The objective was to seek evidence of hypoxia in early human tendinopathy and thereafter, to explore mechanisms whereby tissue hypoxia may regulate apoptosis, inflammatory mediators and matrix regulation in human tenocytes. Fifteen torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from 10 patients undergoing arthroscopic stabilisation surgery. Markers of hypoxia were quantified by immunohistochemical methods. Human tendon-derived primary cells were derived from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The impact of hypoxia upon tenocyte biology ex vivo was measured using quantitative RT-PCR, multiplex cytokine assays, apoptotic proteomic profiling, immunohistochemistry and annexin V FACS staining. Increased expression of HIF 1a, Bcl-2 and clusterin (hypoxic and apoptotic markers) was detected in subscapularis tendon samples compared to both matched torn samples and non matched control samples (p<0.01). Hypoxic tenocytes exhibited increased production of proinflammatory cytokines (p<0.001), altered matrix regulation (p<0.01) with increased production of Collagen type III operating through a MAPK dependent pathway. Finally, hypoxia increased expression of several mediators of apoptosis and thereby promoted tenocyte apoptosis. Hypoxia promotes expression of proinflammatory cytokines, key apoptotic mediators and drives matrix component synthesis towards a collagen type III profile by human tenocytes. We propose hypoxic cell injury as a critical pathophysiological mechanism in early tendinopathy offering novel therapeutic opportunities in the management of tendon disorders

Adult chondrocytes experience a hypoxic environment in vivo. Culturing chondrocytes under oxygen tension that more closely resembles the in vivo situation, i.e. hypoxic conditions, has been shown to have positive effects on matrix synthesis. During redifferentiation of expanded chondrocytes, hypoxia increased collagen type II expression. However, the mechanism by which hypoxia enhances redifferentiation is still incompletely elucidated. We employed micro-bioreactor technology to elucidate the contribution of TGF-β superfamily ligands to the chondrocyte differentiation process under hypoxic conditions in vitro. Dedifferentiated chondrocytes in alginate were cultured for 48 hours under hypoxic (1% pO2) or normoxic (20%) conditions, using specialized bioreactor technology. Gene expression of chondrocyte-specific markers (SOX9, COL2A1, COL1A1, AGC1 and MMP13) as well as established hypoxia-controlled genes (GDF1-, PHD3, HAS2, VEGF, COX2) and components of the TGF-β superfamily signaling pathways were analyzed by qPCR and protein expression after 48 hours in combination with TGF-β superfamily ligand-specific siRNA as well as selected TGF-β superfamily receptor inhibitors. Hypoxic culture showed robust upregulation of the selected hypoxia-specific marker genes. In addition, well-established chondrocyte-specific markers like SOX9 and collagen type II were upregulated. TGF-β isoforms were selectively upregulated under hypoxia on both mRNA and protein level. In addition, both Activin receptor-like kinases, ALK1 and ALK5, were upregulated under hypoxia, while respective type II and III receptors were unresponsive. The hypoxia-induced COL2 expression was abrogated by TGF-β2 siRNA, as was ALK5 inhibition. Our data strongly indicates that TGF-β superfamily signaling pathways are involved in chondrocyte redifferentiation under low oxygen tension in vitro

We have investigated whether the thigh tourniquet used during total knee replacement (TKR) influenced the development of postoperative wound hypoxia and was a cause of delayed wound healing. We allocated randomly 31 patients (31 TKRs) to one of three groups: 1) no tourniquet; 2) tourniquet inflated at low pressure (about 225 mmHg); and 3) tourniquet inflated to high pressure of about 350 mmHg. Wound oxygenation was measured using transcutaneous oxygen electrodes. In the first week after surgery, patients with a tourniquet inflated to a high pressure had greater wound hypoxia than those with a low pressure. Those without a tourniquet also had wound hypoxia, but the degree and duration were less pronounced than in either of the groups with a tourniquet. Use of a tourniquet during TKR can increase postoperative wound hypoxia, especially when inflated to high pressures. Our findings may be relevant to wound healing and the development of wound infection

