Enthesis Formation Research Articles

The 2024 Kappa Delta Young Investigator Award: Leveraging Insights From Development to Improve Adult Repair: Hedgehog Signaling as a Master Regulator of Enthesis Fibrocartilage Formation

The work in this article summarizes findings from our group on key biochemical cues that govern the formation and repair of tendons and ligaments. Specifically, we summarize the journey that started with a serendipitous discovery that is now being translated into novel therapies to improve tendon-to-bone repair outcomes. This journey began with the discovery that the Hedgehog (Hh) signaling pathway was expressed within the enthesis during development and that its primary role was to promote fibrocartilage production and maturation. Next, we developed an anterior cruciate ligament reconstruction model in novel transgenic mice that allowed us to discover that the Hh pathway promotes fibrocartilaginous tendon-to-bone attachments during the integration process. In addition, we established that the coordinated stages of zonal tendon-to-bone integration after anterior cruciate ligament reconstruction were comparable with the stages required for enthesis formation during development. Now that we have demonstrated that the Hh pathway is a potent therapeutic target, we are currently advancing these findings to develop drug delivery systems to improve tendon-to-bone repair. Ultimately, our group aims to establish key mechanisms that govern tendon and ligament formation that can be leveraged for novel regenerative therapies to improve clinical care.

Aging negatively affects postoperative recovery of biomechanical strength through decreased recruitment of Scx+/Sox9+ enthesis-related progenitors after rotator cuff repair in rats

BackgroundAlthough older adult patients have a higher retear rate after rotator cuff (RC) repair, the influence of aging on the healing process remains unclear. During mouse enthesis development, a multipotent progenitor co-expressing scleraxis (Scx) and SRY-box 9 (Sox9) contributes to enthesis formation. Scx+/Sox9+ cells may act as enthesis progenitors even during postnatal healing, and their number decreases with maturation. However, the pathophysiology of decreased RC healing ability due to aging remains unclear. We aimed to evaluate the effects of aging on tendon-to-bone healing after surgical RC repair in rats through biomechanical and histological analyses of Scx+ and Sox9+ progenitors in a ScxGFP transgenic rat model. MethodsThis was a controlled laboratory study. Adult Sprague-Dawley rats (n = 111) underwent unilateral surgery for supraspinatus tendon repair immediately after transection. The rats were divided into aged (95 weeks old) and control (12 weeks old) groups. The effects of aging were assessed using biomechanical tests at 6 weeks postoperatively and histologically at 2 and 6 weeks postoperatively. ScxGFP transgenic rats were used for the immunohistochemical assessment of Scx- and Sox9-expressing progenitors during the repair process. Sox9, Scx, and tenomodulin expression was assessed using real-time reverse transcription polymerase chain reaction. ResultsIn the biomechanical test at 6 weeks postoperatively, the aged group had lower ultimate load to failure (Control: 20.4 ± 6.1 N, Aged: 14.9 ± 6.6 N, P = 0.007), stiffness (Control: 16.1 ± 6.2 N/mm, Aged: 12.6 ± 5.5 N/mm, P = 0.023), and ultimate stress to failure (Control: 6.0 ± 3.4 N/mm2, Aged: 3.4 ± 2.5 N/mm2, P < 0.001) than the control group. The total histological score of the aged group was lower than that of the control group at 6 weeks postoperatively (Control: 8.8 ± 0.8, Aged: 5.8 ± 0.4, P = 0.029). Immunohistochemistry tests showed that the percentages of Sox9+ (Control: 6.6 ± 1.1, Aged: 2.0 ± 1.0, P = 0.029) and Scx+/Sox9+ (Control: 3.6 ± 0.8, Aged: 1.1 ± 0.6, P = 0.029) cells at the reparative site were lower in the aged group than in the control group at 2 weeks postoperatively. ConclusionIn a rat RC tendon-to-bone healing model, the decreased recruitment of Scx+/Sox9+ enthesis-related progenitor cells in the early phase may be associated with delayed reparative tissue remodeling in aging animals. These findings encourage the development of therapeutic strategies that biologically promote healing and reduce postoperative retears in older adult patients.

