Single nucleus and spatial transcriptomic profiling of healthy human hamstring tendon.

The molecular and cellular basis of health in human tendons remains poorly understood. Among human tendons, hamstring tendon has markedly low pathology and can provide a prototypic healthy tendon reference. The aim of this study was to determine the transcriptomes and location of all cell types in healthy hamstring tendon. Using single nucleus RNA sequencing, we profiled the transcriptomes of 10 533 nuclei from four healthy donors and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and identified cell types previously unreported in tendons, including different skeletal muscle cell types, satellite cells, adipocytes, and undefined nervous system cells. The location of these cell types within tendon was defined using spatial transcriptomics and imaging, and potential transcriptional networks and cell-cell interactions were analyzed. We demonstrate that fibroblasts have the highest number of potential cell-cell interactions in our dataset, are present throughout the tendon, and play an important role in the production and organization of extracellular matrix, thus confirming their role as key regulators of hamstring tendon homeostasis. Overall, our findings underscore the complexity of the cellular networks that underpin healthy human tendon function and the central role of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

Mature primary human osteocytes in mini organotypic cultures secrete FGF23 and PTH1-34-regulated sclerostin.

Introduction: For decades, functional primary human osteocyte cultures have been crucially needed for understanding their role in bone anabolic processes and in endocrine phosphate regulation via the bone-kidney axis. Mature osteocyte proteins (sclerostin, DMP1, Phex and FGF23) play a key role in various systemic diseases and are targeted by successful bone anabolic drugs (anti-sclerostin antibody and teriparatide (PTH1-34)). However, cell lines available to study osteocytes produce very little sclerostin and low levels of mature osteocyte markers. We have developed a primary human 3D organotypic culture system that replicates the formation of mature osteocytes in bone. Methods: Primary human osteoblasts were seeded in a fibrinogen / thrombin gel around 3D-printed hanging posts. Following contraction of the gel around the posts, cells were cultured in osteogenic media and conditioned media was collected for analysis of secreted markers of osteocyte formation. Results: The organoids were viable for at least 6 months, allowing co-culture with different cell types and testing of bone anabolic drugs. Bulk RNAseq data displayed the developing marker trajectory of ossification and human primary osteocyte formation in vitro over an initial 8- week period. Vitamin D3 supplementation increased mineralization and sclerostin secretion, while hypoxia and PTH1-34 modulated sclerostin. Our culture system also secreted FGF23, enabling the future development of a bone-kidney-parathyroid-vascular multi-organoid or organ-on-a-chip system to study disease processes and drug effects using purely human cells. Discussion: This 3D organotypic culture system provides a stable, long-lived, and regulated population of mature human primary osteocytes for a variety of research applications.

A New Method to Sort Differentiating Osteoclasts into Defined Homogeneous Subgroups.

Osteoclasts regulate skeletal development but also drive pathological osteolysis, making them prime therapeutic targets. Osteoclast research is limited by the heterogeneity of osteoclast populations generated in vitro, where the mixture of undifferentiated monocytes, binuclear pre-osteoclasts and multinucleated osteoclasts has by necessity been considered a single osteoclast population. This study describes the differentiation of primary human CD14+ monocyte-derived osteoclasts in 3D collagen gels. These osteoclasts remained small (>95% with ≤5 nuclei) but were viable and active; when released from the gel with collagenase, they fused rapidly when reseeded onto solid substrates and resorbed dentine for 2-3 weeks. 3D-generated osteoclasts expressed cell surface markers of osteoclast differentiation (e.g., CD9, RANK, OSCAR, CD63, CD51/61) which, with their small size, enabled live cell sorting of highly enriched viable subpopulations of human osteoclasts that retained full functional resorption capacity. Low-yield osteoclast preparations were strongly enriched to remove undifferentiated cells (e.g., 13.3% CD51/61+ to 84.2% CD51/61+), and subpopulations of CD9+CD51/61- early osteoclasts and CD9+CD51/61+ mature cells were distinguished. This novel approach allows the study of selected populations of differentiating osteoclasts in vitro and opens the door to in-depth transcriptomic and proteomic analysis of these cells, increasing our ability to study human osteoclast molecular mechanisms relevant to development, aging and disease.

