Erosive effect of different dietary substances on deciduous and permanent teeth.

OBJECTIVES: We investigated the effect of different dietary substances on deciduous and permanent enamel. MATERIALS AND METHODS: Enamel specimens were prepared from human teeth (n = 108 deciduous molars and n = 108 permanent premolars). We measured the chemical parameters (pH, titratable acidity, viscosity, calcium, phosphate, fluoride concentration and degree of saturation) of nine dietary substances. The teeth were immersed in the respective substance (2 × 2 min; 30 °C; shaking), and we measured the baseline surface hardness (SH) in Vickers hardness numbers (VHN), and the changes in SH after 2 min (ΔSH2-0) and the 4 min (ΔSH4-0) immersion. We analysed the differences between deciduous and permanent teeth using the Wilcoxon test and correlated ΔSH to the different chemical parameters. RESULTS: Deciduous teeth were significantly softer (549.53 ± 59.41 VHN) than permanent teeth (590.15 ± 55.31 VHN; p < 0.001) at baseline, but they were not more vulnerable to erosive demineralization. Only orange juice, which presented milder erosive potential, caused significantly more demineralisation in deciduous teeth at ΔSH4-0. Practically all chemical parameters significantly correlated with ΔSH (p < 0.05). Substances with lower pH, higher titratable acidity, lower Ca, higher Pi and lower F concentrations, higher viscosity and more undersaturated solutions presented more erosive demineralisation. CONCLUSION: Different parameters in dietary substances affect erosive demineralisation in deciduous and permanent teeth, but we generally observed no differences in susceptibility to erosion between both types of teeth; only orange juice (less severe acid conditions) caused perceptible differences. CLINICAL RELEVANCE: We observe that permanent teeth are harder than deciduous teeth, but most substances cause no perceptible difference in erosive demineralisation in both types of teeth.

Increased friction coefficient and superficial zone protein expression in patients with advanced osteoarthritis.

OBJECTIVE: To quantify the concentration of superficial zone protein (SZP) in the articular cartilage and synovial fluid of patients with advanced osteoarthritis (OA) and to further correlate the SZP content with the friction coefficient, OA severity, and levels of proinflammatory cytokines. METHODS: Samples of articular cartilage and synovial fluid were obtained from patients undergoing elective total knee replacement surgery. Additional normal samples were obtained from donated body program and tissue bank sources. Regional SZP expression in cartilage obtained from the femoral condyles was quantified by enzyme-linked immunosorbent assay (ELISA) and visualized by immunohistochemistry. Friction coefficient measurements of cartilage plugs slid in the boundary lubrication system were obtained. OA severity was graded using histochemical analyses. The concentrations of SZP and proinflammatory cytokines in synovial fluid were determined by ELISA. RESULTS: A pattern of SZP localization in knee cartilage was identified, with load-bearing regions exhibiting high SZP expression. SZP expression patterns were correlated with friction coefficient and OA severity; however, SZP expression was observed in all samples at the articular surface, regardless of OA severity. SZP expression and aspirate volume of synovial fluid were higher in OA patients than in normal controls. Expression of cytokines was elevated in the synovial fluid of some patients. CONCLUSION: Our findings indicate a mechanochemical coupling in which physical forces regulate OA severity and joint lubrication. The findings of this study also suggest that SZP may be ineffective in reducing joint friction in the boundary lubrication mode at an advanced stage of OA, where other mechanisms may dominate the observed tribological behavior.

Del1: a new protein in the superficial layer of articular cartilage.

