Tendon Histology Research Articles

Poster 101: Evaluation of Novel Treatment Agents on Rotator Cuff Tendon and Muscle Pathology in a Murine Subacromial Impingement Model

Poster 101: Evaluation of Novel Treatment Agents on Rotator Cuff Tendon and Muscle Pathology in a Murine Subacromial Impingement Model

Pathological Tendon Histology in Early and Chronic Human Patellar Tendinopathy.

The present pilot study investigated the extent of histological tissue changes in both chronic tendinopathy and in individuals that display early clinical signs of tendinopathy. The study included 8 individuals of whom 3 were healthy without any tendon symptoms, 2 had early symptoms (1-2 months), and 3 had chronic symptoms (>3 months) from their patellar tendons. Percutaneous needle biopsy samples were obtained from the affected tendon tissue region. Biopsy samples were stained with Haematoxylin & Eosin, and multiplex immunofluorescence staining was performed for markers of inflammation and resolution. Both early and chronic stage patellar tendon biopsy samples from this small patient cohort exhibited expansion of the interfascicular matrix (IFM) and endotenon regions together with increased cellularity and vascularity. These histological observations were moderate in early tendinopathy, whereas they were more pronounced and associated with marked disruption of tissue architecture in chronic tendinopathy. Early stage tendinopathic patellar tendons expressed markers associated with an activated phenotype of fibroblasts (CD90, CD34), macrophages (S100A8), and endothelial cells (ICAM1, VCAM1). These tissues also expressed enzymes implicated in inflammation (PTGS2, 15PGDH) and resolution (ALOX12) and the proresolving receptor ERV1. Immunopositive staining for these markers was predominantly located in the IFM regions. These preliminary findings suggest that mild to moderate structural histological changes including expansion of IFM and endotenon regions are pathological features of early tendinopathy, and support inflammatory and resolving processes are active in early-stage disease. Further investigation of the cellular and molecular basis of early-stage tendinopathy is required to inform therapeutic strategies that prevent the development of irreversible chronic tendon disease.

The Therapeutic Effect of iMSC-Derived Small Extracellular Vesicles on Tendinopathy Related Pain Through Alleviating Inflammation: An in vivo and in vitro Study.

BackgroundTendinopathy is a common cause of tendon pain. However, there is a lack of effective therapies for managing tendinopathy pain, despite the pain being the most common complaint of patients. This study aimed to evaluate the therapeutic effect of small extracellular vesicles released from induced pluripotent stem cell-derived mesenchymal stem cells (iMSC-sEVs) on tendinopathy pain and explore the underlying mechanisms.MethodsRat tendinopathy model was established and underwent the injection of iMSC-sEVs to the quadriceps tendon one week after modeling. Pain-related behaviors were measured for the following four weeks. Tendon histology was assessed four weeks after the injection. To further investigate the potential mechanism, tenocytes were stimulated with IL-1β to mimic tendinopathy in vitro. The effect of iMSC-sEVs on tenocyte proliferation and the expression of proinflammatory cytokines were measured by CCK-8, RT-qPCR, and ELISA. RNA-seq was further performed to systematically analyze the related global changes and underlying mechanisms.ResultsLocal injection of iMSC-sEVs was effective in alleviating pain in the tendinopathy rats compared with the vehicle group. Tendon histology showed ameliorated tendinopathy characteristics. Upon iMSC-sEVs treatment, significantly increased tenocyte proliferation and less expression of proinflammatory cytokines were observed. Transcriptome analysis revealed that iMSC-sEVs treatment upregulated the expression of genes involved in cell proliferation and downregulated the expression of genes involved in inflammation and collagen degeneration.ConclusionCollectively, this study demonstrated iMSC-sEVs protect tenocytes from inflammatory stimulation and promote cell proliferation as well as collagen synthesis, thereby relieving pain derived from tendinopathy. As a cell-free regenerative treatment, iMSC-sEVs might be a promising therapeutic candidate for tendinopathy.

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.

Effects of low-density pulsed ultrasound treatment on transforming growth factor-beta, collagen level, histology, biomechanics, and function in repaired rat tendons.

