Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal, newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker

We undertook a comparative immunolocalisation study on type II collagen, aggrecan and perlecan in a number of 12- to 14-week-old human foetal and postnatal (7–19 months) ovine joints including finger, toe, knee, elbow, hip and shoulder. This demonstrated that perlecan followed a virtually identical immunolocalisation pattern to that of type II collagen in the foetal tissues, but a slightly divergent localisation pattern in adult tissues. Aggrecan was also localised in the cartilaginous joint tissues, which were clearly delineated by toluidine blue staining and the type II collagen immunolocalisations. It was also present in the capsular joint tissues and in ligaments and tendons in the joint, which stained poorly or not at all with toluidine blue. In higher power microscopic views, antibodies to perlecan also stained small blood vessels in the synovial lining tissues of the joint capsule; however, this was not discernable in low power macroscopic views where the immunolocalisation of perlecan to pericellular regions of cells within the cartilaginous rudiments was a predominant feature. Perlecan was also evident in small blood vessels in stromal connective tissues associated with the cartilage rudiments and with occasional nerves in the vicinity of the joint tissues. Perlecan was expressed by rounded cells in the enthesis attachment points of tendons to bone and in rounded cells in the inner third of the meniscus, which stained prominently with type II collagen and aggrecan identifying the chondrogenic background of these cells and local compressive loads. Flattened cells within the tendon and in the surface laminas of articular cartilages and the meniscus did not express perlecan. Collected evidence presented herein, therefore, indicates that besides being a basement membrane component, perlecan is also a marker of chondrogenic cells in prenatal cartilages. In postnatal cartilages, perlecan displayed a pericellular localisation pattern rather than the territorial or interterritorial localisation it displayed in foetal cartilages. This may reflect processing of extracellular perlecan presumably as a consequence of intrinsic biomechanical loading on these tissues or to divergent functions for perlecan and type II collagen in adult compared to prenatal tissues.     

The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages

The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and -9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and -9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.     

Perlecan, the multidomain heparan sulfate proteoglycan of basement membranes, is also a prominent component of the cartilaginous primordia in the developing human fetal spine.

The aim of this study was to localize perlecan in human fetal spine tissues. Human fetal spines (12-20 weeks; n=6) were fixed in either Histochoice or 10% neutral buffered formalin, routinely processed, paraffin-embedded, and 4-microm sagittal sections were cut and stained with toluidine blue, H&E, and von Kossa. Perlecan, types I, II, IV, and X collagen, CD-31, aggrecan core protein, and native and delta-HS 4, 5 hexuronate stub epitopes were immunolocalized. Toluidine blue staining visualized the cartilaginous vertebral body (VB) rudiments and annular lamellae encompassing the nucleus pulposus (NP). Von Kossa staining identified the VB primary center of ossification. Immunolocalization of type IV collagen, CD-31, and perlecan delineated small blood vessels in the outer annulus fibrosus (AF) and large canals deep within the VBs. Perlecan and type X collagen were also prominently expressed by the hypertrophic vertebral growth plate chondrocytes. Aggrecan was extracellularly distributed in the intervertebral disk (IVD) with intense staining in the posterior AF. Notochordal tissue stained strongly for aggrecan but negatively for perlecan and types I and II collagen. Type I collagen was prominent in the outer AF and less abundant in the NP, while type II collagen was localized throughout the IVD and VB. The immunolocalization patterns observed indicated key roles for perlecan in vasculogenic, chondrogenic, and endochondral ossification processes associated with spinal development.     

Perlecan displays variable spatial and temporal immunolocalisation patterns in the articular and growth plate cartilages of the ovine stifle joint

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for fibroblast growth factors 1, 2, 7, 9, binds connective tissue growth factor and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal growth plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to 19-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone growth plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and growth plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with growth factor signalling, cellular proliferation and tissue development.     

