Scaffold-Mediated Immunoengineering as Innovative Strategy for Tendon Regeneration.

Tendon injuries are at the frontier of innovative approaches to public health concerns and sectoral policy objectives. Indeed, these injuries remain difficult to manage due to tendon's poor healing ability ascribable to a hypo-cellularity and low vascularity, leading to the formation of a fibrotic tissue affecting its functionality. Tissue engineering represents a promising solution for the regeneration of damaged tendons with the aim to stimulate tissue regeneration or to produce functional implantable biomaterials. However, any technological advancement must take into consideration the role of the immune system in tissue regeneration and the potential of biomaterial scaffolds to control the immune signaling, creating a pro-regenerative environment. In this context, immunoengineering has emerged as a new discipline, developing innovative strategies for tendon injuries. It aims at designing scaffolds, in combination with engineered bioactive molecules and/or stem cells, able to modulate the interaction between the transplanted biomaterial-scaffold and the host tissue allowing a pro-regenerative immune response, therefore hindering fibrosis occurrence at the injury site and guiding tendon regeneration. Thus, this review is aimed at giving an overview on the role exerted from different tissue engineering actors in leading immunoregeneration by crosstalking with stem and immune cells to generate new paradigms in designing regenerative medicine approaches for tendon injuries.

Species variations in tenocytes' response to inflammation require careful selection of animal models for tendon research.

For research on tendon injury, many different animal models are utilized; however, the extent to which these species simulate the clinical condition and disease pathophysiology has not yet been critically evaluated. Considering the importance of inflammation in tendon disease, this study compared the cellular and molecular features of inflammation in tenocytes of humans and four common model species (mouse, rat, sheep, and horse). While mouse and rat tenocytes most closely equalled human tenocytes' low proliferation capacity and the negligible effect of inflammation on proliferation, the wound closure speed of humans was best approximated by rats and horses. The overall gene expression of human tenocytes was most similar to mice under healthy, to horses under transient and to sheep under constant inflammatory conditions. Humans were best matched by mice and horses in their tendon marker and collagen expression, by horses in extracellular matrix remodelling genes, and by rats in inflammatory mediators. As no single animal model perfectly replicates the clinical condition and sufficiently emulates human tenocytes, fit-for-purpose selection of the model species for each specific research question and combination of data from multiple species will be essential to optimize translational predictive validity.

Selected Clinical Features Fail to Predict Inflammatory Gene Expressions for TNF-α, TNFR1, NSMAF, Casp3 and IL-8 in Tendons of Patients with Rotator Cuff Tendinopathy.

The pathophysiology of rotator cuff tendinopathy is not fully understood, particularly in terms of the local inflammatory process. This study aimed to investigate the expression of selected molecules in the tumour necrosis factor (TNF)-α transduction pathway, including TNF-α, TNF receptor 1 (TNFR1), neutral sphingomyelinase activation associated factor (NSMAF), caspase 3 (Casp3), and interleukin (IL)-8, in patients with rotator cuff tendinopathy that had undergone surgical treatment. We included 44 participants that underwent arthroscopy, due to rotator cuff tendinopathy. Samples from the injured tendon were collected during arthroscopy, and RT-PCR was performed to determine gene expression. Pearson correlation analyses or U-Mann-Whitney test were performed to identify associations with the following parameters: sex, age at admission, body mass index, the presence of night pain, previous treatment (nonsteroidal anti-inflammatory drugs and/or steroids), medical history of the shoulder injury, upper subluxation of the humeral head, and the number of tendons injured. RT-PCR showed that the selected pro-inflammatory factors involved in the TNF-α signalling pathway expression levels were expressed in the tendon tissues. However, the levels of expression varied from patient to patient. Variations were over 250-fold for TNF-α, about 130-fold for TNFR1, NSMAF, and Casp3, and 1000-fold for IL-8. We could not confirm that any of the clinical parameters investigated were associated with the level of gene expression in the TNF-α pathway and IL-8.

Normal human enthesis harbours conventional CD4+ and CD8+ T cells with regulatory features and inducible IL-17A and TNF expression.