Objectives The aim of this study is to compare the effects of low-density pulsed ultrasound (LIPUS) treatment on growth factors/collagen production, histological, biomechanical, and function of rats with Achilles tendon injury. Materials and methods A total of 44 Wistar Albino rats were used in the study between April 2017 and June 2018. The rats were randomized to two treatment groups. Group 1 (n=6) received LIPUS treatment (0.3 Watt/cm2; 1 MHz, 1:5 pulse mode) and Group 2 (n=6)received sham ultrasound (US) treatment following Achilles tendon surgery. Transforming growth factor-beta 1 (TGF-β1) and collagen gene expression levels were evaluated using polymerase chain reaction. The histological evaluation was performed with the Bonar scoring system. The tensile strength was measured by biomechanical testing and the function was evaluated with the Achilles Functional Index (AFI). Results Although TGF-β1 expression and tensile strength evaluation showed a tendency to improve in favor of the LIPUS group, no statistically significant difference was found (p=0.065 and p=0.053, respectively). The COL3 gene expression in the LIPUS group and the COL1 expression in the sham US group were significantly higher. Bonar scores and AFI scores showed a statistically significant improvement in the LIPUS group, compared to the sham US group. Conclusion Our study results show that LIPUS yields positive effects on tendon histology and functional status in repaired Achilles tendon in rats.

Effect Of Mitomycin C in The Prevention of Tendon Adhesion After Surgery and The Effect of Biomechanical Stretching on Tendon Histology

Effect Of Mitomycin C in The Prevention of Tendon Adhesion After Surgery and The Effect of Biomechanical Stretching on Tendon Histology

The pathological features of hip abductor tendon tears \u2013 a cadaveric study

BackgroundThe hip abductors are crucial in maintaining pelvic stability. Tears in these tendons are common and often debilitating. There is uncertainty regarding both the histological and macroscopic features of hip abductor tears. This study aims to clarify both the macroscopic and microscopic features of the tendon and enthesis in hip abductor tendon tears.MethodsThirty-six cadavers with an average age of 81 were dissected, and the hip abductor mechanisms removed en-bloc. The presence, location and size of the tears were recorded and analysed. The samples were processed into histological blocks and viewed using both transmitted and polarised light. Tendon histology was graded using the modified Movin’s score in three sections (deep, middle and superficial layers) and the enthesis graded separately using 5-point criteria. Analysis of variance was used to confirm histological features associated with tears.ResultsTears were found in 24 of 36 samples (67%). The most common finding was an isolated tear in the gluteus minimus (46%), followed by concurrent gluteus medius and gluteus minimus tears (33%). Histology revealed significantly more degeneration in both the tendon (p = 0.0005) and enthesis (p = 0.0011) when tears were present. Furthermore, these changes were concentrated in the deeper layers of the tendon (p = 0.0002) and enthesis (p = 0.003).ConclusionThis study demonstrated degeneration as the primary pathology underlying hip abductor tendon tears. Degenerative changes occur in both the tendon and enthesis, with the deeper layers predominantly affected. These findings are important for guiding surgical repair techniques and to aid the development of novel materials and biologics.

Experimental tendinopathy treatment with SM04755, a topical small molecule inhibitor of the WNT pathway

