Tendon Samples Research Articles

Exploring cellular changes in ruptured human quadriceps tendons at single-cell resolution.

Tendon ruptures in humans have often been studied during the chronic phase of injury, particularly in the context ofrotator cuff disease. However, the early response to acute tendon ruptures remains less investigated. Quadriceps tendons, which require prompt surgical treatment, offer a model to investigate this early response. Therefore, this study aimed to explore the early cellular changes in ruptured compared to healthy human quadriceps tendons. Quadriceps tendon samples were collected from patients undergoing fracture repair (healthy) or tendon repair surgery (collected 7-8days post-injury). Nuclei were isolated for single-nucleus RNA sequencing, and comprehensive transcriptomic analysis was conducted. The transcriptomes of 12,808 nuclei (7268 from healthy and 5540 from ruptured quadriceps tendons) were profiled, revealing 12major cell types and several cell subtypes and states. Rupture samples showed increased expression of genes related to extracellular matrix organisation and cell cycle signalling, and a decrease in expression of genes in lipid metabolism pathways. These changes were predominantly driven by gene expression changes in the fibroblast, vascular endothelial cell (VEC), mural cell, and macrophage populations: fibroblasts shift to an activated phenotype upon rupture and there is an increase in the proportion of capillary and dividing VECs. A diverse immune environment was observed, with a shift from homeostatic to activated macrophages following rupture. Cell-cell interactions increased in number and diversity in rupture, and primarily involved fibroblast and VEC populations. Collectively, this transcriptomic analysis suggests that fibroblasts and endothelial cells are key orchestrators of the early injury response within ruptured quadriceps tendon. KEY POINTS: Tendon ruptures in humans have regularly been studied during the chronic phase of injury, but less is known about the early injury response after acute tendon ruptures. This study explored the early cellular changes in ruptured compared to healthy human quadriceps tendons at single-cell resolution. Fibroblasts and endothelial cells seem to be the key orchestrators of the early injury response within ruptured quadriceps tendon. Therefore, these cell types are obvious targets for interventions to enhance tendon healing. Overall, this study highlights that the development of more effective therapeutic options for tendon injury requires better understanding of the cellular, extracellular, and mechanical landscape of tendon tissue.

14-3-3ζ: an optimal housekeeping protein for western blot analysis in swine rotator cuff tendon studies.

Healthy biomechanics of the shoulder involving rotator cuff muscles and rotator cuff tendon (RCT) is pivotal for joint stability, yet co-morbid conditions like hyperlipidemia and hyperglycemia can lead to degenerative changes jeopardizing tendon integrity. A change in protein expression, the functional moiety for molecular events, may result in altered healing of RCT and prolonged morbidity. Expression and activity of proteins are critical while investigating the underlying molecular and cellular changes involved in tendinopathy. While investigating the changes in the protein expression of various inflammatory mediators, we observed that the Western Blot bands for commonly used housekeeping genes (GAPDH, β-actin, and α-tubulin) were not uniform in different tendon samples. Therefore, we investigated for an optimal housekeeping gene for Western blot analysis in swine RCT under normal and hyperlipidemic conditions, as this is essential for accurate normalization of protein expression. The study evaluated several housekeeping genes-GAPDH, beta-actin, alpha and beta-tubulin, Ubiquitin C, Cyclophilin A, TATA-box binding protein, and 14-3-3ζ-to ensure robust normalization across experimental setups. The results revealed that the protein expression of 14-3-3ζ was uniform in all samples, thereby validating its suitability as a stable housekeeping protein. The findings are important while studying the RCT pathology in a clinically relevant animal model, like swine, which mimics human RCT and provides translationally significant findings. Thus, the 14-3-3ζ protein will be an ideal housekeeping gene in the design of experiments utilizing musculoskeletal tissues.

Reversal of allodynia and enhanced tendon repair in rats via 660 and 808 nm photobiomodulation.

