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Tendon injury and healing involve significant changes to tissue biology and composition. Current techniques often require animal sacrifice or tissue destruction, limiting assessment of dynamic changes in tendons, including treatment response, disease development, rupture risk, and healing progression. Changes in tendon composition, such as altered collagen content, can significantly impact tendon mechanics and function. Analyses of compositional changes typically require ex vivo techniques with animal sacrifice or destruction of the tissue. In vivo evaluation of tendons is critical for longitudinal assessment. We hypothesize that photoacoustic ultrasound detects differences in collagen concentration throughout healing. We utilized photoacoustic ultrasound, a hybrid imaging modality that combines ultrasound and laser-induced photoacoustic signals to create detailed and high-resolution images of tendons, to identify its endogenous collagen composition. We correlated the photoacoustic signal to picrosirius red staining. The results show that the photoacoustic ultrasound-estimated collagen content in tendons correlates well with picrosirius red staining. This study demonstrates that photoacoustic ultrasound can assess injury-induced compositional changes within tendons and is the first study to image these targets in rat Achilles tendon in vivo. [ABSTRACT FROM AUTHOR]

Small leucine‐rich proteoglycans, such as decorin and biglycan, play pivotal roles in collagen fibrillogenesis during development, healing, and aging in tendon. Previous work has shown that the absence of decorin and biglycan affects fibril shape and mechanical properties during tendon healing. However, the roles of decorin and biglycan in the healing process of aged tendons are unclear. Therefore the objective of this study was to evaluate the differential roles of decorin and biglycan during healing of patellar tendon injury in aged mice. Aged (300 days old) female Dcn+/+/Bgn+/+ control (WT, n = 52), Dcnflox/flox (I‐Dcn−/−, n = 36), Bgnflox/flox (I‐Bgn−/−, n = 36), and compound Dcnflox/flox/Bgnflox/flox (I‐Dcn−/−/Bgn−/−, n = 36) mice with a tamoxifen‐inducible Cre were utilized. Targeted gene expression, collagen fibril diameter distributions, mechanical properties, and histological assays were employed to assess the effects of knockdown of decorin and/or biglycan at the time of injury. Knockdown resulted in alterations in fibril diameter distribution and scar area, but surprisingly did not lead to many differences in mechanical properties. Biglycan played a larger role in early healing stages, while decorin is more significant in later stages, particularly in scar remodeling. This study highlights some of the differential roles of biglycan and decorin in the regulation of fibril structure and scar area, as well as influencing gene expression during healing in aged mice. [ABSTRACT FROM AUTHOR]

Collagen V (Col5) is a quantitatively minor component of collagen fibrils comprising tendon, however, plays a crucial role in regulation of development and dynamic healing processes. Clinically, patients with COL5a1 haploinsufficiency, known as classic Ehlers‐Danlos Syndrome (cEDS), present with hyperextensible skin, joint instability and laxity, with females more likely to be affected. Previous studies in Col5‐deficient mice indicated that reduced Col5a1 expression leads to a reduction in stiffness, fibril deposition, and altered fibril structure. Additionally, Col5‐deficient male tendons demonstrated altered healing compared to wild‐type tendons, however female mice have not yet been studied utilizing this model. Along with clinical differences between sexes in cEDS patient populations, differences in hormone physiology may be a factor influencing tendon health. Therefore, the objective of this study was to utilize a Col5a1+/− female mouse model, to determine the effect of Col5 on tendon cell morphology, cell density, tissue composition, and mechanical properties throughout healing. We hypothesized that reduction in Col5 expression would result in an abnormal wound matrix post‐injury, resulting in reduced mechanical properties compared to normal tendons. Following patellar tendon surgery, mice were euthanized at 1, 3, and 6‐week post‐injury. Col5‐deficient tendons demonstrated altered and decreased healing compared to WT tendons. The lack of resolution in cellularity by 6‐week post‐injury in Col5‐deficient tendons influenced the decreased mechanical properties. Stiffness did not increase post‐injury in Col5‐deficient mice, and collagen fiber realignment was delayed during mechanical loading. Therefore, increased Col5a1 expression post‐injury is necessary to re‐establish matrix engagement and cellularity throughout tendon healing. [ABSTRACT FROM AUTHOR]