Aims. Mesenchymal stem cells (MSCs) are usually cultured in a normoxic atmosphere (21%) in vitro, while the oxygen concentrations in human tissues and organs are 1% to 10% when the cells are transplanted in vivo. However, the impact of hypoxia on MSCs has not been deeply studied, especially its translational application. Methods. In the present study, we investigated the characterizations of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in hypoxic (1%) and normoxic (21%) atmospheres with a long-term culture from primary to 30 generations, respectively. The comparison between both atmospheres systematically analyzed the biological functions of MSCs, mainly including stemness maintenance, immune regulation, and resistance to chondrocyte apoptosis, and studied their joint function and anti-inflammatory effects in osteoarthritis (OA) rats constructed by collagenase II. Results. We observed that long-term hypoxic culture surpassed normoxic atmosphere during hUC-MSCs culture in respect of promoting proliferation, anti-tumorigenicity, maintaining normal karyotype and stemness, inhibiting senescence, and improving immunoregulatory function and the role of anti-apoptosis in chondrocytes. Furthermore, we demonstrated that the transplantation of long-term hypoxic hUC-MSCs (Hy-MSCs) had a better therapeutic effect on OA rats compared with the hUC-MSCs cultured in the normoxic atmosphere (No-MSCs) in terms of the improved function and swelling recovery in the joints, and substantially inhibited the secretion of pro-inflammatory factors, which effectively alleviated cartilage damage by reducing the expression of matrix metallopeptidase 13 (MMP-13). Conclusion. Our results demonstrate that Hy-MSCs possess immense potential for clinical applications via promoting stemness maintenance and enhancing immunoregulatory function. Cite this article: Bone Joint Res 2024;13(12):763–777

Background. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a role in the progression of several common solid cancers. Given that osteosarcoma features extensive local invasion and haematogenous metastases, we hypothesised that osteosarcoma cells utilise MT1-MMP to drive these processes. Moreover, since hypoxia regulates MT1-MMP expression in breast cancer we investigated the effects of hypoxia on MT1-MMP expression in osteosarcoma cells. Aims. Examination of MT1-MMP expression in osteosarcoma biopsy tissue in relation to clinical outcome. Assessment of MT1-MMP, together with hypoxia inducible factors HIF-1α and HIF-2α expression in a panel of osteosarcoma cell lines under normoxia and hypoxia. Methods. Immunohistochemistry: Formalin-fixed and paraffin embedded osteosarcoma biopsy samples from 71 patients were immunostained for MT1-MMP, HIF-1α and -2α and the data correlated with patient survival. Confocal microscopy: following 24 hours culture in 20% versus 1% oxygen, a panel of osteosarcoma cell lines were analysed for the subcellular distribution of MT1-MMP, HIF-1α and -2α. Subcellular fractionation: following 48 hours culture in 20% versus 1% oxygen, the U2OS cell line was fractionated and the compartmental lysates immunoblotted for MT1-MMP, HIF-1α and -2α. Results. Immunohistochemistry showed MT1-MMP immunopositive cytoplasmic and nuclear staining. Biopsy samples with the highest MT1-MMP and HIF-2α intranuclear staining correlated with reduced patient survival: HR 16.10; (95% CI: 5.1–40.3); p< 0.0001. In vitro studies confirmed the intranuclear MT1-MMP presence with an increased nuclear fraction in hypoxia and evidence of nuclear co-localisation with HIF-2α. Conclusions. MT1-MMP expression in osteosarcoma tissue correlates with patient survival. The functional significance of the increased intranuclear presence in hypoxia warrants further investigation

