The Effect of Age and Intrinsic Aerobic Exercise Capacity on the Expression of Inflammation and Remodeling Markers in Rat Achilles Tendons

Old age, adiposity, and metabolic disorders are known as risk factors for chronic tendinopathy, which is a common problem in both athletes and the general population. However, the importance of these influencing factors has not yet been well understood. This study investigated alterations in gene expression and histology of Achilles tendons of young (10 weeks) and old (100 weeks) rats bred for low (low capacity runners, LCR) and high (high capacity runners, HCR) intrinsic aerobic exercise capacity. In this rat model, LCR displayed a phenotype of reduced exercise capacity, higher body weight, and metabolic dysfunctions compared to HCR. We hypothesized that the risk factors for tendinopathy in old LCR could lead to more pronounced impairments in Achilles tendon tissue. In quantitative real-time PCR (qPCR), age-related downregulation of tenocyte markers e.g., tenomodulin, genes related to matrix modeling and remodeling (e.g., collagens, elastin, biglycan, fibronectin, tenascin C) as well as transforming growth factor beta 3 (Tgfb3) have been detected. Inflammation marker cyclooxygenase 2 (Cox2) was downregulated in old rats, while microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in old HCR and old LCR. In all groups, interleukin 6 (Il6), interleukin 1 beta (Il1b), and tumor necrosis factor alpha (Tnfa) showed no significant alteration. In histological evaluation, tendons of old rats had fewer and more elongated tenocyte nuclei than young rats. Even though a higher content of glycosaminoglycans, a sign of degeneration, was found in old HCR and LCR, no further signs of tendinopathy were detectable in tendons of old rats by histological evaluation. Low intrinsic aerobic exercise capacity and the associated phenotype did not show significant effects on gene expression and tendon histology. These findings indicate that aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue and suggests that other risk factors associated with intrinsic aerobic exercise capacity are less influential in this rat model.

Combination of Hyaluronan and Lyophilized Progenitor Cell Derivatives: Stabilization of Functional Hydrogel Products for Therapeutic Management of Tendinous Tissue Disorders

Cultured progenitor cells and derivatives have been used in various homologous applications of cutaneous and musculoskeletal regenerative medicine. Active pharmaceutical ingredients (API) in the form of progenitor cell derivatives such as lysates and lyophilizates were shown to retain function in controlled cellular models of wound repair. On the other hand, hyaluronan-based hydrogels are widely used as functional vehicles in therapeutic products for tendon tissue disorders. The aim of this study was the experimental characterization of formulations containing progenitor tenocyte-derived APIs and hyaluronan, for the assessment of ingredient compatibility and stability in view of eventual therapeutic applications in tendinopathies. Lyophilized APIs were determined to contain relatively low quantities of proteins and growth factors, while being physicochemically stable and possessing significant intrinsic antioxidant properties. Physical and rheological quantifications of the combination formulas were performed after hydrogen peroxide challenge, outlining significantly improved evolutive viscoelasticity values in accelerated degradation settings. Thus, potent effects of physicochemical protection or stability enhancement of hyaluronan by the incorporated APIs were observed. Finally, combination formulas were found to be easily injectable into ex vivo tendon tissues, confirming their compatibility with further translational clinical approaches. Overall, this study provides the technical bases for the development of progenitor tenocyte derivative-based injectable therapeutic products or devices, to potentially be applied in tendinous tissue disorders.

Secretome from In Vitro Mechanically Loaded Myoblasts Induces Tenocyte Migration, Transition to a Fibroblastic Phenotype and Suppression of Collagen Production

It is known that mechanical loading of muscles increases the strength of healing tendon tissue, but the mechanism involved remains elusive. We hypothesized that the secretome from myoblasts in co-culture with tenocytes affects tenocyte migration, cell phenotype, and collagen (Col) production and that the effect is dependent on different types of mechanical loading of myoblasts. To test this, we used an in vitro indirect transwell co-culture system. Myoblasts were mechanically loaded using the FlexCell® Tension system. Tenocyte cell migration, proliferation, apoptosis, collagen production, and several tenocyte markers were measured. The secretome from myoblasts decreased the Col I/III ratio and increased the expression of tenocyte specific markers as compared with tenocytes cultured alone. The secretome from statically loaded myoblasts significantly enhanced tenocyte migration and Col I/III ratio as compared with dynamic loading and controls. In addition, the secretome from statically loaded myoblasts induced tenocytes towards a myofibroblast-like phenotype. Taken together, these results demonstrate that the secretome from statically loaded myoblasts has a profound influence on tenocytes, affecting parameters that are related to the tendon healing process.