Osteoblast-Osteoclast Coculture Amplifies Inhibitory Effects of FG-4592 on Human Osteoclastogenesis and Reduces Bone Resorption.

The link between bone and blood vessels is regulated by hypoxia and the hypoxia-inducible transcription factor, HIF, which drives both osteogenesis and angiogenesis. The recent clinical approval of PHD enzyme inhibitors, which stabilize HIF protein, introduces the potential for a new clinical strategy to treat osteolytic conditions such as osteoporosis, osteonecrosis, and skeletal fracture and nonunion. However, bone-resorbing osteoclasts also play a central role in bone remodeling and pathological osteolysis, and HIF promotes osteoclast activation and bone loss in vitro. It is therefore likely that the result of PHD enzyme inhibition in vivo would be mediated by a balance between increased bone formation and increased bone resorption. It is essential that we improve our understanding of the effects of HIF on osteoclast formation and function and consider the potential contribution of inhibitory interactions with other musculoskeletal cells. The PHD enzyme inhibitor FG-4592 stabilized HIF protein and stimulated osteoclast-mediated bone resorption, but inhibited differentiation of human CD14+ monocytes into osteoclasts. Formation of osteoclasts in a more physiologically relevant 3D collagen gel did not affect the sensitivity of osteoclastogenesis to FG-4592, but increased sensitivity to reduced concentrations of RANKL. Coculture with osteoblasts amplified inhibition of osteoclastogenesis by FG-4592, whether the osteoblasts were proliferating, differentiating, or in the presence of exogenous M-CSF and RANKL. Osteoblast coculture dampened the ability of high concentrations of FG-4592 to increase bone resorption. These data provide support for the therapeutic use of PHD enzyme inhibitors to improve bone formation and/or reduce bone loss for the treatment of osteolytic pathologies and indicate that FG-4592 might act in vivo to inhibit the formation and activity of the osteoclasts that drive osteolysis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Organotypic Culture of Bone-Like Structures Using Composite Ceramic-Fibrin Scaffolds.

We have developed an organotypic culture system that allows the production of bone tissue features on a centimeter scale. A composite, calcium phosphate-strained fibrin gel system is able to organize itself in the presence of osteoblastic cells, creating basic hierarchical units as seen in vivo, and can be modified to produce a range of other tissues that require such directional structuring. Constructs evolve over time into multi-compositional structures containing a high mineral content and terminally differentiated, osteocyte-like cells. These tissues can be cultured over extended durations (exceeding 1 year) and are responsive to a variety of chemical and biological agents. The platform can reduce the number of animals used in experimentation by acting as an intermediate stage in which more personalized research conditions can be generated. We provide a thorough description of the protocol used to successfully culture and modify this system, as well as guidance on compositional characterization. © 2019 by John Wiley & Sons, Inc.

Platelet rich Plasma in Achilles Tendon Healing 2 (PATH-2) trial: protocol for a multicentre, participant and assessor-blinded, parallel-group randomised clinical trial comparing platelet-rich plasma (PRP) injection versus placebo injection for Achilles tendon rupture.

BACKGROUND: Achilles tendon injuries give rise to substantial long-lasting morbidity and pose considerable challenges for clinicians and patients during the lengthy healing period. Current treatment strategies struggle to curb the burden of this injury on health systems and society due to lengthy rehabilitation, work absence and reinjury risk. Platelet-rich plasma (PRP) is an autologous preparation that has been shown to improve the mechanobiological properties of tendons in laboratory and animal studies. The use of PRP in musculoskeletal injuries is on the increase despite the lack of adequately powered clinical studies. METHODS AND DESIGN: This is a multicentre randomised controlled trial to evaluate the efficacy and mechanism of PRP in patients with acute Achilles tendon rupture (ATR). All adults with acute ATR presenting within 12 days of the injury who are to be treated non-operatively are eligible. A total of 230 consenting patients will be randomly allocated via a remote web-based service to receive PRP injection or placebo injection to the site of the injury. All participants will be blinded to the intervention and will receive standardised rehabilitation to reduce efficacy interference.Participants will be followed up with blinded assessments of muscle-tendon function, quality of life, pain and overall patient's functional goals at 4, 7, 13, 24 weeks and 24 months post-treatment. The primary outcome is the heel-rise endurance test (HRET), which will be supervised by a blinded assessor at 24 weeks. A subgroup of 16 participants in one centre will have needle biopsy under ultrasound guidance at 6 weeks. Blood and PRP will be analysed for cell count, platelet activation and growth factor concentrations. ETHICS AND DISSEMINATION: The protocol has been approved by the Oxfordshire Research Ethics Committee (Oxfordshire Research Ethics Committee A, reference no 14/SC/1333). The trial will be reported in accordance with the CONSORT statement and published in peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: ISRCTN: 54992179, assigned 12 January 2015. ClinicalTrials.gov: NCT02302664, received 18 November 2014. UK Clinical Research Network Study Portfolio Database: ID 17850.

