The anterolateral ligament of the knee is not a solid structure in human fetuses.

PURPOSE: The purpose of this study was to investigate if the anterolateral ligament of the knee (ALL) is present in the human fetus and describe its topography along with other structures of the region. METHODS: Forty human fetuses knee joints, at mean age 34 weeks (± 2.57 weeks), fixed in 10% formalin, were submitted to cross-sectional dissection and mesoscopic analysis. RESULTS: The ALL was not identified, although the usual topography of the region was identified in all specimens: skin, subcutaneous tissue, iliotibial tract (ITT), fibular collateral ligament, popliteal muscle tendon, lateral meniscus, patellar ligament, infrapatellar fat pad, lateral patellar retinaculum, knee joint capsule, lateral inferior genicular vessels, and the biceps femoris tendon. The ITT reveals anterior (n = 12) and lateral thickening (n = 17) in some specimens. This thickening was found in both knees of the same subject in 6/20 specimens. CONCLUSION: The anterolateral ligament of the knee is not a congenital or solid structure. Our results suggest that the ALL may be a deep layer of the ITT or part of the knee joint capsule, or its identification is evaluator dependent.

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.

Mechanobiological simulations of prenatal joint morphogenesis.

Joint morphogenesis is the process in which prenatal joints acquire their reciprocal and interlocking shapes. Despite the clinical importance of the process, it remains unclear how joints acquire their shapes. In this study, we simulate 3D mechanobiological joint morphogenesis for which the effects of a range of movements (or lack of movement) and different initial joint shapes are explored. We propose that static hydrostatic compression inhibits cartilage growth while dynamic hydrostatic compression promotes cartilage growth. Both pre-cavitational (no muscle contractions) and post-cavitational (with muscle contractions) phases of joint development were simulated. Our results showed that for hinge type motion (planar motion from 45° to 120°) the proximal joint surface developed a convex profile in the posterior region and the distal joint surface developed a slightly concave profile. When 3D movements from 40° to -40° in two planes were applied, simulating a rotational movement, the proximal joint surface developed a concave profile whereas the distal joint surface rudiment acquire a rounded convex profile, showing an interlocking shape typical of a ball and socket joint. The significance of this research is that it provides new and important insights into normal and abnormal joint development, and contributes to our understanding of the mechanical factors driving very early joint morphogenesis. An enhanced understanding of how prenatal joints form is critical for developing strategies for early diagnosis and preventative treatments for congenital musculoskeletal abnormalities such as developmental dysplasia of the hip.

The zinc finger transcription factors Osr1 and Osr2 control synovial joint formation.

Synovial joints enable smooth articulations between different skeletal elements and are essential for the motility of vertebrates. Despite decades of extensive studies of the molecular and cellular mechanisms of limb and skeletal development, the molecular mechanisms governing synovial joint formation are still poorly understood. In particular, whereas several signaling pathways have been shown to play critical roles in joint maintenance, the mechanism controlling joint initiation is unknown. Here we report that Osr1 and Osr2, the mammalian homologs of the odd-skipped family of zinc finger transcription factors that are required for leg joint formation in Drosophila, are both strongly expressed in the developing synovial joint cells in mice. Whereas Osr1(-/-) mutant mice died at midgestation and Osr2(-/-) mutant mice had only subtle defects in synovial joint development, tissue-specific inactivation of Osr1 in the developing limb mesenchyme in Osr2(-/-) mutant mice caused fusion of multiple joints. We found that Osr1 and Osr2 function is required for maintenance of expression of signaling molecules critical for joint formation, including Gdf5, Wnt4 and Wnt9b. In addition, joint cells in the double mutants failed to upregulate expression of the articular cartilage marker gene Prg4. These data indicate that Osr1 and Osr2 function redundantly to control synovial joint formation.

Proteolytic cleavage of versican during limb joint development.

Versican is highly expressed in developing joint interzones during limb morphogenesis. This study was undertaken to examine whether proteolytic cleavage of versican occurs that could potentially impact its function during the process of embryonic synovial joint formation. Using an antibody to the DPEAAE neoepitope generated by ADAMTS proteolysis, versican amino terminal cleavage fragments were detected in joint interzones at 12-16 days post coitum (dpc). ADAMTS-1 localization overlapped that of DPEAAE-reactive versican fragments suggesting it as one possible protease activity involved in processing of versican in the interzone. Results show that increased cleavage of versican in the interzone accompanies cavitation and suggests that proteolytic modification of versican may be important during the process of synovial joint maturation.

