Bletilla striata Polysaccharide-Containing Carboxymethyl Cellulose Bilayer Structure Membrane for Prevention of Postoperative Adhesion and Achilles Tendon Repair.

Postoperative tissue adhesion and poor tendon healing are major clinical problems associated with tendon surgery. To avoid postoperative adhesion and promote tendon healing, we developed and synthesized a membrane to wrap the surgical site after tendon suturing. The bilayer-structured porous membrane comprised an outer layer [1,4-butanediol diglycidyl ether cross-linked with carboxymethyl cellulose (CX)] and an inner layer [1,4-butanediol diglycidyl ether cross-linked with Bletilla striata polysaccharides and carboxymethyl cellulose (CXB)]. The morphology, chemical functional groups, and membrane structure were determined. In vitro experiments revealed that the CX/CXB membrane demonstrated good biosafety and biodegradability, promoted tenocyte proliferation and migration, and exhibited low cell attachment and anti-inflammatory effects. Furthermore, in in vivo animal study, the CX/CXB membrane effectively reduced postoperative tendon-peripheral tissue adhesion and improved tendon repair, downregulating inflammatory cytokines in the tendon tissue at the surgical site, which ultimately increased tendon strength by 54% after 4 weeks.

Anti-Inflammatory and Antioxidant Properties of a New Mixture of Vitamin C, Collagen Peptides, Resveratrol, and Astaxanthin in Tenocytes: Molecular Basis for Future Applications in Tendinopathies.

Tendinopathy is one of the most frequent musculoskeletal disorders characterized by sustained tissue inflammation and oxidative stress, accompanied by extracellular matrix remodeling. Patients suffering from this pathology frequently experience pain, swelling, stiffness, and muscle weakness. Current pharmacological interventions are based on nonsteroidal anti-inflammatory drugs; however, the effectiveness of these strategies remains ambiguous. Accumulating evidence supports that oral supplementation of natural compounds can provide preventive, and possibly curative, effects. Vitamin C (Vit C), collagen peptides (Coll), resveratrol (Res), and astaxanthin (Asx) were reported to be endowed with potential beneficial effects based on their anti-inflammatory and antioxidant activities. Here, we analyzed the efficacy of a novel combination of these compounds (Mix) in counteracting proinflammatory (IL-1ß) and prooxidant (H2O2) stimuli in human tenocytes. We demonstrated that Mix significantly impairs IL-6-induced IL-1ß secretion, NF-κB nuclear translocation, and MMP-2 production; notably, a synergistic effect of Mix over the single compounds could be observed. Moreover, Mix was able to significantly counteract H2O2-triggered ROS production. Together, these results point out that Mix, a novel combination of Vit C, Coll, Resv, and Asx, significantly impairs proinflammatory and prooxidant stimuli in tenocytes, mechanisms that contribute to the onset of tendinopathies.

Oxidized Phospholipids Regulate Tenocyte Function via Induction of Amphiregulin in Dendritic Cells.

Inflammation is a driving force of tendinopathy. The oxidation of phospholipids by free radicals is a consequence of inflammatory reactions and is an important indicator of tissue damage. Here, we have studied the impact of oxidized phospholipids (OxPAPC) on the function of human tenocytes. We observed that treatment with OxPAPC did not alter the morphology, growth and capacity to produce collagen in healthy or diseased tenocytes. However, since OxPAPC is a known modulator of the function of immune cells, we analyzed whether OxPAPC-treated immune cells might influence the fate of tenocytes. Co-culture of tenocytes with immature, monocyte-derived dendritic cells treated with OxPAPC (Ox-DCs) was found to enhance the proliferation of tenocytes, particularly those from diseased tendons. Using transcriptional profiling of Ox-DCs, we identified amphiregulin (AREG), a ligand for EGFR, as a possible mediator of this proliferation enhancing effect, which we could confirm using recombinant AREG. Of note, diseased tenocytes were found to express higher levels of EGFR compared to tenocytes isolated from healthy donors and show a stronger proliferative response upon co-culture with Ox-DCs, as well as AREG treatment. In summary, we identify an AREG-EGFR axis as a mediator of a DC-tenocyte crosstalk, leading to increased tenocyte proliferation and possibly tendon regeneration.

The dipeptide prolyl-hydroxyproline promotes cellular homeostasis and lamellipodia-driven motility via active ß1-integrin in adult tendon cells.

Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal-regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active ß1-integrin-containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp-mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5ß1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.

