Tendon Maturation 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.

Early intervention of extracorporeal shockwave therapy sustained positive long-term effect on rotator cuff healing: A randomized controlled trial with 3-year follow-up

BackgroundThe long-term effects of extracorporeal shockwave therapy (ESWT) on rotator cuff repair are unknown. ObjectivesTo investigate the functional outcomes and structural changes of ESWT at 3-year follow-up after rotator cuff repair. MethodsA randomized clinical trial was conducted, including patients who underwent rotator cuff repair. The patients were assigned to two groups based on whether they underwent radial ESWT 3 months postoperatively. The ESWT Group received 5 weeks of rehabilitation and ESWT weekly, whereas the CONTROL Group received only rehabilitation. Visual analog scale (VAS) pain score and functional scores were analyzed at 3 months (baseline), 6 months, and 3 years follow-up. In addition, MRI and ultrasonography were used to assess tendon maturation, integrity, tendon quality, acromiohumeral distance (AHD), and muscle fatty infiltration. ResultsFinally, 32 participants completed all the assessments. At the final follow-up, 2 patients in the ESWT Group (16.5 %) versus one patient in the CONTROL Group (6.25 %) had rotator cuff failure (P = 0.176). The ESWT treatment showed similar clinical outcomes in VAS-pain score, functional scores, tendon quality, AHD, and muscle fatty infiltration versus the CONTROL Group (Ps > 0.05). MRI analysis indicated improved tendon healing after ESWT treatment at both 6-month (P = 0.036) and 3-year follow-up (P = 0.028). ConclusionEarly intervention with radial ESWT sustained long-term effects on the healing of the repaired rotator cuff and similar functional outcomes at long-term follow-up compared with standard rehabilitation.

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.

Janus Membranes Patch Achieves High-Quality Tendon Repair: Inhibiting Exogenous Healing and Promoting Endogenous Healing.

The imbalance between endogenous and exogenous healing is the fundamental reason for the poor tendon healing. In this study, a Janus patch was developed to promote endogenous healing and inhibit exogenous healing, leading to improved tendon repair. The upper layer of the patch is a poly(dl-lactide-co-glycolide)/polycaprolactone (PLGA/PCL) nanomembrane (PMCP-NM) modified with poly(2-methylacryloxyethyl phosphocholine) (PMPC), which created a lubricated and antifouling surface, preventing cell invasion and mechanical activation. The lower layer is a PLGA/PCL fiber membrane loaded with fibrin (Fb) (Fb-NM), serving as a temporary chemotactic scaffold to regulate the regenerative microenvironment. In vitro, the Janus patch effectively reduced 92.41% cell adhesion and 79.89% motion friction. In vivo, the patch inhibited tendon adhesion through the TGF-β/Smad signaling pathway and promoted tendon maturation. This Janus patch is expected to provide a practical basis and theoretical guidance for high-quality soft tissue repair.

ADVANCED TENDON REPAIR STRATEGIES: FROM EVS TO MRNA-BASED APPROACHES

Approximately 30% of general practice consultations for musculoskeletal pain are related to tendon disorders, causing substantial personal suffering and enormous related healthcare costs. Treatments are often prone to long rehabilitation times, incomplete functional recovery, and secondary complications following surgical repair. Overall, due to their hypocellular and hypovascular nature, the regenerative capacity of tendons is very poor and intrinsically a disorganized scar tissue with inferior biomechanical properties forms after injury. Therefore, advanced therapeutic modalities need to be developed to enable functional tissue regeneration within a degenerative environment, moving beyond pure mechanical repair and overcoming the natural biological limits of tendon healing.Our recent studies have focused on developing biologically augmented treatment strategies for tendon injuries, aiming at restoring a physiological microenvironment and boosting endogenous tissue repair. Along these lines, we have demonstrated that the local application of mesenchymal stromal cell-derived small extracellular vesicles (sEVs) has the potential to improve rotator cuff tendon repair by modulating local inflammation and reduce fibrotic scarring. In another approach, we investigated if the local delivery of the tendon ECM protein SPARC, which we previously demonstrated to be essential for tendon maturation and tissue homeostasis, has the potential to enhance tendon healing. Finally, I will present results demonstrating the utility of nanoparticle-delivered, chemically modified mRNAs (cmRNA) to improve tendon repair.

