Rat Achilles Tendon Research Articles

Micro-groove patterned PCL patches with DOPA for rat Achilles tendon regeneration

Achilles tendon regeneration using current tissue engineering techniques requires morphometrically mimetic tendon biomaterials to attain the required microstructural tendon tissue and to achieve the necessary mechanical properties. In this study, we used nano- (400 nm) and micro-groove (5000 nm) patterned polycaprolactone patches (PCL-400, PCL-5000), which offer adequate elastic modulus and biocompatibility. The PCL patches were fabricated via capillary force lithography and were surface modified with 3,4-dihydroxy-l-phenylalanine (DOPA) for increased hydrophilicity. We hypothesized that the resulting biologically surface-modified elastic groove patterned patches would enhance the cell behaviors in vitro and the rat Achilles tendon regeneration in vivo. The micro-groove patterned PCL patches with DOPA coating (DOPA-PCL-5000) exhibited excellent cell elongation, tenogenic differentiation and YAP expression of the mesenchymal stem cells in our in vitro study and these results were further confirmed by application to the rat Achilles tendon rupture model in our in vivo study. DOPA-PCL-5000 induced dramatic regeneration of rat Achilles tendon compared to the other groups, which was further confirmed by the Achilles functional index and histological analysis at 8 weeks. In conclusion, DOPA-PCL-5000 can be used not only for damaged Achilles tendon, but also for various functional tendon regeneration techniques such as rotator cuff, patellar tendon, and flexor tendon.

Effects of hypoxia on Achilles tendon repair using adipose tissue-derived mesenchymal stem cells seeded small intestinal submucosa

BackgroundThe study was performed to evaluate the feasibility of utilizing small intestinal submucosa (SIS) scaffolds seeded with adipose-derived mesenchymal stem cells (ADMSCs) for engineered tendon repairing rat Achilles tendon defects and to compare the effects of preconditioning treatments (hypoxic vs. normoxic) on the tendon healing.MethodsFifty SD rats were randomized into five groups. Group A received sham operation (blank control). In other groups, the Achilles tendon was resected and filled with the original tendon (Group B, autograft), cell-free SIS (Group C), or SIS seeded with ADMSCs preconditioned under normoxic conditions (Group D) or hypoxic conditions (Group E). Samples were collected 4 weeks after operation and analyzed by histology, immunohistochemistry, and tensile testing.ResultsHistologically, compared with Groups C and D, Group E showed a significant improvement in extracellular matrix production and a higher compactness of collagen fibers. Group E also exhibited a significantly higher peak tensile load than Groups D and C. Additionally, Group D had a significantly higher peak load than Group C. Immunohistochemically, Group E exhibited a significantly higher percentage of MKX + cells than Group D. The proportion of ADMSCs simultaneously positive for both MKX and CM-Dil observed from Group E was also greater than that in Group D.ConclusionsIn this animal model, the engineered tendon grafts created by seeding ADMSCs on SIS were superior to cell-free SIS. The hypoxic precondition further improved the expression of tendon-related genes in the seeded cells and increased the rupture load after grafting in the Achilles tendon defects.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

CD146 Delineates an Interfascicular Cell Sub-Population in Tendon That Is Recruited during Injury through Its Ligand Laminin-α4

The interfascicular matrix (IFM) binds tendon fascicles and contains a population of morphologically distinct cells. However, the role of IFM-localised cell populations in tendon repair remains to be determined. The basement membrane protein laminin-α4 also localises to the IFM. Laminin-α4 is a ligand for several cell surface receptors, including CD146, a marker of pericyte and progenitor cells. We used a needle injury model in the rat Achilles tendon to test the hypothesis that the IFM is a niche for CD146+ cells that are mobilised in response to tendon damage. We also aimed to establish how expression patterns of circulating non-coding RNAs alter with tendon injury and identify potential RNA-based markers of tendon disease. The results demonstrate the formation of a focal lesion at the injury site, which increased in size and cellularity for up to 21 days post injury. In healthy tendon, CD146+ cells localised to the IFM, compared with injury, where CD146+ cells migrated towards the lesion at days 4 and 7, and populated the lesion 21 days post injury. This was accompanied by increased laminin-α4, suggesting that laminin-α4 facilitates CD146+ cell recruitment at injury sites. We also identified a panel of circulating microRNAs that are dysregulated with tendon injury. We propose that the IFM cell niche mediates the intrinsic response to injury, whereby an injury stimulus induces CD146+ cell migration. Further work is required to fully characterise CD146+ subpopulations within the IFM and establish their precise roles during tendon healing.