Hypoxia-inducible factor 1-alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl-4-hydroxylase 2.

Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature osteoclasts. Phd2+/- murine osteoclasts also exhibited enhanced bone resorption, associated with increased expression of resorption-associated Acp5, in comparison with wild-type cells from littermate controls. Phd3-/- bone marrow precursors displayed accelerated early fusion, mirroring results with HIF-1α siRNA. In vivo, Phd2+/- and Phd3-/- mice exhibited reduced trabecular bone mass, associated with reduced mineralization by Phd2+/- osteoblasts. These data indicate that HIF predominantly functions as a regulator of osteoclast-mediated bone resorption, with little effect on osteoclast differentiation. Inhibition of HIF might therefore represent an alternative strategy to treat diseases characterized by pathological levels of osteolysis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Co-expression of DKK-1 and Sclerostin in Subchondral Bone of the Proximal Femoral Heads from Osteoarthritic Hips.

BACKGROUND: Osteoarthritis (OA) is a progressively degenerative joint disease influenced by structural and metabolic factors. There is growing evidence that subchondral bone is involved in both symptomatic and structural progression in OA. The Wnt pathway has been implicated in the progression of OA but the expression and function of the Wnt inhibitors, Dikkopf (DKK-1) and sclerostin (SOST), are unclear. METHODS: We examined the regional distribution of DKK-1 and SOST in subchondral bone of the femoral head using resection specimens following arthroplasty in patients presenting with end-stage OA. Cylindrical cores for immunohistochemistry were taken through midpoint of full thickness cartilage defect, partial cartilage defect, through base of osteophyte and through macroscopically normal cartilage. RESULTS: Subchondral bone was thickest in cores taken from regions with full cartilage defect and thinnest in cores taken from osteophyte regions. In subchondral bone, expression of both DKK-1 and SOST was observed exclusively in osteocytes. Expression was highest in subchondral bone in cores taken from regions with partial but not full thickness cartilage defects. DKK-1 but not SOST was expressed by chondrocytes in cores with macroscopically normal cartilage. CONCLUSION: The current study describes the regional cellular distribution of SOST and DKK-1 in hip OA. Expression was highest in the osteocytes in bone underlying partial thickness cartilage defects. It is however not clear if this is a cause or a consequence of alterations in the overlying cartilage. However, it is suggestive of an active remodeling process which might be targeted by disease-modifying agents.

25-Hydroxy- and 1α,25-Dihydroxycholecalciferol Have Greater Potencies than 25-Hydroxy- and 1α,25-Dihydroxyergocalciferol in Modulating Cultured Human and Mouse Osteoblast Activities.

