A Tendon-Specific Double Reporter Transgenic Mouse Enables Tracking Cell Lineage and Functions Alteration In Vitro and In Vivo.

We generated and characterized a transgenic mouse line with the tendon-specific expression of a double fluorescent reporter system, which will fulfill an unmet need for animal models to support real-time monitoring cell behaviors during tendon development, growth, and repair in vitro and in vivo. The mScarlet red fluorescent protein is driven by the Scleraxis (Scx) promoter to report the cell lineage alteration. The blue fluorescent protein reporter is expressed under the control of the 3.6kb Collagen Type I Alpha 1 Chain (Col1a1) proximal promoter. In this promoter, the existence of two promoter regions named tendon-specific cis-acting elements (TSE1, TSE2) ensure the specific expression of blue fluorescent protein (BFP) in tendon tissue. Collagen I is a crucial marker for tendon regeneration that is a major component of healthy tendons. Thus, the alteration of function during tendon repair can be estimated by BFP expression. After mechanical stimulation, the expression of mScarlet and BFP increased in adipose-derived mesenchymal stem cells (ADMSCs) from our transgenic mouse line, and there was a rising trend on tendon key markers. These results suggest that our tendon-specific double reporter system is a novel model used to study cell re-differentiation and extracellular matrix alteration in vitro and in vivo.

Factors Involved in Morphogenesis in the Muscle-Tendon-Bone Complex.

A decline in the body's motor functions has been linked to decreased muscle mass and function in the oral cavity and throat; however, aging of the junctions of the muscles and bones has also been identified as an associated factor. Basic and clinical studies on the muscles, tendons and bones, each considered independently, have been published. In recent years, however, research has focused on muscle attachment as the muscle-tendon-bone complex from various perspectives, and there is a growing body of knowledge on SRY-box9 (Sox9) and Mohawk(Mkx), which has been identified as a common controlling factor and a key element. Myostatin, a factor that inhibits muscle growth, has been identified as a potential key element in the mechanisms of lifetime structural maintenance of the muscle-tendon-bone complex. Findings in recent studies have also uncovered aspects of the mechanisms of motor organ complex morphostasis in the superaged society of today and will lay the groundwork for treatments to prevent motor function decline in older adults.

Transcriptional profiling of mESC-derived tendon and fibrocartilage cell fate switch.

The transcriptional regulators underlying induction and differentiation of dense connective tissues such as tendon and related fibrocartilaginous tissues (meniscus and annulus fibrosus) remain largely unknown. Using an iterative approach informed by developmental cues and single cell RNA sequencing (scRNA-seq), we establish directed differentiation models to generate tendon and fibrocartilage cells from mouse embryonic stem cells (mESCs) by activation of TGFß and hedgehog pathways, achieving 90% induction efficiency. Transcriptional signatures of the mESC-derived cells recapitulate embryonic tendon and fibrocartilage signatures from the mouse tail. scRNA-seq further identify retinoic acid signaling as a critical regulator of cell fate switch between TGFß-induced tendon and fibrocartilage lineages. Trajectory analysis by RNA sequencing define transcriptional modules underlying tendon and fibrocartilage fate induction and identify molecules associated with lineage-specific differentiation. Finally, we successfully generate 3-dimensional engineered tissues using these differentiation protocols and show activation of mechanotransduction markers with dynamic tensile loading. These findings provide a serum-free approach to generate tendon and fibrocartilage cells and tissues at high efficiency for modeling development and disease.

Combinatorial effect of plasma treatment, fiber alignment and fiber scale of poly (ε-caprolactone)/collagen multiscale fibers in inducing tenogenesis in non-tenogenic media.