Calebin A, a Compound of Turmeric, Down-Regulates Inflammation in Tenocytes by NF-κB/Scleraxis Signaling.

Calebin A (CA) is one of the active constituents of turmeric and has anti-inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are the main causes of tendinitis and tendinopathies. However, the role of CA in tendinitis is still unclear and needs to be studied in detail. Tenocytes in monolayer or 3D-alginate cultures in the multicellular tendinitis microenvironment (fibroblast cells) with T-lymphocytes (TN-ME) or with TNF-α or TNF-ß, were kept without treatment or treated with CA to study their range of actions in inflammation. We determined that CA blocked TNF-ß-, similar to TNF-α-induced adhesiveness of T-lymphocytes to tenocytes. Moreover, immunofluorescence and immunoblotting showed that CA, similar to BMS-345541 (specific IKK-inhibitor), suppressed T-lymphocytes, or the TNF-α- or TNF-ß-induced down-regulation of Collagen I, Tenomodulin, tenocyte-specific transcription factor (Scleraxis) and the up-regulation of NF-κB phosphorylation; thus, its translocation to the nucleus as well as various NF-κB-regulated proteins was implicated in inflammatory and degradative processes. Furthermore, CA significantly suppressed T-lymphocyte-induced signaling, similar to TNF-ß-induced signaling, and NF-κB activation by inhibiting the phosphorylation and degradation of IκBα (an NF-κB inhibitor) and IκB-kinase activity. Finally, inflammatory TN-ME induced the functional linkage between NF-κB and Scleraxis, proposing that a synergistic interaction between the two transcription factors is required for the initiation of tendinitis, whereas CA strongly attenuated this linkage and subsequent inflammation. For the first time, we suggest that CA modulates TN-ME-promoted inflammation in tenocytes, at least in part, via NF-κB/Scleraxis signaling. Thus, CA seems to be a potential bioactive compound for the prevention and treatment of tendinitis.

Toxicity of tranexamic acid (TXA) to intra-articular tissue in orthopaedic surgery: a scoping review.

PURPOSE: Intra-articular administration of tranexamic acid (TXA) in orthopaedic arthroplasty and arthroscopic procedures has become increasingly common over the past decade. However, several recent reports have shown that TXA has the potential to be cytotoxic to cartilage, tendon and synovium. Our aim was to review the literature for evidence of toxic effects from TXA exposure to intra-articular tissue. METHODS: A scoping review methodology was used to search for studies assessing the toxic effects of TXA exposure to intra-articular tissues. MEDLINE, EMBASE, SCOPUS and The Cochrane Library were searched. Relevant information was extracted and synthesis of the retrieved data followed a basic content analytical approach. RESULTS: A total of 15 laboratory studies were retrieved. No clinical studies reporting a toxic effect of TXA on intra-articular tissue were identified in our search. Studies were analysed according to species of origin, tissue of origin and study setting (in vitro, ex vivo, or in vivo). There was increasing cytotoxicity to chondrocytes, tenocytes, synoviocytes and periosteum-derived cells with TXA concentrations beyond 20 mg/ml. Monolayer cell cultures appear more susceptible to TXA exposure, than three-dimensional and explant culture models. In vivo studies have not demonstrated a major toxic effect. CONCLUSIONS: Current evidence suggests a dose-dependent toxic effect on cartilage, tendon, and synovial tissue. Concentrations of 20 mg/ml or less are expected to be safe. There is a significant body of evidence to suggest the need for caution with intraarticular administration of TXA. There is a need for further human clinical trials in order to clarify the long-term safety of TXA topical application.

Exogenous micro-RNA and antagomir modulate osteogenic gene expression in tenocytes.

Tendinopathy is a common and disabling condition that is difficult to treat. The pathomolecular events behind tendinopathy remain uncertain. Micro-RNAs (miRNAs, miRs) are short non-coding RNAs that regulate gene expression and may play a role in tendinopathy development. Tenocytes were obtained from human patellar tendons in patients undergoing anterior cruciate ligament (ACL) reconstruction. Micro-RNA mimics and antagomirs for miR-30d, 26a, and 29a were separately transfected into tenocyte culture. Gene expression for scleraxis, collagen 1 alpha 1 (COL1A1), collagen 3 alpha 1 (COL3A1), interleukin-1-beta (IL-1ß), interleukin-6 (IL-6), bone morphogenic protein 2 (BMP2), bone morphogenic protein 12 (BMP12), and osteocalcin was determined for each miRNA mimic and antagomir transfection using real-time quantitative PCR (qPCR). The results showed that exogenous miR-29a downregulated BMP2 and BMP12, while miR-26a and miR-30d did not have a significant effect on tenocyte gene expression. These findings suggest miR-29a contributes to tendon homeostasis and can serve as a potential therapeutic target in treating tendinopathy.

