Tendon Development Research Articles

Embryo movement is required for limb tendon maturation

IntroductionFollowing early cell specification and tenocyte differentiation at the sites of future tendons, very little is known about how tendon maturation into robust load-bearing tissue is regulated. Between embryonic day (E)16 and E18 in the chick, there is a rapid change in mechanical properties which is dependent on normal embryo movement. However, the tissue, cellular and molecular changes that contribute to this transition are not well defined.MethodsHere we profiled aspects of late tendon development (collagen fibre alignment, cell organisation and Yap pathway activity), describing changes that coincide with tissue maturation. We compared effects of rigid (constant static loading) and flaccid (no loading) immobilisation to gain insight into developmental steps influenced by mechanical cues.ResultsWe show that YAP signalling is active and responsive to movement in late tendon. Collagen fibre alignment increased over time and under static loading. Cells organise into end-to-end stacked columns with increased distance between adjacent columns, where collagen fibres are deposited; this organisation was lost following both types of immobilisation.DiscussionWe conclude that specific aspects of tendon maturation require controlled levels of dynamic muscle-generated stimulation. Such a developmental approach to understanding how tendons are constructed will inform future work to engineer improved tensile load-bearing tissues.

Structural determinants of tendon multiscale mechanics and their sensitivity to mechanical stimulation during development in an embryonic chick model

There is an abrupt increase in the multiscale mechanical properties and load-bearing capabilities of tendon during development. While prior work has identified numerous changes that occur within the collagenous structure during this developmental period, the primary structural elements that give rise to this abrupt increase in mechanical functionality, and their mechanobiological sensitivity, remain unclear. To address this knowledge gap, we used a shear lag model along with ultrastructural imaging, biochemical/thermodynamic assays, and multiscale mechanical testing to investigate the dynamic structure-function relationships during late-stage embryonic chick development and to establish their sensitivity to mechanical stimulation. Mechanical testing and modeling suggested that the rapid increase in multiscale mechanics can be explained by increases in fibril length, intrafibrillar crosslinking, and fibril area fraction. To partially test this, we inhibited collagen crosslinking during development and observed a drastic reduction in multiscale mechanical behavior that was explained by a reduction in both fibril modulus and length. Using muscle paralysis to investigate mechanosensitivity, we observed a significantly impaired multiscale mechanical response despite minimal changes in fibril diameter and fibril area fraction. Additionally, the shear lag model found a trend toward lower fibril lengths with paralysis and experimental data found decreased crosslinking and fibril modulus values following flaccid paralysis. Together, these data suggest that both intrafibrillar crosslink formation and fibril elongation are critical to the formation of load-bearing capabilities in tenogenesis and are sensitive to mechanical loading. These findings provide critical insights into the biological and structural mechanisms that give rise to tensile load-bearing soft tissue. Statement of SignificanceDespite prior work investigating the structural and mechanical changes that occur during tendon development, there has not been a comprehensive analysis of how these simultaneous changes in structure and function are connected. In this study, we performed a comprehensive battery of mechanical and structural assessments of embryonic chick tendons and input these data into a shear lag model to estimate the individual importance of each structural change to the tendon mechanical properties. Additionally, we inhibited muscle activity in the embryos to evaluate the impact of mechanical stimulation on these evolving structure-function relationships during tendon development. These data provide insight into the primary structural elements that produce the tensile load-bearing capabilities of tendon, which will inform efforts to produce tissue engineered tendon replacements.

Skeletal Muscle Satellite Cells Co-Opt the Tenogenic Gene Scleraxis to Instruct Regeneration.

Skeletal muscles connect bones and tendons for locomotion and posture. Understanding the regenerative processes of muscle, bone and tendon is of importance to basic research and clinical applications. Despite their interconnections, distinct transcription factors have been reported to orchestrate each tissue's developmental and regenerative processes. Here we show that Scx expression is not detectable in adult muscle stem cells (also known as satellite cells, SCs) during quiescence. Scx expression begins in activated SCs and continues throughout regenerative myogenesis after injury. By SC-specific Scx gene inactivation (ScxcKO), we show that Scx function is required for SC expansion/renewal and robust new myofiber formation after injury. We combined single-cell RNA-sequencing and CUT&RUN to identify direct Scx target genes during muscle regeneration. These target genes help explain the muscle regeneration defects of ScxcKO, and are not overlapping with Scx -target genes identified in tendon development. Together with a recent finding of a subpopulation of Scx -expressing connective tissue fibroblasts with myogenic potential during early embryogenesis, we propose that regenerative and developmental myogenesis co-opt the Scx gene via different mechanisms.