Comparative analysis of Achilles tendon healing outcomes after open tenotomy versus percutaneous tenotomy: An experimental study in rats.

There is growing interest in conservative treatment of Achilles tendon rupture. However, the majority of experimental studies of Achilles tendon have been performed by open tenotomy. More appropriate model of conservative treatment of Achilles tendon rupture is required. We performed an experimental study to evaluate whether outcomes differ between open tenotomy and percutaneous tenotomy of the Achilles tendon in rats. The Achilles tendons of 48 rats were transected. The animals were divided into two groups according to surgical technique: open tenotomy or microscopy-assisted percutaneous tenotomy. After 1, 2, and 4 weeks, functional, biomechanical, and histological analyses were performed. Western blot was performed for quantitative molecular analysis at 1 week. The Achilles functional index was superior in the percutaneous tenotomy group, compared with the open tenotomy group, at 1 week. The cross-sectional area was significantly larger in the percutaneous tenotomy group than in the open tenotomy group at 4 weeks. Relative to the native tendons, load to failure and stiffness yielded comparable results at 2 weeks in the percutaneous tenotomy group and at 4 weeks in the open tenotomy group. The histological score was significantly better in the percutaneous tenotomy group than in the open tenotomy group at 1 week. At 1 week, interleukin-1β expression in the open tenotomy group was higher than in the percutaneous tenotomy group. In summary, Achilles tendon healing was substantially affected by the tenotomy method. We presume that our percutaneous tenotomy method might constitute a useful experimental animal model for conservative treatment of Achilles tendon rupture.

Altered Differentiation of Tendon-Derived Stem Cells in Diabetic Conditions Mediated by Macrophage Migration Inhibitory Factor.

The purpose of our study was to evaluate the role of macrophage migration inhibitory factor (MIF) in the differentiation of tendon-derived stem cells (TdSCs) under hyperglycemic conditions. In the in vivo experiment, rats were classified into diabetic (DM) and non-DM groups depending on the intraperitoneal streptozotocin (STZ) or saline injection. Twelve-week after STZ injection, the supraspinatus tendon was harvested and prepared for histological evaluation and real-time reverse transcription polymerase chain reaction for osteochondrogenic (aggrecan, BMP-2, and Sox9) and tenogenic (Egr1, Mkx, scleraxis, type 1 collagen, and Tnmd) markers. For the in vitro experiment, TdSCs were isolated from healthy rat Achilles tendons. Cultured TdSCs were treated with methylglyoxal and recombinant MIF or MIF gene knockdown to determine the effect of hyperglycemic conditions and MIF on the differentiation function of TdSCs. These conditions were classified into four groups: hyperglycemic-control group, hyperglycemic-recombinant-MIF group, hyperglycemic-knockdown-MIF group, and normal-control group. The mRNA expression of osteochondrogenic and tenogenic markers was compared among the groups. In the in vivo experiment, the mRNA expression of all osteochondrogenic and tenogenic differentiation markers in the DM group was significantly higher and lower than that in the non-DM group, respectively. Similarly, in the in vitro experiments, the expression of all osteochondrogenic and tenogenic differentiation markers was significantly upregulated and downregulated, respectively, in the hyperglycemic-control group compared to that in the normal-control group. The hyperglycemic-knockdown-MIF group demonstrated significantly decreased expression of all osteochondrogenic differentiation markers and increased expression of only some tenogenic differentiation markers compared with the hyperglycemic-control group. In contrast, the hyperglycemic-recombinant-MIF group showed significantly increased expression of all osteochondrogenic differentiation markers, but no significant difference in any tenogenic marker level, compared to the hyperglycemic-control group. These results suggest that tendon homeostasis could be affected by hyperglycemic conditions, and MIF appears to alter the differentiation of TdSCs via enhancement of the osteochondrogenic differentiation in hyperglycemic conditions. These are preliminary findings, and must be confirmed in a further study.