Purpose: It is known that postoperative delirium and hypoxia are factors that make mobilisation of elderly patients more difficult. Our purpose was to estimate the rate of hypoxia and delirium postoperatively, and their possible correlation, in elderly people suffering from a hip fracture. Material: One hundred-seventeen patients (35 male, 82 female) over 65 years old were studied. Sixty two of them, who had a fracture of the neck of the femur and 15 with an intertrochanteric fracture were treated with a hemiarthroplasty, and 40 who sustained an intertrochanteric fracture were treated with internal fixation with a gliding screw and a plate. Spinal anaesthesia was used in all patients. Their age was 65–97 (average 79, 7). Patients suffering from dementia or Alzheimer were excluded. Method: We counted satO2 preoperatively and until the fourth postoperative day. Postoperative delirium was assessed with the Confusion Assessment Method (CAM). Postoperatively, O2 was given at patients with satO2< 90 or when saturation was 5% lower than the preoperative. Results: Hypoxia occurred in 60 patients (51,28%), which was usually reduced until the 4th pop day. In two patients who suffered from chronic respiratory failure, hypoxia persisted until the 10th pop day. Postoperative delirium occurred in 31 patients (26,5%). Seventeen of then had also hypoxia. Two patients didn’t recover until their discharge from the hospital. In one of them the delirium persists 3 months pop. Correlation between delirium and hypoxia was not noticed (p< 0,0024), neither the delirium was better after the administration of O2 in hypoxaemic patients, altought satO2 was made better. Conclusion: Hypoxia, postoperatively, in elderly patients, sufering from hip fracture is usual, but it is made better after the 4th pop day. Postoperative delirium in these patients is also usual. In our study there was no correlation between delirium and hypoxia. Delirium wasn’t any better after administration of O2

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff. We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis). The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears. These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment

The aim of this study was to understand the role tissue hypoxia and apoptosis have on a human model of rotator cuff failure. We studied twenty seven patients with no tear mild impingment (3), no tear moderate impingment (3), no tear severe impingment (3), partial tear (3), small tear (3), moderate tear (3), large tear (3), massive tear (3) and control (3). A supraspinatus tendon biopsy was taken during debridement/repair in all cases (ethics no. C01.071). Control tendon was obtained from the subscapularis tendon of patients undergoing stabilization surgery. Biopsies were analysed using two immunocytological techniques. A monoclonal antibody against BNIP-III (a marker of hypoxia) and TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling – an apoptotic detection process). An immunoflorescent counterstain DAPI (4′,6-diamidino-2-phenylindol) was used to stain all cells. Positive cells and total cell number were then counted in 10 high powered fields. The results showed a significant increase in BNIP-III expression in the cuff tears compared with intact tendons. This increase was least in the massive tears. Apoptosis increases from mild impingment to massive cuff tears (mean 7.3% to 21%) In conclusion, as tear size increases, the viability of the tendon reduces with increasing hypoxia and apoptosis