BACKGROUND: The human enthesis conventional T cells are poorly characterised. OBJECTIVES: To study the biology of the conventional T cells in human enthesis. METHODS: CD4+ and CD8+ T cells were investigated in 25 enthesis samples using immunofluorescence, cytometrically, bulk RNAseq and quantitative real-time PCR following anti-CD3/CD28 bead stimulation to determine interleukin (IL)-17A and tumour necrosis factor (TNF) levels. T-cell receptor (TCR) repertoires were characterised and a search for putative T-cell reactivity was carried out using TCR3 database. The impact of pharmacological antagonism with retinoic acid receptor-related orphan nuclear receptor gamma t inhibitor (RORγti), methotrexate and phosphodiesterase type 4 inhibitor (PDE4i) was investigated. RESULTS: Immunofluorescence and cytometry suggested entheseal resident CD4+ and CD8+ T cells with a resident memory phenotype (CD69+/CD45RA-) and tissue residency gene transcripts (higher NR4A1/AhR and lower KLF2/T-bet transcripts). Both CD4+ and CD8+ T cells showed increased expression of immunomodulatory genes including IL-10 and TGF-ß compared with peripheral blood T cells with entheseal CD8+ T cells having higher CD103, CD49a and lower SIPR1 transcript that matched CD4+ T cells. Following stimulation, CD4+ T cells produced more TNF than CD8+ T cells and IL-17A was produced exclusively by CD4+ T cells. RNAseq suggested both Cytomegalovirus and influenza A virus entheseal resident T-cell clonotype reactivity. TNF and IL-17A production from CD4+ T cells was effectively inhibited by PDE4i, while RORγti only reduced IL-17A secretion. CONCLUSIONS: Healthy human entheseal CD4+ and CD8+ T cells exhibit regulatory characteristics and are predicted to exhibit antiviral reactivity with CD8+ T cells expressing higher levels of transcripts suggestive of tissue residency. Inducible IL-17A and TNF production can be robustly inhibited in vitro.

A systematic review of inflammatory cells and markers in human tendinopathy.

BACKGROUND: This article systematically reviews the current evidence regarding inflammation in Tendinopathy with the aim to increase understanding of a potential common pathophysiology. METHODS: Following the PRISMA statements, the terms: (tendinopathy OR (tendons AND rupture)) AND (inflammation OR (inflammation AND cells) OR immune system OR inflammation mediators OR bacteria) were used. One thousand four hundred thirty-one articles were identified which was screened down to 53. RESULTS: 39/53 studies mentioned inflammatory cells but had contradicting conclusions. Macrophages were the most common cell type and inflammatory markers were detectable in all the articles which measure them. CONCLUSIONS: The included studies show different conclusions, but this heterogeneity is not unexpected since the clinical criteria of 'tendinopathy' encompass a huge clinical spectrum. Different 'tendinopathy' conditions may have different pathophysiology, and even the same clinical condition may be at different disease stages during sampling, which can alter the histological and biochemical picture. Control specimen sampling was suboptimal since the healthy areas of the pathological-tendon may actually be sub-clinically diseased, as could the contralateral tendon in the same subject. Detection of inflammatory cells is most sensitive using immunohistochemistry targeting the cluster of differentiation markers, especially when compared to the conventional haematoxylin and eosin staining methods. The identified inflammatory cell types favour a chronic inflammatory process; which suggests a persistent stimulus. This means NSAID and glucocorticoids may be useful since they suppress inflammation, but it is noted that they may hinder tendon healing and cause long term problems. This systematic review demonstrates a diversity of data and conclusions in regard to inflammation as part of the pathogenesis of Tendinopathy, ranging from ongoing or chronic inflammation to non-inflammatory degeneration and chronic infection. Whilst various inflammatory markers are present in two thirds of the reviewed articles, the heterogenicity of data and lack of comparable studies means we cannot conclude a common pathophysiology from this systematic review.

Pulsed Electromagnetic Field Modulates Tendon Cells Response in IL-1ß-Conditioned Environment.