A role for TGFβ signaling in Gli1+ tendon and enthesis cells.

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFβ signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFβ signaling in Gli1+ cell function, the receptor for TGFβ, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFβ signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFβ signaling reduced proliferation and increased apoptosis. Invitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFβ controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFβ directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

LEVERAGING THE HEDGEHOG SIGNALLING PATHWAY TO IMPROVE TENDON-TO-BONE INTEGRATION

Despite extensive research aimed at improving surgical outcomes of enthesis injuries, re-tears remain a common problem, as the repairs often lead to fibrovascular scar as opposed to a zonal enthesis. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage [1]. During development, the hedgehog signaling pathway promotes the formation and maturation of fibrocartilage within the zonal tendon-to-bone enthesis [1-4]. However, whether this pathway has a similar role in adult zonal tendon-to-bone repair is not known. Therefore, we developed a murine anterior cruciate ligament (ACL) reconstruction model [5] to better understand the zonal tendon-to-bone repair process and perturb key developmental regulators to determine the extent to which they can promote successful repair in the adult. In doing so, we activated the hedgehog signaling pathway both genetically using transgenic mice and pharmacologically via agonist injections. We demonstrated that both treatments improved the formation of zonal attachments and tunnel integration strength [6]. These improved outcomes were due in part to hedgehog signaling's positive role in proliferation of the bone marrow stromal cell (bMSC) progenitor pool and subsequent fibrocartilage production of bMSC progeny cells that form the attachments. These results suggest that, similar to growth and development, hedgehog signaling promotes the production and maturation of fibrocartilage during tendon-to-bone integration in adults. Lastly, we developed localized drug delivery systems to further improve the treatment of these debilitating injuries in future translational studies.Acknowledgements: This work was supported by NIH R01AR076381, R21AR078429, R00AR067283, F31AR079840, T32AR007132, and P30AR069619, in addition to the McCabe Fund Pilot Award at the University of Pennsylvania.

A test of the New Coimbra method of recording entheseal changes as applied to the foot skeleton

AbstractPrior studies have indicated that entheseal changes throughout the skeleton are largely reflective of age and body mass with mixed results concerning activity patterns. To add to this debate, this study tests the effectiveness of employing the New Coimbra method to record entheseal changes of the calcaneus (Calcaneal tendon enthesis), hallucal metatarsal (Peroneus longus m. tendon, and Tibialis anterior m. tendon entheses), and fifth metatarsal (Peroneus brevis m. tendon enthesis) to understand past activity patterns. Presence and severity of entheseal changes were scored for 71 white adults from the William M. Bass Donated Skeletal Collection, and these scores were contrasted according to age, sex, stature, body mass (BMI), and occupation. Spearman's rank correlations show that age has the greatest impact on changes for the Calcaneal tendon enthesis, the Peroneus brevis m. tendon enthesis, and the Tibialis anterior m. tendon enthesis. For all of these entheses, entheseal scores increase as age increases. Stature is the largest contributing factor to changes in the Peroneus longus m. tendon enthesis, and this relationship is negative: entheseal scores decrease as stature increases. BMI has a limited effect on entheseal changes in the foot skeleton, and this relationship may instead be mediated by gait differences related to stature. No statistically significant relationships between entheseal changes and occupational standing ranks were found, although this may be related to the ways in which occupations were recorded and ranked. It is possible that statistically significant effects of physical activity patterns on the expression of entheseal changes could be documented using other measurements of activity patterns beyond occupational standing. These findings support prior studies that established age as a major contributor to entheseal changes but show that stature also contributes to enthesis formation in the foot.

Enthesis maturation in engineered ligaments is differentially driven by loads that mimic slow growth elongation and rapid cyclic muscle movement

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our tissue engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date. Statement of significanceEntheses attach ligaments to bone and are critical to load transfer; however, entheses do not regenerate with repair or replacement, contributing to high re-tear rates. Mechanical cues are critical to enthesis development in vivo but their individual contribution to maturation is largely unknown and they have not been widely explored in engineered replacements. Here, using a novel culture system, we provide new insight into how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, differentially affect enthesis maturation in engineered ligament-to-bone tissues, ultimately producing some of the most organized entheses to date. This system is a promising platform to explore cues regulating enthesis formation so to produce functional engineered replacements and better drive regeneration following repair.