Our study aimed to explore the potential of Photobiomodulation (PBM) as a treatment for Tendinopathy (TP), a chronic condition characterized by tendon degeneration, inflammation, and pain, for which current treatment options often yield unsatisfactory outcomes. PBM, utilizing low-level laser, has shown promise in reducing inflammatory markers, stimulating cell proliferation, and alleviating pain, thus presenting itself as a promising alternative therapy for TP. Using an experimental rat model of tendinopathy, male Wistar rats weighing 250-370 g were induced with TP through the injection of type I collagenase into the calcaneal tendon. Animals were divided into four equal groups (control, TP, TP + 660 nm, TP + 808 nm), with 10 animals in each group for the nociceptive behavioral tests and 5 in each for the biomolecular analysis. Fifteen days later, PBM was administered to the tendons once daily for seven consecutive sessions using two wavelengths (660 and 808 nm), with 3 J for 30 s, irradiating an area of 0.028 cm2 totaling 107.14 J/cm2. Mechanical nociception and thermal cold sensitivity were assessed, and tendon samples were collected for analysis of collagen types and total cell count. Spinal cord samples were also collected and immunostained to evaluate specific markers. Our results demonstrated that collagenase injection induced mechanical allodynia and cold hypersensitivity, both of which were reversed by PBM at both 660 and 808 nm wavelengths (**p < 0.005 and *p < 0.05). Additionally, TP led to an increase in collagen type III and a decrease in collagen type I expression, with these effects being reversed by PBM treatment (*p < 0.05). Moreover, TP increased astrocyte immunoreactivity, while PBM at both wavelengths led to a decrease in astrocyte and IL-1β expression. In conclusion, PBM emerged as an effective treatment for TP, as it not only alleviated pain symptoms but also modulated collagen expression and reduced astrocyte and IL-1β expression (**p < 0.01). These findings suggest that PBM could serve as a beneficial adjunctive therapy for analgesia and regeneration in TP management.

Sirolimus as a repurposed drug for tendinopathy: A systems biology approach combining computational and experimental methods.

Sirolimus as a repurposed drug for tendinopathy: A systems biology approach combining computational and experimental methods.

AI-driven histologic analysis of human Achilles tendinopathy provides a roadmap to unravel pathogenesis.

Achilles tendinopathy is a common source of pain and dysfunction, yet its pathogenesis remains poorly understood. Research on human tendons is hampered by lack of standardisation in tissue sample validation, making interpretation of results challenging. We sought to develop an automated and operator-independent approach to histologically score human tendons. We assembled a cohort of 15 tendinopathic and 10 normal control Achilles tendon samples. We stained longitudinal sections with haematoxylin and eosin and Alcian blue and developed a low temperature epitope-retrieval protocol for immunostaining of blood vessels. Histologic sections were scored by pathologists using the current gold standard Bonar score. Whole sections were then analysed with open-source software (QuPath). Histologic features were automatically quantified across the entire section and summarised in the BonAIr score. Tissue from the same patients was subsequently analysed by quantitative polymerase chain reaction and flow cytometry to validate elements of the BonAIr score. We observed increased cell roundness, collagen disarrangement, ground substance, and vascularity in tendinopathy using both the Bonar and BonAIr scores. Increased cellularity was only detected by the BonAIr score. Cellular and transcriptomic analyses corroborated tendinopathic shifts in all elements of the BonAIr score and further identified elevated THY1/CD90 expression in tendinopathy. CD90+ cells were found to localise to areas of low collagen alignment. These results align with the concept of stromal cell dysregulation in tendinopathy. Automated analysis of whole tendon sections refines conventional histopathologic scoring and predicts cellular and molecular changes found in tendinopathy. The BonAIr score should be further developed for standardised assessment of tendons samples across other anatomical locations and different research centres.

Dichroism-sensitive photoacoustic imaging for in-depth estimation of the optic axis in fibrous tissue.

Dichroism-sensitive photoacoustic imaging for in-depth estimation of the optic axis in fibrous tissue.

Changes in Gene Expression of the Extracellular Matrix in Patients with Full-Thickness Rotator Cuff Tears of Varying Sizes.