Abstract. Objectives. Acute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency and can have detrimental outcomes for patients if not treated optimally. The current problem is that there is no clear diagnostic threshold for ACS or guidance as to when fasciotomies should be performed. A new diagnostic method(s) is necessary to provide real-time information about the extent of muscle ischaemia in ACS. Given that lactic acid is produced by cells through anaerobic respiration, it may be possible to measure H+ ion concentration and to use this as a measure of ischaemia within muscle. Although we are familiar with the key biochemical metabolites involved in ischaemia; and the use of viability dyes in cell culture to distinguish between living or dead cells is well recognised; research has not been undertaken to correlate the biochemical and histological findings of ischaemia in skeletal muscle biopsies. Our primary aim was to investigate the potential for viability dyes to be used on live skeletal muscle biopsies (explants). Our secondary aim was to correlate the intramuscular pH readings with muscle biopsy viability. Methods. Nine euthanised Wistar rats were used. A pH catheter was inserted into one exposed gluteus medius muscles to record real-time pH levels and muscle biopsies were taken from the contralateral gluteus medius at the start of experiment and subsequently at every 0.1 of pH unit drop. Prior to muscle biopsy, the surface of the gluteus medius was painted with a layer of 50µmol/l Brilliant blue FCF solution to facilitate biopsy orientation. A 4mm punch biopsy tool was used to take biopsies. Each muscle biopsy was placed in a base mould filled with 4% ultra-low melting point agarose. The agarose embedded tissue block was sectioned to generate 400 micron thick tissue slices with a vibratome. The tissue slices were then placed in the staining solution with Hoechst 33342, Ethidium homodimer-1 and Calcein am. The tissue slices were imaged with Zeiss LSM880 confocal microscope's Z stack function. A dead muscle control was created by adding TritonX-100 to other tissue slices. For quantitative analyses, the images were analysed in Image J using the selection tool. This permitted individual cells to be identified and the mean grey value of each channel to be defined. Using the dead control, we were able to identify the threshold value for living cells using the Calcein AM channel. Results. Viability dyes, used primarily for cell cultures, can be used with skeletal muscle explants. Our study also showed that despite a significant reduction in tissue pH concentration over time, that almost 100% of muscle cells were still viable at pH 6.0, suggesting that skeletal muscle cells are robust to hypoxic insult in the absence of reperfusion. Conclusions. Viability dyes can be used on skeletal muscle biopsies. Further research investigating the likely associations between direct measured pH using a pH catheter, the concentrations of key cellular metabolic markers, and muscle tissue histology using vitality dyes in response to ischaemia, rather than hypoxia, is warranted. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Degenerate disc disease is a major cause of low back pain, yet its aetiology is still poorly understood. The intervertebral disc is the largest avascular structure in the body. Cells of the nucleus pulposus, therefore, rely on diffusion of oxygen & nutrients down concentration gradients from peripheral vessels in the cartilage end-plates. Thus, there is a low oxygen tension and cellular respiration is largely anaerobic. The purpose of this study was to examine the effects of inflammation, hypoxia and acidosis on degeneration and pro-inflammatory mediator production in virgin porcine nucleus pulposus cultures. Intervertebral discs were harvested from normal 6-month old agricultural pigs slaughtered for other purposes. Nucleus pulposus was contained within the annulus until further dissection under sterile conditions in the laboratory was performed. Nucleus pulposus was harvested, diced and divided into 200mg samples. Samples were incubated under optimal conditions. Discs were cultured in 5μg/ml E. coli lipopolysaccharide, in a hypoxic environment or at low pH. IL-6, IL-8 and LDH assays were performed by ELISA, in accordance with manufacturer’s instructions. Time and dose-response curves were generated for each experiment (results not shown). Results at 72 hours incubation are tabulated below:. These results confirm that nucleus pulposus is a biochemically active tissue capable of producing pro-inflammatory mediators in response to environmental stresses. IL-6 and IL-8 are both involved in the inflammatory cascade, causing chemotaxis of neutrophils and macrophages to the area. IL-8 itself causes hyperalgesia. Acidotic and inflammatory conditions, but not hypoxia, stimulated cytokine release. This may indicate a protective reduction in cellular activity in reduced oxygen environments. Necrosis, as measured by LDH production, was negligible

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

This is quite an innovative study that should lead to a multicentre validation trial. We have developed an FDG-PET/MRI texture-based model for the prediction of lung metastases (LM) in newly diagnosed patients with soft-tissue sarcomas (STSs) using retrospective analysis. In this work, we assess the model performance using a new prospective STS cohort. We also investigate whether incorporating hypoxia and perfusion biomarkers derived from FMISO-PET and DCE-MRI scans can further enhance the predictive power of the model. A total of 66 patients with histologically confirmed STSs were used in this study and divided into two groups: a retrospective cohort of 51 patients (19 LM) used for training the model, and a prospective cohort of 15 patients (two patients with LM, one patient with bone metastases and suspicious lung nodules) for testing the model. In the training phase, a model of four texture features characterising tumour sub-region size and intensity heterogeneities was developed for LM prediction from pre-treatment FDG-PET and MRI scans (T1-weighted, T2-weighted with fat saturation) of the retrospective cohort, using imbalance-adjusted bootstrap statistical resampling and logistic regression multivariable modeling. In the testing phase, this multivariable model was applied to predict the distant metastasis status of the prospective cohort. The predictive power of the obtained model response was assessed using the area under the receiver-operating characteristic curve (AUC). In the exploratory phase of the study, we extracted two heterogeneity metrics from the prospective cohort: the area under the intensity-volume histogram of pre-treatment DCE-MRI volume transfer constant parametric maps and FMISO-PET hypoxia maps (AU-IVH-Ktrans, AU-IVH-FMISO). The impact of the addition of these two individual metrics to the texture-based model response obtained in the testing phase was first investigated using Spearman's correlation (rs), and lastly using logistic regression and leave-one-out cross-validation (LOO-CV) to account for overfitting bias. First, the texture-based model reached an AUC of 0.94, a sensitivity of 1, a specificity of 0.83 and an accuracy of 0.87 when tested in the prospective cohort. In the exploratory phase, the addition of AU-IVH-FMISO did not improve predictive power, yielding a correlation of rs = −0.42 (p = 0.12) with lung metastases, and a relative change in validation AUC of 0% in comparison with the texture-based model response alone in LOO-CV experiments. In contrast, the addition of AU-IVH-Ktrans improved predictive power, yielding a correlation of rs = −0.54 (p = 0.04) with lung metastases, and a change in validation AUC of +10%. Our results demonstrate that texture-based models extracted from pre-treatment FDG-PET and MRI anatomical scans could be successfully used to predict distant metastases in STS cancer. Our results also suggest that the addition of perfusion heterogeneity metrics may contribute to improving model prediction performance

Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test. Results. Chondrocytes cultured under hypoxic conditions showed an upregulation of all matrix related genes compared to normoxic conditions noted most markedly in col II, COMP and SOX9 expression. There were similar numbers of chondrocytes between hypoxic and normoxic groups (P=0.68) but the chondrocytes in the hypoxic group produced more GAG per cell (P= 0.052). Viable cells were seen throughout the matrix in both groups. Conclusion. Important matrix related genes (col II, COMP, SOX9) were most significantly upregulated in hypoxic conditions compared to normoxic conditions. This was supported by an increase in GAG production per cell in hypoxic conditions. The results indicate that hypoxia induces an upregulation in the production of extracellular matrix components typical of AC with only modest increases in col I (possibly related to the col I based scaffold used in this experiment). These results indicate that hypoxic conditions are important for the maintenance of chondrocyte phenotype even when the cells are cultured in a 3D environment. In conclusion, hypoxic culture conditions should be used to help maintain chondrocyte phenotype even when culturing these cells in a 3D scaffold

Introduction. Differing levels of tendon retraction are found in full-thickness rotator cuff tears. The pathophysiology of tendon degeneration and retraction is unclear. Neoangiogenesis in tendon parenchyma indicates degeneration. Hypoxia inducible factor 1(HIF) and vascular endothelial growth factor (VEGF) are important inducers of neoangiogenesis. Rotator cuff tendons rupture leads to fatty muscle infiltration (FI) and muscle atrophy (MA). The aim of this study is to clarify the relationship between HIF and VEGF expression, neoangiogenesis, FI, and MA in tendon retraction found in full-thickness rotator cuff tears. Methods. Rotator cuff tendon samples of 33 patients with full-thickness medium-sized rotator cuff tears were harvested during reconstructive surgery. The samples were dehydrated and paraffin embedded. For immunohistological determination of VEGF and HIF expression, sample slices were strained with VEGF and HIF antibody dilution. Vessel density and vessel size were determined after Masson-Goldner staining of sample slices. The extent of tendon retraction was determined intraoperatively according to Patte's classification. Patients were assigned to 4 categories based upon Patte tendon retraction grade, including one control group. FI and MA were measured on standardized preoperative shoulder MRI. Results. HIF and VEGF expression, FI, and MA were significantly higher in torn cuff samples compared with healthy tissue (p<0.05). HIF and VEGF expression, and vessel density significantly increased with extent of tendon retraction (p<0.04). A correlation between HIF/VEGF expression and FI and MA could be found (p<0.04). There was no significant correlation between HIF/VEGF expression and neovascularity (p>0.05). Conclusion. Tendon retraction in full-thickness medium-sized rotator cuff tears is characterized by neovascularity, increased VEGF/HIF expression, FI, and MA. VEGF expression and neovascularity may be effective monitoring tools to assess tendon degeneration

