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

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

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

Introduction. The use of platelet-rich concentrate (PRC) to enhance the healing response in tendon repair is currently an area of considerable interest. Activated platelets release a cocktail of growth factors and ECM regulating molecules. Previous work suggests that tenocytes are activated by contact with these clot-derived molecules. Our studies on tenocytes and PRC aim to establish the direct molecular and functional effects of PRC on tenocytes and to support the clinical research on Achilles tendon repair taking place within our group. We hypothesise that applying PRC to human tenocytes in culture will increase proliferation rate and survival by activating relevant signalling pathways. Materials and Methods. Using a centrifugation method, PRC was extracted from fresh human whole blood. The PRC was immediately clotted and left in medium overnight to release biological factors (at least 95% of presynthesized growth factors are secreted in the first hour of activation). 1. Human tenocytes derived from explanted healthy hamstring were used for up to three passages. Cells were treated with varying concentrations of PRC-conditioned medium and assessed for viable cell number (Alamar Blue™ fluorescence) and proliferation (Ziva™ Ultrasensitive BrdU assay) after 72hrs. For western blotting, cells were treated with 10% PRC for 5 or 30 minutes. Antibodies to P-ERK and P-Akt detected the active protein state on the blot, followed by membrane stripping and re-probing with pan antibodies. Quantification was achieved by densitometry using Visionworks software v. 6.7.1. Results. PRC-conditioned medium affected tenocytes in a dose-dependent manner. Viable number of tenocytes was significantly increased by 10% PRC-conditioned medium compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.001) after 72hrs. 10% PRC-conditioned medium also demonstrated time-dependence with viable tenocyte number significantly increasing between 24 and 72hrs (One-way ANOVA, Bonferroni's post-hoc test P<0.001). After 72hrs, tenocyte proliferation significantly increased in the presence of 5% and 10% PRC-conditioned media compared to controls (One-way ANOVA, Tukey's post-hoc test P<0.05 and P<0.001 respectively). ERK and Akt phosphorylation was strongly stimulated by treatment with 10% PRC-conditioned medium for 5 minutes compared to controls, and remained high after a 30 minute application time. Discussion and Conclusions. Factors released by activated PRC act upon human tendon cells to strongly increase viable cell number and proliferation, which would, in vivo, directly support the healing response, independent of any additional beneficial effects on vascular repair. Both ERK and Akt are pivotal kinases in signalling pathways that favour survival and proliferation. It is clear that both signalling pathways are immediately and strongly activated by PRC, suggesting a clear benefit via both stimulated cell cycle and cell survival in the environmentally compromised conditions of a healing ruptured tendon. This conclusion is strongly supported by previous work on platelet releasates and ERK signalling in other cell types

Objectives

Osteoporosis is a chronic disease. The aim of this study was to identify key genes in osteoporosis.

Objectives

Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs.

Intermittently administered parathyroid hormone (PTH 1-34) has been shown to promote bone formation in both human and animal studies. The hormone and its analogues stimulate both bone formation and resorption, and as such at low doses are now in clinical use for the treatment of severe osteoporosis. By varying the duration of exposure, parathyroid hormone can modulate genes leading to increased bone formation within a so-called ‘anabolic window’. The osteogenic mechanisms involved are multiple, affecting the stimulation of osteoprogenitor cells, osteoblasts, osteocytes and the stem cell niche, and ultimately leading to increased osteoblast activation, reduced osteoblast apoptosis, upregulation of Wnt/β-catenin signalling, increased stem cell mobilisation, and mediation of the RANKL/OPG pathway. Ongoing investigation into their effect on bone formation through ‘coupled’ and ‘uncoupled’ mechanisms further underlines the impact of intermittent PTH on both cortical and cancellous bone. Given the principally catabolic actions of continuous PTH, this article reviews the skeletal actions of intermittent PTH 1-34 and the mechanisms underlying its effect.

Cite this article: L. Osagie-Clouard, A. Sanghani, M. Coathup, T. Briggs, M. Bostrom, G. Blunn. Parathyroid hormone 1-34 and skeletal anabolic action: The use of parathyroid hormone in bone formation. Bone Joint Res 2017;6:14–21. DOI: 10.1302/2046-3758.61.BJR-2016-0085.R1.

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage.

Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test.

Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02).

We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue.