Periprosthetic joint infections (PJI) are increasing in prevalence and are recognised as one of the most common modes of failure of joint replacements. Osteomyelitis arising from PJI is challenging to treat, difficult to cure and increases patient mortality 5-fold. PJI can have subtle symptoms and lie dormant or go undiagnosed for many years, suggesting persistent bacterial infection. Staphylococcus aureus is the most common pathogen causing PJI. Osteocytes are the most numerous and long-lived cell type in hard bone tissue. Our recent work has shown that S. aureus can infect and reside in human osteocytes without causing cell death, both experimentally and in bone samples from patients with PJI. Osteocytes respond to infection by the differential regulation of a large number of genes, suggesting previously unknown immune functions of this important cell type. S. aureus adapts during intracellular infection of osteocytes by adopting a quasi-dormant, small colony variant (SCV) phenotype, a property of several bacterial species known to cause PJI, which could contribute to persistent or silent infection. These findings shed new light on the aetiology of PJI and osteomyelitis in general. Further elucidation of the role of osteocytes in bone infection will hopefully lead to improved disease detection and management

Abstract. Objectives. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Methods. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab. Results. Low mRNA expression of PIEZO1 in unloaded cells was upregulated 5-fold following 1-hr of mechanical load (p=0.003). In addition, the transcription factor NFATc1, a known regulator of Piezo1 mechanotransduction, was also upregulated by load (2.4-fold; p=0.03). Y201 cells differentiated in gels expressed the osteocyte marker, SOST. Yoda1 upregulated PIEZO1 (1.7-fold; p=0.057), the early mechanical response gene, cFOS (4-fold; p=0.006), COL1A1 (3.9-fold; p=0.052), and IL-6 expression (7.7-fold; p=0.001). Discussion. This study reveals PIEZO1 as an important mechanosenser in osteocytes. Piezo 1 mediated increases in the bone matrix protein, type I collagen, and IL-6, a cytokine that drives inflammation and bone resorption. This provides a direct link between mechanical activation of Piezo 1, bone remodelling and inflammation, which may contribute to mechanically-induced joint degeneration in osteoarthritis. Mechanistically, we hypothesise this may occur through promoting Ca2+ influx and activation of the NFAT1 signalling pathway

Objectives. Osteoporosis and osteomalacia lead to increased fracture risk. Previous studies documented dysregulated osteoblast and osteoclast activity, leading to a high-turnover phenotype, reduced bone mass and low bone mineral content. Osteocytes, the most abundant bone cell type, are involved in bone metabolism by enabling cell to cell interaction. Osteocytes presence and viability are crucial for bone tissue homeostasis and mechanical integrity. Osseo-integration and implant degradation are the main problems in developing biomaterials for systemically diseased bone. This study examines osteocyte localisation, morphology and on the implant surface and at the implant bone interface. Furthermore, the study investigates ECM proteins regulation correlated to osteocytes and mechanical competence in an ovariectomised rat model with a critical size metaphyseal defect. Methodology. After induction of osteoporosis, 60 female Sprague-Dawley rats were randomised into five groups: SrCPC (n=15), CPC (n=15), ScB30 (n=15), ScB30Sr20 (n=15) and empty defect (n=15). The left femur of all animals underwent a 4mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with the above mentioned implants or left empty. After six weeks, histomorphometric analysis showed a statistically significant increase in bone formation at the tissue-implant interface in the SrCPC group compared to the other groups (p<0.01). Osteocyte morphology and networks were detected using silver and staining. ECM proteins were investigated through immunohistochemistry. Cellular populations were tested using enzyme histochemistry. Mineralisation was assessed using time of flight secondary ion mass spectrometry (TOF-SIMS). Statistical analysis was performed using Mann Whitney U test with Bonferroni correction. Results. In the SrCPC and compared to other test groups, osteocytes presence and morphology was enhanced. An increased osteocytic activity was also seen in ScB30Sr20 when compared to SCB30 alone. Local osteomalatic lesions characterised by the presence of excessive unmineralised osteoid as revealed by the VKVG staining in the intact bone was also seen. A regular pattern of osteocytes distribution reflecting a better bone maturation was also seen in case of the Sr substituted cements. Whereas in case of the ScB30 degenerated osteocytes with a comparatively irregular arrangement were seen. Nonetheless, ECM proteins indicating discrepant bone turnover (RANKL, OPG, BMP2, OCN; ASMA) were noticed to increase within these regions and were accompanied by the presence of apoptotic osteocytes. Interestingly, osteocytes were also localised near the blood vessels within the newly formed woven bone. On the other hand, osteocytes allocation at implant bone interface and on the implant surface were qualitatively better in the Sr substituted groups when compared to the other test groups. Furthermore, this correlates with healing enhancement and implant retention results obtained from the histomorphometry (BV/TV and Osteoclasts count). The first qualitative results of the sclerostin visualisation showed a lower expression in the Sr supplemented biomaterials compared to the Sr free ones. Conclusion. Osteoblasts, osteoclast and osteocytes are the key players to bone metabolism through production and mineralisation of ECM or resorption. The current study indicates the importance in therapeutically targeting osteocytes to regulate bone metabolism in osteoporotic/osteomalatic bone. Sr inhibits osteoclast activity which is important for implant degradation. However, in osteoporotic bone osteoclasts inhibition is crucial to enhance the healing. Our data suggest that osteocytes allocation at the bone implant interface and on the implant surface is aiding in implant degradation through osteocytes dependent resorption. Currently, discrepancies in mechanosensitivity, proliferation and fibrotic tissue formation are being investigated together with several anchorage proteins to quench further effects of osteocyte presence at the implant bone interface

