Altered mechanics and histomorphometry of canine tibial cartilage following joint immobilization.

Joint immobilization is associated with altered cartilage biosynthesis and catabolism that may affect cartilage mechanics and joint function. In this study, the mechanical behavior of articular cartilage was studied in an experimental model of joint immobilization, in which the canine knee was cast-immobilized at 90 degrees of flexion for 4 weeks. Articular cartilage from the medial tibial plateau was tested in compression and in shear. Biochemical assays for water and glycosaminoglycan content and histomorphometric grading were performed on site-matched samples. Significant decreases in the equilibrium and dynamic shear moduli, but not compressive moduli, were observed in cartilage after 4 weeks of joint immobilization as compared to cartilage from a separate control population. Importantly, there was also evidence of a decrease in the compressive and shear moduli of tibial cartilage from the contralateral knee joints compared to control joints that were not immobilized. No significant effect of immobilization on the biochemical parameters or histomorphometric scores was detected, expect for a significant loss of proteoglycan staining following immobilization. These findings for changes in the tibial cartilage following cast immobilization are consistent with a mild form of cartilage degeneration.

Effect of 1alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on metalloproteinase activity and cell maturation in growth plate cartilage in vivo.

Recent studies indicate that 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and 24R,25-dihydroxyvitamim D3 (24R,25[OH]2D3) differentially regulate proliferation, differentiation, and matrix synthesis of growth plate chondrocytes. To determine whether both metabolites play the same or different roles in vivo, we used the vitamin D-deficient rat as a model. Rickets was induced and then reversed by administering a single dose of ergocalciferol, 1alpha,25(OH)2D3, or 24R,25(OH)2D3 and euthanizing the animals after 4, 24, 48, or 72 h. Growth plates were either processed for histology and histomorphometry or extracted with buffered guanidine-HCl. Neutral metalloproteinase activity in the extracts was measured by use of aggrecan-containing beads, and collagenase activity was determined by use of radioactive type I collagen. The levels of tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator were also determined. The morphology of the growth plate varied as a function of treatment. While 24R,25(OH)2D3 appeared to affect cell maturation and 1alpha,25(OH)2D3 appeared to affect terminal differentiation and calcification, response to ergocalciferol was indicative of the combined responses to the individual metabolites. Enzyme activity was regulated in a differential manner. Treatment with ergocalciferol produced a rapid decline in both neutral metalloproteinase and collagenase activities that was statistically significant by 4 h. By contrast, 1alpha,25(OH)2D3 had no effect on neutral metalloproteinase activity but caused a significant decrease in both active and total collagenase activity by 4 h, while 24R,25(OH)2D3 decreased neutral metalloproteinase activity by 48 h and had no effect on collagenase activity. Ergocalciferol had no effect on TIMP levels at any time examined, whereas 1alpha,25(OH)2D3 caused an increase at 48 and 72 h and 24R,25(OH)2D3 completely blocked TIMP production at 4 and 24 h. By contrast, plasminogen activator activity by ergocalciferol was decreased at 4 h, increased by 1alpha,25(OH)2D3 at 4 and 24 h, and decreased by 24R,25(OH)2D3 at all time points examined. These in vivo results confirm our previous cell culture observations showing that growth plate chondrocytes are differentially regulated by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. Moreover, they show definitively that these two vitamin D metabolites play distinct roles not only in regulating neutral metalloproteinase and collagenase activities in growth plate cartilage but in cell maturation and calcification of this tissue in vivo.

Basic calcium phosphate crystals up-regulate metalloproteinases but down-regulate tissue inhibitor of metalloproteinase-1 and -2 in human fibroblasts.

OBJECTIVE: To examine the effect of basic calcium phosphate (BCP) crystals on expression of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in human fibroblasts. METHOD: Using a semi-quantitative reverse transcription-polymerase chain reaction method and phosphocitrate (PC), a specific inhibitor of the biological effects of BCP crystals, we examined the effects of BCP on the steady state transcript levels of metalloproteinase (MMP)-1, -3, -9 and -13 and TIMP-1 and -2 in human fibroblasts. DNA primers against elongation factor were used as internal controls. RNAs isolated from human fibroblasts treated with BCP crystals (50 microg/ml) in the presence or absence of PC (10(-3) M) were used as templates, and RNA from untreated control cultures and cultures treated with Interleukin-1-beta (IL-1beta) were used as negative and positive controls, respectively. RESULTS: We observed increases in MMP-1, -3, -9 and -13 transcripts by BCP crystals. BCP crystal down-regulated TIMP-1 and -2 over untreated controls. Western blot analysis confirmed that BCP crystals down-regulate the synthesis of TIMP-1 and -2. While IL-1beta up-regulated MMP-1, -3, -9 and -13, it had no significant effect on expression of either TIMP. In all cases, PC specifically reversed the differential regulation of MMPs and TIMPs by BCP crystals but had no effect on IL-1beta induction of MMP expression. CONCLUSION: The ability of BCP to induce the synthesis of degradative MMPs while down-regulating the synthesis of the naturally occurring counterpart TIMPs may explain the changes consistent with a role of BCP crystal in the pathogenesis of degenerative changes in osteoarthritis. The ability of PC to reverse both degradative effects of BCP crystal suggests that PC can be a potential therapeutic agent for BCP crystal deposition diseases.

Perturbations in factors that modulate osteoblast functions in vitamin B6 deficiency.

It was hypothesized that the widespread structural defect of collagen in connective tissue of vitamin B6 deficient-animals and the consequent alteration in bone biomechanical properties cause an additional stress to their inflamed swollen tibiotarsometatarsal joints. The present study showed a 32% elevation (P < 0.02) in mean plasma free cortisol concentration. Vitamin D metabolism was impaired but without changing plasma calcium homeostasis and bone mineral content. Mean plasma calcitriol [1,25(OH)2D] concentration was significantly reduced (P < 0.001). Because plasma calcidiol concentration did not change, we speculated that either renal 25-hydroxycalciferol-1alpha-hydroxylase activity was reduced or 1,25(OH)2D turnover was increased. Plasma osteocalcin, an index of osteoblast function related to bone formation, was significantly decreased (P < 0.05). This adverse effect on osteoblasts was consistent with the reduction of bone specific alkaline phosphatase activity (another index of bone formation) found in a previous study. The excess of cortisol may have impaired these bone cells functions directly and (or) indirectly via the decline in calcitriol synthesis. Plasma hydroxyproline concentrations in B6-deficient animals were found to be significantly reduced (P < 0.001), suggesting that cortisol in excess had also a suppressive effect on another hydroxylase, namely tissue (mainly bone and liver) prolyl hydroxylase. The bone uncoupling (in formation and resorption) associated with vitamin B6 deficiency seems to be due to secondary hypercortisolism and (or) another unknown factors but not related to a change in bone modulators such as IGF-1 and eicosanoids.

Wild type and mutant p53 differentially regulate the gene expression of human collagenase-3 (hMMP-13).

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that can degrade all the proteins of the extracellular matrix and have been implicated in many abnormal physiological conditions including arthritis and cancer metastasis. Recently we have shown for the first time that the human MMP-1 gene is a p53 target gene subject to repression by wild type p53 (Sun, Y., Sun, Y. I., Wenger, L., Rutter, J. L., Brinckerhoff, C. E., and Cheung, H. S. (1999) J. Biol. Chem. 274, 11535-11540). Here, we report that cotransfection of fibroblast-like synoviocytes with p53 expression and hMMP13CAT reporter plasmids revealed that (i) hMMP13, another member of the human MMP family, was down-regulated by wild type p53, whereas all six of the p53 mutants tested lost the wild type p53 repressor activity in fibroblast-like synoviocytes; (ii) this repression of hMMP-13 gene expression by wild type p53 could be reversed by overexpression of p53 mutants p53-143A, p53-248W, p53-273H, and p53-281G; (iii) the dominant effect of p53 mutants over wild type p53 appears to be a promoter- and mutant-specific effect. An intriguing finding was that p53 mutant p53-281G could conversely stimulate the promoter activity of hMMP13 up to 2-4-fold and that it was dominant over wild type p53. Northern analysis confirmed these findings. Although the significance of these findings is currently unknown, they suggest that in addition to the effect of cytokines activation, the gene expression of hMMP13 could be dysregulated during the disease progression of rheumatoid arthritis (or cancer) associated with p53 inactivation. Since hMMP13 is 5-10 times as active as hMMP1 in its ability to digest type II collagen, the dysregulation or up-modulation of MMP13 gene expression due to the inactivation of p53 may contribute to the joint degeneration in rheumatoid arthritis.

