Widespread Vascularization and Correlation of Glycosaminoglycan Accumulation to Tendon Pain in Human Plantar Fascia Tendinopathy.

BACKGROUND: Plantar fasciitis is a painful tendinous condition (tendinopathy) with a high prevalence in athletes. While a healthy tendon has limited blood flow, ultrasound has indicated elevated blood flow in tendinopathy, but it is unknown if this is related to a de facto increase in the tendon vasculature. Likewise, an accumulation of glycosaminoglycans (GAGs) is observed in tendinopathy, but its relationship to clinical pain is unknown. PURPOSE: To explore to what extent vascularization, inflammation, and fat infiltration were present in patients with plantar fasciitis and if they were related to clinical symptoms. STUDY DESIGN: Descriptive laboratory study. METHODS: Biopsy specimens from tendinopathic plantar fascia tissue were obtained per-operatively from both the primary site of tendon pain and tissue swelling ("proximal") and a region that appeared macroscopically healthy at 1 to 2 cm away from the primary site ("distal") in 22 patients. Biopsy specimens were examined with immunofluorescence for markers of blood vessels, tissue cell density, fat infiltration, and macrophage level. In addition, pain during the first step in the morning (registered during an earlier study) was correlated with the content of collagen and GAGs in tissue. RESULTS: High vascularization (and cellularity) was present in both the proximal (0.89%) and the distal (0.96%) plantar fascia samples, whereas inconsistent but not significantly different fat infiltration and macrophage levels were observed. The collagen content was similar in the 2 plantar fascia regions, whereas the GAG content was higher in the proximal region (3.2% in proximal and 2.8% in distal; P = .027). The GAG content in the proximal region was positively correlated with the subjective morning pain score in the patients with tendinopathy (n = 17). CONCLUSION: In patients with plantar fasciitis, marked tissue vascularization was present in both the painful focal region and a neighboring nonsymptomatic area. In contrast, the accumulation of hydrophilic GAGs was greater in the symptomatic region and was positively correlated with increased clinical pain levels in daily life. CLINICAL RELEVANCE: The accumulation of GAGs in tissue rather than the extent of vascularization appears to be linked with the clinical degree of pain symptoms of the disease.

Rotator Cuff Tendinopathy: Pathways of Apoptosis.

Rotator cuff repair is usually successful, but retear is not uncommon. It has been previously identified that there is a higher incidence of apoptosis in the edges of the torn supraspinatus tendon. A prospective cohort study was conducted with 28 patients-14 rotator cuff tear patients, 5 instability patients, and 9 Anterior cruciate ligament reconstruction patients to determine whether there was any increase in several genes implicated in apoptosis, including Fas receptor (FasR), Fas ligand, Aifm-1, Bcl-2, Fadd, Bax, and caspase-3. There was a significant expression of Bax (P=0.2) and FasR (P=0.005) in the edges of torn supraspinatus tendons, and in intact subscapularis tendons, there was a significant expression of caspase-3 (P=0.02) compared with samples from the torn supraspinatus tendon (P=0.04). The cytochrome c pathway, with its subsequent activation of caspase-3, as well as the TRAIL-receptor signaling pathway involving FasR have both been implicated. The elevated expression of Bax supported the model that the Bax to Bcl-2 expression ratio represents a cell death switch. The elevated expression of Bax in the intact subscapularis tissue from rotator cuff tear patients also may confirm that tendinopathy is an ongoing molecular process.

TRIM54 alleviates inflammation and apoptosis by stabilizing YOD1 in rat tendon-derived stem cells.

Tendinopathy is a disorder of musculoskeletal system that primarily affects athletes and the elderly. Current treatment options are generally comprised of various exercise and loading programs, therapeutic modalities, and surgical interventions and are limited to pain management. This study is to understand the role of TRIM54 (tripartite motif containing 54) in tendonitis through in vitro modeling with tendon-derived stem cells (TDSCs) and in vivo using rat tendon injury model. Initially, we observed that TRIM54 overexpression in TDSCs model increased stemness and decreased apoptosis. Additionally, it rescued cells from tumor necrosis factor α-induced inflammation, migration, and tenogenic differentiation. Further, through immunoprecipitation studies, we identified that TRIM54 regulates inflammation in TDSCs by binding to and ubiquitinating YOD1. Further, overexpression of TRIM54 improved the histopathological score of tendon injury as well as the failure load, stiffness, and young modulus in vivo. These results indicated that TRIM54 played a critical role in reducing the effects of tendon injury. Consequently, these results shed light on potential therapeutic alternatives for treating tendinopathy.