Osteoarthritis (OA) is mainly caused by ageing, strain, trauma, and congenital joint abnormalities, resulting in articular cartilage degeneration. During the pathogenesis of OA, the changes in subchondral bone (SB) are not only secondary manifestations of OA, but also an active part of the disease, and are closely associated with the severity of OA. In different stages of OA, there were microstructural changes in SB. Osteocytes, osteoblasts, and osteoclasts in SB are important in the pathogenesis of OA. The signal transduction mechanism in SB is necessary to maintain the balance of a stable phenotype, extracellular matrix (ECM) synthesis, and bone remodelling between articular cartilage and SB. An imbalance in signal transduction can lead to reduced cartilage quality and SB thickening, which leads to the progression of OA. By understanding changes in SB in OA, researchers are exploring drugs that can regulate these changes, which will help to provide new ideas for the treatment of OA. Cite this article: Bone Joint Res 2023;12(9):536–545

Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway

Osteocytes (OCY) are the end stage differentiation cells of the osteoblast lineage, and are incorporated in the bone matrix during bone formation. In doing so, OCY control the mineralisation of osteoid. OCY form a dense inter-connected network of cell bodies and cell processes throughout the mineralised matrix of bone. OCY viability depends on interstitial fluid flow along the OCY canaliculi, driven by pulsatile blood flow and loading of the skeleton. Maintenance of the density and viability of OCY are essential for bone health because OCY perform many important functions in bone. Firstly, OCY appear to initiate bone repair of bone microdamage. Secondly, OCY are almost certainly the cells, which initiate new bone formation in response to increased loading of bone. Thirdly, OCY are able to regulate the amount of new bone formation in bone remodelling cycles, at least in part by the production of a molecule called sclerostin (SCL). Mutations in the SCL gene, or deletion of the SCL gene in transgenic mice, are associated with particularly dense, fracture resistant bones. This information has led to development of anti-SCL antibodies as a potential anabolic therapy for bones. Bone loss in ovariectomised aged rats was shown recently to be reversed by treatment with neutralising SCL antibodies. There is also some data to suggest that these antibodies may promote fracture healing. Reduced OCY viability and/or density have been reported in association with osteoporotic fracture. OCY viability seems to be dependent on skeletal loading, adequate skeletal blood flow and estrogen in females. OCY viability is adversely affected by hypoxia, unloading of the skeleton and pharmacobiology, such as chronic exposure to glucocorticoids. Both micro and macro-fractures result in disruption of the OCY network, as do procedures such as drilling and cutting of bone. Because of the important roles of OCY in bone, new approaches to bone health may require the identification of agents to protect these cells from harmful influences in disease and ageing

