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. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions.

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 Gdf5CreERT2 (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. Zone-specific increases in cell volume were major drivers of tissue thickening, while cell proliferation or death played minor roles. Second harmonic generation with 2-photon microscopy showed that the collagen matrix went from being isotropic and scattered at young stages to being anisotropic and aligned along the cell stacks in adults. Progenitor tracing at prenatal or juvenile stages showed that joint injury provoked a massive and rapid increase in synovial Prg4+ and CD44+/P75+ cells some of which filling the injury site, while neighboring chondrocytes appeared unresponsive. Our data indicate that local cell populations produce distinct joint tissues and that articular cartilage growth and zonal organization are mainly brought about by cell volume expansion and topographical cell rearrangement. Synovial Prg4+ lineage progenitors are exquisitely responsive to acute injury and may represent pioneers in joint tissue repair.

Articular cartilage and joint development from embryogenesis to adulthood.

Within each synovial joint, the articular cartilage is uniquely adapted to bear dynamic compressive loads and shear forces throughout the joint's range of motion. Injury and age-related degeneration of the articular cartilage often lead to significant pain and disability, as the intrinsic repair capability of the tissue is extremely limited. Current surgical and biological treatment options have been unable to restore cartilage de novo. Before successful clinical cartilage restoration strategies can be developed, a better understanding of how the cartilage forms during normal development is essential. This review focuses on recent progress made towards addressing key questions about articular cartilage morphogenesis, including the origin of synovial joint progenitor cells, postnatal development and growth of the tissue. These advances have provided novel insight into fundamental questions about the developmental biology of articular cartilage, as well as potential cell sources that may participate in joint response to injury.

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. In the presumptive layer of mutant glenoid fossa's articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.

Articular Cartilage: Structural and Developmental Intricacies and Questions.

Articular cartilage has obvious and fundamental roles in joint function and body movement. Much is known about its organization, extracellular matrix, and phenotypic properties of its cells, but less is known about its developmental biology. Incipient articular cartilage in late embryos and neonates is a thin tissue with scanty matrix and small cells, while adult tissue is thick and zonal and contains large cells and abundant matrix. What remains unclear is not only how incipient articular cartilage forms, but how it then grows and matures into a functional, complex, and multifaceted structure. This review focuses on recent and exciting discoveries on the developmental biology and growth of articular cartilage, frames them within the context of classic studies, and points to lingering questions and research goals. Advances in this research area will have significant relevance to basic science, and also considerable translational value to design superior cartilage repair and regeneration strategies.

Mouse limb skeletal growth and synovial joint development are coordinately enhanced by Kartogenin.

Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule - we named Kartogenin (KGN) - that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGFß superfamily members, particularly TFGß1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGFß1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGFß1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGFß and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGFß signaling, working in addition to and/or in concert with the filamin A/CBFß/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.

Genesis and morphogenesis of limb synovial joints and articular cartilage.

Limb synovial joints are intricate structures composed of articular cartilage, synovial membranes, ligaments and an articular capsule. Together, these tissues give each joint its unique shape, organization and biomechanical function. Articular cartilage itself is rather complex and organized in distinct zones, including the superficial zone that produces lubricants and contains stem/progenitor cells. For many years there has been great interest in deciphering the mechanisms by which the joints form and come to acquire such unique structural features and diversity. Decades ago, classic embryologists discovered that the first overt sign of joint formation at each prescribed limb site was the appearance of a dense and compact population of mesenchymal cells collectively called the interzone. Work carried out since then by several groups has provided evidence that the interzone cells actively participate in joint tissue formation over developmental time. This minireview provides a succinct but comprehensive description of the many important recent advances in this field of research. These include studies using various conditional reporter mice to genetically trace and track the origin, fate and possible function of joint progenitor cells; studies on the involvement and roles in signaling pathways and transcription factors in joint cell determination and functioning; and studies using advanced methods of gene expression analyses to uncover novel genetic determinants of joint formation and diversity. The overall advances are impressive, and the findings are not only of obvious interest and importance but also have major implications in the conception of future translational medicine tools to repair and regenerate defective, overused or aging joints.