Collagen In Cell Cultures Research Articles

Collagen Isolated from Human Adipose Tissue and Its Cellular Affinity.

The use of collagen in cell cultures promotes cell proliferation and differentiation, and it has been commercialized. In this study, we separated and purified collagen from adipose tissue discarded during liposuction and prepared collagen-coated dishes. After collagen was identified from human adipose tissue, type identification and quantification were performed using SDS-PAGE and FPLC. Collagen type I was used to coat culture dishes. Human skin fibroblasts and human adipose tissue-derived stem cells were seeded at a density of 2.5×105 cells/mL on prepared dishes at a collagen concentration of 3 mg/mL and cultured for 7 days. Cell viability was then measured and analyzed. The WST-1 assay was used to evaluate the results. The amount of collagen in 300 g of adipose tissue was 25.5 mg for type I, 41.4 mg for type III, 10.6 mg for type IV, 6.5 mg for type V, and 15 mg for type VI. The highest rates were observed for adipose stem cells cultured on human adipose tissue-derived collagen-coated dishes. In cell cultures, cell affinity was higher when cells and the substrate used were of the same origin, and affinity was stronger when the tissue of origin was the same.

Jellyfish collagen biomaterials: Next generation collagen for cell culture and med-tech products

Jellyfish collagen biomaterials: Next generation collagen for cell culture and med-tech products

The Novel Small Leucine-rich Protein Chondroadherin-like (CHADL) Is Expressed in Cartilage and Modulates Chondrocyte Differentiation

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers. Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

Selective retention and degradation of molecules with a single mutant α1(I) chain in the Brtl IV mouse model of OI

We investigated the secretion, matrix incorporation and interactions of molecules with one and two mutant α1(I) collagen chains in the Brtl IV murine model for Osteogenesis Imperfecta, carrying a Gly-349 to Cys substitution in one col1a1 allele. We detected a significant deviation from the expected 25 and 50% content of the molecules with no (37–46%) and one (26–40%) mutant chains in skin and bone as well as in fibroblast and osteoblast cell culture media. Steady-state labeling with 35S-Cys demonstrated incomplete secretion of the mutant collagen in cell culture, particularly molecules containing one mutant chain. Pulse and pulse-chase experiments revealed slower secretion of the latter. An enlargement of endoplasmic reticulum in skin fibroblasts from Brtl IV mice, clearly visible by electron microscopy, supported the abnormal secretion identified by biochemical studies. We observed increased susceptibility of molecules with one mutant chain to proteolytic degradation in vitro, but we did not detect significant selective degradation in cell culture media. Mutant collagen molecules incorporated from the media into newly deposited fibers and into fully crosslinked and mature matrix in the same ratio as they were secreted. Specific labeling of reactive –SH demonstrated that about half of the Cys349–SH groups in the mutant molecules were exposed and potentially available for aberrant interactions with other molecules inside or outside the cells. Based on these and our previous findings, we argue that the outcome in Brtl IV may be significantly affected by cellular stress and malfunction caused by the retention and degradation of newly synthesized mutant collagen.

Collagen-coated polylactide microspheres as chondrocyte microcarriers

Polylactide (PLA) microspheres were coated with collagen for cell culture and injectable cell carriers. Utilizing a method of emulsion-solvent evaporation, PLA microspheres with diameter ranging from 180 to 280 μm were prepared, followed with aminolysis in hexanediamine/ n-propanol solution to introduce free amino groups on their surfaces. After the amino groups were transferred into aldehyde groups by a treatment of glutaraldehyde, collagen type I was covalently coupled via Schiff base formation between the aldehyde groups and the amino groups on collagen molecules. Meanwhile, physically entangled collagen molecules were retained following a grafting-coating protocol to yield microspheres coated with larger amount of collagen. Aminolysis resulted in weight loss of the microspheres following a linear relationship with the aminolysis time. The NH 2 and collagen contents existed on the microsphere surface were quantitatively determined by ninhydrin and hydroproline (Hyp) analyses, respectively. Larger amount of collagen was immobilized on the microspheres with higher content of NH 2. In vitro chondrocyte culture revealed that the cells could attach, proliferate and spread on these PLA microspheres, in particular on the ones having higher content of collagen. These results show that the collagen-coated PLA microspheres are promising candidate as cell microcarriers.

Experimental model for collagen estimation in cell culture

In the study of Plastic Surgery, cell culture may be used at experimental level in researches concerning biosynthesis functions of skin cells such as fibroblasts, keratinocytes, adipocytes, chondrocytes and osteocytes. The present study reports an experimental model for estimation of collagen in cell cultures using chromogenic precipitation reaction with an especific dye (Sirius Red).

Gas chromatography-mass spectrometry determination of 18O2 in 18O-labelled 4-hydroxyproline for measurement of collagen synthesis and intracellular degradation.

The use of gas chromatography-mass spectrometry (GC-MS) and 18O2, a stable isotope which is incorporated into collagen during the post-translational conversion of proline to hydroxyproline, offers the potential advantages of high levels of sensitivity and specificity as compared to other techniques for measuring rates of collagen synthesis and degradation in vitro and vivo. Trifluoracetylation and methanol esterification of hydroxyproline yields two derivatives of hydroxyproline: N,O-trifluoroacetyl methyl 4-hydroxy-L-proline (N,O-TFA-Hyp) and N-trifluoroacetyl methyl 4-hydroxy-L-proline (N-TFA-Hyp). In the past, N-TFA-Hyp, which yields the 16O/18O-containing m/z 182/184 ion pair [M-COOH3]+ when analyzed by electron impact ionization GC-MS, has been proposed for analysis of 18O-enriched collagen. Although N,O-TFA-Hyp can be converted to N-TFA-Hyp by solvolysis, we find that this leads to degradation of the chromatography in GC-MS and demonstrate here that this extra chemical step is unnecessary if the m/z 278/280 ion pair (representing the [M-COOCH3]+. fragment) is measured by selected ion monitoring. By labelling fibroblasts in culture with 18O2, a sample of isotope-enriched collagen was obtained which was used to calibrate the GC-MS over the range 0.5-49% atom percent enrichment (APE). The greater sensitivity of 18O2 versus [15N]proline for labelling newly synthesized collagen was demonstrated by the finding of a ten-fold higher enrichment in the former isotope when administered to cell cultures at the same precursor APE. Thus, the approach described herein permits the determination of total hydroxyproline and APE on the same sample avoiding additional processing steps while maintaining the quality of chromatography and the sensitivity of detection. Measurement of absolute rates of both collagen synthesis and intracellular degradation of newly synthesized collagen in cell cultures is thus possible. Preliminary results comparing collagen metabolism in pairs of fibroblasts from hypertrophic scars and normal skin in post-burn patients are presented.

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.

Quantitative assay of types I and III collagen synthesized by keloid biopsies and fibroblasts

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts, However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.

Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts.

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.