Purpose: Tendinopathy is an inflammatory and degenerative disorder caused by injuries and/or overuse. Untreated, affected tendons become fibrotic, with micro tears that can lead to pain and rupture. Current therapeutic options treat symptoms and not underlying causes. Stem cell- and growth factor-based treatments are under investigation, but have not established safety or efficacy. The Wnt pathway is upregulated in chronic tendinopathy and involved in inflammation, tenocyte differentiation and fibrosis. SM04755, a novel, topical, small molecule Wnt pathway inhibitor, has previously been shown to inhibit inflammation, reduce fibrosis and increase tenocyte differentiation in nonclinical models (Deshmukh et al. Arth Rheum 2016). Two further experiments are presented: 1. Dose responses of SM04755 in an acute tendinopathy model; 2. A delayed SM04755 treatment model was studied to examine its effects in established tendon injury. Methods: SM04755 was assessed in intra-tendon collagenase-induced rodent Achilles tendinopathy models. Tendinopathy was induced by collagenase injection (500 μg). In the acute dose response model, a single injection of collagenase or sham per animal on Day -4, followed on Day 0 by daily topical delivery of vehicle, 0.3 mg/cm2, or 0.9 mg/cm2SM04755. Achilles tendons were harvested on Days 7, 14, and 21. In the delayed treatment model, collagenase injections were given at Days -28 and -14, followed on Day 0 with daily vehicle or 0.3 mg/cm2 SM04755. Achilles tendons were harvested on Days 7, 14, 21 and 28. Histological analyses were performed by blinded observers and tendons scored based on linearity, tendon cell shape, tendon cell density, inflammation, and hemorrhage (range 5-20). Animal studies were approved by the Samumed, LLC Animal Committee and performed in accordance with the U.S. Department of Agriculture’s Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Statistical analyses used one-way ANOVA for multiple group comparisons and t-tests for comparison between two groups. Results: In the acute dose response model, SM04755 improved tendon health from baseline compared to vehicle as assessed by tendon histology scores. Tendon health scores for vehicle were 10.77 [±1.46], 10.44 [±0.66], and 10.31 [±1.02] at Days 7, 14, and 21, respectively. In the 0.3 mg/cm2 SM04755 dose group, tendon health scores were 12.30 [±0.62] at Day 7 (NS), 10.45 [±1.29] at Day 14 (NS), and 14.37 [±0.82] at Day 21 (P<0.05). In the 0.9 mg/cm2 SM04755 dose group, tendon health scores were 12.22 [±1.02] at Day 7 (NS), 14.57 [±0.41] at Day 14 (P<0.05), and 14.67 [±0.76] at Day 21 (P<0.05) (Fig. 1). In the delayed treatment model, scores for vehicle were 12.35 [±0.30], 10.09 [±0.76], 11.92 [±0.77] and 13.72 [±0.35] at Days 7, 14, 21, and 28, respectively. The 0.3 mg/cm2 dose group scores were 11.86 [±2.13] at Day 7 (NS), 9.44 [±0.48] at Day 14 (NS), 14.61 [±0.77] at Day 21 (P<0.05), and 14.93 [±0.46] at Day 28 (NS) (Fig. 2). Conclusions: In the acute dose response model, the 0.9 mg/cm2 SM04755 dose resulted in improved tendon health from baseline compared to vehicle at both Day 14 and 21. The 0.9 mg/cm2 dose showed statistically meaningful improvement at Day 14 while the 0.3 mg/cm2 dose did not, indicating a faster response at the higher dose. Additionally, 0.3 mg/cm2 SM04755 promoted tendon healing in an established, repeat collagenase, tendon injury model with delayed intervention. Limitations of the collagenase model were absent fibrotic changes, allowing spontaneous healing in the vehicle group by Day 28, possibly reducing effect size. Chronic tendinopathy currently has limited therapeutic options, which alleviate symptoms, mainly pain. SM04755 demonstrated accelerated improvement of tendon histology compared to vehicle in acute and established injury models, suggesting that topically applied SM04755 has potential as a therapeutic intervention for tendinopathy. Clinical studies are planned.

Rat supraspinatus tendon responds acutely and chronically to exercise.