A cartilage matrix deficiency experimentally induced by vitamin B6 deficiency.

A vitamin B6-deficiency-induced disorder in avian articular cartilage resembling osteoarthritis has been further characterized. We measured several parameters of proteoglycan (PG) metabolism, i.e., fixed charge density and sulfated glycosaminoglycans (S-GAG) content in PN-deficient versus control articular cartilage and synovial fluid from the knee joint. Statistically significant changes were: 1) decreased content and increased extractability of total sulfated PGs from articular cartilage with guanidine HCl; 2) elevation of S-GAG concentration in synovial fluid; 3) increased plasma cystathionine (sulfur amino acid) levels. PG synthesis as assessed by 35SO4 incorporation into S-GAGs was not impaired. A lack of cartilage swelling in 0.15 M saline and the normal water content indicated that although disturbed, the collagen network was not disrupted. This finding was in agreement with a previous microscopic study that revealed no fissures in the articular cartilage. Previous findings of a normal aggregating PG size-distribution and absence of elevated metalloproteases made a disturbance of aggregating PG metabolism unlikely. Escape into the synovial fluid of small PGs, normally bound to articular collagen, was believed to result from an alteration in collagen molecular organization that could be secondary to elevated circulating SH-compounds.

Regulation of the rat interstitial collagenase promoter by IL-1 beta, c-Jun, and Ras-dependent signaling in growth plate chondrocytes.

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5'-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1 beta-inducible reporter gene activity. Cells stimulated by IL-1 beta (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1 beta regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation.

Interleukin-1alpha and beta in growth plate cartilage are regulated by vitamin D metabolites in vivo.

Matrix remodeling plays a prominent role in growth plate calcification. Since interleukin-1 (IL-1) has been implicated in stimulating proteinase production and inhibiting matrix synthesis in articular cartilage, we examined whether IL-1 was present in growth plate and whether the vitamin D metabolites, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 1,25) and 24,25(OH)2D3 (24,25), regulate the level of IL-1 found in this tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreated, injected 24 h prior to euthanasia with 24,25 (-VDP+24,25 rats) or 1,25 (-VDP+1,25 rats), or were given ergocalciferol (Ergo rats) orally, 48 h prior to euthanasia. Growth plates were harvested and extracted in buffer containing 1 M guanidine. IL-1 activity was measured by adding authentic cytokine or growth plate extracts to cultures of lapine articular cartilage and assaying release of glycosaminoglycans (GAGs) and changes in collagenase and neutral metalloproteinase activity. Neutralization of activity in the extracts was performed using polyclonal antisera to IL-1alpha or IL-1beta. An ELISA was used to determine levels of IL-1alpha and beta in the extracts. All extracts contained IL-1alpha and beta, as determined by ELISA. Levels of IL-1beta, but not IL-1alpha, were affected by the vitamin D status of the animal. Extracts from -VDP+24,25 animals contained significantly more IL-1beta than any of the other treatment groups, with the level found in these animals being 3-fold higher than normal and 2-fold higher than -VDP. Extracts were also tested in the bioassay to determine the level of active cytokine present. All growth plate extracts contained activity which altered GAG and proteinase release by lapine articular cartilage. Extracts from -VDP-, -VDP+1,25-, and -VDP+Ergo-treated rats stimulated a 40% increase in glycosaminoglycan release compared with extracts from normal rats. In contrast, extracts from -VDP+24,25-treated rats stimulated a 300% increase in glycosaminoglycan release. Both collagenase and neutral metalloproteinase activity of lapine cartilage were increased after incubation with the growth plate extracts. Collagenase activity was significantly increased 8- to 13-fold by the addition of extracts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral metalloproteinase activity was similarly increased by 4- to 10-fold. To characterize this activity further, growth plate extracts were incubated with neutralizing antibody to IL-1alpha or beta prior to addition to the lapine articular cartilage cultures. When antibodies were used separately, only partial inhibition was observed; incubation with both antibodies blocked 25% of the glycosaminoglycan release observed without antibody and greater than 80% of the enzyme activity released by the articular cartilage cultures. The results of this study show that growth plate cartilage contains both IL-1alpha and beta and indicate that vitamin D regulates the level of IL-1 in this tissue.

Mechanical behavior and biochemical composition of canine knee cartilage following periods of joint disuse and disuse with remobilization.

The mechanical behavior and biochemical composition of articular cartilage were studied in an experimental model of joint disuse, in which the canine knee was immobilized in a sling at 90 degrees of flexion. Articular cartilage from the surface zone of the femur was tested in an isometric tensile test and full-thickness cartilage on the tibial plateau was tested in a compressive indentation test. Water, proteoglycan and collagen contents were measured in site-matched samples. Site-specific increases in the tensile moduli (approximately 88% above control values in distal femoral groove) were observed in cartilage after 8 weeks of joint disuse, and after 3 weeks of remobilization following either 4 (approximately 140%, distal and proximal femoral groove) or 8 weeks (approximately 140%, distal femoral groove) of joint disuse. In contrast, the compressive properties of cartilage determined in the indentation test exhibited no change from control values with joint disuse or disuse followed by remobilization. Water contents increased at some sites on the tibia after 8 weeks of joint disuse (approximately 6% of tissue wet weight, posterior site), but not in the surface zone tissue of the femur. Proteoglycan/collagen and cartilage thickness were not found to change with disuse or disuse followed by remobilization. Reduced values for the ratio of proteoglycan:water were observed in the surface zone tissue of the femur (approximately 23%, distal femoral groove) and in the full-thickness tissue of the tibia (approximately 21%, anterior and posterior sites) after periods of joint disuse. In this study, the measured material properties suggest that the articular surface remains intact following periods of disuse or disuse with remobilization. This finding suggests one important difference between this model of joint disuse and other experimental models in which cartilage changes are both progressive and degenerative, such as surgically-induced joint instability.

Loss of decorin from the surface zone of articular cartilage in a chick model of osteoarthritis.

The objective of this study was to immunolocalize decorin and to assess changes as a result of pyridoxine (PN) deficiency in chick articular cartilage from femoral condyles. After maintenance on a normal diet for the first two weeks after hatching, 15 broiler chickens were deprived of this vitamin for 6 weeks. It was previously shown that the ankle joints of PN-deficient animals are swollen with effusions. They also present an abnormal gait, enlarged bony margins, and fissuring of the articular cartilages. Milder changes (no fissures) were also shown in the knee joints. Data from a previous study were suggestive that sulfated glycosaminoglycans are lost from the knee cartilage surface into synovial fluid. The current study was focused on the small proteoglycan, decorin, which coats the surface of collagen fibrils and may regulate their morphology. To examine decorin in normal and PN-deficient articular cartilage, a monoclonal antibody to an epitope on the protein core of decorin was used for immunohistochemical staining of tissue sections and for Western Blot analysis of cartilage extracts. Reduction of staining with the antibody was demonstrated in the tangential surface zone of PN-deficient cartilage, and Western Blot analysis showed reduced intensity of decorin bands compared to normal controls. These data suggest that a lack of decorin may play a role in the enlargement of collagen bundles in the tangential zone of PN-deficient articular cartilage as observed in a previous electron microscopic study.

Regulation of rat interstitial collagenase gene expression in growth cartilage and chondrocytes by vitamin D3, interleukin-1 beta, and okadaic acid.

The interstitial collagenase produced by the rat growth plate chondrocytes is the homologue of the human collagenase-3, or matrix metalloproteinase-13. This enzyme is responsible for the loss of collagen during hypertrophy of chondrocytes and for the degradation of transverse septa in long bone growth. Rachitic rats (42 days, male Sprague-Dawley) had an 8-fold higher level of collagenase mRNA in the hypertrophic versus proliferative zone of growth plate cartilage. Intramuscular injection of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3; 1.0 micrograms/kg body weight) in rachitic rats increased collagenase mRNA another 1.5-fold in the hypertrophic zone. The regulation of collagenase gene by 1,25-(OH)2D3 and interleukin (IL)-1 beta in cultured proliferative chondrocytes was studied by means of steady-state mRNA and half-life determination of mRNA using the transcriptional inhibitor actinomycin D, and nuclear run-on transcription analyses. Treatment of cells with 1,25-(OH)2D3 (10(-6) M) and IL-1 beta (2 ng/ml) increased collagenase mRNA 8- and 13-fold, respectively. Additionally, the collagenase mRNA half-life was increased by 1,25-(OH)2D3 and IL-1 beta. In the presence of a protein kinase C inhibitor, staurosporine, 1,25-(OH)2 D3 induction of collagenase mRNA was blocked. Here the addition of phorbol 12-myrisate 13-acetate (PMA) to activate protein kinase C increased collagenase mRNA 10-fold. However, in the presence of staurosporine (50 nM), PMA induction was blocked, whereas IL-1 beta was not. IL-1 beta is known to activate several phosphorylation pathways. Okadaic acid (500 nM), a protein phosphatase inhibitor, increased the relative collagenase mRNA abundance 10-fold. The rate of the rat collagenase gene transcription in nuclei was increased with 1,25-(OH)2D3, IL-1 beta and okadaic acid. In separate experiments, the collagenase promoter was ligated to a reporter plasmid and the plasmid was transfected into chondrocytes. The results showed that 1,25-(OH)2D3, IL-1 beta, and PMA increased reporter activity 2.5-, 2.8-, and 3.27-fold, respectively. Thus, there are multiple nuclear and cytoplasmic mechanisms by which cartilage modulators regulate rat interstitial collagenase gene expression.

Hepatocyte growth factor and its actions in growth plate chondrocytes.

Hepatocyte growth factor (HGF) has been implicated as a paracrine regulator of organogenesis and repair in many tissues. Here we have studied the expression and actions of HGF in intact rachitic rat growth plate and derived cultures of proliferative zone chondrocytes. In vivo and in vitro chondrocytes express HGF mRNA; 1,25(OH)2 has a three-fold maximal stimulatory effect, which can be blocked by H-7, an inhibitor of protein kinase C. Although HGF elaboration and action generally follow a paracrine model, chondrocytes appear capable of both expressing and responding to HGF. mRNA encoding the HGF receptor (c-met) was detected in both growth cartilage and derived chondrocyte cultures. HGF addition to chondrocyte cultures increased collagen II mRNA and alkaline phosphatase enzymatic activity to degrees comparable to that observed for active vitamin D metabolites. Combining HGF and 1,25-D evoked a synergistic response (ninefold) of alkaline phosphatase activity. To assess whether a similar stimulatory effect might be seen with bioactive peptides and HGF, we investigated the effect of HGF pretreatment on acute responses of chondrocytes to synthetic human calcitonin, an anabolic chondrocyte regulator whose skeletal action are mediated principally by cAMP elevation and subsequent protein kinase A activation. CT's maximal activation of protein kinase A was increased by prior HGF treatment from 56% to 78%. In concert, our findings indicate that in addition to HGF's classical paracrine role during skeletal growth, this growth factor may modulate hormonal sensitivity of the chondrocyte during proliferation, differentiation, and/or apoptosis.

