MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.     

Structural characterization of pyrrolic cross-links in collagen using a biotinylated Ehrlich's reagent

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.     

Structural and micromechanical characterization of type I collagen gels

In this paper we report a study where we use a novel optical tweezers technique to measure the local viscoelastic properties of type I collagen solutions spanning the sol-to-gel transition. We use phase contrast optical microscopy to reveal dense and sparse regions of the rigid fibril networks, and find that the spatial variations in the mechanical properties of the collagen gels closely follow the structural properties. Within the dense phase of the connected network in the gel samples, there are regions that exhibit drastically different viscoelastic properties. Within the sparse regions of the gel samples, no evidence of elasticity is found. In type I collagen gels, we find a high degree of structural inhomogeneity. The inhomogeneity in the structural properties of collagen gels and the corresponding viscoelastic properties provide benchmark measurements for the behavior of desirable biological materials, or tissue equivalents.     

Loss of growth inhibitory activity of TGF-beta toward normal human mammary epithelial cells grown within collagen gel matrix

TGF-beta at concentrations in the range from 0.1 to 10 ng/ml gave significant growth inhibition of nonmalignant human mammary epithelial cells (HMEC) but not of malignant HMEC grown in monolayer cultures in serum-free medium. However, no growth inhibition of the nonmalignant cells was observed when the cells were cultivated within a type-I collagen gel matrix either adhering to a plastic substratum or floating on the medium. Within floating collagen gels, both nonmalignant and malignant HMEC formed a cell mass having radial extensions, and TGF-beta at 1 or 10 ng/ml prevented the formation of extensions only in the nonmalignant HMEC.     

Electrophysiological properties of tissue cultured heart cells grown in a linear array

Summary Embryonic chick heart cells were grown in tissue culture on an oriented substrate (channels cut in an agar coated slide), so that they formed narrow (5–100 ) strands of arbitrary length. The electrical properties of these strands were examined using intracellular microelectrodes ac and dc cable studies were performed to determine the passive cable parameters. Quantitative histology, using light and electronmicroscopy, permitted calculation of intrinsic capacitances and resistivities.Electrical coupling between polarizing and recording electrodes was ubiquitous, falling off exponentially with distance. It was concluded that individual cells were electrically connected, since coupling was observed at distances greater than 3mm, and the maximum cell length was estimated to be less that 300 . The strands were usually spontaneously active, with phase 4 depolarization (pacemaker potential) occurring almost simultaneously in all cells of a strand.The passive electrical properties determined during phase 4 were: core resistivity (cytoplasm plus cell-to-cell resistance), 245 ohm/cm; membrane capacitance, 1.46F/cm². The membrane resistance increased from 16 to 136 kohm/cm², during phase 4. The space and time constants showed commensurate changes, from 0.95 to 3.2 mm, and from 29 to 269 msec, respectively. The input resistance also increased, from 1.1 to 3.8 Mohm.     

Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells

Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granule-like inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.     

Unilocular fat cells in three-dimensional collagen gel matrix culture

Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture.     

Interactions of extracellular collagen and corneal fibroblasts: Morphologic and biochemical changes of rabbit corneal cells cultured in a collagen matrix

Summary Corneal fibroblasts, also known as keratocytes are surrounded by an extracellular matrix of collagen in vivo. To understand the physiology and pathology of these corneal fibroblasts, it is important to study their interactions with this extracellular matrix. We cultured rabbit corneal fibroblasts on tissue culture plastic dishes or in a hydrated collagen gel and compared the changes in morphology and mitotic activity. Corneal fibroblasts on plastic dishes were flattened and widely spread, whereas those in collagen gel became spindle-shaped with long processes. Examination with an electron microscope revealed that the corneal fibroblasts in collagen gel formed gap junctions with neighboring cells. Gap junctions were hardly ever observed between corneal fibroblasts cultured on plastic dishes. Corneal fibroblasts cultured in a collagen matrix showed much less incorporation of [³H]thymidine than did corneal fibroblasts cultured on plastic, and this incorporation decreased with increasing concentration of collagen. Our present results suggest that the morphologic and biochemical characteristics of corneal fibroblasts cultured in collagen gel are different from those cultured on plastic. This research was supported in part by grants from the Ministry of Education, Science and Culture of Japan, by a grant from Osaka Eye Bank, Osaka, Japan, and by an intramural research fund of Kinki University. Part of this research was presented at the annual meeting of the Japanese Ophthalmological Society (May 1985) at Kyoto, Japan, and at the annual meeting of the Association for Research in Vision and Ophthalmology (May 1987) at Sarasota, FL.     

Electrophysiological properties of tissue cultured heart cells grown in a linear array.