Biglycan, a small leucine‐rich proteoglycan (SLRP), is involved in collagen fibrillogenesis and also acts as a signaling molecule. Although decorin has been considered as the primary SLRP in developing and maintaining tendon structure and mechanics, more recent work using inducible knockdown models suggests that biglycan is involved in tendon homeostasis. The purpose of the study was to determine the role of biglycan in tendon homeostasis to maintain mechanical and structural integrity in aged mice. Aged (485 days old) female Bgn+/+ control (wild type [WT], n = 16) and 16 bitransgenic conditional Bgnflox/flox mice (I‐Bgn−/−, n = 16) with a tamoxifen‐inducible Cre (driven by ROSA) were utilized. After biglycan knockdown, the transgenic model demonstrated effective knockdown of the target gene without any compensation from other SLRPs or type I collagen. Patellar tendon cellularity was not modified after biglycan knockdown. However, biglycan knockdown had an impact on collagen fibrillogenesis with a higher percentage of small diameter fibrils (25–45 nm) and a lower percentage of medium size fibrils (150–165 nm) in I‐Bgn−/− tendons. Biglycan knockdown also induced a reduction in the midsubstance modulus and maximum stress compared to WT. Stress relaxation was reduced at 4% strain in I‐Bgn−/− tendons but no changes were observed in dynamic modulus and tan delta. As in mature tendons (120 days old), this study showed significant effects of biglycan knockdown on mechanical and structural properties of aged tendons only 30 days after knockdown. These data suggest that biglycan has a major role in maintaining homeostasis in aged tendon. [ABSTRACT FROM AUTHOR]

A growing interest in the mechanisms that govern tendon healing has resulted in the develop-ment of tools, such as the tamoxifen-inducible mouse knockdown model, to address these questions. However, tamoxifen is a selective estrogen receptor modulator and may interfere with the tendon healing process. The objective of this study was to evaluate the effects of tamoxifen on post-injury tendon mechanics in wild-type mice. The mice underwent treatment at the time of injury using an established mouse injury model and the injured tendons were evaluated 3 weeks post-injury. The treatment contained tamoxifen suspended in corn oil and was compared to a treatment with only corn oil, as well as mice with no treatment. Tendons were evaluated by measuring the quasi-static and viscoelastic mechanics, collagen fiber realignment, cellularity, and nuclear morphology. Mechanical testing of the tendons post-injury revealed no changes to viscoelastic mechanics, quasi-static mechanics, or collagen realignment during loading after tamoxifen treatment with the dosage regimen utilized (three daily injections of 4.5 mg/40 g body weight). Additionally, histological analysis revealed no changes to cellularity or cell nuclear shape. Overall, this study revealed that tamoxifen treatment at the time of tendon injury did not result in changes to tendon mechanics or the histological parameters at 3 weeks post-injury. [ABSTRACT FROM AUTHOR]

Rotator cuff repair failure remains common due to poor tendon healing, particularly at the enthesis. We previously showed that pulsed electromagnetic field (PEMF) therapy improved the mechanical properties of the rat supraspinatus tendon postoperatively. However, little is known about the mechanisms behind PEMF‐dependent contributions to improved healing in this injury model. The objective of this study was to determine the influence of PEMF treatment on tendon gene expression and cell composition, as well as bone microarchitecture and dynamic bone metabolism during early stages of healing. We hypothesized that PEMF treatment would amplify tendon‐healing related signaling pathways while mitigating inflammation and improve bone metabolism at the repair site. Rats underwent rotator cuff injury and repair followed by assignment to either control (non‐PEMF) or PEMF treatment groups. Gene and protein expression as well as tendon and bone histological assessments were performed 3, 7, 14, 21, and 28 days after injury. Gene expression data demonstrated an upregulation in the bone morphogenetic protein 2 signaling pathway and increases in pro‐osteogenic genes at the insertion, supporting important processes to re‐establish the tendon‐bone interface. PEMF also downregulated genes related to a fibrotic healing response. Anti‐inflammatory effects were demonstrated by both gene expression and macrophage phenotype. PEMF significantly increased the rate of kinetic bone formation directly adjacent to the tendon enthesis as well as the number of cuboidal surface osteoblasts (active osteoblasts) in the humeral head. This study has provided insight into how PEMF affects cellular and molecular processes in the supraspinatus tendon and adjacent bone after injury and repair. [ABSTRACT FROM AUTHOR]

