Rat Achilles Tendon Research Articles

Effects of low molecular weight heparin and rivaroxaban on rat Achilles tendon healing.

This study aims to compare the histopathological and biomechanical effects of low molecular weight heparin (LMWH) and rivaroxaban, which are frequently used in orthopedic surgery for thromboembolic prophylaxis, on rat Achilles tendon healing. In the study, 36 adult, male Sprague Dawley rats weighing between 300 g and 400 g were used. Three groups were formed including 12 rats in each. Achilles tendons of all rats were cut and repaired. Nadroparin calcium was administered subcutaneously for 21 days at a dose of 170 IU AXa to the first group (LMWH group). Rivaroxaban was administered daily at a dose of 3 mg/kg for 21 days as gastric lavage to the second group (rivaroxaban group). The third group was identified as the control group and no medication was administered in this group. At the end of three weeks, tendons extracted from the groups were examined histopathologically and biomechanically. Bonar's and Movin's scores obtained as a result of histopathological examination were statistically significantly higher in the control group (p=0.003 and p=0.004, respectively) (high scores indicate that tendon healing is not sufficient). When type I and type III collagen ratios were examined, type I collagen ratio, which should be found at a higher ratio in mature tendon, was statistically significantly higher in rivaroxaban and LMWH groups compared with the control group (p=0.002). As a result of biomechanical examination, higher mean maximum force values were obtained from the rivaroxaban group compared with the LMWH group (p=0.31). Mean maximum force values obtained from the control group were higher than those obtained from the LMWH group (p=0.03) and the rivaroxaban group (p=0.18). Histopathological examination revealed that both LMWH and rivaroxaban have positive effects on tendon healing. However, the same positive effects were not detected in biomechanical examination.

Collagen Fibers in the Healing Process of Rat Achilles Tendon Rupture Using Different Times of Ultrasound Therapy.

Objective: The aim of this study was to evaluate the organization of collagen fibers in the healing process of rat Achilles tendon rupture using different times of ultrasound therapy (TUS). Approach: Forty Wistar rats were selected. Among these, 32 were submitted to total tenotomy of the calcaneous tendon and divided into 5 groups: control group (CG, n = 8), without tenotomy or any treatment; tenotomy group (n = 8), with tenotomy and without treatment; TUS groups-TUS3 (n = 8), TUS4 (n = 8), and TUS5 (n = 8)-submitted to tenotomy and treated with TUS for 3, 4, and 5 min per effective radiating area (ERA), respectively. The animals were sacrificed on the 12th postoperative day. The tendons were surgically removed for analysis of the collagen fiber organization using the birefringence technique (OR, optical retardation). Results: The collagen fibers exhibited better aggregation and organization in the UST3, TUS4, and TUS5 groups compared with CG (p < 0.05). The TUS5 group had better response rates in intergroup comparison. Innovation: The dose response of therapeutic TUS is influenced by many variables. The scientific evidence to support the dosimetry is insufficient. The application time is an important variable to be considered in TUS. In this study, the longer the application time, the better for organization and aggregation of collagen fibers in the rat tendon. Conclusion: TUS applied for 5 min per ERA presented higher dose response to the organization of collagen fibers in the healing process of rat Achilles tendon rupture.

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily. Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily. Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Interleukin-6 Promotes Proliferation but Inhibits Tenogenic Differentiation via the Janus Kinase/Signal Transducers and Activators of Transcription 3 (JAK/STAT3) Pathway in Tendon-Derived Stem Cells.

