Although epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, has various pharmacological and biological activities including anti-carcinogenic, anti-thrombotic and anti-inflammatory effects, relatively a little is known about its beneficial effects on the non-frozen preservation of mammalian cells and tissues. In the present study, a storage solution containing EGCG was employed to testify the hypothesis that cold preservation of osteochondral allografts was attributed to EGCG-mediated reversible regulation of cell cycle. Human articular cartilages were obtained from knee joints of 10 patients (58 – 86 years old) undergoing total knee arthroplasty at Marunouchi Hospital, Matsumoto, Japan. Cartilage specimens were procured by osteotome under sterile conditions from the donor and placed in saline for 1 hr until the end of surgery. Immediately after surgery, the specimens were transferred in a storage solution (serum-free RPMI 1640 media with 1% antibiotic-antimycotic solution without or with 1 mM EGCG) and kept at 4°C. The specimens were then delivered to the senior author (Prof. Hyon) within 1 day from procurement. Additionally, fresh cartilages were delivered in a complete media with 10% fetal bovine serum at room temperature after procurement from the donor. Because of this necessary processing delay between tissue procurement from the donor and its delivery, 1 day was set as the data point for the fresh specimen. All procedures involving human subjects received prior approval from Marunouchi Hospital, Osaka City University Graduate School of Medicine and the Institutional Review Board of Institute for Frontier Medical Sciences, Kyoto University, and all subjects providing written informed consent. On receipt of the cartilage tissues, the specimens were replaced with either 20 ml of a storage solution without or with EGCG and then stored at 4°C for 1, 2 and 4 wk. At the end of each storage period, chondrocyte viability (CCK-8 assay), biochemical and immunohistochemical composition [glycosaminoglycans (GAG) and (type II) collagen], and biomechanical property (compressive elastic modulus) were assessed. The regulatory effects of EGCG on cell cycle distribution as well as expression levels of cyclins (CCNs) and nuclear factor-κB (NF-κB) were also investigated in articular chondrocytes. Chondrocyte viability of cartilages preserved with EGCG was significantly well-maintained for at least 2 weeks with high contents of GAG and total collagen. These beneficial effects of EGCG were confirmed by histological and immunohistochemical observations showing well-preserved cartilaginous structures and delayed denaturation of extracellular matrices. The compressive elastic modulus of cartilages preserved with EGCG was almost in the same range as that of fresh ones. Moreover, the penetration of FITC-conjugated EGCG into the matrices of cartilages and its incorporation into the cytosol of cells in lacunae were observed. Increased cell population at the G0/G1 phase by EGCG returned to the normal level after EGCG removal, whereas decrease at the G2/M phase did not. Negatively regulated expression of CCND1, CCNE2 or NF-κB in EGCG-treated cells was restored by removing EGCG, but not CCNA2 and CCNB1. It is suggested that EGCG play effective roles in preserving articular cartilages by reversibly regulating cell cycle at G0/G1 phase and NF-κB expression

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

Aims. The involvement of cyclin D1 in the proliferation of microglia, and the generation and maintenance of bone cancer pain (BCP), have not yet been clarified. We investigated the expression of microglia and cyclin D1, and the influences of cyclin D1 on pain threshold. Methods. Female Sprague Dawley (SD) rats were used to establish a rat model of BCP, and the messenger RNA (mRNA) and protein expression of ionized calcium binding adaptor molecule 1 (IBA1) and cyclin D1 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. The proliferation of spinal microglia was detected by immunohistochemistry. The pain behaviour test was assessed by quantification of spontaneous flinches, limb use, and guarding during forced ambulation, mechanical paw withdrawal threshold, and thermal paw withdrawal latency. Results. IBA1 and cyclin D1 in the ipsilateral spinal horn increased in a time-dependent fashion. Spinal microglia proliferated in BCP rats. The microglia inhibitor minocycline attenuated the pain behaviour in BCP rats. The cyclin-dependent kinase inhibitor flavopiridol inhibited the proliferation of spinal microglia, and was associated with an improvement in pain behaviour in BCP rats. Conclusion. Our results revealed that the inhibition of spinal microglial proliferation was associated with a decrease in pain behaviour in a rat model of BCP. Cyclin D1 acts as a key regulator of the proliferation of spinal microglia in a rat model of BCP. Disruption of cyclin D1, the restriction-point control of cell cycle, inhibited the proliferation of microglia and attenuated the pain behaviours in BCP rats. Cyclin D1 and the proliferation of spinal microglia may be potential targets for the clinical treatment of BCP. Cite this article: Bone Joint Res 2022;11(11):803–813

