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Vitamin A and its congeners, collectively called retinoids, are known to have teratogenic potential and have induced craniofacial and limb malformations in numerous animal species. More importantly, retinoids are recognized as teratogenic to fetuses of pregnant women who have taken such preparations for dermatologic disorders. Information gathered from the study of animal models suggests that retinoids interfere with cartilage differentiation. If chondrogenesis in limb development is disturbed it may contribute to limb reductions and malformations. In vitro studies using various animal systems have shown that cartilage matrix macromolecules are altered to resemble those secreted by mesenchymal cells. The response of human chondrocytes to retinoids in vitro is not known. Culture of human chondrocytes in agarose maintains the cartilage phenotype and therefore serves as a model system to evaluate the influence of retinoids directly on human chondrogenesis. The studies presented in this paper were done to determine if the expression of specific matrix macromolecules of human chondrocytes in agarose culture is altered by retinol treatment. Immunocytochemistry demonstrated enhanced labeling of type I collagen while type II collagen labeling was reduced in cultures treated with retinol. In addition, morphometric analyses indicated a decrease in the size and number of chondrogenic clusters and that individual cells synthesized less alcian blue matrix when compared to parallel control cultures. The size of the proteoglycan monomers, glycosaminoglycan side chains as well as the disaccharide composition were not affected. However, there was a reduction in the quantity of proteoglycan monomers produced.
Anat Rec 1992 Jan
PMID:Influence of retinol on human chondrocytes in agarose culture. 153 65

Eight artificial matrices (AMs) were evaluated for the ability to restrict the passage of diffusion probes. Three AMs were composed exclusively of interstitial type I collagen (Col I) and differed from each other in thickness only. Four AMs consisted of reconstituted basement membrane (RBM) -coated polycarbonate filters (containing 10 microns diameter pores) and also only differed in thickness. One AM consisted of an uncoated 10 microns pore polycarbonate filter. The diffusion probes were uncharged fluorescein isothiocyanate-labeled dextrans, having molecular weights of 17,900, 42,000, 71,200, and 148,900 and negatively charged latex microspheres, having diameters of 0.08, 0.30, and 0.95 microns. Probes were applied to the AMs, incubated for 72 hr at 37 degrees C, and then analyzed spectrophotometrically. Dextran passage was increasingly restricted for Col I matrices as either molecular weight or collagen thickness increased (range 7% to 0.7%). Thin RBM-coated filters were more permeable to dextrans (range 100% to 30%) than Col I matrices. The diffusion rate of microspheres for Col I matrices (range 3.5% to 0) was similar to both thick and thin RBM-coated filters (range 4% to 0). The uncoated filter permitted the most diffusion for both dextrans and microspheres (range 100% to 7%). These data demonstrate that the AMs presented in this study will allow direct observation of the degradative and migratory potential of cells in vitro as they interact with various extracellular matrices.
Anat Rec 1990 Sep
PMID:Artificial matrix barriers: a diffusion study utilizing dextrans and microspheres. 170 Jun 47

The distribution of type I collagen, fibronectin, laminin, and heparan sulfate was studied in marrow of rats by indirect immunofluorescence. Most of the type I collagen of marrow is associated with large blood vessels and connective tissue trabeculae, but type I collagen was also localized in a delicate meshwork throughout the marrow and in the basement membrane of the sinusoidal endothelium. Fibronectin is partially co-distributed with type I collagen, but is much more widely distributed. Sheets or septa of fibronectin-rich material divide the marrow into small compartments that contain and appear to separate clusters of developing blood cells. These septa may serve as a substrate for anchorage and migration of blood cells. Labeling of laminin was observed in the basement membranes of blood vessels, of fat cells, and of the sinusoidal wall, but only scattered labeling was seen in other extracellular materials. Heparan sulfate proteoglycan was poorly labeled in the extracellular matrix of marrow.
Anat Rec 1991 Oct
PMID:Immunochemical localization of extracellular materials in bone marrow of rats. 174 22