Podoplanin is expressed by a sub-population of human foetal rib and knee joint rudiment chondrocytes

The aim of this study was to determine if podoplanin was expressed by rudiment chondrocytes in human foetal cartilages. Podoplanin was immunolocalised in first trimester human foetal rib and knee joint rudiments to a sub-population of chondrocytes deep in the rib rudiments, tibial and femoral growth plates and cells associated with the cartilage canals of the foetal knee joint rudiments. Lymphatic vessels in the loose stromal tissues surrounding the developing rudiments were also demonstrated on the same histology slides using antipodoplanin (MAb D2-40) and anti-LYVE-1 and differentiated from CD-31 positive blood vessels confirming the discriminative capability of the antibody preparations used. The D2-40 positive rib and knee rudiment chondrocytes were not stained with antibodies to LYVE-1, CD-31 or CD-34 however perlecan was a prominent pericellular proteoglycan around these cells confirming their chondrogenic phenotype. Discernable differences were evident between the surface and deep rudiment chondrocytes in terms of their antigen reactivities detected with MAb D2-40 or antiperlecan antibodies. Binding of the cytoplasmic tail of PDPN to the ERM proteins ezrin, radixin and moeisin may result in changes in cytoskeletal organisation which alter the phenotype of this central population of rudiment cells. This may contribute to morphological changes in the rudiment cartilages which lead to establishment of the primary ossification centres and is consistent with their roles as transient developmental scaffolds during tissue development.     

Perlecan, the "jack of all trades" proteoglycan of cartilaginous weight-bearing connective tissues

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.     

Topographical variation in the distributions of versican,aggrecan and perlecan in the foetal human spine reflects their diverse functional roles in spinal development

We evaluated the immunohistochemical distribution of three major proteoglycans of cartilage, i.e., aggrecan, versican and perlecan vis-a-vis collagens I and II in the developing human spine of first-trimester foetuses. Aggrecan and perlecan were prominently immunolocalised in the cartilaginous vertebral body rudiments and to a lesser extent within the foetal intervertebral disc. In contrast, versican was only expressed in the developing intervertebral disc interspace. Using domain-specific monoclonal antibodies against the various modules of versican, we discovered the V0 isoform as the predominant form present. Versican immunolocalisations conducted with antibodies directed to epitopes in its N and C termini and GAG-α and GAG-β core protein domains provided evidence that versican in the nucleus pulposus was either synthesised devoid of a G3 domain or this domain was proteolytically removed in situ. The V0 versican isoform was localised with prominent fibrillar components in the annular lamellae of the outer annulus fibrosus. Perlecan was a notable pericellular proteoglycan in the annulus fibrosus and nucleus pulposus but poorly immunolocalised in the marginal tissues of the developing intervertebral disc, apparently delineating the intervertebral disc–vertebral body interface region destined to become the cartilaginous endplate in the mature intervertebral disc. The distribution of collagens I and II in the foetal spine was mutually exclusive with type I present in the outer annulus fibrosus, marginal tissues around the vertebral body rudiment and throughout the developing intervertebral disc, and type II prominent in the vertebral rudiment, absent in the outer annulus fibrosus and diffusely distributed in the inner annulus fibrosus and nucleus pulposus. Collectively, our findings suggest the existence of an intricate and finely balanced interplay between various proteoglycans and collagens and the spinal cell populations which synthesise and assemble these components during spinal development.     

Colocalization in vivo and association in vitro of perlecan and elastin

We have colocalized elastin and fibrillin-1 with perlecan in extracellular matrix of tensional and weight-bearing connective tissues. Elastin and fibrillin-1 were identified as prominent components of paraspinal blood vessels, and posterior longitudinal ligament in the human fetal spine and outer annulus fibrosus of the fetal intervertebral disc. We also colocalized perlecan with a synovial elastic basal lamina, where the attached synovial cells were observed to produce perlecan. Elastin, fibrillin-1 and perlecan were co-localized in the intima and media of small blood vessels in the synovium and in human fetal paraspinal blood vessels. Elastic fibers were observed at the insertion point of the anterior cruciate ligament to bone in the ovine stifle joint where they colocalized with perlecan. Elastin has not previously been reported to be spatially associated with perlecan in these tissues. Interactions between the tropoelastin and perlecan heparan sulfate chains were demonstrated using quartz crystal microbalance with dissipation solid phase binding studies. Electrostatic interactions through the heparan sulfate chains of perlecan and core protein mediated the interactions with tropoelastin, and were both important in the coacervation of tropoelastin and deposition of elastin onto perlecan immobilized on the chip surface. This may help us to understand the interactions which are expected to occur in vivo between the tropoelastin and perlecan to facilitate the deposition of elastin and formation of elastic microfibrils in situ and would be consistent with the observed distributions of these components in a number of connective tissues.     