Uremic Toxins and Ciprofloxacin Affect Human Tenocytes In Vitro.

Tendinopathy is a rare but serious complication of quinolone therapy. Risk factors associated with quinolone-induced tendon disorders include chronic kidney disease accompanied by the accumulation of uremic toxins. Hence, the present study explored the effects of the representative uremic toxins phenylacetic acid (PAA) and quinolinic acid (QA), both alone and in combination with ciprofloxacin (CPX), on human tenocytes in vitro. Tenocytes incubated with uremic toxins +/- CPX were investigated for metabolic activity, vitality, expression of the dominant extracellular tendon matrix (ECM) protein type I collagen, cell-matrix receptor ß1-integrin, proinflammatory interleukin (IL)-1ß, and the ECM-degrading enzyme matrix metalloproteinase (MMP)-1. CPX, when administered at high concentrations (100 mM), suppressed tenocyte metabolism after 8 h exposure and at therapeutic concentrations after 72 h exposure. PAA reduced tenocyte metabolism only after 72 h exposure to very high doses and when combined with CPX. QA, when administered alone, led to scarcely any cytotoxic effect. Combinations of CPX with PAA or QA did not cause greater cytotoxicity than incubation with CPX alone. Gene expression of the pro-inflammatory cytokine IL-1ß was reduced by CPX but up-regulated by PAA and QA. Protein levels of type I collagen decreased in response to high CPX doses, whereas PAA and QA did not affect its synthesis significantly. MMP-1 mRNA levels were increased by CPX. This effect became more pronounced in the form of a synergism following exposure to a combination of CPX and PAA. CPX was more tenotoxic than the uremic toxins PAA and QA, which showed only distinct suppressive effects.

Supporting Cell-Based Tendon Therapy: Effect of PDGF-BB and Ascorbic Acid on Rabbit Achilles Tenocytes in Vitro.

Cell-based tendon therapies with tenocytes as a cell source need effective tenocyte in vitro expansion before application for tendinopathies and tendon injuries. Supplementation of tenocyte culture with biomolecules that can boost proliferation and matrix synthesis is one viable option for supporting cell expansion. In this in vitro study, the impacts of ascorbic acid or PDGF-BB supplementation on rabbit Achilles tenocyte culture were studied. Namely, cell proliferation, changes in gene expression of several ECM and tendon markers (collagen I, collagen III, fibronectin, aggrecan, biglycan, decorin, ki67, tenascin-C, tenomodulin, Mohawk, α-SMA, MMP-2, MMP-9, TIMP1, and TIMP2) and ECM deposition (collagen I and fibronectin) were assessed. Ascorbic acid and PDGF-BB enhanced tenocyte proliferation, while ascorbic acid significantly accelerated the deposition of collagen I. Both biomolecules led to different changes in the gene expression profile of the cultured tenocytes, where upregulation of collagen I, Mohawk, decorin, MMP-2, and TIMP-2 was observed with ascorbic acid, while these markers were downregulated by PDGF-BB supplementation. Vice versa, there was an upregulation of fibronectin, biglycan and tenascin-C by PDGF-BB supplementation, while ascorbic acid led to a downregulation of these markers. However, both biomolecules are promising candidates for improving and accelerating the in vitro expansion of tenocytes, which is vital for various tendon tissue engineering approaches or cell-based tendon therapy.

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.

The Effect of the Repression of Oxidative Stress on Tenocyte Differentiation: A Preliminary Study of a Rat Cell Model Using a Novel Differential Tensile Strain Bioreactor.