Tendon Transection Healing Can Be Improved With Adipose-Derived Stem Cells Cultured With Growth Differentiation Factor 5 and Platelet-Derived Growth Factor.

As hand surgeons, tendon injuries and lacerations are a particularly difficult problem to treat, as poor healing potential and adhesions hamper optimal recovery. Adipose-derived stem cells (ADSCs) have been shown to aid in rat Achilles tendon healing after a puncture defect, and this model can be used to study tendon healing in the upper extremity. We hypothesized that ADSCs cultured with growth differentiation factor 5 (GDF5) and platelet-derived growth factor (PDGF) would improve tendon healing after a transection injury. Rat Achilles tendons were transected and then left either unrepaired or repaired. Both groups were treated with a hydrogel alone, a hydrogel with ADSCs, or a hydrogel with ADSCs that were cultured with GDF5 and PDGF prior to implantation. Tissue harvested from the tendons was evaluated for gene expression of several genes known to play an important role in successful tendon healing. Histological examination of the tendon healing was also performed. In both repaired and unrepaired tendons, those treated with ADSCs cultured with GDF5/PDGF prior to implantation showed the best tendon fiber organization, the smallest gaps, and the most organized blood vessels. Treatment with GDF5/PDGF increased expression of the protenogenesis gene SOX9, promoted cell-to-cell connections, improved cellular proliferation, and enhanced tissue remodeling. Adipose-derived stem cells cultured with GDF5/PDGF prior to implantation can promote tendon repair by improving cellular proliferation, tenogenesis, and vascular infiltration. This effect results in a greater degree of organized tendon healing.

The twisted structure of the rat Achilles tendon.

The rat is frequently used as a model to study the characteristics, aetiology and pathology of the Achilles tendon. However, though the structure of the human Achilles tendon has been extensively investigated, the anatomical structure of the rat Achilles tendon remains unclear, which impedes the ability to use rats to study Achilles tendinopathy. The purpose of this study was to reveal the structure of the rat Achilles tendon and to explore its similarities with the human Achilles tendon through an anatomical dissection of 80 rat Achilles tendons (40 female, 40 male). This study found that the subtendons of the rat Achilles tendon originating from the triceps surae muscle were twisted, and each subtendon also had its own torsion. The extent of these two types of torsion could be very different between rats. Alterations in this torsion may result in distinct stress fields in the Achilles tendon, which may play a critical role in the pathogenesis of Achilles tendinopathy. This study provides an important basis to support the use of rats as model animals to investigate the characteristics of the human Achilles tendon and Achilles tendinopathy.

Biomechanical Effects of Δ9-Tetrahydrocannabinol (THC) and Cannabidiol (CBD), the Major Constituents of Cannabis, in a Sprague Dawley Rat Achilles Tendon Surgical Repair Model: A Pilot Study

Background: The use of cannabis is common among athletes and the US population at large. Cannabinoids are currently being evaluated as alternatives to opioid medications for chronic pain management. However, the effects of recreational and/or medical use of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on musculoskeletal injury and healing remain largely unknown. Hypothesis/Purpose: The purpose of this study was to evaluate the biomechanical effects of CBD and THC on tendon-to-tendon healing in a rat Achilles tendon repair model. The hypothesis was that rats administered CBD would demonstrate decreased tensile load to failure of surgically repaired Achilles tendons compared with the THC and control groups. Study Design: Controlled laboratory study. Methods: A total of 33 Sprague Dawley rats underwent Achilles tendon surgical transection and repair and were randomized to receive subcutaneous injection of THC, CBD, or vehicle once daily starting on the day of surgery and for 5 total days. After sacrifice, biomechanical tensile load-displacement testing was performed to determine Achilles tendon load to failure and stiffness. Data were analyzed by 1-way analysis of variance. Results: The THC group demonstrated the highest median load to failure, 18.7 N (95% CI, 15.3-19.2 N); the CBD group had the second highest at 16.9 N (95% CI, 15.1-19.8 N), and the control group had the lowest at 14.4 N (95% CI, 12.1-18.3 N). Stiffness was highest in the THC group at 4.1 N/mm (95% CI, 2.7-5.1 N/mm) compared with 3.6 N/mm (95% CI, 2.9-4.1 N/mm) for the CBD group and 3.6 N/mm (95% CI, 2.8-4.3 N/mm) for the control group. No statistically significant differences for strength and stiffness were observed between the groups. Conclusion: In this pilot study using an animal tendon-to-tendon repair model, neither THC nor CBD resulted in altered biomechanical characteristics compared to control. Clinical Relevance: Cannabinoids do not appear to adversely affect Achilles tendon healing.

