ARTICLE |
Correspondence to: James Melrose, Raymond Purves Bone and Joint Research Laboratories, Inst. of Bone and Joint Research, Level 5, The University Clinic, Building B26, The Royal North Shore Hospital, St. Leonards NSW 2065, Australia. E-mail: jmelrose@mail.usyd.edu.au
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Summary |
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The aim of this study was to localize perlecan in human fetal spine tissues. Human fetal spines (1220 weeks; n=6) were fixed in either Histochoice or 10% neutral buffered formalin, routinely processed, paraffin-embedded, and 4-µm 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 -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.
(J Histochem Cytochem 51:13311341, 2003)
Key Words: perlecan, HS proteoglycan, human fetal cartilage, development, vertebral growth plate, hypertrophic growth plate, chondrocytes
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Introduction |
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PERLECAN is a member of a group of heparan sulfate (HS)/chondroitin sulfate (CS)-substituted proteoglycans of basement membranes that are implicated in cell growth and differentiation (
The human perlecan gene encodes a large core protein of 467 kD, which consists of five discrete domains (IV) with homology to the laminin A-chain, low-density lipoprotein receptor, neural cell adhesion molecule, and epidermal growth factor (
Perlecan has previously been demonstrated in a range of embryonic and newborn mouse and rat tissues including the spine and intervertebral disc (
Perlecan was first localized in cartilage and chondrosarcoma by
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Materials and Methods |
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All materials and suppliers were as noted previously (
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Preparation of Human Fetal Tissues for Histology and Histological Procedures
Six 1220-week-old human fetuses were obtained at termination of pregnancy after ethical approval by The Human Care and Ethics Review Board of The Royal North Shore Hospital. The posterior elements were dissected from the fetal spines and they were longitudinally bisected in a mid-vertical plane. Half of the spines were fixed in Histochoice for 24 hr and the remaining specimens were fixed in 10% (v/v) neutral buffered formalin for 24 hr. Selected specimens were also briefly decalcified in 10% formic acid for up to 24 hr. The tissues were dehydrated in graded alcohols, embedded in Paraplast, and 4-µm sections were cut and mounted on StarfrostPlus glass microscope slides (Menzel-Glaser; Heidelburg, Germany). The tissue sections were then de-paraffinized in xylene before rehydration through graded ethanol washes to water.
Histochemistry
Neutral formalin-fixed tissue sections were routinely stained for 10 min with 0.04% (w/v) toluidine blue in 0.1 M sodium acetate buffer, pH 4.0, to visualize the tissue proteoglycans. This was followed by 2-min counterstaining in an aqueous 0.1% (w/v) fast green FCF stain which aids in the differentiation of the areas stained for proteoglycan. Hematoxylin and eosin-stained tissue sections were used to examine cell morphology. Mineral deposition in the vertebral center of ossification was visualized in non-decalcified mid-sagittal sections of human fetal spinal tissues using a further modification developed in house at the Department of Anatomical Pathology, Royal North Shore Hospital (NSW, Australia) of the modification of the von Kossa procedure (
Immunohistochemistry
The immunolocalizations were undertaken using a Sequenza vertical cover-plate immunostaining system (Immunon) as described earlier (-4,5 uronate HS stub epitopes identified by this antibody. MAb 10-E-4, in contrast, identifies native HS chains, and heparitinase digestion destroys 10-E-4 reactivity (
-HS stub epitope, and CD-31. After the pre-digestion procedures the sections were rinsed twice in TBS-T and blocked with 10% swine serum for 10 min at RT before addition of primary antibody. Incubation with primary antibodies was performed overnight at 4C using the dilutions specified in Table 1. Appropriate biotinylated secondary antibodies and horseradish peroxidase-conjugated streptavidin were used for visualization with Nova Red substrate for color development. Control sections were also prepared in which an irrelevant isotype-matched primary antibody was substituted for the primary antibody of interest. Commercial (DAKO) isotype-matched mouse IgG (DAKO code X931) or IgM (DAKO code X942) control antibodies (as appropriate) were used for this step. The DAKO products X931 and X942 are mouse monoclonal IgG1 (clone DAK-GO1) and monoclonal IgM (clone DAK-GO8) antibodies directed against Aspergillus niger glucose oxidase, an enzyme that is neither present nor inducible in mammalian tissues. In the case of the 3-G-10 antibody, the heparitinase digestion step was omitted in the control sample, and for the 10-E-4 antibody the control sample was treated with heparitinase.
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Results |
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Immunolocalization of aggrecan, perlecan, and types I and II collagen in longitudinal mid-sagittal sections of human fetal spinal tissues showed some interesting differences in the spatial distributions of these matrix components (Fig 1A1H). Toluidine blue and von Kossa staining clearly showed the cartilaginous nature of the primordial vertebral bodies and the extent of mineralization in the ossifying center at the 1220-week stage of spinal development (Fig 1A and Fig 2C). Type I collagen was confined mainly to the intervertebral disk, with diminished staining evident in the NP (Fig 1B). Strong staining of the anterior and posterior longitudinal ligaments was also observed but no staining of the cartilaginous vertebral body rudiment (Fig 1B). This contrasted with type II collagen, which was more extensively distributed throughout the cartilaginous vertebral body rudiment and intervertebral disk (Fig 1C). However, the outer AF, together with the anterior and posterior longitudinal ligaments, showed no significant staining for type II collagen (Fig 1C). Aggrecan was detected primarily in the intervertebral disk and very little staining was evident for this proteoglycan in the cartilaginous vertebral rudiment (Fig 1D). Staining for aggrecan was generally stronger in the posterior AF. Some cell-associated aggrecan staining in the hypertrophic vertebral growth plate chondrocytes was also evident (Fig 1D). Further examination of the outer AF confirmed the spatial localization patterns for the aforementioned matrix components (Fig 1E1H). Heparan sulfate was diffusely localized throughout the outer AF and displayed somewhat higher levels evident at the cranial and caudal margins of the AF, where it merged with the vertebral growth plates. The extreme periphery of the outer AF lamellae stained negatively for type I collagen (Fig 1F). However, the outer AF stained positively for type I collagen. Type II collagen staining, in contrast, was negative throughout the outer AF except at its margins, where it merged with the vertebral growth plate. Type II collagen staining intensity increased steadily in moving through the inner AF towards the NP (Fig 1G). Aggrecan was again strongly localized in the outer AF tissues examined (Fig 1G).
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Perlecan was produced abundantly by the hypertrophic chondrocytes of the vertebral growth plate (Fig 2A). The most terminally differentiated hypertrophic chondrocytes of the vertebral growth plate located adjacent to the vertebral center of ossification also strongly expressed type X collagen, confirming their differential state (Fig 2B). On closer inspection of the vertebral growth plate, a pericellular localization of HS and perlecan around small columns of hypertrophic vertebral growth plate chondrocytes was apparent, although their distributions were not identical (Fig 2D and Fig 2E). Perlecan was also localized diffusely throughout the cartilaginous vertebral body rudiment and was associated pericellularly with intervertebral disk cells in the NP and in the inner and outer AF (Fig 1F). Perlecan displayed a similar distribution to that previously documented for type IIA collagen in developing intervertebral disk tissues (
Type IV collagen was also extracellularly localized to the outer AF and to many of the basal laminae of blood vessels in the outermost annular lamellae (Fig 3A and Fig 3C). The CD31, perlecan, and -HS MAbs used in the present study also were localized to the blood vessels in the outer AF (Fig 3B, Fig 3D and Fig 3E). Small arterioles and venules were also discernable in many cases in the outer AF of these developing spinal tissues (Fig 3A3E).
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Cartilage canals in the cartilaginous vertebral body rudiments were also prominently featured in longitudinal parasagittal sections of the human fetal spines examined. Several basal lamina components were also immunolocalized to the lining tissues of these cartilage canals, including native and -HS hexuronate stub epitope (Fig 4A and Fig 4C), perlecan (Fig 4E), and type IV collagen (Fig 4F). Higher-power assessment of individual cartilage canals demonstrated the presence of endothelium-like cells on the basal lamina of the canal. These cells also stained positively with the MAb to CD-31 (Fig 4D). Occasional clumps of red blood cells were also observed entrapped within these cartilage canals, suggesting that these structures may have some important nutritional role in these rapidly developing cartilaginous primordial spinal tissues.
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Further immunolocalization of perlecan, types I and II collagen, and aggrecan in parasagittal sections of additional 1214-week-old human fetal spinal tissues confirmed that perlecan was a prominent component of the NP and inner AF. However, perlecan was not present in the notochordal tissue, but some staining of the notochordal sheath for perlecan was noted (Fig 5A5C). Type II collagen was also localized to both the inner AF and NP but, like type I collagen, it was not identified in the notochordal tissue (Fig 5D and Fig 5E). Interestingly, this same tissue stained strongly with the anti-aggrecan antibody (Fig 5 F).
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Discussion |
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The formation of the primordial spinal cartilages that will act as developmental scaffolds and eventually become the ossified vertebral bodies is a complex process. Initially, this involves the migration of precursor cells from remote places to form mesenchymal cell aggregates. Sclerotome cells located lateral to the neural tube also migrate ventrally towards the notochord. This eventually leads to the formation of the primordial cartilage around the notochord, which will become the vertebral body (
An earlier study from our laboratory also examined the distribution of perlecan and other major ECM components (types I, II, VI, and X collagen, aggrecan) in the newborn ovine intervertebral disk (
The vascularization of the human spine has been the subject of anatomic and microanatomic studies for almost a century, and most attention has focused on the external segmental blood supply and drainage of the vertebral bodies (
The present study has demonstrated perlecan as a prominent basal lamina component of small peripheral blood vessels in the outer AF and of cartilage canals within the vertebral body rudiment. Basal lamina components have also been immunolocalized to developing epiphyseal cartilage canals (
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Acknowledgments |
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Supported in part by the National Health and Medical Research Council of Australia (project grant 211266) and by a Seeding Grant from The Lincoln Centre for Research into Bone and Joint Disorders (Royal North Shore Private Hospital; St Leonards, NSW, Australia).
Dr G. Gibson (Henry Ford Hospital, Detroit, MI) is thanked for his kind gift of antibodies to type X collagen.
Received for publication November 25, 2002; accepted April 17, 2003.
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