Articular cartilage contains four distinct zones, extending from the surface to the subchondral bone. Freshly isolated chondrocytes from the superficial zone of articular cartilage retain a collagenase-P-resistant cell-associated matrix. In the studies described here, the protein Del1 was identified as a component of the cell-associated matrix of superficial zone chondrocytes from adult bovine articular cartilage. Very little Del1 was associated with freshly isolated deep zone chondrocytes. Western blot analysis of articular cartilage cell and tissue extracts using polyclonal antibodies specific for Del1 showed Del1 was present in an insoluble cell-associated fraction. Extracts of the superficial zone of articular cartilage were found to be enriched in Del1 compared to the deeper layers of the tissue. Immunohistochemical staining of full-thickness articular cartilage with anti-Del1 antibodies also showed an enrichment of Del1 in the superficial zone. These observations are the first to describe the protein Del1 in a nonendothelial, nonfetal tissue.

Type X collagen and other up-regulated components of the avian hypertrophic cartilage program.

Elucidating the cellular and molecular processes involved in growth and remodeling of skeletal elements is important for our understanding of congenital limb deformities. These processes can be advantageously studied in the epiphyseal growth zone, the region in which all of the increase in length of a developing long bone is achieved. Here, young chondrocytes divide, mature, become hypertrophic, and ultimately are removed. During cartilage hypertrophy, a number of changes occur, including the acquisition of synthesis of new components, the most studied being type X collagen. In this review, which is based largely on our own work, we will first examine the structure and properties of the type X collagen molecule. We then will describe the supramolecular forms into which the molecule becomes assembled within tissues, and how this changes its physical properties, such as thermal stability. Certain of these studies involve a novel, immunohistochemical approach that utilizes an antitype X collagen monoclonal antibody that detects the native conformation of the molecule. We describe the developmental acquisition of the molecule, and its transcriptional regulation as deduced by in vivo footprinting, transient transfection, and gel-shift assays. We provide evidence that the molecule has unique diffusion and regulatory properties that combine to alter the hypertrophic cartilage matrix. These conclusions are derived from an in vitro system in which exogenously added type X collagen moves rapidly through the cartilage matrix and subsequently produces certain changes mimicking ones that have been shown normally to occur in vivo. These include altering the cartilage collagen fibrils and effecting changes in proteoglycans. Last, we describe the subtractive hybridization, isolation, and characterization of other genes up-regulated during cartilage hypertrophy, with specific emphasis on one of these--transglutaminase.

Incomplete processing of type II procollagen by a rat chondrosarcoma cell line.

The Swarm rat chondrosarcoma cell line, RCS-LTC, deposits an extracellular matrix that contains the typical type II, IX, and XI collagen phenotype of hyaline cartilage, but the fibrils appear abnormally thin. By N-terminal sequence analysis, the type II collagen from the matrix was shown to have retained its N-propeptides with no evidence of normal processing to type II collagen. Amplification and sequencing of cDNA prepared from the pro alpha1(II) mRNA of these cells showed a normal N-propeptide cleavage site. Furthermore, the type II N-procollagen could be processed to type II collagen by incubation with culture medium from normal chondrocytes. The findings indicate that the RCS-LTC cell line fails to express an active type II procollagen N-proteinase and, therefore, offers a useful culture system in which to study the role of N-propeptide removal in fibrillogenesis.

Tetracycline derivatives inhibit cartilage degradation in cultured embryonic chick tibiae.

Tetracyclines have been used extensively as antibiotics and growth promoters in the poultry industry. However, they can inhibit angiogenesis and matrix degradation, both of which are essential for normal growth plate cartilage development. The purpose of this research was to test the ability of several tetracyclines to inhibit cartilage degradation in cultured embryonic chick tibiae. Based on gross observations and biochemical quantitation of collagen release into the media, minocycline, doxycycline, oxytetracycline, and tetracycline inhibited cartilage degradation at 20, 40, 60, and 80 micrograms ml-1 respectively. Chlortetracycline did not inhibit cartilage degradation at concentrations tested. The ability of the tetracycline derivative to inhibit cartilage degradation was in general related to its hydrophobicity. Since a majority of the cartilage in the embryonic chick tibia will develop into the post hatched growth plate, it may be important to determine if any of the tetracyclines used as antibiotics could cause problems in in vivo growth plate cartilage development.