Despite differences in the phamacokinetics of 25-hydroxycholecalciferol (25(OH)D3) and 25-hydroxyergocalciferol (25(OH)D2) in man, the effects of these and their 1α-hydroxylated forms (1,25(OH)2D3 and 1,25(OH)2D2) on cellular activity of vitamin D-responsive cells have hardly been compared. We studied differences in the effects of these metabolites on cell number, gene transcription, protein expression and mineralisation of cultured human bone marrow-derived stromal cells (hBMSC) and rapidly mineralising mouse 2T3 osteoblasts. 50-1000 nM 25(OH) and 0.05-10 nM 1,25(OH)2 metabolites were used. At high concentrations, 25(OH)D2/D3 and 1,25(OH)2D2/D3 suppressed cell number in both human and mouse cells. The suppression was greater with cholecalciferol (D3) metabolites than with those of ergocalciferol (D2). In both cell types, 25(OH)D2 and 25(OH)D3 increased the expression of osteopontin, osteocalcin, collagen-1, receptor activator of nuclear factor kappa-B ligand, vitamin D receptor, CYP24A1 and CYP27B1 genes. Whereas there was little or no difference between the effects of 25(OH)D2 and 25(OH)D3 in hBMSCs, differences were observed in the magnitude of the effects of these metabolites on the expression of most studied genes in 2T3 cells. Alkaline phosphatase (ALP) activity was increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 in hBMSC and 2T3 cells, and the increase was greater with the D3 metabolites at high concentrations. In hBMSCs, mineralisation was also increased by 25(OH)D2/D3 and 1,25(OH)2D2/D3 at high concentrations, with D3 metabolites exerting a greater influence. In 2T3 cells, the effects of these compounds on mineralisation were stimulatory at low concentrations and inhibitory when high concentrations were used. The suppression at high concentrations was greater with the D3 metabolites. These findings suggest that there are differences in the effects of 25-hydroxy and 1α,25(OH)2 metabolites of D3 and D2 on human preosteoblasts and mouse osteoblasts, with the D3 metabolites being more potent in suppressing cell number, increasing ALP activity and influencing mineralisation.

Using Fluorescence Recovery After Photobleaching to Study Gap Junctional Communication In Vitro.

Fluorescence recovery after photobleaching (FRAP) is a microscopy-based technique to study the movement of fluorescent molecules inside a cell. Although initially developed to investigate intracellular mobility, FRAP can be also used to measure intercellular dynamics. This chapter describes how to perform FRAP experiment to study gap junctional communication in living cells. The procedures described here can be carried out with a laser-scanning confocal microscope and any in vitro cultured cells known to communicate via gap junctions. In addition, the method can be easily adjusted to measure gap junction function in 3D cell cultures as well as ex vivo tissue.

Cell proliferation is a key determinant of the outcome of FOXO3a activation.

The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail.

Lovastatin-Mediated Changes in Human Tendon Cells.

Statins are among the most widely prescribed drugs worldwide. Numerous studies have shown their beneficial effects in prevention of cardiovascular disease through cholesterol-lowering and anti-atherosclerotic properties. Although some statin patients may experience muscle-related symptoms, severe side effects of statin therapy are rare, primarily due to extensive first-pass metabolism in the liver. Skeletal muscles appear to be the main site of side effects; however, recently some statin-related adverse effects have been described in tendon. The mechanism behind these side effects remains unknown. This is the first study that explores tendon-specific effects of statins in human primary tenocytes. The cells were cultured with different concentrations of lovastatin for up to 1 week. No changes in cell viability or morphology were observed in tenocytes incubated with therapeutic doses. Short-term exposure to lovastatin concentrations outside the therapeutic range had no effect on tenocyte viability; however, cell migration was reduced. Simvastatin and atorvastatin, two other drug family members, also reduced the migratory properties of the cells. Prolonged exposure to high concentrations of lovastatin induced changes in cytoskeleton leading to cell rounding and decreased levels of mRNA for matrix proteins, but increased BMP-2 expression. Gap junctional communication was impaired but due to cell shape change and separation rather than direct gap junction inhibition. These effects were accompanied by inhibition of prenylation of Rap1a small GTPase. Collectively, we showed that statins in a dose-dependent manner decrease migration of human tendon cells, alter their expression profile and impair the functional network, but do not inhibit gap junction function.

Enhanced osteoblastogenesis in three-dimensional collagen gels.

