Cultured Tendon Cells Research Articles

Characterization of TGFβ1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Tendon cell and tissue culture: Perspectives and recommendations.

The wide variety of cell and tissue culture systems used to study and engineer tendons can make it difficult to choose the best approach and "optimal" culture conditions to test a given hypothesis. Therefore, a breakout session was organized at the 2022 ORS Tendon Section Meeting that focused on establishing a set of guidelines for conducting cell and tissue culture studies of tendon. This paper summarizes the outcomes of that discussion and presents recommendations for future studies. In the case of studying tendon cell behavior, cell and tissue culture systems are reductionist models in which the culture conditions should be strictly defined to approximate the in vivo condition as closely as possible. In contrast, for tissue engineering tendon replacements, the culture conditions do not need to replicate native tendon, but the outcome measures for success should be narrowly defined for the specific clinical application. Common recommendations for both applications are that researchers should perform a baseline phenotypic characterization of the cells that are ultimately used for experimentation. For models of tendon cell behavior, culture conditions should be well justified by existing literature and meticulously reported, tissue explant viability should be assessed, and comparisons to in vivo conditions should be made to determine baseline physiological relevance. For tissue engineering applications, the functional/structural/compositional outcome targets should be defined by the specific tendons they seek to replace, with key biologic and material properties prioritized for construct assessment. Lastly, when engineering tendon replacements, researchers should utilize clinically approved cGMP materials to facilitate clinical translation.

Tendon-Specific Activation of Tenogenic Transcription Factors Enables Keeping Tenocytes' Identity In Vitro.

We generated a novel tetracycline-inducible transgenic mouse line with the tendon-specific expression of a series of tendon-critical transcription factors. Primary tenocytes derived from this mouse line consistently expressed green fluorescent protein reporter transcription factors in response to doxycycline. The tenocytes maintained their tendon cell properties for a longer time after the transient induction in the absence of growth factors and mechanical stress. Four key transcription factors for tendon development and the green fluorescent protein reporter were linked with different viral 2A self-cleaving peptides. They were expressed under the control of the tet-responsive element. In combination with the expression of BFP, which reports on the tendon-specific collagen I, and mScarlet, which reports on the tendon-specific transcription factor Scleraxis (Scx), we observed the more extended maintenance of the tendon cell identity of in vitro cultured tendon cells and Achilles tendon explants. This means that the Scleraxis bHLH transcription factor (Scx), mohawk homeobox (Mkx), early growth response 1 (Egr1) and early growth response 2 (Egr2) contributed to the maintenance of tenocytes' identity in vitro, providing a new model for studying extracellular matrix alterations and identifying alternative biomaterials in vitro.

Lidocaine Inhibited Tendon Cell Proliferation and Extracellular Matrix Production by Down Regulation of Cyclin A, CDK2, Type I and Type III Collagen Expression.

Lidocaine injection is a common treatment for tendon injuries. However, the evidence suggests that lidocaine is toxic to tendon cells. This study investigated the effects of lidocaine on cultured tendon cells, focusing on the molecular mechanisms underlying cell proliferation and extracellular matrix (ECM) production. Tendon cells cultured from rat Achilles tendons were treated with 0.5, 1.0, or 1.5 mg/mL lidocaine for 24 h. Cell proliferation was evaluated by Cell Counting Kit 8 (CCK-8) assay and bromodeoxyuridine (BrdU) assay. Cell apoptosis was assessed by Annexin V and propidium iodide (PI) stain. Cell cycle progression and cell mitosis were assessed through flow cytometry and immunofluorescence staining, respectively. The expression of cyclin E, cyclin A, cyclin-dependent kinase 2 (CDK2), p21, p27, p53, matrix metalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9), type I collagen, and type III collagen were examined through Western blotting, and the enzymatic activity of MMP-9 was determined through gelatin zymography. Lidocaine reduced cell proliferation and reduced G1/S transition and cell mitosis. Lidocaine did not have a significant negative effect on cell apoptosis. Lidocaine significantly inhibited cyclin A and CDK2 expression but promoted p21, p27, and p53 expression. Furthermore, the expression of MMP-2 and MMP-9 increased, whereas that of type I and type III collagen decreased. Lidocaine also increased the enzymatic activity of MMP-9. Our findings support the premise that lidocaine inhibits tendon cell proliferation by changing the expression of cell-cycle-related proteins and reduces ECM production by altering levels of MMPs and collagens.

