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Fractal Dimension Characteristics of Human Mesenchymal Stem Cell Proliferation

Fractal Dimension Characteristics of Human Mesenchymal Stem Cell Proliferation

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Published by: mohsen.rabbani3684 on May 08, 2010
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Fractal dimension characteristics of human mesenchymal stem cell proliferation
M. Rabbani
, M. Tafazzoli-Shadpour 
, Z. Goli Malekabadi
and M. Janmaleki
Amirkabir University of Technology, Biomedical Engineering Department, Tehran, Iran
Nanomedicine and Tissue Engineering Research Center, Shahid Beheshti University (M.C.), Tehran, Iran
 — Fractal Dimension (FD) provides informationabout cell complexity and self-similarity. Cell FD is a usefulfeature for automatic cell classification. Recently, proliferation and differentiation of human mesenchymalstem cells (hMSCs) have been of concern of biologicalreserch. This paper aims in evaluating variation of FDduring proliferation of these cells. The cell images of 5 cellculture flasks were acquired, and processed by
 program utilizing box counting method. The value of FDincreased with days in culture during cell growth or  proliferation with a constant correlation factor. Coefficientof Cellular Expansion (CCE) is another fractal parameter explaining cell chaotic dynamics. In spite of FD variation,cell coverage area variable was not consistent with days inculture for different samples. As a novel method, FDincreasing rate for hMSC images expressed by cell fractalcharacteristics was modeled as,
 FD = 0.1215 ln (Day) +1.1852, R
= 0.9267.
It is concluded that fractal dimensionis a suitable parameter for quantitative morphologicalevaluation of hMSCs.
 — fractal dimension, cell morphology, mesenchymalstem cell, chaotic attractor
Cell is a self-organizing, self-regulating, self-replicating,catalytic, dynamic, thermodynamic (i.e. isothermal andopen), and super-molecular system operating on the principal of complementary (Lehninger, 1981). Fractaldimension appears to be the common feature of allinteractive bio-systems [1]. Fractal dimension is aquantitative measure of morphological complexity [2]. Itmay change over time depending on cell differentiation or  proliferation [2]. This provides a precise and theoreticallyappropriate approximation of cell structural properties andespecially their shape complexity [3].The aim of our study is to evaluate alterations of FD inhuman mesenchymal stem cells (hMSCs) during cellgrowth/proliferation.
Cell culture
Human mesenchymal stem cells (hMSCs) obtained fromIranian Blood Transfer Association were used from passages 6 to 9. Cells were cultured in Dulbecco's ModifiedEagle Medium (DMEM- Gibco) containing 450 mgglucose/L, 3.7 g/L NaHCO3, 10% fetal bovine serum (FBS-Biosera), 100 U/ml penicillin, and 100 mg/L Streptomycin.Cells were trypsinized near confluency by using 0.05%trypsin/1 mM ethylenediaminetetraacetic acid and frozen in5% dimethyl sulfoxide (DMSO)/FBS for further use.Medium was replaced on day 3 after each passage.
 Image processing 
In each flask, five separated spots were selected for imaging in different days. Average of image characteristicsof these points referred to each flask for each day. Totally25 average data were calculated for five flasks of hMSCscultured during days after passage to reach confluency on 6to 8
days in culture. Figure 1 demonstrates cell imagesduring proliferation. An optical inverted microscope(Nikon, TE2000-U) with 10X magnification for both ocular and objective lenses was applied. By a digital Sony camera(Coolpix, Japan), 6 Mega pixel cell images (2816*2112Pixels) were captured and imported into image processing program, i.e. IMAGEJ.The image processing algorithm was performed on a PCusing a public domain NIH image program (ImageJ),developed at the US National Institute of Health. The image processing protocol included gray scale (8-bit), subtract background (rolling ball radius, 25-30 pixels), binary (black and white), filters/median (radius, 2-5 pixels). Fraction of covered area with cells was calculated byImageJ/analyze/measurement. Final images are obtained byan optimized filtration and image processing methods whichshow cells as black segments in a white background moreaccurate.
ig. 1 Images of cultured hMSCs after second (A), third (B) and fifth(C) days of culture
 Fractal analysis
The Euclidean dimension describes objects in space asintegers. Thus a straight line, plane and volume havedimensions of one, two and three respectively. Fractaldimension is simply a number that reflects a particulaaspect of a geometric form. It is called dimension because it provides a measure of how completely an object fills space.The dimension values is called fractal as it is a fraction not awhole number [4]. Since cells do not completely cover thetwo-dimensional area, they cannot be adequatelycharacterized by Euclidean geometry and we can use FD for classification of their images. As natural objects are scaleinvariant rather than self-similar within an upper and lower limits due to slight differences in detail between iterationlevels [4], cell magnified image could be applied for cellfractal analysis.Box-counting method in fractal analysis is used todetermine the FD of both scale invariant and self-similar objects. Also in many studies this method have been utilizedfor cell morphology analysis [4]. Using ImageJ program,FD is calculated for cell images (ImageJ/ analyze/ tools/fractal box-counting (box sizes, 2,3,4,6,8,12,16,32,64)).Coefficient of cellular expansion (CCE) is defined,
S=(b/w) – 1
, in which
is the cell image fractal and
isthe integer dimension of space (i.e. 2).
Fractal dimension shows an acceptable curve fitregression for all flasks containing hMSCs during 3
to 6
days of culture (Table 1).T
able 1 box-counting FD of hMSC during cell proliferation,
 FD = a × ln (Day) + b
1 0.1208 1.2037 0.73452 0.1316 1.1828 0.9643 0.1329 1.1485 0.87524 0.0941 1.2506 0.8875 0.1102 1.1798 0.983Flasks Ave. 0.1215 1.1852 0.926
Fig. 2 B
ox-counting FD for hMSC during 3 to 6
days in culture
As shown in Fig. 2, the Box-counting FD of hMSCimages increases during culture days after the third day. Itcan be seen that the curve has a constant rate. Obviouslythis phenomenon is not related to cell number or cells area(Fig. 3) but related to cells morphological complexity. As itis demonstrated in Fig.2 and Table 1, for all flasks FD is

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