Influence of glucocorticoids on some morphological and biochemical aspects of rat small intestinal mucosa.

Glucocorticoid administration (5 mg/day per 100 g of body weight) to month-old rats elicited a reduction of maltase and alkaline phosphatase. Corticotrophic stimulation on month-old rats elicited a specific rise in maltase and alkaline phosphatase activities, total protein content remaining unchanged. Immunological, histological, radioautographical and biochemical studies have shown that these two opposing phenomena do not depend on enzyme activation, on membrane stabilisation, or on modifications of proliferative parameters of the intestinal epithelium. They appear rather to derive from the same origin, i.e. the action of glucocorticoids on the enterocyte differentiation.     

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.     

Effects of extracellular matrix elements on pseudopodial activity of rat keratinocytes

Effects of extracellular matrix elements on the migration activity of keratinocytes have been studied in the primary culture obtained from newborn rats. Collagen of type I, matrigel, its fractions and the matrix produced by fibroblasts were used as substrata for cultivation. A method of migration activity estimation using latex spheres 0.8 mkm in diameter was first used. We have revealed that keratinocytes from the primary culture do not migrate on matrigel and fibroblast matrix, though displaying some pseudopodial activity. This activity dramatically increases on type I collagen, and a weak migration ability appears correlating with a particular structure of the actin cytoskeleton, i.e. with the appearance of special lamellopodia-connected filopodia.     

Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes

Summary Some effects of culturing adult rat hepatocytes on each of four different substrates—laminin (LN), collagen type I (C-I), collagen type IV (C-IV), and fibronectin (FN)—have been investigated under defined conditions. No differential effect on the attachment of the cells to the various substrates was noted; however, the spreading of hepatocytes shortly after initial plating was most strikingly enhanced by FN, whereas LN exhibited little or no such enhancement. The two collagen substrates enhanced the spreading of hepatocytes more than did LN, but less than FN. The different substrates had no differential effect on the induction of tyrosine aminotransferase by dexamethasone and glucagon for at least the first 10 d in culture. The longevity of the hepatocytes was not changed significantly by any of the substrates, at least through the 14th d of culture. During the culture periods the hepatocytes at high cell density were maintained as confluent monolayers, regardless of the substrate on which they had been cultured. After 14 d of culture, γ-glutamyltranspeptidase activity was highest in cells cultured on C-IV, and lowest in those on FN. DNA synthesis in cultured hepatocytes at a low cell density was highest in cells cultured on FN, with decreasing levels of this parameter in cells cultured on C-IV, C-I, and LN, respectively. These results demonstrate that specific components of the extracellular matrix modulate both differentiated functions and the replication of hepatocytes cultured in serum-free medium. This work was supported in part by grants (CA-07175, CA-09135, CA-22484) from the National Cancer Institute, Bethesda MD. N. Sawada was supported by a Cancer Research Campaign Grant D (U.K.) from the International Union Against Cancer.     

Exosomes from tendon stem cells promote injury tendon healing through balancing synthesis and degradation of the tendon extracellular matrix

Tendon injuries are common musculoskeletal system disorders in clinical, but the regeneration ability of tendon is limited. Tendon stem cells (TSCs) have shown promising effect on tissue engineering and been used for the treatment of tendon injury. Exosomes that serve as genetic information carriers have been implicated in many diseases and physiological processes, but effect of exosomes from TSCs on tendon injury repair is unclear. The aim of this study is to make clear that the effect of exosomes from TSCs on tendon injury healing. Exosomes were harvested from conditioned culture media of TSCs by a sequential centrifugation process. Rat Achilles tendon tendinopathy model was established by collagenase‐I injection. This was followed by intra‐Achilles‐tendon injection with TSCs or exosomes. Tendon healing and matrix degradation were evaluated by histology analysis and biomechanical test at the post‐injury 5 weeks. In vitro, TSCs treated with interleukin 1 beta were added by conditioned medium including exosomes or not, or by exosomes or not. Tendon matrix related markers and tenogenesis related markers were measured by immunostaining and western blot. We found that TSCs injection and exosomes injection significantly decreased matrix metalloproteinases (MMP)‐3 expression, increased expression of tissue inhibitor of metalloproteinase‐3 (TIMP‐3) and Col‐1a1, and increased biomechanical properties of the ultimate stress and maximum loading. In vitro, conditioned medium with exosomes and exosomes also significantly decreased MMP‐3, and increased expression of tenomodulin, Col‐1a1 and TIMP‐3. Exosomes from TSCs could be an ideal therapeutic strategy in tendon injury healing for its balancing tendon extracellular matrix and promoting the tenogenesis of TSCs.     

