Matrix (Stuttgart, Germany)最新文献

Addition of chondroitin sulfate (CS) or cartilage proteoglycan to metastable calcium phosphate solutions inhibits the formation of hydroxyapatite (HA). However, pre-equilibration of CS or proteoglycan with calcium prior to the addition of phosphate results in higher levels of HA precipitation compared to control solutions of identical calcium and phosphate activity. These findings indicate that the inhibition of HA formation by proteoglycans and CS is largely due to calcium binding. Further, its ability to bind calcium ions reversibly suggests that proteoglycan may act as a promoter, not an inhibitor, of calcification in cartilage.

Hydroxylysyl pyridinoline (HP) is a nonreducible collagen crosslink derived from three posttranslationally modified lysyl residues. Neonatal rat aorta smooth muscle cell cultures (NNRSMC) produce mg amounts of insoluble collagen. The purpose of the present study was to evaluate the capability of NNRSMC to produce collagen containing HP crosslinks. Cultures were pulsed with [¹⁴C]-lysine and then chased for five weeks. Insoluble collagen was prepared by digestion of the cell layer material with porcine pancreatic elastase and trypsin. After acid hydrolysis and cation-exchange chromatography, purified HP was isolated by reversed phase ion-paired chromatography. The material eluting from the HPLC was monitored continuously at 295 nm and the ultraviolet absorption spectrum was recorded every 21 msec. The ultraviolet spectrum of the HP peak was virtually identical to that of standard HP run on the HPLC. The HP exhibited a homogeneity of 97.3% when the ultraviolet spectrum of the apex of the peak was compared with the spectra of the shoulders of the peak. The radioactive HP also exhibited the expected fluorescence emission spectrum. We calculate a mean of 0.40 ± 0.03 nmol HP/nmol collagen in the three experiments as compared with reported values of 0.57 ± 0.1 for rabbit aorta. This is the first report of cell culture biosynthesis of chemically measurable amounts of HP. Using such pulse-chase techniques one can study the maturation of intermediate collagen crosslinks into HP. HP can also be used as a marker to study the metabolism of mature collagen molecules during normal and pathologic states.

High-performance liquid chromatography methods were developed to measure the concentration of hydroxypyridinium crosslinks in the intramuscular collagen and tendinous parts of functionally different skeletal muscles at different ages. A significant increase in pyridinoline concentration took place during maturation reaching 0.32 + 0.07 (moUmol collagen) in soleus (slow plantar flexor) and 0.28 ± 0.07 in plantaris (fast "mixed" plantar flexor) at the age of 4 months. In medial and lateral gastrocnemius (fast "mixed" plantar flexors) the pyridinoline concentrations (mol/mol collagen) reached 0.24 + 0.06 and 0.19 + 0.04, respectively, similar to those in both the extensor digitorum longus (fast "mixed" dorsi flexor) and rectus femoris (fast "mixed" knee extensor) muscles, but higher than in the fast "mixed" dorsi flexor muscle, anterior tibialis (0.11 ± 0.05 moUmol). By comparison, pyridinoline concentrations of 0.33 moll mol collagen (± 0.10) was measured from longissimus dorsi, a slow-twitch back posture muscle. After maturation the most significant increase in pyridinoline concentration was measured in soleus and gastrocnemius muscles. No differences in the crosslinking between different parts of muscle belly were noticed at any time-point. However, significantly fewer pyridinoline crosslinks were found in tendinous parts of soleus, extensor digitorum longus and anterior tibialis than in intramuscular collagen. The concentration of pyridinoline crosslinks tended to be highest in slow-twitch postural muscles, soleus and longissimus dorsi, and generally higher in plantar flexors which are exposed to higher stretch than dorsal flexors. The reasons for the unexpectedly low concentrations of pyridinoline crosslinks in the tendinous parts of muscles remain to be clarified.

Proteoglycans (PGs) were analyzed and compared in the media of the thoracic aorta, abdominal aorta, left carotid artery and superior mesenteric artery of age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Two ages were examined; 10 week old, during the development of hypertension and 28 week old, when hypertension is well established in the SHR. Large chondroitin sulfate PG, large heparan sulfate PG and biglycan (PGI) and decorin (PGII) small PGs were identified. Biglycan was the predominant small PG found in all arteries. Newly synthesized PGs were labelled in vitro with 35SO4 for quantitation. The synthesis of large and small PGs was similar in the media of the thoracic aorta, abdominal aorta, left carotid artery, and superior mesenteric artery. The large to small ratio value, a measure of the artery PG composition, was also similar among the four arteries but was highest in the mesenteric artery. In both WKY and SHR arteries there was significantly decreased PG synthesis in the 28-week old compared to 10-week old animals. This was especially true for large PG. Hypertensive changes in PG synthesis were seen mainly in the carotid artery. In this artery, synthesis of both large and small PG was increased in the SHR, at both ages. The ratio of large to small PG was not significantly different between SHR and WKY arteries. We conclude that 28-week old WKY and SHR rat arteries synthesize less large and small PG than 10-week old arteries. The most prominent change seen in hypertensive rats is an increase in PG synthesis in the carotid artery.

