Human Tendon Cells Research Articles

Immunomodulatory Behavior of Tendon Magnetic Cell Sheets can be Modulated in Hypoxic Environments under Magnetic Stimulus.

Tissue environments play a crucial role in orchestrating cell behavior, guided by a complex interplay of various factors. Long lasting inflammatory signals compromise tendon homeostasis and promote tissue degeneration, while tissue oxygen levels affect local cells' responses with hypoxic environments influencing apoptosis, inflammatory mediators, and matrix production. Recent works have unveiled the therapeutic potential of pulsed electromagnetic field (PEMF) in modulating inflammatory signals expressed by human tendon cells (hTDCs), and in mitigating the hypoxia-induced effects on the regulation of inflammatory cytokines. Thus, we sought to investigate the role of hypoxic environments, namely, 1 and 2% oxygen tension, in the inflammatory profiles of magnetic cell sheets (magCSs) formed by magnetic nanoparticles internalized in contiguous hTDCs with intact cell-cell junctions and deposited matrix. We also aimed to explore the impact of PEMF over hypoxia-treated magCSs, including IL-1β-primed-magCSs, with the objective of harnessing magnetic stimulation to guide abnormal inflammatory cell responses toward efficient treatments supporting tendon regenerative potential. Our findings revealed that low oxygen tensions amplified the expression of hypoxia-associated genes and of inflammatory markers in IL-1β-primed-magCSs with an involvement of the NF-κB signaling pathway. Encouragingly, when PEMF was applied to IL-1β-primed-magCSs under hypoxic conditions, it successfully modulated inflammatory cues by favoring IL-10 and IL-4, via the NF-κB pathway. These results signify the remarkable potential of PEMF in driving proregenerative strategies and opens up new approaches in tendon therapies, highlighting the transformative impact of immunomodulatory magnetic cell sheets.

MRGPRX2-mediated mast cell activation by substance P from overloaded human tenocytes induces inflammatory and degenerative responses in tendons

Mast cells are immune cells minimally present in normal tendon tissue. The increased abundance of mast cells in tendinopathy biopsies and at the sites of tendon injury suggests an unexplored role of this cell population in overuse tendon injuries. Mast cells are particularly present in tendon biopsies from patients with more chronic symptom duration and a history of intensive mechanical loading. This study, therefore, examined the cross talk between mast cells and human tendon cells in either static or mechanically active conditions in order to explore the potential mechanistic roles of mast cells in overuse tendon injuries. A coculture of isolated human tenocytes and mast cells (HMC-1) combined with Flexcell Tension System for cyclic stretching of tenocytes was used. Additionally, human tenocytes were exposed to agonists and antagonists of substance P (SP) receptors. Mast cell degranulation was assessed by measuring β-hexosaminidase activity. Transwell and cell adhesion assays were used to evaluate mast cell migration and binding to tendon extracellular matrix components (collagen and fibronectin), respectively. Gene expressions were analyzed using real time qRT-PCR. Our results indicate that mechanical stimulation of human tenocytes leads to release of SP which, in turn, activates mast cells through the Mas-related G-protein-coupled receptor X2 (MRGPRX2). The degranulation and migration of mast cells in response to MRGPRX2 activation subsequently cause human tenocytes to increase their expression of inflammatory factors, matrix proteins and matrix metalloproteinase enzymes. These observations may be important in understanding the mechanisms by which tendons become tendinopathic in response to repetitive mechanical stimulation.

Cytotoxicity of local anaesthetics and protective effects of platelet rich plasma on equine tenocytes: An in vitro study

Local anaesthetics (LAs) can have detrimental effects on rat, bovine, canine, and human tendon tissues and cells. Currently, there has been no available data on the impact of these drugs on equine tenocytes. Even if LA injection for managing painful tendon conditions in horses is limited, it is usually used via intra-articular, intrasynovial, perineural, and intrathecal as well as for lameness examinations. In this in vitro study, the cytotoxic effects of LAs, including lidocaine, mepivacaine, and bupivacaine on equine tenocytes, in the presence and absence of platelet rich plasma (PRP), were investigated. PRP accelerates tissue healing and can exert cytoprotective effects on different cell types exposed to different stressful conditions, including drugs. Results indicated that the exposure to LAs significantly reduced tenocytes viability in dose- and time-dependent manners while PRP was able to counteract their cytotoxic effects. Furthermore, microscopy and flow cytometry analyses revealed apoptosis and necrosis in equine tenocytes exposed to these drugs, that were both reduced when PRP was in the medium. These findings highlight the importance of considering the tenocyte toxicity associated with intrathecal and intraneural LA injections, as they might affect tenocytes or reduce the efficacy of associated therapies. Moreover, this study also highlights the protective effects of PRP, which could make LA injections safer.

