inflammatory cytokines and the RANK/RANKL/OPG axis in VBs and IVDs of patients with VO. This finding suggests that common immuno-metabolic pathways are involved in mechanisms leading to tissue degradation in VBs and IVDs during VO

Undifferentiated pleomorphic sarcoma (UPS) is one of the most common and aggressive adult soft tissue sarcomas (STS). Once metastatic, UPS is rapidly fatal. Most STS, including UPS, are resistant to conventional immunotherapies as these tumours have low numbers of spontaneous tumour infiltrating lymphocytes (TILs) and are densely populated with immune suppressive macrophages. Intra-tumoural activation of the STimulator of INterferon Genes (STING) pathway is a novel immunotherapeutic strategy to recruit anti-tumour TILs into the tumour microenvironment. In a murine model of UPS, we have demonstrated that intra-tumoural injection of a murine-specific STING agonist, DMXAA, results in profound immune mediated tumour clearance. Recently, molecules capable of activating both human and mouse STING pathways have been developed. In pursuit of clinically relevant therapeutic opportunities, the purpose of this study is to evaluate the anti-tumour potential of two agonists of the human and murine STING receptors: ADU-S100 and MSA-2 as monotherapies and in combination with the immune checkpoint inhibitor, anti-PD1 in a murine model of UPS. Immune competent mice were engrafted with murine UPS cells in the hindlimb muscle. Once palpable, mice in the monotherapy group were treated with a single intra-tumoural dose of 1) ADU-S100 or 2) MSA-2 or 3) DMXAA. In additional experimental groups, mice were treated with the different STING agonists and monoclonal anti-PD1. Tumour volume measurements and tumour bioluminescence were measured over time. To quantify dynamic changes in immune populations and in the tumour immune microenvironment, STING treated UPS tumours were evaluated using flow cytometry and mRNA quantification at various timepoints after therapy. DMXAA monotherapy produced complete tumour eradication in 50% of mice, whereas both ADU-S100 or MSA-2 monotherapy only extended survival but did not result in complete tumour clearance. Flow cytometry and transcriptional profiling of tumours at multiple timepoints post-treatment showed similar inflammatory changes and increased TILs numbers across all STING agonists. The addition of anti-PD1 treatment to STING therapy significantly extended survival times with both ADU-S100 and MSA-2, and resulted in 14% complete tumour clearance with ADU-S100. No complete survivors were observed with MSA-2-anti-PD1 combinations therapy. STING activation is a promising immunotherapeutic strategy for UPS. Recently developed human STING agonists are not as effective as DMXAA despite similar immunologic responses to treatment. STING and anti-PD-1 treatment were therapeutically synergistic for both human STING agonists. These results justify further research around STING activation as a therapeutic modality for STS. DMXAA may possess additional off-target therapeutic properties beyond STING activation which warrants further investigation. Elucidating these differences may be critical to further optimize STING therapy for human STS

