Bio-Medical Materials and Engineering 25 (2015) S121–S135 DOI 10.3233/BME-141231 IOS Press
S121
Fabrication, characterization and cell cultures on a novel chitosan scaffold Zheng Guan a,b , Songtao Shi c , Buncha Samruajbenjakun a and Suttatip Kamolmatyakul a,∗ a
Prince of Songkla University, Preventive Dentistry, Hadyai, Songkhla, Thailand Biomedical Research Center, The First People’s Hospital of Kunming, Yunnan, China c University of Southern California, Center for Craniofacial Molecular Biology, Los Angeles, CA, USA b
Abstract. Chitosan has been used as scaffolds with various methods of fabrication including expensive commercial available ones for tissue engineering. The objective of this study is to assemble our novel method of chitosan scaffold fabrication in economical and uncomplicated way that suitable for dental pulp stem cell (DPSC) and stem cells of human exfoliated deciduous teeth (SHED). Chitosan scaffolds (2% and 3%) were fabricated in an uncomplicated procedure, including centrifugation and freeze-drying steps. The chitosan scaffolds were compared and the pore size, swelling and degradation were assessed. In addition, the cytocompatibility was assessed of chitosan scaffolds seeded with DPSC and SHED. The pore size of 2% and 3% chitosan scaffolds were similar being 188.71 ± 51.90 µm and 195.30 ± 67.21 µm, respectively. Swelling ratios of 3% chitosan scaffolds were significantly lower than those of 2% chitosan scaffolds. Dimension of scaffolds changed in first 5 minutes. After that, those scaffolds could maintain their dimension. Chitosan scaffolds degraded as from day 7. No differences were found between 2% and 3% chitosan scaffolds. The scaffolds were shown to be non-toxic and to promote DPSCs and SHED growth. The viability of DPSCs and SHED on 2% scaffolds proved to be higher than that of the 3% scaffold group. This study suggested that chitosan scaffolds fabricated with our novel method were suitable for the growth and survival of DPSC and SHED. Keywords: Biocompatibility, cell culture, chitosan scaffold, dental-derived stem cell
Abbreviations DPSC SHED DD Mw FBS PBS SBF
dental pulp stem cell; stem cells of human exfoliated deciduous teeth; deacetylation degree; molecular weight; fetal bovine serum; phosphate buffered saline; simulated body fluid.
1. Introduction Human dental pulp stem cell (DPSCs) [1] and stem cells of human exfoliated deciduous teeth (SHED) [2] are considered to facilitate pulp tissue engineering. Stem cells play an important role in regenerating * Address for correspondence: Associate Professor Suttatip Kamolmatyakul, D.Sc.D., Preventive Dentistry, Faculty of Dentistry, Prince of Songkla University, Hadyai, Songkhla, 90112, Thailand. Tel.: +66 74 287510, Fax: +66 74 212922; E-mail: [email protected].