This study aimed to investigate changes in gene expression related to matrix synthesis in individuals with full-thickness rotator cuff tears (RCTs) and normal tendon tissues. The study also aimed to examine the differences in gene expression according to 4 distinct tear sizes. A total of 12 patients with full-thickness RCTs were included in the study, all of whom underwent arthroscopic rotator cuff repair. The RCTs were stratified by size into small, medium, large, and massive. Tendon samples were harvested from the midpoint between the lateral end of the torn tendon and the musculotendinous junction. Subsequent analysis of the tissue samples revealed the mRNA expression levels of 11 collagen types, 6 proteoglycans, and 8 glycoproteins through real-time polymerase chain reaction techniques. For control purposes, supraspinatus tendon tissue was sourced from 3 patients who had proximal humerus fractures but did not present with RCTs. Among the 11 collagens and 14 non-collagenous protein (NCP) genes examined in this study, COL3A1 and COL10A1 showed a significant increase, whereas COL4A1 and COL14A1 showed a tendency to decrease compared to those in the normal group. ACAN significantly increased by 8.92-fold (p < 0.001) compared to that in the normal group, whereas DCN and LUM showed a tendency to decrease. FN1 and TNC increased significantly by 3.47-fold (p = 0.003) and 5.38-fold (p = 0.005), respectively, and the genes ELN, LAMA2, and THBS1 were all significantly reduced compared to those in the normal group. In the NCPs, almost all the genes with increased expression levels had the highest level in small size RCTs, and gene expression decreased as the size increased. The 3 proteoglycans (ACAN, BGN, and FMOD) showed the highest levels of expression in small size RCTs compared to those in the normal group, and 5 glycoproteins (COMP, FBN1, FN1, HAPLN1, and TNC) also showed the highest expression in small size RCTs. We confirmed that most of the detected extracellular matrix gene expression changes were related to the size of the full-thickness RCTs. In NCPs, gene expression was increased in small-size tears, and gene expression levels were significantly reduced when the size increased.

Mueller matrix polarimetry for quantitative evaluation of the Achilles tendon injury recovery

Achilles tendon injuries, as a widely existing disease, have attracted a lot of research interest. Mueller matrix polarimetry, as a novel label-free quantitative imaging method, has been widely used in various applications of lesion identification and pathological diagnosis. However, focusing on the recovery process of Achilles tendon injuries, current optical imaging methods have not yet achieved the label-free precise identification and quantitative evaluation. In this study, using Mueller matrix polarimetry, various Achilles tendon injury samples were characterized specifically, and the efficacy of different recovery schemes was evaluated accordingly. Experiments indicate that injured Achilles tendons show less phase retardance, larger diattenuation, and relatively disordered orientation. The combination of experiments with Monte Carlo simulation results illustrate the microscopic mechanism of the Achilles tendon recovery process from three aspects, that is, the increased fiber diameter, a more consistent fiber orientation, and greater birefringence induced by more collagen protein. Finally, based on the statistical distribution of polarization measurements, a polarization specific characterization parameter was extracted to construct a label-free image, which cannot only intuitively show the injury and recovery of Achilles tendon samples, but also give a quantitative evaluation of the treatment.Graphical

Biomechanical enhancement in rotator cuff repairs: the impact of innovative nanofiber technology

Rotator cuff repair surgeries often face high failure rates, particularly in cases involving tendon degeneration. Traditional repair techniques and devices frequently fail to adequately restore a healthy native enthesis and strong tendon-bone integration. This study investigates the efficacy of a novel, fully synthetic, bioresorbable nanofiber scaffold in restoring the native enthesis and enhancing the biomechanical properties and overall success of rotator cuff repairs, particularly in the context of chronically degenerated tendons. This study used an ovine model to simulate chronic tendon degeneration with subsequent rotator cuff transection and repair. All repairs were performed using the standard double-row configuration with suture tape; half of the repairs were augmented with the bioresorbable nanofiber scaffold. Nondestructive biomechanical testing was conducted to assess the strength of the repair constructs, followed by histological analysis of all tendon samples to evaluate tissue regeneration and integration at the repair site. Results demonstrated that the scaffold group achieved significantly improved biomechanical properties (peak force, peak stress, equilibrium force, and equilibrium stress) compared to the suture only group, indicating enhanced repair strength and native enthesis restoration. Scaffold samples exhibited significantly decreased cross-sectional areas (ie, less fibrosis) which were similar to healthy tendons. Histological findings indicated the scaffold did not impede re-establishment of Sharpey-like fibers at the tendon insertion. This study provides compelling evidence that the use of a fully synthetic, bioresorbable nanofiber scaffold in rotator cuff repair significantly improves biomechanical outcomes and enthesis regeneration. These improvements were achieved while retaining close to native tendon thickness. The findings suggest that this scaffold represents a significant advancement in rotator cuff repair technology, offering a promising solution to enhance repair strength and quality of bone-tendon integration, especially in challenging cases of tendon degeneration.