Confocal microscopy demonstrates association of LTBP-2 in fibrillin-1 microfibrils and colocalisation with perlecan in the disc cell pericellular matrix

Comparative immunolocalisations of latent transforming growth factor-beta-1 binding protein (LTBP)-2, fibrillin-1, versican and perlecan were undertaken in foetal human and wild type C57BL/6 mouse and Hspg2 exon 3 null HS deficient mouse intervertebral discs (IVDs). LTBP-2 was a prominent pericellular component of annular fibrochondrocytes in the posterior annulus fibrosus (AF), interstitial matrix adjacent to nucleus pulposus (NP) cells and to fibrillar and cell associated material in the anterior AF of the human foetal IVD and also displayed a pericellular localisation pattern in murine IVDs. Perlecan and LTBP-2 displayed strong pericellular colocalisation patterns in the posterior AF and to fibrillar material in the outer anterior AF in the foetal human IVD. Versican was a prominent fibril-associated component in the posterior and anterior AF, localised in close proximity to fibrillin-1 in fibrillar arrangements in the cartilaginous vertebral rudiments around paraspinal blood vessels, to major collagen fibre bundles in the anterior and posterior AF and shorter fibres in the NP. Fibrillin-1 was prominent in the outer anterior AF of the human foetal IVD and in fibres extending from the AF into the cartilaginous vertebral rudiments. LTBP-2 was prominently associated with annular fibrils containing fibrillin-1, versican was localised in close proximity to these but not specifically with LTBP-2. The similar deposition levels of LTBP-2 observed in the AF of the Hspg2 exon 3 null and wild type murine IVDs indicated that perlecan HS was not essential for LTBP-2 deposition but colocalisation of LTBP-2 with perlecan in the foetal human IVD was consistent with HS mediated interactions which have already been demonstrated in-vitro.     

Division and death of cells in developing synovial joints and long bones

Articular chondrocytes are a unique set of cells from the time the cellular condensations that become the anlagen of the long bones develop in the embryo. In the presumptive joint the cells of the opposing bones are packed very closely together, but at cavitation, the central, flattened cells move apart to form the articular surfaces. As the articular cartilage develops the cells are pushed further apart by the cartilaginous matrix. To determine the contributions of cell proliferation and death to cavitation and the subsequent development and growth of articular cartilage, direct observations were made to identify mitotic cells and those with apoptotic bodies in haematoxylin-stained sections of developing joints, and growing and ageing articular cartilage of the rabbit knee. These observations were extended using antibodies to the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labelling (TUNEL) on corresponding sections. Low levels of cell division do occur in the articular cartilage up to 6 weeks postnatally, but matrix formation makes the major contribution to the increase in size of the cartilage. Cell death is not observed during cavitation, nor during the development of the articular cartilage proper. Apoptosis is essential, however, for the removal of the epiphyseal cartilage during ossification of the epiphyses and in the growth plate.     

Ossification communalities of the hand and other body parts: their implication to skeletal assessment

Correlating the age at appearance of 71 postnatal ossification centers (OC's) with every other OC, 4,970 correlations in all, then grouping correlations by body part, the hand does not exhibit usefully higher communality (mean r) than the foot, shoulder, hip, elbow or knee. While low communality round bones and ossification sequence polymorphisms together account for the fact that no one body part adequately represents the entire skeleton, it is also true that ossification communality throughout the skeleton is low unless OC's of maximum predictive value are separately employed.     