Giant cell tumors of bone (GCTs) are locally aggressive tumors with recurrence potential that represent up to 10% of primary tumors of the bone. GCTs pathogenesis is driven by neoplastic mononuclear stromal cells that overexpress receptor activator of nuclear factor kappa-B/ligand (RANKL). Treatment with specific anti-RANKL antibody (denosumab) was recently introduced, used either as a neo-adjuvant in resectable tumors or as a stand-alone treatment in unresectable tumors. While denosumab has been increasingly used, a percentage of patients do not improve after treatment. Here, we aim to determine molecular and histological patterns that would help predicting GCTs response to denosumab to improve personalized treatment. Nine pre-treatment biopsies of patients with spinal GCT were collected at 2 centres. In 4 patients denosumab was used as a neo-adjuvant, 3 as a stand-alone and 2 received denosumab as adjuvant treatment. Clinical data was extracted retrospectively. Total mRNA was extracted by using a formalin-fixed paraffin-embedded extraction kit and we determined the transcript profile of 730 immune-oncology related genes by using the Pan Cancer Immune Profiling panel (Nanostring). The gene expression was compared between patients with good and poor response to Denosumab treatment by using the nSolver Analysis Software (Nanostring). Immunohistochemistry was performed in the tissue slides to characterize cell populations and immune response in CGTs. Two out of 9 patients showed poor clinical response with tumor progression and metastasis. Our analysis using unsupervised hierarchical clustering determined differences in gene expression between poor responders and good responders before denosumab treatment. Poor responding lesions are characterized by increased expression of inflammatory cytokines as IL8, IL1, interferon a and g, among a myriad of cytokines and chemokines (CCL25, IL5, IL26, IL25, IL13, CCL20, IL24, IL22, etc.), while good responders are characterized by elevated expression of platelets (CD31 and PECAM), coagulation (CD74, F13A1), and complement classic pathway (C1QB, C1R, C1QBP, C1S, C2) markers, together with extracellular matrix proteins (COL3A1, FN1,. Interestingly the T-cell response is also different between groups. Poor responding lesions have increased Th1 and Th2 component, but good responders have an increased Th17 component. Interestingly, the checkpoint inhibitor of the immune response PD1 (PDCD1) is increased ~10 fold in poor responders. This preliminary study using a novel experimental approach revealed differences in the immune response in GCTs associated with clinical response to denosumab. The increased activity of checkpoint inhibitor PD1 in poor responders to denosumab treatment may have implications for therapy, raising the potential to investigate immunotherapy as is currently used in other neoplasms. Further validation using a larger independent cohort will be required but these results could potentially identify the patients who would most benefit from denosumab therapy

The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

Introduction. Macrophages phagocytes implant wear debris and produce various cytokines to evoke inflammation and periprosthetic osteolysis of aseptic loosening. It had been reported that expression of Toll-like receptor (TLR) 2 and other TLRs increased in periprosthetic tissues of aseptic loosening. Pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) have been known as ligands of TLRs and considered to be involved in the osteolytic reactions via TLRs. Another type of immune sensors, nucleotide-binding and oligomerization domain (NOD)-like receptors (NLR) with a pyrin domain 3 (NLRP3) can also recognize PAMPs and DAMPs as their lignds, which has been presumed to participate in the local host response of macrophage cascade via phagocytosis of implant wear particles. However, the contribution of NLRP3 in periprosthetic tissues of aseptic loosening and the correlation between TLR2 and NLRP3 are still unclear. Materials and methods. TLR1, TLR2, TLR6, NLRP3, TNF-α and IL-1β of macrophages in aseptic loose periprosthetic tissues were immnohistorically evaluated and compared to osteoarthritic synovium. RAW264.7 cells, macrophagic cell line, were stimulated by titanium particles (Ti) and lipoteichoic acid (LTA)-coated Ti. The celluar reaction associated with TLR2 and NLRP3 and the correlation of them were analyzed at mRNA expression levels with small-interfering RNA of Irak2, one of adaptor molecules in TLR2 cascades. Results. Macrophages, which expressed abundant TLR2, NLRP3, TNF-α and IL-1β, were observed dominantly in foreign body granuloma of aseptic periprosthetic tissues. The features of abundant expression were quite different from osteoarthritic synovium. In vitro experiment of RAW264.7, mRNA levels of NLRP3 and TNF-α increased after stimulation of Ti. mRNA levels of TLR2, NLRP3, TNF-α and IL-1β were enhanced by LTA-coated Ti. mRNA expression level of NLRP3 were suppressed by silencing Irak2. Discussion and conclusion. This study indicated that innate immune sensors, TLR2 and NLRP3, could respond to foreign body particles in aseptic loose periprosthetic connective tissues. In this process, mRNA expression levels of TLR2 and NLRP3 in RAW264.7 were increased by phagocytosis of Ti particles, especially by LTA-coated Ti stimulation. Suppressed mRNA expression level of NLRP3 by knocked down of Irak2 indicated that TLR2 cascade could enhance activation NLRP3 cascade and/or free LTA may stimulate NLRP3 cascade directly. It may be possible that TLR2 and NLRP3 cascades in macrophages recognising PAMPs and/or DAMPs are activated each other and they play an important role of the pathogenesis of wear debris around loose hip joints