The histone H3K9 methyltransferase G9a regulates tendon formation during development

G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9Cre/+ mice with G9afl/fl mice to generate G9afl/fl; Sox9Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.

Focal adhesion kinase regulates tendon cell mechanoresponse and physiological tendon development.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both invitro and invivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in invivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of insitu tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

Fetal development and growth of extensor tendons and their sheaths in the dorsal side of the wrist and hand: a histological study.

To understand considerable variations in number of adult extensor ten-dons at the dorsal side of the wrist and hand. We examined histological sections from 30 human fetuses of gestational age 7-39 weeks and crown-rump length 22-323 mm. At the carpal level, earlier or smaller fetuses showed a simpler configuration with fewer tendon slips, whereas later or larger fetuses had a greater number of tendon slips with considerable variations in number and topographical relationships. Tendon slips of the early extensor digitorum to the middle and ring fingers were always 1 or 2, but were seven or more at late term. A tendon of the extensor digitorum to the little fin-ger could not be distinguished from other tendons at the carpal bone level. At the meta-carpal bone level, however, it extended from the ring finger tendon toward two slips of the extensor digiti minimi tendon. At the distal carpal level at midterm and late term, in which the tendon sheath was lost, each of the extensor digitorum tendon slips further divided to provide a mediolateral linear cluster of thin bundles. This large number of tendon components joined and united together to provide a single tight tendon at the level of the metacarpophalangeal joints. The extensor pollicis longs tendon usually lost a membranous septation when it crossed the extensor carpi radialis brevis tendon. Tendon slips in the fourth and fifth canals of the sheath were most likely to reduce in number after birth depending on the mechanical demand.

The role of mechanotransduction in tendon.

Tendons play an important role in the maintenance of motor function by connecting muscles and bones and transmitting forces. Particularly, the role of mechanical stress has primarily focused on the key mechanism of tendon homeostasis, with much research on this topic. With the recent development of molecular biological techniques, the mechanisms of mechanical stress sensing and signal transduction have been gradually elucidated with the identification of mechanosensor in tendon cells and the master regulator in tendon development. This review provides a comprehensive overview of the structure and function of tendon tissue, including the role for physical performance and the detailed mechanism of mechanotransduction in its regulation. An important lesson is that the role of mechanotransduction in tendon tissue is only partially clarified, indicating the complexity of the mechanisms of motor function and fueling increasing interest in uncovering these mechanisms.

Stem Cell‐Based Therapies and Tissue Engineering Innovations for Tendinopathy: A Comprehensive Review of Current Strategies and Future Directions

AbstractTendon diseases commonly lead to physical disability, exerting a profound impact on the routine of affected patients. These conditions respond poorly to existing treatments, presenting a substantial challenge for orthopedic scientists. Research into clinical translational therapy has yet to yield highly versatile interventions capable of effectively addressing tendon diseases, including tendinopathy. Stem cell‐based therapies have emerged as a promising avenue for modifying the biological milieu through the secretion of regenerative and immunomodulatory factors. The current review provides an overview of the intricate tendon microenvironment, encompassing various tendon stem progenitor cells within distinct tendon sublocations, gene regulation, and pathways pertinent to tendon development, and the pathology of tendon diseases. Subsequently, the advantages of stem cell‐based therapies are discussed that utilize distinct types of autologous and allogeneic stem cells for tendon regeneration at the translational level. Moreover, this review outlines the challenges, gaps, and future innovations to propose a consolidated stem cell‐based therapy to treat tendinopathy. Finally, regenerative soluble therapies, insoluble bio‐active therapies, along with insoluble bio‐active therapies, and implantable 3D scaffolds for tendon tissue engineering are discussed, thereby presenting a pathway toward enhanced tissue regeneration and engineering.