Irisin inhibits tenocyte response to inflammation in vitro: New insights into tendon-muscle cross-talk.

Tendinopathy is one of the most common musculoskeletal disorders with significant repercussions on quality of life and sport activities. Physical exercise (PE) is considered the first-line approach to treat tendinopathy due renowned mechanobiological effects on tenocytes. Irisin, a recently identified myokine released during PE, has been recognized for several beneficial effects towards muscle, cartilage, bone, and intervertebral disc tissues. The aim of this study was to evaluate the effects of irisin on human primary tenocytes (hTCs) in vitro. Human tendons were harvested from specimens of patients undergoing anterior cruciate ligament reconstruction (n = 4). After isolation and expansion, hTCs were treated with RPMI medium (negative control), interleukin (IL)-1ß or tumor necrosis factor-α (TNF-α) (positive controls; 10 ng/mL), irisin (5, 10, 25 ng/mL), IL-1ß or TNF-α pretreatment and subsequent co-treatment with irisin, pretreatment with irisin and subsequent co-treatment with IL-1ß or TNF-α. hTC metabolic activity, proliferation, and nitrite production were evaluated. Detection of unphosphorylated and phosphorylated p38 and ERK was performed. Tissue samples were analyzed by histology and immunohistochemistry to evaluate irisin αVß5 receptor expression. Irisin significantly increased hTC proliferation and metabolic activity, while reducing the production of nitrites both before and after the addition of IL-1ß and TNF-α. Interestingly, irisin reduced p-p38 and pERK levels in inflamed hTCs. The αVß5 receptor was uniformly expressed on hTC plasma membranes, supporting the potential binding of irisin. This is the first study reporting the capacity of irisin to target hTCs and modulating their response to inflammatory stresses, possibly orchestrating a biological crosstalk between the muscle and tendon.

Tumour necrosis factor alpha, interleukin 1 beta and interferon gamma have detrimental effects on equine tenocytes that cannot be rescued by IL-1RA or mesenchymal stromal cell-derived factors.

Tendon injuries occur commonly in both human and equine athletes, and poor tendon regeneration leads to functionally deficient scar tissue and an increased frequency of re-injury. Despite evidence suggesting inadequate resolution of inflammation leads to fibrotic healing, our understanding of the inflammatory pathways implicated in tendinopathy remains poorly understood, meaning successful targeted treatments are lacking. Here, we demonstrate IL-1ß, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for equine tenocytes than when used individually. This includes altering tendon associated and matrix metalloproteinase gene expression and impairing the cells' ability to contract a 3-D collagen gel, a culture technique which more closely resembles the in vivo environment. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1ß using IL-1RA or factors produced by BM-MSCs. Furthermore, we provide evidence that NF-κB, but not JNK, P38 MAPK or STAT 1, is translocated to the nucleus and able to bind to DNA in tenocytes following TNFα and IL-1ß stimulation, suggesting this signalling cascade may be responsible for the adverse downstream consequences of these inflammatory cytokines. We suggest a superior approach for treatment of tendinopathy may therefore be to target specific signalling pathways such as NF-κB.

A combination of omega-3 and exercise reduces experimental Achilles tendinopathy induced with a type-1 collagenase in rats.

This study aimed to evaluate the effectiveness of omega-3 supplementation with exercise in a collagenase-induced Achilles tendinopathy (AT) rat model. Experimental groups (healthy control (HC), AT, exercise (Ex), omega-3 (W), and Ex+W) were randomly allocated. After a week of adaptation, oral omega-3 was initiated for 8 weeks (5 days/week). The exercise groups performed treadmill running for 30 min/day (5 days/week, 20 m/min, 8 weeks) following one week of adaptation (10 m/min, 15 min/day). Matrix metalloproteinase-13 (MMP-13), interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and total antioxidant-oxidant status (TAS) levels were determined in serum samples. Tendon samples were obtained for biomechanical, histopathological, and immunohistochemical assessments. Ultimate tensile force, yield force, stiffness values, collagen type-I alpha 1 expression, and serum TAS significantly decreased (P < 0.05) in AT vs. HC. These values and expression significantly increased in the Ex+W group vs. AT. Serum MMP-13, IL-1ß, and TNF-α levels decreased in all treatment groups vs. AT. The most significant decrease was found in the Ex+W group (P < 0.01). Histopathologically, the improvement in degeneration was statistically significant in the Ex+W group (P < 0.05). Immunohistochemically, MMP-13, IL-1ß, TNF-α, and nitric oxide synthase-2 expression was decreased in all treatment groups vs. AT. In conclusion, omega-3 and exercise might be recommended in AT patients.

