The appearance of free hydroxyproline as the major product of degradation of newly synthesized collagen in cell culture

Embryonic lung fibroblasts and rabbit vascular smooth muscle cells have the ability to degrade newly synthesized collagen. Analysis of 24-h pulse media from cultures given [¹⁴C]proline demonstrates that greater than 90% of the degraded collagen is represented by free hydroxyproline rather than the peptide-bound imino acid. The addition of cycloheximide or α-α-dipyridyl to the culture medium during the pulse period severely diminished the formation of the free hydroxyproline demonstrating its enzymatic and protein (collagen) origin. It is proposed that assessment of free hydroxyproline formation may allow us to distinguish between intracellular and extracellular collagen degradation.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

The stability of the collagen phenotype during stimulated collagen,glycosaminoglycan, and DNA synthesis by articular cartilage organ cultures

Rabbit articular cartilage slices were grown in organ culture for 9 weeks. Eightfold increases in the synthesis of both glycosaminoglycan and collagen were observed at 1 and 3 weeks, respectively. These levels of synthesis gradually declined in parallel to fourfold at 9 weeks. DNA synthesis was stimulated more than 30-fold at 3 weeks and then declined to sevenfold at 9 weeks. In contrast, the content of glycosaminoglycans and collagen per milligram of original wet slices did not vary significantly, while the number of cells increased 1.7-fold by the end of the study. The collagen phenotype of these cultures was determined by sodium dodecyl sulfate electrophoresis of recently synthesized, [³H]proline-labeled intact collagen chains and CNBr peptides. Throughout the study the major collagen synthesized was type II, ranging from 95 to 68% of the collagen synthesized at 0 and 5 weeks, respectively. Increases in the proportions of X₂Y and type III collagen were first observed at 3 weeks in culture. The synthesis of type I collagen was detected only after 5 weeks in culture and never represented more than 11% of the total collagen synthesized. The synthesis of type I trimer could not be verified at any time. This study demonstrates that in vitro organ culture of articular cartilage slices allows chondrocytes to maintain the normal chondrocyte collagen phenotype of predominantly type II synthesis while stimulating their proliferation and matrix synthesis.     

Localization of collagen prolyl hydroxylase to the hepatocyte : Studies in primary monolayer cultures of parenchymal cells from adult rat liver

Significant levels of prolyl hydroxylase activity (prolyl-glycyl-peptide, 2-oxoglutarate: oxygen oxidoreductase; EC 1.14.11.2) have been found in freshly isolated hepatocytes prepared from normal or regenerated adult rat liver and primary non-proliferating monolayer cultures of these cells. Four days after partial hepatectomy, the intact regenerated liver contained two times the normal level of prolyl hydroxylase activity. Freshly isolated hepatocytes contained 24% of the total prolyl hydroxylase activity in normal liver and 47% of that in regenerated liver. Upon incubation of hepatocytes for 24 h in a chemically defined culture medium containing insulin, prolyl hydroxylase activity rose 2- to 3-fold, and gradually declined during the next 48 h. The rise in prolyl hydroxylase activity was blocked by addition of cycloheximide to the culture medium. The presence of prolyl hydroxylase activity in hepatocyte cultures was not likely due to contamination with non-parenchymal liver cells. The latter cells contained less than 20% of the total enzyme activity recovered in all cells isolated from the liver. Furthermore, prolyl hydroxylase was localized by immunofluorescence uniformly to the hepatocytes in culture. Cultured hepatocytes converted [¹⁴C]proline to [¹⁴C]hydroxyproline at rates comparable to those reported for whole liver. However, only a small portion of the hydroxyproline containing product was present as collagen protein, suggesting its rapid degradation in culture. We conclude that the liver parenchymal cell may actively participate in collagen synthesis and possibly in collagen degradation.     

Differentiation and mineralization of murine mesenchymal C3H10T1/2 cells in micromass culture

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4 mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4 mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. ⁴⁵Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of ⁴⁵Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1 mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.     