Ossification of the human fetal ilium.

Ossification of the ilium is similar to that of a long bone. It possesses three cartilaginous epiphyses and one cartilaginous process. Moreover, it undergoes peculiar osteoclastic resorption, comparable with that of the cranium bones. Asymmetrical ossification of the ilium, haversian bone remodelling and apposition of chondroid tissue posterosuperiorly to the acetabulum most probably emphasize the importance of mechanical factors in the morphogenesis of the hip bone during fetal life.     

Role of perlecan in skeletal development and diseases

Perlecan, a large heparan sulfate proteoglycan (HSPG), is present in the basement membrane and other extracellular matrices. Its protein core is 400 kDa in size and consists of five distinct structural domains. A number of in vitro studies suggest multiple functions of perlecan in cell growth and differentiation and tissue organization. Recent studies with gene knockout mice and human diseases revealed critical in vivo roles of perlecan in cartilage development and neuromuscular junction activity. Published in 2003.     

Epigenetic mechanical factors in the evolution of long bone epiphyses

In developing vertebrate long bones in which endochondral ossification occurs, it is preceded or accompanied by perichondral ossification. The speed and extent of perichondral apposition relative to endochondral ossification varies in different taxa. Perichondral ossification dominates early long bone development in extinct basal tetrapods and dinosaurs, extant bony fish, amphibians, and birds. In mammals and lizards, perichondral and endochondral ossification proceed more synchronously. One of the most important epigenetic factors in skeletogenesis is mechanical loading caused by muscle contractions which begin in utero or in ovo . It has been previously shown that the stress distributions created perinatally in the chondroepiphysis during human skeletal development can influence the appearance of secondary ossification centres. Using finite element computer models representing bones near birth or hatching, we demonstrate that in vertebrates in which perichondral ossification significantly precedes endochondral ossification, the distribution of mechanical stresses in the ossifying cartilage anlagen tends to inhibit the appearance of secondary ossification centres in the ends of long bones. In models representing vertebrates in which endochondral ossification keeps pace with perichondral apposition, the appearance of secondary centres is promoted. The appearance of secondary centres leads to the formation of bony epiphyses and growth plates, which are most common in mammals and extant lizards. We postulate that genotypic factors influencing the relative speed and extent of perichondral and endochondral ossification interact with mechanical epigenetic factors early in development to account for many of the morphological differences observed in vertebrate skeletons.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

Relationship between the chondrocyte maturation cycle and the endochondral ossification in the diaphyseal and epiphyseal ossification centers

The chondrocyte maturation cycle and endochondral ossification were studied in human, fetal cartilage Anlagen and in postnatal meta‐epiphyses. The relationship between the lacunar area, the inter‐territorial fibril network variations, and calcium phosphorus nucleation in primary and secondary ossification centers were assessed using light microscopy and scanning electron microscopy (SEM) morphometry. The Anlage topographic, zonal classification was derived from the anatomical nomenclature of the completely developed long bone (diaphysis, metaphyses and epiphyses). A significant increase in the chondrocyte lacunar area was documented in the Anlage of epiphyseal zones 4 and 3 to zone 2 (metaphysis) and zone 1 (diaphysis), with the highest variation from zone 2 to zone 1. An inverse reduction in the intercellular matrix area and matrix interfibrillar empty space was also documented. These findings are consistent with the osmotic passage of free cartilage water from the interfibrillar space into the swelling chondrocytes, which increased the ion concentrations to a critical threshold for mineral precipitation in the matrix. The mineralized cartilage served as a scaffold for osteoblast apposition both in primary and secondary ossification centers and in the metaphyseal growth plate cartilage, though at different periods of bone Anlage development and with distinct patterns for each zone. All developmental processes shared a common initial pathway but progressed at different rates, modes and organization in diaphysis, metaphysis and epiphysis. In the ossification phase the developing vascular supply appeared to play a key role in determining the cortical or trabecular structure of the long bones. J. Morphol. 277:1187–1198, 2016. © 2016 Wiley Periodicals, Inc.     