BackgroundPrevious studies demonstrated that tendon-derived stem cells (TDSCs) were vital healing cells and that mRNA expression of anti-inflammatory cytokine IL-6 was significantly upregulated in injured tendons. The aim of the present study was to investigate the effects of IL-6 on the TDSCs in vitro.Material/MethodsTDSCs isolated from the Achilles tendons in SD rats were co-cultured with various concentrations of IL-6. Cell proliferation, cell cycle analysis, quantitative real-time PCR, western blotting analysis, and statistical analysis were used in the study.ResultsThe result showed that IL-6 strongly increased proliferation capability, and induced cell cycle activation and transition into G2/M phase from G1 phase in TDSCs. However, IL-6 treatment strongly inhibited gene expression of Scleraxis, Collagen 1, Tenomodulin, Collagen 3, Early Growth Response Protein 1, Decorin, Lumican, Biglycan and Fibromodulin in TDSCs. It also strongly inhibited protein expression of tendon cell markers like scleraxis, collagen 1, collagen 3, and tenomodulin. IL-6 treatment strongly activated the JAK/Stat3 signaling pathway in TDSCs. Furthermore, WP1066, a JAK/Stat3 signaling pathway inhibitor, abrogated the effects of IL-6 on TDSCs.ConclusionsThese findings indicated that IL-6 might exert dual effects on TDSCs in vitro: strongly enhancing their proliferation but inhibiting their tenogenic differentiation via the JAK/Stat3 pathway.

Investigation of the biomechanical and histopathological effects of autologous conditioned serum on healing of Achilles tendon

ObjectiveThe aim of this study to evaluate the effects of autologous conditioned serum (ACS) on the healing of transected rat Achilles tendons via the assessment of biomechanical and histological parameters. MethodsThe study was conducted on 45 male Sprague–Dawley rats. Five rats were used as donors for ACS preparation. Animals were randomly assigned to the experimental or control group. In both groups, the Achilles tendon was cut transversally and then sutured. In the placebo control and ACS-treated groups, saline or ACS, respectively, was injected into the repair zone three times after surgery. Ten rats from each group (ACS group, n = 20; control group, n = 20) were euthanized at days 15 and 30 after surgery for histopathological (n = 5) and biomechanical (n = 5) testing. The histopathological findings were interpreted using the Bonar and Movin scales. Tendon remodelling was evaluated via the immunohistochemical staining of collagen type 3. Biomechanical effects were assessed by tensile testing. ResultsThe Bonar and Movin scale scores were significantly better in the ACS-treated group on both day 15 (p = 0.003 and p = 0.003, respectively) and day 30 (p = 0.005 and p = 0.004, respectively). The immunohistochemical density of collagen type 3 was significantly lower in the ACS-treated group on day 30 (p = 0.018). The type 1/3 collagen ratios of the groups were similar on days 15 and 30, as determined by Sirius Red staining (p = 0.910 and p = 0.133, respectively). In the biomechanical assessment results, the ACS-treated group's maximum load to failure values were significantly higher on day 15 (p = 0.049). ConclusionInjection of ACS had a positive effect on the histopathological healing of rat Achilles tendons on days 15 and 30 and on biomechanical healing on day 15. ACS treatment contributed to lowering the collagen type 3 density by day 30. According to our study, ACS may be favourable for the treatment of human Achilles tendon injuries and tendinopathies.

Effects of maturation and advanced glycation on tensile mechanics of collagen fibrils from rat tail and Achilles tendons

Connective tissues are ubiquitous throughout the body and consequently affect the function of many organs. In load bearing connective tissues like tendon, the mechanical functionality is provided almost exclusively by collagen fibrils that in turn are stabilized by covalent cross-links. Functionally distinct tendons display different cross-link patterns, which also change with maturation, but these differences have not been studied in detail at the fibril level. In the present study, a custom built nanomechanical test platform was designed and fabricated to measure tensile mechanics of individual fibrils from rat tendons. The influence of animal maturity (4 vs. 16 week old rats) and functionally different tendons (tail vs. Achilles tendons) were examined. Additionally the effect of methylglyoxal (MG) treatment in vitro to form advanced glycation end products (AGEs) was investigated. Age and tissue type had no significant effect on fibril mechanics, but MG treatment increased strength and stiffness without inducing brittleness and gave rise to a distinct three-phase mechanical response corroborating that previously reported in human patellar tendon fibrils. That age and tissue had little mechanical effect, tentatively suggest that variations in enzymatic cross-links may play a minor role after initial tissue formation. Statement of significanceTendons are connective tissues that connect muscle to bone and carry some of the greatest mechanical loads in the body, which makes them common sites of injury. A tendon is essentially a biological rope formed by thin strands called fibrils made of the protein collagen. Tendon function relies on the strength of these fibrils, which in turn depends on naturally occurring cross-links between collagen molecules, but the mechanical influence of these cross-links have not been measured before. It is believed that beneficial cross-linking occurs with maturation while additional cross-linking with aging may lead to brittleness, but this study provides evidence that maturation has little effect on mechanical function and that age-related cross-linking does not result in brittle collagen fibrils.