ADVANCING THE FABRICATION OF 3D MINI-TISSUE ENTHESIS CONSTRUCTS: CHARACTERIZATION OF BONE, TENDON, AND BMSC SPHEROIDS

AbstractObjectivesThe enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production.MethodsCell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×104, 5×104, 1×105, 2×105 in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope.3D mini-tissue constructs15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix.ResultsRTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×105 RTFs and 3×105 BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage.ConclusionsThis study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model.Declaration of Interest(a) fully declare any financial or other potential conflict of interest

Dynamic Superior Capsular Reconstruction for Irreparable Massive Rotator Cuff Tears: Histologic Analysis in a Rat Model and Short-term Clinical Evaluation

Background: Superior capsular reconstruction (SCR) has been demonstrated to be a valuable treatment for patients with irreparable massive rotator cuff tears (IMRCTs). However, the torn medial supraspinatus (SSP) tendons, which acted as dynamic stabilizers, were left untreated in conventional SCR, and the dynamic force from the SSP tendon was not restored. Purpose: To evaluate the effect of dynamic SCR (dSCR) on fascia-to-bone healing in a rat model, and to compare the short-term clinical effectiveness of dSCR and SCR using autologous fascia lata (FL) in patients with IMRCTs. Study Design: Controlled laboratory study and cohort study; Level of evidence, 3. Methods: A total of 50 rats were divided randomly into 2 groups: the dSCR group and the SCR group (25 rats per group). First, chronic IMRCTs were created, and then the torn tendons in both groups were subjected to SCR using autologous thoracolumbar fascial (TLF) grafts. The remnant of the SSP tendon was sutured to the medial part of the TLF graft in the dSCR group but not in the SCR group. Histologic sections were assessed at 1, 2, 4, 8, and 16 weeks postoperatively. In the clinical study, 22 patients (9 SCR, 13 dSCR) were analyzed. The recovery of shoulder function, including the active range of motion (ROM), visual analog scale (VAS), American Shoulder and Elbow Surgeons score, Constant score, and University of California Los Angeles score, acromiohumeral distance (AHD), and fatty infiltration, was evaluated before surgery and at the last follow-up. Results: Histologic analysis of the fascia-to-bone junction in the rat model showed that the TLF gradually migrated into tendon-like tissue over the rotator cuff defects in both groups, and the modified tendon maturation score of the fascia-to-bone interface in the dSCR group was higher than that in the SCR group at 4 weeks (12.20 ± 1.30 vs 14.60 ± 1.52; P = .004), 8 weeks (19.60 ± 1.14 vs 22.20 ± 1.10; P = .019), and 16 weeks (23.80 ± 0.84 vs 26.20 ± 0.84 P = .024). The dSCR group showed earlier fibrocartilage cell formation and angiogenesis. In the clinical study, all 22 patients completed a minimum of 12 months of follow-up after surgery, and the mean follow-up duration was 22.89 ± 7.59 months in the SCR group and 25.62 ± 7.32 months in the dSCR group. The patients in both groups showed significant improvements in terms of ROM, shoulder function scores, and AHD. At the last follow-up, abduction (56.67°± 27.39° vs 86.54°± 30.37°; P = .029), external rotation (25.00°± 9.35° vs 33.08°± 8.55°; P = .049), internal rotation cone rank (–2.78 ± 2.44 vs –4.38 ± 1.12; P = .049), VAS (–3.00 ± 0.87 vs –3.92 ± 0.95; P = .031) and Constant (47.89 ± 15.39 vs 59.15 ± 9.74; P = .048) scores, and the AHD improvement degree (3.06 ± 1.41 mm vs 4.38 ± 1.35 mm; P = .039) in the dSCR group were significantly improved compared with those in the SCR group. The results of fatty infiltration at the last follow-up showed that there was significant improvement compared with the preoperative results in both the conventional SCR (P = .036) and the dSCR (P = .001) groups. However, there were no significant differences between the 2 groups (P = .511). Conclusion: dSCR can promote faster fascia-to-bone healing in a rat model, and the dSCR technique could provide a preferable treatment option for patients with IMRCTs. Clinical Relevance: dSCR might restore the dynamic of SSP in some sense and then improve the fatty infiltration in the SSP.