The objective of this study was to identify acute responses and chronic adaptations of supraspinatus tendon to noninjurious exercise. We hypothesized that chronic exercise (EX) increases tendon mechanical properties, and a single exercise bout increases matrix metalloproteinase (MMP) activity acutely. Rats were divided into acute or chronic EX or cage activity groups. Animals in acute EX groups were euthanized, 3, 12, 24, 48, or 72 h upon completion of a single bout of exercise (10 m/min, 1 h) on a flat treadmill. Animals in chronic EX groups walked on a flat treadmill for 3 days or 1, 2, or 8 wk. Tendon histology, MMP activity, and mechanics were measured. A single bout of exercise trended toward reducing tendon mechanical properties, but 2 or 8 wk of chronic EX increased tendon mechanics. Cell density was not affected. Cells became rounder with chronic EX. All tendons were highly organized. MMP activity decreased after a single bout of exercise and returned to baseline by 72 h. MMP activity decreased after 8 wk of chronic EX. Decreased MMP activity may indicate an anabolic instead of catabolic response in contrast to injury. Results suggest that mild, acute decreases in MMP activity and tendon mechanics following a single exercise bout lead to enhanced tendon mechanical adaptations with repeated exercise bouts. This study defines acute and chronic changes of MMP activity, mechanical properties, and histology of the rat supraspinatus tendon in response to beneficial exercise and proposes a mechanism by which acute responses translate to chronic adaptations.NEW & NOTEWORTHY The line between beneficial exercise and overuse has not been elucidated. This study defines the acute and chronic temporal response to exercise of supraspinatus tendon in an in vivo model. We found that decreased matrix metalloproteinase activity and tendon mechanics after a single bout of exercise are followed by beneficial chronic adaptations of the tendon with repeated bouts. How the acute responses to exercise lead to chronic adaptations may distinguish beneficial exercise from overuse.

Low-Level Laser Therapy and Cryotherapy as Mono- and Adjunctive Therapies for Achilles Tendinopathy in Rats.

Low-level laser therapy (LLLT) and cryotherapy are widely used treatments in the acute phase of tendon injury. The aim of this study was to investigate the interaction of these two treatments on tendon inflammation and mechanical properties. Six groups of six Wistar rats were used in this study. The Achilles tendons of the healthy control group were not subjected to injury or treatment. The tendons of the injured nontreated group (ING) were injured, but not treated. The remaining four groups were injured and subjected to LLLT, cryotherapy, LLLT first/cryotherapy, or cryotherapy first/LLLT. All treatments were performed at 1 h post-trauma. Inflammatory mediators, tendon histology, and biomechanical properties were assessed at 24 h post-trauma by comparing the treatment groups with the ING. In all treatment groups, the inflammatory process shifted in an anti-inflammatory direction compared with the ING. Significant alterations in cytokine expression were found in only the LLLT group (↓IL-1β) and the combined intervention groups (↓IL-1β, ↓TNF-α, ↑IL-6). It was also found that cryotherapy followed by LLLT was the only treatment that significantly (p < 0.05) improved the biomechanical parameters of force (N) and displacement (mm) at the tendon rupture and corresponded with the best histological scores of all of the treatment groups. Our results demonstrate that cryotherapy in combination with LLLT can produce an anti-inflammatory "add-on" effect. The order of therapy administration seems essential, as superior histology and biomechanical results were found in the cryotherapy first/LLLT group.

The neurohistology ofpainful and pain-free rotator cuff tendons: a case control study

