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Compatible Polymers Journal of Bioactive and

DOI: 10.1177/0883911509102710 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 ZHENG1 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 GUO4 4College of Medicine Laboratory, Wenzhou

Medical College, Wenzhou 325035, Zhejiang Province, PR. China

JIAQUAN HOU5 5Building Design and Research Institute of the General

Logistics Department of PLA, Beijing 100036, PR. China

YIZAO WAN6 AND CHUAN GAO6 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 degra- dation 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

0883-9115/09/01(s) 0137–9 $10.00/0 DOI: 10.1177/0883911509102710  SAGE Publications 2009

Los Angeles, London, New Delhi and Singapore

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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, biocompat- ibility, bone tissue engineering, tissue engineering.

INTRODUCTION

Bone defect treatment is a common clinical practice that involvessurgical 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 composed of a glucose molecular chain connected by a b-(1-4)-glucosidic bond. As a natural polymer, BC has been clinically applied as high- quality 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 tissue- engineered 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 degra- dation of scaffold materials derived from extracellular matrix (ECM). This is a novel, sensitive, accurate, and safe method [11,12], 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

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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.

MATERIALS AND METHODS

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 spectrophotometer of Hitachi U-3310. Field emission scanning electronmicroscope (FE-SEM) was used to 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 24 h; the induced media (DMEM, contained 15%FBS, 100U/mL of penicillin, 100U/mL of streptomycin, 10mM of b-glycerophosphate, 50 mg/mL of

In Vitro Cytotoxicity of Bacterial Cellulose Scaffolds 139

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L-ascorbic acid, and 108mmol 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 24 h. 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 4 h at 378C in 5% CO2. After incubation, all of the media was replaced with dimethyl sulfoxide minimum 99.5% GC (DMSO) solution. The absorbance (OD) of the solution was measured at 570 nm using a scanning multi-well spectrophotometer. The relative growth rate (RGR) was calculated as follows:

RGR ¼ OD1=OD0  100% ð1Þ

where OD1¼ experimental group, OD0¼ control group. According to the national standard scoring method, the RGR value includes six parts: 100, 75–99, 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.

RESULTS AND DISCUSSION

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

The concentration of reducing sugar indicates the degree of degrada- tion 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

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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 differentia- tion of cells [16].

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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. The corresponding RGR values of the OB co-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].

CONCLUSIONS

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

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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.

ACKNOWLEDGMENTS

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.

REFERENCES

1. Lieberman, J.R. and Daluiski, A. (2002). The Role of Growth Factors in the Repair of Bone. Biology and Clinical Applications, J. Bone Joint Surg. Am., 84(A): 1032–1044.

2. Kyriakidou, K. and Lucarini, G. (2008). Dynamic Co-seeding of Osteoblast and Endothelial Cells on 3D Polycaprolactone Scaffolds for Enhanced Bone Tissue Engineering, J. Bioact. Compat. Polym., 23(3): 227–243.

3. Yan, Y.N. and Wang, X.H. (2005). Direct Construction of a Three- dimensional Structure with Cells and Hydrogel, J. Bioact. Compat. Polym., 20(3): 259–269.

4. Kneser, U. and Schaefer, D.J. (2006). Tissue Engineering of Bone: The Reconstructive Surgeon’s Point of View, J. Cell. Mol. Med., 10(1): 7–19.

5. Lei, Y. and Rai, B. (2007). In Vitro Degradation of Novel Bioactive Polycaprolactone-20% Tricalcium Phosphate Composite Scaffolds for Bone Engineering, Mater. Sci. Eng. C, 27: 293–298.

6. Wu, L.B. and Ding, J.D. (2004). In Vitro Degradation of Three-dimensional Porous Poly (D,L-lactide-coglycolide) Scaffolds for Tissue Engineering, Biomaterials, 25: 5821–5830.

7. Lee, J.W. and Deng, F. (2001). Direct Incorporation of Glucosamine and N-acetylglucosamine into Exopolymers by Gluconacetobacter xylinus (¼Acetobacter xylinum) ATCC 10245: Production of Chitosan–cellulose and Chitin–cellulose Exopolymers, Appl. Environ. Microbiol., 67: 3970–3975.

8. Wan, Y.Z. and Huang, Y. (2007). Biomimetic Synthesis of Hydroxyapatite/ Bacterial Cellulose Nanocomposites for Biomedical Applications, Mater. Sci. Eng. C, 27: 855–864.

9. Svensson, A. and Nicklasson, E. (2005). Bacterial Cellulose as a Potential Scaffold for Tissue Engineering of Cartilage, Biomaterials, 26: 419–431.

10. Backdahl, H. and Helenius, G. (2006). Mechanical Properties of Bacterial Cellulose and Interactions with Smooth Muscle Cells, Biomaterials, 27: 2141–2149.

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11. Record, R.D. and Hillegonds, D. (2001). In Vivo Degradation of 14C-labeled Small Intestinal Submucosa (SIS) When Used for Urinary Bladder Repair, Biomaterials, 22: 2653–2659.

12. Gilbert, T.W. and Stewart-Akers, A.M. (2007). A Quantitative Method for Evaluating the Degradation of Biologic Scaffold Materials, Biomaterials, 28: 147–150.

13. Nel, A. and Xia, T. (2006). Toxic Potential of Materials at the Nanolevel, Science, 311: 622.

14. Report (2005). Environmentalists and Industry Insiders Alike Urge Major Investments to Maintain the Emerging Technology’s Spotless Safety Record. Calls Rise for More Research on Toxicology of Nanomaterials, Science, 310: 1609.

15. Wu, G.P. and Yang, K. (2005). Cytotoxicity Study of Hydroxyapatite– Silicone Composite, J. Plast. Reconstr. Surg., 2(4): 228-232.

16. Yue, J. and Lei, D.L. (2005). Material Science of a Scaffold for Tissue Engineered Bone: Cytotoxicity of Short Rod-shaped Nano-hydroxyapatite, Chin. J. Clin. Rehabil., 9(2): 32–33.

17. Woo, K.M. and Jun, J.H. (2007). Nano-fibrous Scaffolding Promotes Osteoblast Differentiation andBiomineralization,Biomaterials, 28: 335–343.

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