Evaluating the role of type 2 diabetes mellitus in rotator cuff tendinopathy: Development and analysis of a novel rat model.

Objective: To establish and validate an intact rotator cuff rat model for exploring the pathophysiological effects of type 2 diabetes on the rotator cuff tendon in vivo. Methods: A total of 45 adult male rats were randomly divided into a control group (n = 9) and type 2 diabetes group (n=36). The rats were sacrificed at 2 weeks (T2DM-2w group, n=9), 4 weeks (T2DM-4w group, n=9), 8 weeks (T2DM-8w group, n=9), and 12 weeks (T2DM-12w group, n=9) after successful modeling of type 2 diabetes. Bilateral shoulder samples were collected for gross observation and measurement, protein expression(enzyme-linked immunosorbent assay,ELISA), histological evaluation, biomechanical testing, and gene expression (real-time quantitative polymerase chain reaction, qRT-PCR). Results: Protein expression showed that the expression of IL-6 and Advanced glycation end products (AGEs)in serum increased in type 2 diabetic group compared with the non-diabetic group. Histologically, collagen fibers in rotator cuff tendons of type 2 diabetic rats were disorganized, ruptured, and with scar hyperplasia, neovascularization, and extracellular matrix disturbances, while Bonar score showed significant and continuously aggravated tendinopathy over 12 weeks. The biomechanical evaluation showed that the ultimate load of rotator cuff tendons in type 2 diabetic rats gradually decreased, and the ultimate load was negatively correlated with AGEs content. Gene expression analysis showed increased expression of genes associated with matrix remodeling (COL-1A1), tendon development (TNC), and fatty infiltration (FABP4) in tendon specimens from the type 2 diabetic group. Conclusion: Persistent type 2 diabetes is associated with the rupture of collagen fiber structure, disturbance in the extracellular matrix, and biomechanical decline of the rotator cuff tendon. The establishment of this new rat model of rotator cuff tendinopathy provides a valuable research basis for studying the cellular and molecular mechanisms of diabetes-induced rotator cuff tendinopathy.

Hyaluronic Acid Alleviates Oxidative Stress and Apoptosis in Human Tenocytes via Caspase 3 and 7.

Rotator cuff tendinopathy (RCT) is the primary reason for shoulder surgery and its clinical management is still challenging. Hyaluronic acid (HA) has been shown to have anti-inflammatory effects in vitro and in vivo under RCT conditions, characterized by an exaggerated oxidative stress (OS). However, molecular mechanisms underlying HA-related effects are still partially disclosed. With these aims, a cell model of RCT was established by exposing primary human tenocytes to H2O2 for up to 72 h. Four different HAs by molecular weight were administered to measure nitric oxide (NO) and OS, apoptosis, and collagen 1 expression. In parallel, the well-known antioxidant ascorbic acid was administered for comparison. The present study highlights that HAs characterized by a low molecular weight are able to counteract the H2O2-induced OS by decreasing the percentage of apoptotic cells and reversing the activation of caspase 3 and 7. Likewise, NO intracellular levels are comparable to the ones of controls. In parallel, collagen 1 expression was ameliorated by HAs characterized by higher molecular weights compared to AA. These findings confirm that HA plays an antioxidant role comparable to AA depending on the molecular weight, and highlight the molecular mechanisms underlying the HA anti-apoptotic effects.

Biomolecules Related to Rotator Cuff Pain: A Scoping Review.

The pathophysiology of pain in patients suffering from rotator cuff (RC) tendinopathy or tears has been examined in various ways. Several molecules from tissue samples taken from the subacromial bursa, supraspinatus tendon, glenohumeral joint fluid, and synovium as well as from peripheral blood have been investigated. This article explores these studies, the assessed biomarkers, and groups their results according to the status of tendon integrity (tendinopathy or tear). Through a structured PubMed database search, 9 out of 658 articles were reviewed. Interleukins, mostly IL-1b and its antagonist, IL-1ra, matrix Metalloproteinases (MMPs), the vascular endothelial growth factor (VEGF) and TNF-a are biomarkers directly searched for correlation to pain level. Most studies agree that IL-1b is directly positively correlated to the degree of pain in patients with RC tendinopathy, especially when the examined sample is taken from the subacromial bursa. VEGF, and TNF-a have been related to shoulder pain preoperatively and TNF-a has also been linked with sleep disturbance. Further studies pointing to more biomarkers taken from the subacromial bursa or tendon directly relating to pain degree are warranted.

Comparative Effects of Exosomes and Ectosomes Isolated From Adipose-Derived Mesenchymal Stem Cells on Achilles Tendinopathy in a Rat Model.

BACKGROUND: Extracellular vesicles derived from mesenchymal stem cells (MSC-EVs) have gained momentum as a treatment for tendinopathy. Multiple studies have demonstrated significant differences in cargo composition between the 2 subtypes of MSC-EVs (ie, exosomes and ectosomes), which may result in different therapeutic effects. However, the effects of the 2 EV subtypes on tendinopathy have not yet been compared. PURPOSE: To compare the effects of adipose stem cell-derived exosomes (ASC-Exos) and ectosomes (ASC-Ectos) on Achilles tendinopathy. STUDY DESIGN: Controlled laboratory study. METHODS: Rats were administered collagenase injections to generate a model of Achilles tendinopathy. A week later, 36 rats were randomly assigned to 3 groups. In each group, Achilles tendons were injected with equal volumes of ASC-Exos, ASC-Ectos, or saline (12 legs/group). The healing outcomes were evaluated by magnetic resonance imaging, histology, immunohistochemistry, transmission electron microscopy, and biomechanical testing at 3 and 5 weeks after collagenase injection. RESULTS: At 3 and 5 weeks, the ASC-Exo group had better histological scores (P = .0036 and P = .0276, respectively), a lower fibril density (P < .0001 and P = .0310, respectively), and a larger collagen diameter (P = .0052 and P < .0001, respectively) than the ASC-Ecto group. At 5 weeks, the expression of collagen type 1 and CD206 in the ASC-Exo group was significantly higher than that in the ASC-Ecto group (P = .0025 and P = .0010, respectively). Regarding biomechanical testing, the ASC-Exo group showed higher failure load (P = .0005), tensile stress (P < .0001), and elastic modulus (P < .0001) than the ASC-Ecto group. CONCLUSION: ASC-Exos had more beneficial effects on tendon repair than ASC-Ectos in a rat model of Achilles tendinopathy. CLINICAL RELEVANCE: Administration of ASC-EVs may have the potential to treat Achilles tendinopathy, and delivery of ASC-Exos could provide additional benefits. It is necessary to compare the healing responses caused by different EV subtypes to further understand their effects on tendinopathy and to aid clinical decision making.

Scleraxis and collagen I expression increase following pilot isometric loading experiments in a rodent model of patellar tendinopathy.

The effect of mechanical load on tendinopathic tissue is usually studied in the context of identifying mechanisms responsible for tendon degradation. However, loading is also one of the most common treatments for tendinopathy. It is therefore possible that different loads result in different cellular responses within a tendon. To test this hypothesis, we first established a rodent model of tendinopathy that has a transcriptional signature similar to human tendinopathy. Tendinopathy was modeled in the rat by producing a lesion in the central core of the patellar tendon using a biopsy punch, followed by two weeks to allow scar formation. We performed 3' Tag RNA-Seq to identify genes that were differentially expressed between the native and scarred rat patellar tendon. Genes involved in extracellular matrix (ECM) structure and turnover were increased, mitochondrial genes were decreased, and there was no inflammatory signature in the tendinopathic tissue. These transcriptional changes phenocopy previously published whole transcriptome analysis in human tendinopathy. After validating the model, the initial response to injury and loading was determined. Two weeks after creation of the patellar tendon lesion, the tendon was loaded using either 4 × 30s isometric or a time-under-tension matched (360 × 0.33s) dynamic protocol. Injured +/- loading and contralateral control tendons were collected eighteen hours after loading, RNA was extracted, and gene expression was quantified using qRT-PCR of the scar with or without loading. The expression of scleraxis and type I collagen increased following isometric loading relative to those loaded dynamically. By contrast, the expression of type II collagen increased in the dynamic samples relative to those loaded isometrically. These data suggest that dynamic loading of a central core tendon injury increases fibrocartilage markers, whereas long isometric loads stimulate markers of tendon regeneration.