Braided suture-reinforced fibrous yarn bundles as a scaffold for tendon tissue engineering in extensor digitorum tendon repair

In this study, we intend to develop an effective tendon tissue engineering scaffold that can provide mechanical stability and tendon regeneration ability. Using a novel electrospinning process, a biodegradable suture was continuously covered with aligned polycaprolactone fibers in core/sheath structure to produce a single yarn. The single yarn was braided together to fabricate a multi yarn (MY) scaffold, which can be surface modified with oxygen plasma and conjugated with heparin. The fibroblast growth factor 2 (FGF2) was bound to MY through bioaffinity between heparin and FGF2 to generate a functional scaffold (MY-FGF2) suitable for extensor digitorum tendon (EDT) repair. The physico-chemical properties of the scaffolds were characterized throughout the modification steps using microscopy, spectroscopy and mechanical testing. In vitro static culture using rabbit tendon-derived fibroblasts (rTFs) indicates combined effects of FGF2 and fiber alignment can enhance cell proliferation and extracellular matrix synthesis rates, as well as fasten tendon maturation. The cytoskeleton staining further endorses aligned morphology of fibers direct cell growth and collagen fiber deposition along the fiber axial direction, mimicking native tendon features. The dynamic culture in a bioreactor under uniaxial cyclic tensile loading authenticates 5 % mechanical stimulation can further increase cell proliferation and tenogenic differentiation rates compared to static culture. After mechanical stimulation for 7 days in vitro, the MY-FGF2/rTFs construct was used for repair of EDT defects in rabbits. The retrieved MY-FGF2/rTFs sample 6-week post-implantation shows superior mechanical properties and tendon regeneration abilities over acellular MY-FGF2. Taken together, we demonstrate a combinatory approach with MY-FGF2 where chemical and physical cues provided by FGF2, fiber alignment and dynamic stimulation contribute to tendon regeneration with a specific focus on EDT repair.

Tendon-Specific Dicer Deficient Mice Exhibit Hypoplastic Tendon Through the Downregulation of Tendon-Related Genes and MicroRNAs.

Tendon is a fibrous connective tissue, that is, transmitting the forces that permit body movement. However, tendon/ligament biology is still not fully understood and especially, the role of miRNAs in tendon/ligament is sparse and uncharacterized in in vivo models. The objectives of this study were to address the function of DICER using mice with tendon/ligament-specific deletion of Dicer (Dicer conditional knockout; cKO), and to identify key miRNAs in tendon/ligament. Dicer cKO mice exhibited hypoplastic tendons through structurally abnormal collagen fibrils with downregulation of tendon-related genes. The fragility of tendon did not significantly affect the tensile strength of tendon in Dicer cKO mice, but they showed larger dorsiflexion angle in gait compared with Control mice. We identified two miRNAs, miR-135a and miR-1247, which were highly expressed in the Achilles tendon of Control mice and were downregulated in the Achilles tendon of Dicer cKO mice compared with Control mice. miR-135a mimic increased the expression of tendon-related genes in injured Achilles tendon-derived fibroblasts. In this study, Dicer cKO mice exhibited immature tendons in which collagen fibrils have small diameter with the downregulation of tendon-related genes such as transcriptional factor, extracellular matrix, and miRNAs. Thus, DICER plays an important role in tendon maturation, and miR-135a may have the potential to become key miRNA for tendon maturation and healing.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

A Cd9+Cd271+ stem/progenitor population and the SHP2 pathway contribute to neonatal-to-adult switching that regulates tendon maturation

Tendon maturation lays the foundation for postnatal tendon development, its proper mechanical function, and regeneration, but the critical cell populations and the entangled mechanisms remain poorly understood. Here, by integrating the structural, mechanical, and molecular properties, we show that post-natal days 7-14 are the crucial transitional stage for mouse tendon maturation. We decode the cellular and molecular regulatory networks at the single-cell level. We find that a nerve growth factor (NGF)-secreting Cd9+Cd271+ tendon stem/progenitor cell population mainly prompts conversion from neonate to adult tendon. Through single-cell gene regulatory network analysis, invitro inhibitor identification, and invivo tendon-specific Shp2 deletion, we find that SHP2 signaling is a regulator for tendon maturation. Our research comprehensively reveals the dynamic cell population transition during tendon maturation, implementing insights into the critical roles of the maturation-related stem cell population and SHP2 signaling pathway during tendon differentiation and regeneration.

