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.

Ontogenetic growth and the development of a unique fibrocartilage entheses in Macropus fuliginosus.

Here we examine the bone histology of the femora and humeri of the Western Grey Kangaroo, Macropus fuliginosus. Our results reveal that bone modelling in response to ontogenetic growth and the development of tuberosities on the femur, and especially in the humerus, lead to a highly complex histology. We propose that the alternating fast and slow rates of bone deposition are seasonal, and are likely correlated with heterothermy related to ecological constraints during the summer months. In females, after the fourth growth mark in the femur, there is a distinctive change to a more lamellar textured bone deposition with sparse vascularisation, directly indicating a slowdown in growth. However, in males, the zones remain woven textured and well vascularised, which is indicative of continued fast growth. Here we also report the novel occurrence of a fibrocartilaginous entheses for the attachment of the m. quadratus femoris to the caudal femoral tuberosity. Using a combination of methodologies, we show that perimeter measurements of growth marks provide a reasonable estimation of the age of kangaroos. Additionally, we observed large individuals that have ceased diaphyseal appositional growth of the femur and the humerus, as well as fusion of the distal epiphyses of both bones, though the proximal epiphyses may remain unfused.

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.

Electrospinning of highly porous yet mechanically functional microfibrillar scaffolds at the human scale for ligament and tendon tissue engineering.

Electrospun fibers offer tremendous potential for tendon and ligament tissue engineering, yet developing porous scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge. Previous studies have rolled, braided, or stacked electrospun sheets to produce three-dimensional (3D) scaffolds with tailored sizes and mechanical properties. A common limitation with such approaches is the development of low porosity scaffolds that impede cellular infiltration into the body of the implant, thereby limiting their regenerative potential. Here, we demonstrate how varying the rotational speed of the collecting mandrel during the electrospinning of poly(ε-caprolactone) (PCL) can be used to limit inter-fiber fusion (or fiber welding). Increasing the fraction of unfused fibers reduced the flexural rigidity of the electrospun sheets, which in turn allowed us to bundle the fibers into 3D scaffolds with similar dimensions to the human anterior cruciate ligament (ACL). These unfused fibers allowed for higher levels of porosity (up to 95%) that facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. Mechanical testing demonstrated that the fiber-bundles possessed a Young's modulus approaching that of the native human ACL. The scaffolds were also capable of supporting the differentiation of MSCs towards either the fibrocartilage or ligament/tendon lineage. This novel electrospinning strategy could be used to produce mechanically functional, yet porous, scaffolds for a wide range of biomedical applications.

Application of a Demineralized Cortical Bone Matrix and Bone Marrow-Derived Mesenchymal Stem Cells in a Model of Chronic Rotator Cuff Degeneration.

BACKGROUND: The success of rotator cuff repair is primarily dependent on tendon-bone healing. Failure is common because weak scar tissue replaces the native enthesis, rendering it prone to reruptures. A demineralized bone matrix (DBM) consists of a network of collagen fibers that provide a sustained release of growth factors such as bone morphogenetic proteins. Previous studies have demonstrated that it can regenerate a fibrocartilaginous enthesis. HYPOTHESIS: The use of a DBM and mesenchymal stem cells (MSCs) at the healing enthesis will result in a higher bone mineral density at the tendon insertion and will enhance the regeneration of a morphologically superior enthesis when compared with an acellular human dermal matrix. STUDY DESIGN: Controlled laboratory study. METHODS: Eighteen female Wistar rats underwent unilateral detachment of the supraspinatus tendon. Three weeks later, tendon repair was carried out in animals randomized into 3 groups: group 1 received augmentation of the repair with a cortical allogenic DBM (n = 6); group 2 received augmentation with a nonmeshed, ultrathick, acellular human dermal matrix (n = 6); and group 3 underwent tendon-bone repair without a scaffold (n = 6). All animals received 1 × 106 MSCs delivered in fibrin glue to the repair site. Specimens were retrieved at 6 weeks postoperatively for histological analysis and the evaluation of bone mineral density. RESULTS: All groups demonstrated closure of the tendon-bone gap with a fibrocartilaginous enthesis. Although there were no significant differences in the enthesis maturation and modified Movin scores, repair augmented with a dermal matrix + MSCs exhibited a disorganized enthesis, abnormal collagen fiber arrangement, and greater cellularity compared with other MSC groups. Only repairs augmented with a DBM + MSCs reached a bone mineral density not significantly lower than nonoperated controls. CONCLUSION: A DBM enhanced with MSCs can augment rotator cuff healing at 6 weeks and restore bone mineral density at the enthesis to its preinjury levels. CLINICAL RELEVANCE: Biological augmentation of rotator cuff repair with a DBM and MSCs may reduce the incidence of retears, although further studies are required to determine its effectiveness.