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S420 756 THE NEUROHISTOLOGY OFPAINFUL AND PAIN-FREE ROTATOR CUFF TENDONS: A CASE CONTROL STUDY B.J. Dean, S.L. Franklin, R.J. Murphy, R. Benson, K. Javaid, A.J. Carr. Univ. of Oxford, Oxford, United Kingdom Purpose:Our main purpose was to compare the tendon neurohistology in two groups of patients; one with significant pain prior to undergoing SAD and one with resolved pain over 6 years post SAD. Methods: Supraspinatus tendon specimens were obtained using an ultrasound guided biopsy technique from 9 patients with painful RCT resistant to conservative management (painful group) and 9 pain-free patients at over 6 years (median 6 years 11 months) following SAD (pain-free group). Pain symptoms were measured using the validated Oxford Shoulder Score (OSS). Structural tendon integrity was assessed ultrasonographically. The tendon tissue was analysed using basic histological techniques (Haematoxylin and Eosin, and Alcian Blue) and Immunohistochemistry. Image analysis was performed by two blinded observers using Image-J to quantify the amount of 3,3’-diaminobenzidine staining present. Isotype controls were also processed. Mann-Whitney U tests were carried out using SPSS with significance levels set at a minimum of p < 0.05. Results: The groups were similar in terms of age, sex and structural tendon abnormality. The painful group consisted of 7 males and 2 females, the pain-free group of 6 males and 3 females. The mean age of the painful group was 50 years (range 38 to 61) and that of the pain-free group was 53 years (range 39 to 65). The median OSS in the painful group was 32 (range 23 to 34) and this was significantly lower (p 1⁄4 0.0002) than the median OSS in the pain-free group (all 48). There were two partial thickness tears in both groups and no full thickness tears. The modified Bonar scores in the painful and the pain-free groups were comparable. There were no significant differences between groups in terms of cellularity, vascularity, proliferation and hypoxia induciblefactor 1a expression. The leucocyte count (CD45 positive cells) and macrophage count (CD68 positive cells) were increased in the painful group versus pain-free (p 1⁄4 0.01 and 0.05 respectively). The expression of the metabotropic glutamate receptor 7 (mGluR7) was reduced in the painful group versus pain-free (p 1⁄4 0.008 and 0.002 respectively). PGP 9.5 (a nerve marker) expression was increased in the painful group versus pain-free (p 1⁄4 0.008). There were no significant differences in glutamate, the inotropic glutamate receptor (NMDAR1) and the metabotropic glutamate receptors (mGluR1, 2 and 5) between groups. Conclusions: This study has shown that specific characteristics of tendon histology are associated with a resolution of shoulder pain over six years following SAD. This provides strong evidence that the rotator cuff tendon is of key importance in the symptomatology of RCT. The mechanism behind these tendon differences remains unclear. These findings are novel and improve our understanding of pain in RCT, and may help provide novel therapeutic targets. 757 NEGATIVE CORRELATION BETWEEN THE POPULATION OF CD105 POSITIVE SUBSET OF MONOCYTES/MACROPHAGES IN SYNOVIAL FLUID AND THE SEVERITY OF JOINT PAIN AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION SURGERY K. Nakamura y, K. Tsuji z, H. Katagiri y, M. Inoue x, K. Abula y, I. Sekiya k, T. Muneta y. yDept. of Joint Surgery and Sports Med., Tokyo Med. and Dental Univ., Tokyo, Japan; zDept. of Cartilage Regeneration, Tokyo Med. and Dental Univ., Tokyo, Japan; xDept. of Plastic Reconstructive and Aesthetic Surgery, Tokyo Med. and Dental Univ., Tokyo, Japan; kCtr. for Stem Cell and Regenerative Med., Tokyo Med. and Dental Univ., Tokyo,

Novel procedure for high‐fidelity tendon histology

A novel method to obtain high-quality histologic sections of rat tendon for light microscopic study is presented. This approach utilizes non-deleterious dehydrating and clearing solvents and a hydrophobic acrylic (methyl methacrylate) resin. This methodology avoids processing and microtomy artifacts common with routine paraffin wax techniques and overcomes specimen size limitations associated with hydrophilic (e.g., glycol methacrylate) resin histologic methods. These novel histologic processing techniques facilitate the reliable assessment of tendons' cellular and matrix components and can be readily adapted to morphologic studies of damage, healing, and repair.

Histologic determination of ontogenetic patterns and processes in hadrosaurian ossified tendons

ABSTRACT Development and metaplasia of ossified tendons in two hadrosaurine dinosaurs (Maiasaura peeblesorum and Brachylophosaurus canadensis) are described by comparison with the known developmental processes of ossified tendons in turkeys (Meleagris gallopavo). Mineralized primary tendon tissue and replacement patterns suggest that ossified tendons in hadrosaurs grew initially in a manner similar to those in turkeys. That is, biomineralization begins with apatite deposition and is followed by resorption of mineralized primary tissue and subsequent bone formation. Earlier ontogenetic onset of tendon ossification and later offset among ossified tendons of hadrosaurs differentiates them from tendons of turkeys. In addition, an external fundamental system (EFS) in hadrosaurian tendons indicates that they grew radially beyond the original tendon boundary, a process unknown among avian tendons. Tendon histology indicates that hadrosaurian ossified tendons developed in step with the skeletal system and that ossification was not induced by biomechanical stresses.