Experimental model for collagen estimation in cell culture 1

The manipulation of cutaneous compartment in Plastic Surgery is of primary importance since the healing quality is one of the criteria used for evaluating the results of esthetic and reconstructive surgical procedures. The behavior of cutaneous cells can be investigated with the use of culture of skin cells as fibroblasts and keratinocytes. Among several methods in vitro, fibroblast culture is an important tool for evaluation of the connective tissue, considering cell proliferation kinetics and synthesis of several components of the extra-cellular matrix. Some physical-chemical methods are used to measure the amount of substances produced by fibroblasts in culture chromogenic reactions (specific dyes), molecular weight comparisons (poliacrilamide or agarose gel electrophoresis and chromatography), immunological reactions (specific antigen-antibody reactions) and radioactive procedures (radioactive isotopes markers).


Introduction
The manipulation of cutaneous compartment in Plastic Surgery is of primary importance since the healing quality is one of the criteria used for evaluating the results of esthetic and reconstructive surgical procedures.
The behavior of cutaneous cells can be investigated with the use of culture of skin cells as fibroblasts and keratinocytes.
Among several methods in vitro, fibroblast culture is an important tool for evaluation of the connective tissue, considering cell proliferation kinetics and synthesis of several components of the extra-cellular matrix.Some physical-chemical methods are used to measure the amount of substances produced by fibroblasts in culture -chromogenic reactions (specific dyes), molecular weight comparisons (poliacrilamide or agarose gel electrophoresis and chromatography), immunological reactions (specific antigen-antibody reactions) and radioactive procedures (radioactive isotopes markers).

Proposition
The proposition of the present study is to describe an application of a collagen mensuration method by means of a chromogenic reaction between the molecule of this protein and an already described1,2,3 specific dye 4,5 Sirius Red F3B in fibroblast culture.

Method description
The description of this experimental model will be described as follows -(1) harvesting of collagen from the cellular layer and the culture medium of a fibroblast culture; and (2) reaction of chromogenic precipitation between collagen and the dye Sirius Red F3B.
Collagen extraction from cellular layer and culture medium.
Extraction of total protein and collagen from the cellular layer (FIGURE 1).Total protein and collagen from the cellular layer was obtained with the addition of a protein extraction solution after rinsing the culture flasks with PBS.This solution consisted of a protease inhibitor cocktail in PBS (5,0 ml) -EDTA 10 mM (Aldrich Chemical Co., Milwaukee, USA), N-etilmaleimide 10 mM (Sigma-Aldrich Chemical Co., Saint Louis, USA), and PMSF 1mM (Sigma-Aldrich Chemical Co., Saint Louis, USA).Then cellular layer was harvested by scraping it at 4°C.

SCRAPPED CELLULAR LAYER
Total protein from the cellular layer was obtained by sonication of the cellular layer extract (3 x 15 s, with 30 s intervals, 20 Hz, 4°C).
The sonicated was submitted to a collagen precipitation reaction with 25% saturated ammonium sulfate, for 24 hours at 4°C, under constant agitation 6 .Then collagen was isolated by centrifugation (40.000 x g, 30 minutes, 4°C).The supernatant was disposed and the pellet was solubilized in 2,0 ml of acetic acid 0,5 M (Merck, Darmstadt, Germany), consisting the aliquots of collagen from the cellular layer.
Extraction of collagen from the culture medium (FIGURE 2).Collagen from the culture medium was precipitated, as in the cellular layer, with 25% saturated ammonium sulfate, for 24 hours at 4°C, under constant agitation 6 .Then this solution was centrifuged (40.000 x g, 30 min, 4°C).
The pellet was dissolved in 2,0 ml acetic acid 0,5 M, which consisted of the collagen aliquot from the culture medium.Collagen chromogenic precipitation with Sirius Red (TABLE 1, FIGURES 3, 4, 5).Small amounts (100 µl) of collagen aliquots from the cellular layer and the culture medium were put in 1,8 ml conic tubes and the collagen was precipitated with 1 ml of a solution of dye Sirius Red (Sigma-Aldrich Chemical Co., Saint Louis, EUA) 50 µM (69 µg/ml) in acetic acid 0,5 M.After shaking, the tubes were maintained in rest for 30 minutes at environment temperature and then centrifuged for 30 minutes at 30.000xg.

Σ of ALIQUOTS OF CULTURE MEDIUM
The supernatant was disposed and the pellet was eluted in 1 mL potassium hydroxid -KOH 0,1 N (Merck, Darmstadt, Germany) for 15 minutes, in environment temperature (FIGURE 3).
Then, absorbance of the eluted was determined in spectrophotometer of 540 nm wavelength.Optic densities obtained were interpolated in a curve of absorbance, using collagen type I from rat tail soluble in acetic acid (Merck, Darmstadt, Germany) as standard (FIGURE 3, TABLE 1).TABLE 1 -Absorbance (540 nm) of the eluted from the collagen precipitation reaction with the dye Sirius Red (collagen type I from rat tail soluble in acetic acid -standard curve).

FIGURE 1 -
FIGURE 1 -Process of total protein and collagen extraction from the cellular layer.[+]aliquots of total protein from the cellular layer.

FIGURE 2 -
FIGURE 2 -Process of collagen extration from the culture medium.

FIGURE 3 -
FIGURE 3 -Standard curve of absorbance (540 nm) of the eluted from the collagen precipitation reaction with the dye Sirius Red (collagen type I from rat tail soluble in acetic acid.