Age-dependence of semitendinosus tendon properties used for anterior cruciate ligament reconstruction differs in males and females

Age-dependence of semitendinosus tendon properties used for anterior cruciate ligament reconstruction differs in males and females

Investıgatıon of the effects of treatment with enoxaparın sodıum and hyperbarıc oxygen therapy on the recovery of rats wıth achılles tendon rupture

PurposeIn this study, we aimed to investigate the effects of hyperbaric oxygen therapy and enoxaparin sodium, which are known to accelerate bone tissue healing as well as tendon and soft tissue healing, on the healing of Achilles tendon rupture.MethodsThirty-six rats were used in the present study. All rats were divided into groups of nine. The groups were the enoxaparin sodium group, enoxaparin sodium and hyperbaric oxygen group, hyperbaric oxygen group and control group. After 21 days, the process was completed, and the rats were sacrificed. Achilles tendon samples were evaluated histopathologically.ResultsThe groups were compared according to the results of statistical analysis based on the histopathological data. There was no significant difference between the groups in terms of acute inflammation (p = 0.785) or chronic inflammation (p = 0.827) scores, but there were significant differences in neovascularization (p = 0.009), proliferation (p < 0.001) and fibrosis (p = 0.006) scores.ConclusionOur study showed that the use of enoxaparin sodium and hyperbaric oxygen had a positive effect on the healing of the Achilles tendon. Based on these results, we believe that the use of enoxaparin sodium and hyperbaric oxygen therapy after Achilles tendon rupture will be beneficial for healing and preventing complications.

Insulin-like growth factor-1 infusion in preterm piglets does not affect growth parameters of skeletal muscle or tendon tissue.

Prematurity has physical consequences, such as lower birth weight, decreased muscle mass and increased risk of adult-onset metabolic disease. Insulin-like growth factor 1 (IGF-1) has therapeutic potential to improve the growth and quality of muscle and tendon in premature births, and thus attenuate some of these sequalae. We investigated the effect of IGF-1 on extensor carpi radialis muscle and biceps brachii tendon of preterm piglets. The preterm group consisted of 19-day-old preterm (10 days early) piglets, treated with either IGF-1 or vehicle. Term controls consisted of groups of 9-day-old piglets (D9) and 19-day-old piglets (D19). Muscle samples were analysed by immunofluorescence to determine the cross-sectional area (CSA) of muscle fibres, fibre type composition, satellite cell content and central nuclei-containing fibres in the muscle. Tendon samples were analysed for CSA, collagen content and maturation, and vascularization. Gene expression of the tendon was measured by RT-qPCR. Across all endpoints, we found no significant effect of IGF-1 treatment on preterm piglets. Preterm piglets had smaller muscle fibre CSA compared to D9 and D19 control group. Satellite cell content was similar across all groups. For tendon, we found an effect of age on tendon CSA, and mRNA levels of COL1A1, tenomodulin and scleraxis. Immunoreactivity for elastin and CD31, and several markers of tendon maturation, were increased in D9 compared to the preterm piglets. Collagen content was similar across groups. IGF-1 treatment of preterm-born piglets does not influence the growth and maturation of skeletal muscle and tendon.