Macrophages (Mφ) are immune cells that play a crucial role in both innate and adaptive immunity as they are involved in a wide range of physiological and pathological processes. Depending on the microenvironment and signals present, Mφ can polarize into either M1 or M2 phenotypes, with M1 macrophages exhibiting pro-inflammatory and cytotoxic effects, while M2 macrophages having immunosuppressive and tissue repair properties. Macrophages have been shown to play key roles in the development and progression or inhibition of various diseases, including cancer. For example, macrophages can stimulate tumor progression by promoting immunosuppression, angiogenesis, invasion, and metastasis. This work aimed to investigate the effect of extracellular vesicles (EVs)-derived from polarized macrophages on an osteosarcoma cell line. Monocytes were extracted from buffy coats and cultured in RPMI medium with platelet lysate or M-CSF. After 6 days of seeding, Mφ were differentiated into M1 and M2 with INF-γ/LPS and IL-4/IL-13, respectively. The medium with M1 or M2 derived EVs was collected and EVs were isolated by differential centrifugation and size exclusion chromatography and its morphology and size were characterized with SEM and NTA, respectively. The presence of typical EVs markers (CD9, CD63) was assessed by Western Blot. Finally, EVs from M1 or M2-polarized Mφ were added onto osteosarcoma cell cultures and their effect on cell viability and cell cycle, proliferation, and gene expression was assessed. The EVs showed the typical shape, size and surface markers of EVs. Overall, we observed that osteosarcoma cells responded differentially to EVs isolated from the M1 and M2-polarized Mφ. In summary, the use of Mφ-derived EVs for the treatment of osteosarcoma and other cancers deserves further study as it could benefit from interesting traits of EVs such as low immunogenicity, nontoxicity, and ability to pass through tissue barriers. Acknowledgements: Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Objectives. Activation of the leptin pathway is closely correlated with human knee cartilage degeneration. However, the role of the long form of the leptin receptor (Ob-Rb) in cartilage degeneration needs further study. The aim of this study was to determine the effect of increasing the expression of Ob-Rb on chondrocytes using a lentiviral vector containing Ob-Rb. Methods. The medial and lateral cartilage samples of the tibial plateau from 12 osteoarthritis (OA) patients were collected. Ob-Rb messenger RNA (mRNA) was detected in these samples. The Ob-Rb-overexpressing chondrocytes and controls were treated with different doses of leptin for two days. The activation of the p53/p21 pathway and the number of senescence-associated β-galactosidase (SA-β-gal)-positive cells were evaluated. The mammalian target of rapamycin (mTOR) signalling pathway and autophagy were detected after the chondrocytes were treated with a high dose of leptin. Results. In total, 12 cases were found to have severe medial cartilage wear compared with the lateral cartilage. Immunofluorescence showed that the expression of Ob-Rb in the medial cartilage of the tibial plateau was high. High levels of leptin led to cell cycle arrest and inhibited autophagy. After overexpression of Ob-Rb, the physiological dose of leptin induced cell senescence in the chondrocytes. High doses of leptin inhibited autophagy by activating the mTOR signalling pathway. Blockade of the mTOR signalling pathway could restore autophagy and partially reverse senescence induced by leptin in chondrocytes. Conclusion. In summary, the present study demonstrated that high doses of leptin induce cell senescence by activating the mTOR pathway in chondrocytes from OA cartilage. Highly expressed Ob-Rb accelerates chondrocyte senescence by activating the leptin pathway in OA. Cite this article: X. Zhao, P. Huang, G. Li, L. Zhendong, G. Hu, Q. Xu. Activation of the leptin pathway by high expression of the long form of the leptin receptor (Ob-Rb) accelerates chondrocyte senescence in osteoarthritis. Bone Joint Res 2019;8:425–436. DOI: 10.1302/2046-3758.89.BJR-2018-0325.R2