Tendon being a hypocellular, low vascularized tissue often requires assistance for restoration after complete tear. Tendon tissue engineering aims in the development of suitable scaffold that could support the regeneration of tendon after damage. The success of such scaffolds is dependent on its integration with the native tissue which in turn is influenced by the cell-material interaction. In this work aligned poly(ε-caprolactone)/collagen (PCL/collagen) multiscale fibers were developed and plasma treatment using argon, nitrogen and its combination was accessed for inducing tenogenic differentiation in mesenchymal stem cells. The developed fibers mimicked tendon extracellular matrix (ECM) which upon plasma treatment maintained moderate hydrophilicity. Oxygen and nitrogen containing groups were observed to be incorporated after argon and nitrogen treatment respectively. Statistically significant (p < 0.001) enhancement was observed in average and root mean square (RMS) roughness after plasma treatment with the maximum in argon treated fibers. Vitronectin was competitively (statistically significant, p < 0.05) adsorbed after argon and combination treatment whereas nitrogen treatment led to the competitive adsorption of fibronectin (statistically significant, p < 0.05). Human mesenchymal stem cells (hMSCs) showed enhanced proliferation and attachment on plasma treated fibers. Increased porosity due to the presence of sacrificial collagen nanofibers improved cell infiltration which was further enhanced upon plasma treatment. RhoA activation was observed (statistically significant, p < 0.05) on aligned PCL/collagen multiscale fibers and PCL microfibers, which proved its impact on tenogenic differentiation. Further enhancement in rhoA expression was observed on argon (p < 0.01) and combination plasma (p < 0.05) treated fibers. Tenogenic differentiation of hMSCs was enhanced (statistically significant) on argon plasma treated aligned fibers which was confirmed by the expression of scleraxis, mohawk (early markers) and tenomodulin (late marker) at protein level and mohawk, collagen I, collagen III (early markers), thrombospondin 4 and tenascin C (late markers) at gene level. Thus argon plasma treatment on aligned fibers is an effective method to induce tenogenesis even in non-tenogenic media.

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments.

BACKGROUND: The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. RESULTS: We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. CONCLUSION: Taken together, we discovered that Fgf9 may play an influential role in muscle-bone cross-talk during embryonic and postnatal development.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation.

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Inhibition of WNT/ß-catenin is necessary and sufficient to induce Scx expression in developing tendons of chicken limb.

The cell differentiation of the musculoskeletal system is highly coordinated during limb development. In the distal-most region of the limb, WNT and FGF released from the apical ectodermal ridge maintain mesenchymal cells in the undifferentiated stage. Once the cells stop receiving WNT and FGF, they respond to differentiation signals. Particularly during tendon development, mesenchymal cells enter the cell differentiation program once Scleraxis (Scx) gene expression occurs. Among the signals that trigger the cell differentiation programs, TGFß signaling has been found to be closely involved in tendon differentiation. However, whether Scx gene expression depends merely on TGFß signaling or other signals is still not fully understood. In the present study, considering that WNT/ß-catenin is an inhibitory signal of cell differentiation, we speculated possible antagonistic or additive effects between canonical Wnt/ß-catenin and TGFß/SMAD signaling pathways to control Scx gene expression. We found that the blockade of WNT/ß-catenin promoted Scx gene expression. In contrast, the inhibition of TGFß/SMAD signaling did not maintain Scx gene expression. Interestingly, the blockade of both WNT/ß-catenin and TGFß/SMAD signaling at the same time promoted Scx gene expression. Thus the inhibition of WNT/ß-catenin signaling appears to be necessary and sufficient to induce Scx gene expression.

Collagen XII mediated cellular and extracellular mechanisms regulate establishment of tendon structure and function.

Tendons have a uniaxially aligned structure with a hierarchical organization of collagen fibrils crucial for tendon function. Collagen XII is expressed in tendons and has been implicated in the regulation of fibrillogenesis. It is a non-fibrillar collagen belonging to the Fibril-Associated Collagens with Interrupted Triple Helices (FACIT) family. Mutations in COL12A1 cause myopathic Ehlers Danlos Syndrome with a clinical phenotype involving both joints and tendons supporting critical role(s) for collagen XII in tendon development and function. Here we demonstrate the molecular function of collagen XII during tendon development using a Col12a1 null mouse model. Col12a1 deficiency altered tenocyte shape, formation of interacting cell processes, and organization resulting in impaired cell-cell communication and disruption of hierarchal structure as well as decreased tissue stiffness. Immuno-localization revealed that collagen XII accumulated on the tenocyte surface and connected adjacent tenocytes by building matrix bridges between the cells, suggesting that collagen XII regulates intercellular communication. In addition, there was a decrease in fibrillar collagen I in collagen XII deficient tenocyte cultures compared with controls suggesting collagen XII signaling specifically alters tenocyte biosynthesis. This suggests that collagen XII provides feedback to tenocytes regulating extracellular collagen I. Together, the data indicate dual roles for collagen XII in determination of tendon structure and function. Through association with fibrils it functions in fibril packing, fiber assembly and stability. In addition, collagen XII influences tenocyte organization required for assembly of higher order structure; intercellular communication necessary to coordinate long range order and feedback on tenocytes influencing collagen synthesis. Integration of both regulatory roles is required for the acquisition of hierarchal structure and mechanical properties.