Collagen Organization of Mouse Tendons and Mechanotransduction Gene Expression of Mouse Tendon Fibroblasts are Dependent on HIF1-alpha and Oxygen tension

Tendons are essential for transmitting skeletal muscle loads to bone yet tendons are unable to regenerate following load-induced injury.1–3 A major challenge in regenerating tendons lies in their low cell number, poor vascularization, and extracellular matrix (ECM) dense environment. The primary cells in tendons are fibroblasts (TFs) which form and organize the nascent collagen-rich ECM necessary for mechanical function and mechanotransduction. Eventually, TFs become embedded in this dense ECM with limited access to nutrients, where these cells also experience low levels of turnover. Development and elongation of tendon depends on the transcription factor, Scleraxis (Scx), which regulates the proliferation and differentiation of TF progenitors under mechanical loading while also promoting collagen deposition and organization.4-9 Collagen deposition is also controlled by hypoxia inducible factor-1a ( Hif1a), and we have recently shown that mouse Achilles tendons are under hypoxic stress during embryonic development, and the maturation of tendon enthesis relies on expression of Hif1a10,11. Hif1a regulates expression of Sox9 and genes associated with ECM deposition, such as Col1a1 and Col1a2.12 Yet, if and how hypoxia and Hif1a influences tendon organization and mechanotransductive gene expression remains unknown. In this study, we tested the hypothesis that both hypoxia and loss of Hif1a contribute to ECM organization in vivo and mechanotransductive gene expression in vitro. Hif1afl/wt wildtype (WT) and Hif1afl/fl; ScxCre+ ( Hif1acKO) mice were generated to study tendon ECM organization of developing (Postnatal day 14, P14) and mature (P56) Achilles tendons. Mouse hindlimbs were fixed, decalcified, paraffn sectioned, and stained with Picrosirius red to visualize collagen organization (n=3/genotype at both P14 and P56). Midsubstance Achilles tendons were imaged using brightfield and circular polarized light microscopy on an epifluorescent microscope (Leica). Images were acquired using a polarization camera and ThorCam software (ThorLabs Inc.) for quantitative polarized light microscopy (qPLI). Gene expression related to ECM ( Col1a1, Col1a2), progenitor markers ( Scx, Sox9), hypoxia ( Hif1a, Hif2a, Hilpda, Plin2), and mechanotransduction (Igfbp2, Igfbp3, and Igfbp9) was assessed using isolated and cultured mouse tail TFs from adult Hif1acKO and WT mice (n=3-4/group, age 4-6 months of age). Cells were cultured in normoxic (20% O2) or hypoxic (1% O2) conditions (for 1-wk) or were treated with a HIF stabilizer Roxadustat (FG-4592) to induce ‘pseudo-hypoxia’ or treated with a vehicle (DMSO) for 16h. RNA was isolated after 16h or 1-wk in culture and cDNA was used in SYBR-based quantitative PCR (qPCR). Data were normalized to 20% O2 conditions for fold change comparisons (2−ΔΔCq; Actb and Polr2a reference genes). Hif1acKO Achilles tendons had poor ECM organization compared to WT tendons at both ages. TFs from Hif1acKO mice had lower Hif1a expression compared to WT cells in both 20% and 1% O2. For WT TFs, Scx and Igfbp9 were downregulated in hypoxia compared to 20% O2 conditions, and the effect of hypoxia on Hif1acKO cells further downregulated Igfbp9 compared to 20% O2. Loss of Hif1a reduced Hilpda expression compared to WT TFs in hypoxia. FG treatment also resulted in increased Hilpda expression compared to hypoxia. Our findings support our hypothesis that Hif1a and hypoxia are regulators of ECM organization and mechanotransduction in mouse tendons. Identifying regulators of tendon ECM deposition and organization will inform therapeutic strategies for guided regeneration. REFERENCES: 1Fu C, et al. J Orthop Res. 2021.2Leong NL, et al. J Orthop Res. 2020. 3Shah SA, et al. J Orthop Res. 2017. 4Murchison ND, et al. Development. 2007. 5Killian ML, et al. FASEB j. 2016. 6Ideo K, et al. PLoS ONE. 2020.7Blitz E, et al. Development. 2013. 8Huang AH, et al. Development. 2019. 9Schweitzer R, et al. Development. 2001. 10Tata Z, et al. 2022, Proceedings of the ORS Annual Meeting. 11Phillips, et al. 2023, Proceedings of the ORS Annual Meeting.12Amarilio R, et al. Development. 2007. ACKNOWLEDGEMENTS: NIH NIAMS (R01AR079367, MLK; P30AR069620), NSF (CAREER 1944448, MLK). 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.

Two-Photon Microscopy for the Study of Tendons.