Background and Aims: Low back pain has been attributed to degeneration of the intervertebral disc (IVD). Increased evidence of senescence biomarkers, including the protein caveolin-1, during IVD degeneration has been demonstrated and linked with disease development rather than ageing per se, suggesting that a particular type of senescence, stress-induced premature senescence (SIPS), occurs in disc degeneration. SIPS can be induced by cytokines such as interleukin-1 (IL-1 Since IL-1 is known to be an important mediator of the catabolic events in IVD degeneration we sought to investigate whether IL-1 induces expression of the senescence biomarker caveolin-1 in IVD cells and whether its induction is associated with markers of cell senescence. Methods: Human nucleus pulposus (NP) cells cultured in monolayer were treated for 24 hours with 10ng/ml IL-1 Quantitative real-time RT-PCR was used to assess gene expression for caveolin-1 and cell cycle inhibitors p53, p21 and p16INK4a. Cells were stained for senescence-associated-galactosidase and flow cytometry performed to analyse cell cycle position. Results: IL-1 treatment induced transcription of caveolin-1 at 8 hours after the start of treatment. This coincided with increased expression of the cell cycle inhibitors p21 and p16INK4a expression at 2 hours and p21 and p53 at 8 hours. Flow cytometry revealed that IL-1 treatment caused a shift away from the S phase of the cell cycle and treated cells exhibited senescence-associated-galactosidase staining. Conclusion: Our findings indicate that IL-1 induces caveolin expression and features of cellular senescence in human NP cells suggesting a role for IL-1 and caveolin-1 in SIPS within the human IVD. Conflicts of Interest: None. Source of Funding: Furlong Research Charitable Foundation

Introduction: The mitochondrial Translocator Protein 18 kDa (TSPO, previously named as the peripheral benzodiazepine receptor - PBR) is involved in cellular respiration, steroidogenesis and apoptosis. In our recent study we reported on the role of the synthetic pharmacological ligands to the TSPO in enhancing human osteoblast catabolism. There is also a previous evidence of the existence of an endogenous ligands to the TSPO, but their role in the human osteoblast physiology hasn’t been verified yet. Porphyrine IX has been found having affinity to the TSPO. Therefore we hypothesize that human osteoblast metabolism might be mediated by the porphyrine IX and the mode of its action is similar the synthetic ligand to the TSPO. Methods: Cell cycle of the cultured human derived osteoblast- like cells, following exposure to Porphyrine IX, endogenous ligand to TSPO, and N,N-di-n-hexyl 2-(4- fluorophenyl)indole-3-acetamide (FGIN-1–27), synthetic ligand to the TSPO, was determined by flow cytometry (FACS). These ligands’ affect on cell number, metabolic activity, i.e. cellular fluorodeoxyglucose ([. 18. F]-FDG) incorporation and alkaline phosphatase activity, and cell death rate, i.e. LDH activity in the culture media, were assayed. The semi-quantitative response of TSPO to exposure to these ligands was estimated by Western blotting. Six samples of cultured cells for each condition were used. The t test was implemented for the statistical analyses. P values below.05 considered as statistically significant. Results: Cell count significantly decreased following exposure to FGIN-1–27 or porphyrine IX. Cellular [. 18. F]-FDG incorporation and alkaline phosphatase activity were suppressed by both ligands. Cell cycle analysis showed a significant decrease in the fraction of cells in the G1 and G2/M phases when exposed to each ligand with a higher proportion of necrotic and apoptotic cells. Western blotting showed a decrease in TSPO abundance following treatment by both ligands. LDH activity in culture media significantly increased following exposure to FGIN-1–27 or porphyrine IX. Discussion: We show that FGIN-1–27 and porphyrine IX have a similar cell death inducing affect on human osteoblast-like cell in vitro. This affect is parallel to the inhibition of the cellular metabolism. Since both ligands similarly reduce the availability of TSPO we postulate that their mode of action is similar by affecting this mitochondrial structure with sub sequential induction of cell death, i.e. apoptosis and necrosis. Therefore we suggest that human osteoblast metabolism and cell cycle are mediated through TSPO and that porphyrine IX might be an active endogenous ligand to the TSPO having a regulatory affect on the human bone cell cycle