Cultured neonatal rat aortic smooth muscle cells are active in synthesizing and depositing large amounts of elastin in their extracellular matrix, making this an ideal system for studying elastogenesis. In this study, the ability of individual cells to synthesize tropoelastin was examined by in-situ hybridization methods. One-micron semi-thin epoxy resin-embedded transverse sections of cells cultured 1, 2, 3 and 4 weeks showed an increase with time in both the number of cells with hybridization signal and the signal intensity; tropoelastin mRNA hybridization signal intensity decreased thereafter up to 8 weeks in culture. In longitudinal sections through the early cultures (1-week), we observed mitotic cells with no detectable hybridization signal, and nonmitotic cells with either no, little or high signal intensity. These data suggest that mitotic cells do not synthesize tropoelastin, and that there is a strong correlation between the hybridization signal intensity and the rate of tropoelastin synthesis. These data also suggest in-situ hybridization methods can detect which cell(s) contain tropoelastin mRNA, their location in the multilayer, and variations in signal intensity. We conclude it is possible to correlate hybridization signal intensity with varitions of tropoelastin mRNA levels within individual cells of the cultured smooth muscle cell multilayer.

Ectopic endochondral ossification is the inevitable consequence of midpoint tenotomy of the rat Achilles tendon. After tenotomy, the tendon stumps retract and the intervening space fills with granulation tissue. The initiation of chondrogenesis is indicated by pre-chondrocytec cells forming a "whorled" pattern, both at the tendon stumps and within the granulation tissue and later clearly differentiating into cartilage nodules. The chondrocytes rapidly "hypertrophy" exhibiting an orientation similar to that in epiphyseal growth plates. The nodules of cartilage are then replaced, by bone. During this total process, a temporal and spatial pattern of new collagen synthesis can be demonstrated, both biochemically and immunocytochemically. Both the cartilage and the subsequent bone closely resemble the tissue in developing long bones enabling this model to be used to study the initial switching on of normal chondrogenesis and osteogenesis in a system not normally programmed to do so.

A Columbian mammoth, Mammuthus columbi, was excavated at an elevation of 9000 feet in Huntington Canyon, Emery County, Utah. Radiocarbon dates on the skeleton indicated death approximately 11200 years ago. The skeleton was removed from postglacial, Late Quaternary, lake sediments deposited as glacial runoff approximately 9500 years ago. The bones and teeth were especially well preserved in a saturated lake bed. After excavation the bones and teeth were preserved by controlled desiccation, without hardeners, over a period of 9 months. Microradiography, light and electron microscopy, medium and high angle X-ray diffraction, amino acid analysis and cyanogen bromide peptide mapping were undertaken to evaluate the packing, organization, and preservation of collagen in bone and dentin of this mammoth. Microradiography and light microscopy showed that the bone consisted of especially well preserved compact and trabecular bone, and electron microscopy of demineralized bone and tusk showed that the matrix consisted of lamellae of densely packed cylindrical collagen fibrils. Cell remnants with intact nuclei, with or without a nucleolus, as well as variable lengths of plasma membrane were occasionally present on the surface of bony trabecula. Remnants of odontoblast processes were present in some dentin tubules. High and low angle X-ray diffraction demonstrated that the demineralized matrix contained native collagen molecules and amino acid analysis showed that the composition was comparable to that of type I collagen. Cyanogen bromide peptide mapping indicated that the major peptides of type I collagen were present and had the same electrophoretic mobility as that of type I collagen of demineralized Asian elephant bone and rat tail tendon. The excellent quality of preservation of this specimen provided an unusual opportunity to compare the collagen in a matrix that has been preserved for 11200 years to that of modern bone and tooth.

We investigated the ability of murine bone organ cultures and osteoblast-like bone cells to produce 72- and 92-kDa gelatinase. 4-6 day newborn mouse calvaria cultures were found to release gelatinase activity into their conditioned medium (CM). This activity was increased by four stimulators of resorption, tumor necrosis factor alpha (TNF), interleukin-1 alpha (IL-1), parathyroid hormone (PTH) and the active phorbol ester, 12-0-tetradecanoylphorbol-13acetate (TPA). Both the 72- and 92-kDa forms of gelatinase were produced by murine bone cultures. In unstimulated bones 72-kDa gelatinase activity was approximately equal to that of the 92-kDa enzyme. IL-1, TNF, PTH and TPA all increased 92-kDa gelatinase activity in the CM of the bone cultures by about 2- to 2.5-fold. In addition TPA and IL-1 also increased 72-kDa gelatinase activity.

In unstimulated osteoblast-like MC3T3-E1 cell cultures 72-kDa gelatinase enzyme activity was much greater than 92-kDa activity and was not substantially regulated (40% change) by IL-1, TNF or PTH. In contrast, these agents stimulated 92-kDa gelatinase activity by 2- to 5-fold.

As with the MC3T3-E1 cells, primary cells constitutively produced both 72-kDa and 92-kDa gelatinase. This was true for cells with both the most differentiated osteoblast-like phenotype (populations 3 and 4) and the least osteoblast-like phenotype (populations 1 and 2). In unstimulated cultures of all 4-primary populations, 92-kDa gelatinase production was less than 72-kDa and IL-1, TNF and PTH had only small effects on 72-kDa production in any of the populations (60% change). However, IL-1, and TNF-stimulated 92-kDa gelatinase production in all populations by 2.7 to 5.7 fold; while PTH completely inhibited 92-kDa gelatinase activity in population 1 and had no effect on populations 2-4.

These results demonstrate that gelatinase activity is released by bone organ cultures and osteoblast-like cells in a regulated manner. This finding suggests that these enzymes are involved in the mechanisms reizulatina bone turnover.

Collagen fibrils from young turkey-leg tendons, just beginning to mineralize, were stained with uranyl acetate and examined by electron microscopy. Small needle-like mineral crystals were observed and located, in relation to the collagen banding pattern, as originating at the e band in the gap region and near the surface of the fibrils. These are evidently the sites of crystal nucleation. They lie near binding locations on collagen fibrils of two glycosylated proteins believed to be implicated in the mineralization process, as well as the sites of early crystals in embryonic fowl bones.