Effects of extracellular matrix on the malignant phenotype

Extracellular matrix molecules, including collagen, glycosaminoglycans (usually linked to a protein core as proteoglycan), elastin, and glycoproteins, influence the initiation and maintenance of differentiation of a variety of cell types. These molecules bind to the cell surface at specific sites and nonspecifically by electrostatic forces. Such interactions may alter the cell's response to growth and differentiation factors. After neoplastic transformation, most cells retain some dependence on these factors. This paper reviews the influence of matrix components on the phenotype of a variety of malignant cells and concludes that in vitro studies of malignant cell behavior require the utilization of an appropriate microenvironment.     

The effects of stress enhancement on the extracellular matrix and fibroblasts in the patellar tendon

The purpose of the present study is to determine the effect of the stress enhancement and intrinsic fibroblasts on the extracellular matrix of the patellar tendon. Thirty-two female Japanese White rabbits were divided into four groups. In Group 1, the patellar tendon underwent the in situ freeze-thaw treatment to kill intrinsic fibroblasts of the patellar tendon and the patellar tendon underwent the wrapping treatment with nylon membrane filters to inhibit extrinsic cell infiltration. In Group 2, the medial and the lateral portions of the frozen-thawed patellar tendon were resected to enhance the stress, and then the central two-thirds of the patellar tendon underwent the wrapping treatment. In Group 3, the patellar tendon without the freeze/thaw treatment underwent the wrapping treatment. In Group 4, the patellar tendon was narrowed and wrapped in the same manner. All rabbits were killed 6 weeks after surgery. While the elastic modulus and the tensile strength of the patellar tendon in Group 2 were significantly less than those in Group 1, we could not find any significant differences in these parameters between Groups 3 and 4. Histologically, while no fibroblasts were observed in Groups 1 and 2, fibroblasts were found in Groups 3 and 4. This study revealed that stress enhancement decreases the elastic modulus and the tensile strength of the extracellular matrix of the patellar tendon and that intrinsic fibroblasts prevent the detrimental effect of stress enhancement on mechanical properties of the patellar tendon.     

A correlated morphological and biochemical study on the rat adrenal steroidogenesis

The ultrastructural and biochemical alterations produced by an hypocholesterolemic drug, 17 alpha-ethinyl estradiol, on the rat adrenal cortex were studied. Male rats aged two months and with approximately 200 g in weight were injected subcutaneously with 10 mg/kg/day of ethinyl estradiol during 9 days; rats injected with 1 ml propylene glycol were used as controls. The animals were sacrificed on the 10th day, and the adrenals from some of them were processed for electron microscopy. The adrenals from the remaining rats were used for measurements of the glands cholesterol and corticosterone, which were also measured in the blood. In estradiol-treated rats the zona fasciculata cells exhibited numerous microvilli, increase in the size of mitochondria and decrease in the number of lipid droplets. The quantitative analysis showed a significant increase of the volumetric density of mitochondria and microvilli and a significant decrease of the lipid droplets in the treated rats, when compared with normal ones. In treated rats, the concentration of cholesterol and corticosterone in the gland and blood were significantly decreased. These data show that hypocholesterolemia produced by estradiol has a remarkable effect on adrenal steroidogenesis, depletes the pool of adrenal cholesteryl esters, and evidences the role of plasma cholesterol in the corticosteroidogenesis.     

Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells

Summary Fibroblasts of the synovium of sheathed tendons were isolated, and their biochemical properties were compared with those of the fibroblasts of the remaining tendon. The synovial cells had a lower attachment efficiency than did the tendon cells. On the day of cell isolation the synovial cells synthesized collagen as 10% of their total protein, whereas the tendon cells synthesized 30% collagen. After growth in fetal bovine serum (FBS), the percentage of collagen synthesized by both populations decreased; however, the synovial cells still made less collagen than did the tendon cells (5 versus 11%). On the basis of cyanogen bromide peptide analysis, the synovial cells were found to synthesize Types I and III collagen in primary culture, whereas the tendon cells synthesized only Type I. The synovial cells aslo synthesized two to three times less sulfated glycosaminoglycans in culture than did the tendon cells. Thus, the two cell, populations differed in attachment efficiency and in their biosynthesis of collagen and sulfated glycosaminoglycans. These differences reflect extracellular matrix differences that have been observed in the tendon in vivo. In addition, the results augment existing data showing that not all fibroblasts have identical phenotypes. This investigation was supported by National Institutes of Health Grant AM 25749.     

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

A novel human glioma-associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athymic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]-leucine- and [3H]-glucosamine-labeled ECM from the human glioma cell line U-251MG has shown that GMEM is a high-molecular-weight macromolecule (Mr approximately 1,000,000) composed of Mr approximately 230,000 disulfide-bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one-dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U-251MG.     

Attachment and extracellular matrix differences between tendon and synovial fibroblastic cells

Fibroblasts of the synovium of sheathed tendons were isolated, and their biochemical properties were compared with those of the fibroblasts of the remaining tendon. The synovial cells had a lower attachment efficiency than did the tendon cells. On the day of cell isolation the synovial cells synthesized collagen as 10% of their total protein, whereas the tendon cells synthesized 30% collagen. After growth in fetal bovine serum (FBS), the percentage of collagen synthesized by both populations decreased; however, the synovial cells still made less collagen than did the tendon cells (5 versus 11%). On the basis of cyanogen bromide peptide analysis, the synovial cells were found to synthesize Types I and III collagen in primary culture, whereas the tendon cells synthesized only Type I. The synovial cells also synthesized two to three times less sulfated glycosaminoglycans in culture than did the tendon cells. Thus, the two cell populations differed in attachment efficiency and in their biosynthesis of collagen and sulfated glycosaminoglycans. These differences reflect extracellular matrix differences that have been observed in the tendon in vivo. In addition, the results augment existing data showing that not all fibroblasts have identical phenotypes.     

Biochemical and biophysical aspects of collagen nanostructure in the extracellular matrix

ECM is composed of different collagenous and non-collagenous proteins. Collagen nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, including bone, skin, tendon, blood vessels, and cartilage. Artificial collagen nanofibers are increasingly significant in numerous tissue engineering applications and seem to be ideal scaffolds for cell growth and proliferation. The modern tissue engineering task is to develop three-dimensional scaffolds of appropriate biological and biomechanical properties, at the same time mimicking the natural extracellular matrix and promoting tissue regeneration. Furthermore, it should be biodegradable, bioresorbable and non-inflammatory, should provide sufficient nutrient supply and have appropriate viscoelasticity and strength. Attributed to collagen features mentioned above, collagen fibers represent an obvious appropriate material for tissue engineering scaffolds. The aim of this minireview is, besides encapsulation of the basic biochemical and biophysical properties of collagen, to summarize the most promising modern methods and technologies for production of collagen nanofibers and scaffolds for artificial tissue development.     

Low level laser therapy accelerates the extracellular matrix reorganization of inflamed tendon

In tendon lesions, inflammation indicates the beginning of tissue repair and influences cell proliferation and the remodeling of the extracellular matrix (ECM). Low level laser (LLL) therapy has been an important method to induce tissue repair, and several studies have sought to better understand the therapeutic possibilities of this modality. This study analyzed the effect of LLL on the ECM of rat tendons during the early phase of the inflammatory process. Wistar rats received an intratendinous application of carrageenan adjacent to the osteotendinous region in the right paw. The animals were divided into the following groups: G1—intact, G2—animals with no treatment after the inflammation induction, G3—animals treated with LLL 1 and 3 h after induction of inflammation (4 J/cm² continuous). After 4 h of application, the animals of the two groups were euthanized with isoflurane overdose. Our results demonstrate that LLL therapy can promote decrease in non-collagenous protein and glycosaminoglycans content, as well as an increase in metalloproteinases ?9, which proved, for the first time, that LLL therapy promotes alterations in the inflamed tendons even when analyzed only four hours after this process occur and could be a useful tool to improve the balance in inflamed tissues.     