Forced chondrocyte expression of sonic hedgehog impairs joint formation affecting proliferation and apoptosis.

Proliferation and apoptosis are two fundamental processes that occur during limb development, and in particular in joint formation. To study the role of hedgehog proteins in limbs, we have misexpressed Sonic Hedgehog specifically in chondrocytes. We found that the appendicular skeleton was severely misshapen while pelvic and shoulder girdles developed normally. In particular, we detected fusion of the elbow/knee joint, no definite carpal/tarsal, metacarpal/metatarsal bones and absence of distinct phalanges, fused in a continuous cartilaginous rod. Molecular markers of joints, such as Gdf5 and sFrp2 were absent at presumptive joint sites and Tenascin C, a molecule associated with joint formation and expressed in permanent cartilage, was expressed in a wider region in transgenic animals as compared to the wild type. The ratio of proliferating to non-proliferating chondrocytes was about two times higher in transgenic developing cartilage as compared to the wild type. Accordingly, the proapoptotic gene Bax was barely detectable in the growth plate of transgenic mice and Tunel assay showed the absence of apoptosis in presumptive joints at E15.5. Taken together, these results suggest that misexpression of Sonic Hedgehog causes apoptosis and proliferation defects leading to the lack of joint cavity and fusion of selected limb skeletal elements.     

Permanence of proliferating cells in developing,juvenile and adult knee epiphyses of lizards in relation to bone growth and regeneration

The distribution of long‐labelling‐retaining cells, putative progenitor or stem cells, in the developing knees of embryo, juvenile and adult lizards has been analysed using H3‐thymidine autoradiography and 5BrdU immunohistochemistry. Proliferating cells are present in developing cartilaginous femur and tibia, especially in the epiphyses where a higher cell multiplication likely determines their typical enlarged shape in comparison with the diaphyses where chondroblast proliferation is low to absent. Sparse 5BrdU‐labelled cells remain in the articular and growth plate cartilages of the epiphyses in older stages of development and are still detected in developing epiphyses 13 days after injection of 5BrdU. This indicates they are slow‐cycling cells, a typical characteristic for progenitor or stem cells. Long retaining 5BrdU‐labelled cells remain in the articular surface also during adult life where they likely sustain the growth of long bones. Adult epiphyses show secondary ossification centres where the articular cartilage is partially or largely replaced by bone trabeculae. The damage in the epiphysis of lizards stimulates the proliferation of progenitor cells for the regeneration of new cartilaginous epiphyses. The localization of cells capable of proliferation in the epiphyses of adult femur and tibia pre‐adapts these lizards to cartilage regeneration in case of injury.     

Covariance in human limb joint articular morphology

Objectives

Limb synovial joints exhibit complex shapes that must accommodate often-antagonistic demands of function, mobility, and stability. These demands presumably dictate coordination among joint articular shapes, but the structure of morphological covariance within and among joints is unknown. This study analyzes the human shoulder, elbow, hip, and knee to determine how articular covariance is structured in relation to joint structure, accessory cartilage, and function.

Materials and Methods

Surface models were created from the CT scans of 200 modern skeletons from the University of Tennessee Donated Skeletal Collection. Three-dimensional landmarks were collected on the shoulder, elbow, hip, and knee joints. Two-block partial least squares were conducted to determine associations between surfaces of conarticular shapes, functionally analogous articulations, and articulations belonging to the same bone.

Results

Except for the components of the shoulder, all conarticular pairs exhibit covariance, though the strength of these relationships appears unrelated to the amount of accessory cartilage in the joint. Only the analogous articulations of the humerus and femur exhibit significant covariance, but it is unlikely that this pattern is due to function alone. Stronger covariance within the lower limb than the upper limb is consistent broader primate patterns of within-limb integration.

Discussion

With the exception of the elbow, complementary joint function does not appear to promote strong covariance between articulations. Analogous humeral and femoral surfaces are also serially homologous, which may result in the articular associations observed between these bones. Broadly, these patterns highlight the indirect relationship between joint congruence and covariance.