Two-photon microscopy has emerged as a potent tool for evaluating deep tissue cells and characterizing the alignment of the extracellular matrix (ECM) in various biological systems. This technique relies on nonlinear light-matter interactions to detect two distinct signals: the second harmonic generated (SHG) diffusion signal, which facilitates the visualization of collagen fibers and their orientation, and the near-infrared excitation signal for imaging ultraviolet excited autofluorescence. SHG imaging proves especially effective in visualizing collagen fibers due to the non-centrosymmetric crystalline structure of fibrillar collagen I. Given that tendons are matrix-rich tissues with a limited number of cells, their high collagen content makes them ideal candidates for analysis using two-photon microscopy. Consequently, two-photon microscopy offers a valuable means to analyze and characterize collagen abnormalities in tendons. Its application extends to studying tendon development, injuries, healing, and aging, enabling the comprehensive characterization of tendon cells and their interactions with the ECM under various conditions using two-photon microscopy tools. This protocol outlines the use of two-photon microscopy in tendon biology and presents an adapted methodology to achieve effective imaging and characterization of tendon cells during development and after injury. The method allows the utilization of thin microscopic sections to create a comprehensive image of the ECM within tendons and the cells that interact with this matrix. Most notably, the article showcases a technique to generate 3D images using two-photon microscopy in animal models.

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension.

Today's challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

Guiding Stem Cell Tenogenesis by Modulation of Growth Factor Signaling and Cell‐Scale Biophysical Cues in Bioengineered Constructs

AbstractTendon injuries and tendinopathies are increasingly prevalent health problems currently lacking effective treatments. Tissue engineering offers promising strategies to boost the low innate regenerative ability of tendons. Within this context, the simultaneous leveraging of both physical and biochemical cues by engineered scaffolding systems can be explored to promote a stronger tenogenic response from stem cells. Here, molecularly imprinted polymeric nanoparticles (MINPs) against transforming growth factor (TGF)‐β3 are combined with bioinspired anisotropic hydrogels to produce tenogenesis‐inductive constructs. MINPs are first solid phase‐imprinted against a TGF‐β3 epitope, achieving an affinity comparable to monoclonal antibodies. MINPs and magnetically‐responsive microfibers are then encapsulated together with adipose‐derived stem cells within gelatin‐based hydrogels, applying a magnetostatic field during gelation to align the microfibers. The created anisotropic microstructure guides cell growth and elongation unidirectionally, while MINPs act as artificial receptors for TGF‐β3, potentiating its paracrine action in the cellular microenvironment. The combination of both stimuli proves effective at increasing TGF‐β signaling, which promotes the expression of tendon‐associated genes and corresponding protein synthesis, suggesting that microstructural cues and biomolecule sequestration act in tandem to direct cell fate commitment. Overall, this system recapitulates several elements of tendon development, constituting a promising strategy for the regeneration of this tissue.

3D WRITING OF MULTICELLULAR TENDON-ON-CNC-CHIP MODELS

Relevant in vitro models emulating tendinopathies are highly needed to study these diseases and develop better treatments. We have recently proposed a new strategy that allows the automated 3D writing of microphysiological systems (MPS) embedded into its own biomimetic fibrillar support platform based on the self-assembling of cellulose nanocrystals (CNCs). Here, we explored this CNC platform for writing humanized in vitro tendon models using tendon decellularized extracellular matrix (dECM)-based bioinks to closely recapitulate the biophysical and biochemical cues of tendon cell niche and self-induce the tenogenic differentiation of stem cells. The proposed concept was further explored to study the crosstalk between the tendon core and vascular compartment.Porcine flexor tendons were decellularized to produce the dECM bioink hydrogel. hASCs were used as cell source and the bioink was directly printed within the CNC fluid gel. Tendon constructs were co-printed with compartmentalized microvascular structures to evaluate the cellular crosstalk with endothelial cells. The tendon-on-chip models showed high cell viability and proliferation during culture up to 21 days, and the synergy between dECM cues and printed patterns induced anisotropic cell organization similar to tendon tissues. Gene and protein analysis showed upregulation of the most important tendon related markers on tendon constructs, demonstrating that the biophysical and biochemical cues of dECM induced hASCs commitment toward tenogenic phenotype. In co-culture system, chemotaxis induced endothelial cells migration toward the tendon compartment, but without significant infiltration. Gene and protein expression results suggest that the cellular crosstalk established in this MPS with endothelial cells boosted hASCs tenogenesis, emulating tendon development stages.Overall, the proposed system might be promising for the automated fabrication of organotypic tendon-on-chip models that will be a valuable new tool to study tendon physiology, pathology, or the effect of drugs for the treatment of tendinopathy.Acknowledgments: EU H2020 for ERC-2017-CoG-772817; ERC-PoC-BioCHIPs-101069302; FCT/MCTES for 2022.05526.PTDC, 2020.03410.CEECIND, and PD/BD/129403/2017

Exploring the role of mechanical forces on tendon development using in vivo model: A scoping review.