Recombinant human osteogenic protein 1 is a potent stimulator of the synthesis of cartilage proteoglycans and collagens by human articular chondrocytes.

OBJECTIVE: To study the effects of recombinant human osteogenic protein-1 (rHuOP-1; bone morphogenetic protein-7) on proteoglycan and collagen synthesis by human articular chondrocytes. METHODS: Articular chondrocytes from fetal, adolescent, and adult human donors were cultured in alginate beads for 4 days in a mixture of Ham's F-12, Dulbecco's modified Eagle's medium, 10% fetal bovine serum (FBS), then for an additional 3-10 days in the presence and absence of rHuOP-1, with and without FBS. Chondrocyte synthetic activity was measured as the amount of incorporation of 35S-sulfate into proteoglycans and 3H-proline into hydroxyproline. Sieve chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed to identify specific proteoglycans and collagens. RESULTS: Recombinant human OP-1 markedly stimulated the synthesis of proteoglycans (mostly aggrecan) and collagens (predominantly type II) by all chondrocyte preparations. This did not require the presence of FBS and was associated with continued expression of the chondrocyte phenotype. CONCLUSION: Recombinant human OP-1 is a more potent stimulator of the synthesis of cartilage-specific molecules by human articular chondrocytes than are other factors tested for comparison, including TGF beta 1 and activin A.

Doxycycline inhibits type X collagen synthesis in avian hypertrophic chondrocyte cultures.

Doxycycline, a member of the tetracycline family, has been shown to reduce a type X collagen epitope as detected by immunohistochemistry with a monoclonal antibody in an avian explant culture system (). It was also shown to decrease collagenase and gelatinase activities and thus matrix degradation. This study investigates the effect of doxycycline on type X collagen synthesis in monolayer cultures of hypertrophic chondrocytes. Protein synthesis was evaluated by radioisotopic labeling during doxycycline, tetracycline, or minocycline treatment. Radiolabeled proteins were analyzed by gel electrophoresis, and total collagen was quantitated by hydroxyproline analysis. Additionally, the synthesis of type X collagen was measured by immunoprecipitation. Doxycycline was found to inhibit type X production more effectively than either of the other tetracyclines at comparable dose levels. Furthermore, type X collagen was inhibited more than other collagens, non-collagenous proteins and proteoglycans, with maximal inhibition at 80 microg/ml and an IC50 of 7 microg/ml. This inhibition by doxycycline was specific for type X collagen at 10 microg/ml, and the pattern was distinct from cycloheximide, a recognized inhibitor of protein translation. This suppression of type X collagen could not be overcome by excess extracellular calcium, conditions that have been demonstrated to induce synthesis of this protein (2).

Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: comparison of two distinct matrix compartments.

We have characterized immunohistochemically and biochemically the collagens accumulating in two compartments of the matrix formed by mature bovine articular chondrocytes in alginate beads. At all times of the 28-day culture period, more than 90% of the collagen molecules were recovered from the rim of cell-associated matrix (CM) which encapsulates individual chondrocytes and chondrocyte clusters. Both the total amount and concentration of collagens in this matrix compartment rose progressively with time. The ratio of collagen/proteoglycan remained relatively constant with time and was always five to seven times higher in the CM than in the interterritorial matrix compartment further removed from the cells. In the CM, collagen types II, IX and XI were present on Day 28 in relative proportions (95/l/3) similar to those in adult cartilage. A higher proportion of newly synthesized collagen type XI than types II or IX molecules did not become incorporated into the pericellular rim of matrix but accumulated in the further removed matrix. Although collagen type I was synthesized in small amounts by flattened cells at the surface of the beads, it did not become incorporated as heterotrimers or homotrimers in the matrix. Mature pyridinium crosslinks, principally pyridinoline, were detected as early as Day 7 of culture but became much more abundant between Days 15 and 28, especially in the CM which contained at all times more than 90% of the crosslinks formed. The codistribution of collagen types II, IX and XI and mature collagen-specific crosslinks support the contention that mature chondrocytes cultured in alginate matrix surround themselves with a protective shell whose composition is very similar to that which encapsulated the cells in vivo.

