Thomas M. Schmid

Developmental acquisition of type × collagen in the embryonic chick tibiotarsus☆

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.

Recombinant osteogenic protein-1 upregulates extracellular matrix metabolism by rabbit annulus fibrosus and nucleus pulposus cells cultured in alginate beads.

This study was performed to determine if recombinant human osteogenic protein‐1 (rhOP‐1) is effective in promoting matrix synthesis and matrix formation by rabbit nucleus pulposus (NP) and annulus fibrosus (AF) cells cultured in alginate beads. The effects of culturing the cells in the presence of various concentrations of rhOP‐1 were assessed by measuring changes in cell proliferation, proteoglycan (PG) and collagen synthesis and mRNA expression, and in the matrix contents of PG and collagen, as indicators of matrix accumulation. At high concentrations, rhOP‐1 had a moderate mitogenic effect on both NP and AF cells. It also stimulated the synthesis of PG and collagen in a dose‐dependent manner: this was associated with a corresponding increase in the expression of mRNA for aggrecan core protein and collagen type II. The stimulatory effect of rhOP‐1 on PG synthesis was more pronounced than that on collagen synthesis. Continuous treatment with rhOP‐1 led to an increase in the total DNA, PG and collagen contents in both NP and AF cultures. The results presented here provide evidence of the ability of rhOP‐1 to stimulate the metabolism of both rabbit AF and NP cells cultured in alginate beads.

Basic Fibroblast Growth Factor Stimulates Matrix Metalloproteinase-13 via the Molecular Cross-talk between the Mitogen-activated Protein Kinases and Protein Kinase Cδ Pathways in Human Adult Articular Chondrocytes

Excessive release of basic fibroblast growth factor (bFGF) during loading and/or injury of the cartilage matrix may contribute to the onset or progression of osteoarthritis. This pathological role may be related to the ability of bFGF to decrease proteoglycan synthesis and to antagonize the activity of anabolic growth factors in cartilage such as insulin-like growth factor-1 and bone morphogenetic protein 7 (BMP7 or OP-1). Matrix metalloproteinase-13 (MMP-13), a catabolic cartilage-degrading enzyme, is dramatically up-regulated by inflammatory cytokines or by fibronectin fragments in articular chondrocytes. In this study, we investigated MMP-13 production by bFGF using human articular chondrocytes. Endogenous concentration of bFGF in synovial fluids collected from arthritis patients and asymptomatic subjects showed a good linear correlation with the endogenous levels of MMP-13. bFGF stimulation of MMP-13 was mediated at the transcriptional level and, at least in part, by stimulation of interleukin-1 production. Also, our findings suggest that bFGF stimulation of MMP-13 required the activation of multiple MAPKs (ERK, p38, and JNK) by bFGF, and more importantly, bFGF activation of protein kinase C (PKC) δ played a key role in the MMP-13 stimulation. Indeed, PKCδ is the only isoform associated with MMP-13 stimulation among the PKC isoforms tested. PKCδ controls the bFGF response by regulating multiple MAPK pathways. Our results suggest that PKCδ activation is a principal rate-limiting event in the bFGF-dependent stimulation of MMP-13 in human adult articular chondrocytes. We propose that deregulation of cross-talk between MAPK and PKCδ signaling may contribute to the etiology of osteoarthritis in human patients.

Detection of Superficial Zone Protein in Human and Animal Body Fluids by Cross-Species Monoclonal Antibodies Specific to Superficial Zone Protein

In this report we describe the purification of human superficial zone protein (SZP), the generation of cross-species monoclonal antibodies (MAbs) and the detection of this protein in human and animal body fluids. Human SZPs, used as immunizing antigens, were purified either from culture media of human cartilage organ cultures or from human synovial fluids. The immunizing antigens were mixed with RIBI adjuvant in one of three forms: nonmodified SZP, superficial zone protein-keyhole limpet hemocyanin conjugate (SZP-KLH), or a mixture of superficial zone protein and hyaluronic acid (SZP-HA). A panel of MAbs including GW4.23, S6.79, S13.52, S13.233, and S17.109 were generated and characterized. Monoclonal antibody (MAb) S6.79, an IgG2b with K(D) 3.14 x 10(-9) M from SZP-KLH immunization, is of particular interest. It reacts strongly to a large molecular weight form of SZP in both enzyme-linked immunosorbent assay (ELISA) and Western blotting. It stains the most superficial layer of articular cartilage in immunohistochemistry, whereas the middle and deep zones of cartilage are not stained. When MAb S6.79 was applied to Western blots of human body fluids, a strong 345-kDa band was detected in samples of synovial fluid and weaker bands of similar size were detected in samples of plasma and serum. MAb S6.79 also showed cross-species immunoreactivity with SZP in samples of synovial fluids harvested from bovine, dog, guinea pig, and rabbit, as demonstrated by Western blotting and antibody absorption experiments. This cross-species MAb will be a useful tool in human and animal model studies for monitoring SZP levels and tissue distribution. It may help define the roles of SZP in normal articular joints and may be of diagnostic or prognostic value for the measurement of SZP in pathological conditions such as osteoarthritis, rheumatoid arthritis, and camptodactyly-arthropathy-coxa vara-pericarditis.

Hypertrophic cartilage matrix. Type X collagen, supramolecular assembly, and calcification.

During endochondral bone formation, individual chondrocytes undergo a progression from young cells undergoing rapid division, to mature cells having the greatest capacity for synthesizing matrix components, to hypertrophic cells, becoming greatly enlarged and eventually removed. Concomitant with, and subsequent to, the process of cellular hypertrophy, major changes occur in the cartilage matrix. These include: (a) enlargement of lacunae to accommodate the great increase in volume of the individual chondrocytes; ’ (b) calcification of the cartilage matrix, providing a substratum for bone deposition; and (c) removal of both calcified and uncalcified cartilage matrix, resulting in formation of a marrow cavity. In young, prehypertrophic cartilage the chondrocytes synthesize a mixture of collagen types 11, IX, and XI, which become coassembled into heterotypic fibrils. Once the cells have initiated hypertrophy, they add type X to their biosynthetic repertoire. Quantitatively, type X collagen may be a minor component of the growth plate; but during the latter stages of hypertrophy, chondrocytes devote much of their biosynthetic potential to its synthesis. As the hypertrophic program progresses, the synthesis of this collagen increases, with a concomitant decrease in synthesis of the others.* This, coupled with the morphological events occurring in the cells and matrix during hypertrophy, have prompted us to hypothesize that: (a) the molecule may represent a component added to the matrix to facilitate turnover; or (b) it may participate in the calcification of the cartilage matrix.

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckels cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)

Lubricin distribution in the goat infraspinatus tendon: a basis for interfascicular lubrication.

BACKGROUND This study was motivated by the need to better understand the tribology of the rotator cuff, as it could provide insights into degenerative processes and suggest therapeutic approaches to cuff disorders. The objective was to evaluate the distribution of a known lubricating protein in the infraspinatus tendon of the rotator cuff in adult goats. The hypothesis was that lubricin, also known as superficial zone protein or proteoglycan 4, serves as an interfascicular lubricant. METHODS Eight infraspinatus tendons were resected from eight Spanish goats, and five patellar tendons and articular cartilage samples were also resected from five of the goats. Samples were processed for immunolocalization of lubricin with use of a purified monoclonal antibody and for histological analysis with Masson trichrome staining for collagen. The locations of lubricin within the tendon were documented, and measurements were made of the distance to which lubricin was detected in the tendon, relative to the humeral insertion site, and the depth of lubricin staining into the fibrocartilage of the fascicle bordering the humeral joint space. Images from polarized light microscopy were used to measure the fascicle diameter and the crimp length. RESULTS Lubricin was prominent in layers separating the fascicles in the infraspinatus tendon, with occasional intrafascicular staining. The fibrocartilaginous portion of the fascicle bordering the humeral joint space displayed diffuse lubricin staining. The Masson trichrome staining of the collagen in this lubricin-containing fibrocartilage indicated that it was not being tensioned at the time of fixation, whereas the collagen contained in the body of the tendon was under tension. The crimp of the fascicles near the humeral joint side of the tendon displayed a shorter peak-to-peak length than the crimp of the fascicles in the superior region of the tendon. Only the surface of two of the five patellar tendon samples stained for lubricin; there was no staining within these ligaments. CONCLUSIONS The sheaths of the fascicles of the infraspinatus tendon near the bone insertion site contain lubricin, indicating that this lubricating protein may be facilitating interfascicular movement. The fact that the crimp pattern of fascicles changes with location in the tendon provides support for the supposition that fascicles move relative to one another as the tendon is loaded, underscoring the importance of a lubricating protein in the layer separating the fascicles.