MicroRNA engineered umbilical cord stem cell-derived exosomes direct tendon regeneration by mTOR signaling

BackgroundExosomes are extracellular vesicles of nano-structures and represent an emerging nano-scale acellular therapy in recent years. Tendon regeneration is a sophisticated process in the field of microsurgery due to its poor natural healing ability. To date, no successful long-term solution has been provided for the healing of tendon injuries. Functional recovery requires advanced treatment strategies. Human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos) are considered as promising cell-free therapeutic agents. However, few studies reported their potential in the tendon repair previously. In this study, we explored the roles and underlying mechanisms of HUMSC-Exos in the tendon regeneration.ResultsExpression of tendon‐specific markers in, and collagen deposition by, tendon-derived stem cells (TDSCs) treated with HUMSC-Exos increased in vitro. In a rat Achilles tendon injury model, treatment with HUMSC-Exos improved the histological structure, enhanced tendon-specific matrix components, and optimized biomechanical properties of the Achilles tendon. Findings in miRNA sequencing indicated a significant increase in miR-29a-3p in HUMSC-Exo-treated Achilles tendons. Next, luciferase assay in combination with western blot identified phosphatase and tensin homolog (PTEN) as the specific target of miR-29a-3p. Furthermore, we applied a miR-29a-3p-specific agonist to engineer HUMSC-Exos. These HUMSC-Exos overexpressing miR-29a-3p amplified the gain effects of HUMSC-Exos on tendon healing in vivo. To explore the underlying mechanisms, a transforming growth factor-β1 (TGF-β1) inhibitor (SB-431542), mTOR inhibitor (rapamycin), and engineered HUMSC-Exos were employed. The results showed that TGF-β1 and mTOR signaling were involved in the beneficial effects of HUMSC-Exos on tendon regeneration.ConclusionThe findings in our study suggest that PTEN/mTOR/TGF-β1 signaling cascades may be a potential pathway for HUMSC-Exos to deliver miR-29a-3p for tendon healing and implicate a novel therapeutic strategy for tendon regeneration via engineered stem cell-derived exosomes.Graphic abstract

In vitro and in vivo tenocyte-protective effectiveness of dehydroepiandrosterone against high glucose-induced oxidative stress

BackgroundDehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes. This study aimed to investigate the in vitro and in vivo protective effects of DHEA against high glucose-induced oxidative stress in tenocytes and tendons.MethodsTenocytes from normal Sprague-Dawley rats were cultured in low-glucose (LG) or high-glucose (HG) medium with or without DHEA. The experimental groups were: control group (LG without DHEA), LG with DHEA, HG without DHEA, and HG with DHEA. Reactive oxygen species (ROS) production, apoptosis, and messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, and interleukin-6 (IL-6) were determined. Further, diabetic rats were divided into a control group and a DHEA-injected group (DHEA group). NOX1 and NOX4 protein expression and mRNA expression of NOX1, NOX4, IL-6, matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-2, and type I and III collagens in the Achilles tendon were determined.ResultsIn rat tenocytes, DHEA decreased the expression of NOX1 and IL-6, ROS accumulation, and apoptotic cells. In the diabetic rat Achilles tendon, NOX1 protein expression and mRNA expression of NOX1, IL-6, MMP-2, TIMP-2, and type III collagen were significantly lower while type I collagen expression was significantly higher in the DHEA group than in the control group.ConclusionsDHEA showed antioxidant and anti-inflammatory effects both in vitro and in vivo. Moreover, DHEA improved tendon matrix synthesis and turnover, which are affected by hyperglycemic conditions. DHEA is a potential preventive drug for diabetic tendinopathy.

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.