Tendons transmit the muscle contraction forces to bones and drive joint movement throughout life. While extensive researchhave indicated the essentiality of mechanical forces on tendon development, a comprehensive understanding of the fundamental role of mechanical forces still needs to be impaerted. This scoping review aimed to summarize the current knowledge about the role of mechanical forces during the tendon developmental phase. The electronic database search using PubMed, performed in May 2023, yielded 651 articles, of which 16 met the prespecified inclusion criteria. We summarized and divided the methods to reduce the mechanical force into three groups: loss of muscle, muscle dysfunction, and weight-bearing regulation. In contrast, there were few studies to analyze the increased mechanical force model. Most studies suggested that mechanical force has some roles in tendon development in the embryo to postnatal phase. However, we identified species variability and methodological heterogeneity to modulatemechanical force. To establish a comprehensive understanding, methodological commonality to modulatethe mechanical force is needed in this field. Additionally, summarizing chronological changes in developmental processes across animal species helps to understand the essence of developmental tendon mechanobiology. Weexpect that the findings summarized in the current review serve as a groundwork for future study in the fields of tendon developmantal biology and mechanobiology.

Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat anterior cruciate ligament reconstruction model

When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.

The Functions and Mechanisms of Tendon Stem/Progenitor Cells in Tendon Healing.

Tendon injury is one of the prevalent disorders of the musculoskeletal system in orthopedics and is characterized by pain and limitation of joint function. Due to the difficulty of spontaneous tendon healing, and the scar tissue and low mechanical properties that usually develops after healing. Therefore, the healing of tendon injury remains a clinical challenge. Although there are a multitude of approaches to treating tendon injury, the therapeutic effects have not been satisfactory to date. Recent studies have shown that stem cell therapy has a facilitative effect on tendon healing. In particular, tendon stem/progenitor cells (TSPCs), a type of stem cell from tendon tissue, play an important role not only in tendon development and tendon homeostasis, but also in tendon healing. Compared to other stem cells, TSPCs have the potential to spontaneously differentiate into tenocytes and express higher levels of tendon-related genes. TSPCs promote tendon healing by three mechanisms: modulating the inflammatory response, promoting tenocyte proliferation, and accelerating collagen production and balancing extracellular matrix remodeling. However, current investigations have shown that TSPCs also have a negative effect on tendon healing. For example, misdifferentiation of TSPCs leads to a "failed healing response," which in turn leads to the development of chronic tendon injury (tendinopathy). The focus of this paper is to describe the characteristics of TSPCs and tenocytes, to demonstrate the roles of TSPCs in tendon healing, while discussing the approaches used to culture and differentiate TSPCs. In addition, the limitations of TSPCs in clinical application and their potential therapeutic strategies are elucidated.

Development of Fiber-Bragg-Grating-Integrated Artificial Embedded Tendon for Multifunctional Assessment of Temperature, Strain, and Curvature.

This paper presents the development and application of an optical fiber-embedded tendon based on biomimetic multifunctional structures. The tendon was fabricated using a thermocure resin (polyurethane) and the three optical fibers with one fiber Bragg grating (FBG) inscribed in each fiber. The first step in the FBG-integrated artificial tendon analysis is the mechanical properties assessment through stress-strain curves, which indicated the customization of the proposed device, since it is possible to tailor the Young's modulus and strain limit of the tendon as a function of the integrated optical fibers, where the coated and uncoated fibers lead to differences in both parameters, i.e., strain limits and Young's modulus. Then, the artificial tendon integrated with FBG sensors undergoes three types of characterization, which assesses the influence of temperature, single-axis strain, and curvature. Results show similarities in the temperature responses in all analyzed FBGs, where the variations are related to the heterogeneity on the polyurethane matrix distribution. In contrast, the FBGs embedded in the tendon presented a reduction in the strain sensitivity when compared with the bare FBGs (i.e., without the integration in the artificial tendon). Such results demonstrated a reduction in the sensitivity as high as 77% when compared with the bare FBGs, which is related to strain field distributions in the FBGs when embedded in the tendon. In addition, the curvature tests indicated variations in both optical power and wavelength shift, where both parameters are used on the angle estimation using the proposed multifunctional artificial tendon. To that extent, root mean squared error of around 3.25° is obtained when both spectral features are considered. Therefore, the proposed approach indicates a suitable method for the development of smart structures in which the multifunctional capability of the device leads to the possibility of using not only as a structural element in tendon-driven actuators and devices, but also as a sensor element for the different structures.