Growth and differentiation of osteoblasts are often studied in cell cultures. In vivo, however, osteoblasts are embedded within a complex three-dimensional (3D) microenvironment, which bears little relation to standard culture flasks. Our study characterizes osteoblast-like cells cultured in 3D collagen gels and compares them with cells in two-dimensional (2D) cultures. Primary rat osteoblasts and MC3T3-E1 cells were seeded within type I collagen gels, and differentiation was determined by mineral staining and gene expression analysis. Cells growing in 3D gels showed positive mineral staining and induction of osteoblast marker genes earlier than cells growing in 2D. A number of genes, including osteocalcin, bone sialoprotein, alkaline phosphatase and dentin matrix protein 1, were already highly upregulated in 3D cultures 24 h after seeding. The early expression of osteoblast genes was dependent on the 3D structure and was not induced in cells growing on collagen-coated dishes in 2D. Comparison of thymidine incorporation between cells in 3D and 2D cultures treated with agents that induce proliferation-transforming growth factor ß, platelet-derived growth factor and lactoferrin-showed a much greater response in 3D gels. Cells in 3D cultures were also much more sensitive to inhibition of proliferation by the protein kinase inhibitor imatinib mesylate. The 3D collagen gels better represent the physiological bone environment and offer a number of technical advantages for the study of osteoblasts in vitro. These studies have additional practical implications as 3D collagen gels are considered as a scaffold material in regenerative medicine for the repair of bone defects.

Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching.

Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 ß-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures.

Glucocorticoids induce senescence in primary human tenocytes by inhibition of sirtuin 1 and activation of the p53/p21 pathway: in vivo and in vitro evidence.

UNLABELLED: Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. OBJECTIVE: To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. METHODS: Features of senescence (ß-galactosidase activity at pH 6 (SA-ß-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. RESULTS: Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-ßgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. CONCLUSION: As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue.

Regulation of hypoxia-induced cell death in human tenocytes.

Degenerate shoulder tendons display evidence of hypoxia. However tendons are relatively avascular and not considered to have high oxygen requirements and the vulnerability of tendon cells to hypoxia is unclear. Cultured human tenocytes were exposed to hypoxia and the cellular response detected using QPCR, Western blotting, viability, and ELISA assays. We find that tenocytes respond to hypoxia in vitro by activating classical HIF-1α-driven pathways. Total hypoxia caused significant tenocyte apoptosis. Transcription factors typically involved in hypoxic response, HIF-1α and FOXO3A, were upregulated. Hypoxia caused sustained upregulation of several proapoptotic proteins known to mediate hypoxia-induced apoptosis, such as Bnip3 and Nix, but others were unchanged although they were reportedly hypoxia-sensitive in other cell types. Antiapoptotic proteins Bcl2 and Bcl-xL were unchanged by hypoxia. Normal human tenocytes expressed all isoforms of the hypoxia-induced vascular growth factor VEGF except VEGF-D. Hypoxia markedly upregulated VEGF-A mRNA, followed by increased VEGF protein secretion. However treatment with VEGF did not improve tenocyte survival. As a protective strategy for tenocytes at risk of hypoxic death we added prosurvival growth factors insulin or platelet rich plasma (PRP). Both agents strongly protected tenocytes from hypoxia-induced death over 48 h, suggesting possible efficacy in the acute postrupture tendon or integrating graft.

Platelet-rich plasma protects tenocytes from adverse side effects of dexamethasone and ciprofloxacin.

BACKGROUND: Ruptured tendons heal very slowly and complete recovery from injury is uncertain. Platelet-rich plasma (PRP), a rich source of growth factors, is currently being widely tested as a soft tissue healing agent and may accelerate tendon repair. The authors assessed the ability of PRP to prevent in vitro adverse effects of 2 drugs commonly linked to tendon rupture and tendinopathy, glucocorticoids and fluoroquinolone antibiotics. HYPOTHESIS: The pro-healing response induced by PRP protects human tenocytes against the cytotoxic effects of dexamethasone and ciprofloxacin. STUDY DESIGN: Controlled laboratory study. METHODS: Human primary hamstring tenocytes were exposed to different doses of ciprofloxacin and dexamethasone with and without PRP. AlamarBlue, ß-galactosidase assay, and live/dead stain were used to measure, respectively, viability, senescence, and death in tenocyte culture. RESULTS: The viability of cells exposed to high doses of ciprofloxacin was significantly decreased compared with controls, with no induced senescence but increased cell death. Dexamethasone reduced viable cell number without inducing overt cell death, but the number of senescent cells increased considerably. After co-treatment with 10% PRP, viable cell number increased significantly in both conditions and the number of dead cells decreased in ciprofloxacin-treated cultures. Moreover, dexamethasone-induced senescence was markedly reduced by co-treatment with 10% PRP. CONCLUSION: This study demonstrates that ciprofloxacin and dexamethasone have differing adverse effects on human tenocytes, with ciprofloxacin inducing cell death while dexamethasone primarily induces senescence. The authors showed that PRP can protect cultured human tenocytes against cell death or senescence induced by these drugs. CLINICAL RELEVANCE: Both ciprofloxacin and dexamethasone are highly effective in treatment of inflammatory and infectious conditions, therefore new strategies to minimize their adverse effects are of strong interest. These findings suggest the potential for local administration of PRP to enhance tendon healing in patients undergoing glucocorticoid or fluoroquinolone treatment.