Small extracellular vesicles from iPSC-derived mesenchymal stem cells ameliorate tendinopathy pain by inhibiting mast cell activation.

Aim: This study aimed to explore the effect of small extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells (iMSC-sEVs) on acute pain and investigate the underlying mechanisms. Materials & methods: The pathology of tendons was accessed by hematoxylin and eosin staining, immunohistochemical and immunofluorescent staining. The pain degree was measured by pain-related behaviors. In vitro, we performed ß-hexosaminidase release assay, RT-qPCR, toluidine blue staining, ELISA and RNA sequencing. Results: iMSC-sEVs effectively alleviated acute pain in tendinopathy as well as inhibiting activated mast cell infiltration and interactions with nerve fibers in vivo. In vitro, iMSC-sEVs reduced the degranulation of mast cells and the expression of proinflammatory cytokines and genes involved in the HIF-1 signaling pathway. Conclusion: This study demonstrated that iMSC-sEVs relieved tendinopathy-related pain through inhibiting mast cell activation via the HIF-1 signaling pathway.

Mesenchymal Stromal Cells Adapt to Chronic Tendon Disease Environment with an Initial Reduction in Matrix Remodeling.

Tendon lesions are common sporting injuries in humans and horses alike. The healing process of acute tendon lesions frequently results in fibrosis and chronic disease. In horses, local mesenchymal stromal cell (MSC) injection is an accepted therapeutic strategy with positive influence on acute lesions. Concerning the use of MSCs in chronic tendon disease, data are scarce but suggest less therapeutic benefit. However, it has been shown that MSCs can have a positive effect on fibrotic tissue. Therefore, we aimed to elucidate the interplay of MSCs and healthy or chronically diseased tendon matrix. Equine MSCs were cultured either as cell aggregates or on scaffolds from healthy or diseased equine tendons. Higher expression of tendon-related matrix genes and tissue inhibitors of metalloproteinases (TIMPs) was found in aggregate cultures. However, the tenogenic transcription factor scleraxis was upregulated on healthy and diseased tendon scaffolds. Matrix metalloproteinase (MMPs) expression and activity were highest in healthy scaffold cultures but showed a strong transient decrease in diseased scaffold cultures. The release of glycosaminoglycan and collagen was also higher in scaffold cultures, even more so in those with tendon disease. This study points to an early suppression of MSC matrix remodeling activity by diseased tendon matrix, while tenogenic differentiation remained unaffected.

Adipose micro-grafts enhance tendinopathy healing in ovine model: An in vivo experimental perspective study.

In Europe, approximatively 100 000 to 500 000 tendon repairs are performed every year. These procedures are associated with a considerable rate of postoperative complications (from 6% to 11%). Autologous micro-grafts (AAMG) and stromal vascular fraction (SVF) have been shown to improve tendon healing in 60% to 70% of treated rodents. The purpose of this study was to evaluate the effects of AAMG in a sheep model with tendinopathy. We used sheep models because, as a large animal, they are more comparable to humans. The hypothesis was that SVF injection would improve tendon healing compared with the control group, reducing inflammatory and matrix degrading, while increasing anti-inflammatory expression and collagen synthesis in the early stage of tendon injury. Sixteen Apennine sheep aged 2 to 5 years underwent 500 UI type I collagenase injection into both common calcaneal tendons (CCT) to induce tendinopathy. After 15 days (T0), one CCT in every ovine underwent randomly to 2.5 mL of AAMG obtained by mechanical disruption and the contralateral CCTs received no treatment. Clinical, ecographic, and sonographic evaluations were performed after 4 weeks (T1) and 8 weeks (T2). Histological, immunohistochemical, real-time polymerase chain reaction (RT-PCR), and biomechanical evaluations were performed at T2. At T2, the treated group showed a final tendon diameter (9.1 ± 1.4 mm) and a hardness expression (62%) that were similar to the original healthy tendon (8.1 ± 1.1 mm; 100%), with a significant recovery compared with the control group (9.5 ± 1.7 mm; 39%). Moreover, histological analysis of the treated group revealed an improvement in the fiber orientation score, fiber edema score, infiltrative-inflammatory process, and necrosis score (4.3 ± 3.3) compared with control group (8.8 ± 2.9). Immunohistochemically, the treated group showed high expression of collagen 1, Factor VIII and significantly low expression of collagen 3. These data were confirmed by RT-PCR analysis. The study findings suggested that AAMGs obtained through mechanical disruption present a safe, efficient, and reliable technique, enhancing tendon healing.