Glycine improves the remodeling process of tenocytes in vitro.

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

Controlled release of collagen-binding SDF-1α from the collagen scaffold promoted tendon regeneration in a rat Achilles tendon defect model

It had been demonstrated that stromal cell-derived factor-1α (SDF-1α) could promote in situ tendon regeneration by recruiting endogenous cells. However, native SDF-1α diffuses too fast in vivo, reducing its local concentration and efficacy. In this study, we prepared a recombinant SDF-1α containing a collagen-binding domain (CBD-SDF-1α) and developed a functional collagen scaffold by tethering CBD-SDF-1α on the collagen scaffold for in situ tendon regeneration. CBD-SDF-1α could induce the migration of mesenchymal stem cells, dermal fibroblasts and Achilles tendon fibroblasts in vitro, and achieve controlled release from the collagen scaffold. In a rat Achilles tendon defect model, the functional scaffold could increase the recruitment of CXCR4 positive fibroblast-like cells and the deposition of Tenascin-C at 7 days after implantation. After 4 and 12 weeks, the functional collagen scaffold could promote the expression of type I collagen, increase the diameters of collagen fibrils and improve the mechanical properties of regenerated tendons. Hence, the functional scaffold increased the efficacy of tendon regeneration by controlling release of SDF-1α, enhancing the recruitment of fibroblast-like cells and providing instructive microenvironment and mechanical support for tendon regeneration. Therefore, CBD-SDF-1α-modified collagen scaffold could serve as a practical application for tendon regeneration.

Bevacizumab Improves Achilles Tendon Repair in a Rat Model

Background/Aims: Effective wound-healing generally requires efficient re-vascularization after injury, ensuring sufficient supply with oxygen, nutrients, and various cell populations. While this applies to most tissues, tendons are mostly avascular in nature and harbor relatively few cells, probably contributing to their poor regenerative capacity. Considering the minimal vascularization of healthy tendons, we hypothesize that controlling angiogenesis in early tendon healing is beneficial for repair tissue quality and function. Methods: To address this hypothesis, Bevacizumab, a monoclonal antibody blocking VEGF-A signaling, was locally injected into the defect area of a complete tenotomy in rat Achilles tendon. At 28 days post-surgery, the defect region was investigated using immunohistochemistry against vascular and lymphatic epitopes. Polarization microscopy and biomechanical testing was used to determine tendon integrity and gait analysis for functional testing in treated vs non-treated animals. Results: Angiogenesis was found to be significantly reduced in the Bevacizumab treated repair tissue, accompanied by significantly reduced cross sectional area, improved matrix organization, increased stiffness and Young’s modulus, maximum load and stress. Further, we observed an improved gait pattern when compared to the vehicle injected control group. Conclusion: Based on the results of this study we propose that reducing angiogenesis after tendon injury can improve tendon repair, potentially representing a novel treatment-option.

EFFECTS OF HIGH-DOSE VITAMIN C AND HYALURONIC ACID ON TENDON HEALING.