Histologic and biomechanical evaluation of the thoracolumbar fascia graft for massive rotator cuff tears in a rat model.

Fascial autografts, which are easily available grafts, have provided a promising option in patients with massive rotator cuff tears. However, no fascial autografts other than the fascia lata have been reported, and the exact healing process of the fascia-to-bone interface is not well understood. The objective of this study is to histologically and biomechanically evaluate the effect of the thoracolumbar fascia (TLF) on fascia-to-bone healing. A total of 88 rats were used in this study. Eight rats were killed at the beginning to form an intact control group, and the other rats were divided randomly into 2 groups (40 rats per group): the TLF augmentation group (TLF group) and the repair group (R group). The right supraspinatus was detached, and a 3 × 5 mm defect of the supraspinatus was created. The TLF was used to augment the torn supraspinatus in the TLF group, whereas in the R group, the torn supraspinatus was repaired in only a transosseous manner. Histology and biomechanics were assessed at 1, 2, 4, 8, and 16 weeks postoperatively. The modified tendon maturation score of the TLF group was higher than that of the R group at 8 weeks (23.00 ± 0.71 vs. 24.40 ± 0.89, P = .025) and 16 weeks (24.60 ± 0.55 vs. 26.40 ± 0.55, P ≤ .001). The TLF group showed a rapid vascular reaction, and the peak value appeared at 1 week. Later, the capillary density decreased, and almost no angiogenesis was observed at 8 weeks postoperatively. Immunohistochemistry results demonstrated a significantly higher percentage of collagen I in the TLF group at 4, 8, and 16 weeks (24.78% ± 2.76% vs. 20.67% ± 2.11% at 4 weeks, P = .046; 25.46% ± 1.77% vs. 21.49% ± 2.33% at 8 weeks, P= .026; 34.77% ± 2.25% vs. 30.01% ± 3.17% at 16 weeks, P = .040) postoperatively. Biomechanical tests revealed that the ultimate failure force in the TLF group was significantly higher than that in the R group at the final evaluation (29.13 ± 2.49 N vs. 23.10 ± 3.47 N, P = .022). The TLF autograft can promote a faster biological healing process and a better fixation strength. It could be used as an alternative reinforcement or bridging patch when the fascia lata is not appropriate or available for superior capsule reconstruction (SCR).

Development and Regeneration of Muscle, Tendon, and Myotendinous Junctions in Striated Skeletal Muscle.

Owing to a rapid increase in aging population in recent years, the deterioration of motor function in older adults has become an important social problem, and several studies have aimed to investigate the mechanisms underlying muscle function decline. Furthermore, structural maintenance of the muscle–tendon–bone complexes in the muscle attachment sites is important for motor function, particularly for joints; however, the development and regeneration of these complexes have not been studied thoroughly and require further elucidation. Recent studies have provided insights into the roles of mesenchymal progenitors in the development and regeneration of muscles and myotendinous junctions. In particular, studies on muscles and myotendinous junctions have—through the use of the recently developed scRNA-seq—reported the presence of syncytia, thereby suggesting that fibroblasts may be transformed into myoblasts in a BMP-dependent manner. In addition, the high mobility group box 1—a DNA-binding protein found in nuclei—is reportedly involved in muscle regeneration. Furthermore, studies have identified several factors required for the formation of locomotor apparatuses, e.g., tenomodulin (Tnmd) and mohawk (Mkx), which are essential for tendon maturation.

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.

Stretch-Induced Tenomodulin Expression Promotes Tenocyte Migration via F-Actin and Chromatin Remodeling.

The mechanosensitive gene tenomodulin (Tnmd) is implicated in tendon maturation and repair. However, the mechanism by which mechanical loading regulates Tnmd’s expression and its role in tenocyte migration is yet to be defined. Here, we show that Tnmd and migration were upregulated in uniaxial cyclic stress-stimulated tenocytes. The knockdown of Tnmd reduced cell migration in the presence and absence of mechanical loading, suggesting that Tnmd is involved in tenocyte migration. Moreover, the treatment of stress-stimulated tenocytes with the actin inhibitor latrunculin (Lat A), histone acetyltransferase inhibitor anacardic acid (ANA), or histone demethylases inhibitor GSK-J4 suppressed Tnmd expression and tenocyte migration. These results show that actin stress fiber formation and chromatin decondensation regulates Tnmd expression, which might then regulate tenocyte migration. Thus, this study proposes the involvement of the actin and chromatin mechanotransduction pathway in the regulation of Tnmd and reveals a novel role of Tnmd in tenocyte migration. The identification of Tnmd function in tenocyte migration provides insight into the molecular mechanisms involved in Tnmd-mediated tendon repair.