Common tendon injuries impair healing, leading to debilitation and an increased re-rupture risk. The impact of oxygen-sensing pathways on repair mechanisms, vital in regulating inflammation and fibrosis, remains unclear despite their relevance in tendon pathologies. Recent studies show that pulsed electromagnetic field (PEMF) reduce inflammation in human tendon cells (hTDCs) and in hypoxia-induced inflammation. We investigated the hypoxia's impact (1% and 2% oxygen tension) using magnetic cell sheet constructs (IL-1β-magCSs) primed with IL-1β. IL-1β-magCSs were exposed to low OT (1h, 4h,6h) in a hypoxic chamber. To confirm the role of PEMF (5Hz, 4mT, 50% duty cycle) on hypoxia modulation, IL-1β-magCSs, previously exposed to OT, were 1h-stimulated with PEMF. Our results show a significant increase in HIF- 1a and HIF-2a expression on IL-1β-magCSs after exposure to 2%-OT at all time points, compared to 1%- OT and normoxia. TNFa, IL-6, and IL-8 expression increased after 6 hours of 1%-OT exposure. PEMF stimulation of hypoxic IL-1β-magCSs led to decreased pro-inflammatory genes and increased anti-inflammatory (IL-4,IL-10) expression compared to unstimulated magCSs. IFN-g, TNF-α, and IL-6 release increased after 6 hours, regardless of %-OT, while IL-10 levels tended to rise after PEMF stimulation at 2%-OT. Also, NFkB expression was increased on IL-1β-magCSs exposed to 4 h and 6 h of 2%-OT, suggesting a link between NFkB and the production of pro-inflammatory factors. Moreover, PEMF stimulation showed a significantly decreased NFkB level in IL-1β-magCSs. Overall, low OT enhances expression of hypoxia-associated genes and inflammatory markers in IL-1β-magCSs with the involvement of NFkB. PEMF modulates the response of magCSs, previously conditioned to hypoxia and to inflammatory triggers, favouring expression of anti-inflammatory genes and proteins, supporting PEMF impact in pro-regenerative tendon strategies. Acknowledgements: ERC CoG MagTendon(No.772817), FCT under the Scientific Employment Stimulus-2020.01157.CEECIND. Thanks to Hospital da Prelada for providing tendon tissue samples (Portugal), and TERM. RES Hub (Norte-01-0145-FEDER-022190)

The rotator cuff tendinopathy is one of the most common shoulder problems leading to full-thickness rotator cuff tendon tear and, eventually, to degenerative arthritis. Recent research on rotator cuff tendon degeneration has focused on its relationship to cell death. The types of cell death known to be associated with rotator cuff tendon degeneration are apoptosis, necrosis, and autophagic cell death. The increased incidence of cell death in degenerative tendon tissue may affect the rates of collagen synthesis and repair, possibly weakening tendon tissue and increasing the risk of tendon rupture. The biomolecular mechanisms of the degenerative changes leading to apoptotic cell death in rotator cuff tenofibroblasts have been identified as oxidative-stress-related cascade mechanisms. Furthermore, apoptosis, necrosis, and autophagic cell death are all known to be mediated by oxidative stress, a condition in which ROS (reactive oxygen species) are overproduced. Lower levels of oxidative stress trigger apoptosis; higher levels mediate necrosis. Although the signaltransduction pathway leading to autophagy has not yet been fully established, ROS are known to be essential to autophagy. A neuronal theory regarding rotator cuff degeneration has been developed from the findings that glutamate, a neural transmitter, is present in increased concentrations in tendon tissues with tendinopathy and that it induces rat supraspinatus tendon cell death. Recent studies have reported that hypoxia involved in rotator cuff tendon degeneration. Because antioxidants are known to scavenge for intracellular ROS, some studies have been conducted to determine whether antioxidants can reduce cell death in rotator cuff tendon-origin fibroblasts. The first study reported that an antioxidant has the ability to reduce apoptosis in oxidative-stressed rotator cuff tenofibroblasts. The second study reported that antioxidants have both antiapoptotic effects and antinecrotic effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The third study reported that an antioxidant has antiautophagic-cell-death effects on rotator cuff tendon-origin fibroblasts exposed to an oxidative stimulus. The fourth study reported that glutamate markedly increases cell death in rotator cuff tendonorigin fibroblasts. The glutamate-induced cytotoxic effects were reduced by an antioxidant, demonstrating its cytoprotective effects against glutamate-induced tenofibroblast cell death. The fifth study reported that hypoxia significantly increases intracellular ROS and apoptosis. The hypoxia-induced cytotoxic effects were markedly attenuated by antioxidants, demonstrating their cytoprotective effects against hypoxia-induced tenofibroblast cell death. In conclusion, antioxidants have cytoprotective effects on tenofibroblasts exposed in vitro to an oxidative stressor, a neurotransmitter, or hypoxia. These cytoprotective effects result from antiapoptotic, antinecrotic, and antiautophagic actions involving the inhibition of ROS formation. These findings suggest that antioxidants may have therapeutic potential for rotator cuff tendinopathy. Further studies must be conducted in order to apply these in vitro findings to clinical situations