Evaluating the Effect of Electrospun Polyvinyl Alcohol Nanofiber Containing Eucalyptus globules Extract on the Healing of Experimental Achilles Tendon Injury in Rat

Injury and degeneration of tendons can be highly debilitating and can result in substantial pain, disability, and healthcare costs. Nano-sized fibers have a much wider surface area than conventionally produced fibers, which can hold composite materials more compactly and thus provide greater mechanical capabilities. In this study, the injured tendon was treated by electrospun PVA mats containing eucalyptus extract and histopathological results of healing were evaluated. For this study, 45 male Wistar rats were prepared and a partial thickness tenotomy was created on right hindlimbs. All rats were divided into three groups (n = 15) and three sub-groups (n = 5) including, eucalyptus extract-loaded nanofibers, PVA nanofibers, and without any treatment as a control group. Histological samples were taken on days 14, 28, and 42. The histological analysis on day 14 indicated no significant difference was observed between all groups (p > 0.05). While on days 28 and 42 post-rupture indicated a higher regenerating activity and capacity in the eucalyptus extract-loaded nanofibers than PVA nanofiber and control groups (p ≤ 0.05). In summary, these results suggest that the eucalyptus extract-loaded nanofibers mats promoted the healing process of damaged Achilles tendon in rats.

MSC-derived immunomodulatory extracellular matrix functionalized electrospun fibers for mitigating foreign-body reaction and tendon adhesion

Adhesion formation during tendon healing remains a severe problem in clinical practice. Multiple factors contribute to postoperative adhesion formation, and macrophage-driven inflammation is thought to be greatly involved in this process. We hypothesize that reducing macrophage-mediated inflammation in the injured tendon by regulating M1 to M2 macrophage polarization may effectively inhibit adhesion formation. Here, we developed an acellular immunomodulatory biomaterial consisting of an electrospun polycaprolactone/silk fibroin (PCL/SF) composite fibrous scaffold functionalized with mesenchymal stem cell (MSC)-derived extracellular matrix (ECM). To enhance the immunoregulatory potential of MSCs, we performed inflammatory licensing with IFN-γ to obtain immunomodulatory ECM (iECM). Proteomic analyses of MSCs and their secreted ECM components from different culture conditions revealed the MSC-ECM molecular signatures and the potential mechanism of ECM immunoregulation. Then, the immunoregulatory potential of the iECM-modified scaffold was evaluated in vitro and in vivo. Relative to the PCL/SF fibrous scaffold, the iECM-functionalized scaffold facilitated M2 macrophage polarization and inhibited the expression of multiple cytokines (IL-1β, IL-6, CXCL11, IL-10, IL-1R2, and TGF-β1) in vitro, strongly suggesting the immunosuppressive ability of iECM derived from inflammatory licensed MSCs. Consistent with the in vitro findings, the results of rat subcutaneous implantation indicated that a markedly lower foreign-body reaction (FBR) was obtained in the PCL/SF-iECM group than in the other groups, as evidenced by thinner fibrotic capsule formation, less type I collagen production and more M2-type macrophage polarization. In the rat Achilles tendon injury model, the PCL/SF-iECM scaffold greatly mitigated tendon adhesion with clear sheath space formation between the tendon and the scaffold. These data highlight the immunomodulatory potential of iECM-functionalized fibrous scaffolds to attenuate FBR by modulating M2 macrophage polarization, thereby preventing tendon adhesion. Statement of significanceElectrospun PCL/SF fibrous scaffolds functionalized with ECM secreted by MSCs stimulated by inflammatory factor IFN-γ was developed that combined physical barrier and immunomodulatory functions to prevent tendon adhesion formation. PCL/SF micro-nanoscale bimodal fibrous scaffolds prepared by emulsion electrospinning possess high porosity and a large pore size beneficial for nutrient transport to promote intrinsic healing; moreover, surface modification with immunomodulatory ECM (iECM) mitigates the FBR of fibrous scaffolds to prevent tendon adhesion. The iECM-functionalized electrospun scaffolds exhibit powerful immunomodulatory potency in vitro and in vivo. Moreover, the iECM-modified scaffolds, as an anti-adhesion physical barrier with immunomodulatory ability, have an excellent performance in a rat Achilles tendon adhesion model. MSC secretome-based therapeutics, as an acellular regenerative medicine strategy, are expected to be applied to other inflammatory diseases due to its strong immunoregulatory potential.