Mechanobiology in Tendon, Ligament, and Skeletal Muscle Tissue Engineering.

Tendon, ligament, and skeletal muscle are highly organized tissues that largely rely on a hierarchical collagenous matrix to withstand high tensile loads experienced in activities of daily life. This critical biomechanical role predisposes these tissues to injury, and current treatments fail to recapitulate the biomechanical function of native tissue. This has prompted researchers to pursue engineering functional tissue replacements, or dysfunction/disease/development models, by emulating in vivo stimuli within in vitro tissue engineering platforms-specifically mechanical stimulation, as well as active contraction in skeletal muscle. Mechanical loading is critical for matrix production and organization in the development, maturation, and maintenance of native tendon, ligament, and skeletal muscle, as well as their interfaces. Tissue engineers seek to harness these mechanobiological benefits using bioreactors to apply both static and dynamic mechanical stimulation to tissue constructs, and induce active contraction in engineered skeletal muscle. The vast majority of engineering approaches in these tissues are scaffold-based, providing interim structure and support to engineered constructs, and sufficient integrity to withstand mechanical loading. Alternatively, some recent studies have employed developmentally inspired scaffold-free techniques, relying on cellular self-assembly and matrix production to form tissue constructs. Whether utilizing a scaffold or not, incorporation of mechanobiological stimuli has been shown to improve the composition, structure, and biomechanical function of engineered tendon, ligament, and skeletal muscle. Together, these findings highlight the importance of mechanobiology and suggest how it can be leveraged to engineer these tissues and their interfaces, and to create functional multitissue constructs.

The effect of estrogen-like compound on rotator cuff tendon healing in a murine model.

Estrogen deficiency has been shown to negatively influence rotator cuff tendon healing. Therefore, the addition of an estrogen-like-compound (ELC) in a nonestrogen-deficient animal may improve the quality of a rotator cuff repair. The purpose of this study was to evaluate the effects of an ELC, diethylstilbestrol (DES), on tendon healing in a murine rotator cuff repair model. Thirty-three male wild-type mice (C57BL/6NJ) were randomly divided into three study groups. Group 1-unoperated mice with normal rotator cuff tendons. Groups 2 and 3 consisted of surgically repaired rotator cuff tendons; Group 2 (repair-only) was the standard repair group (no DES injected), whereas Group 3 (repair + DES) was the experimental repair group (injected with DES). Comparing the maximal thickness of calcified fibrocartilage to uncalcified fibrocartilage, the ratios for the control (intact tendon), repair-only, and repair + DES groups were 2:1, 0.9:1, and 1.7:1. RNA expression data demonstrated upregulation of chondrogenic, angiogenic, and tendon modulation genes in the repair- only group compared to the control (intact tendon) group (p < 0.04 for all), and that addition of DES further increased the osteogenic, angiogenic, and tendon modulation gene expression compared to the repair-only group (p < 0.02). Immunohistochemical analysis indicated that the addition of DES further increased osteogenic, angiogenic, and tendon maturation protein expression at the enthesis compared to standard repairs.

Effect of microfracture combined with biomimetic hydrogel scaffold on rotator cuff tendon-to-bone healing in rabbits