Osteocytes direct bone adaptation to mechanical loading (e.g., exercise), but the ways in which osteocytes detect loading remain unclear. We recently showed that osteocytes develop repairable plasma membrane disruptions (PMD) in response to treadmill-running exercise, and that these PMD initiate mechanotransduction. As treadmill running is a non-voluntary activity for rodents, our current goal was to determine whether osteocytes develop PMD with voluntary wheel running as a better model of physiological exercise. Male and female Hsd:ICR mice from lines selectively bred (>75 generations) to demonstrate high voluntary wheel running (“High Runners”) or non-selected control lines (“Control”) were studied (n=9 to 12 mice per sex per line, 4 lines each). At 12 weeks of age, half of the animals within each group were provided access to running wheels for 6 days while remaining mice had no wheel access. Tibias were collected at sacrifice and bone mineral density was analyzed by DXA. Osteocyte PMD were quantified by immunochemistry for intracellular albumin. Groups were compared with 3-factor ANOVA. Voluntary exercise (wheel access) significantly increased osteocyte PMD (+16.4%, p=0.013). PMD-labelled osteocytes did not differ between sexes (p=0.415). Male mice had significantly greater BMD (p=0.0007) and BMC (<0.0001) than females. Interestingly, mice with wheel access had significantly lower BMD and BMC compared to mice without wheel access (p<0.004), and high runner lines had significantly lower BMD (p=0.001) and BMC (p<0.0001) than control lines. This may reflect new bone formation in the exercising mice, as newly formed bone is less mineralized than older bone. Data from this experiment support the idea that loading-induced disruptions develop in the osteocyte plasma membrane during both voluntary (wheel running) and forced (treadmill, shown previously) physical activity. These studies support the role of plasma membrane disruptions as a mechanosensation mechanism in osteocytes

Aim

Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria.

Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception.

Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception.

Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test.

All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation.

Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression.

Abstract

Abstract

Aims

To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism.

Aims

Osteoarthritis (OA) is a common degenerative joint disease. The osteocyte transcriptome is highly relevant to osteocyte biology. This study aimed to explore the osteocyte transcriptome in subchondral bone affected by OA.

Clinical use of glucocorticoids engenders deleterious changes in bone fragility and initiates apoptosis in osteoblasts and osteocytes. The pathways leading to corticosteroid-induced death in bone remain unclear. Similarly little is known about the effects of ‘bone sparing’ bisphosphonates on osteocytes in vivo. We investigated the effects of bisphosphonates (BPs) on dexamethasone (Dex)-induced apoptosis in the murine osteocyte cell line, MLO-Y4 and studied the putative pathways involved by intervention with inhibitors of signalling molecules, such as p42/44 MAPK and protein kinase A (PKA). Cells were preincubated with N- & non N-containing BPs and/or inhibitors before insult with Dex or H₂O₂ for 5 hrs. Apoptotic morphology was revealed by acridine orange staining. Activation of p42/44 was identified using Western blotting and in situ immunocytochemistry in the presence or absence of serum.

Both N- & non N-containing BPs were shown to protect against cell death. The addition of inhibitors of p42/44 and PKA blocked the action of Dex. H₂O₂-induced apoptosis was not blocked by BPs or by any of the inhibitors. Dex appeared to activate p42/44 only in serum supplemented cultures. These data suggest that glucocorticoid but not oxidant-induced osteocyte apoptosis involves activation of p42/44 and that bisphosphonate engendered cell rescue is brought about by inhibition of these MAPK’s. Studies using truncated BPs that lack anti-resorptive activity, and therefore do not interrupt bone remodelling showed that these BPs were also able to protect osteocytes from glucocorticoid-induced death. The ability of bisphosphonates to influence MAPK activation and cell death in the osteocyte opens up exciting possibilities for pharmaceutical intervention during age and steroid hormone related osteocyte loss.