3D representation of the developing chick knee joint: a novel approach integrating multiple components.

The knee joint has a highly complex 3-dimensional (3D) morphology that is sculpted at the interface of the forming long bones as they are generated in the embryo. Although it is clear that regulatory genes guide joint formation, the mechanisms that are responsible for morphogenesis of the knee are poorly understood. Certainly the process involves integration across several tissues and physical/mechanical influences from neighbouring tissues are important. We describe the acquisition of shape in the chick knee joint in detail and show that by HH34 the joint already displays shape characteristics of the adult structure. Through imaging developing cartilage, tendons, ligaments and muscle across developmental stages from HH28-34 we have built 3D representations of the forming structure including the various components important in knee formation. We describe the timing of muscle and tendon development in parallel with the refinement of cartilage shape, showing when and where (tendon attachment points) muscle forces are applied to the cartilage elements. Shape begins to emerge as the tendons are forming (HH30-32) but is fully refined (HH34) in the presence of tendons. The resulting integrated 3D representations of the developing knee across time will serve as the foundation for computational analysis of the mechanical environment, and experimental approaches to investigating morphogenetic mechanisms.

Examination of the accessory tendons of extensor hallucis longus muscle in fetuses.

There are various data about the incidence of accessory tendons (AT) of extensor hallucis longus (EHL) muscle; however, their function is unknown. This study aimed to determine the incidence and morphometric features of the AT of EHL muscle in fetuses in order to provide more information to discuss its possible function. Forty-five fetuses (26 female and 19 male) were used in this study. Fetuses were grouped as Group A (16-21 weeks), Group B (22-27 weeks), and Group C (28-34 weeks) according to their age. In 23 (51%) out of 45 fetuses, there were AT. These were bilateral in 15 fetuses (65%) and unilateral in eight fetuses (35%). Fifty-two percent of the fetuses in group A, 43% in group B, and 67% in group C had AT. AT were observed in 14 female (54%) and 9 male (47%) fetuses. In all cases, the AT were always diverging to the medial side of the main EHL tendons and attached to the metatarsophalangeal joint capsule distal to the joint space. Significant correlations were observed in this study between EHL and AT widths as well as between EHL width and EHL-AT distance on both sides. The present study is the first to provide morphometric data about the AT of EHL muscle in fetuses which will be of use in understanding their function, particularly in biomechanics of the great toe.

[Mechanisms of synovial joint formation].

Synovial joints are comprised of relatively simple biomechanical structures including articular cartilage, synovial membrane, synovial fluid, ligaments and a fibrous capsule; they are fundamentally important for function and quality of life. Recent studies described the spatio-temporal expression patterns of signaling molecules and transcription factors in the developing synovial joints. Though few in number, the gain and/or loss of function-experiments demonstrated direct involvement of these molecules in joint formation. This review focuses on recent advances in understanding the mechanisms of synovial joint formation in the limbs.

Comparative expression pattern of Odd-skipped related genes Osr1 and Osr2 in chick embryonic development.

Odd-skipped genes encode zinc-finger transcription factors with widespread roles in embryonic development. In Drosophila, odd-skipped acts as a pair-rule gene, while its orthologous gene in Caenorhabditis elegans is involved in gut development. In mammals two paralogs exist, Osr1 and Osr2, with functions described in heart and urogenital, and in secondary palate development, respectively. As the chicken embryo is a widely used system for analysing gene function in vivo, we determined the expression pattern of the two chicken orthologues, cOsr1 and cOsr2, during embryonic development. We demonstrate expression of both genes in a variety of organs and structures, such as kidney, eye, branchial arches and dermis. Both genes show a highly dynamic expression pattern with partially overlapping, but mostly distinct domains of expression. Special emphasis in this study was laid on the investigation of cOsr1 and cOsr2 in limb development, where we compared their expression pattern with the expression of Osr1 and Osr2 in the mouse.

Innervation and functional characteristics of connective tissues, especially elastic fibers, in human fetal thoracic intervertebral articular capsule and its surroundings.