Platelet-Rich Plasma Releasate Promotes Early Healing in Tendon After Acute Injury.

Background:Acute tendon injury can limit motion and thereby inhibit tendon healing. Positive results have been found after the use of platelet-rich plasma (PRP) to treat tendon injury; however, the early effects of PRP on tendon regeneration are not known.Purpose/Hypothesis:The purpose of this study was to evaluate the effects of PRP releasate (PRPr) on the early stages of tendon healing in a rat partial tenotomy model. It was hypothesized that PRPr can promote early healing of an Achilles tendon in rats.Study Design:Controlled laboratory study.Methods:PRP was prepared by a 2-step method of manual platelet concentration from 10 rats. PRPr was isolated from the clotted preparation after activation by thrombin and was applied to an Achilles tendon on 1 side of 30 rats on the second day after partial tenotomy, with normal saline used as the control on the other side. Achilles tendon samples were harvested 5 and 10 days after tenotomy. At each time point, 15 Achilles tendon samples were obtained, of which 5 samples were evaluated by Masson trichrome staining, apoptosis, and cell proliferation, while the other 10 samples were tested for tensile strength using a material testing machine.Results:Compared with saline-treated control tendons, the PRPr-treated tendons showed increased collagen synthesis near the cut edge and fewer apoptotic cells (P = .01). An immunohistochemical analysis revealed more Ki-67–positive cells but fewer cluster of differentiation (CD) 68+ (ED1+) macrophages in PRPr tendons compared with saline-treated tendons (P < .01). Tendons treated with PRPr also showed higher ultimate tensile strength than those treated with saline (P = .03).Conclusion:PRPr treatment promotes tissue recovery in the early phase of tendon healing by stimulating tendon cell proliferation and collagen production while inhibiting cell apoptosis and CD68+ (ED1+) macrophage infiltration.Clinical Relevance:These findings suggest that with PRPr treatment, higher loads can be applied to the healing tendon at an earlier time, which can help the patient resume activity earlier.

Photobiomodulation therapy increases collagen II after tendon experimental injury.

A tendon is a mechanosensitive tissue that transmits muscle-derived forces to bones. Photobiomodulation (PBM), also known as low-level laser therapy (LLLT), has been used in therapeutic approaches in tendon lesions, but uncertainties regarding its mechanisms of action have prevented its widespread use. We investigated the response of PBM therapy in experimental lesions of the Achilles tendon in rats. Thirty adult male Wistar rats weighing 250 to 300 g were surgically submitted to bilateral partial transverse section of the Achilles tendon. The right tendon was treated with PBM, whereas the left tendon served as a control. On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each, which were treated with PBM (Konf, Aculas - HB 750), 780 nm and 80 mW for 20 seconds, three times/week for 7, 14 and 28 days. The rats were sacrificed at the end of the therapeutic time period. The Sca-1 was examined by immunohistochemistry and histomorphometry, and COLA1, COLA2 and COLA3 gene expression was examined by qRT-PCR. COLA2 gene expression was higher in PBM treated tendons than in the control group. The histomorphometric analysis coincided with increased number of mesenchymal cells, characterized by Sca-1 expression in the lesion region (p<0.001). PBM effectively interferes in tendon tissue repair after injury by stimulating mesenchymal cell proliferation and the synthesis of collagen type II, which is suggested to provide structural support to the interstitial tissues during the healing process of the Achilles tendon. Further studies are needed to confirm the role of PBM in tendon healing.

Comparison between dry needling and focused ultrasound on the mechanical properties of the rat Achilles tendon: A pilot study