To assess the effect of microfracture and biomimetic hydrogel scaffold on tendon-to-bone healing in a rabbit rotator cuff tear model. Gelatin and methacrylic anhydride were used to synthesize gelatin methacryloyl (GelMA). Then the GelMA were treated with ultraviolet rays and vacuum freeze-drying method to obtain a biomimetic hydrogel scaffold. The morphology of the scaffold was observed by gross observation and scanning electron microscope. Degradation of the scaffold was determined at different time points. Twenty-four adult New Zealand rabbits, weighting 2.8-3.5 kg and male or female, were surgically created the bilateral acute rotator cuff tear models. One shoulder was treated with microfractures on the footprint and transosseous suture (control group, n=24). The other shoulder was treated with the same way, except for putting the scaffold on the footprint before transosseous suture (experimental group, n=24). The general conditions of rabbits were observed postoperatively. Tendon-to-bone healing was evaluated by gross observation, Micro-CT, HE staining, and bio-mechanical testing at 4 and 8 weeks after operation. The scaffold was white and has a porous structure with pore size of 31.7-89.9 μm, which degraded slowly in PBS solution. The degradation rate was about 95% at 18 days. All the rabbits survived to the completion of the experiment. Micro-CT showed that there was no obvious defect and re-tear at the tendon-to-bone interface in both groups. No difference was found in bone mineral density (BMD), tissue mineral density (TMD), and bone volume/total volume (BV/TV) between the two groups at 4 and 8 weeks postoperatively ( P>0.05). HE staining showed that the fibrous scar tissue was the main component at the tendon-to-bone interface in the control group at 4 and 8 weeks postoperatively; the disorderly arranged mineralized cartilage and fibrocartilage formation were observed at the tendon-to-bone interface in the experimental group at 4 weeks, and the orderly arranged cartilage formation was observed at 8 weeks. Besides, the tendon maturation scores of the experimental group were significantly higher than those of the control group at 4 and 8 weeks ( P<0.05). There was no significant difference in the ultimate load to failure and stiffness between the two groups at 4 weeks ( P>0.05); the ultimate load to failure at 8 weeks was significantly higher in the experiment group than in the control group ( t=4.162, P=0.009), and no significant difference was found in stiffness between the two groups at 8 weeks ( t=2.286, P=0.071). Compared with microfracture alone, microfracture combined with biomimetic hydrogel scaffold can enhance tendon-to-bone healing and improve the ultimate load to failure in rabbits.

Effect of kartogenin-loaded gelatin methacryloyl hydrogel scaffold with bone marrow stimulation for enthesis healing in rotator cuff repair

Strategies involving microfracture, biomaterials, growth factors, and chemical agents have been evaluated for improving enthesis healing. Kartogenin (KGN) promotes selective differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes. Gelatin methacryloyl (GelMA) is a promising biomaterial for engineering scaffolds and drug carriers. Herein, we investigated KGN-loaded GelMA hydrogel scaffolds with a bone marrow-stimulating technique for the repair of rotator cuff tear. KGN-loaded GelMA hydrogel scaffolds were obtained by ultraviolet GelMA crosslinking and vacuum freeze-drying. Fifty-four New Zealand rabbits were randomly divided into (1) repair only (control), (2) microfracture + repair (BMS), and (3) microfracture + repair augmentation with a KGN-loaded GelMA hydrogel scaffold (combined) groups. Tendons were repaired by transosseous sutures. The structure, degradation, and invitro KGN release of the scaffolds were characterized. Animals were euthanized 4, 8, and 12 weeks after repair. Enthesis healing was evaluated by macroscopy, microcomputed tomography, histology, and biomechanical tests. The KGN-loaded GelMA hydrogel scaffolds are porous with a 60.4 ± 28.2-μm average pore size, and they degrade quickly in 2.5 units/mL collagenase solution. Nearly 81% of KGN was released into phosphate-buffered saline within 12 hours, whereas the remaining KGN was released in 7 days. Macroscopically, the repaired tendons were attached to the footprint. No differences were detected postoperatively in microcomputed tomography analysis among groups. Fibrous scar tissue was the main component at the tendon-to-bone interface in the control group. Disorderly arranged cartilage formation was observed at the tendon-to-bone interface in the BMS and combined groups 4 weeks after repair; the combined group exhibited relatively more cartilage. The combined group showed improved cartilage regeneration 8 and 12 weeks after repair. Similar results were found in tendon maturation scores. The ultimate load to failure and stiffness of the repaired tendon increased in all 3 groups. At 4 weeks after repair, the BMS and combined groups exhibited greater ultimate load to failure than the control group, although there was no difference in stiffness among groups. The BMS and combined groups exhibited greater ultimate load to failure and stiffness than the control group, and the combined group exhibited better values than the BMS group at 8 and 12 weeks after repair. Compared with the bone marrow-stimulating technique, the KGN-loaded GelMA hydrogel scaffold with bone marrow stimulation improved enthesis healing by promoting fibrocartilage formation and improving the mechanical properties.

Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons from early specification. Scleraxis-null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)-a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2–5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.—Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin-Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.