ABSTRACTObjective:To assess the histopathologic and biomechanical effects of hyaluronic acid (HA) and high-dose vitamin C (VC) on rat Achilles tendon healing. Methods:Forty-eight Sprague-Dawley rats were randomized to HA and VC and control groups with equal numbers. Each group was further divided into two subgroups to be sacrificed on Day 15 (n=8) and Day 30 (n=8). The Achilles tendons were cut and repaired. While the control rats remained untreated, HA and VC were administered after repair. The repaired tendons were removed for biomechanical and histopathologic analyses. In the biomechanical tests, the tendons were stretched to failure and maximum forces were measured. For histopathologic examination, the specimens were interpreted semiquantitatively using Movin’s grading scale and Bonar scores. Results:The highest mean forces were obtained in the HA group on Day 15 and in the VC group on Day 30, with a significant difference between HA and VC on Day 15 between control and VC on Day 30 (p<0.05). Histological examination showed both Movin and Bonar scores decreased in all groups on Day 30, with significant improvements in the HA and VC groups (p<0.05). Conclusion:Our results demonstrated that both VC and HA had therapeutic effects on tendon healing, especially in the late phase. Level of Evidence I; High quality randomized trial with statistically significant difference.

Sub-failure Stretch Injury Response in Rat Achilles Tendon

Sub-failure ligament and tendon injuries are of great concern to athletes and the general population because they account for a significant proportion of all musculoskeletal injuries per year. The goals of this manuscript were to design and evaluate the performance of a reproducible small animal model for tendon sub-failure injury and investigate the tissue response to a singular, controlled sub-failure stretch damage in rat Achilles tendon matrices using biochemical, biomechanical, and image analyses. Utilizing a new tendon stretch injury device, we showed that sub-failure stretch lead to significant changes in the elastic moduli of the toe region of the stress-strain curves of tendons. Scanning electron microscopy was used to examine the ultrastructural damage and disorganization of the normally aligned collagen matrix of the tendon. Additionally, ex vivo biomechanical testing confirmed the persistence of compromised biomechanical properties for up to 7 days post-injury in the healing Achilles tendon. Furthermore, in vivo magnetic resonance imaging and histological analysis showed strong evidence of the injury during repair. Lastly, immunohistochemical staining revealed increased expression of myofibroblast markers elastin and smooth muscle actin, indicative of matrix remodeling facilitated by migrating tissue fibroblasts. These results support the clinical utility of this new in vivo model for the study of sub-failure injury of tendon. Taken together, these findings provide clinically relevant information about the most common type of ligament and tendon injury. Sub-failure sprain and strain injuries to ligaments and tendons are common injuries of the musculoskeletal system. We set out to design a device that could accurately model the response of a sub-failure tendon stretch injury in rat. Using 3D-printed parts, we developed a new injury device and tested the biological and biomechanical response of stretch injury in rat cadavers and live rats for up to 1 week. Our results showed sub-failure stretch of the Achilles tendon led to changes in the biomechanics, most notably the toe region modulus under tension. We saw persistence of compromised biomechanics for up to 1 week, accompanied by a cellular response led by migrating tendon fibroblasts. Future studies will evaluate the long-term response of the stretch injury in rat, in addition to adapting the device for larger animal models.

Non-uniform displacement and strain between the soleus and gastrocnemius subtendons of rat Achilles tendon.

Achilles tendon (AT) comprises of 3 subtendons arising from the soleus (SOL) and the lateral (LG) and medial (MG) heads of the gastrocnemius muscle. While recent human studies show differential displacement within AT, these displacements have not been attributed to specific subtendons. We tested the hypothesis that the SOL and LG subtendons show differential displacement and strain during various combinations of SOL, LG, and MG excitations. Movement of knots, sutured onto SOL and LG subtendons of 12 Wistar rats, was videotaped, while the muscles were stimulated intramuscularly and ankle torque was assessed. When SOL only was stimulated, the plantar flexion torque was the smallest among the different conditions (P<.001). In this condition, from passive to active state, the displacement (0.57 vs 0.47mm, P=.002) and strain (8.4% vs 2.4%, P<.001) in the SOL subtendon were greater than in LG subtendon. When LG only was stimulated, a higher ankle torque was measured as compared to SOL stimulation (P<.001); the displacement was similar in both subtendons (~0.6mm), while the strain was greater in LG than in SOL (4.7% vs 1.7%, P<.001). When all 3 muscles were stimulated simultaneously, ankle torque was highest and the displacement (0.79 vs 0.74mm, P=.002) and strain (7.7% vs 4.4%, P=.003) were greater in SOL than in LG. These data show that the different subtendons of AT can experience relative displacement and differential strains. Together with anatomical dissections, the results revealed that such uniformities may be due to a lower stiffness of SOL subtendon compared to LG.

Fos Promotes Early Stage Teno-Lineage Differentiation of Tendon Stem/Progenitor Cells in Tendon

Stem cells have been widely used in tendon tissue engineering. The lack of refined and controlled differentiation strategy hampers the tendon repair and regeneration. This study aimed to find new effective differentiation factors for stepwise tenogenic differentiation. By microarray screening, the transcript factor Fos was found to be expressed in significantly higher amounts in postnatal Achilles tendon tissue derived from 1 day as compared with 7‐days‐old rats. It was further confirmed that expression of Fos decreased with time in postnatal rat Achilles tendon, which was accompanied with the decreased expression of multiply tendon markers. The expression of Fos also declined during regular in vitro cell culture, which corresponded to the loss of tendon phenotype. In a cell‐sheet and a three‐dimensional cell culture model, the expression of Fos was upregulated as compared with in regular cell culture, together with the recovery of tendon phenotype. In addition, significant higher expression of tendon markers was found in Fos‐overexpressed tendon stem/progenitor cells (TSPCs), and Fos knock‐down gave opposite results. In situ rat tendon repair experiments found more normal tendon‐like tissue formed and higher tendon markers expression at 4 weeks postimplantation of Fos‐overexpressed TSPCs derived nonscaffold engineering tendon (cell‐sheet), as compared with the control group. This study identifies Fos as a new marker and functional driver in the early stage teno‐lineage differentiation of tendon, which paves the way for effective stepwise tendon differentiation and future tendon regeneration. Stem Cells Translational Medicine 2017;6:2009–2019

Laserterapia para tratamento de tendinopatia induzida em ratos wistar

O objetivo desse estudo foi avaliar os efeitos da laserterapia na lesão do tendão calcanearcomum de ratos Wistar. Foram utilizados 12 ratos machos adultos, distribuídosaleatoriamente em dois grupos (L=laser e C=controle). Todos foram submetidos àtendinopatia unilateral (escolha aleatória) mediante compressão transversal do tendão (10segundos) com pinça Halstead mosquito, assim como escarificações (com bisturi). Após 24horas da indução da lesão os animais do grupo L receberam laser (904 nm/3 J/cm²/9s) por 20dias. Os do grupo C foram manipulados como se fossem receber a radiação. Após 3, 7, 14 e21 dias da realização da lesão, três ratos de cada grupo foram submetidos à eutanásia, e ostendões obtidos para análise histomorfométrica. As amostras foram processadas como derotina e os fragmentos corados com hematoxilina-eosina, tricrômico de Masson e PicrosiriusRed. Os dados foram analisados por ANOVA, a 5% de probabilidade e análise de regressão.Não houve diferença entre grupos nem entre tempos para as características hemorragia,angiogênese, espessamento do epitendão. Independente do tratamento ocorreu diminuição(p=0,0129) da formação de aderência fibrinosa (do 3o ao 21o dias). Por outro lado, a avaliaçãomorfométrica revelou maiores (p=0,0120) quantidades de fibroblastos no grupo que recebeulaserterapia, não havendo efeito de tempo. Avaliação semiquantitativa, revelou maiores(p=0,0000) quantidades de fibroblastos no grupo tratado, porém nessa análise, a quantidadedessas células aumentou com o tempo (p=0,0001) em ambos os grupos. Diferentemente,ANOVA revelou redução do infiltrado inflamatório do 3o ao 21o dia em ambos os grupos(histologia: p=0,0003; morfometria: p=0,0000). Embora não tenha ocorrido diferença entregrupos na quantidade de fibras colágenas (I e III), a morfometria revelou que os ratos dogrupo L apresentaram maiores (p=0,0096) quantidades de fibras colágenas do que do grupoC. Para essa característica, não ocorreu efeito do tempo, ainda que tenha sido observada maior(p=0,0000) organização da deposição de colágeno (7o dia). A quantidade de fibras colágenasdeterminada por histologia revelou efeito apenas do tempo (p=0,0005), independente dotratamento, ocorrendo aumento dessa variável até o 14o dia, com posterior redução. Alaserterapia iniciada 24 horas após indução cirúrgica de lesão no tendão calcanear de ratosWistar apresenta a vantagem de aumentar em qualidade e quantidade as fibras colágenas,assim como os fibroblastos, células fundamentais na síntese dos colágenos.