The application of human periodontal ligament stem cells and biomimetic silk scaffold for in situ tendon regeneration

Background With the development of tissue engineering, enhanced tendon regeneration could be achieved by exploiting suitable cell types and biomaterials. The accessibility, robust cell amplification ability, superior tendon differentiation potential, and immunomodulatory effects of human periodontal ligament stem cells (hPDLSCs) indicate their potential as ideal seed cells for tendon tissue engineering. Nevertheless, there are currently no reports of using PDLSCs as seed cells. Previous studies have confirmed the potential of silk scaffold for tendon tissue engineering. However, the biomimetic silk scaffold with tendon extracellular matrix (ECM)-like structure has not been systematically studied for in situ tendon regeneration. Therefore, this study aims to evaluate the effects of hPDLSCs and biomimetic silk scaffold on in situ tendon regeneration. Methods Human PDLSCs were isolated from extracted wisdom teeth. The differentiation potential of hPDLSCs towards osteo-, chondro-, and adipo-lineage was examined by cultured in different inducing media. Aligned and random silk scaffolds were fabricated by the controlled directional freezing technique. Scaffolds were characterized including surface structure, water contact angle, swelling ratio, degradation speed and mechanical properties. The biocompatibility of silk scaffolds was evaluated by live/dead staining, SEM observation, cell proliferation determination and immunofluorescent staining of deposited collagen type I. Subsequently, hPDLSCs were seeded on the aligned silk scaffold and transplanted into the ruptured rat Achilles tendon. Scaffolds without cells served as control groups. After 4 weeks, histology evaluation was carried out and macrophage polarization was examined to check the repair effects and immunomodulatory effects. Results Human PDLSCs were successfully isolated, and their multi-differentiation potential was confirmed. Compared with random scaffold, aligned silk scaffold had more elongated and aligned pores and promoted the proliferation and ordered arrangement of hPDLSCs. After implantation into rat Achilles tendon defect, hPDLSCs seeded aligned silk scaffold enhanced tendon repair with more tendon-like tissue formation after 4 weeks, as compared to the scaffold-only groups. Higher expression of CD206 and lower expression of iNOS, IL-1β and TNF-α were found in the hPDLSCs seeded aligned silk scaffold group, which revealed its modulation effect of macrophage polarization from M1 to M2 phenotype. Conclusions In summary, this study demonstrates the efficacy of hPDLSCs as seed cells and aligned silk scaffold as a tendon-mimetic scaffold for enhanced tendon tissue engineering, which may have broad implications for future tendon tissue engineering and regenerative medicine researches.

Amniotic Epithelial Stem Cells Counteract Acidic Degradation By-Products of Electrospun PLGA Scaffold by Improving Their Immunomodulatory Profile In Vitro

Electrospun poly(lactic-co-glycolic acid) (PLGA) scaffolds with highly aligned fibers (ha-PLGA) represent promising materials in the field of tendon tissue engineering (TE) due to their characteristics in mimicking fibrous extracellular matrix (ECM) of tendon native tissue. Among these properties, scaffold biodegradability must be controlled allowing its replacement by a neo-formed native tendon tissue in a controlled manner. In this study, ha-PLGA were subjected to hydrolytic degradation up to 20 weeks, under di-H2O and PBS conditions according to ISO 10993-13:2010. These were then characterized for their physical, morphological, and mechanical features. In vitro cytotoxicity tests were conducted on ovine amniotic epithelial stem cells (oAECs), up to 7 days, to assess the effect of non-buffered and buffered PLGA by-products at different concentrations on cell viability and their stimuli on oAECs’ immunomodulatory properties. The ha-PLGA scaffolds degraded slowly as evidenced by a slight decrease in mass loss (14%) and average molecular weight (35%), with estimated degradation half-time of about 40 weeks under di-H2O. The ultrastructure morphology of the scaffolds showed no significant fiber degradation even after 20 weeks, but alteration of fiber alignment was already evident at week 1. Moreover, mechanical properties decreased throughout the degradation times under wet as well as dry PBS conditions. The influence of acid degradation media on oAECs was dose-dependent, with a considerable effect at 7 days’ culture point. This effect was notably reduced by using buffered media. To a certain level, cells were able to compensate the generated inflammation-like microenvironment by upregulating IL-10 gene expression and favoring an anti-inflammatory rather than pro-inflammatory response. These in vitro results are essential to better understand the degradation behavior of ha-PLGA in vivo and the effect of their degradation by-products on affecting cell performance. Indeed, buffering the degradation milieu could represent a promising strategy to balance scaffold degradation. These findings give good hope with reference to the in vivo condition characterized by physiological buffering systems.