Introduction. Some patients complain ingrown pain or discomfort after implanting Co-Cr conventional endprosthesis of the hip. Some of this complaint may be attributable for effect on cartilage metabolism. It have been reported that ceramic is bioinert for biological tissue. On the other hand, metal including cobalt-chrome (Co-Cr) have some detrimental effect on biological tissue. However, there is no report concerning acetabular cartilage metabolism after hip endprosthesis implantation. In the present study, we hypothesized that ceramic head have small detrimental effect on cartilage cell metabolism. Specific aim of the study is to compare the protein level of inflammation related cytokines, amount of hyaluronic acid (HA) in culture media, and cartilage mRNA expression in organ culture model of hip end prosthesis implanted using ceramic head and Co-Cr head. Materials and Methods. Six acetabulum of 3 matured crossbred pig (average weight: 36 +/− 3.6kg) was retrieved. Animal experiment was performed under the rules of ethical committee of animal experiment. Average diameter of pig acetabulum was 26.3 +/− 0.6 mm. Just after sacrifice, mechanical loading using Instron testing machine with 26mm diameter of Co-Cr in right hip and Ceramic heads in left hip was performed in culture media. Ten thousand cycles of cyclic compression and rotation load (1.5kN to 0.15kN of compression and 12 degrees of rotation) to cartilage was applied at 1Hz (Figure 1). Culture media was analyzed for protein levels of inflammation related cytokines and amount of HA. Relative quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) from acetabular cartilage was performed as previously reported using specific primer sets for type II collagen, aggrecan, TNF-alpha, Interleukine-1 and 6, and MMP-1, 3, 13. Results. IL-1 beta protein level from culture media was significantly higher in Co-Cr than that in Ceramic (155+/−25.2 pg/ml vs. 86.3+/−9.6 pg/ml respectively). MMP-3 protein level had tendency to be higher in culture media from Co-Cr than that from Ceramic (16.3+/−10.6 ng/ml vs. 10.0+/−0.1 ng/ml respectively, p<0.05), however there was no significant difference. There were no significant differences of protein levels from culture media in MMP-1, IL-1a, and TNF between two groups. Amount of HA from culture media of Co-Cr group was significantly higher than that from Ceramic group (337+/−38.4 mg/ml versus 257+/−11.1 mg/ml respectively, p<0.05). Type II collagen mRNA expression was 3 times higher in Ceramic group than that in Co-Cr group. IL-1 beta mRNA expression was 4 times higher in Co-Cr group than that in Ceramic group. Other gene expression had no significant differences. Discussion. The present study showed that Co-Cr affects cartilage metabolism than Ceramic. Co-Cr group had higher protein level and mRNA expression of inflammation related cytokine, IL-1 beta, and higher HA. Concerning the mRNA expression from cartilage, type II collagen was significantly higher in Ceramic group. It has been reported that HA level is high in osteoarthritic joint. These report and our results showed that ceramic head have small detrimental effect on cartilage cell metabolism. There are limitations of the present study. Firstly, the sample size is small. Secondly, we did not evaluate synovial membrane metabolism

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols

The purpose of this study was to profile the mRNA expression for the 23 known matrix metalloproteinases (MMPs), 4 tissue inhibitor of metalloproteinases (TIMPs) and 19 ADAMTSs (a disintegrin and metalloproteinase with thrombospontin motif) in Dupuytren's Disease and normal palmar fascia. Dupuytren's Disease (DD) is a fibroproliferative disorder affecting the palmar fascia, leading to contractures. The MMPs and ADAMTSs are related enzymes collectively responsible for turnover of the extracellular matrix. The balance between the proteolytic action of the MMPs and ADAMTSs and their inhibition by the TIMPs underpins many pathological processes. Deviation in favour of proteolysis is seen in e.g. invasive carcinomata, whereas an imbalance towards inhibition causes e.g. fibrosis. A group of patients with end-stage gastric carcinoma was treated with a broad spectrum MMP inhibitor in an attempt to reduce the rate of carcinoma advancement; a proportion developed a ‘musculoskeletal syndrome’ resembling DD. Tissue samples were obtained from patients undergoing surgery to correct contractures caused by DD and from healthy controls undergoing carpal tunnel decompression. The DD tissue was separated macroscopically into cord and nodule. Total RNA was extracted and mRNA expression analysed by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), normalised to 18S rRNA. Comparing across all genes, the DD nodule, DD cord and normal palmar fascia samples each had a distinct mRNA expression profile. Statistically significant (p<0.05) differences in mRNA expression included: higher MMP-2, -7 and ADAMTS-3 levels in both cord and nodule; higher MMP-1, -14, TIMP-1 and ADAMTS-4 and -5 in nodule alone, lower MMP-3 in nodule and cord and lower TIMP-2, -3 and -4 and ADAMTS-1 and -8 levels in nodule alone. The distinct mRNA profile of each group suggests differences in extracellular proteolytic activity which may underlie the process of fascial remodelling in DD

Aim. The diagnosis of periprosthetic joint infection (PJI) in total joint arthroplasty (TJA) remains a serious clinical challenge. Nowadays, limited biomarkers associated with PJI are available. We investigated therefore the utility of gene expression pattern of Toll-like receptors (TLR) and members of interleukin (IL)1/IL1R family, molecules critically involved in the innate immune response to invading pathogens, for detecting PJI in periprosthetic tissues around TJA. Method. Periprosthetic tissues were collected from 37 patients presenting with PJI and 39 patients having an aseptic failure of TJA. mRNA expression of known TLR receptors (TLR1–10) and 21 members of IL-1/IL-1R family was investigated using an innovative Smartchip Real-Time RT-PCR System. *. ; the data were normalized relative to the housekeeping gene GAPDH. Statistical tests were performed using GraphPad Prism. **. and bio-data mining methods. Results. In PJI, elevated mRNA expression levels of TLR1 (P=0.03), TLR4 (P=0.01) and TLR6 (P=0.01) were detected when compared to tissues from aseptic cases. On the contrary, lower mRNA expression of TLR3 (P=0.04) and TLR7 (P=0.047) were detected in PJI than in aseptic loosening. From IL1/IL-1R family, PJI was associated with elevated levels of IL1β (P=0.0004), IL1RN (P=0.05), IL1R1 (P=0.04), IL1R2 (P=0.01), and IL18RAP (P=0.02) comparing to aseptic failure. Multivariate analysis and sophisticated bio-data mining analysis are ongoing to determine the potential of TLRs and IL1/IL1R family as biomarkers of PJI in TJA. Conclusions. Tissue expression of TLRs and IL1/IL-1R family differ in terms of pattern and expression level between septic and aseptic failure of TJA. Our data support the potential of “innate gene” expression panel as candidate biomarker for assessment of PJI. Further studies are required to replicate our data and also to enable valid interpretation of our findings. Grant support: AZV MZ CR VES15-27726A, VES16-131852A, IGA LF UP_2016_011

The purpose of this study was to evaluate whether AGEs induce annulus fibrosus (AF) cell apoptosis and to further explore the mechanism by which this process occurs. AF cells were treated with various concentrations of AGEs for 3 days. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) and EdU incorporation assays. Cell apoptosis was examined by the Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3 and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by RT-PCR. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by JC-1 staining and DCFH-DA fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggest that AGEs induce rabbit AF cell apoptosis and mitochondrial pathways may be involved in AGE-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration

The superficial zone (SFZ) of articular cartilage has unique structural and biomechanical features, and is important for joint long-term function. Previous studies have shown that TGF-β/Alk5 signaling upregulating PRG4 expression maintains articular cartilage homeostasis. However, the exact role and molecular mechanism of TGF-β signaling in SFZ of articular cartilage homeostasis are still lacking. In this study, a combination of in vitro and in vivo approaches were used to elucidate the role of Alk5 signaling in maintaining the SFZ of articular cartilage and preventing osteoarthritis initiation. Mice with inducible cartilage SFZ-specific deletion of Alk5 were generated to assess the role of Alk5 in OA development. Alterations in cartilage structure were evaluated histologically. The chondrocyte apoptosis and cell cycle were detected by TUNEL and Edu staining, respectively. Isolation, culture and treatment of SFZ cells, the expressions of genes associated with articular cartilage homeostasis and TGF-β signaling were analyzed by qRT-PCR. The effects of TGF-β/Alk5 signaling on proliferation and differentiation of SFZ cells were explored by cells count and alcian blue staining. In addition, SFZ cells isolated from C57 mice were cultured in presence of TGF-β1 or SB505124 for 7 days and transplanted subcutaneously in athymic mice. Postnatal cartilage SFZ-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. qRT-PCR and IHC results confirmed that Alk5 gene was effectively deleted in articular cartilage SFZ cells. Next, the PRG4-positive cells in articular cartilage SFZ were significantly decreased in Alk5 cKO mice compared with those in Cre-negative control mice. The mRNA expression of Aggrecan and Col2 were decreased, meanwhile, expression of Mmp13 and Adamts5 were significantly increased in articular cartilage SFZ cells of Alk5 cKO mice. In addition, Edu and TUNEL staining results revealed that slow-cell cycle cell number and increase the apoptosis positive cell in articular cartilage SFZ of Alk5 cKO mice compared with Cre-negative mice, respectively. Furthermore, all groups of SFZ cells formed ectopic solid tissue masses 1 week after transplantation. Histological examination revealed that the TGF-β1-pretreated tissues was composed of small and round cells and was positive for alcian blue staining, while the SB505124-pretreated tissue contained more hypertrophic cells though it did stain with alcian blue. TGF-β/alk5 signaling is an essential regulator of the superficial layer of articular cartilage by maintaining chondrocyte number, its differentiation properties, and lubrication function. Furthermore, it plays a critical role in protecting cartilage from OA initiation

Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

INTRODUCTION. Several reports suggest that low-intensity pulsed ultrasound stimulation (LIPUS) facilitates chondrogenesis. 1). Recently it has been suggested that LIPUS may be transmitted via Integrin: a protein which mediates cellular attachment between cells and extracellular matrix. 2). In this study, the Arg-Gly-Asp (RGD) amino acid sequence, which is a ligand of Integrin, was induced to the fibroin substrates by either gene transfer or physical mixing, and the variation of chndrocyte response to LIPUS was evaluated. EXPERIMENTAL METHODS. Three kinds of culture dishes coated with three diffrent fibroin aqueous solutions were prepared: 1 wild-type, 2 transgenic and 3 mixed. The wild-type aqueous solution was prepared from Bombyx mori silkworm cocoons. The transgenic aqueous solution was prepared from Bombyx mori silkworm cocoons in which RGD was interfused in the fibroin light chain. 3). The mixed aqueous solution was prepared simply by blending RGD peptides with the wild-type fibroin aqueous solution. Chondrocytes were asepically harvested from the joints of 4-week-old Japanese white rabbits and then subcultured on T-flasks and seeded at 2.0 × 10. 5. cells/dish. LIPUS stimulation, with spatial and temporal average intensity of 30 mW/cm. 2. and a frequency of 1.71 MHz with a 200 ms tone burst repeated at 1.0 kHz, was applied to the chondrocytes at 12, 36, 60 hours and administered for 20 minutes each time. GAG production and the number of chondrocytes were measured by the Dimethylmethylene blue (DMMB) method. 4). and the LDH method. 5). , respectively. Extracted mRNA from the chondrocytes was analyzed by using the Syber Green method, where the primers were designed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the house-keeping gene, aggrecan and Sox 9. This data was analyzed using the two-sided Student's t-test. RESULTS and DISCUSSION. In the transgenic group, the number of chondrocytes and GAG production were increased by the LIPUS stimulation in 1 day of culture (Fig. 1,2), and the mRNA expression levels of agrrecan (Fig. 3) and Sox 9 were increased in 2 days of culture. However the mRNA expression level of aggrecan was decreased after 3 days of culture. These LIPUS-derived changes were not found in the wild-type and mixed groups. We previously reported that the adhesive force between chondrocytes and RGD transgenic fibroin surfaces was higher than that for mixed fibroin, suggesting that adhesive force is translated via RGD which bonds covalently to the fibroin proteins for the transgenic group. The present results suggest that the early biological adhesion via RGD on the transgenic fibroin is sensitive to LIPUS