Although homogeneous in appearance, several lines of evidence suggest early (stage 17-19) limb mesenchymal cells are committed to particular cell lineages, e.g., myogenic or chondrogenic. However, subsequent expression of cell or tissue phenotype in the developing limb does not occur in a randomized process but rather in a spatially specific pattern. The potential regulatory mechanisms controlling the "patterned" expression of tissue phenotype in the limb have not been resolved. The purpose of this study was to determine if, prior to the formation of an apical ectodermal ridge, nondissociated limb mesenchyme has inherent morphogenetic potential to form nonrandomized patterns of tissue organization. The hypotheses to be tested were that, if provided a spatially permissive culture environment, 1) mesenchymal cells committed to a particular lineage would segregate into precursor (sub)populations prior to overt expression of phenotype and 2) the ultimate expression of a tissue phenotype may be regulated, in part, by histogenic interactions between the precursor cell groups. For these studies, mesoblasts (intact mesenchyme minus ectoderm) from stage 17-19 hindlimb buds were explanted intact to the surface of a 1-3 mm thick hydrated lattice of repolymerized type I collagen and incubated for 2-11 days. Examination of cultures at variable intervals revealed three distinct temporal sequences (periods) which were arbitrarily termed early morphogenesis (0-3 days), cytodifferentiation (3-5.5 days), and primitive tissue formation (5.5-11 days) based on similarities to in situ limb development. By the end of the first period, the mesenchymal cells had sorted into three distinct precursor populations: 1) an epithelial-like outgrowth of premyogenic and prefibrogenic cells at the surface of the gel lattice (termed the "surface subset") which circumscribed, 2) a centrally positioned prechondrogenic condensate ("central subset"), and overlaid 3) a dispersed, population of free cells that invaded the collagen lattice ("seeded subset"). Subsequent cytodifferentiation led to the appearance of multinucleated myotubes within the surface subset and chondrification of the central subset. Cells of the seeded subset remained dispersed within the collagen lattice. Primitive histogenic events were initiated during the final period of development including 1) at sites where surface cells established boundaries with the central subset, collectives or "bundles" of variable sized myotubes were formed which became partially ensheathed by the attenuated processes of fibroblastlike cells; and 2) a secondary site of chondrogenic activity was initiated within the gel lattice at the boundary between the central and seeded cell populations. Transformation of seeded fibroblasts into chondroblasts accompanied expansion of the secondary chondrogenic element within the gel lattice.(ABSTRACT TRUNCATED AT 400 WORDS)
Anat Rec 1990 Jan
PMID:Morphogenesis of precursor subpopulations of chicken limb mesenchyme in three dimensional collagen gel culture. 229 86

The production of type I collagen by fibroblasts, odontoblasts, and osteoblasts is reviewed on the basis of results obtained by electron microscopy, 3H-proline radioautography, and immunostaining for type I procollagen. In the three cell types, the precursors of type I collagen are processed along the rough endoplasmic reticulum (rER)-Golgi-secretory granule pathway in the same manner as secretory proteins, but the available evidence suggests a few special features: 1) From the rER site of synthesis, the initial collagen precursors, known as pro-alpha chains, are transported to the Golgi apparatus within tubular structures, referred to as intermediate tubules, rather than within vesicles. 2) The pro-alpha chains coil into a triple helix within spherical distensions present along the saccules on the cis side of Golgi stacks. 3) The resulting procollagens are fairly rigid and form bundles that cause spherical distensions to lengthen into cylindrical ones, whereas by an unknown mechanism these distensions become part of the saccules on the trans-side of Golgi stacks. 4) The procollagen-containing cylindrical distensions are released from trans-saccules to become secretory granules, and some procollagen material finds its way into lysosomes. 5) The secretory granules release their procollagen content by exocytosis at the cell surface. 6) The released procollagen is transformed into collagen before or, more probably, after associating with the surface of a collagen fibril.
Anat Rec 1989 Jun
PMID:Synthesis and secretion of collagen by cells of connective tissue, bone, and dentin. 267 80

The anterior part of the mammalian nasal septum (NS) persists throughout the life span as hyaline cartilage, in contrast to cartilage in most parts of the body, which is gradually replaced by bone during development. In this study, we have cultured differentiating rat NS under various experimental conditions in an attempt to gain some insight into the osteogenic potential, if any, of the NS and its surrounding connective tissue. Differentiating NS from E15 and E19 rat embryos were dissected and grown under the following conditions: 1) organ cultured in Waymouth's medium or modified Eagle's medium, with or without serum; 2) cultured on chick chorioallantoic membrane (CAM); 3) implanted under rat kidney capsule (KC). Bone-like substance (BLS) never developed in organ cultures, but was observed in CAM cultures and KC implants after 7 days. The BLS was located external to the perichondrium of the NS and was stained red by the van Gieson's technique, indicating the presence of mature collagen. Further evidence of its bone-like characteristics was demonstrated by the presence of alkaline phosphatase and type I collagen. The CAM and KC represent two experimental conditions under which progenitor cells in the nasal septum area may be induced to synthesize BLS.
Anat Rec 1988 Aug
PMID:Development of bone-like substance in cartilaginous rat nasal septum under experimental conditions. 318 74