Tendons are relatively hypovascular but become hypervascular during both injury and degeneration. This is due to the angiogenic response, or the formation of new blood vessels, to tissue injury. The objective of this study was to evaluate the effect of vascular modulation in the rat Achilles tendons during healing. Fischer rats received a bilateral Achilles incisional injury followed by local injections of vascular endothelial growth factor (VEGF), anti‐VEGF antibody (B20.4‐1‐1), or saline either early or late during the healing process. Vascular modulation and healing were evaluated using multiple in vivo ultrasound imaging modalities, in vivo functional assessment, and ex vivo measures of tendon compositional and mechanical properties. The late delivery of anti‐VEGF antibody, B20, caused a temporary reduction in healing capacity during a time point where vascularity was also decreased, and then an improvement during a later time point where vascularity was increased relative to control. However, VEGF delivery had a minimal impact on healing and vascular changes in both early and late delivery times. This study was the first to evaluate vascular changes using both in vivo imaging methods and ex vivo histological methods, as well as functional and mechanical outcomes associated with these vascular changes. Clinical significance: this study demonstrates that the alteration of vascular response through the delivery of angiogenic growth factors has the ability to alter tendon healing properties. [ABSTRACT FROM AUTHOR]

Nicotine is harmful to many bodily systems; however, the effects of nicotine on intra‐substance tendon healing remain largely unexplored. The purpose of this study was to examine the functional, structural, and biomechanical effects of nicotine on the healing of Achilles tendons in rats after an acute full‐thickness injury. Sixty Sprague‐Dawley rats were enrolled in this study. Half were exposed to 0.9% saline and half to 61 ng/mL of nicotine for 3 months via subcutaneous osmotic pumps. At 3 months, all rats underwent blunt full thickness transection of the left Achilles tendon and were immobilized for one week in plantarflexion. In‐vivo assays were conducted prior to injury, at 21 days, and at 42 days post‐injury and included the following: Functional limb assessment, passive joint mechanics, and vascular evaluation. Rats were sacrificed at 21 and 42 days for biomechanical testing and histologic evaluation. Rats exposed to nicotine demonstrated decreased vascularity, greater alteration in gait mechanics, and increased passive ROM of the ankle joint. Biomechanically, the nicotine tendons failed at lower maximum loads, were less stiff, had smaller cross‐sectional areas and had altered viscoelastic properties. Histologically, nicotine tendons demonstrated decreased vessel density at the injury site. This study demonstrates that nicotine leads to worse functional outcomes and biomechanical properties in tendons. The decreased vascularity in the nicotine group may suggest an underlying mechanism for inferior tendon healing. Patients should be counseled that using nicotine products increase their risk of poor tendon healing and may predispose them to tendon re‐rupture. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [ABSTRACT FROM AUTHOR]

Tendon is a vital musculoskeletal tissue that is prone to degeneration. Proper tendon maintenance requires complex interactions between extracellular matrix components that remain poorly understood. Collagen VI and biglycan are two matrix molecules that localize pericellularly within tendon and are critical regulators of tissue properties. While evidence suggests that collagen VI and biglycan interact within the tendon matrix, the relationship between the two molecules and its impact on tendon function remains unknown. We sought to elucidate potential coordinate roles of collagen VI and biglycan within tendon by defining tendon properties in knockout models of collagen VI, biglycan, or both molecules. We first demonstrated co-expression and co-localization of collagen VI and biglycan within the healing tendon, providing further evidence of cooperation between the two molecules during nascent tendon matrix formation. Deficiency in collagen VI and/or biglycan led to significant reductions in collagen fibril size and tendon mechanical properties. However, collagen VI-null tendons displayed larger reductions in fibril size and mechanics than seen in biglycan-null tendons. Interestingly, knockout of both molecules resulted in similar properties to collagen VI knockout alone. These results indicate distinct and non-additive roles for collagen VI and biglycan within tendon. This work provides better understanding of regulatory interactions between two critical tendon matrix molecules., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V.)