Multiple-reaction cycling: a method for enhancement of the immunochemical signal of monoclonal antibodies.

Most current studies using immunochemical and immunohistochemical procedures to detect antigen-antibody complexes employ some type of indirect method. Such procedures afford signal amplification because several marker-conjugate molecules can bind to each primary antibody molecule. We have observed that for monoclonal antibodies an even greater amplification can be afforded simply by performing two (or more) reaction cycles (i.e., primary antibody, secondary antibody-primary antibody, secondary antibody-etc). In the present report, we demonstrate the utility of this method for immunohistochemical (immunofluorescence) and immunochemical (ELISA: enzyme-linked immunosorbent assay) procedures employing well-characterized monoclonal antibodies directed against avian type IV (basement membrane) collagen.

Cleavage of collagen type X by human synovial collagenase and neutrophil elastase.

Chick-derived native cartilage collagen type X and the pepsin-resistant 45 kDa fragment were susceptible to attack by human synovial collagenase and neutrophil elastase at 25 degrees C and 35 degrees C. Synovial collagenase cleaved type X collagen at two sites which were equally susceptible to the enzyme. In contrast, elastase produced three cleavages, but the sensitive loci showed different susceptibilities as judged by the sequential appearance of specific breakdown products. Both enzymes produced a major, enzyme-resistant fragment of approximately 32 kDa at 35 degrees C, and both of these end-products co-migrated in SDS polyacrylamide gels. Human chondrocyte-derived collagenase also degraded native, 59 kDa collagen type X in a similar manner to that shown by the synovial collagenase. From amino acid sequence data the enzyme cleavages probably occur at three regions of sequence imperfection. The specific cleavages brought about by synovial or chondrocyte collagenase, or neutrophil elastase, may have a functional catabolic role in vivo, and in vitro might provide useful tools with which to further analyse specific properties of the native collagen type X molecule.

Degradation of cartilage collagens type II, IX, X and XI by enzymes derived from human articular chondrocytes.

Conditioned culture medium derived from Interleukin-I alpha-activated human articular chondrocytes contained both collagen- and proteoglycan-degrading activities. Preparations of soluble type I collagen and the cartilage collagens type II, IX, X and XI were all degraded when incubated with the conditioned culture medium at 35 degrees C. Fractionation of the enzymic activities using column chromatography with Ultragel AcA 34 and Heparin-Sepharose allowed the separation and identification of neutral proteinase, collagenolytic and proteoglycan-degrading activities. Eluant fractions which contained type I collagenase activity effectively degraded collagen type II, but these fractions did not correspond precisely with those which degraded collagen types IX, X and XI. These observations indicate that chondrocytes have the potential to produce a conventional interstitial type II collagenase together with other enzymes having some specificity for the minor collagens. Thus IL-1-activated chondrocytes produce a range of collagenolytic and proteoglycan-degrading enzymes which can process most of the structural components of the cartilage matrix.