Single-cell RNA sequencing reveals cellular and molecular heterogeneity in fibrocartilaginous enthesis formation.

The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open-source GSE182997 datasets (three samples) provided by Fang et al., we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And, we furtherly performed single-cell spatial transcriptomic sequencing on postnatal day 1 mouse enthesis, in order to deconvolute bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes.

Anatomical Tissue Engineering of the Anterior Cruciate Ligament Entheses.

The firm integration of anterior cruciate ligament (ACL) grafts into bones remains the most demanding challenge in ACL reconstruction, since graft loosening means graft failure. For a functional-tissue-engineered ACL substitute to be realized in future, robust bone attachment sites (entheses) have to be re-established. The latter comprise four tissue compartments (ligament, non-calcified and calcified fibrocartilage, separated by the tidemark, bone) forming a histological and biomechanical gradient at the attachment interface between the ACL and bone. The ACL enthesis is surrounded by the synovium and exposed to the intra-articular micromilieu. This review will picture and explain the peculiarities of these synovioentheseal complexes at the femoral and tibial attachment sites based on published data. Using this, emerging tissue engineering (TE) strategies addressing them will be discussed. Several material composites (e.g., polycaprolactone and silk fibroin) and manufacturing techniques (e.g., three-dimensional-/bio-printing, electrospinning, braiding and embroidering) have been applied to create zonal cell carriers (bi- or triphasic scaffolds) mimicking the ACL enthesis tissue gradients with appropriate topological parameters for zones. Functionalized or bioactive materials (e.g., collagen, tricalcium phosphate, hydroxyapatite and bioactive glass (BG)) or growth factors (e.g., bone morphogenetic proteins [BMP]-2) have been integrated to achieve the zone-dependent differentiation of precursor cells. However, the ACL entheses comprise individual (loading history) asymmetric and polar histoarchitectures. They result from the unique biomechanical microenvironment of overlapping tensile, compressive and shear forces involved in enthesis formation, maturation and maintenance. This review should provide a road map of key parameters to be considered in future in ACL interface TE approaches.

DLP 3D printing of high-resolution root scaffold with bionic bioactivity and biomechanics for personalized bio-root regeneration

Digital light projection (DLP) printing of hydroxyapatite (HAp) bioceramic provides a promising strategy for fabrication of complex personalized bio-tooth root scaffold with high-resolution. However, it is still a challenge to fabricate bionic bio-tooth root with satisfied bioactivity and biomechanics. This research studied the HAp-based bioceramic scaffold with bionic bioactivity and biomechanics for personalized bio-root regeneration. Compared to natural decellularized dentine (NDD) scaffolds with unitary shape and restricted mechanical properties, those DLP printing bio-tooth roots with natural size, high precision appearance, excellent structure, and a smooth surface were successfully manufactured, which met various shape and structure requirements for personalized bio-tooth regeneration. Moreover, the bioceramic sintering at 1250 °C enhanced the physicochemical properties of HAp and exhibited good elastic modulus (11.72 ± 0.53 GPa), which was almost twice of early NDD (4.76 ± 0.75 GPa). To further improve the surface activity of sintered biomimetic, the nano-HAw (nano-hydroxyapatite whiskers) coating deposited by hydrothermal treatment increased the mechanical properties and surface hydrophilicity, which indicated positive effects on dental follicle stem cells (DFSCs)’ proliferation and enhanced the DFSCs osteoblastic differentiation in vitro. Subcutaneous transplantation in nude mice and in-situ transplantation in rat alveolar fossa proved that the nano-HAw-containing scaffold could promote the DFSCs differentiate into periodontal ligament-like enthesis formation. In conclusion, by combining the optimized sintering temperature and modified nano-HAw interface through hydrothermal treatment, the DLP-printing of HAp-based bioceramic with favorable bioactivity and biomechanics is a promising candidate for personalized bio-root regeneration.

Multiparity and Aging Impact Chondrogenic and Osteogenic Potential at Symphyseal Enthesis: New Insights into Interpubic Joint Remodeling

Pregnancy and childbirth cause adaptations to the birth canal to allow for delivery and fast recovery. To accommodate delivery through the birth canal, the pubic symphysis undergoes changes that lead to the interpubic ligament (IpL) and enthesis formation in primiparous mice. However, successive deliveries influence joint recovery. We aimed to understand tissue morphology and chondrogenic and osteogenic potential at symphyseal enthesis during pregnancy and postpartum in primiparous and multiparous senescent female mice. Morphological and molecular differences were found at the symphyseal enthesis among the study groups. Despite the apparent incapacity to restore cartilage in multiparous senescent animals, the symphyseal enthesis cells are active. However, these cells have reduced expression of chondrogenic and osteogenic markers and are immersed in densely packed collagen fibers contiguous to the persistent IpL. These findings may indicate alterations of key molecules in the progenitor cell population maintenance of the chondrocytic and osteogenic lineages at the symphyseal enthesis in multiparous senescent animals, possibly compromising the mouse joint histoarchitecture recovery. This sheds light on the distention of the birth canal and the pelvic floor that may play a role in pubic symphysis diastasis (PSD) and pelvic organ prolapse (POP), both in orthopedic and urogynecological practice in women.

GLI1 Deficiency Impairs the Tendon-Bone Healing after Anterior Cruciate Ligament Reconstruction: In Vivo Study Using Gli1-Transgenic Mice.

Hedgehog (Hh) signaling plays a fundamental role in the enthesis formation process and GLI-Kruppel family member GLI1 (Gli1) is a key downstream mediator. However, the role of Gli1 in tendon-bone healing after anterior cruciate ligament reconstruction (ACLR) is unknown. To evaluate the tendon-bone healing after ACLR in Gli1LacZ/LacZ (GLI1-NULL) mice, and compare Gli1LacZ/WT (GLI1-HET) and Gli1WT/WT wild type (WT) mice, a total of 45 mice, 15 mice each of GLI1-NULL, GLI1-HET and WT were used in this study. All mice underwent microsurgical ACLR at 12 weeks of age. Mice were euthanized at 4 weeks after surgery and were used for biomechanical testing, histological evaluation, and micro-CT analysis. The GLI1-NULL group had significantly lower biomechanical failure force, poorer histological healing, and lower BV/TV when compared with the WT and GLI1-HET groups. These significant differences were only observed at the femoral tunnel. Immunohistology staining showed positive expression of Indian hedgehog (IHH) and Patched 1(PTCH1) in all three groups, which indicated the activation of the Hh signal pathway. The GLI1 was negative in the GLI1-NULL group, validating the absence of GLI1 protein in these mice. These results proved that activation of the Hh signaling pathway occurs during ACL graft healing, and the function of Gli1 was necessary for tendon-bone healing. Healing in the femoral tunnel is more obviously impaired by Gli1 deficiency. Our findings provide further insight into the molecular mechanism of tendon-bone healing and suggest that Gli1 might represent a novel therapeutic target to improve tendon-bone healing after ACLR.

Driving native-like zonal enthesis formation in engineered ligaments using mechanical boundary conditions and β-tricalcium phosphate

Fibrocartilaginous entheses are structurally complex tissues that translate load from elastic ligaments to stiff bone via complex zonal gradients in the organization, mineralization, and cell phenotype. Currently, these complex gradients necessary for long-term mechanical function are not recreated in soft tissue-to-bone healing or engineered replacements, contributing to high failure rates. Previously, we developed a culture system that guides ligament fibroblasts to develop aligned native-sized collagen fibers using high-density collagen gels and mechanical boundary conditions. These constructs are promising ligament replacements, however functional ligament-to-bone attachments, or entheses, are required for long-term function in vivo. The objective of this study was to investigate the effect of compressive mechanical boundary conditions and the addition of beta-tricalcium phosphate (βTCP), a known osteoconductive agent, on the development of zonal ligament-to-bone entheses. We found that compressive boundary clamps, that restrict cellular contraction and produce a zonal tensile-compressive environment, guide ligament fibroblasts to produce 3 unique zones of collagen organization and zonal accumulation of glycosaminoglycans (GAGs), type II, and type X collagen. Ultimately, by 6 weeks of culture these constructs had similar organization and composition as immature bovine entheses. Further, βTCP applied under the clamp enhanced maturation of these entheses, leading to significantly increased tensile moduli, and zonal GAG accumulation, ALP activity, and calcium-phosphate accumulation, suggesting the initiation of endochondral ossification. This culture system produced some of the most organized entheses to date, closely mirroring early postnatal enthesis development, and provides an in vitro platform to better understand the cues that drive enthesis maturation in vivo. Statement of significanceLigaments are attached to bone via entheses. Entheses are complex tissues with gradients in organization, composition, and cell phenotype. Entheses are necessary for proper transfer of load from ligament-to-bone, but currently are not restored with healing or replacements. Here, we provide new insight into how tensile-compressive boundary conditions and βTCP drive zonal gradients in collagen organization, mineralization, and matrix composition, producing tissues similar to immature ligament-to-bone attachments. Collectively, this culture system uses a bottom-up approach with mechanical and biochemical cues to produce engineered replacements which closely mirror postnatal enthesis development. This culture system is a promising platform to better understanding the cues that regulate enthesis formation so to better drive enthesis regeneration following graft repair and in engineered replacements.

Radiographic and Histopathologic Evidences of Movement versus Immobilization Effects on Enthesis Formation in Rabbits

Enthesis joins tendon, ligament, or joint capsule to the bone, which are two very different tissues. As a result, it is more prone to stress and increased risk of rupture. The purposes of this study were to evaluate the role of limb movement or immobilization in enthesis healing after surgical repair and found out if radiography could be a reliable method for determining its prognosis. Fourteen New Zealand white rabbits were randomly divided into two groups. After detaching the Achilles tendon from the bone surface, the tendon was re-attached to the bone with a modified Kessler suture pattern. The limb of group 1 rabbits was externally fixed with the fiberglass cast. In group 2, however, no cast was used. Radiographs were taken after surgery and in weeks 2, 4, 6, and 8 post-operation. Samples were taken from the area after 8 weeks and stained by the hematoxylin-eosin method. The results were reported descriptively. Bone growth, increased chondrocytes in the cartilaginous region, and increased fibroblasts and fibrocytes were seen in group 1. While erosion, collagen fibers regularity, and tissue maturity were more predominant in group 2. Although early limb movement during the enthesis healing period helps to increase the regularity of collagen fibers in the tendinous zone of the enthesis, it does not increase the amount of callus in the bone and increases the risk of bone erosions and even fractures. While stabilizing the limb in the first two months of enthesis healing protects the bone against the forces and helps bone formation, which has a positive role in healing the enthesis.

Rotator cuff anchor repair: Histological changes associated with the recovering mechanical properties in a rabbit model.

Rotator cuff anchor repair is an increasingly common surgical procedure but the failure rate remains high. In order to improve surgical outcomes, a better understanding of postrepair histological and cellular responses at the tendon-bone attachment site (enthesis) is needed. We examined operated shoulders from 42 New Zealand female white rabbits. The animals underwent unilateral supraspinatus detachment followed by anchor repair a week later. To assess enthesis reformation, fibrocartilage staining area and the number of chondrocytes or nonchondrocytes were quantified at 0, 1, 2, and 4weeks postrepair. Using linear regression, we correlated these results with the load to failure and stiffness recorded during mechanical testing of the tendons. Fibrocartilage staining and chondrocyte number increased during the first 2weeks of enthesis formation. Between 2 and 4weeks, fibrocartilage staining plateaued while chondrocyte number decreased. The presence of nonchondrocytes remained similar between 0- and 1-week postrepair but then decreased abruptly at 2weeks. There was a linear correlation between fibrocartilage staining area and load to failure as well as stiffness. Nonchondrocyte number negatively correlated with stiffness. Early plateau of fibrocartilage staining and decrease in chondrocytes between 2 and 4weeks postrepair suggest a blunted enthesis formation response in our animal model.

Spatiotemporal localization of Scx+/Sox9+ progenitors during postnatal maturation of the fibrocartilaginous enthesis of the supraspinatus tendon in mice

A multipotent cell population co-expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) has been shown to contribute to the formation of entheses during mouse embryonic development. Previously, fibrocartilaginous enthesis was demonstrated to form during the postnatal stage. However, whether Scx+/Sox9+ progenitors participate in the postnatal formation and maturation of the fibrocartilage layer is unclear. This study aimed to investigate the contribution of Scx+/Sox9+ progenitors to fibrocartilaginous enthesis formation by evaluating their spatiotemporal localization during the postnatal maturation of the supraspinatus tendon enthesis using ScxGFP transgenic mice.

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.

Treated dentin matrix‐based scaffolds carrying TGF-β1/BMP4 for functional bio-root regeneration

Biomaterial scaffold-based bioengineered tooth root (bio-root) has been proven to be a prospective tool for the treatment of tooth loss. However, while emulating the mechanical integrity of native tooth root, the regenerated bio-root still has limited capacity to achieve long-term functional stability because of lacking the effective cervical seal. Enlightened by physiological cues from cervical seal development and recovery of native tooth, this study reports a bioinspired approach as incorporation of two pivotal odontogenic growth factors (TGF-β1 and BMP4) into biomaterial scaffold (treated dentin matrix, TDM) embodying a spatial interface gradient for functional enthesis formation to promote functional bio-root regeneration. The biological characteristics of dental follicle stem cells (DFSCs) induced by TGF-β1/BMP4 were studied by relevant gene and protein expressions in vitro as well as ectopic subcutaneous transplantation and orthotopic jaw bone implantation in vivo. We further fabricated artificial crown-TDM/TGF-β1/BMP4/DFSCs in beagle to verify the potential occlusional function of regenerated bio-root. Notably, TGF-β1/BMP4 synergistically activated proliferation, migration and osteoinductivity related to the enthesis differentiation of DFSCs as increasing expressions of Periostin, BSP, OPN, ALP, Vinculin, and Paxillin and ascending numbers of migrated cells. The TDM/TGF-β1/BMP4/DFSCs composites imparted the uniform and persistent inductive interface for neovascularization and canonical enthesis regeneration. During more than 5 months follow-up in beagle model, the effectively functional bio-roots were distinguished by outstanding biomechanical properties and healthy gingiva similar to native teeth. This notion of integrating TGF-β1/BMP4 with TDM and DFSCs provides an alternative strategy for biomimetic bio-root regeneration and other interface tissue engineering.

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.

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5-/- ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5-/- mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5-/- mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5-/- mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5-/- and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5-/- mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.

Engineering the hard-soft tissue interface with random-to-aligned nanofiber scaffolds.

Tendon injuries can be difficult to heal and have high rates of relapse due to stress concentrations caused by scar formation and the sutures used in surgical repair. Regeneration of the tendon/ligament-to-bone interface is critical to provide functional graft integration after injury. The objective of this study is to recreate the tendon-to-bone interface using a gradient scaffold which is fabricated by a one-station electrospinning process. Two cell phenotypes were grown on a poly-ε-caprolactone nanofiber scaffold which possesses a gradual transition from random to aligned nanofiber patterns. We assessed the effects of the polymer concentration, tip-to-collector distance, and electrospinning time on the microfiber diameter and density. Osteosarcoma and fibroblast cells were seeded on the random and aligned sections of scaffolds, respectively. A random-to-aligned cocultured tissue interface which mimicked the native transition in composition of enthesis was created after 96 h culturing. The results showed that the microstructure gradient influenced the cell morphology, tissue topology, and promoted enthesis formation. This study demonstrates a heterogeneous nanofiber scaffold strategy for interfacial tissue regeneration. It provides a potential solution for mimicking transitional interface between distinct tissues, and can be further developed as a heterogeneous cellular composition platform to facilitate the formation of multi-tissue complex systems.