Musculoskeletal Tissue Engineering Using Fibrous Biomaterials.

In tissue engineering, scaffolds should provide the topological and physical cues as native tissues to guide cell adhesion, growth, migration, and differentiation. Fibrous structure is commonly present in human musculoskeletal tissues, including muscles, tendons, ligaments, and cartilage. Biomimetic fibrous scaffolds are thus critical for musculoskeletal tissue engineering. Electrospinning is a versatile technique for fabricating nanofibers from a variety of biomaterials. However, conventional electrospinning can only generate 2D nanofiber mats. Postprocessing methods are often needed to create 3D electrospun nanofiber scaffolds. In this chapter, we present two novel electrospinning-based scaffold fabrication techniques, which can generate 3D nanofiber scaffolds in one-station process: divergence electrospinning and hybrid 3D printing with parallel electrospinning. These techniques can be applied for engineering tissues with aligned fiber structures.

Loss of Smad4 in the scleraxis cell lineage results in postnatal joint contracture.

Growth of the musculoskeletal system requires precise coordination between bone, muscle, and tendon during development. Insufficient elongation of the muscle-tendon unit relative to bone growth results in joint contracture, a condition characterized by reduction or complete loss of joint range of motion. Here we establish a novel murine model of joint contracture by targeting Smad4 for deletion in the tendon cell lineage using Scleraxis-Cre (ScxCre). Smad4ScxCre mutants develop a joint contracture shortly after birth. The contracture is stochastic in direction and increases in severity with age. Smad4ScxCre mutant tendons exhibited a stable reduction in cellularity and a progressive reduction in extracellular matrix volume. Collagen fibril diameters were reduced in the Smad4ScxCre mutants, suggesting a role for Smad4 signaling in the regulation of matrix accumulation. Although ScxCre also has sporadic activity in both cartilage and muscle, we demonstrate an essential role for Smad4 loss in tendons for the development of joint contractures. Disrupting the canonical TGFß-pathway in Smad2;3ScxCre mutants did not result in joint contractures. Conversely, disrupting the BMP pathway by targeting BMP receptors (Alk3ScxCre/Alk6null) recapitulated many features of the Smad4ScxCre contracture phenotype, suggesting that joint contracture in Smad4ScxCre mutants is caused by disruption of BMP signaling. Overall, these results establish a model of murine postnatal joint contracture and a role for BMP signaling in tendon elongation and extracellular matrix accumulation.

Role of Scx+/Sox9+ cells as potential progenitor cells for postnatal supraspinatus enthesis formation and healing after injury in mice.

A multipotent cell population co-expressing a basic-helix-loop-helix transcription factor scleraxis (Scx) and SRY-box 9 (Sox9) has been shown to contribute to the establishment of entheses (tendon attachment sites) during mouse embryonic development. The present study aimed to investigate the involvement of Scx+/Sox9+ cells in the postnatal formation of fibrocartilaginous entheses and in the healing process after injury, using ScxGFP transgenic mice. We demonstrate that Scx+/Sox9+ cells are localized in layers at the insertion site during the postnatal formation of fibrocartilaginous entheses of supraspinatus tendon until postnatal 3 weeks. Further, these cells were rarely seen at postnatal 6 weeks, when mature fibrocartilaginous entheses were formed. Furthermore, we investigated the involvement of Scx+/Sox9+ cells in the healing process after supraspinatus tendon enthesis injury, comparing the responses of 20- and 3-week-old mice. In the healing process of 20-week-old mice with disorganized fibrovascular tissue in response to injury, a small number of Scx+/Sox9+ cells transiently appeared from 1 week after injury, but they were rarely seen at 4 weeks after injury. Meanwhile, in 3-week-old mice, a thin layer of fibrocartilaginous tissue with calcification was formed at healing enthesis at 4 weeks after injury. From 1 to 2 weeks after injury, more Scx+/Sox9+ cells, widely distributed at the injured site, were seen compared with the 20-week-old mice. At 4 weeks after injury, these cells were located near the surface of the recreated fibrocartilaginous layer. This spatiotemporal localization pattern of Scx+/Sox9+ cells at the injured enthesis in our 3-week-old mouse model was similar to that in postnatal fibrocartilaginous enthesis formation. These findings indicate that Scx+/Sox9+ cells may have a role as entheseal progenitor-like cells during postnatal maturation of fibrocartilaginous entheses and healing after injury in a manner similar to that seen in embryonic development.