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with³⁵S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher M_r than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.Supported by a fellowship from the Centre National de la Recherche Scientifique     

Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts

Summary Total insoluble collagen and hydroxyproline formation were examined in lung embryonic fibroblasts (IMR-90) grown in the presence or absence of added ascorbate. As expected, when the cells from both groups (+ and −ascorbate) are pulsed with [¹⁴C]proline in the presence of ascorbate, the percent hydroxylation in a 24-hr period does not vary significantly. However, there are dramatic differences in the quantity and quality of the insoluble collagen fraction produced by those cells grown for a long period of time with added ascorbate. Those cells deprived of continuous addition of ascorbate to the culture medium do not display large quantities of accumulated collagen in the cell layer fractions as measured by the hydroxyproline content, whereas the cells grown in the presence of ascorbate contain significant amounts of accumulated collagen. A new method for examining the extracellular insoluble collagen produced in cell cultures is described in these studies. With the aid of pancreatic elastase relatively pure insoluble collagen can be obtained from cells grown in culture. In those cells grown in the presence of ascorbate, the purified insoluble collagen yeilds appropriately banded fibrils when examined in the electron microscope and has an amino-acid composition that is compatible with pure collagen. On the other hand, those cells grown in the absence of ascorbate do not yield purified insoluble collagen as determined by these same criteria. The elastase procedure for the purification of insoluble collagen in cell cultures is simple, easy to use and allows one to assess additional aspects of collagen biosynthesis.     

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices

The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF- stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.     

Cross-linked collagen surface for cell culture that is stable,uniform, and optically superior to conventional surfaces

Summary A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluenesulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed. This work was supported by the Leopold Schepp Foundation, the Dysautonomia Foundation, National Institutes of Health Grants NS14768 and NS11237, and Institutional Core Grant HD06276.     

Decreased synthesis and increased intracellular degradation of newly synthesized collagen in freshly isolated chick tendon cells incubated with monensin

The synthesis and secretion of procollagen in embryonic chick tendon fibroblasts in suspension culture were inhibited with the carboxylic ionophore monensin. The synthesis of procollagen was inhibited by 50% in a 2-h exposure to 0.1 microM monensin and was inhibited by 70% in a 6-h exposure to 0.1 microM monensin. Secretion of procollagen was inhibited by greater than 90% in the 0.1 microM monensin-treated cultures and was totally inhibited by higher doses of the reagent. A cellular pool of collagenase-digestible peptides was demonstrated in the control cells, the level of which was elevated 3-4 times in the monensin-treated cultures. In order to determine whether the secretory and synthesis block caused by monensin inhibited intracellular degradation of newly synthesized collagen, the hydroxy[14C]proline in degraded collagen fragments present in control and monensin-treated cultures was determined and compared to the total hydroxy[14C]proline synthesized in each culture. The intracellular degradation of newly synthesized, pulse-labeled collagen was shown to proceed at rates comparable to those seen in the control cultures. The monensin-treated cells degraded pulse-labeled newly synthesized collagen nearly twice as long as the controls, resulting in an overall increase in the fraction of newly synthesized collagen that was degraded. These findings suggest that force generation in the activated cross-bridge cycle may occur as a result of an actin-attached cross-bridge transition between these two orientations.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.     

A simplified method for passage and long-term growth of human mammary epithelial cells

Summary A method is described for culturing human mammary epithelial cells in primary culture and allowing more than 50 generations and a 1000-fold increase from starting inocula without need of enzymatic transfers. Organoids dissociated from breast tissue are plated in medium containing 1.05 mM Ca⁺⁺ to effect attachment and growth to monolayer density. Medium is then switched to one containing 0.06 mM Ca⁺⁺ to overcome “renewal inhibition” and to stimulate growth. In low Ca⁺⁺ media, primary cultures become a long-term, continuous source of free-floating viable cells free of fibroblasts. A fundamental requirement for extended growth in primary culture is maintaining calcium levels at approximately 0.06 mM. Above 0.06 mM Ca⁺⁺, cells divide only 3 to 4 times in primary cultures before terminal differentiation occurs. At 0.06 mM Ca⁺⁺, cells continue to divide for periods of time determined partly by feeding schedule, but up to 6 mo. and 50 generations of (linear) growth. Cells released from monolayer were greater than 90% viable and yielded 10⁵ cells/cm² of attached cells every 72 h. Free-floating single cells readily replated and cloned, when transferred, without need of trypsin for dissociation. Long-term free-floating cells were typical mammary epithelium: (a) they formed domes and exhibited renewal inhibition, (b) they produced ductlike formations in collagen gels, (c) they contained epithelium-specific keratin filaments, and (d) they were diploid.     

Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces

Fibroblast cultures were initiated from two distinct regions of the adult bovine deep flexor tendon and synthesis of 35S-labeled proteoglycans by these cultures was investigated. The proximal/tensional region of the tendon was composed of linearly arranged dense collagen bundles, and its glycosaminoglycan hexosamine content was only 0.2% of the dry weight of the tissue. The proteoglycans of this region were predominantly small (Kav = 0.5 on Sepharose CL-4B). Cells placed into culture from this region attached to the substratum readily, and the radiolabeled proteoglycans from these cultures were 90% small proteoglycans. In a more distal region of the tendon that is subjected to compressive forces, the collagen was arranged as a network of fibrils separated from each other by a matrix that stained intensely with Alcian blue. The glycosaminoglycan content of this compressed region was up to 5-fold higher than in the proximal region, and as much as 50% of the proteoglycans were large molecules (eluted from Sepharose CL-4B in the Vo). Cells placed into culture from the distal/compressed region did not attach to the substratum as readily as those from the proximal region and were characterized by the presence of numerous cytoplasmic lipid inclusions. The [35S]proteoglycans synthesized by the distal tendon fibroblast cultures were divided into two approximately equal populations of large and small proteoglycans having elution characteristics similar to the proteoglycans extracted from this tissue. The distinct profiles of proteoglycan production were maintained by the cells in culture for several weeks, although eventually the amount of large proteoglycan synthesized by the distal tendon fibroblast cultures diminished. Both regions of tendon contained predominantly type I collagen, and collagen production was about 10% of the total protein synthesized by both cell cultures. These observations indicate that adult tendon fibroblasts in culture express stable synthesis of proteoglycan populations similar to those found in the region of tendon from which they were derived.     

Phenol degradation and colour removal in submerged culture of Pleurotus sajor-caju with paper mill effluents

The fungus Pleurotus sajor-caju secretes phenol-oxidases that enable the use of recalcitrant compounds as substrates. The residues of paper manufacture contain high lignin levels, which gives the effluents a characteristic brownish colour. To test the potential of P. sajor-caju cultures on reducing these parameters, we used 90% of raw effluents from medium consistency oxygen delignification and bleaching stages plus 10% of mineral solution and different levels of glucose (5–15 g L^?1) as substrate. We observed a greater fungal biomass in cultures using effluent than in controls. Cultures containing 10 to 15 g L^?1 of glucose resulted in about 42% colour reduction. The polyphenol content was also reduced by 58.9% by the 13th day of culture. In addition, we observed the secretion of laccases (211.44 U mL^?1 and 45.98 U mL^?1 using ABTS and syringaldazine, respectively) and peroxidases (6.11 U mL^?1-ABTS) both peaking at the 7th day of culture and with similar kinetics of production in different glucose concentrations.     

Independent regulation of collagen types by chondrocytes during the loss of differentiated function in culture

Collagen phenotypes were determined for rabbit articular chondrocytes in cartilage slices and first through fifth monolayer cultures. During the first 24 hr of slice culture, chondrocytes exhibited the following collagen phenotype: 96% type II, 3% X₂Y and 1% type III. In primary monolayer culture, no other types of collagen were added to this differentiated chondrocyte phenotype; however, the synthesis per cell of each of the expressed collagens was stimulated. By the fifth day of primary culture, X₂Y synthesis increased 10 fold, and by the eighth day, a further 4 fold. In contrast, the synthesis of collagen types II and III showed no change by the fifth day, but increased 7 fold by the eighth day. These results suggest independent regulation of X₂Y in this situation. In a separate experiment, first through fifth cultures were studied. The synthesis per cell of type II collagen declined steadily and essentially ceased by the fifth culture, indicating the loss of differentiated function by these chondrocyte progeny. The loss of type II synthesis was not quantitatively replaced by the synthesis of type I trimer and type I collagen which was first detected in the third culture. While these qualitative changes in phenotype occurred, the stimulated rate of type III collagen synthesis did not change and that of X₂Y declined only slightly. Thus the termination of type II synthesis did not significantly alter the synthesis of the other collagens produced by differentiated chondrocytes. The final “de-differentiated” phenotype was 41% type I, 25% X₂Y, 20% type I trimer, 13% type III and 1% type II.     