Poster 367: Nano-Semiconductor Fibers in a Commercial Compression Sleeve Accelerates Healing of Achilles Tendon Injury in a Rat Model

Objectives: Commercial compression sleeves have been promoted to ameliorate pain secondary to arthritis and tendinopathy. The fabric of one particular sleeve is embedded with nano-semiconductor fibers that reflect thermal energy produced by the body at a photonic wavelength mimicking low-level laser therapy. The device is theorized to accelerate the inflammatory stage of tissue healing to reach the proliferation and remodeling stages at faster rates. Previous clinical studies have shown a significant improvement in patient-reported outcomes between patients who did and did not use this sleeve to treat osteoarthritis. The objective of this investigation was to determine if compression sleeves embedded with nano-semiconductor fibers can accelerate Achilles tendon healing in a rabbit model. Methods: Twelve New Zealand White rabbits—under IACUC approval—were administered intratendinous injections of 0.2% collagenase into the right Achilles tendon to simulate an acute tendon injury. Rabbits were divided into three groups: group I received the commercial compression sleeve without nano-semiconductors (n = 4), group II received the sleeve with nano-semiconductors (n = 4), and group III received an alternate sleeve design also with nano-semiconductors (n = 4). Calf circumference measurements and thermal radiation imaging of the right lower leg of each rabbit were collected days 7, 14, 21, and 28 post-injection. Two rabbits from each group were sacrificed at the 14-day and 28-day marks and the right Achilles tendons were harvested. Histological analysis using picrosirius red staining was performed to evaluate tissue organization. A custom MATLAB program (MathWorks, Natick, MA) was used to quantify the collagen fiber dispersion within the Achilles tendon samples. Results: A statistically significant difference in calf circumference was found at the 28-day mark between group I versus groups II and III: group I had an average calf circumference of 5.2 cm while groups II and III had an average calf circumference of 4.85 cm (p = 0.036) and 4.60 cm (p = 0.021) respectively. Additionally, after 28 days, group I demonstrated an average pixel density intensity on thermal radiation imaging of 1,531,192 while groups II and III yielded average pixel density intensities of 1,363,515 (p = 0.008) and 1,335,456 (p = 0.003) respectively. Histological staining showed observable alterations in collagen architecture, with group I displaying widespread disorganization and degradation of type I and type III collagen fibers near the injection site. Groups II and III displayed greater organization at the 28-day mark in comparison to group I, with signs of tissue proliferation and remodeling evident. No statistically significant differences were found between group I versus groups II and III regarding collagen fiber dispersion at both the 2-week mark (p = 0.145, p = 0.236) and the 4-week mark (p = 0.113, p = 0.268), respectively. Conclusions: The nano-semiconductor fibers embedded within a commercial sleeve significantly decreased swelling and temperature of the lower leg 28 days following acute Achilles tendon injury in a rabbit model. Furthermore, histologic analysis demonstrated greater collagen fiber and extracellular matrix organization as well as greater proliferation of type III collagen suggesting a faster transition to the proliferation stage of tissue healing.

Upregulation of FABP4 induced inflammation in the pathogenesis of chronic tendinopathy

Upregulation of FABP4 induced inflammation in the pathogenesis of chronic tendinopathy

IDENTIFICATION OF HEALTHY AND TENDINOPATHIC CELL SUBTYPES IN FOOT AND ANKLE TENDONS USING SINGLE-CELL TRANSCRIPTOMICS

Tendinopathy can commonly occur around the foot and ankle resulting in isolated rupture, debilitating pain and degenerative foot deformity. The pathophysiology and key cells involved are not fully understood. This is partly because the dense collagen matrix that surrounds relatively few resident cells limits the ability of previous techniques to identify and target those cells of interest. In this study, we apply novel single cell RNA sequencing (CITE-Seq) techniques to healthy and tendinopathic foot/ankle tendons. For the first time we have identified multiple sub-populations of cells in human tendons. These findings challenge the view that there is a single principal tendon cell type and open new avenues for further study. Healthy tendon samples were obtained from patients undergoing tendon transfer procedures; including tibialis posterior and FHL. Diseased tendon samples were obtained during debridement of intractable Achilles and peroneal tendinopathy, and during fusion of degenerative joints. Single cell RNA sequencing with surface proteomic analysis identified 10 sub-populations of human tendon derived cells. These included groups expressing genes associated with fibro-adipogenic progenitors (FAPs) as well as ITGA7+VCAM1- recently described in mouse muscle but, as yet, not human tendon. In addition we have identified previously unrecognised sub-classes of collagen type 1 associated tendon cells. Each sub-class expresses a different set of extra-cellular matrix genes suggesting they each play a unique role in maintaining the structural integrity of normal tendon. Diseased tendon harboured a greater proportion of macrophages and cytotoxic lymphocytes than healthy tendon. This inflammatory response is potentially driven by resident tendon fibroblasts which show increased expression of pro-inflammatory cytokines. Finally, identification of a previously unknown sub-population of cells found predominantly in tendinopathic tissue offers new insight into the underlying pathophysiology. Further work aims to identify novel proteins targets for possible therapeutic pathways.