Collagen XI regulates the acquisition of collagen fibril structure, organization and functional properties in tendon.

Collagen XI is a fibril-forming collagen that regulates collagen fibrillogenesis. Collagen XI is normally associated with collagen II-containing tissues such as cartilage, but it also is expressed broadly during development in collagen I-containing tissues, including tendons. The goals of this study are to define the roles of collagen XI in regulation of tendon fibrillar structure and the relationship to function. A conditional Col11a1-null mouse model was created to permit the spatial and temporal manipulation of Col11a1 expression. We hypothesize that collagen XI functions to regulate fibril assembly, organization and, therefore, tendon function. Previous work using cho mice with ablated Col11a1 alleles supported roles for collagen XI in tendon fibril assembly. Homozygous cho/cho mice have a perinatal lethal phenotype that limited the studies. To circumvent this, a conditional Col11a1flox/flox mouse model was created where exon 3 was flanked with loxP sites. Breeding with Scleraxis-Cre (Scx-Cre) mice yielded a tendon-specific Col11a1-null mouse line, Col11a1Δten/Δten. Col11a1flox/flox mice had no phenotype compared to wild type C57BL/6 mice and other control mice, e.g., Col11a1flox/flox and Scx-Cre. Col11a1flox/flox mice expressed Col11a1 mRNA at levels comparable to wild type and Scx-Cre mice. In contrast, in Col11a1Δten/Δten mice, Col11a1 mRNA expression decreased to baseline in flexor digitorum longus tendons (FDL). Collagen XI protein expression was absent in Col11a1Δten/Δten FDLs, and at ~50% in Col11a1+/Δten compared to controls. Phenotypically, Col11a1Δten/Δten mice had significantly decreased body weights (p < 0.001), grip strengths (p < 0.001), and with age developed gait impairment becoming hypomobile. In the absence of Col11a1, the tendon collagen fibrillar matrix was abnormal when analyzed using transmission electron microscopy. Reducing Col11a1 and, therefore collagen XI content, resulted in abnormal fibril structure, loss of normal fibril diameter control with a significant shift to small diameters and disrupted parallel alignment of fibrils. These alterations in matrix structure were observed in developing (day 4), maturing (day 30) and mature (day 60) mice. Altering the time of knockdown using inducible I-Col11a1-/- mice indicated that the primary regulatory foci for collagen XI was in development. In mature Col11a1Δten/Δten FDLs a significant decrease in the biomechanical properties was observed. The decrease in maximum stress and modulus suggest that fundamental differences in the material properties in the absence of Col11a1 expression underlie the mechanical deficiencies. These data demonstrate an essential role for collagen XI in regulation of tendon fibril assembly and organization occurring primarily during development.

Developmental changes in ACLs and semitendinosus tendons dimensions according to age in children.

PURPOSE: Managing anterior cruciate ligament (ACL) injuries in skeletally immature patients remains difficult. The main aim of this study was to retrospectively compile normative data on the cross-sectional area (CSA) of the semitendinosus tendon (ST) and the diameter of the ACL in children and young adults. METHODS: Knee magnetic resonance imaging (MRI) examinations were performed for a 2-year period in 132 patients (83 female and 49 male patients). The mean age was 14.9 years (8-18 years). Measurements of the ST CSA were performed on axial views in greyscale by two independent researchers. The ACL diameter was measured as well. RESULTS: The results show the CSA of the ST was related to age, and its growth was not linear. The highest growth rate of the CSA of the ST occurred at age 12-13 at the level of the femoral growth plate and at the level of the tibial plateau. The growth of the ACL diameter was linear until 18 years of age. CONCLUSIONS: ST growth (measured in CSA increments) is almost complete at the age of 13, even though the growth is not linear. ACL growth measured in diameter increments proceeds linearly from 8 to 18 years of age. MRI is a clinically useful tool for assessing hamstring tendon grafts preoperatively. LEVEL OF EVIDENCE: Level III, diagnostic studies.