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 quantitative PCR was conducted to identify alterations in the expression of anti- and pro-inflammatory chemokines and cytokines, osteoclastic and osteoblastic factors, in addition to multiplex assays for the quantification of cytokine/chemokine release in cell supernatant in response to HDACi treatments in the presence or absence of TNFα. TNFα stimulated robust production of pro-inflammatory cytokines and chemokines by PBMCs (IL-1β, TNFα, MCP1 and MIP-1α) both at the mRNA and protein level (p < 0 .05). HDACi that target the isoforms HDAC-1 and −2 in combination significantly suppressed the expression or production of these inflammatory factors with greater efficacy than targeting these HDAC isoforms individually. Suppression of HDAC-5 and −7 had no effect on the inflammatory cascade induced by TNFα in monocytes. During osteoclastic differentiation, TNFα stimulated the size and number of active cells, increasing the bone destruction observed on dentine slices (p < 0 .05). Targeting HDAC-1 and −2 significantly reduced bone resorption through modulation of the expression of RANKL signalling factors (NFATc1, TRAF6, CatK, TRAP, and CTR) and fusion factors (DC-STAMP and β3-integerin). Conversely, the anabolic activity of osteoblasts was preserved with HDACi targeting HDAC-5 and −7, significantly increasing their mineralising capacity in the presence of TNFαthrough enhanced RUNX2, OCN and Coll-1a expression. These results identify the therapeutic potential of HDACi through epigenetic regulation of cell activity, critical to the processes of inflammatory bone destruction

Aims

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.

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

The bioactive polyetheretherketone (PEEK) was fabricated by the combination of PEEK and CaO-SiO. 2. particles, which formed hydroxyapatite on its surfaces in simulated body fluid and showed good mechanical propeties. The study revealed osteoblast-like cell proliferation and gene expression on the bioactive PEEK. Materials and Methods. Peek and bioactive PEEK discs (24 mm in diameter and 2 mm in thickness) were prepared. Bioactive PEEk was produced by the combination of 80 vol% Peek powder and 20 vol% CaO-SiO. 2. particles (30CaO · 70SiO. 2. ). Discs were sterilized with ethylene oxide gas. The study was approved by the ethics committee in Chiba University. Human osteoblast-like cells were used in the study. The cells at passage 3–5 were used in the experiments. 2 × 10. 5. cells /disc were culture at 37°C in a humidified atmosphere with 5% CO. 2. , and the media was replaced every 3 days. At days 3, 7, 21, the culture media, cells and discs were collected respectively. Cell attachment assay was performed. Cells were seeded at a density of 4 × 10. 5. cells /well and incubated for 2 hours at 37 C in a humidified atmosphere with 5% CO. 2. The cells on the discs were evaluated by DNA content. The real-time PCR was performed with regard to type I collagen (COLI), osteocalcin (OC), osteonectin (ON), osteopontin (OPN), and GAPDH. The alkaline phosphatase activity (ALP) was measeured at 3, 7, and 21 days, which samples as used in the DNA-content assay. Alizalin Red Staining was performed at day 21 to quantify calcification deposits in discs. Results were analyzed using Student's t-test with at least three samples. The level of siginificance was set at p=0.05. Results. The content of DNA showed similar increases on PEEK and bioactive PEEK in the course of day 3, 7, 21. The cell attachment of bioactive PEEK was two times larger than that of PEEK. Real-time PCR results of human osteoblast-like cells cultured on PEEK and bioactive PEEK discs were shown in Fig. 1. There were no significant differences between cells on PEEK and bioactive PEEK with respect to COL I and ON mRNA expression. However, human osteoblast-like cells on bioactive PEEK presented higher expression of OPN and OCN mRNA at day 21. No significant differences were found in ALP activity of both discs. Calcification deposits were observed only on bioactive PEEK at day 21. Discussion. The bioactive PEEK, with the combination of 80 vol% Peek powder and 20 vol% CaO-SiO. 2. particles (30CaO · 70SiO. 2. ) showed 123.5 MPa and 6.43 GPa in bending strength and Young's modulus, respectively. The bioactive PEEK has the aggregated CaO-SiO. 2. oarticles between the PEEK particles on its surface, which causes hydroxyapatite formation in vivo. The mechanism is considered to enhance the osteoblast attachment ability, and induce OPN and OC mRNA expression, following the calcification of human osteobloast-like cells. Therefore, the study indicated that bioactive PEEK is the most promising for use as an orthopedic implant