Magnetic resonance imaging evaluation of the labrum to predict acetabular development in developmental dysplasia of the hip: A STROBE compliant study.

Recently, more attention has been paid to the role of the acetabular labrum. Therefore, we designed a retrospective cohort study of patients with residual hip dysplasia (RHD) who underwent magnetic resonance imaging (MRI). The objective of this study was to investigate an association between the MRI appearance of the labrum before school age and the natural history of RHD.We retrospectively investigated 45 hips of 40 patients who underwent MRI at about 3 and 4 years of age for RHD and were conservatively followed up with until 6 years of age or older. We evaluated the extent of eversion with a new method that measures the ß angle (MRI ß angle) using landmarks of the Graf method on MRI T2*-weighted images. The outcome measure was the Severin classification at the final follow-up. We compared the radiographic and MRI parameters at approximately 3 and 4 years of age between the good and poor outcome groups. The Student t test or one-way analysis of variance was used to compare the quantitative variables between groups. The Chi-square test was used to perform a ratio comparison.Although there was a significant difference in the center-edge (CE) angle, there was no significant difference in the acetabular index and the ratio of the presence of femoral head necrosis and the break in Shenton line between the good and poor groups. The MRI ß angle was significantly greater in the poor outcome group than in the normal and good outcome groups. The cut-off value of the MRI ß angle to differentiate the good and poor outcome groups was 65°, and its specificity and sensitivity were 92% and 53%, respectively.There was labral eversion on MRI scans in patients with RHD. Acetabular development before adolescence was poorer with greater labral eversion on MRI scans. The specificity for poor acetabular development was high when the MRI ß angle was 65° or more. The MRI ß angle has the potential to predict acetabular development.

Kartogenin with PRP promotes the formation of fibrocartilage zone in the tendon-bone interface.

Treatment of tendon-bone junction injuries is a challenge because tendon-bone interface often heals poorly and the fibrocartilage zone, which reduces stress concentration, at the interface is not formed. In this study, we used a compound called kartogenin (KGN) with platelet-rich plasma (PRP) to induce the formation of fibrocartilage zone in a rat tendon graft-bone tunnel model. The experimental rats received KGN-PRP or PRP injections in the tendon graft-bone tunnel interface. The control group received saline. After 4, 8 and 12 weeks, Safranin O staining of the tendon graft-bone tunnels revealed abundant proteoglycans in the KGN-PRP group indicating the formation of cartilage-like transition zone. Immunohistochemical and immuno-fluorescence staining revealed collagen types I (Col-I) and II (Col-II) in the newly formed fibrocartilage zone. Both fibrocartilage zone formation and maturation were healing time dependent. In contrast, the PRP and saline control groups had no cartilage-like tissues and minimal Col-I and Col-II staining. Some gaps were also present in the saline control group. Finally, pull-out strength in the KGN-PRP-treated group at 8 weeks was 1.4-fold higher than the PRP-treated group and 1.6-fold higher than the saline control group. These findings indicate that KGN, with PRP as a carrier, promotes the formation of fibrocartilage zone between the tendon graft and bone interface. Thus, KGN-PRP may be used as a convenient cell-free therapy in clinics to promote fibrocartilage zone formation in rotator calf repair and anterior cruciate ligament reconstruction, thereby enhancing the mechanical strength of the tendon-bone interface and hence the clinical outcome of these procedures. Copyright © 2017 John Wiley & Sons, Ltd.

The effect of light-emitting diode and laser on mandibular growth in rats.

OBJECTIVE: To evaluate the effect of a light-emitting diode (LED) and/or low-level laser (LLL) with or without the use of anterior bite jumping appliances (also known as functional appliances [FAs]) on mandibular growth in rats. MATERIALS AND METHODS: Thirty-six 8-week-old male Sprague-Dawley rats weighing 200 g were obtained from Charles River Canada (St. Constant, QC, Canada) and were divided into six groups of six animals each. Groups were as follows: group 1: LLL; group 2: LLL + FA; group 3: LED; group 4: LED + FA; group 5: FA; and group 6: control (no treatment). Mandibular growth was evaluated by histomorphometric and micro computed tomographic (microCT) analyses. RESULTS: The LED and LED + FA groups showed an increase in all condylar tissue parameters compared with other groups. CONCLUSION: The LED-treated groups showed more mandibular growth stimulation compared with the laser groups.

PTHrP regulates the modeling of cortical bone surfaces at fibrous insertion sites during growth.

The sites that receive ligament and tendon insertions (entheses) on the cortical surfaces of long bones are poorly understood, particularly regarding modeling and regulation. Entheses are classified as either fibrocartilaginous or fibrous based on their structures. Fibrous entheses typically insert into the metaphysis or diaphysis of a long bone, bear a periosteal component, and are modeled during long-bone growth. This modeling forms a root system by which the insertions attach to the cortical surface. In the case of the medial collateral ligament, modeling drives actual migration of the ligament along the cortical surface in order to accommodate linear growth, whereas in other sites modeling may excavate a deep cortical root system (eg, the teres major insertion) or a shallow root system with a large footprint (eg, the latissimus dorsi insertion). We report here that conditionally deleting parathyroid hormone-related protein (PTHrP) in fibrous entheses via Scleraxis-Cre targeting causes modeling to fail in these three iterations of osteoclast-driven enthesis excavation or migration. These iterations appear to represent formes frustes of a common modeling strategy, presumably differing from each other as a consequence of differences in biomechanical control. In sites in which PTHrP is not induced, either physiologically or because of conditional deletion, modeling does not take place and fibrocartilage is induced. These findings represent the initial genetic evidence that PTHrP regulates periosteal/intramembranous bone cell activity on cortical bone surfaces and indicate that PTHrP serves as a load-induced modeling tool in fibrous insertion sites during linear growth.

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold.

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor ß3 (TGF-ß3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-ß3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-ß3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-ß3 delayed the mineralization process of chondrocytes.

Mesenchymal stem cell-dependent formation of heterotopic tendon-bone insertions (osteotendinous junctions).

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

Indian hedgehog roles in post-natal TMJ development and organization.

Indian hedgehog (Ihh) is essential for embryonic mandibular condylar growth and disc primordium formation. To determine whether it regulates those processes during post-natal life, we ablated Ihh in cartilage of neonatal mice and assessed the consequences on temporomandibular joint (TMJ) growth and organization over age. Ihh deficiency caused condylar disorganization and growth retardation and reduced polymorphic cell layer proliferation. Expression of Sox9, Runx2, and Osterix was low, as was that of collagen II, collagen I, and aggrecan, thus altering the fibrocartilaginous nature of the condyle. Though a disc formed, it exhibited morphological defects, partial fusion with the glenoid bone surface, reduced synovial cavity space, and, unexpectedly, higher lubricin expression. Analysis of the data shows, for the first time, that continuous Ihh action is required for completion of post-natal TMJ growth and organization. Lubricin overexpression in mutants may represent a compensatory response to sustain TMJ movement and function.

Application of bone marrow-derived mesenchymal stem cells in a rotator cuff repair model.

BACKGROUND: Rotator cuff tendons heal to bone with interposed scar tissue, which makes repairs prone to failure. The purpose of this study was to determine if the application of bone marrow-derived mesenchymal stem cells (MSCs) can improve rotator cuff healing after repair. HYPOTHESIS: Application of MSCs to the repair site will result in superior results compared with controls on histologic and biomechanical testing. STUDY DESIGN: Controlled laboratory study. METHODS: Ninety-eight Lewis rats underwent unilateral detachment and repair of the supraspinatus tendon; 10 rats were used for MSC harvest. Eight animals were used for cell tracking with Ad-LacZ. The remaining animals received either 10(6) MSCs in a fibrin carrier, the carrier alone, or nothing at the repair site. Animals were sacrificed at 2 and 4 weeks for histologic analysis to determine the amount of fibrocartilage formation and the collagen organization at the insertion. Biomechanical testing was also performed. RESULTS: Specimens treated with Ad-LacZ-transduced MSCs exhibited more beta-galactosidase activity at the repair site compared with controls at both 2 and 4 weeks, although activity at 4 weeks was less than that at 2 weeks. There were no differences in the amount of new cartilage formation or collagen fiber organization between groups at either time point. There were also no differences in the biomechanical strength of the repairs, the cross-sectional area, peak stress to failure, or stiffness. CONCLUSION: The addition of MSCs to the healing rotator cuff insertion site did not improve the structure, composition, or strength of the healing tendon attachment site despite evidence that they are present and metabolically active. CLINICAL RELEVANCE: A biologic solution to the problem of tendon-to-bone healing in the rotator cuff remains elusive. The repair site may lack the cellular and/or molecular signals necessary to induce appropriate differentiation of transplanted cells. Further studies are needed to determine if cell-based strategies need to be combined with growth and differentiation factors to be effective.

Future perspectives of cell-based therapy for intervertebral disc disease.

Intervertebral disc degeneration is a primary cause of low back pain and has a high societal cost. Research on cell-based therapies for intervertebral disc disease is emerging, along with the interest in biological therapy to treat disc disease without reducing the mobility of the spinal motion segment. Results from animal models have shown promising results under limited conditions; however, future studies are needed to optimise efficacy, methodology, and safety. To advance research on cell-based therapy for intervertebral disc disease, a better understanding of the phenotype and differentiation of disc cells and of their microenvironment is essential. This article reviews current concepts in cell-based therapy for intervertebral disc disease, with updates on potential cell sources tested primarily using animal models, and discusses the hurdles to clinical application. Future perspectives for cell-based therapies for intervertebral disc disease are also discussed.

Age-related changes in expression of tissue inhibitor of metalloproteinases-3 associated with transition from the notochordal nucleus pulposus to the fibrocartilaginous nucleus pulposus in rabbit intervertebral disc.

STUDY DESIGN: Experimental study on age-related changes in expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) associated with transition from notochordal nucleus pulposus (NP) to fibrocartilaginous NP in rabbit intervertebral disc (IVD). OBJECTIVES: To identify roles of notochordal NP in extracellular matrix (ECM) metabolism of IVD. SUMMARY OF BACKGROUND DATA: One of most interesting properties of TIMP-3 is to inhibit aggrecanases in addition to matrix metalloproteinases. Balance of aggrecanase/TIMP-3 is critical to maintain homeostasis of ECM metabolism. METHODS: Four-week-old and 160-week-old male Japanese white rabbits were used. Age-related changes in IVDs were evaluated histologically using previously established grading system. Immunohistochemistry of TIMP-3 and semiquantitative reverse transcriptase-polymerase reaction (RT-PCR) of TIMP-3, a disintegrin and metalloproteinases with thrombospondin motifs (ADAMTS) 4, 5, and transforming growth factor-beta1 (TGF-beta1), were conducted. RESULTS: Semiquantitative assessment of histologic changes indicated that 4-week-old rabbit was equivalent to fetus to 2-year-old human and 160-week-old rabbit was equivalent to 11- to 30-year-old human, particularly 11- to 16-year-old, which corresponds to transition period from notochordal to fibrocartilaginous NP. Immunohistochemistry revealed that TIMP-3 was positive in 4-week-old rabbit only. Semiquantitative RT-PCR revealed that levels of expressions of TGF-beta1 and TIMP-3 mRNAs in 4-week-old were significantly higher than those in 160-week-old rabbits. There was no significant difference in expression of ADAMTS4 mRNA. ADAMTS5 mRNA was not detected or extremely low in both groups. Expression of TIMP-3 mRNA in NP was upregulated by TGF-beta1 but was not affected by IL-1beta. On the contrary, expression of ADAMTS4 mRNA was not upregulated by TGF-beta1 but was upregulated by IL-1beta. CONCLUSIONS: Levels of expression of TIMP-3 in notochordal NP were significantly lower in 160-week-old rabbits than those in 4-week-old rabbits. Decrease in expression of TIMP-3, possibly mediated in part by TGF-beta1, may cause imbalance of ADAMTS4/TIMP-3 ratio at transition period from notochordal to fibrocartilaginous NP.