Introduction: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been reported to suppress bone repair and remodeling in vivo. Our previous studies showed that NSAIDs inhibited osteoblast proliferation and induced cell death in fetal rat osteoblast cultures. However, the NSAIDs effects on the functions of human osteoblasts remain unclear. Newly developed selective cyclo-oxygenase 2 (COX-2) inhibitors, celecoxib and refecoxib, have been reported to have lower risk of gastrointestinal complications than traditional nonsteroidal anti-inflammatory drugs. A recent report showed that refecoxib decreased bone ingrowth in an animal study. However, the effects of COX-2 selective inhibitors on human osteoblasts have rarely been investigated. In this study, the effects of steroid, non-selective, and selective COX-2 inhibitors on proliferation, cell cycle kinetics, and cytotoxicity in cultured human osteoblasts were examined. Materials and Methods: Indomethacin,ketorolac,piroxicam, and diclofenac (10. −5. and 10. −4. M); dexamethasone (10. −7. and 10. −6. M); Celecoxib and DFU, an analogue of rofecoxib, (10. −7. –10. −4. M) were tested for 24 or 48 hr in human osteoblast cultures. Results: In this study, we found that a 24 hour treatment of COX-2 selective inhibitors, celecoxib and DFU, significantly inhibited proliferation, arrested cell cycle, and had cytotoxicity in cultured human osteoblasts. However, the inhibitory effect on proliferation could be reversed if these agents were withdrawn for 24 hours. Indomethacin, ketorolac, diclofenac, and piroxicam also significantly inhibited proliferation and arrested cell cycle at the G. 0. /G. 1. phase, but had no cytotoxic effects on human osteoblasts. Discussion: These results suggest that the COX-2 selective and non-selective NSAIDs may affect osteoblastic functions through different mechanisms

Maintenance of acid-base homeostasis in extracellular fluids and in the cytoplasm is essential for the physiological activities of cells and tissues [1]. However, changes in extracellular pH (pHe) occurs in a variety of physiological and pathological conditions, including hypoxia and inflammation associated with trauma and cancer. Concerning bone tissue, if abnormal acidification occurs, mineral deposition and osteoblast differentiation are inhibited, whereas osteoclast formation and activity are enhanced [2]. Indeed, acidification, that usually occurs in the early phases of fracture repair, has been suggested as a driving force for regeneration via release of growth factors that act on the stem cell fraction of repair bone [3]. However, the effect of low pHe on stemness has been insufficiently explored so far. Thus, in this study, we investigated the role of short term exposure to low pHe (6.5–6.8) on MSC stemness. MSC derived from dental pulps (DPSC) and bone marrow (BM-MSC) were used. To perform the specific assays, culture medium at specific pH (6.5, 6.8, 7.1 and 7.4) was maintained by using different concentrations of sodium bicarbonate according to the Henderson-Hasselbach equation. Changes in osteoblast-related gene expression (COL1A1 and ALPL), and mineral nodule formation were measured by qRT-PCR and Alizarin red staining, respectively. The stem phenotype was analysed by measuring changes in stemness-related genes (SOX2, OCT4, KLF4, c-MYC) expression and spheres forming ability. Additionally, cell number, Ki67 index and cell cycle were analysed to monitor cell proliferation and quiescence. We confirmed that acidic pHe inhibits the osteogenic differentiation of DPSC. Low pHe significantly but transitorily decreased the expression of osteoblast-related genes (COL1A1 and ALPL) and decreased the mineral nodule formation in vitro. Acidic pHe conditions significantly increased the ability of DPSC and BM-MSC to form floating spheres. At acidic pHe spheres were higher but smaller when compared to spheres formed at alkaline pHe conditions. Moreover, acidic pHe increased significantly the expression of stemness-related genes. Finally, low pHe induced a significant decrease of DPSC cell number. Reduction of cell proliferation correlated with a lower number of cycling cells, as revealed by the Ki67 index that significantly decreased in a pH-dependent manner. Cell cycle analysis revealed an accumulation of cells in the G0 phase, when cultured at low pH. In this study, we demonstrated a close relationship between acidic pHe and the regulation of MSC stemness. We therefore suggest that pHe modulation of MSC stemness is a major determinant of skeletal homeostasis and regeneration, and this finding should be considered in bone healing strategies based on cell therapy

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

Aim. In osteosarcoma, local control of the tumour is absolutely critical otherwise the chances of long term survival are <10% and may effectively approach zero. Radiotherapy is used in case of non-resectable tumours. Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation (XRT) by altering numerous molecular pathways. Therefore, we investigated the effect of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on radiation response in osteosarcoma (OS) and rhabdomyosarcoma (RMS) cell lines. Methods. Clonogenic survival, cell cycle analysis, apoptosis and gammaH2AX phosphorylation as a marker of DNA double strand breaks (DSBs) were examined in two OS (KHOS-24OS, SAOS2) and two RMS (A-204, RD) cell lines treated with SAHA alone and SAHA plus XRT, respectively. Protein expression was investigated via immunoblot analysis, cell cycle analysis, measurement of apoptosis and gammaH2AX expression were performed using flow cytometry. Results. In the sarcoma cell lines, SAHA induced an inhibition of cell proliferation and clonogenic survival and lead to a significant radiosensitization. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In both tumour entities, gammaH2AX expression was significantly increased when XRT was combined with SAHA treatment which was correlated with attenuation of radiation-induced DNA repair protein expression. Conclusion. Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair rather than increased apoptosis induction after exposure to HDACIs could be one key mechanism of radiosensitization by HDACIs

The development of multidisciplinary therapy for Ewing’s sarcoma (ES) has increased current long-term survival rates to greater than 50%, but only 20% for patients with clinically detectable metastases at diagnosis, or not responding to therapy or with disease relapse. Anti-bone resorption bisphosphonates (BP) may represent promising adjuvant molecules to limit the osteolytic component of bone tumor. The combination of zoledronic acid (ZOL) and ifosfamide (IFOS) or mafosfamide (MAFOS) was studied in ES models and in 8 human cell lines all expressing the EWS-FLI1 fusion gene. Cell proliferation, viability, apoptosis and cell cycle distribution were analysed. The ES models were developed in immuno-deficient mice by inoculating the human tumor cells either intra-muscular (soft tissue tumor development) or intra-osseous (bone tumor development). Mice were then treated with ZOL (100 μg/kg twice or 4 times/week) and/or ifosfamide (IFOS 30 mg/kg, one to 3 sequences of 3 injections). All the cell lines studied were more or less sensitive to ZOL and MAFOS in terms of cell proliferation. Both drugs induced cell cycle arrest respectively in S and G2M phase and final apoptosis associated to caspase 3 activation. In vivo, ZOL had no effect on soft tumor progression although it dramatically inhibits ES development in bone site. When combined with IFOS, ZOL exerts synergistic effects in the soft tissue model leading to a similar quantitative inhibitory effect when associated with 1 sequence IFOS as compared to 3 sequences of IFOS alone. In the bone model, ZOL prevents tumor recurrence observed with a lonely sequence of IFOS. Combination of ZOL with conventional chemotherapy showed promising results in both ES models and could allow the clinicians to diminish the doses of chemotherapy. Moreover, as ZOL and MAFOS induce cell death by different pathways, respective resistance may be circumvented

Osteosarcoma is the most frequent malignant primary bone tumors. Despite recent improvements in multimodal therapy the problem of non-response to mono-chemotherapy remains. Therefore, novel multi-drug combinations targeting various molecular pathways are needed to decrease the emergence of resistance phenomenon and to potentiate the treatment efficacy. In this context, the effects of RAD001, a new orally available mTOR inhibitor was investigated in vitro and in vivo on osteosarcoma proliferation, both alone and in combination with Zoledronic acid (ZOL). The in vitro effects of ZOL and RAD001 were analyzed on human (MG63), rat (OSRGa) and mouse (POS-1 and MOS-J) osteosarcoma cell lines in terms of cell proliferation (XTT assay, manual cell counting, time-lapse microscopy), cell cycle analysis (flow cytometry analysis) and apoptosis (caspase 1, 3 and 8 activity). RAD001 and ZOL inhibit MG63, OSRGA and POS-1 osteosarcoma cell proliferation in a dose- and time-dependent manner without any modification of cell cycle distribution. In contrast, MOS-J cells are resistant to ZOL and RAD001. In all cell lines assessed, combination of RAD001 and ZOL exerts synergistic effect on the inhibition of cell proliferation and induces a significant decrease of P-mTOR, P-4EBP1 and Ras expression with no accumulation of IPP and ApppI. This drug combination has been then investigated in a mouse osteosarcoma model induced by i.m. inoculation of MOS-J cells in C57BL/6J mice. Clinical relevant doses of RAD001 (5 mg/kg) and ZOL (100 μg/kg) alone have no effect on tumor growth in contrast to combination of both drugs which decreases osteosarcoma progression. ZOL (alone or in combination) strongly increases bone formation. The combination of RAD001 with ZOL improves tissue repair as shown by important area of fibrosis into the residual tumor mass. The present work demonstrates the in vitro and in vivo synergistic effect of mTOR (RAD001) and mevalonate (ZOL) pathway inhibitors and suggests that ZOL potentiates RAD001 activity through Ras molecular pathway

A non-invasive technique for labelling S phase osteoblasts in vitro following immunolabelling of their focal adhesions is proposed. Quantification of cell adhesion area in the S phase (where the cells are most spread) of the cell cycle is then possible with a scanning electron microscope (SEM). Primary calvarial osteoblasts (isolated by migration) were cultured on plastic and implant quality metal discs. S-phase cells were labelled by a pulse of 3H thymidine in the culture medium for 30 min. Cells were cultured for a further 2h in normal media before being processed for immunogold labelling of vinculin. Briefly, cells were permeabilised and fixed in 4% paraformaldehyde. Non specific binding sites were blocked for 30 min. Cells were incubated with mouse anti vinculin for 1h before rinsing and blocking with 5% goat serum for 30 min. Secondary incubation was with goat anti mouse 5nm gold conjugate for 2h. After rinsing, cells were permanently fixed with 2.5% glutaraldehyde. For SEM visualisation, the gold label was enhanced with gold enhance solutions. Postfixation and staining was performed with osmium tetroxide. Samples were dehydrated and critically point dried. The discs were carbon coated and covered with a thin layer of photographic emulsion in a dark room and left in a light tight box at 4°C for 7 days before developing the emulsion. Backscattered electron imaging with the SEM revealed silver grains on the nuclei of S-phase cells, produced by the interaction of radioactive emissions, from the labelled DNA, and the photographic emulsion. Immunolabelled focal adhesions were also observed at higher magnifications on the same cells. This combination of autoradiography and high resolution SEM removes cell cycle variability, which has been a problem with previous in vitro adhesion studies. This method will be applied to quantify osteoblast cell adhesion to various implant materials to evaluate cell/implant interactions