Effects of 32 d of spaceflight on tendon collagen fibril morphology and nanomechanics

INTRODUCTION: Collagen is an essential component of connective tissue and plays a crucial role in elasticity, stability, and force distribution. While the adaptations of tendon to internal stimuli have been extensively researched, the impact of environmental factors (such as microgravity) on its structure and function at the nanoscopic scale (i.e., fibril level) remains largely unexplored. The purpose of this study was to determine the effects of 32 days (d) aboard the International Space Station (ISS) on murine tendon collagen fibril morphology and nanomechanics (Young’s Modulus, YM). We hypothesized there would be a decrease in fibril size and YM after spaceflight. Methods: Quadriceps tendon samples from female C57BL/6J mice from NASA Rodent Research-1 underwent chemical processing and mechanical isolation. Subsequently, collagen fibrils (n=56) were studied via atomic force microscopy (AFM; JPK NanoWizard 4a) to compare the morphology (diameter, height) and nanomechanics (average YM) between spaceflight (SF) and ground control (GC) specimens. The qp-BioAC-CB1 cantilever (spring constant: 0.3 N/m, tip radius: 10μm) was used under quantitative imaging mode (QI) for all force and morphological measurements. The data from 56 micrographs (SF n=30; GC n=26) was processed via the JPK Data Processing Software (Version 6.4.5.), and finally analyzed with Gwyddion (Version 2.62). A 2-way MANOVA compared all measurements. Results: More ‘small fibrils’ (height &lt;50nm) were identified in the SF vs GC samples. SF fibrils averaged 20.3% smaller (width) compared to GC, but this difference was not significant (p&gt;0.05). Additionally, while there was a trend for a lower YM in the SF samples, YM for SF specimens was not statistically different between groups (GC=3.26±1.94 MPa; SF= 2.76±1.44 MPa) (p&gt;0.05). CONCLUSION: To our knowledge, this is the first investigation to use AFM QI-mode on tendon collagen fibrils after spaceflight. While there were no statistically significant changes in mean fibril size and YM between SF and GC, these results suggest that collagen fibrils may require more time to adapt to changes in their environment, and/or more rapid changes may take place at different levels of the collagen hierarchy. Our data provides valuable insight into mammalian tissue adaptations during spaceflight. Future research should continue these investigations with longer-duration missions to build a time-course of tendon adaptations to microgravity. FUNDING: CSUBIOTECH New Investigator Grant (JRB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Anti-inflammatory effects of sericin and swimming exercise in treating experimental Achilles tendinopathy in rat.

The aim of this study was to assess the effectiveness of combining sericin with swimming exercise as a treatment for type-I collagenase-induced Achilles tendinopathy (AT) in rats, with a focus on inflammatory cytokines. An experimental AT model was established using type-I collagenase in male Sprague-Dawley rats, categorized into five groups: Group 1 (Control+Saline), Group 2 (AT), Group 3 (AT+exercise), Group 4 (AT+sericin), and Group 5 (AT+sericin+exercise). Intratendinous sericin administration (0.8g/kg/mL) took place from days 3 to 6, coupled with 30 min daily swimming exercise sessions (5 days/week, 4 weeks). Serum samples were analyzed using ELISA for tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), and total antioxidant-oxidant status (TAS-TOS), alongside histopathological and immunohistochemical assessments of Achilles tendon samples. Elevated TNF-α and IL-1β and decreased IL-10 levels were evident in Group 2; Of these, TNF-α and IL-1β were effectively reduced and IL-10 increased across all treatment groups, particularly groups 4 and 5. Serum TAS was notably lower in Group 2 and significantly increased in Group 5 compared to Group 2. Histopathologically, Group 2 displayed severe degeneration, irregular fibers, and round cell nuclei, while Group 5 exhibited decreased degeneration and spindle-shaped fibers. The Bonar score increased in Group 2 and decreased in groups 4 and 5. Collagen type-I alpha-1 (Col1A1) expression was notably lower in Group 2 (P=0.001) and significantly increased in groups 4 and 5 compared to Group 2 (P=0.011 and 0.028, respectively). This study underscores the potential of sericin and swimming exercises in mitigating inflammation and oxidative stress linked to AT pathogenesis, presenting a promising combined therapeutic strategy.