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

Pyridoxine deficiency affects biomechanical properties of chick tibial bone.

The mechanical integrity of bone is dependent on the bone matrix, which is believed to account for the plastic deformation of the tissue, and the mineral, which is believed to account for the elastic deformation. The validity of this model is shown in this study based on analysis of the bones of vitamin B6-deficient and vitamin B6-replete chick bones. In this model, when B6-deficient and control animals are compared, vitamin B6 deficiency has no effect on the mineral content or composition of cortical bone as measured by ash weight (63 +/- 6 vs. 58 +/- 3); mineral to matrix ratio of the FTIR spectra (4.2 +/- 0.6 vs. 4.5 +/- 0.2), line-broadening analyses of the X-ray diffraction 002 peak (beta 002 = 0.50 +/- 0.1 vs. 0.49 +/- 0.01), or other features of the infrared spectra. In contrast, collagen was significantly more extractable from vitamin B6-deficient chick bones (20 +/- 2% of total hydroxyproline extracted vs. 10 +/- 3% p < or = 0.001). The B6-deficient bones also contained an increased amount of the reducible cross-links DHLNL, dehydro-dihydroxylysinonorleucine, (1.03 +/- 0.07 vs. 0.84 +/- 0.13 p < or = 0.001); and a nonsignificant increase in HLNL, dehydro-hydroxylysinonorleucine, (0.51 +/- 0.03 vs. 0.43 +/- 0.03, p < or = 0.10). There were no significant changes in bone length, bone diameter, or area moment of inertia. In four-point bending, no significant changes in elastic modulus, stiffness, offset yield deflection, or fracture deflection were detected. However, fracture load in the B6-deficient animals was decreased from 203 +/- 35 MPa to 151 +/- 23 MPa, p < or = 0.01, and offset yield load was decreased from 165 +/- 9 MPa to 125 +/- 14 MPa, p < or = 0.05. Since earlier histomorphometric studies had demonstrated that the B6-deficient bones were osteopenic, these data suggest that although proper cortical bone mineralization occurred, the alterations of the collagen resulted in changes to bone mechanical performance.

Vitamin D regulation of metalloproteinase activity in matrix vesicles.

Matrix vesicles (MVs) are enriched in matrix metalloproteinases (MMPs) capable of degrading proteoglycans. The aim of the present study was to identify which MMPs are present in MVs and determine whether these MMPs are regulated by 1,25-(OH)2D3 [1,25] and 24,25-(OH)2D3 [24,25]. To do this, growth zone (GC) and resting zone (RC) chondrocytes were isolated from rate costochondral cartilage and placed into culture. At confluence, GCs were treated with 1,25 and RCs with 24,25 for 24 hours. MVs, plasma membranes (PMs), and conditioned media were then collected from the cultures. RTPCR demonstrated the presence of mRNA for stromelysin-1 and 72 kDa gelatinase in both RCs and GCs, Casein zymography revealed activity at M(r) 48 and 28 kDa in MV, but not PM or conditioned media; Western analysis confirmed that this activity was stromelysin-1. Gelatinolytic activity, at low levels, was also found in MVs, but not PMs or conditioned media. When enzyme activity was measured using a proteoglycan bead assay, it was found that both GCs and RCs produced MVs and PMs containing neutral metalloproteinase. Both cells also produced MVs and PMs containing plasminogen activator. The addition of 1,25 to GCs caused a significant 4- to 5-fold increase in metalloproteinase activity in MVs, but not PMs. In contrast, MVs from cultures of RCs treated with 24,25 contained decreased metalloproteinase activity; enzyme activity in PMs was unaffected by 24,25. Plasminogen activator in MVs from RC was increased by treatment with 24,25, while MV enzyme activity was decreased after treatment of GC cultures with 1,25. This study shows that both RCs and GCs produce stromelysin-1 and 72 kDa gelatinase and that these enzymes are preferentially localized in MVs. Further, MMP and plasminogen activator activities in MVs and PMs are regulated by vitamin D metabolites.