Age-related fragility fractures are highly correlated with the loss of bone integrity and deteriorated morphology of the osteocytes. Previous studies have reported low-magnitude high-frequency vibration(LMHFV) promotes osteoporotic diaphyseal fracture healing to a greater extent than in age-matched normal fracture healing, yet how osteoporotic fractured bone responds to the mechanical signal has not been explored. As osteocytes are prominent for mechanosensing and initiating bone repair, we hypothesized that LMHFV could enhance fracture healing in ovariectomized metaphyseal fracture through morphological changes and mineralisation in the osteocyte Lacuno-canalicular Network(LCN). As most osteoporotic fractures occur primarily at the metaphysis, an osteoporotic metaphyseal fracture model was established.

A total of 72 six-month old female Sprague-Dawley rats (n=72) were obtained(animal ethical approval ref: 16–037-MIS). Half of the rats underwent bilateral ovariectomy(OVX) and kept for 3 months for osteoporosis induction. Metaphyseal fracture on left distal femur was created by osteotomy and fixed by a plate. Rats were then randomized to (1) OVX+LMHFV(20 mins/day and 5 days/week, 35Hz, 0.3g), (2) OVX control, (3) SHAM+LMHFV, (4) SHAM control. Assessments of morphological structural changes, functional markers of the LCN(Scanning Electron Microscopy, FITC-Imaris, immunohistochemistry), mineralization status(EDX, dynamic histomorphometry) and healing outcomes(X-ray, microCT, mechanical testing) were performed at week 1, 2 and 6 post-fracture. One‐way ANOVA with post-hoc test was performed. Statistical significance was set at p < 0.05.

Our results showed LMHFV could significantly enhance the morphology of the LCN. There was a 65.3% increase in dendritic branch points(p=0.03) and 93% increase in canalicular length(p=0.019) in the OVX-LMHFV group at week 2 post-fracture. Besides, a similar trend was also observed in the SHAM+LMHFV group, with a 43.4% increase in branch points and 53% increase in canaliculi length at week 2. A significant increase of E11 and DMP1 was observed in the LMHFV groups, indicating the reconstruction of the LCN. The decreasing sclerostin and increasing FGF23 at week 1 represented the active bone formation phase while the gradual increase at week 6 signified the remodelling phase. Furthermore, Ca/P ratio, mineral apposition rate and bone formation rate were all significantly enhanced in the OVX+LMHFV group. The overall bone mineral density in BV was significantly raised in the OVX+LMHFV group at week 2(p=0.043) and SHAM+LMHFV at week 6(p=0.04). Quantitative analysis of microCT showed BV/TV was significantly increased at week 2 in OVX+LMHFV group(p=0.008) and week 6(p=0.001) in both vibration groups. In addition, biomechanical testing revealed that the OVX+LMHFV group had a significantly higher ultimate load(p=0.03) and stiffness(p=0.02) at week 2.

To our best knowledge, this is the first report to illustrate LMHFV could enhance osteocytes' morphology, mineralisation status and healing outcome in a new osteoporotic metaphyseal fracture animal model. Our cumulative data supports that the mechanosensitivity of bone would not impair due to osteoporosis. The revitalized osteocyte LCN and upregulated osteocytic protein markers implied a better connectivity and transduction of signals between osteocytes, which may foster the osteoporotic fracture healing process through an enhanced mineralisation process. This could stimulate further mechanistic investigations with potential translation of LMHFV to our fragility fracture patients.

Bone metastases are common and severe complications of cancers. It is estimated to occur in 65–75% of breast and prostate cancer patients and cause 80% of breast cancer-related deaths. Metastasised cancer cells have devastating impacts on bone due to their ability to alter bone remodeling by interacting with osteoblasts and osteoclasts. Exercise, often used as an intervention for cancer patients, regulates bone remodeling via osteocytes. Therefore, we hypothesise that bone mechanical loading may regulate bone metastases via osteocytes. This provides novel insights into the impact of exercises on bone metastases. It will assist in designing cancer intervention programs that lowers the risk for bone metastases. Investigating the mechanisms for the observed effects may also identify potential drug targets.