Impact of isolation method on cellular activation and presence of specific tendon cell subpopulations during in vitro culture.

Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell subpopulations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation methods preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior. Statement of clinical significance: The development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.

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.

Cyclical strain improves artificial equine tendon constructs in vitro.

Tendon injuries are a common cause of morbidity in humans. They also occur frequently in horses, and the horse provides a relevant, large animal model in which to test novel therapies. To develop novel cell therapies that can aid tendon regeneration and reduce subsequent reinjury rates, the mechanisms that control tendon tissue regeneration and matrix remodelling need to be better understood. Although a range of chemical cues have been explored (growth factors, media etc.), the influence of the mechanical environment on tendon cell culture has yet to be fully elucidated. To mimic the in vivo environment, in this study, we have utilised a novel and affordable, custom-made bioreactor to apply a cyclical strain to tendon-like constructs generated in three-dimensional (3D) culture by equine tenocytes. Dynamic shear analysis (DSA), dynamic scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy were used to determine the mechanical and chemical properties of the resulting tendon-like constructs. Our results demonstrate that equine tenocytes exposed to a 10% cyclical strain have an increased amount of collagen gel contraction after 7 and 8 days of culture compared with cells cultured in 3D in the absence of external strain. While all the tendon-like constructs have a very similar chemical composition to native tendon, the application of strain improves their mechanical properties. We envisage that these results will contribute towards the development of improved biomimetic artificial tendon models for the development of novel strategies for equine regenerative therapies.

Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-β1 Stimulated Tenocyte Culture.

The natural healing process for tendon repair is associated with high upregulation of collagen type III, leading to scar tissue and tendon adhesions with functionally deficient tendons. Gene delivery systems are widely reported as potential nanotherapeutics to treat diseases, providing a promising approach to modulate collagen type III synthesis. This work investigates a proof-of-concept four-arm cationic polymer-siRNA polyplex to mediate a transient downregulation of collagen type III expression in a tendon cell culture system. The tendon culture system was first supplemented with TGF-β1 to stimulate the upregulation of collagen type III prior to silencing experiments. The four-arm poly[2-(dimethylamino) ethyl acrylate] (PDMAEA) polymer was successfully synthesized via RAFT polymerization and then mixed with siRNA to formulate the PDMAEA-siRNA polyplexes. The formation of the polyplex was optimized for the N:P ratio (10:1) and confirmed by agarose gel electrophoresis. The size and solution behavior of the polyplex were analyzed by dynamic light scattering and zeta potential, showing a hydrodynamic diameter of 155 ± 21 nm and overall positive charge of +30 mV at physiological pH. All the polyplex concentrations used had a minimal effect on the metabolic activity of cultured cells, indicating good biocompatibility. The dose and time effects of the TGF-β1 on collagen type III gene expressions were analyzed by qPCR, showing an optimal dose of 10 ng mL–1 TGF-β1 and 3-fold increase of COL3α1 expression at 48 h in cultured tenocytes. The PDMAEA-siRNA polyplex concept observed a limited yet successful and promising efficiency in silencing collagen type III at 48 h compared to PEI-siRNA. Therefore, this concept is a promising approach to reduce tissue scarring and adhesion following injuries.

Paper #32 - Effects of aging on the molecular profile of cultured tendon cells

Paper #32 - Effects of aging on the molecular profile of cultured tendon cells

Bone Morphogenetic Protein-7 Regulates Differentially the mRNA Expression of Bone Morphogenetic Proteins and Their Receptors in Rat Achilles and Patellar Tendon Cell Cultures. Yeh, L.-C. C., Tsai, A. D. and Lee, J. C.