Mechanical behavior of articular cartilage in shear is altered by transection of the anterior cruciate ligament.

The flow-independent viscoelastic and equilibrium behaviors of canine articular cartilage were examined with time after transection of the anterior cruciate ligament. The equilibrium, transient, and dynamic shear behaviors of cartilage were studied in biaxial compression-torsion testing at two time periods after transection of the anterior cruciate ligament and at two sites on the femoral condyle, in order to test for differences between sites of frequent and less frequent contact. Water content also was measured in cartilage at sites corresponding to the areas of mechanical testing. Transection of the anterior cruciate ligament produced significant decreases in all measured moduli of articular cartilage tested in equilibrium and dynamic shear and in equilibrium compression; the values for these moduli were 61, 56, and 77% of the control values, respectively, beginning at 6 weeks following transection of the anterior cruciate ligament. There was evidence of increased energy dissipation of cartilage in shear, with a 13 and 35% increase in tan delta at 6 and 12 weeks after transection of the anterior cruciate ligament, respectively. Changes in the viscoelastic relaxation function of cartilage in shear also were evident at 12 weeks after surgery. In all tissue, there was a significant increase in hydration of approximately 4% at 6 or 12 weeks after surgery. There was little difference between the material parameters for areas considered to be in frequent and less frequent contact, with the exception of hydration, which was greater for areas of less frequent contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative polymerase chain reaction assay for aggrecan and link protein gene expression in cartilage.

A procedure has been developed to quantify the levels of aggrecan and link protein mRNAs in small amounts of various tissues, including cartilage, using the power of PCR to amplify extremely low levels of specific templates. The PCR protocol which was selected allows for a simple assay procedure, with standards in different tubes from the samples. This straightforward procedure is quantitative, inexpensive, and allows for many samples to be analyzed at one time.

Cartilage metalloproteases in disuse atrophy.

A canine knee model of disuse atrophy produced by nonrigid fixation (sling) was characterized in respect to variables of proteoglycan size distribution, as well as biomechanical properties versus controls. Using this model, we found, in addition to the accepted dogma attributing changes to reduced protein synthesis by chondrocytes, that there is elevation of proteases and depression of tissue inhibitor of metalloproteases (TIMP) in atrophic knee cartilage. The findings are suggestive of cartilage remodelling reminiscent of bone remodelling in disuse atrophy reported by others. Whether the abnormal changes of protease-TIMP balance in knee cartilage can be retarded prophylactically by concurrent treatment with pentosan polysulfate and insulin like growth factor 1 remains uncertain.

Connective tissue integrity is lost in vitamin B-6-deficient chicks.

The objective of the present investigation was to characterize further the connective tissue disorder produced by pyridoxine (vitamin B-6) deficiency, as previously evidenced by electron microscopy. Following the second post-natal week, fast growing male chicks were deprived of pyridoxine for a 1-mo period. Six weeks post-natally, blood concentrations in the experimental deficiency group had declined to deficiency levels as registered by low concentrations of pyridoxal phosphate (coenzyme form) in erythrocytes, but did not reach levels associated with neurological symptoms. Light microscopic study showed abnormalities in the extracellular matrix of the connective tissues. Collagen cross-links and the aldehyde contents were not significantly lower in cartilage and tendon collagens of vitamin B-6-deficient animals than in age-matched controls; also, their proteoglycan degrading protease and collagenase activities measured in articular cartilages were not greater. Thus, proteolysis was an unlikely alternative mechanism to account for the loss of connective tissue integrity. These results point to the need for further investigation into adhesive properties of collagen associated proteoglycans or other proteins in vitamin B-6-deficient connective tissue.