The articular capsules between the thoracic vertebrae, which have physiologically different functions from those of other levels of the vertebrae, have yet to be subjected to neuro-anatomical and fine structural analysis. In the present study, we analyzed serial frozen sections of decalcified thoracic vertebrae in human fetuses, and identified the articular capsule tissue with its unique distribution of elastic fibers. The fine structure of the elastic fibers was studied by transmission electron microscopy. In the early-stage fetus, the fibrous membrane forming the lateral intervertebral articular capsule contained abundant thin elastic fibers consisting of microfibrils. In the late-stage fetus, the lateral capsule of fibrous membrane was occupied by thick elastic fibers. A medial articular capsule, namely the ligamenta flava, contained numerous thick elastic fibers in both early and late-stage fetuses. The distributional differences in nerve fibers between early and late-stage fetuses were determined by immunostaining, using antibodies raised against protein gene product 9.5 (PGP 9.5; ubiquitin carboxyl-terminal hydrolase). Innervation by PGP 9.5 immunoreactive fibers was limited to the areas of the articular capsules near the blood vessels, which may indicate their functional relation with blood flow. No PGP 9.5 immunoreactive fibers were found in the ligamenta flava of the late-stage fetus. Innervation might be directly involved in the development of the intervertebral articular capsules in normal human fetuses.

The immature athlete.

The skeletally immature athlete poses unique problems in diagnosis and treatment of injuries to the extensor mechanism of the knee. An accurate and detailed history and physical examination of the knee are essential for making a specific diagnosis and formulating an appropriate treatment plan. This article presents an overview of acute and chronic injuries of the extensor mechanism of the knee that are unique to skeletally immature athletes. The subjects of femoral trochlear dysplasia and medial subluxation of the patella are briefly discussed. The etiopathology, clinical evaluation, and management (non-operative and operative) of sleeve fractures of the patella and avulsion fractures of the tibial tubercle in children and adolescents are discussed. The pathoanatomy, clinical features, and management of synovial plica syndrome, Hoffa's syndrome, Osgood-Schlatter disease, and Sinding-Larsen-Johansson disease are presented.

Developmental morphological and histological studies on structures of the human fetal shoulder joint.

In the present work, morphological changes in the interior structures of the developing human shoulder joint were studied at different prenatal ages (9, 12, 16, 23 and 40 weeks) and were compared with the same structures in the adult joint. It was found that the shoulder joint had gone through important developmental changes during the 12th week of the prenatal life and it is assumed that genetic factors operative during this stage of development were more important than mechanical factors. A subsequent development of the intracapsular glenohumeral ligaments was present at the 16th week. The glenoid labrum, the biceps tendon and the three glenohumeral ligaments formed a complete ring around the glenoid fossa which constituted a functional unit, which seemed to have a role in stabilizing the joint. In the present work, histological prenatal studies were done on sagittal and radial sections from the glenoid fossa and its associated structures and the results were compared with the same structures in adults. At a crown-rump length of 30 mm (9 weeks), intermingling of the collagen fibres of the superior labrum and the biceps tendon was observed and the superior labrum could be considered as an extension of the biceps tendon. While the superior and inferior parts of the labrum appeared fibrous, the posterior labrum appeared as a primitive cellular condensation. At the 12th week, it became a fibrocellular structure and changed to a fibrocartilaginous structure at the 16th week. But until full term, no definitive fibrocartilage was found due to its hypercellularity compared to the adult. It was found that at all ages, the capsule was formed of cellular and fibrous elements, its collagenous content was progressively increased with age and at full term, it became generally fibrous but was still different compared to adults. In all stages of development, the synovial tissue of different regions of the same joint exhibited marked variations in thickness, vascularity, cellular density and collagenous content. It lined the capsule, surrounded the biceps tendon and reflected on the labrum. Its cell density as well as their vascular and collagenous contents were progressively increased with age. At full term, the synovial tissue was thickest at its inferior reflection and forming large folds. The synovial tissue lining the capsule was thinner than the synovial tissue at its reflection from the labrum, many villi and processes arising from it and projecting into the joint cavity.

Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton.

The long bones of the vertebrate appendicular skeleton arise from initially continuous condensations of mesenchymal cells that subsequently segment and cavitate to form discrete elements separated by synovial joints. Little is known, however, about the molecular mechanisms of joint formation. We present evidence that Wnt-14 plays a central role in initiating synovial joint formation in the chick limb. Wnt-14 is expressed in joint-forming regions prior to the segmentation of the cartilage elements, and local misexpression of Wnt-14 induces morphological and molecular changes characteristic of the first steps of joint formation. Induction of an ectopic joint-like region by Wnt-14 suppresses the formation of the immediately adjacent endogenous joint, potentially providing insight into the spacing of joints.