[Do calcium and zinc ions influence matrix molecule synthesis of chondrocytes?]. / Haben Kalzium- und Zinkionen einen Einfluss auf die Matrixmolekülsynthese von Chondrozyten?

The experiments described here tested the effect of various calcium (Ca) and Zinc (Zn) concentrations on cell proliferation and matrix molecule synthesis of fetal and adult bovine chondrocytes in monolayer cultures. Levels of Ca < 0.2 mM in a culture medium or the addition of Zn (0.1-50 microM) selectively promoted the production of collagen but did not affect significantly synthesis of proteoglycans. No change in proliferation of fetal and adult chondrocytes could be observed. In contrast 10 mM Ca promoted the hypertrophic differentiation of chondrocytes (e.g. expression of collagen type X). The results are related to calcium channel configurations in chondrocytes in the discussion.

Type X collagen isolated from the hypertrophic cartilage of embryonic chick tibiae contains both hydroxylysyl- and lysylpyridinoline cross-links.

Hypertrophic cartilage from the tibiotarsus of Day 20 chick embryonic tibiae was found to contain an unusually high concentration of lysylpyridinoline (LP), a nonreducible collagen cross-link normally found only in bone and dentin but not in cartilage. Since type X collagen is abundant in this cartilage, research was conducted to see if type X was the primary source of LP. The 45-kDa pepsin-resistant form of type X was purified by immunoaffinity chromatography. It contained a high concentration of the LP cross-link while type II contained primarily hydroxylysylpyridinoline (HP), the predominant cross-link in cartilage. This, to our knowledge is the first time that type X has been shown directly to form nonreducible cross-links and that a collagen other than type I has a high level of LP. Also, it is interesting that the HP and LP cross-links are found in a collagen that is degraded so rapidly. Possible explanations for these findings are discussed.

Collagen and proteoglycan production by bovine fetal and adult chondrocytes under low levels of calcium and zinc ions.

The experiments described herein tested the effects of CaCl2 and ZnCl2, added at various concentrations in the culture medium, upon the synthesis of collagen and proteoglycan by adult and fetal (articular, epiphyseal and hypertrophic) bovine chondrocytes maintained in high density multilayer cultures. CaCl2 concentrations below 0.5 mM or the addition of 1-50 microM ZnCl2 to the medium selectively promoted the production of collagen by all four populations of chondrocytes but had no effect on fibroblasts. Further, these changes had no statistically significant effect on the incorporation of 35S-sulfate into macromolecules or on the synthesis of gelatinase A, measured by gelatin zymography. The addition of CaCl2 and ZnCl2 at these concentrations did not result in a change in the relative proportion of non-crosslinked 3H-collagen molecules (synthesized in the presence of beta-aminopropionitrile) partitioning in the cell layer and medium compartments, and did not appreciably alter the pattern of collagens synthesized by any of the cell populations. The hypertrophic cells synthesized high levels of collagen type X in the presence as well as absence of exogenously added cations. However, CaCl2 at 10 mM caused a marked upregulation of collagen type X synthesis by a preparation of chondrocytes derived from the entire growth plate, consistent with the view that calcium at that concentration stimulated the differentiation of some of the cells into hypertrophic chondrocytes.

Complete degradation of type X collagen requires the combined action of interstitial collagenase and osteoclast-derived cathepsin-B.

We have studied the degradation of type X collagen by metalloproteinases, cathepsin B, and osteoclast-derived lysates. We had previously shown (Welgus, H. G., C. J. Fliszar, J. L. Seltzer, T. M. Schmid, and J. J. Jeffrey. 1990. J. Biol. Chem. 265:13521-13527) that interstitial collagenase rapidly attacks the native 59-kD type X molecule at two sites, rendering a final product of 32 kD. This 32-kD fragment, however, has a Tm of 43 degrees C due to a very high amino acid content, and thus remains helical at physiologic core temperature. We now report that the 32-kD product resists any further attack by several matrix metalloproteinases including interstitial collagenase, 92-kD gelatinase, and matrilysin. However, this collagenase-generated fragment can be readily degraded to completion by cathepsin B at 37 degrees C and pH 4.4. Interestingly, even under acidic conditions, cathepsin B cannot effectively attack the whole 59-kD type X molecule at 37 degrees C, but only the 32-kD collagenase-generated fragment. Most importantly, the 32-kD fragment was also degraded at acid pH by cell lysates isolated from murine osteoclasts. Degradation of the 32-kD type X collagen fragment by osteoclast lysates exhibited the following properties: (a) cleavage occurred only at acidic pH (4.4) and not at neutral pH; (b) the cysteine proteinase inhibitors E64 and leupeptin completely blocked degradation; and (c) specific antibody to cathepsin B was able to inhibit much of the lysate-derived activity. Based upon these data, we postulate that during in vivo endochondral bone formation type X collagen is first degraded at neutral pH by interstitial collagenase secreted by resorbing cartilage-derived cells. The resulting 32-kD fragment is stable at core temperature and further degradation requires osteoclast-derived cathepsin B supplied by invading bone.

Doxycycline disrupts chondrocyte differentiation and inhibits cartilage matrix degradation.

OBJECTIVE: The effects of doxycycline were tested in an in vitro system in which the cartilages of embryonic avian tibias are completely degraded. METHODS: Tibias were cultured with 5, 20, or 40 microgram/ml doxycycline. Control tibias were cultured without doxycycline. Conditioned media and tissue sections were examined for enzyme activity and matrix loss. RESULTS: Cartilages were not resorbed in the presence of doxycycline, whereas control cartilages were completely degraded. Collagen degradation was reduced in association with treatment with doxycycline at all doses studied. Higher concentrations of doxycycline reduced collagenase and gelatinase activity and prevented proteoglycan loss, cell death, and deposition of type X collagen in the cartilage matrix; in addition, treatment with doxycycline at higher concentrations caused increases in the length of the hypertrophic region. CONCLUSION: The effects of doxycycline extend beyond inhibition of the proteolytic enzymes by stimulating cartilage growth and disrupting the terminal differentiation of chondrocytes.

A novel proteoglycan synthesized and secreted by chondrocytes of the superficial zone of articular cartilage.

A novel proteoglycan (PG) has been identified in culture medium from thin slices of the superficial zone of bovine articular cartilage. This PG is synthesized and secreted selectively by chondrocytes of this zone but has not been demonstrated in culture medium from slices deeper in the same tissue. There is little, if any, incorporation of this PG into the extracellular matrix. The PG has been partially purified by isopycnic CsCl density gradient ultracentrifugation, ion-exchange chromatography on DEAE Sephacel, and gel filtration chromatography on Sepharose CL-2B. It migrates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of approximately 345 kDa. The molecule is degraded by papain, trypsin, or pronase; however, limited pepsin treatment performed at 4 degrees C only decreases its molecular weight to approximately 315 kDa. The molecule is substituted with keratan sulfate and chondroitin sulfate, which are largely removed by limited pepsin treatment. In addition, this PG, or a very similar molecule, has been demonstrated in synovial fluid. This novel PG may serve as a functional metabolic marker for chondrocytes of the superficial zone of articular cartilage.

Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads.

Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the 'interterritorial' matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a significant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteoglycans as usually occurs when chondrocytes lose their phenotypic stability. After this extended period of time in culture, the cells were present as two populations exhibiting differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found throughout the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tangential to the surface.

Type X collagen degradation in long-term serum-free culture of the embryonic chick tibia following production of active collagenase and gelatinase.