0959-2989/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved
S122
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
new tissue [3]. Frequently stem cells are seeded on a scaffold prior to their use. As a carrier, different types of scaffolds can be selected for tooth engineering. Several scaffolds have been tested for regeneration of bone, and these scaffolds are possibly also suitable for regeneration of dental tissue. However, regenerating dental tissue is not the same as regenerating bone. Pulp in the canal space has its specific locations; therefore, any scaffold system that is osteo-inductive such as hydroxyapatite and tricalcium phosphate is in fact not appropriate for pulp regeneration [4]. On the other hand, when pulp tissue engineering is used in vital pulp therapy, the material should possess preferably anti-microbial properties and should be shaped easily in order to fit the pulp space. Natural occurring materials such as collagen and chitosan have a good biocompatibility, and these materials can be shaped in any configuration. Collagen-based scaffolds have been studied with DPSC, and they were shown to support cell growth and differentiation in vivo [5]. Yet, the collagen scaffold used did not have anti-microbial properties, and in general collagen scaffolds often loose shape and size because of a fairly rapid degradation when contacted with body fluid [6]. Huang and coworkers reported a considerable contraction of the collagen scaffold by pulp cells; the scaffold decreased to half of its original size by 3–15 days [7]. In other words, three-dimensional space of collagen scaffold will decrease which may result in loss of space for pulp cells to proliferate. Therefore, a collagen matrix may not be a suitable scaffold for pulp tissue engineering [7]. There is, however, an alternative: chitosan. Chitosan is a natural polymer, is obtained from shell of shellfish, and the wastes of the seafood industry, and it is a deacetylated derivative of chitin. Chitosan scaffolds possess some special properties for application in tissue engineering. First, it can be easily molded in any form [8]. Second, it possesses excellent properties to form porous structures [9]. The microstructure of porous chitosan scaffolds allows swelling of the material, which stimulates cell attachment, easy nutrient supply and a proper 3-D structure. Third, the cationic nature of chitosan provides the means to bind cytokines and growth factors. This property is beneficial for tissue engineering purposes [10]. Another distinct property of chitosan is its antibacterial activity against a broad spectrum of bacteria [11]. Fourth, the degradation rate of the chitosan can be manipulated by the incorporation of deacetylation degree (DD) and by varying its molecular weight (Mw). Degradation has been shown to increase as DD decreases. Moreover, high Mw contribute to prolonged degradation [12]. Provided the many advantages of chitosan, scaffolds of this material were studied in various tissue engineering applications such as skin, bone, cartilage, liver, nerve and blood vessel [11]. We assume that such chitosan scaffolds are suitable also for vital pulp therapy and the restoration of dental structure. Therefore, we analyzed in the present study the cytocompatibility of dental pulp-derived stem cells seed on different types of chitosan scaffolds. 2. Materials and methods 2.1. Cell culture Primary and permanent teeth were collected under the approved guideline of the Ethical Committee of Prince of Songkla University. Human primary exfoliated teeth were collected from 6 to 12-year-old children (n = 6). Informed consent was obtained from the parents. Permanent teeth were obtained from adult donors (29 years old, n = 6). Impacted third molars and bicuspids were extracted due to orthodontic considerations. All of these teeth contained a normal healthy pulp and were confirmed by clinical and radiographic examination. SHED and DPSC were isolated as described by Gronthos et al. [1]. Tooth surfaces were cleaned by 70% alcohol and cut around the cementum–enamel junction by using sterilized dental fissure burs to
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S123
reveal the pulp chamber. The pulp tissue was gently separated and minced. The minced pulp tissues were digested in a mixture of 3 mg/ml collagenase type I and 4 mg/ml dispase (Dissolved in PBS, Sigma, St. Louis, Mo., USA) for 30–60 min at 37°C water-bath. Cell suspensions were obtained by passing the digested tissues through a 70 µm cell strainer (Becton/Dickinson, Franklin Lakes, NJ, USA). Single cell suspensions were seeded in 100 mm culture plates (Nunc, Denmark) containing DMEM (Life Technologies/GIBCO BRL) supplemented with 20% fetal bovine serum (FBS, Biochrom AG, Germany), 2 mM L-glutamine (Gibco Invitrogen, USA), 100 U/ml penicillin-G, 100 µg/ml streptomycin, 50 U/ml mycostatin and 100 µg/ml kanamycin and maintained under 5% CO2 at 37°C. Those cells were allowed to grow for 10 to 12 days, and then were collected by assessing their colony-forming efficiency (CFU-F) [13]. Cells aggregated in groups of less than 50 cells were scratched from the bottom of the plate and removed by phosphate buffered saline (PBS). The other colonies (50 cells) were transferred to T-75 cultural flasks (TPP, Switzerland) and were cultured up to 70–80% confluence, and then they were passaged at 1 : 3 ratios for experiment or storage. Cells at passage 3–5 were used in the experiments. 2.2. Fabrication of chitosan scaffolds The chitosan (Sea Fresh Chitosan (Lab) Co., Thailand) used had a DD of 85% and an Mw of 57,000 Dalton. To construct the scaffolds [14–16], chitosan was dissolved in 0.2 M acetic acid in final concentrations of 2 and 3% (w/v). Then they were injected with syringe into 1 M NaOH. Under these conditions a fibril-like chitosan was formed. The fibril-like chitosan was filtered though a sheet cloth, and placed in 15 ml centrifuge tubes and centrifuged at 3,000 rpm for 5 min, then kept at 4°C for 24 h, and subsequently frozen at −20°C. After 24 h the scaffolds were stabilized by immersing them in 96% alcohol for 1 h, 1 M NaOH for 5 min, and 70% alcohol for 12 h. The thus formed scaffolds were sectioned into slices with 5 mm diameter and 2 mm thickness (used for characterization of the scaffolds); or with 1 mm thickness (used for cell seeding). They were submerged into liquid nitrogen for a few seconds and then placed into 24-well plates and dried at 37°C for 2 days. 2.3. Analysis of swelling Swelling of the scaffolds was studied by analyzing water uptake [17] and the subsequent dimensional changes of the scaffolds. Simulated body fluid (SBF) was prepared according to the method described by Kokubo et al. [18]. Appropriate quantities of precursor chemicals were dissolved in deionized water, with final ion concentrations nearly equal to those of the inorganic constituents of human blood plasma. 2.3.1. Swelling ratio of scaffolds Chitosan scaffolds (n = 9 of both types of scaffold) were weighted using an electronic balance and placed in SBF for 5 minutes. After this period all excessive water was removed and the scaffolds were weighted again. Swelling ratios were determined by using the following equation: Swelling ratio = (W − W0 )/W0 , W0 represents initial dry weight and W denotes wet weight of scaffold. 2.3.2. Dimension changes of chitosan scaffolds Chitosan scaffolds (n = 9 of both types of scaffold) were immersed in SBF for a period of 21 days. After 5 min, 7, 14 and 21 days, of each specimen micrographs were taken using a converted microscope. The surface area of the scaffold was measured using Program Image Frame Work v.0.9.9.
S124
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
2.4. Degradation study To analyze the degradation of the scaffold, they were incubated in a solution containing lysozyme, an enzyme taken to be essential in the dissolvement of chitosan [19]. Scaffolds (n = 9 of both types of scaffold) were incubated for 7, 14 and 21 days in 1 × 104 U/ml lysozyme in PBS (pH 7.4) at 37°C. At the different time intervals, the scaffolds were washed with double distilled water and freeze-dried. The level of degradation was determined by assessing the weight loss by using the following formula: Percentage weight loss = (W0 − Wt )/W0 , W0 denotes the original weight, and Wt represents the weight at the different time intervals. 2.5. Cytotoxicity test The cytotoxicity of the scaffolds was evaluated by adding conditioned medium obtained from chitosan scaffolds to cultured cells (ISO/EN 109935 guidelines [20]). In brief, sterilized 2% and 3% chitosan scaffolds were immersed in culture medium consisting of DMED with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin-G, 100 µg/ml streptomycin, 50 U/ml mycostatin and 100 µg/ml kanamycin at 37°C. After 24 h the conditioned media were collected in sterilized tubes and kept at 4°C until use. SHED and DPSC were seeded at a density of 5 × 104 cells/well in 96-well-plates with the 150 µl of normal growth medium for 24 h (5% CO2 , 37°C) in order to establish an 80% confluent monolayer. After the removal of normal growth medium, the scaffold-conditioned medium was added to the different cell types. As a control cells were kept in non-conditioned medium. After a culture period of 24, 48, 72 h, the cells were evaluated by light microscopy, and after 72 h, the cell viability was performed assessed by an MTT assay. Culture medium was removed from 96-well-plate, and then the plate was washed by PBS twice. After 100 µl of medium and 10 µl of MTT solution (5 mg/ml, Sigma, USA) were added to each well, the plate was incubated for 4 h (5%, 37°C), then removed above solution and added 100 µl of DMSO (Dimethyl sulfoxide, Amresco, USA). The plate was incubated for 15 min at room temperature. Absorbance of the colored solution at wavelength 572 nm by plate reader (Biotrak II, Amersham Biosciences). 2.6. Morphology and viability of cells seeded in the chitosan scaffolds 2.6.1. Cell seeding in scaffolds Before cell seeding, chitosan scaffolds were immersed in 70% alcohol for 1 h, then thoroughly washed with sterilized distilled water several times, and then washed in PBS. The scaffolds were immersed in PBS and sterilized by an overnight ultraviolet light treatment. After removal of PBS, the sterilized scaffolds were placed in 48-well plates. 500 µl D-MEM was added supplemented with 10% FBS, glutamine and antibiotics (see above) for 24 h at 37°C. SHED and DPSC were counted by cell counter (Coutess, Invitrogen) and seeded with a density of 5 × 104 cells/scaffold and kept for 3 h (37°C, 5% CO2 ). After this period, the media were replaced by phenol red free DMEM with high glucose (Gibco) and the same additions as mentioned above. 2.6.2. Cell viability in the scaffolds The viability of the cells in the scaffolds was performed by using a WST-1 assay [21]. WST-1 assay is based on the finding that living cells are capable of cleaving slightly red colored WST salt to dark red colored water-soluble formazan by mitochondrial dehydrogenases [22]. Absorbance of water soluble
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S125
formazan was measured under 450 nm. Optical density (OD) is in proportion to cell number. The WST1 assay (Roche, Mannheim, Germany) was performed at day 8, 15 and 21 according to the protocol of the manufacturer. Absorbance of formazan was measured at 450 nm with 620 nm as reference and corrected to blank values (scaffolds without cells). 2.6.3. Scanning electron microscopy of cells and scaffolds Cell-loaded and non-cell-loaded scaffolds were prepared to observe the cell morphology on scaffold and microstructure of scaffold. Specimens were washed with phosphate buffer (PB) and fixed in 2.5% glutaraldehyde (Sigma) at room temperature for 2 h. After washing in PB, the cell-loaded scaffolds were dehydrated in ethanol and critically point dried and coated with gold. The specimens were observed by SEM (JSM-5800LV, JEOL). 2.7. Data analysis Statistical analyses were performed by using SPSS software (Version 16.0, Standard Software Package Inc., USA). The data was presented as mean ± SD. Differences among groups or differences among time intervals were analyzed using one-way analysis of variance (ANOVA). When a difference was statistically significant at P < 0.05, a multiple comparison test was performed. If the variances of the data were normal, the Scheffe method was used. If the variances of the data were not normal, the Dunnette T3 method was used. Significant differences were set at 95% confidence.
3. Results 3.1. Characterization of chitosan scaffolds 3.1.1. Morphology of chitosan scaffolds We measured the pore size in different position of the 15 ml tube after centrifugal step (Fig. 1). Specimens of 2–2.5 cm and 3–3.5 cm from 2% (Fig. 1(a)) and 3% (Fig. 1(b)) chitosan scaffolds were chosen for these measurements, and their pore size were 188.71 ± 51.90 µm and 195.30 ± 67.21 µm, respec-
Fig. 1. Pore size of chitosan scaffolds in different positions of 15 ml tube (a) 2% chitosan scaffolds; (b) 3% chitosan scaffolds. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141231.)
S126
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Fig. 2. SEM micrographs showed the microstructures of chitosan scaffolds: (a) Cross-section of 2% chitosan scaffolds; (b) Longitudinal-section of 2% chitosan scaffolds; (c) Cross-section of 3% chitosan scaffolds; (d) Longitudinal-section of 3% chitosan scaffolds.
tively (Fig. 2, NS, analyzed by non-parametric analysis, Mann–Whitney U test, n = 9). Microstructures of scaffolds revealed the presence of interconnected micropores. 3.1.2. Swelling study As shown in Fig. 3, swelling ratio of 3% chitosan scaffolds was significantly lower than that of 2% scaffolds (738.47 ± 18.27% and 883.89 ± 20.92%, respectively; P < 0.05, Student’s t-test, n = 9). For a further investigation on the dimensional changes of chitosan scaffolds, the specimens were immersed in the SBF buffer for 21 days. The longitudinal (Fig. 4(a)) and cross (Fig. 4(b)) sectional sizes of the scaffolds only significantly changed in the first 5 minutes. Scaffolds maintained their dimension after that and there were no significant differences between the 2% and 3% scaffolds. 3.1.3. Degradation of chitosan scaffolds Compared to day 0, 2% chitosan scaffolds had a 2.12 ± 1.25% weight loss and 3% scaffolds had a 2.85 ± 2.38% weight loss on day 7. The weight loss of the scaffolds was significantly increased during day 7, day 14 and day 21 (P < 0.05, one-way ANOVA, multiple comparisons, n = 9) (Table 1). However, there were no significant differences in degradation between 2% and 3% chitosan scaffolds at any measured time point (P > 0.05, Student’s t-test, n = 9) (Table 1, Fig. 5).
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S127
Fig. 3. Swelling ratios (738.47 ± 18.27) of 3% chitosan scaffolds were significantly lower than that (883.89 ± 20.92) of 2% chitosan scaffolds (P < 0.05, analyzed by Student t-test, n = 9).
Fig. 4. Dimensional changes of 2% and 3% chitosan scaffolds in the longitudinal-section (a) and in cross-section (b).
S128
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold Table 1 In vitro degradation (%) of chitosan scaffolds 2% 3%
Day 7 2.88 ± 1.75 2.85 ± 0.79
Day 14 20.86 ± 2.32 14.67 ± 3.44
Day 21 30.42 ± 3.20 24.15 ± 1.23
Notes: Between different time point, P < 0.05, analyzed by Mann–Whitney U test, n = 9.
Fig. 5. In vitro degradation of chitosan scaffolds, Error bars represent means ± SD. Comparisons among time points: P > 0.05, data was analyzed by Student t-test, n = 9; comparisons between 2% and 3% chitosan scaffolds P < 0.05, were analyzed by one-way ANOVA, multiple comparisons, n = 9.
3.2. Cytocompatibility 3.2.1. Cytotoxicity test After 24, 48 and 72 h, the response of the cells to scaffold-conditioned medium was evaluated microscopically, and no obvious differences in cellular morphology were observed. Table 2 and Fig. 6 demonstrates the cell viability after cells exposed to scaffold-conditioned medium for 72 h (one-way ANOVA, multiple comparisons, n = 6). The viability of DPSC incubated with scaffold-conditioned medium of 2% and 3% groups were not significantly different, but they were significantly higher than cell viability of the normal media control group (P < 0.01). For SHED, cell viabilities of 2% groups were significantly higher than control groups (P < 0.01), but cell viability of 3% group was not significantly different from those of normal media control group and 2% group. 3.2.2. Morphology of cells seeded in scaffolds Cells were found to attach to the bottom (Fig. 7(c)) and wall (Fig. 7(d)) of the pores in scaffolds. They attached on the wall, and the pseudopods could be seen clearly (Fig. 7(a)). When the cells were confluent, they could cross the border of the pores (Fig. 7(b)) and were shown to have contact with each other. 3.2.3. Cell viability on scaffolds The cell viability as analyzed by the WST-1 assay of DPSC and SHED in chitosan scaffolds is shown in Table 3. The optical densities of DPSC in the 2% and 3% scaffold groups were significantly higher than those of the controls on day 8, 15 and 21. Particularly on day 21, the optical density of 2% scaffold group was not only higher than that of the control group but also higher than that of 3% scaffold group
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S129
Table 2 Cytotoxicity test by MTT assay DPSC SHED
Control 0.430 ± 0.044 0.366 ± 0.021
2% chitosan 0.680 ± 0.055∗ 0.454 ± 0.022∗
3% chitosan 0.627 ± 0.043∗ 0.417 ± 0.016
Notes: Means ± SD of optical density in different cell groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.01).
Fig. 6. Cytotoxicity test by MTT assay: OD (A = 572 nm) was expressed as a measure of cell viability after exposure to the 2% and 3% extraction media after 72 h, and the cells cultured with normal media were set as a control, n = 6. Error bars represent means ± SD. Data was analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.01).
