teste de citotoxidade em scaffolds de CB

teste de citotoxidade em scaffolds de CB

(Parte 1 de 3)

http://jbc.sagepub.com Compatible Polymers

Journal of Bioactive and

DOI: 10.17/0883911509102710 2009; 24; 137 Journal of Bioactive and Compatible Polymers

Chuan Gao

Y.M. Chen, Tingfei Xi, Yudong Zheng, Tingting Guo, Jiaquan Hou, Yizao Wan and Tissue-engineered Bone

Cytotoxicity of Bacterial Cellulose Scaffolds Used forIn Vitro http://jbc.sagepub.com/cgi/content/abstract/24/1_suppl/137 The online version of this article can be found at:

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In Vitro Cytotoxicity of Bacterial

Cellulose Scaffolds Used for Tissue-engineered Bone

Y.M. CHEN,1 TINGFEI XI1,2,3,* AND YUDONG ZHENG 1

1School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing 100083, P.R. China

2National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, P.R. China

3Shenzhen Institute, Peking University, Shenzhen 518057, P.R. China

TINGTING GUO 4

4College of Medicine Laboratory, Wenzhou Medical College, Wenzhou 325035, Zhejiang Province, PR. China

JIAQUAN HOU 5

5Building Design and Research Institute of the General Logistics Department of PLA, Beijing 100036, PR. China

YIZAO WAN6 AND CHUAN GAO 6

6School of Materials Science and Engineering Tianjin University, Tianjin 300072, PR China

ABSTRACT: The in vitro degradation and cytotoxicity of bacterial cellulose (BC) and its degradation products were studied for potential applications in bone tissue engineering. Emission scanning electron microscope was used to observe the morphology of original materials and their degradation products. The degradation was evaluated by measuring the concentration of reducing sugar by using ultraviolet spectrophotometer. Bone forming osteoblast (OB) cells and infinite culture cell line L929 fibroblasts were used to measure the cytotoxicity of materials using the MTT assay. Both types of cells proliferated normally with the BC and its degradation products with a cytotoxicity graded of 0–1. Nevertheless,

*Author to whom correspondence should be addressed. E-mail: xitingfei@tom.com Figures 2–4 appear in color online: http://jbc.sagepub.com

Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol. 24—May 2009 137

SAGE Publications 2009 Los Angeles, London, New Delhi and Singapore at CAPES on July 24, 2009 http://jbc.sagepub.comDownloaded from the bone-forming target OB cells were more susceptible to cytotoxicity than the infinite culture fibroblast cells L929 fibroblasts. The results indicate that the BC is not very cytotoxic and that tissue functional cells are more suitable for evaluating the cytotoxicity of biomedical materials.

KEY WORDS: bacterial cellulose (BC), osteoblast, degradability, biocompatibility, bone tissue engineering, tissue engineering.

Bone defect treatment is a common clinical practice that involves surgical therapy. Implant materials are available for bone regen- eration that initiate and regulate the remodeling process [1]. Osteoblast (OB) cells, appropriate 3D scaffolds, and growth factors are three of the important elements for tissue engineering bone. OB cells are seeded on 3D scaffolds, such as polycaprolactone scaffolds, as part of the tissue engineering process [2]. The scaffolds act as a matrix within the tissue and provide support for nutrients, cytokines, growth factors, and cells [3–5]. Therefore, scaffold materials for tissue engineering must provide biocompatibility, surface activity, mechanical strength, porosity, and timely degradation. Among these, the degradation rate of porous scaffolds is important for the success of tissue-engineered bone. The scaffolds must degrade slowly to maintain structural support during the initial stage of bone formation [6].

Bacterial cellulose (BC), which is secreted by Acetobacter xylinum, is composedof a glucose molecularchain connectedby a b-(1-4)-glucosidic bond. As a natural polymer, BC has been clinically applied as highquality audio membranes, electronic paper, and dressing for artificial skin. In addition, BC is a novel scaffold material in tissue engineering due to it’s high crystallinity, high mechanical strength (tensile strength 42GPa) and Young’s modulus (138GPa), biodegradability, porosity, and fine web-like network structures [7,8]. Presently, BC used in tissueengineered cartilage and blood vessels has been reported, but no literature refers to its usage in tissue-engineered bone [9,10].

Glucose is the degradation product of BC, consequently, in vivo degradation is not manageable or detectable. Recently, researchers introduced radioactive 14C labeling to quantitatively evaluate the degradation of scaffold materials derived from extracellular matrix (ECM). This is a novel, sensitive, accurate, and safe method [1,12], but requires a costly accelerator mass spectrometry for analysis. Therefore, in vitro degradation is usually used in research on cellulose tissue engineering scaffolds. For in vitro cytotoxicity, L929 cells, a form of fibroblast cells, are widely employed to evaluate biomaterials. However, the L929 cells

138 Y. M. CHEN ET AL.

at CAPES on July 24, 2009 http://jbc.sagepub.comDownloaded from results are considered ineffective and unauthentic because they possess tumor cell features that can proliferate infinitely.

In this article, the degradation performance and an extended in vitro cytotoxicity of cellulose scaffolds for tissue-engineered bone was investigated. Using definite implanting positions, we introduced OB cells to evaluate the cytotoxicity of bone tissue engineering scaffolds materials to improve the evaluation method of in vitro cytotoxicity.

The intact materials of BC were obtained cooperatively from Tianjin

University. The bacterial strain of Acetobacter xylinum X-2 was incubated for 7 days in a static culture containing 0.3% (w/w) green tea powder and 5% (w/w) sucrose. After purification and surface modification, BC was obtained with a 3D network.

Degradation of Biomaterials

The materials with the length of 5mm 0.5mm and 5mm 0.5mm wide were immersed in 0.1mol/L phosphate buffered saline (PBS) at pH 7.25, kept in an incubator at 378C. Vestigial materials taken out from PBS periodically were reserved to detect their influence on the relative generation of cells and solution to measure the concentration of reducing sugar (glucose) by using ultraviolet spectrophotometerof Hitachi U-3310. Fieldemissionscanningelectronmicroscope(FE-SEM)wasusedto observe the morphology of original materials and their degradation products.

OB Cultures

Stromal osteoblastic cells were obtained from the marrow of young adult male Wistar rats. The primary OBs were extracted from stromal marrow by the method of isopycnic gradient centrifugation using 6% Pecoll solution. The primary media was Dulbecco’s Modified Eagle Medium (DMEM) containing 15% fetal bovine serum (FBS), 100U/mL penicillin and 100U/mL streptomycin. The obtained cell pellets were resuspended in primary media at the concentration of 2 105/mL, and incubated in a 378C and 5% CO2 incubator for 4 days. After incubation, the hematopoietic cells and unattached cells were removed from the flasks by repeated washes with DMEM. The primary cells were digested and passaged when they were 80% confluent. After the third passage, the cells were inoculated at the concentration of 5 104/mL for 24h; the induced media (DMEM, contained 15%FBS, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of b-glycerophosphate, 50mg/mL of

In Vitro Cytotoxicity of Bacterial Cellulose Scaffolds 139 at CAPES on July 24, 2009 http://jbc.sagepub.comDownloaded from

L-ascorbic acid, and 10 8mmol of L-dexamethasone) was added to promote the OB phenotype marrow stromal cells. The induced OB cells then used to detect in vitro cytotoxicity of materials.

In Vitro Cytotoxicity Evaluation of Materials (MTT Assay)

In this assay, indirect contact method (leaching liquor method) was used to evaluate the cytotoxicity of initial materials and direct contact method to evaluate that of materials taken-out-of degrading solution (degradation products). Bone-forming OB cells and the infinite culture L929 cell line fibroblast cells are adopted in this assay.

The cell suspension (density of 4 104 cells/mL) was seeded in microtiter 96-wells plates for 24h. After the plates were inoculated for 2 and 4 days, respectively, the MTT reagent (5mg/mL, Sigma) was added to the plates and incubated for 4h at 378Ci n5 %C O2. After incubation, all of the media was replaced with dimethyl sulfoxide minimum 9.5%

GC (DMSO) solution. The absorbance (OD) of the solution was measured at 570nm using a scanning multi-well spectrophotometer. The relative growth rate (RGR) was calculated as follows:

where OD1¼experimental group, OD0¼control group. According to the national standard scoring method, the RGR value includes six parts:

100, 75–9, 50–74, 25–49, 1–24, 0, corresponding to grade 0, grade 1, grade 2, grade 3, grade 4, and grade 5, respectively. The parts exceeding 75% was considered valid.

Morphology of BC and the Degradation Residue

The morphology of BC and the degradation products is shown in

Figure 1. As seen in Figure 1(a), the primary self-assembled BC possess excellent network interconnecting structure that formed uniform pores. In Figure 1(b), fragmentation has occurred on the BC fibril after being immersed for 8 weeks. After 12 weeks, the BC had degraded to form fuzzy aggregates (Figure 1(c)).

Concentration of Reducing Sugar in Degradation Solution

(Parte 1 de 3)

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