Embryonic chick heart cells were grown in tissue culture on an oriented substrate (channels cut in an agar coated slide), so that they formed narrow(5-100mu) strands of arbitrary length. The electrical properties of these strands were examined using intracellular microelectrodes. ac and dc cable studies were performed to determine the passive cable parameters. Quantitative histology, using light and electronmicroscopy, permitted calculation of intrinsic capacitances and resistivities. Electrical coupling between polarizing and recording electrodes was ubiquitous, falling off exponentially with distance. It was concluded that individual cells were electrically connected, since coupling was observed at distances greater than 3 mm, and the maximum cell length was estimated to be less that 300 mu. The strands were usually spontaneously active, with phase 4 depolarization (pacemaker potential) occurring almost simultaneously in all cells of a strand. The passive electrical properties determined during phase 4 were: core resistivity (cytoplasm plus cell-to-cell resistance), 245 ohm/cm; membrane capacitance, 1.46 muF/CM2. The membrane resistance increased from 16 to 136 kohm/cm2 during phase 4. The space and time constants showed commensurate changes, from 0.95 to 3.2 mm, and from 29 to 269 msec, respectively. The input resistance also increased, from 1.1 to 3.8 Mohm.     

Ultrastructural features of bovine mammary epithelial cells grown on collagen gels

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.     

DNA as a matrix of collagen fibrils

In this paper we demonstrate that DNA binds to collagen directly to form DNA–collagen complex. Our model suggests that DNA, containing well-arranged phosphate groups, helps the collagen to make ordered aggregates—fibrils. During this process hydration shell of collagen triple helix destroys and stabilizes hydration shell of ds-DNA.     

The formation of hydroxyproline in collagen by cells grown in the absence of serum

L-929 and 3T6 cells were conditioned to grow in a chemically defined medium lacking serum and ascorbate. Serum, when added, had a small stimulatory effect on the growth rate of the cells, but ascorbate had no effect either on the growth rate or on the rate of protein synthesis. These cells were also shown to lack gulonolactone oxidase activity and therefore could not synthesize their own ascorbate. Nevertheless, in the absence of serum and ascorbate both cell types were able to hydroxylate peptidyl proline to an appreciable extent. This suggest that reductant other than ascorbate can at least partially satisfy the requirement for a reductant in the prolyl hydroxylase reaction in vivo.     

Formation of iodinin by a strain ofAcidithiobacillus ferrooxidans grown on elemental sulfur

The presence of the pigment iodinin, anAcidithiobacillus ferrooxidans culture metabolite, was demonstrated after growth of bacteria on elemental sulfur. The structure of iodinin was confirmed by X-ray structure analysis; its physiological role is discussed.     

In vitro cytodifferentiation of perinatal rat islet cells within a tridimensional matrix of collagen

Summary Cell suspensions prepared by collagenase digestion of pancreases obtained from rat fetuses (21.5 d old) and newborns (2.5 d old) were mixed with a collagen solution and inoculated on a collagen base layer. At the onset of the culture, most acinar cells became necrotic, whereas other epithelial cells proliferated. Most of the cell clusters arranged themselves into simple polarozed structures composed of epithelial cells forming hollow spheres, and from these budded neoformed endocrine islets. Scarce fibroblasts were located close to these structures. Immunocytochemical localization of insulin and glucagon, as well as ultrastructural characteristics of the cell types revealed an intrainsular distribution similar to the in vivo localization. Tridimensional matrix of collagen offers, to perinatal pancreatic cells in culture, an environment close to the in vivo conditions: cells reorganize themselves in tissuelike structures and cell interactions concerned in the cytodifferentiation of pancreatic islets occur. This system allows for the study of undifferentiated epithelial cells—the presumed stem cells—differentiating and differentiated endocrine cells in the same preparation. B.A. is supported by a doctoral scholarship from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture, Brussels. This work was supported by grants from the Fonds National de la Recherche Scientifique, Brussels, and from Petrofina S.A., Brussels.     

In vitro carcinogenesis of mammary epithelial cells byN-nitroso-N-methylurea using a collagen gel matrix culture

Summary Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of “microtumors” in collagen gels, induced by 7,12-dimethylbenz(a)anthracene. In the present study the direct acting water soluble, mammary carcinogen,N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiated cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these “tumors” was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.     

Culture of human amniotic fluid stem cells in 3D collagen matrix

Most of the researchers attribute amniotic fluid stem cells (AF SCs) to mesenchymal stem cells (MSCs). However, AF SCs express both mesenchymal and epithelial markers, which distinguishes them from postnatal MSCs. Cultivation in the three-dimensional (3D) matrix provides a different look at the nature of the cells. We showed that in 3D collagen gel AF SCs form epithelial structures (tubules and cysts). The active contraction of the gel during the first days of cultivation, which is characteristic of mesenchymal cells, does not occur. Electron microscopic study showed that adherent junctions typical to epithelial cells are formed between AF SCs. On the other hand, during culturing in the gel AF SCs continue to express MSCs markers. Thus, AF SCs may be not true mesenchymal cells because they can display properties of epithelial cells. Perhaps these cells undergo epithelial-mesenchymal transition, a process which actively takes place during embryogenesis.     

Cell shape and arrangement of cultured aortic smooth muscle cells grown on collagen gels

Aortic smooth muscle cells (SMC) grown on conventional plastic culture dishes have morphological and functional properties of dedifferentiated cells in sub-culture. We examined the influence of collagen gels on the cell shape and arrangement. The cells grown on collagen gels showed a multilayered growth with formation of nodules. When the edge of the collagen gels was detached from the culture dish, the shape and arrangement of cells on the edge differed from that of the central, still attached region. The cells grown on floating collagen gels exhibited a spindle-like shape and were arranged in concentric circles. These findings suggest that the physical property of the substrate influences the cell shape and arrangement.