In the U.S., approximately 14 million tendon and ligament injuries are reported each year. Dry needling (DN) is a conservative treatment introduced to alleviate pain and restore function; however, it is invasive and has mixed success. Focused ultrasound (fUS) is a non-invasive technology that directs ultrasound energy into a well-defined focal volume. fUS induces thermal and/or mechanical bioeffects which can be controlled by the choice of ultrasound parameters. fUS could be an alternative to DN for treatment of tendon injuries, but the bioeffects must be established. Thus, the purpose of this pilot study was to compare the effect of DN and fUS on the mechanical properties and cell morphology of 30 ex vivo rat Achilles tendons. Tendons were randomly assigned to sham, DN, or fUS, with 10 tendons per group. Within each group, 5 tendons were evaluated mechanically, and 5 tendons were analyzed histologically. Elastic modulus in the DN (74.05 ± 15.0 MPa) group was significantly lower than sham (149.84 ± 59.1 MPa; p = 0.0094) and fUS (128.84 ± 28.3 MPa; p = 0.0453) groups. Stiffness in DN (329.05 ± 236.8 N/mm; p = 0.0034) and fUS (315.26 ± 68.9 N/mm; p = 0.0027) groups were significantly lower than sham (786.10 ± 238.7 N/mm) group. Histologically, localized necrosis was observed in 3 out of 5 tendons exposed to fUS, with surrounding tissue unharmed; no evidence of cellular injury was observed in DN or sham groups. These results suggest that fUS preserves the mechanical properties of tendon better than DN. Further studies are needed to evaluate fUS as an alternative, noninvasive treatment modality for tendon injuries.

Effect of a bovine collagen matrix on tendon adhesions during the repair of rat Achilles tendon

Adhesion after a tendon injury is one of the major problems following upper extremity surgery. In the present study, we evaluated a new material that is clinically usable as an adhesion barrier. Twenty-four male Wistar albino rats were used in the study. These rats (48 legs) were divided into three groups: sham, control, and experimental. No surgical intervention was performed in the sham group. After making a full-thickness cut through the right Achilles tendon, the tendon was repaired using the modified Kessler technique in the control group, while bovine collagen matrix was wrapped around the surgically repaired tendon using the modified Kessler technique in the experimental group. Two months after surgery, the operated and non-operated tendons were resected and analyzed through biomechanical, macroscopic, and histopathological examinations. The results of the biomechanical testing did not differ significantly between the control and experimental groups. Macroscopic examination of the adhesions revealed less adhesions in the experimental group but this difference was not statistically significant. Moreover, the results of the histopathological examination, which was performed based on five criteria, did not differ significantly between the two groups. Our study’s results indicate that a bovine collagen matrix can be used to prevent tendon adhesion; however, larger studies are needed to verify these findings.

Melatonin Promotes Heterotopic Ossification Through Regulation of Endothelial-Mesenchymal Transition in Injured Achilles Tendons in Rats.

Although heterotopic ossification (HO) has been reported to be a common complication of the posttraumatic healing process, the underlying mechanism remains unknown. Endothelial-mesenchymal transition (EndMT) is known to play a role in HO, and our recent study observed that neuroendocrine signals can promote HO by modulating EndMT. Melatonin, a neuroendocrine hormone secreted mainly by the pineal gland, has been documented to perform its function in the skeletal system. This study aimed at describing the expression of melatonin during the formation of HO in rat models of Achilles tendon injury and to further investigate its role in regulating EndMT in HO. Histological staining revealed the expression of melatonin throughout the formation of heterotopic bone in injured Achilles tendons, and the serum melatonin levels were increased after the initial injury. Double immunofluorescence showed that the MT2 melatonin receptor was notably expressed at the sites of injury. Micro-CT showed the enhancement of heterotopic bone volume and calcified areas in rats treated with melatonin. Additionally, our data showed that melatonin induced EndMT in primary rat aortic endothelial cells (RAOECs), which acquired traits including migratory function, invasive function and EndMT and MSC marker gene and protein expression. Furthermore, our data exhibited that melatonin promoted the osteogenic differentiation of RAOECs undergoing EndMT in vitro. Importantly, inhibition of the melatonin-MT2 pathway by using the MT2 selective inhibitor 4-P-PDOT inhibited melatonin-induced EndMT and osteogenesis both in vivo and in vitro. In conclusion, these findings demonstrated that melatonin promoted HO through the regulation of EndMT in injured Achilles tendons in rats, and these findings might provide additional directions for the management of HO.

The efficiency of Polytendon Complex (PC) and St. John’s Wort oil (Hypericum perforatum) on healthy Achilles tendon in rats

The efficiency of Polytendon Complex (PC) and St. John’s Wort oil (Hypericum perforatum) on healthy Achilles tendon in rats