IMMUNOMODULATORY POTENTIAL OF MAGNETICALLY ASSISTED CELL SHEET UNDER HYPOXIC ENVIRONMENTS UPON MAGNETIC STIMULATION

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 TERMRES Hub (Norte-01-0145-FEDER-022190).

HIF-1A INITIATES VASCULAR RECRUITMENT AND IRREVERSIBLE FIBROSIS IN TENDON

Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation.

Exposure to oxLDL impairs TGF-β activity in human tendon cells

BackgroundPrevious studies have shown that patients with hypercholesterolemia experience elevated levels of oxidized LDL (oxLDL), a molecule which triggers inflammation and collagenase activity. In this study we discovered novel mechanistic effects of oxLDL on tendon cells and the mediators regulating matrix remodeling by analyzing the expression and activity of related proteins and enzymes. These effects may contribute to tendon damage in patients with high cholesterol.MethodsIsolated human tendon cells (male and female donors age 28 ± 1.4 age 37 ± 5.7, respectively) were incubated in the presence or absence of oxLDL. The influence of oxLDL on the expression level of key mRNA and proteins was examined using real time quantitative PCR, ELISA and Western blots. The activities of enzymes relevant to collagen synthesis and breakdown (lysyl oxidase and matrix metalloproteinases) were quantified using fluorometry. Finally, the isolated human tendon cells in a 3D construct were exposed to combinations of oxLDL and TGF-β to examine their interacting effects on collagen matrix remodeling.ResultsThe one-way ANOVA of gene expression indicates that key mRNAs including TGFB, COL1A1, DCN, and LOX were significantly reduced in human tendon cells by oxLDL while MMPs were increased. The oxLDL reduced the activity of LOX at 50 µg/ml, whereas conversely MMP activities were induced at 25 µg/ml (P ≤ 0.01). COL1A1 synthesis and TGF-β secretion were also inhibited (P ≤ 0.05). Adding recombinant TGF-β reversed the effects of oxLDL on the expression of collagens and LOX. OxLDL also impaired collagen matrix remodeling (P ≤ 0.01), and adding TGF-β restored the native phenotype.ConclusionExposure to oxLDL in patients with hypercholesterolemia may adversely affect the mechanical and structural properties of tendon tissue through a direct action of oxLDL on tendon cells, including impairment of TGF-β expression. This impairment leads to disturbed matrix remodeling and synthesis, thereby potentially leading to increased risk of acute or chronic tendon injury. Our discovery may provide an opportunity for developing effective treatments for tendon injury in hypercholesterolemia patients by targeting the TGF-β pathway.

Single cell multi-omics characterise discrete human tendon cells populations that persist in vitro and on fibrous scaffolds.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

Bioengineered 3D Living Fibers as In Vitro Human Tissue Models of Tendon Physiology and Pathology.

Clinically relevant in vitro models of human tissue's health and disease are urgently needed for a better understanding of biological mechanisms essential for the development of novel therapies. Herein, physiological (healthy) and pathological (disease) tendon states are bioengineered by coupling the biological signaling of platelet lysate components with controlled 3D architectures of electrospun microfibers to drive the fate of human tendon cells in different composite living fibers (CLFs). In the CLFs-healthy model, tendon cells adopt a high cytoskeleton alignment and elongation, express tendon-related markers (scleraxis, tenomodulin, and mohawk) and deposit a dense tenogenic matrix. In contrast, cell crowding with low preferential orientation, high matrix deposition, and phenotypic drift leading to increased expression of nontendon related and fibrotic markers, are characteristics of the CLFs-diseased model. This diseased-like profile, also reflected in the increase of COL3/COL1 ratio, is further evident by the imbalance between matrix remodeling and degradation effectors, characteristic of tendinopathy. In summary, microengineered 3D in vitro models of human tendon healthy and diseased states are successfully fabricated. Most importantly, these innovative and versatile microphysiological models offer major advantages over currently used systems, holding promise for drugs screening and development of new therapies.

VEGF-D-mediated signaling in tendon cells is involved in degenerative processes.

Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

Targeting the CCR6/CCL20 Axis in Entheseal and Cutaneous Inflammation.

To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target. First, we quantified CCL20 levels in peripheral blood and synovial fluid from PsA patients and examined the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an interleukin-23 minicircle DNA (IL-23 MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression as well as the preventive and therapeutic effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL-1β to assess the production of CCL20 by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated using a Transwell system. We observed an up-regulation of both CCR6 and CCL20 in the enthesis of IL-23 MC-treated mice, which was confirmed in human biopsy specimens. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL-23 MC-treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and, accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD. Our study highlights the pathogenic role of the CCR6/CCL20 axis in enthesitis and introduces the prospect of a novel therapeutic approach for treating patients with PsO and PsA.

Platelet-rich Plasma and Its Application in Clinical Trial

Platelet-rich Plasma and Its Application in Clinical Trial

Proteomics identifies differences in fibrotic potential of extracellular vesicles from human tendon and muscle fibroblasts

BackgroundFibroblasts are the powerhouses responsible for the production and assembly of extracellular matrix (ECM). Their activity needs to be tightly controlled especially within the musculoskeletal system, where changes to ECM composition affect force transmission and mechanical loading that are required for effective movement of the body. Extracellular vesicles (EVs) are a mode of cell-cell communication within and between tissues, which has been largely characterised in cancer. However, it is unclear what the role of healthy fibroblast-derived EVs is during tissue homeostasis.MethodsHere, we performed proteomic analysis of small EVs derived from primary human muscle and tendon cells to identify the potential functions of healthy fibroblast-derived EVs.ResultsMass spectrometry-based proteomics revealed comprehensive profiles for small EVs released from healthy human fibroblasts from different tissues. We found that fibroblast-derived EVs were more similar than EVs from differentiating myoblasts, but there were significant differences between tendon fibroblast and muscle fibroblast EVs. Small EVs from tendon fibroblasts contained higher levels of proteins that support ECM synthesis, including TGFβ1, and muscle fibroblast EVs contained proteins that support myofiber function and components of the skeletal muscle matrix.ConclusionsOur data demonstrates a marked heterogeneity among healthy fibroblast-derived EVs, indicating shared tasks between EVs of skeletal muscle myoblasts and fibroblasts, whereas tendon fibroblast EVs could play a fibrotic role in human tendon tissue. These findings suggest an important role for EVs in tissue homeostasis of both tendon and skeletal muscle in humans.EueS9_TM9G2gnPvs8i76-XVideo abstract

Conjugation with Methylsulfonylmethane Improves Hyaluronic Acid Anti-Inflammatory Activity in a Hydrogen Peroxide-Exposed Tenocyte Culture In Vitro Model.

Rotator cuff tears (RCTs) and rotator cuff disease (RCD) are important causes of disability in middle-aged individuals affected by nontraumatic shoulder dysfunctions. Our previous studies have demonstrated that four different hyaluronic acid preparations (HAPs), including Artrosulfur® hyaluronic acid (HA) (Alfakjn S.r.l., Garlasco, Italy), may exert a protective effect in human RCT-derived tendon cells undergoing oxidative stress damage. Recently, methylsulfonylmethane (MSM) (Barentz, Paderno Dugnano, Italy) has proven to have anti-inflammatory properties and to cause pain relief in patients affected by tendinopathies. This study aims at evaluating three preparations (Artrosulfur® HA, MSM, and Artrosulfur® MSM + HA) in the recovery from hydrogen peroxide-induced oxidative stress damage in human tenocyte. Cell proliferation, Lactate Dehydrogenase (LDH) release, and inducible nitric oxide synthases (iNOS) and prostaglandin E2 (PGE2) modulation were investigated. In parallel, expression of metalloproteinases 2 (MMP2) and 14 (MMP14) and collagen types I and III were also examined. Results demonstrate that Artrosulfur® MSM + HA improves cell escape from oxidative stress by decreasing cytotoxicity and by reducing iNOS and PGE2 secretion. Furthermore, it differentially modulates MMP2 and MMP14 levels and enhances collagen III expression after 24 h, proteins globally related to rapid acceleration of the extracellular matrix (ECM) remodelling and thus tendon healing. By improving the anti-cytotoxic effect of HA, the supplementation of MSM may represent a feasible strategy to ameliorate cuff tendinopathies.

A2A adenosine receptors are involved in the reparative response of tendon cells to pulsed electromagnetic fields.