The transcription factor scleraxis differentially regulates gene expression in tenocytes isolated at different developmental stages.

The transcription factor scleraxis (SCX) is expressed throughout tendon development and plays a key role in directing tendon wound healing. However, little is known regarding its role in fetal or young postnatal tendons, stages in development that are known for their enhanced regenerative capabilities. Here we used RNA-sequencing to compare the transcriptome of adult and fetal tenocytes following SCX knockdown. SCX knockdown had a larger effect on gene expression in fetal tenocytes, affecting 477 genes in comparison to the 183 genes affected in adult tenocytes, indicating that scleraxis-dependent processes may differ in these two developmental stages. Gene ontology, network and pathway analysis revealed an overrepresentation of extracellular matrix (ECM) remodelling processes within both comparisons. These included several matrix metalloproteinases, proteoglycans and collagens, some of which were also investigated in SCX knockdown tenocytes from young postnatal foals. Using chromatin immunoprecipitation, we also identified novel genes that SCX differentially interacts with in adult and fetal tenocytes. These results indicate a role for SCX in modulating ECM synthesis and breakdown and provide a useful dataset for further study into SCX gene regulation.

Tendon Regeneration After Partial-Thickness Peroneus Longus Tendon Harvesting: Magnetic Resonance Imaging Evaluation and In Vivo Animal Study.

BACKGROUND: In recent years, the use of the anterior half of the peroneus longus tendon (AHPLT) as an autograft source for ligament reconstruction has gained popularity. However, no reports are available regarding tendon regeneration after harvesting of the AHPLT. HYPOTHESIS: When half of the tendon is preserved during tendon harvesting, the quality of the regenerated tendon is better than that of the regenerated tendon after full-thickness harvesting. STUDY DESIGN: Case series; Level of evidence, 4; controlled laboratory study. METHODS: A total of 21 patients who underwent AHPLT harvesting for lower extremity ligament reconstruction participated in the magnetic resonance imaging (MRI) study to evaluate tendon regeneration 1 year after the harvesting. An in vivo animal study was performed to compare the quality of the regenerated tendon after partial-thickness and full-thickness tendon harvesting. A total of 30 adult female Sprague-Dawley rats were allocated to 2 groups-15 rats underwent partial-thickness Achilles tendon harvesting (partial-thickness harvesting [PTH] group), and 15 rats underwent full-thickness Achilles tendon harvesting (full-thickness harvesting [FTH] group). The quality of the regenerated tendons was compared 180 days after tendon harvesting. RESULTS: All 21 patients showed regeneration of the peroneus longus tendon (PLT) (homogeneously dark on both T1- and T2-weighted sequences). The cross-sectional area of the regenerated tendon divided by that of the preoperative tendon was 92.6% and 84.5% at 4 cm and 9 cm proximal to the tip of the distal fibula, respectively. In the animal study, the mean histologic score was better for the PTH group compared with the FTH group (9.17 ± 1.35 vs 14.72 ± 0.74; P < .001). The ultimate strength and the stiffness of the regenerated Achilles tendon were significantly higher for the PTH group compared with the FTH group (35.5 ± 8.3 vs 22.4 ± 8.3 N, P = .004; and 31.6 ± 7.7 vs 23.5 ± 4.8 N/mm, P = .016). CONCLUSION: The PLT was found to regenerate after partial-thickness harvesting on MRI. In the animal study, the quality of the regenerated tendon when half of the tendon was preserved during tendon harvesting was better than that after full-thickness tendon harvesting.

Switching of Sox9 expression during musculoskeletal system development.