Journal of Cellular BiochemistryVolume 117, Issue 10 p. 2435-2435 ErratumFree Access Bone Morphogenetic Protein-7 Regulates Differentially the mRNA Expression of Bone Morphogenetic Proteins and Their Receptors in Rat Achilles and Patellar Tendon Cell Cultures. Yeh, L.-C. C., Tsai, A. D. and Lee, J. C. This article corrects the following: Bone morphogenetic protein-7 regulates differentially the mRNA expression of bone morphogenetic proteins and their receptors in rat achilles and patellar tendon cell cultures Lee-Chuan C. Yeh, Alicia D. Tsai, John C. Lee, Volume 104Issue 6Journal of Cellular Biochemistry pages: 2107-2122 First Published online: April 7, 2008 First published: 18 July 2016 https://doi.org/10.1002/jcb.25599AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Fig. 7B in the published paper is a duplicate of Fig. 8B. Please replace it with the new Fig. 7B. REFERENCE Yeh L.-C. C., Tsai A. D., Lee J. C. 2008. Bone morphogenetic protein-7 regulates differentially the mRNA expression of bone morphogenetic proteins and their receptors in rat achilles and patellar tendon cell cultures. J Cell Biochem 104: 2107– 2122. doi: 10.1002/jcb.21768 Volume117, Issue10October 2016Pages 2435-2435 ReferencesRelatedInformation

TGF-β rescues extracellular matrix turnover in rotator cuff pathology

Introduction: Tendinopathies are the most frequent causes of chronic shoulder pain. Long head of the biceps (LHB) tendon lesions are often associated with massive rotator cuff (RC) tears. Palliative LHB tenotomy decreases RC disease patient’s pain and disability. The aim of this work was to identify the biological changes of LHB in RC disease and assess its association with clinical manifestations. Methods: RC disease patients submitted to LHB tenotomy were evaluated using a clinical protocol in order to retrieve information regarding shoulder pain duration and intensity (visual analogue scale) and shoulder function (Constant score). LHB tendon samples from these patients were compared with cadaver controls. Tendon tissue was qualitatively studied by conventional histology and immunohistochemistry was used to access semi quantitatively the presence of substance P and calcitonin gene-related peptide (CGRP). Tendon cell cultures were used to determine the gene expression of several extracellular matrix genes with and without stimulation with transforming growth factor (TGF)-β, TNF, IL-10 or dexamethasone. Results: Histologically, LHB tendon from RC patients and cadaver controls had similar characteristics. RC patients had a significantly higher CGRP immunohistochemistry score as compared to controls (p=0.010) but there was no correlation with patient clinical features. On the contrary, regarding substance P no differences were found between RC patients and controls immunohistochemistry score but a correlation with shoulder pain (r=0.828, p=0.021) was identified. Through gene expression analysis we found a downregulation of the extracellular matrix genes type I collagen and thrombospondin 4, as well as vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) in Manuscript Click here to download Manuscript manuscript.docx 2 patients with RC disease. However, in vitro stimulation of RC tenocytes with TGF-β rescued their ability to produce type I collagen and VEGF. Conclusion: LHB tendon from RC disease patients had neurotransmitter disturbances that could be related to shoulder pain. Moreover, we demonstrated that LHB from RC disease patients had a downregulation of extracellular matrix genes, as well as of VEGF and NGF genes. We showed that TGF-β can partially normalize the expression of these genes, suggesting that modulating TGF-β could be a therapeutic opportunity for improving tendon quality in the context of chronic tendinopathies.

Methods of Assessing Human Tendon Metabolism and Tissue Properties in Response to Changes in Mechanical Loading.

In recent years a number of methodological developments have improved the opportunities to study human tendon. Microdialysis enables sampling of interstitial fluid in the peritendon tissue, while sampling of human tendon biopsies allows direct analysis of tendon tissue for gene- and protein expression as well as protein synthesis rate. Further the (14)C bomb-pulse method has provided data on long-term tissue turnover in human tendon. Non-invasive techniques allow measurement of tendon metabolism (positron emission tomography (PET)), tendon morphology (magnetic resonance imaging (MRI)), and tendon mechanical properties (ultrasonography combined with force measurement during movement). Finally, 3D cell cultures of human tendon cells provide the opportunity to investigate cell-matrix interactions in response to various interventions.