Because of limitations in the current understanding of the exact pathogenesis of tendinopathy, and the lack of an optimal experimental model, effective therapy for the disease is currently unavailable. This study aims to prove that repression of oxidative stress modulates the differentiation of tendon-derived cells (TDCs) sustaining excessive tensile strains, and proposes a novel bioreactor capable of applying differential tensile strains to cultured cells simultaneously. TDCs, including tendon-derived stem cells, tenoblasts, tenocytes, and fibroblasts, were isolated from the patellar tendons of Sprague‒Dawley rats. Cyclic uniaxial stretching with 4% or 8% strain at 0.5 Hz for 8 h was applied to TDCs. TDCs subjected to 8% strain were treated with epigallocatechin gallate (EGCG), piracetam, or no medication. Genes representing non-tenocyte lineage (Pparg, Sox9, and Runx2) and type I and type III collagen were analyzed by quantitative polymerase chain reaction. The 8% strain group showed increased expression of non-tenocyte lineage genes and type III/type I collagen ratios compared with the control and 4% strain groups, and the increased expression was ameliorated with addition of EGCG and piracetam. The model developed in this work could be applied to future research on the pathophysiology of tendinopathy and development of treatment options for the disease. Repression of oxidative stress diminishes the expression of genes indicating aberrant differentiation in a rat cell model, which indicates potential therapeutic intervention of tendinopathy, the often relentlessly degenerate condition.

Pioglitazone-Primed Mesenchymal Stem Cells Stimulate Cell Proliferation, Collagen Synthesis and Matrix Gene Expression in Tenocytes.

Various therapeutic effects of mesenchymal stem cells (MSCs) have been reported. However, the rapid clearance of these cells in vivo, difficulties in identifying their therapeutic mechanism of action, and insufficient production levels remain to be resolved. We investigated whether a pioglitazone pre-treatment of MSCs (Pio-MSCs) would stimulate the proliferation of co-cultured tenocytes. Pioglitazone increased the proliferation of MSCs and enhanced the secretion of VEGF (vascular endothelial growth factor) and collagen in these cells. We then examined the effects of Pio-MSCs on tenocytes using an indirect transwell culture system. A significant increase in tenocyte proliferation and cell cycle progression was observed in these co-cultures. Significant increases were observed in wound scratch closure by tenocytes from a Pio-MSC co-culture. Pio-MSCs also enhanced the secretion of collagen from tenocytes. A higher mRNA level of collagen type 1 (Col 1) and type 3 (Col 3), scleraxis (Scx), and tenascin C (TnC) was found in the tenocytes in Pio-MSC co-cultures compared with monocultured cells or tenocytes cultured with non-treated MSCs. Our results indicate that pioglitazone enhances the therapeutic effects of MSCs on tendon repair.

Photochemical tissue bonding promotes the proliferation and migration of injured tenocytes through ROS/RhoA/NF-κB/Dynamin 2 signaling pathway.

Photochemical tissue bonding (PTB) has been found to promote the healing of Achilles tendon tissue injury and to reduce postoperative complications. However, the underlying cellular and molecular mechanisms are not clear. In this study, the cell proliferation, ROS generation, migration and the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF in tenocytes after PTB treatment were measured by CCK-8, flow cytometry, Transwell and western blot assay, respectively. And those in tenocytes after DNM2 silencing or overexpressing or treatment with inhibitors of NF-κB, ROS and RhoA were also measured. Our results showed that 10 mW PTB treatment for 80 and 120 s significantly increased cell proliferation and increased ROS generation in tenocytes. 10 mW PTB treatment for 40 and 80 s significantly activated RhoA and increased the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF, but 10 mW PTB treatment for 120 s decreased the protein expression of those. DNM2 silencing significantly suppressed cell migration and the expression of DNM2, TGF-ß1, and VEGF in tenocytes after PTB treatment (10 mW, 80 s), which was inhibited by DNM2 overexpression. Individual treatment with inhibitor of NF-κB, ROS, and RhoA in tenocytes showed decreased protein expression of DNM2, TGF-ß1, and VEGF. Moreover, in vivo experiment found that PTB treatment significantly inhibited cell apoptosis and the expression of DNM2, NF-κB p65, RhoA, TGF-ß1, and VEGF in a time-dependent manner. Taken together, our results suggest that PTB promotes the proliferation and migration of injured tenocytes through ROS/RhoA/NF-κB/DNM2 signaling pathway.

Glycine improves the remodeling process of tenocytes in vitro.

Tendinitis changes the biochemical and morphological properties of the tendon, promoting an increase of activity of metalloproteinases and disorganization of collagen bundles. Tenocytes, the primary cells in tendon, are scattered throughout the collagenic fibers, and are responsible of tendon remodeling and tissue repair in pathological condition. In vivo, glycine, component of the typical Gly-X-Y collagen tripeptide, showed beneficial effects in biochemical and biomechanical properties of Achilles tendon with tendinitis. In this study, we analyzed the effect of glycine in tenocytes subjected to inflammation. Tenocytes from Achilles tendon of rats were treated with TNF-α (10 ng/mL) with and without previous treatment with glycine (20 mM). Cell proliferation and migration were evaluated, as well as the expression of matrix molecules such as glycosaminoglycans, metalloproteinases (MMPs), TIMPs, and collagen I. Glycine can revert the inflammation due to the action of TNF-α by controlling the MMPs quantity and activity. These data indicated that the molecules involved to remodeling process of extracellular matrix are modulated both by TNF-α and the availability of collagen precursors; in fact, this study indicates the glycine can be useful for treatment of inflammation and for modulating tenocytes metabolism in tendons.

The effect of autologous platelet rich plasma on tenocytes of the human rotator cuff.

BACKGROUND: Platelet rich plasma (PRP) is widely used in rotator cuff repairs but its effect on the healing process is unclear. Several cell culture studies on the effect of allogenic PRP have reported promising results but are not transferable to clinical practice. The aim of the present study is to assess the possible effect of autologous PRP on rotator cuff tendon cells. The amount of growth factors involved with tendon-bone healing (PDGF-AB, IGF-1, TGF-ß1, BMP-7 and -12) is quantified. METHODS: Rotator cuff tissue samples were obtained from (n = 24) patients grouped by age (>/< 65 years) and sex into four groups and cells were isolated and characterized. Later, autologous PRP preparations were obtained and the effect was analyzed by means of cell proliferation, collagen I synthesis and expression of collagen I and III. Furthermore, the PRPs were quantified for growth factor content by means of platelet-derived growth factor (PDGF-AB), insulin-like growth factor (IGF-1), transforming growth factor (TGF-ß1), as well as bone morphogenetic protein (BMP) -7 and - 12. RESULTS: Cell proliferation and absolute synthesis of collagen I were positively affected by PRP exposure compared to controls (p < 0.05), but expression and relative synthesis of collagen I (normalized to cell proliferation) were significantly reduced. PRP contained high amounts of IGF-1 and lower levels of TGF-ß1 and PDGF-AB. The amounts of BMP-7 and -12 were below the detection limits. CONCLUSIONS: PRP is a source of growth factors such involved with tendon-bone healing. PRP had an anabolic effect on the human rotator cuff tenocytes of the same individual in vitro by means of cell proliferation and absolute, but not relative collagen I synthesis. These results encourage further studies on clinical outcomes with more comparable standards in terms of preparation and application methods. LEVEL OF EVIDENCE: Controlled laboratory study.

Exploring the In Vivo Anti-Inflammatory Actions of Simvastatin-Loaded Porous Microspheres on Inflamed Tenocytes in a Collagenase-Induced Animal Model of Achilles Tendinitis.

Tendon rupture induces an inflammatory response characterized by release of pro-inflammatory cytokines and impaired tendon performance. This study sought to investigate the therapeutic effects of simvastatin-loaded porous microspheres (SIM/PMSs) on inflamed tenocytes in vitro and collagenase-induced Achilles tendinitis in vivo. The treatment of SIM/PMSs in lipopolysaccharide (LPS)-treated tenocytes reduced the mRNA expressions of pro-inflammatory cytokines (Matrix metalloproteinase-3 (MMP-3), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). In addition, the local injection of SIM/PMSs into the tendons of collagenase-induced Achilles tendinitis rat models suppressed pro-inflammatory cytokines (MMP-3, COX-2, IL-6, TNF-α, and MMP-13). This local treatment also upregulated anti-inflammatory cytokines (IL-4, IL-10, and IL-13). Furthermore, treatment with SIM/PMSs also improved the alignment of collagen fibrils and effectively prevented collagen disruption in a dose-dependent manner. Therefore, SIM/PMSs treatment resulted in an incremental increase in the collagen content, stiffness, and tensile strength in tendons. This study suggests that SIM/PMSs have great potential for tendon healing and restoration in Achilles tendinitis.

The selective cysteinyl leukotriene receptor 1 (CysLT1R) antagonist montelukast regulates extracellular matrix remodeling.

