teste de citotoxidade em scaffolds de celulose bacteriana

teste de citotoxidade em scaffolds de celulose bacteriana

(Parte 2 de 3)

The concentration of reducing sugar indicates the degree of degradation of carbohydrates, since a reducing sugar is the end product of the

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degradation of these materials. A five-point standard curve of glucose solutions was constructed to evaluate the Abs-concentration of glucose relationship. The degradation curve obtained was coincident with a single linear relationship. Using this method, we measured the concentration of the reducing sugar in solution after the residue materials were removed (Figure 2). The concentration of reducing sugar obtained by the degradation of BC increased with the time, however, the degradation rate was always58% over the 12 weeks.

Cytotoxicity of the Original BC

For the co-culture of infusion cells for 2, 4, and 7 days, the cellular

RGR values obtained are shown in Figure 3. All of the RGR values were 475% which indicated that the cytotoxicity of BC was a 0–1 grade. However, the OB cells were affected by the original material and the

1 Time of degradation (weeks)

Concentration (ug/mL) 0

De gradation rate (%)

Concentration Degradation rate

246 10 12 Figure 2. Concentration of glucose in degradation solution.

(a) (b) (c)

Figure 1. SEM images of (a) BC and degradation products of (b) 8 weeks, (c) 12 weeks, respectively.

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RGR values were always lower than those with L929 cells. During the co-culture for 4 days, the RGR values of the OB cells decreased slightly, however, based on the OB 7 day growth curve, the cells commenced to grow rapidly on the forth day. Thus, compared with normal growth in culture medium, the materials seem to influence cell growth. Furthermore, the OB cells are more sensitive for evaluating the cytotoxicity of materials than L929. Currently, researchers are more concerned with the bio-safety of nano-materials, particularly, the new nano-materials being developed for medical applications may possess potential cytoxicity [13,14]. Therefore, the cytotoxicity of materials is a major requisite in evaluating a novel biomedical material.

Presently, the L929 cell line, which is separated and extracted from subcutaneous Jimpy mouse tissue is widely used to evaluate the cytotoxicity of materials. However, the L929 cells possess tumor cells, that proliferate infinitely. Consequently, for evaluating the cytotoxicity of biomedical materials, the sensitivity and accuracy of L929 are considered suspect [15]. Furthermore, functional tissue cells, based on the implant position, should be used to evaluate the cytotoxicity of biomedical materials. The infusion interaction with functional cells of implanted position reflects not only the cytotoxicity of materials, but also their influence on the proliferation and differentiation of cells [16].

Relative growth rate (%)

2d 4d 7d

Figure 3. The RGR values of two types of cells affected by BC infusion.

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Cytotoxicity of Degradation Residue of BC

After being immersed in PBS, the degradation products were taken out to study their cytotoxicity on OB cells by direct contact for 4, 8, and 12 weeks,respectively.Thecorresponding RGR valuesofthe OBco-cultured with the degradation materials are graphically presented in Figure 4. The cytotoxicity of all products was graded as 0–1. With the time of immersion, the RGR values of OB, as BC degraded, increased and the cytotoxicity decreased. Moreover, the RGR values of the degradation materials increased with the time of co-culture was consistent with the cellular growth curve. One could consider that the degradation products of BC could promote cellular proliferation. For example, it has been reported that 3D nano-fibril scaffolds are better than orthodox nonfibril scaffolds for promoting cellular differentiation and proliferation [17].

With time of immersion in PBS, BC degradation increased slowly with the appearance of fragments and fuzzy aggregates. The MTT assay indicated that the BC and its degradation products are not very cytotoxic and are biocompatible. Nevertheless, bone-forming OB cells, as target cells, are more sensitive for cytotoxicity evaluations than the cultured

Relative growth rate (%)

2d 4d

Figure 4. The RGR values of the OB cells co-cultured with degradation products.

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L929 fibroblast cells. However, further work must be performed in order to determine the mechanism of degradation of BC and the effects of longterm degradation products of BC on organelle of cells.

This work was funded by National 973 project of China, No.2007

CB936101. Grateful acknowledgments should be given to Professor Yizao Wan, who offered the original materials used in this work.

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(Parte 2 de 3)