Developmental morphological and histological studies on structures of the human fetal elbow joint.

In the present work, morphological changes in the developing human elbow joint were studied at different prenatal ages (8, 12, 16, 20, 29 and 40 weeks) and were compared with the same structures in the adult joint. The elbow joint had gone through its most important developmental changes during the 20th week of prenatal life, probably due to the direct dynamic effect of the newly developed fetal movement. During later prenatal development, the articular surfaces of the lower end of humerus and the upper ends of radius and ulna developed their characteristic congruencies, so that the highly curved convexities always articulate with the highly curved concavities. That process progressed postnatally and even till adult age. In full-term infants it was found that the lower end of humerus had acquired its adult shape, while the shape of the upper ends of radius and ulna were still not fully developed. They continued development in postnatal life even till adult age. In the present work, histological prenatal studies were done on longitudinal sections from the back of the capsule and synovial tissue, early (8 weeks) and late in full term, and the results were also compared with the same structures in adults. It was found that at all ages, the capsules were formed of cellular and fibrous elements, but at early prenatal age (8 weeks), this cellular condensation was more massive and prominent while in full-term infants, it became generally more fibrous, but was still different compared to adults. Basic cellular structures of the synovial tissue changed very little during the late prenatal developmental stage, as it did not become more fibrous than cellular during these periods, but differences in vascularity became more obvious. The cartilaginous content of the articular surface at 8 weeks was highly cellular with very little intercellular matrix. In contrast to that of full term, this cartilage became fully chondrogenous with a notable decrease in cellular density and massive increase in matrix content.

A scientific basis for the biologic regeneration of synovial joints.

Temporomandibular joint disorders represent a large group of conditions involving a local or more generalized musculoskeletal disease process. The disorders have many features and many causes and may result in limited or more severe damage of the joint associated tissues including the disks, the articular surface, the underlying bone, and the ligamentous structures. As our understanding of the molecular processes guiding skeletal tissue formation progresses, new opportunities arise in the field of skeletal tissue and joint repair. Reconstruction of the temporomandibular joint by means of scientifically designed approaches will revolutionize surgical treatment modalities.

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing.

The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.

Collagen gene expression during development of avian synovial joints: transient expression of types II and XI collagen genes in the joint capsule.

The developmental sequence of the embryonic joint has been well studied morphologically. There are, however, no definitive studies of cell function during joint development. In order to begin to understand the differentiation events that contribute to joint formation, we examined the expression of collagen mRNAs encoding types I, IIA, IIB, and XI. In situ hybridization was performed on chicken embryo hind limb buds and digits from day 7 to day 18 (Hamburger and Hamilton stages 31-44). In the day 7 (stage 31) limb bud, there was a condensation of mesenchyme forming the primitive tarsal and metatarsal bones that showed abundant expression of type IIA procollagen message, but no type IIB or type alpha 1(XI) message. By day 8 (stage 33), co-expression of types IIA, and type XI procollagen mRNAs was observed in the condensations, with expression of IIB restricted to early chondrocytes with metachromatically staining matrix. At this stage, DNA fragmentation characteristic of apoptosis was observed in cells near the midline of the interzone region between the developing anlagen, and in areas between and around the individual digits of the paddle. The presumptive apoptotic cells were more numerous at day 9 (stage 35), and were not found in the developing joint at subsequent time points, including the initiation of spatial cavitation of the joint. From days 11-18, type IIA procollagen mRNA was expressed in flattened cells at the surface of the anlagen, and in the perichondrium and in the developing joint capsule; type IIB mRNA message was found only in chondrocytes. Type XI mRNA was expressed by all type II-expressing cells. Alpha 1(I) mRNA was expressed early by cells of the interzone and capsule, but as cavitation progressed, the type I expressing cells of the interzone merged with the superficial layer of the articular surface. Thus, at the time of joint cavitation, there was a distinct pattern of expression of procollagen messages at the articular surface, with type I being outermost, followed by morphologically similar cells expressing type IIA, then chondrocytes expressing type IIB. The progenitor cells expressing type IIA message define a new population of cells. These cell populations contribute to the molecular heterogeneity of the articular cartilage, and these same populations likely exist in the developing joints of other species. The transient transcription of type II and type XI collagen genes, characteristic of chondrocytes, by cells in the joint capsule demonstrates that these cells may have chondrogenic potential.