The rat Achilles and patellar tendons have similar increases in mechanical properties but become transcriptionally divergent during postnatal development.

The molecular events that drive post-natal tendon development are poorly characterized. In this study, we profiled morphological, mechanical, and transcriptional changes in the rat Achilles and patellar tendon before walking (P7), shortly after onset of walking (P14), and at motor maturity (P28). The Achilles and patellar tendons increased collagen content and mechanical strength similarly throughout post-natal development. However, at P28 the patellar tendon tended to display a higher maximal tensile load (MTL) (P=0.0524) than the Achilles tendon, but a similar ultimate tensile strength (UTS; load relative to cross-sectional area) probably due to its increased cross-sectional area during development. The tendons started transcriptionally similar, with overlapping PCA clusters at P7 and P14, before becoming transcriptionally distinct at P28. In both tendons, there was an increase in extracellular matrix (ECM) gene expression and a concomitant decrease in cell cycle and mitochondrial gene expression. The transcriptional divergence at P28 suggested that STAT signalling was lower in the patellar tendon where MTL increased the most. Treating engineered human ligaments with the STAT inhibitor itacitinib increased collagen content and MTL. Our results suggest that during post-natal development, cellular resources are initially allocated towards cell proliferation before shifting towards extracellular matrix development following the onset of mechanical load and provide potential targets for improving tendon function. KEY POINTS: Little is known about mechanisms of post-natal tendon growth. We characterized morphological, mechanical, and transcriptional changes that occur before (P7), and early (P14) and late after (P28) rats begin to walk. From P7 to P28, the Achilles tendon increased in length, whereas the patellar tendon increased in cross-sectional area. Mechanical and material properties of the Achilles and patellar tendon increased from P7 to P28. From P7 to P28, the Achilles and patellar tendons increased expression of ECM genes and decreased mitochondrial and cell cycle gene expression. Ribosomal gene expression also significantly decreased in the Achilles and tended to decrease in the patellar tendon. At P28, STAT1 signalling tended to be lower in the patellar tendon which had grown by increasing cross-sectional area and inhibiting STAT activation in vitro improved mechanical properties in engineered human ligaments.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Association of Severe Histological Degeneration of the Torn Supraspinatus Tendon and Retear After Arthroscopic Repair of Full-Thickness Rotator Cuff Tears Using the Suture Bridge Technique

Background: Previous studies have demonstrated several prognostic factors for retear after arthroscopic rotator cuff repair (ARCR). However, studies that histologically evaluate the quality of the torn rotator cuff (RC) tendon and its association with postoperative outcomes are limited. Purpose: To investigate factors associated with retear after ARCR using the suture bridge (SB) technique, including the degree of histological degeneration of the RC tendon edge. Study Design: Case-control study; Level of evidence, 3. Methods: The authors retrospectively evaluated 187 patients who underwent ARCR for full-thickness tears using the SB technique; intraoperative biopsy samples were taken to assess the degree of histological degeneration using the Bonar score. The cohort was divided into healed (n = 165) and retear (n = 22) groups according to magnetic resonance imaging results obtained ≥6 months postoperatively. The evaluation included preoperative patient data (age, sex, symptom duration, trauma history, history of heavy manual work, smoking habit, hypertension, diabetes mellitus, and hyperlipidemia) and radiological data (Hamada classification, Patte classification, Goutallier classification, and global fatty degeneration index [GFDI]). Additionally, intraoperative data (anteroposterior tear size, Lafosse classification for concomitant subscapularis tendon tear, and long head of biceps injury) and preoperative and postoperative clinical findings (active range of motion, University of California, Los Angeles [UCLA], score) were evaluated. Results: The retear rate was 11.8%. The retear group had a higher percentage of men (P = .031), higher Bonar score (P < .001), higher mean GFDI value (P = .002), higher rate of tear retraction degree (P = .010), and larger anteroposterior tear size (P = .020) than the healed group. The retear group had lower postoperative internal rotation (P = .031) and lower UCLA score (P < .001). Multivariate logistic regression analysis with a stepwise variable selection revealed anteroposterior tear size (odds ratio [OR], 2.4; 95% CI, 1.3-4.5; P = .004) and Bonar score (OR, 1.7; 95% CI, 1.3-2.4; P < .001) as independent predictors for a retear. Conclusion: The results indicate that end-stage severe tendon degeneration might affect retear. Therefore, further investigation on the progression mechanisms of tendon degeneration and development of methods to assess degenerative tissue might improve clinical outcomes after ARCR.