Anisotropic properties of acoustically induced electric polarization in soft fibrous biological tissues

Acoustically induced electric polarization and its anisotropy in soft fibrous biological tissues were investigated under wet conditions. Assuming that fibrous tissues have polar uniaxial symmetry, stress-induced polarization should occur in the direction of fiber orientation in the non-shear terms of the piezoelectric tensor. Using the acoustically stimulated electromagnetic method, we measured the anisotropic properties of acoustically induced polarization in wet samples of Achilles tendon, skeletal muscle, and aortic wall. In all these tissues, the major non-shear term was confirmed to be d33, with polarization occurring along the fibrous direction. In Achilles tendon, which contains highly oriented collagen fibers, the uniaxially symmetric fiber structure explains the anisotropic polarization well. However, substantial polarization perpendicular to the fiber orientation (the d11 term) was observed in skeletal muscle and aortic wall, suggesting that the presence of fiber crimps and complex extracellular matrix produces polarization that does not occur in the uniaxially symmetric structures.

Successive tendon injury in an in vivo rat overload model induces early damage and acute healing responses.

Introduction: Tendinopathy is a degenerative condition resulting from tendons experiencing abnormal levels of multi-scale damage over time, impairing their ability to repair. However, the damage markers associated with the initiation of tendinopathy are poorly understood, as the disease is largely characterized by end-stage clinical phenotypes. Thus, this study aimed to evaluate the acute tendon responses to successive fatigue bouts of tendon overload using an in vivo passive ankle dorsiflexion system. Methods: Sprague Dawley female rats underwent fatigue overloading to their Achilles tendons for 1, 2, or 3 loading bouts, with two days of rest in between each bout. Mechanical, structural, and biological assays were performed on tendon samples to evaluate the innate acute healing response to overload injuries. Results: Here, we show that fatigue overloading significantly reduces in vivo functional and mechanical properties, with reductions in hysteresis, peak stress, and loading and unloading moduli. Multi-scale structural damage on cellular, fibril, and fiber levels demonstrated accumulated micro-damage that may have induced a reparative response to successive loading bouts. The acute healing response resulted in alterations in matrix turnover and early inflammatory upregulations associated with matrix remodeling and acute responses to injuries. Discussion: This work demonstrates accumulated damage and acute changes to the tendon healing response caused by successive bouts of in vivo fatigue overloads. These results provide the avenue for future investigations of long-term evaluations of tendon overload in the context of tendinopathy.

Can the Sterilization Protocol Be Improved to Enhance the Healing of Allograft Tendons? An In Vivo Study in Rabbit Tendons.

