Tgfβ signaling stimulates glycolysis to promote the genesis of synovial joint interzone in developing mouse embryonic limbs.

The initial interzone cells for synovial joints originate from chondrocytes, but such critical transition is minimally understood. With single-cell RNA sequencing (scRNA-seq) of murine embryonic knee joint primordia, we discovered that heightened expression of glycolysis genes characterized developing interzone cells when compared to flanking chondrocytes. Conditional deletion of the glucose transporters and/or , in either the incipient pre-skeletal mesenchyme with or in chondrocytes with , disrupted interzone formation dose-dependently.

Reduction in postnatal weight-bearing does not alter the trajectory of murine meniscus growth and maturation.

The early postnatal period represents a critical window for the maturation and development of orthopedic tissues, including those within the knee joint. To understand how mechanical loading impacts the maturational trajectory of the meniscus and other tissues of the hindlimb, perturbation of postnatal weight bearing was achieved through surgical resection of the sciatic nerve in neonatal mice at 1 or 14 days old. Sciatic nerve resection (SNR) produced significant and persistent disruptions in gait, leading to reduced tibial length and reductions in Achilles tendon mechanical properties.

Rapid specialization and stiffening of the primitive matrix in developing articular cartilage and meniscus.

Understanding early patterning events in the extracellular matrix (ECM) formation can provide a blueprint for regenerative strategies to better recapitulate the function of native tissues. Currently, there is little knowledge on the initial, incipient ECM of articular cartilage and meniscus, two load-bearing counterparts of the knee joint. This study elucidated distinctive traits of their developing ECMs by studying the composition and biomechanics of these two tissues in mice from mid-gestation (embryonic day 15.

Synovial joint cavitation initiates with microcavities in interzone and is coupled to skeletal flexion and elongation in developing mouse embryo limbs.

The synovial cavity and its fluid are essential for joint function and lubrication, but their developmental biology remains largely obscure. Here, we analyzed E12.5 to E18.

Primary Cilia Direct Murine Articular Cartilage Tidemark Patterning Through Hedgehog Signaling and Ambulatory Load.

Articular cartilage (AC) is essential for body movement but is highly susceptible to degenerative diseases and has poor self-repair capacity. To improve current subpar regenerative treatments, developmental mechanisms of AC should be clarified and, specifically, how its postnatal multizone organization is acquired. Primary cilia are cell surface organelles crucial for mammalian tissue morphogenesis.

SAG therapy restores bone growth and reduces enchondroma incidence in a model of skeletal chondrodysplasias caused by Ihh deficiency.

Inactivation mutations in the Indian hedgehog (Ihh) gene in humans cause numerous skeletal chondrodysplasias, including acrocapitofemoral dysplasia, brachydactyly type A1, and human short stature. The lack of an appropriate human-relevant model to accurately represent these chondrodysplasias has hampered the identification of clinically effective treatments. Here, we established a mouse model of human skeletal dysplasia induced by Ihh gene mutations via ablation of Ihh in Aggrecan-positive (Acan+) cells using Aggrecan (Acan)-creERT transgenic mice.

Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid.

This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood.

The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1 cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1 cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers.

Hox11 expression characterizes developing zeugopod synovial joints and is coupled to postnatal articular cartilage morphogenesis into functional zones in mice.

Previous studies on mouse embryo limbs have established that interzone mesenchymal progenitor cells emerging at each prescribed joint site give rise to joint tissues over fetal time. These incipient tissues undergo structural maturation and morphogenesis postnatally, but underlying mechanisms of regulation remain unknown. Hox11 genes dictate overall zeugopod musculoskeletal patterning and skeletal element identities during development.

Premature Growth Plate Closure Caused by a Hedgehog Cancer Drug Is Preventable by Co-Administration of a Retinoid Antagonist in Mice.

The growth plates are key engines of skeletal development and growth and contain a top reserve zone followed by maturation zones of proliferating, prehypertrophic, and hypertrophic/mineralizing chondrocytes. Trauma or drug treatment of certain disorders can derange the growth plates and cause accelerated maturation and premature closure, one example being anti-hedgehog drugs such as LDE225 (Sonidegib) used against pediatric brain malignancies. Here we tested whether such acceleration and closure in LDE225-treated mice could be prevented by co-administration of a selective retinoid antagonist, based on previous studies showing that retinoid antagonists can slow down chondrocyte maturation rates.

Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3).

The Roles of Indian Hedgehog Signaling in TMJ Formation.

The temporomandibular joint (TMJ) is an intricate structure composed of the mandibular condyle, articular disc, and glenoid fossa in the temporal bone. Apical condylar cartilage is classified as a secondary cartilage, is fibrocartilaginous in nature, and is structurally distinct from growth plate and articular cartilage in long bones. Condylar cartilage is organized in distinct cellular layers that include a superficial layer that produces lubricants, a polymorphic/progenitor layer that contains stem/progenitor cells, and underlying layers of flattened and hypertrophic chondrocytes.

Joints in the appendicular skeleton: Developmental mechanisms and evolutionary influences.

The joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed Tomato ) than underlying epiphyseal chondrocytes (termed Tomato ).

Retinoic Receptor Signaling Regulates Hypertrophic Chondrocyte-specific Gene Expression.

Background/aim: Retinoid signaling is important for the maturation of growth-plate chondrocytes. The effect of retinoid receptor gamma (RARγ) signaling on the expression of genes in hypertrophic chondrocytes is unclear. This study investigated the role of RARγ signaling in regulation of hypertrophic chondrocyte-specific genes.

Roles of Ihh signaling in chondroprogenitor function in postnatal condylar cartilage.

Condylar articular cartilage in mouse temporomandibular joint develops from progenitor cells near the articulating surface that proliferate, undergo chondrogenesis and mature into hypertrophic chondrocytes. However, it remains unclear how these processes are regulated, particularly postnatally. Here we focused on the apical polymorphic layer rich in progenitors and asked whether the phenotype and fate of the cells require signaling by Indian hedgehog (Ihh) previously studied in developing long bones.

Biomechanical properties of murine TMJ articular disc and condyle cartilage via AFM-nanoindentation.

This study aims to quantify the biomechanical properties of murine temporomandibular joint (TMJ) articular disc and condyle cartilage using AFM-nanoindentation. For skeletally mature, 3-month old mice, the surface of condyle cartilage was found to be significantly stiffer (306±84kPa, mean±95% CI) than those of the superior (85±23kPa) and inferior (45±12kPa) sides of the articular disc. On the disc surface, significant heterogeneity was also detected across multiple anatomical sites, with the posterior end being the stiffest and central region being the softest.

Unsuspected osteochondroma-like outgrowths in the cranial base of Hereditary Multiple Exostoses patients and modeling and treatment with a BMP antagonist in mice.

Hereditary Multiple Exostoses (HME) is a rare pediatric disorder caused by loss-of-function mutations in the genes encoding the heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. HME is characterized by formation of cartilaginous outgrowths-called osteochondromas- next to the growth plates of many axial and appendicular skeletal elements. Surprisingly, it is not known whether such tumors also form in endochondral elements of the craniofacial skeleton.

Cell origin, volume and arrangement are drivers of articular cartilage formation, morphogenesis and response to injury in mouse limbs.

Limb synovial joints are composed of distinct tissues, but it is unclear which progenitors produce those tissues and how articular cartilage acquires its functional postnatal organization characterized by chondrocyte columns, zone-specific cell volumes and anisotropic matrix. Using novel Gdf5 (Gdf5-CE), Prg4-CE and Dkk3-CE mice mated to R26-Confetti or single-color reporters, we found that knee joint progenitors produced small non-migratory progenies and distinct local tissues over prenatal and postnatal time. Stereological imaging and quantification indicated that the columns present in juvenile-adult tibial articular cartilage consisted of non-daughter, partially overlapping lineage cells, likely reflecting cell rearrangement and stacking.

Epiprofin Regulates Enamel Formation and Tooth Morphogenesis by Controlling Epithelial-Mesenchymal Interactions During Tooth Development.

The synchronization of cell proliferation and cytodifferentiation between dental epithelial and mesenchymal cells is required for the morphogenesis of teeth with the correct functional shapes and optimum sizes. Epiprofin (Epfn), a transcription factor belonging to the Sp family, regulates dental epithelial cell proliferation and is essential for ameloblast and odontoblast differentiation. Epfn deficiency results in the lack of enamel and ironically the formation of extra teeth.

Osteophyte formation and matrix mineralization in a TMJ osteoarthritis mouse model are associated with ectopic hedgehog signaling.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa.