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Current Stem Cell Research & Therapy, 2014, 9, 00-00 1

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Novel Chemically Modified Bacterial Cellulose Nanocomposite as Potential Biomaterial for Stem Cell Therapy Applications

Gerson Arisoly Xavier Acasigua1, Gabriel Molina de Olyveira

2,*, Ligia Maria Manzine Costa

3,

Daikelly Iglesias Braghirolli4,5

, Anna Christina Medeiros Fossati1, Antonio Carlos Guastaldi

2,

Patricia Pranke4,5,6

, Gildásio de Cerqueira Daltro7 and Pierre Basmaji

8

1Post-graduate Program in Dentistry, Federal University of Rio Grande do Sul-UFRGS, Porto Alegre-RS, 90610-000,

Brazil; 2Department of Physical Chemistry- UNESP/Araraquara-SP, 14800-900, Brazil;

3Department of Nanoscience

and Advanced Materials- UFABC/Santo André-SP, 09210-170, Brazil; 4Hematology and Stem Cell Laboratory, Faculty

of Pharmacy, Federal University of Rio Grande do Sul- UFRGS, Porto Alegre-RS, 90610-000, Brazil.; 5Post-graduate

Program in Physiology, Federal University of Rio Grande do Sul-UFRGS , Porto Alegre-RS, 90610-000, Brazil; 6Stem

Cell Research Institute (SCRI), Porto Alegre-RS, 90610-000, Brazil; 7College Hospital Complex Prof. Edgard Santos

(COM-HUPES); UFBA, Salvador, 40110-910, Brazil; 8Innovatec's - Biotechnology Research and Development, São

Carlos-SP, 13560-042, Brazil

Abstract: Bacterial cellulose (BC) has become established as a remarkably versatile biomaterial and can be used in a

wide variety of applied scientific applications, especially for medical devices. In this work, the bacterial cellulose fermen-

tation process is modified by the addition of hyaluronic acid and gelatin (1% w/w) to the culture medium before the bacte-

ria is inoculated. Hyaluronic acid and gelatin influence in bacterial cellulose was analyzed using Transmission Infrared

Spectroscopy (FTIR) and Scanning Electron Microscopy (SEM). Adhesion and viability studies with human dental pulp

stem cells using natural bacterial cellulose/hyaluronic acid as scaffolds for regenerative medicine are presented for the

first time in this work. MTT viability assays show higher cell adhesion in bacterial cellulose/gelatin and bacterial cellu-

lose/hyaluronic acid scaffolds over time with differences due to fiber agglomeration in bacterial cellulose/gelatin. Confo-

cal microscopy images showed that the cell were adhered and well distributed within the fibers in both types of scaffolds.

Keywords: Bacterial cellulose, cell viability study, Nanoskin®

, natural nanocomposites, regenerative medicine, stem cells.

1. INTRODUCTION

Gluconacetobacter xylinus (bacterial cellulose, BC) is an emerging biomaterial with great potential in several applica-tions due its high purity, ultra-fine network structure and high mechanical properties in dry state [1]. These features allow its application as scaffolds for tissue regeneration, medical applications and nanocomposites. Some studies have used bacterial cellulose mats to reinforce polymeric matrices and scaffolds with wound healing properties. BC is a natural cellulose produced by bacterial synthesis by biochemical steps and self-assembling of the secreted cellulose fibrils on the medium. Shaping of BC materials in the culture medium can be controlled by the type of cultivation that changes chain size, origin of strains which produces different propor-tions of crystalline phase of BC and the kind of bioreactor. BC hydrogel or BC in dry state is then obtained by methods, such as freeze-drying [2]. Although chemically identical to plant cellulose, the cellulose synthesized by the bacteria has a fibrillar nanostructure, which determines its physical and mechanical properties, necessary characteristics for modern medicine and biomedical research [3]. The structural features of microbial cellulose, its properties and compatibility as a

*Address correspondence to this author at the Department of Physical

Chemistry- UNESP/Araraquara-SP, 14800-900, Brazil;

Tel: (55)1149012998; E-mail: [email protected]

biomaterial for regenerative medicine can be changed by modifying its culture medium [4] or surface modification by physical [5, 6]; chemical methods [7] and genetic modifica-tions [8] to obtain a biomaterial with less rejection when in contact to the cell and cell interaction.

Different gelatin formulations have been studied to evaluate the drug loading capacity and release rate. Like other hydrogels, drug release profiles obtained from gelatin hydrogels can be readily adjusted by changing the network cross-linking density. Because gelatin has a sol-gel transition temperature around 30ºC, it should be cross-linked chemi-cally to avoid dissolution at body temperature [9]. Gelatin nanofibers play a dominant role in maintaining the biological and structural integrity of various tissues and organs, includ-ing bone, skin, tendon, blood vessels and cartilage. There are several commercially available gelatin based carriers for drug delivery that are being applied in tissue engineering [10]. Physical and chemical permeation enhancers can be used in conjunction with cellulose bacterial membrane (Nanoskin

®) to affect the desired level of delivery. Early

results also showed that hyaluronic acid (HA) was effective in protecting retinal damage during ophthalmic surgery, re-ducing scarring, preventing post-operative adhesions and reducing pain while increasing mobility in arthritic joints [11]. In addition, HA also provides important structural sup-

2 Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2 Acasigua et al.

port to the extracellular matrix (ECM). Hyaluronan-binding proteins, called hyaladherins, mediate its interaction with various extracellular components, including proteoglycans, collagen and fibrin, which stabilizes both HA and ECM [12].

However, the success of the scaffolds 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 [13, 14].

The interaction of cells with the surfaces of materials is of extreme importance for the effectiveness of medical im-plants [15] and may define the degree of acceptance or rejec-tion. Knowledge of basic mechanisms of cell-material inter-action 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 [16]. Cells identify the exposed surface topography and nanofiber features, such as porous matrices and align-ment, influence the adhesion, spreading, proliferation and gene expression of the cells seeded onto them.

Stem cells are a non-specialized cell type which can self-renew and remain for a long period of time with the potential to produce different cell lineages or tissues with specialized functions. Tissue-specific stem cells, or adult stem cells, have been considered as an alternative for the use of embry-onic stem cells, due to their availability, ease of acquisition and growth in comparison with mesenchymal stem cells from bone marrow [17]. ESCs also encounter some problems related to safety issues such as ESC rejection, the risk of tumorigenicity and government moratorium, which has re-stricted research on this type of cell [18, 19]. Mesenchymal stem cells have thus been studied as a group of cells with plasticity similar to embryonic stem cells; this has been the target of numerous researchers.

Several nanocomposites were tested specifically with stem cell adhesion and differentiation. Cell adherence and proliferation ability of hBMSCs (human bone mesenchymal stem cells) on scaffolds were improved by coating with HA/PLLA nanocomposites [20]. Another study investigated the effects of nanophase hydroxyapatite (nano-HA), nano-HA/poly(lactide-co-glycolide) (PLGA) composites and a small peptide derived from bone morphogenetic protein (BMP-7) on osteogenic differentiation of hMSCs (Human mesenchymal stem cells). The nanocomposites provided promising alternatives in controlling the adhesion and differ-entiation of hMSCs without osteogenic factors from the cul-ture media and,thus, should be further studied for clinical translation and the development of novel nanocomposite-guided stem cell therapies [21].

The use of poly(L-Lactide) and nanohydroxiapatite was studied in another work. The culture of bone mesenchymal stem cells indicated that the composite nanofibrous poly(L-lactide) scaffold with 50 wt % nanohydroxyapatite showed the highest cell viability among various poly(L-lac-tide)-based scaffolds [22] Other nanocomposites and electrospun nanocomposites succeeded in testing the interaction with cells [23-26]. Besides this, collagen-based nanocomposites incorporating nanobioactive glass (Col/nBG) were developed as a scaffold matrix for dentin–pulp regeneration. The effects of the novel matrix on the proliferation of human dental pulp

cells (hDPCs) and their differentiation into odontoblastic lineage were investigated. In conclusion, the nanocomposite Col/nBG matrix induced the growth and odontogenic differ-entiation more effectively than Col alone, providing a prom-ising scaffold condition for regeneration of dentin–pulp complex tissue [27].

It is of particular interest that stem cells from deciduous teeth have certain advantages. Miura and colleagues attrib-uted to these cells significantly greater potential for prolif-eration and clonogenicity when compared to pulp stem cells from permanent teeth and stem cells from bone marrow [28].

In this work, novel studies of natural nanocomposites with bacterial cellulose for functional dental materials is re-ported. In order to produce scaffolds with drug delivery abil-ity, porous structure and better cell adhesion, fermentation changes in bacterial cellulose with gelatin and hyaluronic acid were performed. A link has been established between fermentation, morphological surface and cell attachment.

2. MATERIALS AND METHODS

2.1. Materials

The bacterial cellulose raw material (Nanoskin) was pro-vided from Innovatec´s (São Carlos SP, Brazil). Gelatin from porcine skin and hyaluronic acid sodium salt from Streptococcus equi (bacterial glycosaminoglycan polysaccharide) were purchased from Sigma Aldrich.

2.2. Methods

2.2.1. Synthesis of Bacterial Cellulose/Gelatin and

Hyaluronic Acid

The acetic fermentation process was achieved by using glucose as a carbohydrate source. Results of this process are vinegar and a nanobiocellulose biomass. The modifying process is based on the addition of hyaluronic acid and gela-tin (1% w/w) to the culture medium before the bacteria is inoculated. After being added to the culture medium, the medium is autoclaved at 100

oC degree. Bacterial cellulose

(BC) is produced by Gram-negative bacteria Gluconaceto-bacter xylinus, which can be obtained from the culture me-dium in the pure 3-D structure, consisting of an ultra fine network of cellulose nanofibers [29].

2.2.2. Bionanocomposite Preparation

In the present study, a novel biomaterial has been ex-plored and different bacterial cellulose nanocomposites have been prepared; 1) BC/hyaluronic acid, 2) BC/gelatin.

2.3. Samples of Pulp Tissue from Deciduous Teeth

In order to isolate the cells from the pulp tissue and es-tablish their culture, dental pulp was removed from decidu-ous teeth in the resorption process. After extraction, the teeth was immersed in 1 mL culture medium DMEM/Hepes (Sigma Aldrich), 10 % fetal bovine serum (GIBCO), 100U/mL penicillin, 100μg/mL streptomycin (Gibco) and 0.45μg/mL gentamicin (Gibco) at room temperature for transport to the laminar flow. Those responsible for the pa-tient signed a consent form approved by the Ethics Commit-tee of the Federal University of Rio Grande do Sul, registra-tion number 19273.

Novel Chemically Modified Bacterial Cellulose Nanocomposite Current Stem Cell Research & Therapy, 2014, Vol. 9, No. 2 3

2.3.1. Cell Culture

The handling of the pulp tissue removed was performed following the protocol established in the laboratory [30]. Cell suspension in the culture medium was seeded onto a 12 well culture plate and then incubated at 37˚C in a humidified at-mosphere 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 con-fluence of approximately 90% when it was then held in its first passage. The cells in culture were harvested with tryp-sin-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 5

th 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 perfomed as follows.

2.4. Characterization

2.4.1. 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.

2.4.2. Transmission Infra-Red Spectroscopy (FTIR, Perkin

Elmer Spectrum 1000)

Influences of hyaluronic acid and gelatin in bacterial cel-lulose were analyzed in the range between 250 and 4000 cm

-

1 and with 2 cm

-1 resolution with samples.

2.4.3. Cell Viability

For the study of cell viability during the 28 days of cul-ture, as performed for the cell adhesion essay, the cells were seeded onto each type of scaffold in triplicate and then incu-bated at 37˚C in a humidified atmosphere of 5% CO2. To collect the initial viability of the seeded cells, the viability of 5 x 10

4 cells was analyzed, 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 tre-

tazolyum method, a colorimetric assay using bromide 3 - (4,5 - dimethylthiazol -2-yl ) -2,5 – diphenyltetrazolium bromide (MTT). After the experiment time, the culture me-dium was removed and 200 L MTT solution (0.25 mg/mL) was added and maintained for 2 hours. The MTT was then removed and 200μL 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 ana-lyzed by a spectrophotometer (Wallac EnVision - Perkin Elmer). The data was calculated using the difference in ab-sorbance 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 main-tained by the same experimental period and the same proce-dures for data collection were performed.

Cell Adhesion - 5 x 10 4

cells in 50 μL 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 non-adherent cells on the scaffolds. The cells attached to the scaffolds were then fixed with 4 % paraformaldehyde for 20 minutes. Following this, staining was performed with 0.5 mg/mL 4,6-diamidino-2-phenylindole (DAPI), a fluorescent marker which binds strongly to DNA. Confocal microscopy images were then obtained, corresponding to different randomly distributed microscopic fields.

3. RESULTS AND DISCUSSION

3.1. Bacterial Cellulose Nanocomposite Mats

Bacterial cellulose nanocomposite mats were character-ized by SEM. Fig. (1a and 1b) and Fig. (2a and 2b) show SEM images of bacterial cellulose/hyaluronic acid and bacte-rial cellulose/gelatin nanocomposites, respectively. Different morphological surface with gelatin and hyaluronic acid addi-tion to fermentation medium can be observed. A connection has been established between fermentation changes and bac-terial cellulose biosynthesis to explain such behavior.

Bacterial cellulose contains a thin peptidoglycan layer adjacent to the cytoplasmic membrane. In addition, it con-tains an outer membrane composed of phospholipids and lipopolysaccharides with invaginations of the cell membrane,

Fig. (1). (a, b) Scanning electron microscopy (SEM) of bacterial cellulose/hyaluronic acid.


which can be either simple folds, such as vesicular or tubular structures. Several functions have been observed, such as the role of bacterial cellulose in cell division and respiration of the bacteria .In general, aerobic respiration uses glucose more efficiently to produce energy (i.e. electrons), when compared to the fermentation process for energy production [31, 32].

It is believed that the tubular fibrils become positioned within tunneling distance of the cofactors with little conse-quence to denaturation. The combination of symbioses with redox active enzymes would appear to offer an excellent and convenient platform for a fundamental understanding of bio-logical redox reactions [33].

The biosynthesis of the sugar in cell structures begins by the synthesis of the units of sugar nucleotides. The supply of the sugars for the biosynthesis is dependent on the intracellu-lar sugar nucleotide levels that are influenced by the activi-ties of the intracellular enzymes involved in their biosynthe-sis [34].

The size of the cellulose molecules is normally expressed in terms of their polymerization degree (PD), this is the number of anhydroglucose units present in a chain. How-ever, the conformational analysis of cellulose indicates that cellobiose (4-O- -D-glucopyranosyl- -D-glucopyranose) is its basic structural unit [35]. The conformation of the repeat-ing unit of cellulose can be explained if the model proposed for the biosynthesis of glucose is considered [36].

The active site of the enzyme cellulose synthase, respon-sible for the synthesis of cellulose, contains two consecutive sites for binding to the uridine-diphosphoglucose precursor (UDP-glucose), positioned at 180°C from each other and a binding site at the non-reducing end of the -glucan. The hydroxyl at C-5 of glucose residues linked to these sites is activated by a mechanism of general base catalysis by pro-moting the dephosphorylation of UDP-glucose units and establishing new links (1-4). The resulting -glucan, which has little affinity with the binding sites for UDP-glucose, moves to the binding site for -glucan, which is a better loca-tion. The synthesis can then be continued with the addition of two new units of UDP-glucose [35].

Bacterial cellulose has cell division and DNA synthesis within the cells. DNA is organized into long structures called

chromosomes. During cell division these chromosomes are duplicated in the process of DNA replication, providing each cell with its own complete set of chromosomes, it will then subsequently assemble the -1,4-glucan chains outside the cell in a precise, hierarchical process. The production of cel-lulose characterizes this bacteria both in liquid medium, where it produces a thick pellicle on the air–liquid interface, and isolated cells where a cellulose ribbon can be clearly seen attached to the long side of the cell [35].

In this scope, hyaluronic acid structure is similar to bacte-rial cellulose and may derive from Streptococcus bacterium. Biosynthesis with bacterial cellulose and hyaluronic acid was then produced more successfully in fibers structure than bac-terial cellulose/gelatin, as observed in Figs. (1 and 2).

3.2. Interaction Between Bacterial Cellulose with Hyaluronic Acid and Gelatin

Influences of gelatin and hyaluronic acid in bacterial cel-lulose were analyzed in the range between 250 and 4,000 cm

-

1 and with resolution of 2 cm

-1 with FTIR analysis. The main

features of the bacterial cellulose in infrared spectroscopy is: 3,500 cm

-1:OH stretching, 2,900 cm

-1:CH stretching of al-

kane and asymmetric CH2 stretching, 2,700 cm-1

:CH2 sym-metric stretching, 1,640 cm

-1:OH deformation, 1,400 cm

-

1:CH2 deformation, 1,370 cm

-1:CH3 deformation, 1,340 cm

-

1:OH deformation and 1,320-1,030 cm

-1:CO deformation

[37].

It can be observed in Figs. (3 and 4) that the intensity of transmittance of the bio nanocomposites is smaller than that of bacterial cellulose, which means that exposed groups on the bacterial cellulose molecules are interacting with other components and in comparison, hyaluronic acid is interact-ing more than gelatin with bacterial cellulose (Fig. 5). More OH stretching (at 2,900 cm

-1) can be observed, in bacterial

cellulose/hyaluronic nanocomposites than with gelatin com-posites, mainly because of NH2 interaction with hydroxyl groups (Fig. 5). Besides this, changes can be observed in the symmetrical stretching of CH2 bonds of bacterial cellulose structures in the absorption peak of 1,640cm

-1. Another ab-

sorption peak was obtained in the range of 1,490 cm 1 in both samples, which shows the presence of a carbonyl group in the bacterial cellulose together with bonds corresponding

Fig. (2). (a, b) Scanning electron microscopy (SEM) of bacterial cellulose/gelatin.


to those of glycoside, including C–O–C at 1,162 cm1 (as in

the case of natural cellulose) [38]. These results clearly show one possible interaction between bacterial cellulose and gela-tin/hyaluronic acid, mainly by hydrogen interactions between hydroxyl and carbonyl groups.

Fig. (3). FTIR spectra of bacterial cellulose/gelatin nanocomposites.

Fig. (4). FTIR spectra of bacterial cellulose/hyaluronic acid.

Fig. (5). FTIR spectra of bacterial cellulose/hyaluronic acid and

bacterial cellulose/gelatin.

3.3. Cell Adhesion and Viability

The cells used in this study were characterized as mesen-chymal stem cells, keeping the profile positive for CD29, CD44 and CD90 (>95%) and with a low expression of CD34, CD45, CD146, STRO-1 and HLA-DR (<2%), show-ing ability to differentiate into adipocytes, osteoblasts and chondrocytes.

For the successful application of scaffolds in tissue engi-neering, a crucial feature is that the matrices promote cell adhesion. According to Andrews and colleagues [39], cell adhesion is mediated by the adsorption of extracellular ma-trix proteins produced by cells on the surface of the scaffold. The signalling pathways are then activated and cell adhesion occurs in the mould by means of receptors. Therefore, ac-commodation and cell behavior is strongly affected by the structure of the scaffolds and cell adhesion assay becoming important in order to determine whether the scaffolds have a good structure for the initial interaction with cells.

The metabolic activity was assessed by measuring the ac-tivity of the enzyme mitochondrial succinate dehydrogenase (MTT assay), which is widely used in in vitro evaluation of cell viability [40, 41]. It was tested for analysis of cell per-formance over time, allowing the monitoring of cell viability during the experimental period.

Through the MTT assay, it was found that after 1 day of experiment, the three groups (samples I and II and control) showed similar behavior, with no statistical difference. At seven days, similar behavior was observed between the groups, with no statistical difference. It was noted that the absorbance increased over the first day in three groups, indi-cating that the proliferation was similar between the two nanocomposites and the control group. On the 15

th day, a

statistically significant difference in the absorbance was ob-served between the test groups and the control group. The control group had an absorbance greater than the two test groups, indicating greater proliferation of cells in the control group. The test groups maintained with similar absorbance until the seventh day, indicating that the number of cells re-mained constant from the seventh to the fifteenth day of cul-ture - which is nonetheless a good result (Fig. 6a). This shows that the fibers prepared for the study provide an initial adhesion and increase of viability in the initial stage and that they have ability to promote maintenance of viability in the long-term.

It can, therefore, be concluded that gelatin nanocompo-sites have lower cell adhesion over time because of the fiber agglomeration during bacterial cellulose biosynthesis Based on recent literature, there are other variables which have in-fluence on adhesion and cell viability, such as cell dispersion [42] and different types of stem cell cultured on poly-D-lysine, poly-L-lysine and collagen, in order to improve cell differentiation [43]. Besides this, factors such as the deposi-tion of apatite using SBF (simulated body fluid) will be tested in future experiments [44, 45].

Through images obtained by confocal microscope, it can be observed that 1 day after the cells had been seeded onto the scaffolds, they were distributed in a homogenous manner over the entire scaffold structure. By applying digital zoom on the images, it was possible to observe that the cells ad-hered on the nanocomposite surface and exhibited a spread


morphology in the red colored area in Figs. (6b and 6c). Confocal images in Fig. (6b), bacterial cellulose/hyaluronic acid and Fig. (6c), bacterial cellulose/gelatin, confirmed that after 1 day in culture the cells assumed a fusiform aspect, showing that the surface of nanocomposites promote and favor cell adhesion and development. These results demon-strate the quality of the nanocomposites and how they allow cell adhesion and maintenance in a similar way to the culture well plates.

Fig. (6a). Cell viability assay over a time period of 1, 7 and 15days

in bacterial cellulose/hyaluronic acid (sample I), bacterial cellu-

lose/gelatin (sample II) and control group.

Figs. (6). Confocal microscopy images obtained to check cell adhe-

sion and morphology:(b) bacterial cellulose/hyaluronic acid; (c)

bacterial cellulose/gelatine

Furthermore, it can be observed that fermentation pro-duced different surface morphologies with the addition of hyaluronic acid/gelatin and there was more agglomeration of the fiber formation process in the bacterial cellulose/gelatine than the bacterial cellulose/hyaluronic acid, which has a det-rimental effect on cellular adhesion, as illustrated in Fig. (6c).

CONCLUSION

Bacterial cellulose was successfully modified by chang-ing the fermentation medium as shown with SEM and FTIR, which produced scaffolds with different surface morphology but similar cell adhesion and attachment. Natural scaffolds with bacterial cellulose and bacterial cellulose nanocompo-sites had good cell adhesion over time between tested sam-ples, being an extremely effective material for tissue regen-eration. Such nanocomposites present adhesion behavior similar with that which can be seen in the literature. How-ever, a better controlled development in methods for produc-tion, purification and surface morphology is essential for widespread use of these scaffolds.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGEMENTS

Nanoskin – Bacterial cellulose produced by Innovatec's - Biotechnology Research and Development- Brazil.

LIST OF ABBREVIATIONS

BC = Bacterial cellulose.

DMEM = Dulbecco´s Modified Eagle Medium

ECM = Extracellular matrix

EDTA = Ethylenediamine Tetraacetic acid

ESC = Embryonic stem cell

REFERENCES

[1] Olyveira GM, Acasigua GA, Costa LM, et al. Human dental pulp

stem cell behavior using natural nanotolith/bacterial cellulose scaf-folds for regenerative medicine. J Biomed Nanotechnol 2013; 9: 1-

8. [2] Olyveira GM, Costa, LMM, Basmaji P, Filho LX. Bacterial Nano-

cellulose for medicine regenerative. J Nanotech Eng Med 2011; 2(3): 34001-9.

[3] Xavier Filho L, Olyveira GM, Basmaji P, Costa LM. Novel elec-trospun nanotholits/PHB scaffolds for bone tissue regeneration. J

Nanosci Nanotechnol 2013; 13(7): 4715-9. [4] Costa LMM, Olyveira GM, Basmaji P, Filho LX. Bacterial cellu-

lose towards functional medical materials. J Biomater Tissue Eng 2012; 2: 185-96.

[5] Olyveira GM, Costa, LMM, Basmaji P. Physically Modified Bacte-rial Cellulose as Alternative Routes for Transdermal Drug Deliv-

ery. J. Biomater. Tissue Eng 2013; 2(3): 1-6. [6] Costa LMM Olyveira GM Basmaji P Filho LX. Nanopores struc-

ture in electrospun bacterial cellulose. J Biomater Nanobiotech 2012; 3: 92-6.

[7] Cherian BM, Olyveira GM, Costa LMM, Leão AL, Souza SF. Protein Based Polymer Nanocomposites for Regenerative Medi-

cine. Royal Society of Chemistry (RSC Green Chemistry), Edited by John J Maya and Thomas Sabu,No. 17,Natural Polymers, Vol-

ume 2: Nanocomposites 2012; 255-293.


[8] Gois PBP, Olyveira GM, Costa LMM, et al. Influence of symbio-

ses culture between microorganisms/ yeast strain on cellulose syn-thesis. Int Rev Biophys Chem 2013; 3(3): 48-54.

[9] Kuijpers AJ, Engbers GHM, Krijgsveld J. Cross-Linking and char-acterization of gelatin matrices for biomedical applications. J Bio-

mater Sci Polym Ed 2000; 11(3): 225-43. [10] Ponticiello MS, Schinagl RM, Kadiyala S. Gelatin based resorbable

sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res 2000; 52: 246-

55. [11] Denlinger JL, Balazs EA. Replacement of the liquid vitreus with

sodium hyaluronate in monkeys. I. Short-term evaluation. Exp Eye Res 1980; 31: 81-99.

[12] Toole, B.P. Hyaluronan in morphogenesis. Semin Cell Dev Biol 2001; 12: 79-87.

[13] Dee KC, Andersen TT, Bizios R. Design and function of novel osteoblast-adhesive peptides for chemical modification of biomate-

rials. J Biomed Mater Res 1998; 40: 371-7. [14] Lauffenburger DA, Horwitz AF. Cell migration: A physically in-

tegrated molecular process. Cell, 1996,84: 359-69. [15] Toole BP, Linsenmayer TF. Newer knowledge of skeletogenesis:

macromolecular transitions in the extracellular matrix. Clin Orthop Relat Res 1977; 129: 258-78.

[16] Teixeira AI, .Nealey PF, Murphy CJ. Responses of human kerato-cytes to micro- and nanostructured substrates. J Biomed Mater Res

2004; 71: 369-76. [17] Seo BM, Miura M, Gronthos S, et al. Investigation of multipotent

postnatal stem cells from human periodontal ligament. Lancet 2004; 364: 149-55.

[18] Wang, HS, Hung,SC, Peng,ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord. Stem Cells 2004;

22(7): 1330-7. [19] Bunting KD, Hawley RG. Integrative molecular and developmental

biology of adult stem cells. Biol Cell 2003; 95: 563-78. [20] Nie L, Chen D, Yang Q, et al. Hydroxiapatite/poly-L-lactide nano-

composites coating improves the adherence and proliferation of human bone mesenchymal stem cells on porous biphasic calcium

phosphate scaffolds. Materials lett 2013; 92: 25-8. [21] Lock J, Nguyen TY, Liu H. Nanophase hydroxiapatite and

poly(lactide-co-glycolide) composites promote human mesenchi-mal stem cell adhesion and osteogenic differentiation in vitro.

J.Mater sci Mater Med 2012; 23: 2543-52. [22] Han W, Zhao J, Tu M, Zeng R, Zha Z, Zhou C. Preparation and

characterization of nanohydroxiapatite strengthening nanofibrous Poly(L-lactide) scaffold for bone tissue engineering. J Appl Polym

Sci 2012; 128(3): 1332-8. [23] Hung HS, Tang CM, Lin CH, et al. Biocompatibility and favorable

response of mesenchymal stem cells on fibronectin-gold nanocom-posites. PLoS One 2013; 8(6): e65738.

[24] Hild N, Fuhrer R, Mohn D, et al. Nanocomposites of high density polyethylene with amorphous calcium phosphate: in vitro biomin-

eralization and cytocompatibility oh human mesenchymal stem cells. Biomed Mater 2012; 7: 054103-10.

[25] Lü LX, Zhang XF, Wang YY, et al. Effects of hydroxyapatite-containing composite nanofibers on osteogenesis of mesenchymal

stem cells in vitro and bone regeneration in vivo. ACS Appl. Mater Interfaces 2013; 5: 319-30.

[26] Zhou C, Shi Q, Guo W, et al. Electrospun Bio-nanocomposite scaffolds for bone tissue engineering by cellulose nanocrystals rein-

forcing maleic anhydride grafted PLA. ACS Appl Mater Interfaces 2013; 5: 3847-54.

[27] Bae WJ, Min KS, Kim JJ, Kim JJ, Kim HW, Kim EC. Odontogenic

responses of human dental pulp cells to collagen/nanobioactive glass nanocomposites. Dent Mater 2012; 28(12): 1271-9.

[28] Miura M, Gronthos S, Zhao M, et al. SHED: stem cells from hu-man exfoliated deciduous teeth. Proc Natl Acad Sci USA 2003,

100: 5807-12.

[29] Costa LMM, Olyveira GM, Basmaji P, Filho LX. Bacterial Cellu-

lose Towards Functional Green Composites Materials. J Bionanos-cience 2011; 5: 167-72.

[30] Bernardi L, Luisi SB, Fernandes R, et al. The isolation of stem cells from human deciduous teeth pulp is related to the physiologi-

cal process of resorption. J Endod 2011; 37(7): 973-9.

[31] Barbara PF, .Meyer TJ, Ratner MA.. Contemporary Issues in Elec-

tron Transfer Research. J Phys Chem 1996; 100(31): 13148-68. [32] Dong, S.J.and Wang, B.Q.Electrochemical Biosensing in Extreme

Environment. Electroanalysis 2002; 14: 7-16. [33] Gilardi, G and Fantuzzi, A. Manipulating redox systems: applica-

tion to nanotechnology. Trends Biotechnol 2001; 19: 468-76. [34] Koyama M, Helbert W, Imai T, Sugiyama J, Henrissat B. Parallel-

up structure evidences the molecular directionality during biosyn-thesis of bacterial cellulose. Proc Natl Acad Sci USA 1997; 94:

9091-5. [35] Cherian, B.M; Leão, A.L; Souza, S.F, Olyveira, G.M; Cos-

ta;L.M.M; Brandão;C.V.S Narine, S.S.Bacterial nanocellulose for medical implants. Springer Berlin Heidelberg, Advances in Natural

Polymers, Advanced Structured Materials, Edited by S.Thomas, P.M.Visakh, A.P.Mathew, 2013; 337-359.

[36] Zugenmaier, P. Conformation and packing of various crystalline cellulose fibers. Prog Polym Sci 2001; 26: 1341-417.

[37] Zhbanko RG. Infrared Spectra of Cellulose and Its Derivates. Ed-ited by B.I. Stepanov. Translated from the Russian by A.B. Den-

sham. Consultants Bureau:New York 1966; 325-33. [38] Kacuráková M, Smith AC, Gidley MJ, Wilson RH. Molecular

interactions in bacterial cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR spectroscopy. Carbohydr Res 2012; 337:

1145-53. [39] Andrews KD, Hunt,JA, Black RA. Effects of sterilisation method

on surface topography and in-vitro cell behaviour of electrostati-cally spun scaffolds. Biomaterials 2007; 28: 1014-26.

[40] Leong NL, Jiang,J, Lu, HH. Polymer–ceramic composite scaffold induces osteogenic differentiation of human mesenchymal stem

cells. Conf Proc IEEE Eng Med Biol Soc 2006; 1: 2651-2654. [41] Saad B, Abouatta BS, Basha W, et al. Hypericum triquetrifolium—

Derived Factors Downregulate the Production Levels of LPS-Induced Nitric Oxide and Tumor Necrosis Factor-a in THP-1 Cells.

Evid Based Complement Alternat Med 2008; 14: 1-7. [42] Williams SJ, Wang Q, Macgregor RR, Siahaan TJ, Stehno-Bittel L,

Berkland C. Adhesion of Pancreatic Beta Cells to Biopolymer Films. Biopolymers 2009; 91(8): 676-85.

[43] Qian L, Saltzman WM. Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture surface

modification. Biomaterials 2004; 25: 1331-7. [44] Kim HW, Song JH, Kim HE. Bioactive glass nanofiber-collagen

nanocomposite as a novel bone regeneration matrix. J Biomed Ma-ter Res A 2006; 79: 698-705.

[45] Six N, Tompkins K, Septier D, Veis A, Goldberg M. Recruitment and characterization of the cells involved in reparative dentin for-

mation in the exposed rat molar pulp after implantation of amelo-genin gene splice products A + 4 and A 4. Oral Biosci Med 2004;

1: 35-44.

Received: July 20, 2013 Revised: November 4, 2013 Accepted: November 14, 2013

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