Tendinopathy is a degenerative disease in which inflammatory mediators have been found to be sometimes present. The interaction between inflammation and matrix remodeling in human tendon cells (TCs) is supported by the secretion of cytokines such as IL-1β, IL-6 and IL-33. In this context, it has been demonstrated that pulsed electromagnetic fields (PEMFs) were able to reduce inflammation and promote tendon marker synthesis. The aim of this study was to evaluate the anabolic and anti-inflammatory PEMF-mediated response on TCs in an in vitro model of inflammation. Moreover, since PEMFs enhance the anti-inflammatory efficacy of adenosine through the adenosine receptors (ARs), the study also focused on the role of A2AARs. Human TCs were exposed to PEMFs for 48 hours. After stimulation, A2AAR saturation binding experiments were performed. Along with 48 hours PEMF stimulation, TCs were treated with IL-1β and A2AAR agonist CGS-21680. IL-1Ra, IL-6, IL-8, IL-10, IL-33, VEGF, TGF-β1, PGE2 release and SCX, COL1A1, COL3A1, ADORA2A expression were quantified. PEMFs exerted A2AAR modulation on TCs and promoted COL3A1 upregulation and IL-33 secretion. In presence of IL-1β, TCs showed an upregulation of ADORA2A, SCX and COL3A1 expression and an increase of IL-6, IL-8, PGE2 and VEGF secretion. After PEMF and IL-1β exposure, IL-33 was upregulated, whereas IL-6, PGE2 and ADORA2A were downregulated. These findings demonstrated that A2AARs have a role in the promotion of the TC anabolic/reparative response to PEMFs and to IL-1β.

Magnetic responsive materials modulate the inflammatory profile of IL-1β conditioned tendon cells

Tendinopathies represent half of all musculoskeletal injuries worldwide. Inflammatory events contribute to both tendon healing and to tendinopathy conditions but the cellular triggers leading to one or the other are unknown. In previous studies, we showed that magnetic field actuation modulates human tendon cells (hTDCs) behavior in pro-inflammatory environments, and that magnetic responsive membranes could positively influence inflammation responses in a rat ectopic model.Herein, we propose to investigate the potential synergistic action of the magnetic responsive membranes, made of a polymer blend of starch with polycaprolactone incorporating magnetic nanoparticles (magSPCL), and the actuation of pulsed electromagnetic field (PEMF): 5 Hz, 4mT of intensity and 50% of duty cycle, in IL-1β-treated-hTDCs, and in the immunomodulatory response of macrophages.It was found that the expression of pro-inflammatory (TNFα, IL-6, IL-8, COX-2) and ECM remodeling (MMP-1,-2,-3) markers tend to decrease in cells cultured onto magSPCL membranes under PEMF, while the expression of TIMP-1 and anti-inflammatory genes (IL-4, IL-10) increases. Also, CD16++ and CD206+ macrophages were only found on magSPCL membranes with PEMF application.Magnetic responsive membranes show a modulatory effect on the inflammatory profile of hTDCs favoring anti-inflammatory cues which is also supported by the anti-inflammatory/repair markers expressed in macrophages. These results suggest that magnetic responsive magSPCL membranes can contribute for inflammation resolution acting on both resident cell populations and inflammatory cells, and thus significantly contribute to tendon regenerative strategies.Statement of significanceMagnetically-assisted strategies have received great attention in recent years to remotely trigger and guide cell responses. Inflammation plays a key role in tendon healing but persistent pro-inflammatory molecules can contribute to tendon disorders, and therefore provide a therapeutic target for advanced treatments.We have previously reported that magnetic fields modulate the response of human tendon cells (hTDCs) conditioned to pro-inflammatory environments (IL-1β-treated-hTDCs), and that magnetic responsive membranes positively influence immune responses. In the present work, we combined pulsed electromagnetic field (PEMF) and magnetic responsive membranes to guide the inflammatory profile of IL-1β-treated-hTDCs and of macrophages. The results showed that the synergistic action of PEMF and magnetic membranes supports the applicability of magnetically actuated systems to regulate inflammatory events and stimulate tendon regeneration.