MLO-Y4 osteocyte-like cells (gift of Dr. Bonewald, University of Missouri-Kansas City) on glass slides were placed in flow chambers and subjected to oscillatory fluid flow (1Pa; 1Hz; 2 hours). Media were extracted (conditioned media; CM) post-flow. RAW264.7 osteoclast precursors were conditioned in MLO-Y4 CM for 7 days. Migration of MDA-MB-231 breast cancer cells and PC3 prostate cancer cells towards CM was assayed using Transwell. Viability, apoptosis, and proliferation of the cancer cells in the CM were measured with Fixable Viability Dye eFluor 450, APOPercentage, and BrDu, respectively. P-values were calculated using Student's t-test.

Significantly more MDA-MB-231 and PC3 cells migrated towards the CM from MLO-Y4 cells with exposure to flow in comparison to CM from MLO-Y4 cells not exposed to flow. The preferential migration is abolished with anti-VEGF antibodies. MDA-MB-231 cells apoptosis rate was slightly lower in CM from MLO-Y4 cells exposed to flow, while proliferation rate was slightly higher. The current data showed no difference in cancer cells viability and adhesion to collagen between any two groups. On the other hand, it was observed that less MDA-MB-231 cells migrated towards CM from RAW264.7 cells conditioned in CM from MLO-Y4 cells stimulated with flow in comparison to those conditioned in CM from MLO-Y4 cells not stimulated with flow. TRAP staining results confirmed that there were less differentiated osteoclasts when RAW264.7 cells were cultured in CM from MLO-Y4 cells exposed to flow.

Overall, this study suggests that when only osteocytes and cancer cells are involved, osteocytes subjected to mechanical loading can promote metastases due to the increased secretion of VEGF. However, with the incorporation of osteoclasts, mechanical loading on osteocytes seems to reduce MDA-MB-231 cell migration. This is likely because osteocytes reduce osteoclastogenesis in response to mechanical stimulation, and osteoclasts have been shown to support cancer cells. Animal studies will also be conducted to verify the pro- or anti-metastatic effect of mechanical loading that is observed in the in vitro part of this study.

Introduction: Open lower leg fractures are frequently associated with severe soft tissue damage. Cortical bone tissue is thus denudated. Osteomyelitis and impaired circulation with loss of bone tissue and subsequent defects are among the main complications. Necrosis vs. revascularisation are supposed to be reflected by local tissue contents of high energy phosphates.

Methods: 80 inbred white New Zealand rabbits with two groups of 40 animals each were employed. Each animal had a tibial fracture induced in a standardized fashion, stabilized by screw osteosynthesis. The fracture area was freed from soft tissue and periost and the medullary space reamed. After 3 or 7 days (group one or two, respectively), the tissue defect was covered by a local fascia-free gastrocnemius muscle flap. In increasing intervalls from one to 16 weeks, the implants were removed and the animals euthanized. Cortical bone of the fragment created and of the adjacent cortical bone with and without periostal linig was analysed. The bone was removed after euthanisation and analysed histomorphologically. Simultaneously, fragments were deep frozen in liquid nitrogen at −190°C, a two by one centimeter fragment from the unaffected contralateral tibia harvested as control. Analysis of high energy phosphates (ATP) was performed by high pressure liquid chromatography as described by NEES (HPLC). All animals were kept i

Results: The average ATP contents in healthy cortical bone was 0,092 +/− 0,009 nmol/mg dry weight. A muscle flap after three days led to significantly higher concentrations as compared to 7 days with 0,081 +/− 0,011 vs 0,03 +/− 0,008 nml/mg dry weight (mean +/− SEM; p < 0,05, paired t-test), the latter resembling sequestration. Simultaneously, flap covering after three days displayed a lower rate of necroses with 23 vs. 40 % (p < 0,05, paired t-test). Incidence of osteomyelitis was as well higher in the 7-days-group (24%).

Discussion: Delayed plastic covering of open lower leg fractures led to decreased ATP levels, delayed healing and infection in our experimental setting. For the first time, we could determine the contents of ATP by HPLC in cortical bone. Increase in ATP contents reflected the biological quality of the bone investigated, ranging from reconstituted healthy bone to sequesters.