Protection against glucocorticoid-induced damage in human tenocytes by modulation of ERK, Akt, and forkhead signaling.

Antiinflammatory glucocorticoid (GC) injections are extensively used to treat painful tendons. However, GC cause severe tissue wasting in other collagen-producing tissues such as skin and bone. The objective of this study was to determine the effects of GC on tenocytes and to explore strategies to protect against unwanted side effects of GC treatment. Cell survival, collagen production, and the induction of signaling pathways in primary human tenocytes treated with dexamethasone (Dex) were assessed. Antioxidant and growth factor approaches to protection were tested. Dex treatment resulted in reduced viable cell number, cell proliferation, and collagen production. Dex induced reactive oxygen species generation in tenocytes and strongly up-regulated the stress-response transcription factors FOXO1 and FOXO3A. Phosphorylation of ERK and protein kinase B/Akt, which regulate cell proliferation and also inhibit forkhead activity, was decreased. Chemical inhibition of ERK or Akt activity significantly reduced tenocyte cell number. Ameliorating the Dex-induced reduction in ERK or Akt activity by cotreatment with vitamin C or insulin protected against the Dex-induced reduction in cell number. Silencing FOXO1 prevented the Dex-induced reduction in collagen 1α1 expression. Cotreatment with vitamin C or insulin protected against the Dex-induced increase in FOXO and the Dex-induced inhibition of collagen 1α1 expression. Reduced ERK and Akt activation and increased forkhead signaling contribute to the negative effects of GC on tenocytes. Cotreatment therapies that target these signaling pathways are protective. Vitamin C in particular may be a clinically useable co-therapy to reduce connective tissue side effects associated with GC therapy.

The histological features of anteromedial gonarthrosis--the comparison of two grading systems in a human phenotype of osteoarthritis.

Anteromedial gonarthrosis (AMG) displays a well recognised pattern of cartilage damage on the medial tibial plateau. Anteriorly there is a full thickness cartilage defect, with transition to a partial thickness defect, becoming full thickness cartilage in the posterior third of the tibial plateau. The retained posterior cartilage is macroscopically normal. This study characterises the histological changes of AMG and examines the usefulness of two histological assessment tools. Sixteen unicompartmental resection specimens of patients with primary AMG were assessed. Samples were stained with Haematoxylin and Eosin and Safranin-O stains and scored using the modified Mankin grade, and the OOCHAS assessment tool. Each specimen was assessed at five regions along the antero-posterior axis starting from the exposed bone to the region of macroscopically normal cartilage. From anterior to posterior the staining showed a consistent increase in structural integrity and cellularity of the cartilage, matched by a qualitative increase in GAG content. Mean modified Mankin and OOCHAS scores showed a progressive decrease in grade (p < 0.001). The OOCHAS grade had a good correlation with the modified Mankin grade (ρ = 0.886) and there was good intra- and inter-observer variability with both assessment tools. We conclude that there is progressive decrease in histological score from anterior to posterior in AMG and that the macroscopically normal cartilage seen posteriorly is histologically normal. Both the modified Mankin and OOOCHAS assessment tools are useful in histological grading but we found the OOCHAS easier and quicker to use. We propose that AMG represents a spatial model of progressive cartilage damage.