Tendon Cells Derived From The Long Head Of The Biceps And The Supraspinatus Tendons Of Patients Affected By Rotator Cuff Tears Show Different Expression Of Inflammatory Markers

ABSTRACT Aim of the study Tendons are exposed to mechanical stress constantly during movements and thus they are frequently subjected to injuries. Rotator cuff tears are common musculoskeletal disorders, mainly involving the supraspinatus tendon. The characterization of the tenocytes derived from this tendon and the comparison to cells isolated from the long head of the biceps tendon obtained from donors affected by rotator cuff disease may improve the knowledge of the cellular mechanisms involved in the initiation and progression of the pathology. Thus, the aim of the present study was to characterize and compare donor-matched human tendon cells (TCs) isolated from the long head of the biceps (LHB-TCs) and the supraspinatus tendons (SSP-TCs) of patients affected by rotator cuff tears. Methods donor-matched LHB-TCs and SSP-TCs were isolated and cultured up to passage 3. Phenotypic appearance, metabolic activity, DNA content, production of soluble mediators (IL-1Ra, IL-1β, IL-6, and VEGF) and gene expression of tendon markers (SCX, COL1A1, COL3A1), inflammatory (PTGS2), and catabolic enzymes (MMP-1, MMP-3) were evaluated. Results LHB-TCs showed an elongated fibroblast-like shape, while SSP-TCs appeared irregular with jagged membrane. SSP-TCs gene expression revealed an augmented production of PTGS2, a marker of inflammation, whereas they produced a reduced amount of IL-6, in respect to LHB-TCs. Conclusion SSP-TCs showed higher cellular stress and expression of inflammatory markers with respect to donor-matched LHB-TCs, suggesting that addressing the physio-pathological state of supraspinatus tendon cells during treatment of rotator cuff tears could favor tissue healing and possibly prevent relapses.

Magnetic Stimulation Drives Macrophage Polarization in Cell to-Cell Communication with IL-1β Primed Tendon Cells.

Inflammation is part of the natural healing response, but it has been simultaneously associated with tendon disorders, as persistent inflammatory events contribute to physiological changes that compromise tendon functions. The cellular interactions within a niche are extremely important for healing. While human tendon cells (hTDCs) are responsible for the maintenance of tendon matrix and turnover, macrophages regulate healing switching their functional phenotype to environmental stimuli. Thus, insights on the hTDCs and macrophages interactions can provide fundamental contributions on tendon repair mechanisms and on the inflammatory inputs in tendon disorders. We explored the crosstalk between macrophages and hTDCs using co-culture approaches in which hTDCs were previously stimulated with IL-1β. The potential modulatory effect of the pulsed electromagnetic field (PEMF) in macrophage-hTDCs communication was also investigated using the magnetic parameters identified in a previous work. The PEMF influences a macrophage pro-regenerative phenotype and favors the synthesis of anti-inflammatory mediators. These outcomes observed in cell contact co-cultures may be mediated by FAK signaling. The impact of the PEMF overcomes the effect of IL-1β-treated-hTDCs, supporting PEMF immunomodulatory actions on macrophages. This work highlights the relevance of intercellular communication in tendon healing and the beneficial role of the PEMF in guiding inflammatory responses toward regenerative strategies.

The Synergistic Effect of Anabolic Steroid and Loading on Intercellular Calcium Signaling Via Gap Junctions in Human Supraspinatus Tendon Cells

Objective: We hypothesized that anabolic steroid administration would act synergistically with substrate strain in two-dimensional cultures of human supraspinatus tendon cells, to upregulate the expression of connexin-43 and to increase the Ca2+ wave propagation through gap junctions. Methods: Supraspinatus tendon cells were isolated intra-operatively from human specimens during shoulder arthroscopy. Cells were plated in two-dimensional spot cultures and arranged into four experimental groups: 1) non-load, non-steroid (NLNS, n=12 wells); 2) non-load, steroid (NLS, n=12 wells); 3) load, non-steroid (LNS, n=12 wells); and 4) load, steroid (LS, n=12 wells) in order to produce bioartificial tendons (BATs). The load groups were stretched in culture plates and the steroid groups were given nandrolone decanoate. When BATs were macro- and microscopically mature, at five days, they were evaluated with immunocytochemistry for connexin-43 staining, fluorescence microscopy for calcium imaging and mechanical stimulation with a micropipette tip manipulation for calcium propagation. Dose response test was performed in order to establish any relation between nandrolone decanoate dose and calcium signaling response. ATP was applied to the spot culture cells from all groups and all patients to determine if the cells were sensitive to extracellular ATP. Results: Load-steroid group demonstrated the greatest density of cnx43 in comparison to all other groups. There were no significant differences between the groups considering the percentage of cells responding after mechanical stimulation (cell recruitment). The cells of load-steroid group showed a significantly greater mean peak [Ca2+]ic compared to the values of the other groups (p<0.05). The propagation time was significantly decreased in the LS group compared with the other groups (p<0.05). There were no significant differences between the groups considering the number of cells that were responding spontaneously prior to stimulation or the number of responding cells that were oscillating after the stimulation. Conclusion: Nandrolone decanoate and loading seem to have a synergistic effect on the upregulation of the gap junction protein cxn43 enhancing calcium signaling via gap junctions. Consecutively, anabolic steroid administration and load may enhance the formation of a better-organized cytoskeleton and particularly the actin stress monofilaments.