(Fig. 8(a)). SHED showed similar results as DPSC except that between day 15 and day 21, the optical density of the 3% group was somewhat lower (Fig. 8(b)). 4. Discussion The procedure used in the present study for preparation of chitosan scaffold is easy and the used materials are not expensive due to locally availability. We have food grade chitosan produce by a local factory using law material from the sea. We introduced a centrifuge step before the material was freezedried according to the method used by Park et al. [16], and then we obtained 2% chitosan scaffolds with the pore size of 188.71 ± 51.90 µm and 3% scaffolds with the pore size of 195.30 ± 67.21 µm. Viable cell sizes of DPSC and SHED can be read from cell counter that were at the range of 10–40 µm. Mean size range of osteoblasts is 10–30 µm [23]. Kose et al. suggested that the average pore size should be at least three times larger than the size of cells so that a single cell could establish contact with others cells [24]. Fibroblasts have been demonstrated to bind to a wide range of pore sizes from 95 to 150 µm and the viability of the cells would increase with decreasing pore size until no cells could fit into the pores
S130
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Fig. 7. Scanning electron micrographs of cells cultured in chitosan scaffolds for 16 days. (a) On the scaffolds; (b) across the border of the pores; (c) on the bottom of the pores; (d) on the wall of the pores.
Table 3 Cell viability on scaffolds detected by WST-1 assay Control Day 8 Day 15 Day 21
0.777 ± 0.063 1.414 ± 0.275 1.534 ± 0.018
Day 8 Day 15 Day 21
0.787 ± 0.098 1.214 ± 0.129 1.453 ± 0.012
2% chitosan DPSC 1.142 ± 0.112∗ 2.286 ± 0.049∗ 2.854 ± 0.103*,# SHED 1.365 ± 0.067∗ 2.470 ± 0.099∗ 2.905 ± 0.066*,#
3% chitosan 1.105 ± 0.095∗ 2.062 ± 0.101∗ 2.343 ± 0.212∗,# 1.603 ± 0.018∗ 2.150 ± 0.076∗ 2.121 ± 0.178*,#
Notes: Means ± SD of optical density in different cell and chitosan scaffold groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.05 between control and test groups, # P < 0.05 within 2% and 3% scaffold groups).
[25]. Another study suggested that porous scaffold microstructure with minimal pore size ranging from 100 to 150 µm was usually required to allow tissue in growth [26].
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S131
Fig. 8. OD (A = 450 nm) was expressed as a measure of cell ((a) DPSC; (b) SHED) viability in scaffolds on day 8, 15 and 21, n = 6. Error bars represent means ± SD. Means ± SD of optical density in different cell and chitosan scaffold groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level.
Pore size of used scaffolds in our study was approximately three times the size of SHED and DPSC. Therefore, it is reasonable to choose scaffolds with the pore size as found in our study. Figure 7 confirmed that the cells were able to maintain their normal morphology and could get into the pore of scaffolds.