Strategies aiming at controlling and modulating inflammatory cues may offer therapeutic solutions for improving tendon regeneration. This study aims to investigate the modulatory effect of pulsed electromagnetic field (PEMF) on the inflammatory profile of human tendon-derived cells (hTDCs) after supplementation with interleukin-1ß (IL-1ß). IL-1ß was used to artificially induce inflammatory cues associated with injured tendon environments. The PEMF effect was investigated varying the frequency (5 or 17 Hz), intensity (1.5, 4, or 5 mT), and duty-cycle (10% or 50%) parameters to which IL-1ß-treated hTDCs were exposed to. A PEMF actuation with 4 mT, 5 Hz and a 50% duty cycle decreased the production of IL-6 and tumor necrosis factor-α (TNF-α), as well as the expression of TNFα, IL-6, IL-8, COX-2, MMP-1, MMP-2, and MMP-3, while IL-4, IL-10, and TIMP-1 expression increased. These results suggest that PEMF stimulation can modulate hTDCs response in an inflammatory environment holding therapeutic potential for tendon regenerative strategies. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:160-172, 2020.

Process of neovascularisation compared with pain intensity in tendinopathy of the long head of the biceps brachii tendon associated with concomitant shoulder disorders, after arthroscopic treatment. Microscopic evaluation supported by immunohistochemical.

BACKGROUND: Tendinopathy of the long head of the biceps brachii tendon (LHBT) is one of the most common, painful conditions of the anterior part of the shoulder and often coexists with rotator cuff tears. Multifactorial aetiopathology of tendi-nopathy is poorly understood; however, several studies indicated that it is seen predominantly in areas with decreased vascularity of the tissue; the pathology is also characterised by expansive and abundant neovascular in-growth. The aim of the study was to investigate the relationship between the neovascularisation of proximal part of the LHBT and pain along the bicipital groove. MATERIALS AND METHODS: Tissue material was obtained from 28 patients who underwent a shoulder arthroscopy and experienced pain along the bicipital groove measured using Visual-Analog Scale (VAS) score. CD31 and CD34 molecules were visualised by immunohistochemical method to assess biceps tendon neovascula-risation and quantify it based on a Bonar scoring system. RESULTS: Although all patients reported pain prior to arthroscopy (mean VAS score was 7.5), microscopic examination did not reveal neovascularisation in all cases. Immunohistochemical staining for CD31 and CD34 allowed for very precise visualisation and quantification of neovascularisation; however there was also no correlation between vessels in-growth scores and pain. CONCLUSIONS: The obtained data suggest that neovascularisation process in tendino-pathy is not directly related to pain; however, further studies are needed to explain its significance in the LHBT tendinopathy. (Folia Morphol 2018; 77, 2: 378-385).

Macrophage infiltration of electrospun polyester fibers.

Electrospun fibrous polylactide (PLA) membranes have been widely used for peritendinous anti-adhesion, but their degradation may lead to granuloma formation triggered by macrophages as the result of a foreign body reaction. To understand and solve this problem in peritendinous anti-adhesion, the effects of electrospun fibrous PLA membranes (PLA-M) and the mechanism by which macrophages elicit a foreign body reaction should be elaborated. Thus, the purpose of this study was to evaluate the ability of PLA-M and ibuprofen (IBU)-loaded fibrous PLA membranes (IBU/PLA-M) to prevent adhesion/granuloma formation around the tendon, and to understand the mechanism of macrophage infiltration. The results showed that PLA-M and IBU/PLA-M were porous and that IBU could be sustainably released from IBU/PLA-M. The adhesion and proliferation of RAW264.7 macrophages were worse on the surface of IBU/PLA-M than on PLA-M. After implantation around the tendon, IBU/PLA-M had higher anti-adhesion and tendon healing scores than PLA-M. Furthermore, PLA-M was able to promote macrophage infiltration, neovascularization, TNF-α expression and collagen III deposition to a greater extent than IBU/PLA-M. Consequently, these results show that macrophages can be triggered by PLA-M which subsequently leads to inflammation and granuloma formation, while IBU/PLA-M results in enhanced anti-inflammation and anti-adhesion effects when compared to PLA-M, by reducing macrophage infiltration.

Platelet-Rich Plasma Activates Proinflammatory Signaling Pathways and Induces Oxidative Stress in Tendon Fibroblasts.

BACKGROUND: Tendon injuries are one of the most common musculoskeletal conditions in active patients. Platelet-rich plasma (PRP) has shown some promise in the treatment of tendon disorders, but little is known as to the mechanisms by which PRP can improve tendon regeneration. PRP contains numerous different growth factors and cytokines that activate various cellular signaling cascades, but it has been difficult to determine precisely which signaling pathways and cellular responses are activated after PRP treatment. Additionally, macrophages play an important role in modulating tendon regeneration, but the influence of PRP on determining whether macrophages assume a proinflammatory or anti-inflammatory phenotype remains unknown. PURPOSE: To use genome-wide expression profiling, bioinformatics, and protein analysis to determine the cellular pathways activated in fibroblasts treated with PRP. The effect of PRP on macrophage polarization was also evaluated. STUDY DESIGN: Controlled laboratory study. METHODS: Tendon fibroblasts or macrophages from rats were cultured and treated with either platelet-poor plasma (PPP) or PRP. RNA or protein was isolated from cells and analyzed using microarrays, quantitative polymerase chain reaction, immunoblotting, or bioinformatics techniques. RESULTS: Pathway analysis determined that the most highly induced signaling pathways in PRP-treated tendon fibroblasts were TNFα and NFκB pathways. PRP also downregulated the expression of extracellular matrix genes and induced the expression of autophagy-related genes and reactive oxygen species (ROS) genes and protein markers in tendon fibroblasts. PRP failed to have a major effect on markers of macrophage polarization. CONCLUSION: PRP induces an inflammatory response in tendon fibroblasts, which leads to the formation of ROS and the activation of oxidative stress pathways. PRP does not appear to significantly modulate macrophage polarization. CLINICAL RELEVANCE: PRP might act by inducing a transient inflammatory event, which could then trigger a tissue regeneration response.

Harmful Effects of Leukocyte-Rich Platelet-Rich Plasma on Rabbit Tendon Stem Cells In Vitro.

BACKGROUND: Platelet-rich plasma (PRP) is now widely used as a promising treatment for patients with tendinopathy. However, the efficacy of PRP treatment for tendinopathy is controversial mainly because of inconsistent results from human clinical trials and particularly because the concentration and effect of leukocytes in PRP remain largely unknown. HYPOTHESIS: Leukocyte-rich PRP (L-PRP) inhibits growth factor release, decreases proliferation, and induces nontenocyte differentiation of tendon stem cells (TSCs); increases catabolic cytokine concentrations; and causes inflammation and apoptosis. Thus, L-PRP has a detrimental effect on tendon stem/progenitor cells, which impairs injured tendon healing. STUDY DESIGN: Controlled laboratory study. METHODS: Pure PRP (P-PRP) and L-PRP were prepared from the same individual rabbit blood, and platelet numbers in each PRP product were adjusted to reach the same level. The leukocyte level in L-PRP was 4 and 8 times higher than those in whole blood and P-PRP, respectively. The growth factors in both P-PRP and L-PRP were measured by enzyme-linked immunosorbent assay kits. The morphology, stemness, proliferation, and differentiation of TSCs grown in L-PRP and P-PRP were examined by microscopy, immunocytochemistry, population doubling time, quantitative real-time polymerase chain reaction, and histological analysis. RESULTS: L-PRP produced lower levels of growth factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor (TGF)-ß1, and platelet-derived growth factor (PDGF), than did P-PRP. TSC proliferation was significantly decreased in L-PRP in a concentration-dependent manner. Furthermore, TSCs cultured in P-PRP produced more collagen and formed tendon-like tissue; however, TSCs grown in L-PRP differentiated into nontenocytes and produced more inflammatory factors such as membrane-associated prostaglandin synthase (mPGES) and interleukin (IL)-1ß. Moreover, L-PRP was associated with increased apoptosis. CONCLUSION: L-PRP has harmful effects on TSCs. CLINICAL RELEVANCE: This study revealed the direct effects of different compositions of PRP on TSCs and provided basic scientific data to help understand the cellular and molecular mechanisms of the efficacy of PRP treatment in clinical use.

Inflammation activation and resolution in human tendon disease.

Improved understanding of the role of inflammation in tendon disease is required to facilitate therapeutic target discovery. We studied supraspinatus tendons from patients experiencing pain before and after surgical subacromial decompression treatment. Tendons were classified as having early, intermediate, or advanced disease, and inflammation was characterized through activation of pathways mediated by interferon (IFN), nuclear factor κB (NF-κB), glucocorticoid receptor, and signal transducer and activator of transcription 6 (STAT-6). Inflammation signatures revealed expression of genes and proteins induced by IFN and NF-κB in early-stage disease and genes and proteins induced by STAT-6 and glucocorticoid receptor activation in advanced-stage disease. The proresolving proteins FPR2/ALX and ChemR23 were increased in early-stage disease compared to intermediate- to advanced-stage disease. Patients who were pain-free after treatment had tendons with increased expression of CD206 and ALOX15 mRNA compared to tendons from patients who continued to experience pain after treatment, suggesting that these genes and their pathways may moderate tendon pain. Stromal cells from diseased tendons cultured in vitro showed increased expression of NF-κB and IFN target genes after treatment with lipopolysaccharide or IFNγ compared to stromal cells derived from healthy tendons. We identified 15-epi lipoxin A4, a stable lipoxin isoform derived from aspirin treatment, as potentially beneficial in the resolution of tendon inflammation.

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

BACKGROUND: Tendinopathies 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. METHODS: Tendon cells were cultured on monolayer and treated with interleukin-1ß (IL-1ß) 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. RESULTS: This study demonstrates that IL-1ß 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-1ß treatment. Besides, TOL19-001 cotreatment suppresses tendon cell inflammation in vitro, marked by the downregulation of PGE2, MMPs and type III collagen in IL-1ß stimulated-cells. TOL19-001 also represses CIP induced-changes. CONCLUSIONS: These 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.

p38 MAPK signaling in postnatal tendon growth and remodeling.

Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

Platelet-rich plasma modulates the secretion of inflammatory/angiogenic proteins by inflamed tenocytes.

BACKGROUND: Platelet-rich plasma therapies for tendinopathy appear to provide moderate pain reduction. However, the biological mechanisms behind the observed clinical effects remain poorly characterized. QUESTIONS/PURPOSES: The purpose of this study was to explore whether platelet-rich plasma modifies the inflammatory/angiogenic status of already inflamed tenocytes by examining (1) gene expression; (2) modulation of chemokine and interleukin secretion; and (3) differences between healthy and tendinopathic tenocytes. METHODS: Cells from both healthy and tendinopathic tendons were exposed to interleukin (IL)-1ß and after treated with platelet-rich plasma. Modifications in the expression of selected genes were assessed by real-time reverse transcription-polymerase chain reaction and changes in secretion of angiogenic/inflammatory molecules by enzyme-linked immunosorbent assay. Platelet-rich plasma-induced changes in tendinopathic cells were compared with normal after normalizing platelet-rich plasma data against IL-1ß status in each specific sample. RESULTS: In IL-1ß-exposed cells, platelet-rich plasma downregulates expression of IL-6/CXCL-6 (mean, 0.015; 95% confidence interval [CI], 0.005-0.025; p = 0.026), IL-6R (mean, 0.61; 95% CI, 0.27-0.95; p = 0.029), and IL-8/CXCL-8 (mean, 0.02; 95% CI, 0.007-0.023; p = 0.026). Secretion of IL-6/CXCL6, 0.35 (95% CI, 0.3-0.4; p = 0.002), IL-8/CXCL8, 0.55 (95% CI, 0.5-0.7; p = 0.01), and monocyte chemoattractant protein-1/CCL2, 0.40 (95% CI, 0.2-0.6; p = 0.001) was reduced by platelet-rich plasma, whereas vascular endothelial growth factor increased by twofold, (95% CI, 1.7-2.3; p < 0.001). RANTES/CCL5 increased by10-fold (95% CI, 4-17) and hepatocyte growth factor by 21-fold (95% CI, 0.2-42) in tendinopathic and by 2.3-fold (95% CI, 2-3) and threefold (95% CI, 1-5) in normal cells (p = 0.005 for both). CONCLUSIONS: Platelet-rich plasma induces an immunomodulatory and proangiogenic phenotype consistent with healing mechanisms with few differences between tendinopathic and normal cells. CLINICAL RELEVANCE: Platelet-rich plasma injections in pathological and nearby tissue might help to recover tendon homeostasis.