Peracetic acid and irradiation are common sterilization methods for allograft tendons; however, under some conditions, both methods adversely affect the fiber arrangement and ultimate load of the tendon. An in vitro study showed that low-dose peracetic acid combined with irradiation may be less detrimental to allograft tendon structure and properties, possibly because the breakdown of peracetic acid can lead to an enlargement of the interstitial spaces and an increase in porosity. Using a rabbit Achilles tendon model, we asked: What is the effect of peracetic acid-ethanol combined irradiation on (1) the histopathology and fiber diameter of the allograft tendon, (2) tensile creep and load-to-failure biomechanical properties of allograft tendons, and (3) healing of the treated tendon in vivo compared with fresh-frozen allograft and peracetic acid-ethanol sterilization at 4 and 8 weeks? The Achilles tendons used in this study were sourced from euthanized 10-week-old male New Zealand White rabbits previously used for ophthalmic experiments. All allografts were divided into three groups: fresh-frozen group (control group, n = 20), peracetic acid-ethanol sterilization group (n =20), and peracetic acid-ethanol combined irradiation group (n = 20). The sterilization protocols were performed per a predetermined plan. In the peracetic acid-ethanol sterilization group, the tendon tissues were covered with the peracetic acid-ethanol sterilization solution (1% peracetic acid for 30 minutes). In the peracetic acid-ethanol combined irradiation group, the tendon tissues were covered with the peracetic acid-ethanol sterilization solution (0.2% peracetic acid for 30 minutes) and were subjected to 15 kGy gamma irradiation. Thirty 10-week-old male New Zealand White rabbits received bilateral Achilles tendon allografts surgically. Tendon samples from each group were harvested at 4 weeks (n = 30) and 8 weeks (n = 30) postoperatively. For each timepoint, eight tissues were used for histologic staining and electron microscopy, 15 tissues were used for biomechanical testing, and seven tissues were used for hydroxyproline assay and quantitative polymerase chain reaction. Histopathology was determined qualitatively by hematoxylin and eosin and Masson staining, while fiber diameter was measured quantitatively by transmission electron microscopy. Biomechanical properties were measured using cyclic loading tests and load-to-failure tests. The healing outcome was quantitatively judged through healing-related genes and proteins. At 4 weeks and 8 weeks postoperatively, the peracetic acid-ethanol combined irradiation group visually demonstrated the best continuity and minimal peripheral adhesions. Histologic staining showed that tendon fibers in the peracetic acid-ethanol combined irradiation group maintained consistent alignment without notable disruptions or discontinuities, and there was a qualitatively observed increase in the number of infiltrating cells compared with the control group at the 4-week timepoint (444 ± 49 /mm 2 versus 256 ± 43 /mm 2 , mean difference 188 /mm 2 [95% confidence interval 96 to 281]; p < 0.001). At 8 weeks postoperatively, the tendon fiber diameter in the peracetic acid-ethanol combined irradiation groups was similar to that of the control group (0.23 ± 0.04 µm versus 0.21 ± 0.03 µm, mean difference 0.02 µm [95% CI -0.04 to 0.08]; p = 0.56). At 8 weeks postoperatively, the peracetic acid-ethanol combined irradiation group exhibited better properties in terms of both ultimate load (129 ± 15 N versus 89 ± 20 N, mean difference 40 N [95% CI 7 to 73]; p = 0.02) and energy absorption density (17 ± 6 kJ/m 2 versus 8 ± 4 kJ/m 2 , mean difference 8 kJ/m 2 [95% CI 0.7 to 16]; p = 0.004) compared with the control group. Gene expression analysis revealed higher expression levels of COL1A1 (2.1 ± 0.8 versus 1.0 ± 0, mean difference 1.1 [95% CI 0.1 to 2.1]; p = 0.003) and MMP13 (2.0 ± 0.8 versus 1.0 ± 0, mean difference 1.0 [95% CI 0.4 to 1.6]; p = 0.03) in the peracetic acid-ethanol combined irradiation group than in the control group. There was a higher amount of collagen Type I in tendons treated with peracetic acid-ethanol combined irradiation than in the control group (0.36 ± 0.03 versus 0.31 ± 0.04, mean difference 0.05 [95% CI 0.01 to 0.09]; p = 0.02). Treatment with peracetic acid-ethanol combined irradiation did not have any discernible adverse effect on the histology, fiber diameter, enzymatic resistance, collagen content, or biomechanical strength of the allograft tendons compared with the control group. Peracetic acid-ethanol combined irradiation treatment had a positive impact on remodeling of the extracellular matrix and realignment of collagen fibers. This sterilization method could be helpful to expand the scope and frequency with which allogeneic materials are applied. The long-term healing effect and strength of allograft tendons must be tested before clinical use, and it is necessary to conduct comparative studies on autografts and synthetic materials that are currently widely used clinically.