Mandibular condyles of fetal mice 19 to 20 days in utero were kept in an organ culture system for up to 10 days. After 2 days in culture the cartilage of the mandibular condyle appeared to have maintained all its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic. After 5 days in culture no chondroblasts could be seen and, instead, the entire cartilage was occupied by hypertrophic chondrocytes. At the same time, the mesenchymal cells at the chondroprogenitor zone differentiated with osteoblasts which produced osteoid. Light microscopic examinations showed that the newly formed osteoid did not stain with acidic toluidine blue or with alcian blue, but stained intensively with the van Gieson stain and with Periodic acid-Schiff (PAS). The osteoid reacted with antibodies against type I collagen but not with antibodies against type II collagen. Electron microscopic examinations showed that the mineralization appeared to be associated with collagen fibers in bone rather than with matrix vesicles in the cartilage. The process of bone formation progressed with time and by the 10th day new bone replaced almost the entire cartilage, thus forming an expanded layer of membrane bone. This in vitro system represents an experimental model whereby undifferentiated precursor cells transform into osteoblasts with the subsequent formation of a typical membrane bone.
Anat Rec 1983 Aug
PMID:In vitro transformation of chondroprogenitor cells into osteoblasts and the formation of new membrane bone. 662 99

All known bone-derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate osteoinductive proteins from IM bones. To accomplish this, we extracted proteins from bovine frontal bone matrix (intramembranous origin) using methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (< 1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sepharose (HS) affinity chromatography was performed. HS-binding (HS-B) and non-binding proteins (HS-NB) were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin-Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern than a comparable osteoinductive/chondroinductive factor isolated from EC bone.
Anat Rec 1994 Jan
PMID:Intramembranous bone matrix is osteoinductive. 811 88

Using an agarose gel culture system, the response of adult human chondrocytes to prolonged exposure of ascorbic acid was evaluated using histochemical, immunocytochemical and morphological techniques. The response of these cells to ascorbic acid was different from those previously reported in the literature. Many chondrocytes branched within the agarose gel with continued exposure to ascorbic acid while other chondrocytes maintained a round configuration typical of chondrocytes in vivo. Fibronectin and type I collagen were closely associated with the cell processes of the branching cells. Type II collagen and an alcian blue-staining matrix were associated with the rounded cells but not with the branched cells. These data suggest that the chondrocytes are able to express both dedifferentiated and redifferentiated phenotypes with ascorbic acid under these culture conditions. In addition, human chondrocytes were cultured in a collagen gel and began branching within 1 hour of culture. It is possible that an accumulation of type I collagen in the pericellular matrix of ascorbic acid treated cultures may enhance and explain the branching seen in these cultures. Studies by others have indicated that ascorbic acid may enhance, reduce, and/or modify the cartilage matrices produced by chondrocytes. These controversial reports in the literature are presumably due to variations between species and the culture methods employed.
Anat Rec 1994 Jan
PMID:Prolonged exposure of human chondrocytes to ascorbic acid modifies cellular behavior in an agarose gel. 811 89

In addition to the unique feature of retention of unfertilized ova, the oviducts of mares frequently contain large intraluminal masses with a fibrillar component and some cells. The aim of this study was to identify the cells and examine their relationship to the extracellular components of these masses. Intraluminal masses were examined both in situ and flushed from the oviducts. The nature of the contained cells and their relationship to the fibrils were examined by light microscopy and by transmission and scanning electron microscopy. In some mares the large masses distended the oviduct, but neither loss of the oviductal epithelium nor damage to this epithelium was seen. Electron microscopy verified that the principal cellular component was fibroblasts, and that the fibrils were type I collagen. Collagen masses collected shortly after ovulation frequently contained viable fibroblasts with collagen fibrils associated with their cell surfaces and with surface clefts. Although such collagen masses were present in pregnant and nonpregnant mares, masses with viable fibroblasts were chronologically associated with recent ovulation. It was concluded that connective tissue drawn into the oviduct at ovulation is retained, and collagen synthesis continues at least for a few days. Although the fibroblasts eventually disintegrate, the collagen remains and may in some cases aggregate within the oviductal lumen to the extent that oviductal transport and embryonic viability could be affected.
Anat Rec 1998 12
PMID:Possible significance of cells within intraluminal collagen masses in equine oviducts. 984 7


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