Objective. Early cell loss of up to 50% is common to in vitro chondrogenesis of mesenchymal stromal cells (MSC) and stimulation of cell proliferation could compensate for this unwanted effect and improve efficacy and tissue yield for cartilage tissue engineering. We recently demonstrated that proliferation is an essential requirement for successful chondrogenesis of MSC, however, how it is regulated is still completely unknown. We therefore aimed to identify signaling pathways involved in the regulation of proliferation during in vitro chondrogenesis and investigated, whether activation of relevant pathways could stimulate proliferation. Design. Human MSC were subjected to in vitro chondrogenesis for up to 42 days under standard conditions in the presence of 10 ng/ml TGF-β. Cells were or were not additionally treated with inhibitors of bone morphogenetic protein (BMP), insulin-like growth factor (IGF) IGF/PI3K, fibroblast growth factor (FGF) or indian hedgehog (IHH) pathways for two or four weeks. To investigate the stimulation of proliferation by exogenous factors, cells were treated with BMP-4, IGF-1, FGF-18 or purmorphamine (small molecule hedgehog agonist). Proliferation was determined by [3H]-thymidine incorporation. Results and Discussion. Quantitative assessment of proliferation revealed that proliferation arrest occurred during condensation up to day 3 and cell division was re-initiated thereafter with a peak on day 28. To test which pathways are relevant for the restart of proliferation, BMP, IGF/PI3K, FGF or IHH signaling was inhibited up to day 14. All treatments significantly reduced proliferation > 50% and, thus, seemed to participate in the re-entry into the cell cycle. To study whether the same pathways are relevant to maintain cells in a proliferative state later on, inhibitors were supplemented from day 14–28. This resulted in a significant decrease of proliferation in the groups treated with inhibitors of BMP (67% decrease), FGF (70%) and IHH (30%) signaling, while inhibition of IGF/PI3K did not influence late proliferation. Although BMP-4, IGF-1 or FGF-18 are known mitogenic factors in the growth plate, stimulation of cells by exogenous addition of these factors did not enhance proliferation in any differentiation phase. In contrast, stimulation of IHH signaling from day 14–28 significantly increased proliferation by 44%. This is in line with the documented strong mitogenic activity of hedgehog signaling in the proliferative zone of the growth plate. Thus, our data demonstrated that BMP, IGF/PI3K, FGF and IHH essentially participate in the regulation of proliferation during in vitro chondrogenesis. Early or late activation of single pathways by exogenous factors was, however, not sufficient to increase proliferation significantly with the exception of late activation of hedgehog signaling. Optimization of stimulation of the hedgehog pathway with a focus on increased tissue yield will now be the next step

Tissue expansion is a technique used by plastic and restorative surgeons to cause the body to grow additional skin, bone or other tissues. For example, distraction osteogenesis has been widely applied in lower limb surgery (trauma / congenital), and congenital upper limb reconstruction (e.g. radial dysplasia). This complex and tightly regulated expansion process can thus far only be optimised by long-term animal or human experimentation. Here the intent is to develop an in vitro model of tissue expansion that will allow to both optimise the extension regime (µm/h, continuous/ intermittent) and investigate using proteomic techniques which molecular pathways are involved in its regulation. Cells cultured onto sheets of polymer (PCL) can be stretched at very low, adjustable speeds, using a stepper motor and various 3D printed and laser cut designs. The system utilises plastic flow of the polymer, enabling the material to stay extended upon strain being released. Tensile tests have displayed the plastic behaviour of the polymer sheet when stretched, and digital image correlation (DIC) has been used to analyse homogeneity of the strain field. Further analysis involving nuclear localisation of yes-associated protein (YAP) aims to link cell response to this strain field. Nuclear orientation analysis has demonstrated a morphological response to strain (1 mm/day) in comparison to not being stretched, and this is in the process of being linked to nanoscale changes of the substrate (using atomic force microscopy) during the stretching regime. Future work will identify how strain is affecting the cell cycle, before a mass tagging approach is used to identify protein changes induced by strain

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Aims

Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy.

Aims

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation.