Phase Of Tendon Healing Research Articles

Extracellular Vesicles From Primed Adipose-Derived Stem Cells Enhance Achilles Tendon Repair by Reducing Inflammation and Promoting Intrinsic Healing.

Achilles tendon rupture is a common sports-related injury. Even with advanced clinical treatments, many patients suffer from long-term pain and functional deficits. These unsatisfactory outcomes result primarily from an imbalanced injury response with excessive inflammation and inadequate tendon regeneration. Prior studies showed that extracellular vesicles from inflammation-primed adipose-derived stem cells (iEVs) can attenuate early tendon inflammatory response to injury. It remains to be determined if iEVs can both reduce inflammation and promote regeneration in the later phases of tendon healing and the underlying mechanism. Therefore, this study investigated the mechanistic roles of iEVs in regulating tendon injury response using a mouse Achilles tendon injury and repair model in vivo and iEV-macrophage and iEV-tendon cell coculture models in vitro. Results showed that iEVs promoted tendon anti-inflammatory gene expression and reduced mononuclear cell accumulation to the injury site in the remodeling phase of healing. iEVs also increased collagen deposition in the injury center and promoted tendon structural recovery. Accordingly, mice treated with iEVs showed less peritendinous scar formation, much lower incidence of postoperative tendon gap or rupture, and faster functional recovery compared to untreated mice. Further in vitro studies revealed that iEVs both inhibited macrophage M1 polarization and increased tendon cell proliferation and collagen production. The iEV effects were partially mediated by miR-147-3p, which blocked the toll-like receptor 4/NF-κB signaling pathway that activated the M1 phenotype of macrophages. The combined results demonstrate that iEVs are a promising therapeutic agent that can enhance tendon repair by attenuating inflammation and promoting intrinsic healing.

Suspensory Ligament Injuries: Pathogenesis, diagnosis, treatment and rehabilitation

This paper aims to present the causes, clinical signs, diagnosis and treatment of suspensory ligament injuries in horses. Suspensory ligament damage is a major cause of lameness in sports horses. The suspensory ligament is a very strong anatomical structure – an average force of 1338 kg is needed to damage the suspensory apparatus. The diagnosis of lameness is complex, starting with anorthopedic examination and ending with imaging diagnostics. Treatment of damage to the suspensory ligament depends on the phase of tendon healing. Controlled exercise programmes with ultrasound monitoring are the gold standard for horses to return to the pre-injury level of activity.

Physical therapy and precision rehabilitation in shoulder rotator cuff disease.

To describe the main features of the rehabilitation protocols for RC disease based on the phases of tendon healing and to investigate about the use of wearable devices as monitoring systems. We performed a comprehensive search of PubMed, Medline, Cochrane, CINAHL, and EMBASE databases using various combination of the keywords "rotator cuff," "rotator cuff tear," "rehabilitation protocol," "accelerated," "conservative," "stiffness," "exercises," and "wearable devices." All articles concerning precision orthopaedic rehabilitation therapy in rotator cuff disease were retrieved. To date, rehabilitation protocols are not comparable due to wide heterogeneity of RC diseases. Wearable technologies are becoming a revolutionary tool for movement and posture monitoring. Rehabilitation protocols for RC disease should be tailored on the basis of the different phases of tendon healing. Wearable devices hold the promise to offer a new outlook for long-term follow-up during the postoperative period providing information to the clinician about patient's status.

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing.

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.

Radiofrequency preserves histoarchitecture and enhances collagen synthesis in experimental tendon injury.

We investigated the action of radiofrequency (RF) on the healing process after inducing experimental lesions of the Achilles tendon in rats. Wistar rats were surgically subjected to bilateral partial transverse sectioning of the Achilles tendon. The right tendon was treated with radiofrequency (RFT), whereas the left tendon served as a control (CT). On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each, which were treated with monopolar radiofrequency (Tonederm™) until they were sacrificed on the 7th, 14th or 28th days. The histological specimens were studied for inflammatory cell content, collagen types I and III, immunostaining and morphometry. Total collagen were biochemically analyzed and to evalute fibroblast and myofibroblast proliferation by vimentin and α-actin smooth muscle immunohistochemistry methods. Statistical analysis was performed using the Student's t-test, the sign test and the Kruskal-Wallis test to compare tendons treated with radiofrequency with the non-treated tendons (α=5%; α=10%). Larger amounts of collagen I with hydroxyproline content and myofibroblast cells were clearly evident within 7 days (p<0.05). No difference was observed in the inflammatory cell content between the groups. We found better collagen arrangement with RF administration across the entire time studied. Radiofrequency administration preserves histoarchitecture and enhances collagen synthesis during the initial phases of cicatrization, suggesting that the treatment can provide improved stiffness during the most vulnerable phases of tendon healing. Clinical studies may include RF among the therapeutic tools in tendinous lesion management.

Acupuncture increases the diameter and reorganisation of collagen fibrils during rat tendon healing.

Our previous study showed that electroacupuncture (EA) increases the concentration and reorganisation of collagen in a rat model of tendon healing. However, the ultrastructure of collagen fibrils after acupuncture is unknown. To assess the effect of acupuncture protocols on the ultrastructure of collagen fibrils during tendon healing. Sixty-four rats were divided into the following groups: non-tenotomised (normal group), tenotomised (teno group), tenotomised and subjected to manual acupuncture at ST36 (ST36 group), BL57 (BL57 group) and ST36+BL57 (SB group) and EA at ST36+BL57 (EA group). The mass-average diameter (MAD) and the reorganisation of collagen fibril diameters were determined during the three phases of tendon healing (at 7, 14 and 21 days). The MAD increased during the three phases of healing in the SB group. In the EA group, MAD increased initially but was reduced at day 21. The reorganisation of collagen fibrils was improved in the EA and SB groups at days 14 and 21, respectively. EA at day 21 appeared to reduce the reorganisation. These results indicate that the use of EA up to day 14 and manual acupuncture at ST36+BL57 up to day 21 improve the ultrastructure of collagen fibrils, indicating strengthening of the tendon structure. These data suggest a potential role for acupuncture in rehabilitation protocols.

Macrophage Sub-Populations and the Lipoxin A4 Receptor Implicate Active Inflammation during Equine Tendon Repair

Macrophages (Mϕ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mϕ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3–6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mϕ sub-populations were quantified based on surface antigen expression of CD172a (pan Mϕ), CD14highCD206low (pro-inflammatory M1Mϕ), and CD206high (anti-inflammatory M2Mϕ) to assess potential polarised phenotypes. In addition, the Lipoxin A4 receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mϕ and FPR2/ALX. In contrast, M1Mϕ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mϕ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mϕ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A4 (LXA4) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E2. Stimulation with either mediator induced LXA4 release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mϕ.

Tendinose und Ruptur der Achillessehne

Tendinosis of the Achilles tendon is a degenerative-reparative structural change of the tendon with microdefects, increases in cross-section due to cicatricial tendon regeneration, neoangiogenesis and reduction of elasticity. The previously used term tendinitis is only rarely used for the chronic form since signs of inflammation such as redness and hyperthermia or elevated levels of inflammatory parameters on laboratory testing are generally absent. Duplex sonography with visualization of the neovascularization has become a valuable supplement not only for diagnostics but also for therapy planning. The classic, conservative therapy for painful tendinosis consists of oral anti-inflammatory drugs, pain-adapted load reduction, raising the heel, stretching the calf musculature, and various physiotherapeutic interventions. When conservative treatment over a period of 4 - 6 months fails to produce any or non-adequate pain relief, an indication for surgical treatment should be considered. In the therapy for fresh ruptures of the Achilles tendon further developments in minimally invasive techniques have led to a worldwide paradigm change over the past 10 years. The decisive advantage of minimally invasive surgical techniques is the lower risk of wound infection as compared to the sutures of the open technique. When compared with conservative functional therapy the minimally invasive repair has the advantage of being less dependent on the compliance of the patient since, in the early phase of tendon healing the suture prevents a separation of the tendon ends upon controlled movements. However, not every patient with a ruptured Achilles tendon should be treated with a minimally invasive repair. Open tendon reconstruction and functional conservative therapy are still justified when the correct indication is given.

The use of Vicryl™ in extensor tendon repairs

Conventionally, tendons are repaired with the strongest possible suture material, usually nonabsorbable in a variety of techniques using two or more strands between the tendon ends. In the initial phase of tendon healing, the repair is almost entirely dependent on the strength of the suture used and sound tendon suturing techniques. While this holds true for the strong flexor tendon system, we question the validity of this approach in the case of extensor tendons. Many of these are flat, and anecdotal evidence suggests that nonabsorbable sutures tend to promote a foreign-body reaction and cause increased tendon tethering. In this article, we seek to confirm that absorbable sutures such as Vicryl™ constitute a superior repair material for extensor tendons.

Gene expression changes in SNAP-stimulated and iNOS-transfected tenocytes—expression of extracellular matrix genes and its implications for tendon-healing

Nitric oxide (NO) has a variety of physiological roles, including acting as a key mediator in various phases of tendon healing, but its importance as a modulator of gene expression during tendon healing has not been well studied. The current study used microarray analysis to elucidate global gene expression after transfection with inducible nitric oxide synthase (iNOS) in tenocytes isolated from the injured rotator cuff tendons of human patients. We show that the expression of a wide range of genes is affected by NO, with many activated genes having known roles in healing. Of particular significance is that NOS overexpression stimulates the transcription and translation of a range of extracellular matrix genes important to the structure of connective tissues such as tendons, including collagen Ialpha1, collagen IIIalpha1, collagen IValpha5, biglycan, decorin, laminin, and matrix metalloproteinase 10 (MMP10). These genes were also shown to respond to stimulation by the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) in a dose-dependent manner. We further show that varying levels of NO significantly affect cellular adhesion in tenocytes, a critical process during tendon repair. These findings will be of use when optimizing the dose of NO delivery in further work investigating NO as potential treatment of tendon injuries.

1048. Gene-Enhanced Mesenchymal Stem Cells for the Treatment of Tendinitis

Top of pageAbstract INTRODUCTION IGF-I improves tendon healing in vitro.1 Exogenous IGF-I improves healing of tendinitis lesions.2 Autologous mesenchymal stem cells (MSCs) provide a pool of pluripotent cells to enhance tendinitis repair. This study combined these two modalities by examining the therapeutic use of MSCs overexpressing IGF-I ligand. METHODS An adenoviral vector containing IGF-I (AdIGF-I) was assessed in MSCs to determine the optimal dose. MSC monolayers were transduced with AdIGF-I: 100, 250, 500, 750, and 1000 MOI. Monolayers were harvested on day 6 for analysis of IGF-I, collagen type I, and 18s mRNA. Two optimal viral doses were then assessed over time. Monolayers were harvested 2, 4, 8, 14, 21 and 28 days following transduction to determine gene expression of IGF-I, collagen type I, and 18S mRNA. Medium samples were utilized to determine IGF-I ligand levels. RESULTS MSCs transduced with adenoviral IGF-I expressed ligand at all experimental viral doses (Fig. 1). The optimal viral dose for enhanced IGF-I ligand production and minimal cytotoxic effects was 750 to 1000 MOI (Table 1). From the initial experiment, these two doses were examined over time. MSC expression of IGF-I message and ligand was significantly elevated in both viral doses, and remained significantly elevated throughout the course of 28 days in culture (Fig. 2). Medium IGF-I analysis confirmed therapeutic ligand levels. Collagen type I message was significantly elevated in the MSC cultures with AdIGF-I 48 hours post transduction. DISCUSSION MSCs produced therapeutic levels of ligand for 14 days following transduction. IGF-I tissue levels lag for 2 weeks following tendinitis injury.3 Utilizing MSCs as a vehicle for gene transfer and lesion healing is a logical therapeutic option; autogenous cell populations reduce immunoreactivity and extend ligand production. Adenoviral gene therapy in vivo avoids exogenous re-supplementation with recombinant IGF-I. Additionally, AdIGF-I transduced MSCs released IGF-I for 2 weeks, correlating to the initial phases of tendon healing. This study indicates AdIGF-I transduced mesenchymal stem cells may improve tendon healing.