Hormonal regulation of collagen degradation in the uterus: Inhibition of collagenase expression by progesterone and cyclic AMP

Dibutyryl cyclic AMP markedly increases the ability of progesterone to prevent the expression of collagenase activity in cultures of post-partum rat uterus. Dibutyryl cyclic AMP itself and, to a lesser extent, native cyclic AMP, are capable of producing a partial decrease in enzyme activity, but complete abolition is not observed at high cyclic nucleotide concentrations (5 mM) in the culture medium. Theophylline, when added to cultures, mimics the effect of dibutyryl cyclic AMP. Other cyclic nucleotides were without effect on levels of collagenase activity in the uterine cultures.When non-inhibitory concentrations of either dibutyryl cyclic AMP (1 · 10^?4 M) or theophylline (1 · 10^?4 M) are added to cultures together with a non-inhibitory concentration of either progesterone (5 · 10^?6 M) or the potent progesterone analogue Provera (1 · 10^?8 M) the ability of the tissue to produce collagenase is decreased by 40–70%. Collagenase activity is consistently diminished more than additively by combinations of steroid and cyclic nucleotide. Theophylline mimics the effect of dibutyryl cyclic AMP on steroid activity in culture. In the presence of dibutyryl cyclic AMP, diminution of collagenase activity can be observed at concentrations of steroid more than two orders of magnitude lower than the normal minimally inhibitory dose. Reduction of collagenase activity is reflected in all experiments by a concomitant decrease in the normal proteolytic degradation of collagen in the tissue ex-plants. The possibility that progesterone acts in the uterus to raise cyclic AMP levels is suggested by the fact that uterine tissue, when cultured in the presence of progesterone, contains reduced levels of cyclic nucleotide phosphodiesterase.These data suggest that, in some way a cyclic AMP-mediated system is critically involved in the control of collagenase activity by progesterone in the rat uterus.     

Cell growth and hemoglobin synthesis in murine erythroleukemic cells propagated in high density microcapsule culture

Summary A new microencapsulation technology, developed for the encapsulation of living cells, has been demonstrated to be useful for the study of growth and differential gene expression using Friend erythroleukemic cells cultured at high cell densities. Using this technology, cultures of FL Clone 745 cells were encapsulated within semipermeable membranes composed of cross-linked alginic acid and poly-l-lysine. Cell growth studies measuring total cell number demonstrated an average generation time of 8.5 h in 5% (vol/vol) microcapsule cultures vs. 8.0 h in suspension cultures. Similar microcapsule cultures were serially propagated for more than 90 cell generations (13 sequential passages) with no significant change in this growth rate. In addition, final culture densities of greater than 1.0×10⁸ cells/ml of intracapsular volume were attained using a 3% (vol/vol) microcapsule culture in conjunction with a standard refeeding schedule. Comparison of the level of dimethyl sulfoxide-induced hemoglobin production in suspension and microcapsule cultures demonstrated that the total amount of hemoglobin produced on a per cell basis was comparable in both systems. Due to the retention characteristics of the semipermeable membrane, the concentration of detergent-released hemoglobin, relative to other released protein, was approximately twofold higher in microcapsule cultures than in control suspension cultures.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Phenotypic analysis of bovine chondrocytes cultured in 3D collagen sponges: effect of serum substitutes

Repair of damaged cartilage usually requires replacement tissue or substitute material. Tissue engineering is a promising means to produce replacement cartilage from autologous or allogeneic cell sources. Scaffolds provide a three-dimensional (3D) structure that is essential for chondrocyte function and synthesis of cartilage-specific matrix proteins (collagen type II, aggrecan) and sulfated proteoglycans. In this study, we assessed porous, 3D collagen sponges for in vitro engineering of cartilage in both standard and serum-free culture conditions. Bovine articular chondrocytes (bACs) cultured in 3D sponges accumulated and maintained cartilage matrix over 4 weeks, as assessed by quantitative measures of matrix content, synthesis, and gene expression. Chondrogenesis by bACs cultured with Nutridoma as a serum replacement was equivalent or better than control cultures in serum. In contrast, chondrogenesis in insulin-transferrin-selenium (ITS⁺³) serum replacement cultures was poor, apparently due to decreased cell survival. These data indicate that porous 3D collagen sponges maintain chondrocyte viability, shape, and synthetic activity by providing an environment favorable for high-density chondrogenesis. With quantitative assays for cartilage-specific gene expression and biochemical measures of chondrogenesis in these studies, we conclude that the collagen sponges have potential as a scaffold for cartilage tissue engineering.