Despite advances in treating acute spinal cord injury (SCI), measures to mitigate permanent neurological deficits in affected patients are limited. Augmentation of mean arterial blood pressure (MAP) to promote blood flow and oxygen delivery to the injured cord is one of the only currently available treatment options to potentially improve neurological outcomes after acute spinal cord injury (SCI). However, to optimize such hemodynamic management, clinicians require a method to measure and monitor the physiological effects of these MAP alterations within the injured cord in real-time. To address this unmet clinical need, we developed a series of miniaturized optical sensors and a monitoring system based on multi-wavelength near-infrared spectroscopy (MW-NIRS) technique for direct transdural measurement and continuous monitoring of spinal cord hemodynamics and oxygenation in real-time. We conducted a feasibility study in a porcine model of acute SCI. We also completed two separate animal studies to examine the function of the sensor and validity of collected data in an acute experiment and a seven-day post-injury survival experiment. In our first animal experiment, nine Yorkshire pigs underwent a weight-drop T10 vertebral level contusion-compression injury and received episodes of ventilatory hypoxia and alterations in MAP. Spinal cord hemodynamics and oxygenation were monitored throughout by a transdural NIRS sensor prototype, as well as an invasive intraparenchymal (IP) sensor as a comparison. In a second experiment, we studied six Yucatan miniature pigs that underwent a T10 injury. Spinal cord oxygenation and hemodynamics parameters were continuously monitored by an improved NIRS sensor over a long period. Episodes of MAP alteration and hypoxia were performed acutely after injury and at two- and seven-days post-injury to simulate the types of hemodynamic changes patients experience after an acute SCI. All NIRS data were collected in real-time, recorded and analyzed in comparison with IP measures. Noninvasive NIRS parameters of tissue oxygenation were highly correlated with invasive IP measures of tissue oxygenation in both studies. In particular, during periods of hypoxia and MAP alterations, changes of NIRS-derived spinal cord tissue oxygenation percentage were significant and corresponded well with the changes in spinal cord oxygen partial pressures measured by the IP sensors (p < 0.05). Our studies indicate that a novel optical biosensor developed by our team can monitor real-time changes in spinal cord hemodynamics and oxygenation over the first seven days post-injury and can detect local tissue changes that are reflective of systemic hemodynamic changes. Our implantable spinal cord NIRS sensor is intended to help clinicians by providing real-time information about the effects of hemodynamic management on the injured spinal cord. Hence, our novel NIRS system has the near-term potential to impact clinical care and improve neurologic outcomes in acute SCI. To translate our studies from bench to bedside, we have developed an advanced clinical NIRS sensor that is ready to be implanted in the first cohort of acute SCI patients in 2022

Aims. The aims of this study were to determine the indications and frequency of ordering a CT pulmonary angiography (CTPA) following primary arthroplasty of the hip and knee, and to determine the number of positive scans in these patients, the location of emboli and the outcome for patients undergoing CTPA. Patients and Methods. We analyzed the use of CTPA, as an inpatient and up to 90 days as an outpatient, in a cohort of patients and reviewed the medical records and imaging for each patient undergoing CTPA. Results. Out of 11 249 patients, scans were requested in 229 (2.04%) and 86 (38%) were positive. No patient undergoing CTPA died within 90 days. The rate of mortality from pulmonary embolism (PE) overall was 0.08%. CTPA was performed twice as often following total knee arthroplasty (TKA) compared with total hip arthroplasty (THA), and when performed was twice as likely to be positive. Hypoxia was the main indication for a scan, being the indication in 149 scans (65%); and in 23% (11 of 47), the PE was peripheral and unilateral. Three patients suffered complications resulting from therapeutic anticoagulation for possible PE, two of whom had a negative CTPA. Conclusion. CTPA is more likely to be performed following TKA compared with THA. Hypoxia was the main presenting feature of PE. A quarter of PEs which were diagnosed were unilateral and peripheral. Further study may indicate which patients who have a PE after lower limb arthroplasty require treatment, and which can avoid the complications associated with anticoagulation. Cite this article: Bone Joint J 2018;100-B:938–44

Hypoxic Inducible Factor and Hypoxic mimicking agents (HMA) trigger the initiation and promotion of angiogenic-osteogenic cascade events. However, there has been paucity of studies investigating how HIF could be over expressed under chronic hypoxic conditions akin to that seen in sickle cell disease patients to help form a template for tackling the matter of macrocellular avascular necrosis. Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration, and the hypoxia-inducible factor-1 alpha (HIF-1) pathway has been identified as a key component in this process studies have shown. There are still no established experimental models showing how this knowledge can be used for the evaluation of bone implant integration and suggest ways of improving osseointegration in sickle cell disease patients with hip arthroplasty and thereby prevent increased implant loosening. The aim of this study is to help develop an in vitro experimental model which would mimic the in vivo pathologic state in the bone marrow of sickle cell disease patients. It also seeks to establish if the hypoxic inducible factor (HIF) could be over expressed in vitro and thus enhancing osseointegration. MG63 osteoblastic cells were cultured under normoxia and hypoxic conditions (20%; and 1% oxygen saturation) for 48 and 72 hours. Cobalt chloride was introduced to the samples in order to mimic true hypoxia. Cells cultured under normoxic conditions and without cobalt chloride was used as the control in this study. The expression of the hypoxic inducible factor was assessed using the reverse transcriptase qualitative polymerase chain reaction (RT-qPCR). There was increased expression of HIF1-alpha at 72hours as compared to 48hours under the various conditions. The level of expression of HIF increased from 48hrs (mean rank= 4.60) to 72hrs (mean rank =5.60) but this difference was not statistically significant, X2(1) = 0.24, p =0.625. The mean rank fold change of HIF in hypoxic samples decreased compared to the normoxic samples but this difference was not statistically significant, X2(1) = 0.54, p= 0.462. Therefore, the expression of HIF is only increased with prolonged hypoxia as seen in the 72hours samples. The expression of HIF increased in samples with CoCl2 (mean rank=5.17), compared with samples without CoCl2 (mean rank 4.67), however this was not statistically significant, X2(1) = 0.067, p=0.796, p value > 0.05. The over expression of HIF was achieved within a few days (72hours) with the introduction of Cobalt Chloride, which is a mimetic for hypoxia similar to the in vivo environment in sickle cell disease patients. This is an in vitro model which could help investigate osseointergation in such pathologic bone conditions

In the last decades, significant effort has been attempted to salvage the meniscus following injury. Basic science approaches to meniscus repair include procedures for both meniscus regeneration and meniscus healing. Regeneration of meniscal tissue focuses on filling a defect with reparative tissue, which resembles the native structure and function of the meniscus. Procedures for meniscus healing, on the other hand, aim to accomplish adhesion between the margins of a meniscal lesion, with no attempt to regenerate or replace meniscal tissue. Regeneration studies of tissue to fill a defect in the meniscus have shown interesting results, but complete restoration of the native meniscus has not yet been accomplished. Healing of a meniscal lesion has been investigated in different models although none has demonstrated reproducible healing. Therefore, different paths of investigation must be undertaken, and one of these may be the cell-therapy / tissue engineering approach. In a study from our group, we showed the capacity of chondrocyte-seeded cartilaginous scaffold to repair a bucket-handle lesion of the knee meniscus orthotopically in a large animal study. Following studies were done in order to test the potential of other scaffolds and different cell sources for the repair of the meniscal tissue. We have also evaluated the role of hypoxia in meniscal development in vitro as basis for future research in this field, as hypoxia could be be considered as a promoter for meniscal cells maturation, and opens considerably opportunities in the field of meniscus tissue engineering