882. Adenovirus-Mediated Gene Transfer of Human Growth and Differentiation Factor-5 (GDF-5) into the Healing Rat Achilles Tendon

Top of pageAbstract Growth and differentiation factor-5 (GDF-5) protein is known to induce tendon tissue and to stimulate tendon healing. The hypothesis at the basis of this study was that a local adenoviral GDF-5 transfer leads to continuous transgene expression and accelerates healing of transected Achilles tendons in rats. Adenoviruses efficiently transferred GDF-5 into cultured rat tenocytes in vitro. According to Western blotting, two GDF-5 forms were secreted, with molecular weights consistent with the activated GDF-5 dimer and the proform. About 330 ng of GDF-5 protein / 106 cells per 24 h was produced at 2 weeks after transduction. In vivo the right Achilles tendon of 154 Sprague Dawley rats was transected and 3 × 1010 virus particles encoding for GDF-5 or luciferase were injected into the proximal tendon stump, which was left unsutured. According to RT-PCR, in vivo GDF-5 transgene expression lasted for 2 to 12 weeks. Eight weeks after transduction, the GDF-5-treated specimens were significantly thicker than controls (P=0.048), and biomechanical testing revealed a tendency that they were stronger. Histologically, the regenerates in the GDF-5 group were less highly organized and had fewer collagen type-I fibers at 8 and 12 weeks than controls. In conclusion, this study shows successful GDF-5 transfer into the healing Achilles tendon in rats over a 12-week period. GDF-5 seems to be a good candidate to catalyze and improve the early phase of tendon healing. Delayed tendon differentiation as seen after GDF-5 protien administration is however, not overcome by continuous endogenous GDF-5 expression.

Growth Factors and Gene Transfer with DNA Strand Technique in Tendon Healing

Growth factors are likely to be critically involved in all aspects of tendon healing and many studies, in vitro and in vivo, have tried to define the role of growth factors in this process, and to determine appropriate strategies for their use in tissue engineering to improve the healing of tendons. Gene transfer, with DNA strand technique, may offer the optimal vehicle for growth factor delivery to improve tendon healing. In this article, we will discuss phases of tendon healing, review recent work that has shown the role that growth factors play in this process, describe the potential that experimental gene transfer has in delivering these growth factors to the local wound environment, and outline perspectives for improvement of tendon healing.

Temporal expression of nitric oxide synthase isoforms in healing Achilles tendon

We investigated the temporal expressions of the three nitric oxide synthase (NOS) isoforms by semi-quantitative polymerase chain reaction (PCR) assays and by immunoblot analysis, following Achilles tendon transection in rats. Four days after injury, there were increases in the steady-state levels of mRNA for all three NOS isoforms, with peaks for the inducible isoform (iNOS) (23-fold increase) at day 4, the endothelial isoform (eNOS) (24-fold increase) at day 7 and the neuronal isoform (bNOS) (seven-fold increase) at day 21. The temporal expression of NOS isoforms at a protein level was consistent with the results at the mRNA level. We have previously shown a five-fold increase in the NOS activity, as detected by 3H-arginine to 3H-citrulline conversion, at day 7 post-injury. These findings indicate that all three NOS isoforms are expressed during tendon healing with differential expression patterns during the various phases of tendon healing. These findings may prove clinically relevant with respect to strategies for regulating tendon healing.

Expression of mRNA for Vascular Endothelial Growth Factor at the Repair Site of Healing Canine Flexor Tendon

Neovascularization is an important and prominent feature of tendon healing that contributes to wound repair and potentially to adhesion formation. To define the location of cell populations that recruit and organize the angiogenic response during early healing of flexor tendon, we examined the gene expression pattern of the prototypic angiogenic factor, vascular endothelial growth factor, at and around the tenorrhaphy site in a canine model of flexor tendon repair. In situ hybridization with radiolabeled antisense riboprobes was used to identify tendon cell populations that contribute to the neovascularization process by expressing vascular endothelial growth factor and to relate this cell population to the previously described cell populations that participate in matrix synthesis (express type alpha1(I) collagen) and mitotic renewal (express histone H4). The majority of cells (approximately 67%) within the repair site itself express vascular endothelial growth factor mRNA; however, minimal levels accumulate within cells of the epitenon (approximately 10% of cells; p < 0.0002). By contrast, expression of type alpha1(I) collagen and histone H4 does not differ significantly between the epitenon and the repair site (uniformly approximately 30% of cells). Thus, a gradient of cell populations expressing vascular endothelial growth factor exists in the repairing tendon. These data suggest a potential contribution of cells within the repair site to the organization of angiogenesis during the early postoperative phase of tendon healing.