Wnt/β-catenin signaling suppresses expressions of Scx, Mkx, and Tnmd in tendon-derived cells.

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

Different gene response to mechanical loading during early and late phases of rat Achilles tendon healing.

Mechanical loading stimulates tendon healing both when applied in the inflammatory phase and in the early remodeling phase of the process, although not necessarily via the same mechanisms. We investigated the gene response to mechanical loading in these two phases of tendon healing. The right Achilles tendon in rats was transected, and the hindlimbs were unloaded by tail suspension. The rats were exposed to 5 min of treadmill running 3 or 14 days after tendon transection. Thereafter, they were resuspended for 15 min or 3 h until euthanasia. The controls were suspended continuously. Gene analysis was first performed by microarray analysis followed by quantitative RT-PCR on selected genes, focusing on inflammation. Fifteen minutes after loading, the most important genes seemed to be the transcription factors EGR1 and C-FOS, regardless of healing phase. These transcription factors might promote tendon cell proliferation and differentiation, stimulate collagen production, and regulate inflammation. Three hours after loading on day 3, inflammation was strongly affected. Seven inflammation-related genes were upregulated according to PCR: CCL20, CCL7, IL-6, NFIL3, PTX3, SOCS1, and TLR2. These genes can be connected to macrophages, T cells, and recruitment of leukocytes. According to Ingenuity Pathway Analysis, the recruitment of leukocytes was increased by loading on day 3, which also was confirmed by histology. This inflammation-related gene response was not seen on day 14 Our results suggest that the immediate gene response after mechanical loading is similar in the early and late phases of healing but the late gene response is different.NEW & NOTEWORTHY This study investigates the direct effect of mechanical loading on gene expression during different healing phases in tendon healing. One isolated episode of mechanical loading was studied in otherwise unloaded healing tendons. This enabled us to study a time sequence, i.e., which genes were the first ones to be regulated after the loading episode.

Characteristics of Sonography in a Rat Achilles Tendinopathy Model: Possible Non-invasive Predictors of Biomechanics

The purpose of this study was to investigate the dynamic changes of histopathology, biomechanical properties, echo intensity, and ultrasound features in a collagenase-induced tendinopathy model of rat Achilles tendons, and to examine the associations among biomechanical properties, echo intensity, and ultrasound features. Forty-two rats received an ultrasound-guided collagenase injection on their left Achilles tendons, and needle puncture on the right ones as the control. At four, eight, and twelve weeks post-injury, the tendons were examined via measurements of their biomechanical properties, histopathological and ultrasonographic characteristics. The injured tendons showed significantly higher histopathological scores, lower Young’s modulus, and higher ultrasound feature scores than the those of control ones throughout the study period. Up to week 12, all injured tendons showed defective healing. The neovascularization score had a significant negative linear association with the failure stress and Young’s modulus. Maximum normalized echo intensity had a significant positive linear association with maximum strain. Therefore, neovascularization and maximum normalized echo intensity are associated with mechanically altered tendinopathic tendons. Non-invasive ultrasound methodology, including echo intensity and ultrasound feature scores, may provide useful information about biomechanical properties of tendinopathic tendons.

Effect of different intensity treadmill training on repair of micro-injured Achilles tendon in rats

To explore the effect of different intensity treadmill training on the repair of micro-injured Achilles tendon induced by collagenase in rats. Seventy-two 8-week-old male Sprague Dawley rats (weighing, 200-250 g) were selected. After adaptive treadmill training for 1 week, rats were injected with 30 μL type I collagenase solution (10 mg/mL) into both Achilles tendons to make micro-injured Achilles tendon models. After 1 week of cage feeding, the rats were randomly divided into 3 groups: the control group, the low-intensity group, and the high-intensity group, 24 rats each group. The rats in control group could move freely, and the rats underwent daily treadmill training at the intensity of 13 m/min and 20 min/d in the low-intensity group and at the intensity of 17 m/min and 60 min/d in the high-intensity group. At immediate, 1 week, and 4 weeks after training, bilateral Achilles tendons were collected from 8 rats of each group for gross observation, histological analysis, and mechanical testing. At immediate after training, there was no significant difference in the gross observation, histological observation, and biomechanical properties of the Achilles tendon between groups ( P>0.05). The gross observation showed connective tissue hyperplasia near Achilles tendon and lackluster tendon in each group at 1 week; hyperplasia significantly reduced in the low-intensity group when compared with the control group, and there were more connective tissue and a large number of neovascularization in the high-intensity group at 4 weeks. At 1 week, there was no significant difference in the semi-quantitative histological total score between groups ( P>0.05), but there were significant differences in vascularity between low-intensity group or high-intensity group and control group ( P<0.05). At 4 weeks, the semi-quantitative histological total score was significantly higher in high-intensity group than control group and low-intensity group ( P<0.05), and in control group than low-intensity group ( P<0.05). There were significant differences in collagen arrangement, cell morphology, abnormal cells, and vascularity between low-intensity group and high-intensity group or control group ( P<0.05). And there was significant difference in abnormal cells between high-intensity group and control group ( P<0.05). The mechanical testing showed that there was no significant difference in cross-sectional area of the Achilles tendon, the ultimate force, tensile strength, and elastic modulus between groups at 1 week ( P>0.05); the low-intensity group was significantly higher than the control group in the ultimate force and the tensile strength ( P<0.05), and than high-intensity group in the ultimate force and elastic modulus ( P<0.05), but no significant difference was found in the other indexes between groups ( P>0.05) at 4 weeks. Low-intensity treadmill training can promote the repair of rat micro-injured Achilles tendon induced by collagenase.

The influence of chronic IL-6 exposure, in vivo, on rat Achilles tendon extracellular matrix

When compared to placebo, acetaminophen (APAP) reduces tendon stiffness and collagen cross-linking. APAP also enhances the exercise-induced increase in peritendinous levels of IL-6. Elevated levels of IL-6 are associated with tendinopathy, thus we hypothesized that chronic, elevated peritendinous IL-6 would alter tendon extracellular matrix (ECM). IL-6 (∼3000pgml−1) was injected (3dwk−1 for 8-wks) into the Achilles peritendinous region of male Wistar rats (n=16) with the opposite leg serving as a sham. Fractional synthesis rates (FSR) were determined using deuterium oxide. Collagen (hydroxyproline) and hydroxylysl pyridinoline (HP) cross-linking were analyzed by HPLC. ECM and IL-6 related genes were evaluated using qRT-PCR. Relative to sham, collagen (Col) 1a1 but not Col3a1 expression was suppressed (47%) in tendons exposed to IL-6 (p<0.05). Lysyl oxidase (LOX) and MMP-1 expression were also reduced (37%) in IL-6 treated tendons (p<0.05). Relative to sham the expression of MMP-2, -3, -9, and TIMP-1 were not altered by IL-6 treatment (p>0.05). Interleukin-6 receptor subunit beta precursor (IL6st) was lower (16%) in IL-6 treated tendons when compared to sham (p<0.05). Suppressor of cytokine signaling 3 (Socs3), signal transducer and activator of transcription 3 (STAT3), and protein inhibitor of activated STAT 1 (Pias1) were not altered by IL-6 exposure (p>0.05). Neither collagen nor cross-linking content were altered by IL-6 (p>0.05). Additionally, IL-6 treatment did not alter tendon FSR. Chronic treatment with physiologically relevant levels of IL-6 suppresses expression of Col1a1 and LOX while also altering expression of select MMPs but does not alter Achilles tendon collagen synthesis.