Introduction: Evidence exists concerning the anti-oxidant properties of oestrogen in protecting neuronal cells from oxidative stress. The withdrawal of oestrogen after menopause is the major factor determining age related bone loss and apoptotic death of osteocytes. While oestrogen replacement demonstrates clear oestrogen receptor mediated benefits to bone cells little is known regarding oestrogens’ anti-oxidant effects in bone.

Methods: Here we have used MLO-Y4 osteocyte-like cell line to determine whether oestrogen saving effects on osteocytes involves its activities as an anti-oxidant.

MLO-Y4 cells were treated with physiological doses (10⁻⁸)M of either 17-beta E₂ or the oestrogen receptor inactive stereoisomer 17-alpha E₂ with or without the specific oestrogen receptor antagonist ICI 182,780 prior to the addition of 0.4milliM 30% (v/v) H₂O₂. Cellular apoptosis was determined using morphological and biochemical criteria.

Results: H₂O₂ induced an increase in apoptosis of MLO-Y4 (14.3 ± 3 SD vs control 1.4 ± 0.9). Pre-treatment of the cells with 17-beta E₂ significantly reduced H₂O₂ induced apoptosis (2.4 ± 0.96). Pre-treatment of cells with 17-alpha E₂ or ICI 182,780 also reduced oxidant induced apoptosis to 3.4 ± 1.5 SD and 7.0 ± 2.3 respectively.

The cellular production of reactive oxygen species was determined using the free radical indicator 2′7′- dichlorodihydrofluorescein diacetate. H₂O₂ induced increases in the number of ROS positive cells (34.6 ± 9.07 SD vs control 0.22 ± 0.39 SD). In contrast pre-treatment with both 17-beta E₂ and 17-alpha E₂ reduced the number of ROS positive cells associated with H₂O₂ treatment (Fig 1).

Conclusion: These data suggest that oestrogens ability to save osteocytes from oxidant induced death is independent of the oestrogen receptor and may be related to oestrogens known activity as an anti-oxidant. This raises the possibility that loss of osteocytes during oestrogen insufficiency may occur through a failure to suppress the activity of naturally occurring or disease associated production of oxidant molecules.

Introduction and Objective. The osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances that occur during bone remodeling, caused by hormone perturbations or alterations in mechanical loading, can induce bone disease as osteoporosis. Due to limited understanding of the underlying mechanisms, current therapies for osteoporosis cannot adequately address this imbalance because current studies of osteocytes rely on conventional cell culture that cannot recapitulate local in vivo microenvironments for the lack of control of the spatial/temporal distribution of cells and biomolecules. Microfluidics is the science and technology of microscale fluid manipulating and sensing and can help fill this gap. Materials and Methods. We used a microfluidic device to enable the culture of osteocyte-like cells (MLO-Y4 and MLO-A5) in a 3D fashion. Osteocytes were cultured in a perfused and 160 μm high channel and embedded in a bone-like extracellular matrix: osteocytes were embedded in a matrigel- and collagen-based hydrogel enriched with nanostructured hydroxypatite crystals (HA-NP) to mimic bone. To set up the best combination of matrigel enriched with Type I collagen we used fluorescent microspheres and confocal analysis. To evaluate the viability and the expression of osteocytic markers, we used live-dead assay amd immunofluorescent staining and confocal analysis combined with automated quantification. For mineralization, we performed alizarin red staining. Results. Osteocytes in the organ-on-a-chip model showed high viability and, in respect to 2D conventional cell cultures an increased differentiation, as assessed by a live-dead assay and the staining of the osteocytic markers connexin-43 and alkaline phosphatase and the increased mineralization activity. Furthermore, the addition of HA-NP significantly increased the formation of dendrite-like structures spreading through the xyz-axes, as assessed after G-actin immunofluorescence. Conclusions. Using a microfluidic device for MLO-Y4 and MLO-A5 cell cultures, compared to the 2D surfaces, we demonstrated a significant difference in cell differentiation and morphology. In particular, 3D cultures allowed the formation of 3D cell networks and the osteogenic phenotype. As a platform technology, this microfluidic device can function as a novel cell culture model that enables further studies of osteocytes and 3D co-culturing with other bone cells for the screening of anti-osteoporotic drugs

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro. The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane. Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs. This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment