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Bacterial Cellulose/Chondroitin Sulfate for
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  1. 1. Copyright © 2014 American Scientific Publishers All rights reserved Printed in the United States of America Journal of Biomaterials and Tissue Engineering Vol. 4, 1–5, 2014 Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds Gabriel Molina de Olyveira1 ∗ , Márcio Luiz dos Santos1 , Paula Braga Daltro1 , Pierre Basmaji2 , Gildásio de Cerqueira Daltro3 , and Antônio Carlos Guastaldi1 1 2 Department of Physical Chemistry-UNESP, 14800-900, Brazil Innovatec’s—Biotechnology Research and Development, São Carlos-SP, 13560-042, Brazil 3 College Hospital Complex Professor Edgard Santos (COM-HUPES) Bacterial cellulose (BC) has become established as a remarkably versatile biomaterial and can be used in a wide variety of scientific applications, especially for medical devices. In this work, the bacterial cellulose fermentation process is modified by the addition of chondroitin sulfate (1% w/w) to the culture medium before the bacteria are inoculated. Besides, biomimetic precipitation of calcium phosphate of biological interest from simulated body fluid on bacterial cellulose was studied. Chondroitin sulfate influences in bacterial cellulose were analyzed using transmission infrared spectroscopy (FTIR), XRD (X-ray diffraction) and scanning electron microscopy (SEM). FTIR analysis showed interaction between chondroitin sulfate, bacterial cellulose and calcium phosphate and XRD demonstrated amorphous calcium phosphate and carbonated apatite on bacterial cellulose nanocomposites. SEM images confirmed incorporation of calcium phosphate in bacterial cellulose nanocomposite surface and uniform spherical calcium phosphate particles. Future experiments with cells adhesion and viability are in course. Materials. 1. INTRODUCTION The development of materials for tissue repair or replacement of lost tissues or organs has been encouraged by the progress of research in the field of biomaterials, contributing to improving the quality and life expectancy of the population.1 In this way, the advance of research on implants gave incentive for research of biomaterials, and to study the reactions that occur in the tissue-implant interface.2 With rapid development technology has enabled advances in this area, increasing the efficiency of dentistry implants, and an important functional role in the reconstruction/aesthetics of the patient.3 The commercially pure titanium (cpTi) and its alloys are the most widely used biomaterials for the manufacture of dental implants due to its excellent biocompatibility and recognized, low modulus, high strength and corrosion resistance when compared to other metallic biomaterials. The main problem in the production of parts of titanium (Ti) is the high cost of raw materials and their processing.4 ∗ Author to whom correspondence should be addressed. J. Biomater. Tissue Eng. 2014, Vol. 4, No. 2 Nowadays, there is a significant number of metallic biomaterials, ceramics, polymers and composites, as options for use in various fields of dentistry and medicine. Experience with biomaterials represents a major advance in dentistry implant, but still requires studies of the bioactivity and biocompatibility in the human body.5 6 As alternative biomaterials, the ceramics-based materials were highlighted because bioceramics has no local or systemic toxicity, absence of response to a foreign body or inflammation and apparent ability to integrate with the host tissue.7–9 Other biomaterials are biopolymers based on chitosan, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA) and among these bacterial cellulose produced by Acetobacter xylinum.10 Bacterial cellulose (BC), provides an advantage over the conventional material, as the bacterial culture can be directed into the production of cellulose chains exclusively, yielding the purest form of the polymer (containing no lignin or hemicelluloses). Experimentally, it was found that BC also provided the benefit of finer fibrils of the material, suitable for use in composites. In literature, BC is reported as being produced with high crystallinity, fine microstructure (25–50 nm in diameter) and 2157-9083/2014/4/001/005 doi:10.1166/jbt.2014.1155 1 RESEARCH ARTICLE Keywords: Bacterial Cellulose (Nanoskin), Nanocomposites, Scaffolds, Dental Scaffolds
  2. 2. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds RESEARCH ARTICLE long fibre length (∼ 5 m).11–16 Bacterial cellulose has also been found to have a larger degree of polymerization in comparison to that of wood pulp cellulose. Several techniques of surface modification of surgical implants were developed in order to optimize the adhesion of bone tissue and therefore the implant to the bone. Among these, we highlight the process of deposition of coatings on implants that combine features of specific surface coating with structural properties of the substrate. To increase biocompatibility and bioactivity of titanium, the use of hydroxyapatite phase (HA) has been recommended for coating which accelerates the amount of bone fixation and increases the longevity of the surgical implant.17 18 Based on these properties, therefore, a biomaterial should provide consistency with the mechanical properties of the tissue to be regenerated and provide interface stability tissue/implant, as well as being biodegradable during bone regeneration. Several works reported bacterial cellulose nanocomposites with chemical changes in bacterial surface for hydroxyapatite adhesion.19 20 In this work, it is reported chondroitin sulfate/bacterial cellulose for dental materials scaffolds without membrane chemical modification, this proposal aims to obtain nanocomposite biomaterial for implant and bone regeneration with functionality. Future experiments with cells adhesion and viability are in course. 2. MATERIALS AND METHODS 2.1. Materials The bacterial cellulose raw material (Nanoskin) was provided from Innovatec’s (São Carlos SP, Brazil). Chondroitin sulfate was provided by MAPRIC (Brazil). 2.2. Methods 2.2.1. Synthesis of Bacterial Cellulose/Chondroitin Sulfate The acetic fermentation process is achieved by using glucose as a carbohydrate source and green tea as nitrogen source. Results of this process are vinegar and a nanobiocellulose biomass. The modified process is based on the addition of chondroitin sulfate (1% w/w) to the culture medium (green tea) before the bacteria are inoculated. Bacterial cellulose (BC) is produced by Gramnegative bacteria Gluconacetobacter xylinus, which can be obtained from the culture medium in a pure 3-D structure, consisting of an ultra fine network of cellulose nanofibers.21 2.2.2. Bionanocomposite Preparation In the present study, a novel biomaterial has been explored and different bacterial cellulose nanocomposites have been prepared; (1) BC and (2) BC/chondroitin sulfate. After, BC pieces were immersed in 1.5 SBF solution at 37 C for 7 days. The 1.5SBF solution was prepared from a 2 Olyveira et al. Fig. 1. Flow Diagram of bacterial cellulose synthesis and bionanocomposite preparation. protocol developed by Aparecida.22 Finally, the obtained BC nanocomposites were oven dried. In Figure 1 is ilustred flow diagram demonstrating the synthesis of bacterial cellulose as well as bionanocomposite preparation. 2.3. Characterization Scanning Electron Microscopy (SEM) images were performed on a PHILIPS XL30 FEG. The samples were covered with gold and silver paint for electrical contact and to perform the necessary images. Transmission infrared spectroscopy (FTIR, Perkin Elmer Spectrum 1000) Influences of chondroitin sulfate in bacterial cellulose were analyzed in a range between 250 and 4000 cm−1 and with 2 cm−1 resolution with samples. XRD (X-ray diffraction) were performed on a diffractometer Rigaku-DMax/2500PC, Japan ) with Cu–Ka radiation ( = 1 5406 Å), scan speed 0.02 /min in a range of 10–70 . Crystallographica search match software was used to identify the crystal structure of samples. 3. RESULTS AND DISCUSSION 3.1. SEM Pure Bacterial cellulose mats and chondroitin sulfate/bacterial cellulose were characterized by scanning electron microscopy (SEM). Figures 2(a) and (b) shows, as an example, SEM images of both bacterial cellulose mats. It can be observed similar morphology with chondroitin sulfate/(1% w/w) bacterial cellulose sample and pure bacterial cellulose mats which proved the addition of the appropriate amount chondroitin sulfate causes no major morphological changes. It is believed that the primary hydroxyl group of cellulose does not have enough reactivity to grow hydroxyapatite. Therefore, surface modification is needed to stimulate apatite formation on cellulose. The pre-incubation in the J. Biomater. Tissue Eng. 4, 1–5, 2014
  3. 3. Olyveira et al. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds CaCl2 solution is believed to provide the supersaturation of Ca2+ ions around BC through ionic interaction between calcium ions and the negatively charged OH groups available on BC and/or physical entrapment due to the 3-D network structure of the BC with tiny hollow spaces. Then the incorporated calcium ions can bind phosphate ions to form the initial nuclei. Once the apatite nuclei are formed, they grow by uptake of calcium and phosphate ions from the surrounding SBF fluid.17–20 However, biomimetic precipitation of calcium phosphate from simulated body fluid (SBF) on bacterial cellulose (BC) was perfomed and it can be observed by SEM images in Figures 3(a), (b) that calcium phosphate are deposited on bacterial cellulose nanocomposites, which shows that the deposition process do not need surface chemistry for the incorporation of calcium phosphate. 3.2. FTIR The main features of the bacterial cellulose in infrared spectroscopy is: 3500 cm−1 : OH stretching, J. Biomater. Tissue Eng. 4, 1–5, 2014 Fig. 3. SEM aimages of Bacterial cellulose nanocomposites with superficial calcium phosphate. 2900 cm−1 : CH stretching of alkane and asymmetric CH2 stretching, 2700 cm−1 : CH2 symmetric stretching, 1640 cm−1 : OH deformation, 1400 cm−1 : CH2 deformation, 1370 cm−1 : CH3 deformation, 1340 cm−1 : OH deformation and 1320–1030 cm−1 : CO deformation.21 23 It can be observed from Figure 4(a), the transmittance intensity is different of bacterial cellulose and bacterial cellulose nanocomposites, which means the exposed groups are interacting with bacterial cellulose components. Changes in symmetrical stretching CH2 bonds of bacterial cellulose structures in 1640 cm−1 and another absorption peak was obtained in a range of 1490 cm−1 , which shows the presence of a carbonyl group in the bacterial cellulose together with bonds corresponding to those of glycoside, including C O C at 1162 cm−1 (as in case of natural cellulose). These results, clearly, shows one possible interaction between bacterial cellulose and chondroitin sulfate mainly by hydrogen interactions between hydroxyl and carbonyl groups.21 23 3 RESEARCH ARTICLE Fig. 2. (a) SEM image of bacterial cellulose; (b) SEM image of bacterial cellulose/chondroitin sulfate.
  4. 4. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds Olyveira et al. (a) BC BC/SBF Intensity 80 60 40 CH2 Transmittance (u.a) 100 0 4000 C=O C–O–C 20 Bacterial cellulose Bacterial cellulose/chondroitin sulfate 3500 3000 2500 2000 1500 1000 500 10 20 30 Wavenumber (cm–1) 3500 3000 2500 2000 Wavenumber 1500 PO4 P-OH PO4–CO3 Transmittance (u.a) RESEARCH ARTICLE 60 70 4. CONCLUSION BC/SBF BC/chondroitin sulfate SBF 1000 (cm–1) Fig. 4. (a) FTIR spectra from bacterial cellulose and bacterial cellulose nanocomposites; (b) bacterial cellulose and nanocomposites with SBF. In Figure 4(b), sample was subsequently soaked in 1.5 SBF solution in order to promote absorption of calcium phosphate on nanocomposites surface. The bands displayed by IR spectroscopy were characteristic of the groups (PO4 in 454, 544 and 595 cm−1 P OH (879 and 944 to 1095 cm−1 ) and carbonated apatite types A and B (respectively, 1547 and 1311, 1468 cm−1 ). 3.3. XRD It can be seen in Figure 5 behavior with respect to the coating SBF. The peaks observed at 14.4 , 16.9 and 22.5 are attributed to bacterial cellulose, BC was identified as native cellulose (PDF #50-2241) and the characteristic peaks are indexed. Besides, amorphous calcium phosphate (PDF #18-303) and carbonated apatite (PDF #19-272) peaks are indexed in sample with calcium phosphate.22 Then, this indicates the amorphous calcium phosphate and carbonated apatite are formed on the surface of bacterial cellulose. 4 50 Fig. 5. XRD patterns of bacterial cellulose and bacterial cellulose/chondroitin sulfate. (b) 4000 40 2θ Bacterial cellulose was successfully modified by changing the fermentation medium as shown with SEM and FTIR, which produced scaffolds with different surface morphology because of calcium phosphate deposition. Natural scaffolds with bacterial cellulose and bacterial cellulose nanocomposites have good calcium phosphate incorporation over time between tested samples, being an extremely effective material for dental scaffolds application, such nanocomposites present similar behavior without membrane chemical modification with what is seen in the literature. Future experiments with cells adhesion and viability are in course. References and Notes 1. P. I. Branemark. Osseointegration and its experimental background. Journal of Prosthetic Dentistry 50, 399 (1983). 2. D. F. Willians, Concise Encyclopedia of Medical and Dental Materials, Pergamon Press, Oxford (1991). 3. D. F. Willians, Biocompatibility of clinical implant materials, CRC Press, Boca Raton (1981). 4. D. Ratner, Biomaterials Science: An Introduction to Materials in Medicine, Academic Press, San Diego (1996). 5. Y. Abe, T. Kobubo, and T. Yamamuro, Apatite coatings on ceramics, metals and polymers utilising a biological process. J. Mater. Sci. Mater. Med. 1, 233 (1990). 6. P. Ducheyne, Titanium and calcium phosphate ceramic dental implants, surfaces, coatings and interfaces. Journal Oral Implantology 14, 325 (1988). 7. R. Z. Le Geros, Properties of osteoconductive biomaterials:calcium phosphates. Clinical Orthopaedics and Related Research 395, 81 (2002). 8. H. Aoki, Science and Medical Applications of Hydroxyapatite, Takayama Press System Center, Tokio (1999), p. 214. 9. L. X. Filho, G. M. de Olyveira, P. Basmaji, and L. M. M. Costa, Novel electrospun nanotholits/PHB scaffolds for bone tissue regeneration. J. Nanosci. Nanotech. 13, 1 (2013). 10. G. M. Olyveira, G. A. X. Acasigua, L. M. M. Costa, C. R. Scher, L. X. Filho, P. H. L. Pranke, P. Basmaji, Human dental pulp stem cell behavior using natural nanotolith/bacterial cellulose J. Biomater. Tissue Eng. 4, 1–5, 2014
  5. 5. Olyveira et al. 11. 12. 13. 14. 15. 16. Bacterial Cellulose/Chondroitin Sulfate for Dental Materials Scaffolds scaffolds for regenerative medicine. J. Biomed. Nanotech. 9, 1 (2013). L. E. Millon, G. Guhados, and W. Wan, Anisotropic polyvinyl alcohol-Bacterial cellulose nanocomposite for biomedical applications. J. Biomed. Mater. Res. B Appl. Biomater. 86B, 444 (2008). L. M. M. Costa, G. M. Olyveira, P. Basmaji, and L. X. Filho, Bacterial cellulose towards functional medical materials. J. Biomater. Tissue Eng. 2, 185 (2012). G. M. Olyveira, L. M. M. Costa, and P. Basmaji, Physically modified bacterial cellulose as alternative routes for transdermal drug delivery. J. Biomater. Tissue Eng. 2, 31 (2013). D. P. Valido, L. M. M. Costa, G. M. Olyveira, P. B. P. Góis, R. L. A. C. Júnior, L. X. Filho, and P. Basmaji, Novel otholits/bacterial celulose nanocomposites as a potential natural product for direct dental pulp capping. J. Biomater. Tissue Eng. 2, 48 (2012). G. M. Olyveira, L. M. M. Costa, and P. Basmaji, High dispersivity bacterial cellulose/carbon nanotube nanocomposite for sensor applications. J. Biomater. Tissue Eng. 3, 665 (2013). P. Basmaji, G. M.Olyveira, M. L. dos Santos, and A. C. Guastaldi, Novel antimicrobial peptides bacterial cellulose obtained by symbioses culture between polyhexanide biguanide (phmb) and green tea. J. Biomater. Tissue. Eng. 4, 1 (2014). 17. L. Hong, Y. L. Wang, S. R. Jia, Y. Huang, C. Gao, and Y. Z. Wan, Hydroxyapatite/bacterial cellulose composites synthesized via a biomimetic route. Mater. Lett. 60, 1710 (2006). 18. Y. Z. Wan, L. Hong, S. R. Jia, Y. Huang, Y. Zhu, Y. L. Wang, and H. J. Jiang, Synthesis and characterization of hydroxyapatite– bacterial cellulose nanocomposites. Composites Science and Technology 66, 1825 (2006). 19. Y. Z. Wan, Y. Huang, C. D. Yuan, S. Raman, Y. Zhu, H. J. Jiang, F. He, and C. Gao, Biomimetic synthesis of hydroxyapatite/bacterial cellulose nanocomposites for biomedical applications. Materials Science and Engineering C 27, 855 (2007). 20. C. J. Grande, F. G. Torres, C. M. Gomez, and M. C. Baño’, Nanocomposites of bacterial cellulose/hydroxyapatite for biomedical applications. Acta Biomaterialia 5, 1605 (2009). 21. L. M. M. Costa, G. M. Olyveira, P. Basmaji, and L. X. Filho, Bacterial cellulose towards functional green composites materials. Journal of Bionanoscience 5, 167 (2011). 22. A. H. Aparecida, M. V. LFook, and A. C. Guastaldi, Biomimetic apatite formation on Ultra-High molecular weight polyethylene using modified biomimetic solution. Jornal of Materials Science: Materials in Medicine 20, 1215 (2009). 23. R. G. Zhbanko, Infrared Spectra of Cellulose and Its Derivates, edited by B. I. Stepanov, Translated from the Russian by A. B. Densham, Consultants Bureau, New York (1966). pp. 325–333 Received: xx xxxx xxxx. Accepted: xx xxxx xxxx. RESEARCH ARTICLE J. Biomater. Tissue Eng. 4, 1–5, 2014 5