Allogeneic tendon-derived stem cells promote tendon healing and suppress immunoreactions in hosts: in vivo model.

The medium- to long-term healing effect and infiltration of inflammatory cells, after transplantation of allogeneic tendon-derived stem cell (TDSC) to the rat patellar tendon window wound, were examined. Allogeneic patellar TDSCs derived from a green fluorescent protein rat were used. The outcome of tendon healing and the infiltration of inflammatory cells were examined by histology and immunohistochemistry up to week 16 postinjury. The fate of the transplanted cells was examined by ex vivo fluorescent imaging and immunohistochemistry. Our results showed that the transplantation of allogeneic TDSCs promoted tendon healing with no increased risk of ectopic chondro-ossification up to week 16. A low infiltration of T cells, ED1 macrophages, ED2 macrophages, and mast cells in the window wound was obtained. The transplanted TDSCs were found in the window wound at week 1 and 2, but were absent after week 4 postinjury. In conclusion, allogeneic TDSCs promoted tendon repair in the medium to long term and exhibited weak immunoreactions and anti-inflammatory effects in the hosts after transplantation in a rat model. There was no increased risk of ectopic chondro-ossification after TDSC transplantation. The decrease in the number of transplanted cells with time suggested that allogeneic TDSCs did not promote tendon repair through direct differentiation.

Tenocyte activation and regulation of complement factors in response to in vitro cell injury.

Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)α, interleukin (IL)-1ß, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was down-regulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNFα gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses.

Increased serum and musculotendinous fibrogenic proteins following persistent low-grade inflammation in a rat model of long-term upper extremity overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1ß after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed.

Conditioned medium from horse amniotic membrane-derived multipotent progenitor cells: immunomodulatory activity in vitro and first clinical application in tendon and ligament injuries in vivo.

We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases.

IL-23 induces spondyloarthropathy by acting on ROR-γt+ CD3+CD4-CD8- entheseal resident T cells.

The spondyloarthropathies are a group of rheumatic diseases that are associated with inflammation at anatomically distal sites, particularly the tendon-bone attachments (entheses) and the aortic root. Serum concentrations of interleukin-23 (IL-23) are elevated and polymorphisms in the IL-23 receptor are associated with ankyosing spondylitis, however, it remains unclear whether IL-23 acts locally at the enthesis or distally on circulating cell populations. We show here that IL-23 is essential in enthesitis and acts on previously unidentified IL-23 receptor (IL-23R)(+), RAR-related orphan receptor γt (ROR-γt)(+)CD3(+)CD4(-)CD8(-), stem cell antigen 1 (Sca1)(+) entheseal resident T cells. These cells allow entheses to respond to IL-23 in vitro-in the absence of further cellular recruitment--and to elaborate inflammatory mediators including IL-6, IL-17, IL-22 and chemokine (C-X-C motif) ligand 1 (CXCL1). Notably, the in vivo expression of IL-23 is sufficient to phenocopy the human disease, with the specific and characteristic development of enthesitis and entheseal new bone formation in the initial complete absence of synovitis. As in the human condition, inflammation also develops in vivo at the aortic root and valve, which are structurally similar to entheses. The presence of these entheseal resident cells and their production of IL-22, which activates signal transducer and activator of transcription 3 (STAT3)-dependent osteoblast-mediated bone remodeling, explains why dysregulation of IL-23 results in inflammation at this precise anatomical site.