Effect of Mn3O4 Nanoparticles on Lipopolysaccharide-Induced Inflammatory Factors in the Human Tendon Cells and Its Mechanism

Objective. To investigate the effect of Mn3O4 nanoparticles (Mn3O4NPs) on inflammatory factors induced by lipopolysaccharide (LPS) in human tendon cells and its mechanism. Methods. The Mn3O4NPs were synthesized by a hydrothermal method. RT-qPCR was used to detect the expression levels of miRNAs related to inflammation in human tendon cells. The expression level of NLRP1 (NOD-like receptor containing pyrin domain 1) was measured by Western blotting. ELISA assay was used to measure the level of TNF-α, IL-1β, IL-4, and IL-10. The relationship between miR-181a-5p and NLRP1 was verified by dual-luciferase reporter assay. Results. Mn3O4NPs produced in this study were brown spherical particles with an average size of 7-10 nm. Mn3O4NP treatment significantly reduced the levels of TNF-α and IL-1β but increased the levels of IL-4 and IL-10 in the human tendon cells induced by LPS. In addition, Mn3O4NP treatment remarkably increased the expression level of miR-181a-5p. NLRP1 is one of the targets of miR-181a-5p, and miR-181a-5p downregulated its expression. Further study showed that Mn3O4NPs could alleviate the inflammatory response of human tendon cells induced by LPS by upregulating miR-181a-5p and thus downregulating the expression of NLRP1. Conclusion. Mn3O4NPs affect the expression of inflammatory cytokines in the human tendon cells induced by LPS by modulating the molecular axis of miR-181a-5p/NLRP1.

Effects and Mechanism of Berberine on the Dexamethasone-Induced Injury of Human Tendon Cells.

Objective To investigate the effects of berberine (Berb) on dexamethasone- (Dex-) induced injury of human tendon cells and its potential mechanism. Methods CCK-8 assay was used to explore the appropriate concentration of Dex-induced injury of tendon cells and the doses of Berb attenuates Dex cytotoxicity; cell wound healing assay was used to detect the effects (P < 0.05) of Berb and Dex on the migration ability of tendon cells; flow cytometry was used to measure cell apoptosis; DCF DA fluorescent probe was used to measure the ROS activity of cells. Western blotting was used to detect the expression of phenotype related factors including smooth muscle actin α (SMA-α), type I collagen (Col I), col III, apoptosis-related factors, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, and PI3K/AKT. Results CCK-8 assay showed that 1–100 μM Dex significantly inhibited the proliferation of tendon cells in a concentration-dependent manner (P < 0.05), where the inhibitory effect of 100 μM Dex was most significant (P < 0.005), and the pretreatment of 150, 200 μM Berb could reverse those inhibitions (all P < 0.05). Compared with the control group, Dex significantly inhibited cell migration (P < 0.05), while Berb pretreatment could enhance cell migration (P < 0.05). Flow cytometry and ROS assay showed that Dex could induce apoptosis and oxidative stress response of tendon cells (all P < 0.05), while Berb could reverse those responses (P < 0.05). Western blot showed that Dex could inhibit the expression of the col I and III as well as α-SMA (all P < 0.05) and enhance the expression of apoptosis-related factors including cleaved caspase-3 and cleaved caspase-9 (all P < 0.05). Besides, Dex could also inhibit the activation of the PI3K/AKT signaling pathway (all P < 0.05), thus affecting cell function, while Berb treatment significantly reversed the expression of those above proteins (all P < 0.05). Conclusion Berb attenuated DEX induced reduction of proliferation and migration, oxidative stress, and apoptosis of tendon cells by activating the PI3K/AKT signaling pathway and regulated the expression of phenotype related biomarkers in tendon cells. However, further studies are still needed to clarify the protective effects of Berb in vivo.