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Stem cell2

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  • 1. Article Journal of Biomedical Nanotechnology Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America Vol. 9, 1–8, 2013 www.aspbs.com/jbn Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds for Regenerative Medicine Gabriel Molina Olyveira1 3 ∗ , Gerson Arisoly Xavier Acasigua2 , Ligia Maria Manzine Costa1 , Cristiane Regina Scher2 , Lauro Xavier Filho4 , Patricia Pranke5 6 7 , and Pierre Basmaji4 1 Department of Nanoscience and Advanced Materials-UFABC, Rua Santa Adélia, 166, Santo André-SP, Brazil Dentistry Post-Graduation Programme, Federal University of Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil 3 Natural Products Laboratory and Biotechnology, UNIT, Aracaju, Brazil 4 Innovatec’s—Biotechnology Research and Development, São Carlos-SP, 13560-042,, Brazil 5 Material Science Post-Graduation Programme, Federal University of Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil 6 Faculty of Pharmacy, Hematology and Stem Cell Laboratory, Federal University of Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil 7 Stem Cell Research Institute (SCRI), Federal University of Rio Grande do Sul, Av. Ipiranga 2752, Porto Alegre-RS, 90610-000, Brazil 2 Adhesion and Viability study with human dental pulp stem cell using natural nanotolith/bacterial cellulose scaffolds for regenerative medicine are presented at first time in this work. Nanotolith are osteoinductors, i.e., they stimulate bone regeneration, enabling higher cells migration for bone tissue regeneration formation. This is mainly because nanotoliths are rich minerals present in the internal ear of bony fish. In addition, are part of a system which acts as a depth sensor and balance, acting as a sound vibrations detector and considered essential for the bone mineralization process, as in hydroxiapatites. Nanotoliths influence in bacterial cellulose was analyzed using transmission infrared spectroscopy (FTIR). Results shows that fermentation process and nanotoliths agglomeration decrease initial human dental pulp stem cell adhesion however tested bionanocomposite behavior has cell viability increase over time. KEYWORDS: Adhesion and Viability Study, Bacterial Cellulose (Nanoskin), Nanotoliths, Natural Nanocomposites, Regenerative Medicine, Stem Cells. INTRODUCTION Human dental composition is made up of hard tissues (enamel and dentin) and soft tissues (pulp, periodontal ligament and alveolar bone), which are composed of connective tissues and those from the basement membrane.1 2 Soft tissues are composed of cells and extracellular matrix (ECM), where there are many types of cells performing different functions, e.g., biosynthesis of the ECM components, lipid storage, biological protection, maintaining the body homeostasis. The ECM is comprised of many ∗ Author to whom correspondence should be addressed. Email: gabriel.ufabc@gmail.com Received: 14 September 2012 Revised/Accepted: 6 January 2013 J. Biomed. Nanotechnol. 2013, Vol. 9, No. xx different macromolecular structures, which can be grouped into collagens, glycoprotein’s, and proteolysis, attending to their major components.3 4 Nanocollagenous proteins present in oral cavity tissues are involved in several processes, such as the initiation and formation of apatite crystals (mineralized tissues) and the regulation of cell proliferation and tissue growth and development.5–7 The family of collagens accounts for more than 50% of the total tissular protein. Collagens are well-known proteins whose chemical and tridimensional structures are clearly elucidated.8 Bacterial cellulose (BC) is produced by microorganism via biochemical steps and self-assembling of the secreted cellulose fibrils in the medium. Shaping of BC materials 1550-7033/2013/9/001/008 doi:10.1166/jbn.2013.1620 1
  • 2. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds in the culture medium can be controlled by the type of cultivation that changes chain sizes, origin of strains that produce different proportion of crystalline phase of BC and a kind of bioreactor.9 The structural features of microbial cellulose, its properties and compatibility of the biomaterial for regenerative medicine can be changed modifying its culture medium10–12 or surface modification by physical13 14 and chemical methods15 16 to obtain a biomaterial with less rejection when cellular contact and blood cell contact interaction occurs. Among all the calcium phosphates, hydroxyapatite has lowest rate of biodegradation, or absorption by the animal organism, where the sequence is the following order at the rate of resorption. As the main mineral constituent of bone structure, hydroxyapatite has been extensively studied and used as material for permanent inclusion in defective or abnormal bone structure in surgical bone repair depending on his performance as biomaterials (biocompatible, bioactive and osteoconductive).17–19 However, recent studies reported that hydroxyapatite is a carcinogen material for bone tissue regeneration.20 In this context, Innovatec’s, which makes development with natural nanomaterials, discovered a new material with bone tissue regeneration bone called otolith. This material has same characteristics related to hydroxyapatite with lower toxicity because is a natural material. Based on the results with artificial skin, bacterial cellulose was tested in dental tissue regeneration. Olyveira et al.21 reported the first otolith/collagen/bacterialcellulose nanocomposites as a scaffold for bone regeneration. The success of the subsequent transplantation of the engineered in vitro construction is due to the properties of the materials but also to the osteoprogenitor cell sources. In addition, Olyveira et al. reported that otoliths showed biocompatibility with pulp tissues in vivo and the response of pulp tissue was successful.22 It can be concluded that direct pulp capping with the preparation of otoliths, similar to calcium hydroxide, preserves the vitality, stimulates the formation of mineralized tissue barrier and induces reparative pulp response. This biomaterial represents a promising biomaterial for use in human dental medicine in the future. Besides, the success of the scaffold to be used in tissue engineering depends, in part, on the adhesion and growth of cells of interest on its surface. The surface chemistry of the material may define the cellular material and thus affect the adhesion, proliferation, migration and cell function.23–25 The interaction of cells with the surfaces of materials is of extreme importance for the effectiveness of medical implants26 and may define the degree of acceptance or rejection. Knowledge of basic mechanisms of cell-material interaction and a better understanding of processes at the cellular level during the accession may contribute to the development of new biomaterials and the development of new biomedical products.27 Cells identify the exposed 2 Olyveira et al. surface topography and nanofiber features, like porous matrices and alignment influence the adhesion, spreading, proliferation and gene expression of the cells seeded onto them. Different cell behavior was found in several surface topographies obtained from lithography,28 29 phase separation,30 electrospinning;31 nanoimprinting32 and self– assembly.33 Stem cells are a non-specialized cell type, which can self-renew and remain for a long period of time with the potential to derive in a cell lineage or tissue with specialized functions. Tissue-specific stem cells, or adult stem cells, have been considered as an alternative for the use of embryonic stem cells, due to their availability, ease of acquisition and growth.34 35 Thus, the study of populations of adult stem cells with plasticity similar to embryonic stem cells has been the target of numerous researchers. Of particular interest, stem cells from deciduous teeth have certain advantages. Miura et al. attributed to these cells significantly greater potential for proliferation and clonogenisticity when compared to pulp stem cells from permanent teeth and stem cells from bone marrow.36 In order to obtain a bionanocomposite with mechanical properties and an osteoconductive environment that can facilitate cell attachment37 and to increase in vitro proliferation and differentiation of osteoblastic cells as well as the rapid vascularization, it is reported in this work a novel natural nanotoliths/bacterial cellulose for future dental materials is reported with adhesion and viability study using human dental pulp stem cell in bionanocomposite. MATERIALS AND METHODS Materials Bacterial cellulose membranes (Nanoskin), ∼ 500 mm thick, and Otoliths were supplied by Innovatec’s— Biotechnology Research and Development—Brazil. Synthesis of Bacterial Cellulose Bacterial Cellulose (BC) produced by Gram-negative acetic acid bacteria Gluconacetobacter xylinus can be obtained from a culture medium in the pure 3-D structure consisting of an ultra fine gel network of cellulose nanofibres (3–8 nm), highly hydrated (99% in weight), and displaying higher molecular weight, higher cellulose crystallinity (60–90%), enormous mechanical strength and full biocompatibility.38 Nanotoliths Gels The material in this study was prepared with 1 g powder of otolith of Cynoscion acoupa with particle size 60 mesh and addiction 0.25 g of hydrolyzed collagen, diluted in distilled water. The final product was packaged in petri dishes and sterilized in UV rays (25 min). Subsequently, 1.0 g of the otoliths were diluted in 100 mL of distillated water and the pH of the compound was assessed using J. Biomed. Nanotechnol. 9, 1–8, 2013
  • 3. Olyveira et al. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds phmeter Digimed® . Stable gel is formulated with a otolith calcium salt concentration solution. Scaffolds Preparation In the present study, different natural scaffold materials were prepared; (a) Pure BC, (b) BC with nanotoliths. Bacterial cellulose nanocomposite was obtained by immersion of dried bacterial cellulose into nanotolith gels and posterior soft drying at 50 C for 12 hours. Samples of Pulp Tissue from a Deciduous Tooth In order to isolate the cells from the pulp tissue and establish their culture, dental pulp was removed from a deciduous tooth in the process of rhizolysis. After extraction, the tooth was immersed in 1 ml of culture medium DMEM/Hepes (Sigma Aldrich), 10% fetal bovine serum (Laborclin), 100 U/ml penicillin, 100 g/mL streptomycin (Gibco) and 0.45 g/mL gentamicin (Gibco) at room temperature for transport to the laminar flow. Those responsible for the patient signed a consent form approved by the Ethics Committee of Universidade Tiradentes, it being passed on 10.2.2006, under protocol 171 205, in accordance with ethical for the use of laboratory animals, legally stipulated 1153-B 1995. Cell Culture The handling of the pulp tissue removed was performed following the protocol established in the laboratory.39 Cell suspension in the culture medium was seeded onto a culture plate of 12 wells and then incubated at 37 C in a humidified atmosphere of 5% CO2 . The culture medium was changed 24 hours after initial plating and then every 3 days thereafter. The culture was maintained under these conditions until confluence of approximately 90% when it was then held in its first passage. In sub-cultures, the cells in culture were harvested with trypsin—EDTA solution 0.5% (Sigma-Aldrich) and transferred to sub-cultures in their culture medium. The sub-culture was maintained in a monolayer until required for the next raise. When the cells reached approximately 90% of confluence between the 5th passage (P5), cell viability was assessed with trypan blue 4% (Gibco) in a Neubauer chamber and testing to verify the interaction between cells and scaffolds was performed. Characterization Scanning Electron Microscopy (SEM)—Scanning electronic microscopy images were performed on a PHILIPS XL30 FEG. The samples were covered with gold and silver paint for electrical contact and to produce the necessary images. Transmission Electron Microscopy (TEM)— Transmission electron micrographs of bacterial cellulose were taken with a Hitachi-600 transmission electron microscope with an acceleration voltage of 5 kV. J. Biomed. Nanotechnol. 9, 1–8, 2013 Transmission infrared spectroscopy (FTIR, Perkin Elmer Spectrum 1000)—Influences of nanotoliths in bacterial cellulose were analyzed in the range between 250 and 4000 cm−1 and with of 2 cm−1 resolution with samples. Cell Adhesion—5 × 104 cells in 50 L of concentrated culture medium were seeded on each type of scaffold (in triplicate) and incubated at 37 C in a humidified atmosphere of 5% CO2 . After 6 hours of culture, the culture medium was removed and the samples were washed three times with phosphate-buffered saline to remove nonadherent cells on the scaffolds. The cells attached to the scaffolds were then fixed with 4% paraformaldehyde for 20 min. Following this, staining was performed with 0.5 mg/ml 4,6-diamidino-2-phenylindole (DAPI), a fluorescent marker that binds strongly to DNA. From each sample nine images were obtained (Olympus CX50, 400× magnification) corresponding to nine different randomly distributed microscopic fields. Thus, it was possible to quantify the number of cells attached by means of the number of cells/field. As a control group, the cells was seeded in a similar way onto cell culture plates of 24 wells (in triplicate) without scaffolds, and the same procedures for data collection were carried out. Viability cell—For the study of cell viability during the 28 days of culture, similar to that performed for the cell adhesion essay, cells were seeded onto each type of scaffold (in triplicate) and then incubated at 37 C in a humidified atmosphere of 5% of CO2 . To collect the initial viability of the seeded cells, the viability of 5 × 104 cells was analysed, 6 hours after seeding onto the culture dishes. Analysis was then made 7, 14, 21 and 28 days after the start of the cultivation of the cells in the biomaterial. After each trial, cell viability was performed by the salt tretazolyum method, a colorimetric assay using the bromide 3-(4,5-dimethylthiazol-2-yl)-2,5-difeniltetrazolio (MTT). Elapsed times set were removed and the culture medium added to 200 L of MTT (0.25 mg/mL) for 2 hours. The MTT was then removed and 200 L of dimethyl sulfoxide (DMSO) was added to dissolve the crystals formed by the reaction. Using 96-well plates, the absorbance of the final solution was analysed by a spectrophotometer (Wallac EnVision—Perkin Elmer). The data was calculated using the difference in absorbance between the wavelengths (560 nm–630 nm). As a control group, the cells were seeded in a similar way onto 24-well plates (in triplicate) without scaffolds and maintained by the same experimental periods and the same procedures for data collection were performed. RESULTS AND DISCUSSION Pure Bacterial Cellulose and Bacterial Cellulose Nanocomposites Mats Pure Bacterial cellulose mats were characterized by SEM. Figures 1(a) and (b) show, as an example, SEM images and 3
  • 4. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds (a) Olyveira et al. 100 BC BC/nanotoliths 90 Transmitance u.a. 80 70 60 50 40 30 20 10 0 –10 4000 3500 3000 2500 2000 1500 1000 500 Wavelenght (cm–1) (b) Figure 2. FTIR spectra of bacterial cellulose and bacterial cellulose/otoliths nancomposites. (c) features of the bacterial cellulose in infrared spectroscopy is: 3500 cm−1 : OH stretching, 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.40 It can be observed in Figures 2 and 3 that that nanotoliths addiction changes symmetrical stretching CH2 bonds in bacterial cellulose structures in 1640 cm−1 41 Besides in Figure 3 nanotoliths bands at 712 and 874 cm−1 can be observed; characteristic absorption bands of CaCO3 .42 43 The band of 874 cm−1 was just overlapped with the absorption band of Bacterial cellulose from 892 cm−1 . Cell Adhesion and Viability The use of matrices associated with the cells is the target of interest for researchers dedicated to this area.44–48 Thus, the characterization of scaffolds is needed to verify Figure 1. (a), (b) Scanning electron microscopy (SEM) and TEM of Pure bacterial cellulose. (c) Cellulose bacterial/otoliths surface morphology. 80 712 BC BC/otoliths TEM images of pure Bacterial cellulose mats. Figure 1(c) shows SEM images from bacterial cellulose/otoliths surface morphology. It can be observed some changes in bacterial cellulose surface morphology with otolith. A high roughness changes opacity of the film and has influences in superficial properties mainly in adhesion and viability cells as proved by other characterizations in this work. Interaction Between Bacterial Cellulose with Nanotoliths Influences of nanotoliths in bacterial cellulose were analysed in the range between 250 and 4000 cm−1 and with resolution of 2 cm−1 with FTIR analysis. The main 4 Transmittance (a.u.) 70 60 50 40 30 20 874 10 0 –10 1400 1200 1000 800 600 400 Wave Number (cm–1 ) Figure 3. FTIR spectra of the samples BC, BC/otoliths. J. Biomed. Nanotechnol. 9, 1–8, 2013
  • 5. Olyveira et al. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds their structure and determine whether they have the necessary requirements for their application to stem cells from deciduous teeth used in this study. As described by Pham et al.49 the morphology of the nanofibers is the result of the combination of different factors. Normally, the primary teeth will go through the process of resorption of their roots during childhood.50 For the successful application of scaffolds in tissue engineering, a crucial feature is that the matrices promote cell adhesion. According to Andrews,51 cell adhesion is mediated by the adsorption of extracellular matrix proteins produced by cells on the surface of the scaffold. The signaling pathways are then activated and cell adhesion occurs in the mould by means of receptors. Therefore, accommodation and cell behaviour is strongly affected by the structure of the scaffolds and cell adhesion assay becomes important in order to determine whether the scaffolds have a good structure for the initial interaction with cells. The next step was staining with 0.5 mg/ml 4,6diamidino-2-phenylindole (DAPI), a fluorescent marker that binds strongly with DNA. By staining cell nucleus with DAPI, it is possible to verify the capacity of adhesion to SCDT (stem cells from deciduous cells) in different scaffolds. Bacterial cellulose (AP) and bacterial cellulose/nanotholits (A1) was statistically lower than the control condition on the culture plates—25.03, 25.73 and 62.69 cells per field in groups: bacterial cellulose (AP), bacterial cellulose/nanotholits (A1) and control respectively (C), with no statistical difference between the bacterial cellulose groups and the bacterial cellulose/nanotholits (Fig. 5). It can be observed in Figure 4 images obtained from DAPI treatment to verify cell adhesion in the control group (a), bacterial cellulose sample (b), and the bacterial cellulose/nanotholits sample (c). This fact demonstrates the quality of the nanofibers in enabling cells to Figure 4. Images obtained from DAPI treatment to verify cell adhesion: control group (a), bacterial cellulose (b), bacterial cellulose/nanotoliths (c). J. Biomed. Nanotechnol. 9, 1–8, 2013 Figure 5. Statistical analysis of stem cells from a deciduos tooth in the control group (C), bacterial cellulose (AP), bacterial cellulose/nanotoliths (A1). perform adhesion, though even this adhesion was statistically lower than in the control group mainly because of production method of biocomposite using fermentation roughness surface morphology were obtained, illustrated in Figure 4(b). With the addition of nanotoliths there was an agglomeration of nanotoliths on the surface of the scaffolds plus specific cell adhesion and localized punctually, which showed satisfactory results, as illustrated in Figure 4(c). The metabolic activity was assessed by measuring the activity of the mitochondrial enzyme succinate dehydrogenase (MTT assay), which is widely used in in vitro evaluation of cell viability.52–54 It was tested for analysis of cell performance over time, allowing for the monitoring of cell viability during the experimental periods. As can be seen in Figure 6, on the seventh day of culture, cell viability in the control group was statistically higher than in the bacterial cellulose nanocomposite and bacterial cellulose. The test groups did not differ among themselves. At 14 days, there was a statistical difference between the control and test groups, and cell viability of bacterial cellulose nanocomposites showed a lower value than the bacterial cellulose. At 21 and 28 days of culture, there was no difference in the viability of the three groups, with the performing groups testing similarly to the control group. This shows that, although the fibers prepared for the study do not provide an initial adhesion and maintenance of viability in the initial stage, they have the ability to promote a gradual increase in the long-term (21 days) since, after 21 days of culture, the test groups were presented in a manner similar to the control group and the standard for cell culture. Based in recent literature, there is another variables which has influence in adhesion and cells viability like cells dispersion.55 56 In this scope, natural scaffolds with bacterial cellulose and bacterial cellulose nanocomposites had lower cell adhesion at the beginning of the test; however, during the test their response was very positive, being a material extremely effective for bone and tissue regeneration in the body. 5
  • 6. Human Dental Pulp Stem Cell Behavior Using Natural Nanotolith/Bacterial Cellulose Scaffolds Olyveira et al. Figure 6. Viability cell essay over a time period of 7 days, 14 days, 21 days and 28 days in control group (C), bacterial cellulose (AP), and bacterial cellulose/nanotoliths (A1). CONCLUSION Pure Bacterial cellulose and bacterial cellulose nanocomposites showed great response with cell essays over time and these results show BC/BC nanocomposite as potential biomaterial for cell delivery applications, mainly because their natural properties and constitution are like the extracellular matrix. Their degradability, biocompatibility, low cost and intrinsic cellular interaction make them very attractive candidates for biomedical applications. However, a better controlled development in methods for production, fermentation and filler agglomeration control is essential for better surface morphology with higher adhesion and viability cells to widespread use of these scaffolds. However, another variables like adhesion and cells viability will be study in future work. Thus, undoubtedly, natural-origin polymers or nature-inspired materials appear as the natural and desired choice for medical applications. LIST OF ABBREVIATIONS Nanoskin—Bacterial cellulose produced by Innovatec’s— Biotechnology Research and Development—Brazil. BC—Bacterial cellulose. DMEM—Dulbecco’s Modified Eagle Medium. EDTA—Ethylenediamine Tetraacetic acid. ECM—Extracellular matrix. OTL—Otoliths supplied by Innovatec’s—Biotechnology Research and Development-Brazil. 6 Acknowledgments: This work was supported by grants from the National Council for Scientific and Technological Development (CNPq) and FAPERGS (Fundação de Apoio à pesquisa do Estado do Rio Grande do Sul) and Stem Cell Research Institute (SCRI). REFERENCES 1. F. P. Reinholt, K. Hultenby, A. Oldberg, and D. Heinegard, Osteopontin—A possible anchor of osteoclasts to bone. Proc. Natl. Acad. Sci. 87, 4473 (1990). 2. N. E. Waters, Some mechanical and physical properties of teeth. Symp. Soc. Exp. Biol. 34, 99 (1980). 3. A. S. Narayanan and R. C. Page, Connective tissue of the periodontium: A summary of current work. Collagen Rel. Res. 3, 33 (1983). 4. A. J. Fosang and T. E. Hardingham, Matrix proteoglycans, Extracellular Matrix, edited by W. D. Comper, Harwood Academic, Amsterdam (1996), pp. 200–229. 5. S. Pitaru, N. Savion, H. Hekmati, S. Olson, and S. A. Narayanan, Molecular and cellular interactions of a cementum attachment protein with periodontal cells and cementum matrix components. J. Periodontal. Res. 28, 560 (1993). 6. R. L. MacNeil, J. Berry, J. D’Errico, C. Strayhorn, and M. J. Somerman, Localization and expression of osteopontin inmineralized and nonmineralized tissues of the periodontum, Osteopontin: Role in Cell Signalling and Adhesion, Annals of the New York Academy of Sciences, New York, USA (1995), pp. 166–176. 7. S. Johansson, Non-collagenous matrix proteins, Extracellular Matrix, edited by W. D. Comper, Harwood Academic, Amsterdam (1996), pp. 68–94. 8. M. Van der Rest and R. Garrone, Collagen as multidomain proteins. Biochemie. 72, 473 (1990). J. Biomed. Nanotechnol. 9, 1–8, 2013
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