The musculoskeletal system, which comprises muscles, tendons, and bones, is an efficient tissue complex that coordinates body movement and maintains structural stability. The process of its construction into a single functional and complex organization is unclear. SRY-box containing gene 9 (Sox9) is expressed initially in pluripotent cells and subsequently in ectodermal, endodermal, and mesodermal derivatives. This study investigated how Sox9 controls the development of each component of the musculoskeletal system. Sox9 was expressed in MTJ, tendon, and bone progenitor cells at E13 and in bone at E16. We detected Sox9 expression in muscle progenitor cells using double-transgenic mice and myoblastic cell lines. However, we found no Sox9 expression in developed muscle. A decrease in Sox9 expression in muscle-associated connective tissues, tendons, and bones led to hypoplasia of the cartilage and its attachment to tendons and muscle. These results showed that switching on Sox9 expression in each component (muscle, tendon, and bone) is essential for the development of the musculoskeletal system. Sox9 is expressed in not only tendon and bone progenitor cells but also muscle progenitor cells, and it controls musculoskeletal system development.

Connexin 43 Is Necessary for Murine Tendon Enthesis Formation and Response to Loading.

The enthesis is a mineralized fibrocartilage transition that attaches tendon to bone and is vital for musculoskeletal function. Despite recent studies demonstrating the necessity of muscle loading for enthesis formation, the mechanisms that regulate enthesis formation and mechanoresponsiveness remain unclear. Therefore, the current study investigated the role of the gap junction protein connexin 43 in these processes by deleting Gja1 (the Cx43 gene) in the tendon and enthesis. Compared with their wild-type (WT) counterparts, mice lacking Cx43 showed disrupted entheseal cell alignment, reduced mineralized fibrocartilage, and impaired biomechanical properties of the supraspinatus tendon entheses during postnatal development. Cx43-deficient mice also exhibited reduced ability to complete a treadmill running protocol but no apparent deficits in daily activity, metabolic indexes, shoulder muscle size, grip strength, and major trabecular bone properties of the adjacent humeral head. To examine enthesis mechanoresponsiveness, young adult mice were subjected to modest treadmill exercise. Gja1 deficiency in the tendon and enthesis reduced entheseal anabolic responses to treadmill exercise: WT mice had increased expression of Sox9, Ihh, and Gli1 and increased Brdu incorporation, whereas Cx43-deficient mice showed no changes or decreased levels with exercise. Collectively, the results demonstrated an essential role for Cx43 in postnatal tendon enthesis formation, function, and response to loading; results further provided evidence implicating a link between Cx43 function and the hedgehog signaling pathway. © 2020 American Society for Bone and Mineral Research.

An overview of decellularisation techniques of native tissues and tissue engineered products for bone, ligament and tendon regeneration.

Decellularised tissues and organs have been successfully used in a variety of tissue engineering/regenerative medicine applications. Because of the complexity of each tissue (size, porosity, extracellular matrix (ECM) composition etc.), there is no standardised protocol and the decellularisation methods vary widely, thus leading to heterogeneous outcomes. Physical, chemical, and enzymatic methods have been developed and optimised for each specific application and this review describes the most common strategies utilised to achieve decellularisation of soft and hard tissues. While removal of the DNA is the primary goal of decellularisation, it is generally achieved at the expense of ECM preservation due to the harsh chemical or enzymatic processing conditions. As denaturation of the native ECM has been associated with undesired host responses, decellularisation conditions aimed at effectively achieving simultaneous DNA removal and minimal ECM damage will be highlighted. Additionally, the utilisation of decellularised matrices in regenerative medicine is explored, as are the most recent strategies implemented to circumvent challenges in this field. In summary, this review focusses on the latest advancements and future perspectives in the utilisation of natural ECM for the decoration of synthetic porous scaffolds.

Flexor Tendon: Development, Healing, Adhesion Formation, and Contributing Growth Factors.

Management of flexor tendon injuries of the hand remains a major clinical problem. Even with intricate repair, adhesion formation remains a common complication. Significant progress has been made to better understand the mechanisms of healing and adhesion formation. However, there has been slow progress in the clinical prevention and reversal of flexor tendon adhesions. The goal of this article is to discuss recent literature relating to tendon development, tendon healing, and adhesion formation to identify areas in need of further research. Additional research is needed to understand and compare the molecular, cellular, and genetic mechanisms involved in flexor tendon morphogenesis, postoperative healing, and mechanical loading. Such knowledge is critical to determine how to improve repair outcomes and identify new therapeutic strategies to promote tissue regeneration and prevent adhesion formation.

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons.

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.