Scar formation after filtration surgery of glaucoma is mainly caused by excessive synthesis of new extracellular matrix (ECM) and contraction of subconjunctival tissue mediated by human Tenon fibroblasts (HTFs) and the transforming growth factor (TGF-ß1). Montelukast, a potent and specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist, is a licensed drug clinically used for the treatment of bronchial asthma. In this study, we investigated the effects of montelukast on the contractility of HTFs cultured in a three-dimensional collagen gel. We found that cysLT1R was expressed in HTFs. Interestingly, the expression of cysLT1R was increased in response to TGF-ß1 in a dose dependent manner, suggesting its potential role in TGF-ß1 induced fibrosis. Importantly, we found that montelukast inhibited TGF-ß1-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. In addition, TGF-ß1-induced expression of MMP-1 and MMP-3, generation of fibronectin and type I collagen production, focal adhesion kinase (FAK) and paxillin phosphorylation in HTFs were also ameliorated by montelukast in a dose dependent manner. These results suggested that montelukast might provide therapeutic possibilities for inhibition of scar formation after such surgery.

Development of a co-culture device for the study of human tenocytes in response to the combined stimulation of electric field and platelet rich plasma (PRP).

One of the objectives of rotator cuff repairs is to achieve biological healing and recovery in the tendon-bone zone. Some clinical evaluations reported the feasibility of tendon healing based on the stimulations of electric field and platelet-rich plasma (PRP). However, because of lack of appropriate tool for in vitro primary culture under complicated conditions, the efficacy and standard protocol of these healing approaches are still controversial among clinical experts. In this study, a novel co-culture device was developed for the study of tenocytes proliferation under single and combined stimulations of electric field and PRP. The device was a culture well divided into three sub-chambers separated by a barrier and embedded with a pair of parallel plate electrodes. Tenocytes and PRP gel could be respectively loaded into the sub-chambers and cultured with interlinked medium. Hence, tenocytes could concurrently receive a uniform electric field and platelet-derived growth factors by diffusion. Results revealed that the proliferation of tenocytes could be significantly enhanced by these stimulations. The device provides a precise and practical approach for the in vitro study of tendon healing, especially for PRP study. Moreover, optimization of the conditions of electric field and PRP could be determined by in vitro screening procedure before surgery to provide a personalized therapy.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro.

BACKGROUND: Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions. QUESTIONS/PURPOSES: We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration? METHODS: Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration. RESULTS: Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-ß (TGF-ß), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-ß bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes. CONCLUSIONS: Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo. CLINICAL RELEVANCE: If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

Cell Toxicity in Fibroblasts, Tenocytes, and Human Mesenchymal Stem Cells-A Comparison of Necrosis and Apoptosis-Inducing Ability in Ropivacaine, Bupivacaine, and Triamcinolone.

PURPOSE: To analyze the ability of ropivacaine, bupivacaine, and triamcinolone to induce apoptosis and necrosis in fibroblasts, tenocytes, and human mesenchymal stem cells. METHODS: Human dermal fibroblasts, adipose-derived human mesenchymal stem cells (hMSCs), and tenocytes gained from the rotator cuff tendon were seeded with a cell density of 0.5 × 104/cm2. One specimen of ropivacaine, bupivacaine, and triamcinolone was tested separately on the cells with separate concentrations of 0.5%, 0.25%, and 0.125% for each specimen. The negative control received no agent, only a change of medium. The incubation period for each agent was 30 minutes. After a change of medium and 1 hour, 24 hours, and 7 days of incubation, 104 cells were harvested and analyzed via fluorescence-activated cell sorting with double-staining with annexin V and propidium iodide. Statistical analysis to determine significant difference (P < .05) between the groups with SPSS statistics 23 through one-way analysis of variance with a univariate general linear model was performed. RESULTS: Bupivacaine showed necrosis-inducing effects on fibroblasts and tenocytes, with the necrotic effect peaking at 0.5% and 0.25%. Ropivacaine and triamcinolone caused no significant necrosis. Compared with fibroblasts and tenocytes, hMSCs did not show significant necrotic or apoptotic effects after exposure to bupivacaine. Overall, no significant differences in apoptosis were detected between different cell lines, varying concentrations, or time measurements. CONCLUSIONS: Bupivacaine 0.5% and 0.25% have the most necrosis-inducing effects on fibroblasts and tenocytes. Ropivacaine caused less necrosis than bupivaine. Compared with fibroblasts and tenocytes, hMSCs were not affected by necrosis using any of the tested agents. A significant apoptosis-inducing effect could not be detected for the different cell lines. CLINICAL RELEVANCE: Possible cell toxicity raises questions of concern for intra-articular injections using local anesthetics and corticosteroids. The present study demonstrates the necrotic and apoptotic effects of ropivacaine, bupivacaine, and triamcinolone and may give recommendations for intra-articular use of local anesthetics and corticosteroids.