Regional biochemical and morphological characteristics of rat knee meniscus

The biochemical and morphological characteristics of the anterior and posterior regions of the rat knee meniscus were studied. The anterior meniscal horn was thicker and contained a lower concentration of DNA, hydroxyproline, and uronic acid as compared to the posterior region. The calcium concentration in the anterior region, however, was significantly greater than the calcium concentration in the posterior horn. Presence of a significant concentration of calcium in the normal rat knee meniscus is unique to rats and uncommon in other mammalian species.     

Effects of boron derivatives on extracellular matrix formation

Boric acid solution (3%) dramatically improves wound healing through action on the extracellular matrix, a finding that has been obtained in vitro. Consequently, investigations are presently underway to produce boronated compounds having a therapeutical effectiveness similar to that of boric acid. On the basis of experimental results obtained with boric acid, we examined the effects of boron derivatives on extracellular matrix formation and degradation and analyzed their potential toxicity by using two biological models (chick embryo cartilage and human fibroblasts). The four boron derivatives tested in this study (triethanolamine borate; N-diethyl-phosphoramidate-propylboronique acid; 2,2 dimethylhexyl-1,3-propanediol-aminopropylboronate and 1,2 propanediol-aminopropylboronate) mimicked the effects of boric acid. They induced a decrease of intracellular concentrations in extracellular matrix macromolecules (proteoglycans, proteins)-associated with an increase of their release in culture medium and stimulated the activity of intra- and extracellular proteases. Similarly to boric acid, these actions occurred after exposure of the cells to concentrations of all boron derivatives without apparent toxic effects. The compounds were found to be more toxic than boric acid itself when concentrations were calculated according to their molecular weight. Nevertheless, these in vitro preliminary results demonstrate effects of boron derivatives that may be of therapeutic benefit in wound repair.     

Effects of extracellular matrix on the expression of specific ovarian proteins

A unique ovarian follicle cell culture system has been established to analyze the effects of extracellular matrix (ECM) on early granulosa cell differentiation. Primary and early secondary follicles isolated from ovaries of sexually immature rabbits were grown on poly-D-lysine or Englebreth-Holm-Swarm basement membrane biomatrix substrata (EHS) in serum-free, hormonally defined medium. Granulosa cells from these follicles were examined for growth pattern characteristics and for secretory protein synthesis by two-dimensional (2D) PAGE. Whereas some proteins were synthesized by cells on either matrix, the expression of other secreted proteins was markedly affected by the ECM used. Secretion of zona pellucida (ZP) proteins was demonstrated by ELISA assays and immunoblots of one-dimensional (1D) and 2D-PAGE separations of secreted proteins probed with monoclonal and epitope-selected antibodies. Expression of two ZP proteins was altered by ECM: 55-kDa endo-beta-galactosidase (EBGD)-treated ZP glycoprotein (55-kDaEBGD) was secreted by cells grown on either ECM, but a greater amount of 75-kDaEBGD was secreted by cells grown on poly-D-lysine. These studies are the first to show that granulosa cells from early-stage follicles express ZP proteins in vitro in the absence of oocytes, although proper post-translational modification may not occur. They also demonstrate the dramatic effect of ECM on the expression of these and other secretory proteins.     

Growth of neonatal rat uterine luminal epithelium on extracellular matrix

Summary We have developed a tissue culture system using an extract of basement membrane (extracellular matrix) which promotes the in vitro growth and development of uterine luminal epithelium from the 5-day-old rat. Uterine luminal epithelium, free of stroma, was obtained as short tubes by trypsinization of uterine segments followed by mechanical separation. Epithelial segments were grown in a serum-free medium on culture dishes coated with an extracellular matrix. After 2 days, rapid cell growth resulted in monolayer cultures, which subsequently formed organoid structures similar to differentiated uterine glands present in uterine tissue taken from older rats. Electron microscopy of cultures revealed columnar cells with basally located nuclei, apical microvilli, lateral membranes with interdigitations, desmosomes, and secretory Golgi complexes, all features found in functioning uterine epithelium in vivo. This model will allow the in vitro investigation of the development of uterine epithelium-specific functions free of the influence of stromal cell factors.     

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon.

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6-7 months of age, the rats in the two exercise groups performed 40-50 min of treadmill running at 1.2-1.6 miles h-1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P less than 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P less than 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.