Type X collagen has a very limited distribution during skeletal development in regions of hypertrophic cartilage destined for degradation. In solution assay, type X collagen is degraded to a 32-kDa cleavage product which is resistant to further degradation, suggesting this product may have a function in skeletal development. In this study, we have identified the 32-kDa cleavage product of type X collagen present in the conditioned media (CM) during incubation of isolated 12-day chick tibiae in the absence of serum. In this culture system, chondrocytes throughout the tibial cartilages hypertrophied and deposited type X collagen within their matrix. During culture, the cartilage matrix was degraded in two stages. First proteoglycan was lost followed by degradation of the collagenous components. Collagen degradation was accompanied by the release of active interstitial collagenase and gelatinase into the CM. Purified type X collagen incubated in this CM was cleaved to form a 32-kDa product which was resistant to further degradation. This cleavage product has the same electrophoretic mobility as the 32-kDa chain produced by purified human collagenase.

Domains of type X collagen: alteration of cartilage matrix by fibril association and proteoglycan accumulation.

During endochondral bone formation, hypertrophic cartilage is replaced by bone or by a marrow cavity. The matrix of hypertrophic cartilage contains at least one tissue-specific component, type X collagen. Structurally type X collagen contains both a collagenous domain and a COOH-terminal non-collagenous one. However, the function(s) of this molecule have remained largely speculative. To examine the behavior and functions of type X collagen within hypertrophic cartilage, we (Chen, Q., E. Gibney, J. M. Fitch, C. Linsenmayer, T. M. Schmid, and T. F. Linsenmayer. 1990. Proc. Natl. Acad. Sci. USA. 87:8046-8050) recently devised an in vitro system in which exogenous type X collagen rapidly (15 min to several hours) moves into non-hypertrophic cartilage. There the molecule becomes associated with preexisting cartilage collagen fibrils. In the present investigation, we find that the isolated collagenous domain of type X collagen is sufficient for its association with fibrils. Furthermore, when non-hypertrophic cartilage is incubated for a longer time (overnight) with "intact" type X collagen, the molecule is found both in the matrix and inside of the chondrocytes. The properties of the matrix of such type X collagen-infiltrated cartilage become altered. Such changes include: (a) antigenic masking of type X collagen by proteoglycans; (b) loss of the permissiveness for further infiltration by type X collagen; and (c) enhanced accumulation of proteoglycans. Some of these changes are dependent on the presence of the COOH-terminal non-collagenous domain of the molecule. In fact, the isolated collagenous domain of type X collagen appears to exert an opposite effect on proteoglycan accumulation, producing a net decrease in their accumulation, particularly of the light form(s) of proteoglycans. Certain of these matrix alterations are similar to ones that have been observed to occur in vivo. This suggests that within hypertrophic cartilage type X collagen has regulatory as well as structural functions, and that these functions are achieved specifically by its two different domains.

The influence of bone and marrow on cartilage hypertrophy and degradation during 30-day serum-free culture of the embryonic chick tibia.

In this study, an organ culture system is defined which demonstrates complete loss of cartilage matrix from embryonic chick tibiae. Efficient loss of the cartilage matrix occurs within 30 days of serum-free culture only when the intact tibiae containing bone, marrow, and cartilage tissue are cultured. During organ culture nonhypertrophic chondrocytes become hypertrophic and stain positively for type X collagen and alkaline phosphatase. The cartilage loses Safranin O staining, and finally all cartilage matrix disappears leaving the bony collar and marrow cells. If the tibial cartilage is separated from the bony collar and cultured alone in serum-free medium, the nonhypertrophic chondrocytes also hypertrophy; the matrix loses Safranin O staining; however, some components of the matrix including type X collagen still remain after 30 days. In the presence of serum, the chondrocytes will hypertrophy but cartilage degradation is not evident. The results of this study support the conclusions that 1) hypertrophy is inherently programmed in the chondrocyte and 2) while Safranin O staining of cartilage cultured alone is diminished in serum-free organ culture, the degradation of cartilage is complete only when bone and marrow are also present.