Tendon Tissue Repair in Prospective of Drug Delivery, Regenerative Medicines, and Innovative Bioscaffolds

The natural healing capacity of the tendon tissue is limited due to the hypovascular and cellular nature of this tissue. So far, several conventional approaches have been tested for tendon repair to accelerate the healing process, but all these approaches have their own advantages and limitations. Regenerative medicine and tissue engineering are interdisciplinary fields that aspire to develop novel medical devices, innovative bioscaffold, and nanomedicine, by combining different cell sources, biodegradable materials, immune modulators, and nanoparticles for tendon tissue repair. Different studies supported the idea that bioscaffolds can provide an alternative for tendon augmentation with an enormous therapeutic potentiality. However, available data are lacking to allow definitive conclusion on the use of bioscaffolds for tendon regeneration and repairing. In this review, we provide an overview of the current basic understanding and material science in the field of bioscaffolds, nanomedicine, and tissue engineering for tendon repair.

Tension Stimulation of Tenocytes in Aligned Hyaluronic Acid/Platelet-Rich Plasma-Polycaprolactone Core-Sheath Nanofiber Membrane Scaffold for Tendon Tissue Engineering

To recreate the in vivo niche for tendon tissue engineering in vitro, the characteristics of tendon tissue underlines the use of biochemical and biophysical cues during tenocyte culture. Herein, we prepare core-sheath nanofibers with polycaprolactone (PCL) sheath for mechanical support and hyaluronic acid (HA)/platelet-rich plasma (PRP) core for growth factor delivery. Three types of core-sheath nanofiber membrane scaffolds (CSNMS), consisting of random HA-PCL nanofibers (Random), random HA/PRP-PCL nanofibers (Random+) or aligned HA/PRP-PCL (Align+) nanofibers, were used to study response of rabbit tenocytes to biochemical (PRP) and biophysical (fiber alignment) stimulation. The core-sheath structures as well as other pertinent properties of CSNMS have been characterized, with Align+ showing the best mechanical properties. The unidirectional growth of tenocytes, as induced by aligned fiber topography, was confirmed from cell morphology and cytoskeleton expression. The combined effects of PRP and fiber alignment in Align+ CSNMS lead to enhanced cell proliferation rates, as well as upregulated gene expression and marker protein synthesis. Another biophysical cue on tenocytes was introduced by dynamic culture of tenocyte-seeded Align+ in a bioreactor with cyclic tension stimulation. Augmented by this biophysical beacon from mechanical loading, dynamic cell culture could shorten the time for tendon maturation in vitro, with improved cell proliferation rates and tenogenic phenotype maintenance, compared to static culture. Therefore, we successfully demonstrate how combined use of biochemical/topographical cues as well as mechanical stimulation could ameliorate cellular response of tenocytes in CSNMS, which can provide a functional in vitro environmental niche for tendon tissue engineering.

Combination of Shengji ointment and bromelain in the treatment of exposed tendons in diabetic foot ulcers: Study protocol for a non-blind, randomized, positive control clinical trial

Background : Diabetic foot ulcers often affect tendon tissue. Consequently, the infection may spread proximally along the tendon, leading to amputation or even the death of patients. Exposed, degenerated, and necrotic tendons are key factors affecting the healing of diabetic foot ulcers. The effective treatment of the tendon involvement may positively affect the prognosis. In clinical practice, treatment with Shengji ointment and bromelain induces islands of granulation tissue on the denatured tendon surface, which gradually grows and merges. Ideally, the exposed tendon is covered entirely by granulation tissue. This trial aims to assess the effect of a combined treatment regime of Shengji ointment, which has been shown to regenerate muscle tissue and pineapple protease in preventing the loss of function and amputation caused by tendon necrosis. This trial will provide high-quality evidence for the effectiveness of this combination in healing diabetic ulcers with tendon necrosis. Methods: The sample size will be 180 patients who will be randomly assigned 1:1 to a treatment group (90 patients) using Shengji ointment combined with bromelain and a control group (90 patients) using hydrocolloid dressing. Both groups will continue their conventional treatments, such as blood glucose and blood pressure medication, lipid regulation, antiplatelets, and others. The primary outcome will be the wound coverage with granulation tissue. Secondary outcomes will be the wound healing rate, amputation extent (where needed), time to granulation, and the Maryland Foot Score. Other efficacy outcomes will be the time to debridement of necrotic tendon tissue and granulation tissue score. Discussion: This study will treat patients with diabetic foot ulcers with exposed, degenerated, and necrotic tendons with Shengji ointment and bromelain. The trial aims to promote regeneration and healing, to preserve the limb and its function, and to develop a comprehensive and effective protocol that can be applied to promote the healing of exposed tendons in diabetic foot wounds. Trial registration: ChiCTR2000039327; date of registration: 2020-10-23. http://www.chictr.org.cn/com/25/showproj.aspx?proj=62692