Spironolactone Ameliorates Senescence and Calcification by Modulating Autophagy in Rat Tendon-Derived Stem Cells via the NF-κB/MAPK Pathway.

Tendinopathy is a disabling musculoskeletal disease, the pathological process of which is tightly associated with inflammation. Spironolactone (SP) has been widely used as a diuretic in clinical practice. Recently, SP has shown anti-inflammatory features in several diseases. Tendon-derived stem cells (TDSCs), a subset cell type from tendon tissue possessing clonogenic capacity, play a vital role in the pathological process of tendinopathy. In the present study, the protective effect of SP on TDSCs was demonstrated under simulated tendinopathy conditions both in vitro and in vivo. SP contributed to the maintenance of TDSC-specific genes or proteins, while suppressing the interleukin- (IL-) 1ß-induced overexpression of inflammation-mediated factors. Additionally, IL-1ß-induced cellular senescence in TDSCs was inhibited, while autophagy was enhanced, as determined via ß-galactosidase activity, western blot (WB), and quantitative real-time polymerase chain reaction analysis. With the aid of several emerging bioinformatics tools, the mitogen-activated protein kinase (MAPK) pathway likely participated in the effect of SP, which was further validated through WB analysis and the use of MAPK agonist. Immunofluorescence analysis and an NF-κB agonist were used to confirm the inhibitory effect of SP on IL-1ß-induced activation of the NF-κB pathway. X-ray, immunofluorescence, immunohistochemistry, hematoxylin and eosin staining, histological grades, and Masson staining showed that SP ameliorated tendinopathy in an Achilles tenotomy (AT) rat model in vivo. This work elucidates the protective role of SP on the pathological process of tendinopathy both in vitro and in vivo, indicating a potential therapeutic strategy for tendinopathy treatment.

A 3D Dynamic In Vitro Model of Inflammatory Tendon Disease.

Three-dimensional (3D) cell cultures combining multipotent mesenchymal stromal cells (MSC), tendon extracellular matrix scaffolds, and mechanical stimulation by a bioreactor have been used to induce tenogenic differentiation in vitro. Yet, these conditions alone do not mimic the environment of acute inflammatory tendon disease adequately, thus the results of such studies are not representatives for tendon regeneration after acute injury. In this chapter, we describe two different approaches to introduce inflammatory stimuli, comprising co-culture with leukocytes and supplementation with the cytokines IL-1 ß and TNF-α. The presented in vitro model of inflammatory tendon disease could be used to study musculoskeletal pathophysiology and regeneration in more depth.

The Effect of Age and Intrinsic Aerobic Exercise Capacity on the Expression of Inflammation and Remodeling Markers in Rat Achilles Tendons.

Old age, adiposity, and metabolic disorders are known as risk factors for chronic tendinopathy, which is a common problem in both athletes and the general population. However, the importance of these influencing factors has not yet been well understood. This study investigated alterations in gene expression and histology of Achilles tendons of young (10 weeks) and old (100 weeks) rats bred for low (low capacity runners, LCR) and high (high capacity runners, HCR) intrinsic aerobic exercise capacity. In this rat model, LCR displayed a phenotype of reduced exercise capacity, higher body weight, and metabolic dysfunctions compared to HCR. We hypothesized that the risk factors for tendinopathy in old LCR could lead to more pronounced impairments in Achilles tendon tissue. In quantitative real-time PCR (qPCR), age-related downregulation of tenocyte markers e.g., tenomodulin, genes related to matrix modeling and remodeling (e.g., collagens, elastin, biglycan, fibronectin, tenascin C) as well as transforming growth factor beta 3 (Tgfb3) have been detected. Inflammation marker cyclooxygenase 2 (Cox2) was downregulated in old rats, while microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in old HCR and old LCR. In all groups, interleukin 6 (Il6), interleukin 1 beta (Il1b), and tumor necrosis factor alpha (Tnfa) showed no significant alteration. In histological evaluation, tendons of old rats had fewer and more elongated tenocyte nuclei than young rats. Even though a higher content of glycosaminoglycans, a sign of degeneration, was found in old HCR and LCR, no further signs of tendinopathy were detectable in tendons of old rats by histological evaluation. Low intrinsic aerobic exercise capacity and the associated phenotype did not show significant effects on gene expression and tendon histology. These findings indicate that aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue and suggests that other risk factors associated with intrinsic aerobic exercise capacity are less influential in this rat model.