TOL19-001 reduces inflammation and MMP expression in monolayer cultures of tendon cells.

BackgroundTendinopathies are tendon conditions associated with degeneration and disorganization of the matrix collagen fibers, tendon cells apoptosis and inflammation through up-regulation of proinflammatory cytokines, matrix metalloproteinase (MMP) expression, and prostaglandin E2 (PGE2) production. Currently, the pharmacological treatment is mainly based on non-steroidal anti-inflammatory drugs (NSAIDs) use and corticosteroid injections, which both can lead to numerous side effects for patients. TOL19-001 is a diet supplementary composed mostly of spirulina and glucosamine sulfate whose antioxidant properties could be helpful to treat tendinopathies while avoiding taking NSAIDs. In this study we developed an in vitro model of tendinopathy in order to evaluate the therapeutic potential of TOL19-001.MethodsTendon cells were cultured on monolayer and treated with interleukin-1β (IL-1β) or ciprofloxacin (CIP), and then, MMPs, PGE2 and collagen expression was evaluated by RT-PCR or Elisa. In addition, a cotreatment with increased doses of TOL19-001 was done. Toxicity of TOL19-001 was evaluated using a metabolic activity assay.ResultsThis study demonstrates that IL-1β mimics some aspects of tendinopathies with PGE2 induction, MMP expression (mostly MMP1 and MMP3), and increases of type III/I collagen ratio. CIP, meanwhile, leads to an increase of MMP2 and p65 mRNA, whereas it reduces TIMP1 expression. Scleraxis expression was also increased by CIP whereas it was decreased by IL-1β treatment. Besides, TOL19-001 cotreatment suppresses tendon cell inflammation in vitro, marked by the downregulation of PGE2, MMPs and type III collagen in IL-1β stimulated-cells. TOL19-001 also represses CIP induced-changes.ConclusionsThese findings indicate that TOL19-001 exerts anti-inflammatory effects on tendon cells, which might explain why TOL19-001 diet may improve tendon function in patients with tendon injury. Future research is required to determine TOL19-001 effect on injured or overused tendons in vivo.

Wnt5a is expressed in spondyloarthritis and exerts opposite effects on enthesis and bone in murine organ and cell cultures

Spondyloarthritis (SpA) is a chronic inflammatory joint disorder that initiates at the enthesis, where tendons attach to bone through a fibrocartilage zone. At late stages, excessive bone apposition appears within the diseased enthesis. Because Wnt5a participates to normal bone formation and appears related to inflammatory processes, we investigated the role of this Wnt growth factor in inflammation-associated ossification in SpA. The concentration of Wnt5a assessed by enzyme-linked immunosorbent assay in synovial fluids of patients with SpA (2.58 ± 0.98 ng/mL) was higher than in osteoarthritic patients (1.33 ± 0.71 ng/mL). In murine primary cultures of tendon cells, chondrocytes, and osteoblasts and in an organotypic model of mouse ankle, we showed that tumor necrosis factor α reversibly diminished Wnt5a expression and secretion, respectively. Wnt5a decreased gene expression of differentiation markers and mineralization in cultured chondrocytes and reduced alkaline phosphatase activity in Achilles tendon enthesis (-14%) and osteocalcin protein levels released by ankle explants (-36%). On the contrary, Wnt5a stimulated ossification markers' expression in cultured osteoblasts and increased the bone volume of the tibial plateau of the cultured explants (+19%). In conclusion, our results suggest that Wnt5a is expressed locally in the joints of patients with SpA. Wnt5a appears more associated with ossification than with inflammation and tends to inhibit mineralization in chondrocytes and enthesis, whereas it seems to favor the ossification process in osteoblasts and bone. Further studies are needed to decipher the opposing effects observed locally in enthesis and systemically in bone in SpA.

The Effect of 5 FU on the Expression of Transforming Growth Factor Beta-1 (Tgf-1) in Cultured Tendon Cells

The Effect of 5 FU on the Expression of Transforming Growth Factor Beta-1 (Tgf-1) in Cultured Tendon Cells

In vitro functional response of human tendon cells to different dosages of low-frequency pulsed electromagnetic field.

Chronic tendinopathy is a degenerative process causing pain and disability. Current treatments include biophysical therapies, such as pulsed electromagnetic fields (PEMF). The aim of this study was to compare, for the first time, the functional in vitro response of human tendon cells to different dosages of PEMF, varying in field intensity and duration and number of exposures. Tendon cells, isolated from human semitendinosus and gracilis tendons (hTCs; n = 6), were exposed to different PEMF treatments (1.5 or 3 mT for 8 or 12 h, single or repeated treatments). Scleraxis (SCX), COL1A1, COL3A1 and vascular endothelial growth factor-A (VEGF-A) expression and cytokine production were assessed. None of the different dosages provoked apoptotic events. Proliferation of hTCs was enhanced by all treatments, whereas only 3 mT-PEMF treatment increased cell viability. However, the single 1.5 mT-PEMF treatment elicited the highest up-regulation of SCX, VEGF-A and COL1A1 expression, and it significantly reduced COL3A1 expression with respect to untreated cells. The treated hTCs showed a significantly higher release of IL-1β, IL-6, IL-10 and TGF-β. Interestingly, the repeated 1.5 mT-PEMF significantly further increased IL-10 production. 1.5 mT-PEMF treatment was able to give the best results in in vitro healthy human tendon cell culture. Although the clinical relevance is not direct, this investigation should be considered an attempt to clarify the effect of different PEMF protocols on tendon cells, in particular focusing on the potential applicability of this cell source for regenerative medicine purpose, both in surgical and in conservative treatment for tendon disorders.

Soft-Focused Extracorporeal Shock Waves Increase the Expression of Tendon-Specific Markers and the Release of Anti-inflammatory Cytokines in an Adherent Culture Model of Primary Human Tendon Cells

Focused extracorporeal shock waves have been found to upregulate the expression of collagen and to initiate cell proliferation in healthy tenocytes and to positively affect the metabolism of tendons, promoting the healing process. Recently, soft-focused extracorporeal shock waves have also been found to have a significant effect on tissue regeneration. However, very few in vitro reports have dealt with the application of this type of shock wave to cells, and in particular, no previous studies have investigated the response of tendon cells to this impulse. We devised an original model to investigate the in vitro effects of soft-focused shock waves on a heterogeneous population of human resident tendon cells in adherent monolayer culture. Our results indicate that soft-focused extracorporeal shock wave treatment (0.17 mJ/mm2) is able to induce positive modulation of cell viability, proliferation and tendon-specific marker expression, as well as release of anti-inflammatory cytokines. This could prefigure a new rationale for routine employment of soft-focused shock waves to treat the failed healing status that distinguishes tendinopathies.

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 β-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.

Ascorbic acid is essential for significant collagen deposition by human tenocytes in vitro

Objective: We sought to compare L-ascorbic acid (AA), the natural form of vitamin C, and magnesium ascorbyl phosphate (MAP), a stable derivative, for their effect on cell proliferation, culture collagen content and collagen expression in tendon-derived cells. Methods: Tenocytes received these two ascorbate formulations at concentrations between 0.01 mM and 2 M. The effect of supplementation was evaluated in terms of cell growth, collagen expression, collagen secretion, cell morphology and collagen localization. Results: MAP supplementation resulted in dose-dependent increase in growth rates, whilst AA supplementation was toxic to tendon-derived cells from as low as 1 mM. Collagen expression was only mildly modulated by ascorbic acid, but collagen accumulation was significantly increased, supporting the hypothesis of its possible role in secretion. Immunofluorescence staining revealed differential localization of collagen I in vitro with collagen localized outside cells in the presence of ascorbic acid. Multi-photon microscopy further demonstrated that collagen fibrils were only formed in the presence of ascorbic acid. Conclusions: Taken together, these results suggest that ascorbic acid, preferably the magnesium ascorbyl phosphate formulation, should be routinely used as a supplement to tendon cell culture when collagen fibrils are required, especially in the context of tissue engineering and scaffold development.

HGF mediates the anti-inflammatory effects of PRP on injured tendons.

Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2.