Photobiomodulation therapy on expression of HSP70 protein and tissue repair in experimental acute Achilles tendinitis.

The aim of the present study was to investigate the effects of photobiomodulation (PBM) therapy on the expression of heat shock protein 70 (HSP70) and tissue repair in an experimental model of collagenase-induced Achilles tendinitis. Thirty Wistar rats (aged 12 weeks) were randomly distributed among control group (n = 8), tendinitis group (n = 11), and LED group (n = 11). Tendinitis was induced in the tendinitis and LED groups through a peritendinous injection of collagenase (100 µl). The LED group animals received the first irradiation 1 h after injury. A 630 ± 20 nm, 300-mW continuous wave light-emitting diode (LED), spot size 1 cm2, was placed in contact with the skin. One point over the tendon was irradiated for 30 s, delivering 9 J (9 J/cm2). LED irradiation was performed once daily for 7 days, with the total energy delivered being 63 J. The tendons were surgically removed and expression of the HSP70 protein was calculated using semi-quantitative analyses of immunohistochemistry (HSCORE). Number of fibroblasts and amount of collagen were measured using histological and histochemical analyses. An increase in the mean HSCORE for HSP70, in the number of fibroblasts, and in the amount of collagen were found in the LED group compared with those in the tendinitis and control group (P ≤ 0.05). PBM therapy increased the expression of the HSP70, number of fibroblasts, and amount of collagen in the acute Achilles tendinitis in rats.

Moderate and intensive mechanical loading differentially modulate the phenotype of tendon stem/progenitor cells in vivo.

To examine the differential mechanobiological responses of specific resident tendon cells, we developed an in vivo model of whole-body irradiation followed by injection of either tendon stem/progenitor cells (TSCs) expressing green fluorescent protein (GFP-TSCs) or mature tenocytes expressing GFP (GFP-TNCs) into the patellar tendons of wild type C57 mice. Injected mice were subjected to short term (3 weeks) treadmill running, specifically moderate treadmill running (MTR) and intensive treadmill running (ITR). In MTR mice, both GFP-TSC and GFP-TNC injected tendons maintained normal cell morphology with elevated expression of tendon related markers collagen I and tenomodulin. In ITR mice injected with GFP-TNCs, cells also maintained an elongated shape similar to the shape found in normal/untreated control mice, as well as elevated expression of tendon related markers. However, ITR mice injected with GFP-TSCs showed abnormal changes, such as cell morphology transitioning to a round shape, elevated chondrogenic differentiation, and increased gene expression of non-tenocyte related genes LPL, Runx-2, and SOX-9. Increased gene expression data was supported by immunostaining showing elevated expression of SOX-9, Runx-2, and PPARγ. This study provides evidence that while MTR maintains tendon homeostasis by promoting the differentiation of TSCs into TNCs, ITR causes the onset of tendinopathy development by inducing non-tenocyte differentiation of TSCs, which may eventually lead to the formation of non-tendinous tissues in tendon tissue after long term mechanical overloading conditions on the tendon.

Molecular and Structural Effects of Percutaneous Interventions in Chronic Achilles Tendinopathy.

Achilles tendinopathy (AT) is a common problem, especially in people of working age, as well as in the elderly. Although the pathogenesis of tendinopathy is better known, therapeutic management of AT remains challenging. Various percutaneous treatments have been applied to tendon lesions: e.g., injectable treatments, platelet-rich plasma (PRP), corticosteroids, stem cells, MMP inhibitors, and anti-angiogenic agents), as well as percutaneous procedures without any injection (percutaneous soft tissue release and dry needling). In this review, we will describe and comment on data about the molecular and structural effects of these treatments obtained in vitro and in vivo and report their efficacy in clinical trials. Local treatments have some impact on neovascularization, inflammation or tissue remodeling in animal models, but evidence from clinical trials remains too weak to establish an accurate management plan, and further studies will be necessary to evaluate their value.