S132
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Next, we examined the swelling ratio of those chitosan scaffolds. Both chitosan concentrations of 2% and 3% scaffolds possess an equally high swelling ratio. It could preserve a high volume of liquid within the porous structure, maintained their dimensions, and further enhance the penetration of cells into the inner area of the scaffold [17,27,28]. Seda et al. found [14] that swelling ratios of 2% chitosan scaffolds were significantly higher than that of 3% scaffolds since in their study pore size of 2% scaffold was larger than 3% scaffolds, and a larger pore was assumed to contain more liquid. However, when we used the scaffolds with almost equal pore size, we got the same result as theirs. Hsieh et al. suggested that the mechanical property of scaffold increased when the chitosan concentration increased from 1 to 3% [29]. It resulted in 3% scaffold possess stronger compressive strength. When liquid got into scaffolds, the pores of 2% scaffolds would expand more than those of 3% scaffolds even though their sizes were equal. Degradation occurred of both 2% and 3% scaffolds. Chitosan is mainly degraded by lysozyme [19] which is present in various human body fluids and tissues [24]. β-(1-4)-glycosidic bonds between polysaccharide chains are related to the functional activity of lysozyme, and after all β-(1-4)-glycosidic bonds are broken, the degradation will decelerate [29]. Chitosan with more β-(1-4)-glycosidic bonds would decelerate degradation rate. The 2% chitosan scaffold contains about 6% less β-(1-4)-glycosidic bonds than the 3% scaffold. Therefore, the degradation rate of 2% chitosan scaffold was only about 6% higher than those of 3% chitosan scaffold. Moreover, we agree with a previous study, slight increase of chitosan concentration could decelerate the degradation [29], but would not significant impact on the scaffold degradation. The rate of scaffold degradation should mirror the rate of new tissue formation or be adequate for the controlled release of bioactive molecules [12]. According to above, we can easily control the rate of scaffold degradation by adjusting the chitosan concentration. The chitosan scaffold appeared to be non-cytotoxic. After 24, 48 and 72 h, no changes in cellular morphology were detected microscopically in both test and control groups. Toxicity of chitosan is reported to depend on degree of deacetylation (DD). Chitosan with DD higher than 35% showed low toxicity [30]. Chitosan DD in our study was 85%, and the cytotoxicity experiments showed that the viability of SHED and DPSC was not influenced by scaffold-conditioned medium. Hence, those scaffolds were considered to be non-toxic. During scaffold-conditioned medium preparation, some chitosan oligosaccharides may be released into the medium. Those oligosaccharides had been demonstrated to possess biological activities [31]. In line with this possibility, DPSC viability in scaffold-conditioned medium groups and SHED viability in 2% scaffold-conditioned medium group were significantly higher than those of normal medium control group. In addition, the 3% chitosan scaffold possess stronger mechanical force than 2% chitosan scaffold [29], therefore oligosaccharides released from 3% scaffold-conditioned medium were possibly less than those from 2% scaffold-conditioned medium. For this reason, SHED and DPSC viabilities in 2% scaffold-conditioned medium group were higher than those in 3% scaffold-conditioned medium group. On the other hand, a small number of growth factor or components from fetal bovine serum, for example insulin, may bond to chitosan [32] when scaffold-conditioned medium was prepared. Insulin could promote glucose taken up by cells to provide enough energy for cell growth. The 2% chitosan scaffold bond less such components than 3% chitosan scaffold during scaffold-conditioned medium preparation, thus 2% scaffold-conditioned medium contain more such components result in higher cell viabilities in 2% scaffold-conditioned medium. Moreover, Howling et al. found chitosan could stimulate, no effect or inhibit cell proliferation when it cultured with different fibroblast strains or cell lines [31]. In our study,
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S133
DPSC viability of 3% scaffold-conditioned medium group was significantly higher than control group whereas SHED viability of 3% scaffold-conditioned medium group was not significantly different from control group. It suggested that chitosan also effected on stimulation of DPSC and SHED proliferation differently. The cytocompatibility of chitosan has been proved in vitro with myocardial, endothelial and epithellial cells, fibroblasts, hepatocytes, condrocytes, keratinocytes [33] and periodontal ligament cells [34]. Chitosan contains a large amount of amino groups, which give it high positive charges. It has been demonstrated that all vertebrate cells possess unevenly distributed negative surface charges [35]. Cells could bind tightly within chitosan via electrostatic interaction. Figure 7 shows that the cells were able to maintain their normal morphology in the scaffolds. These results indicate that chitosan scaffolds were suitable for DPSC and SHED to adhere to and proliferate. General recommendations for chitosan application in tissue engineering are summarized by Inmaculada et al.: (1) A DD of around 85% is good for cell proliferation and scaffold structure maintenance; (2) A high Mw assists in prolonging biodegradation of scaffolds [12]. We chose the commercial chitosan product of 85% as mentioned above, which is good for cell proliferation. Results showed that SHED and DPSC viabilities in scaffold groups were significant higher than those in control groups, since there was much more culture space in the three-dimensional scaffolds, and cells could continue growing without contacting inhibitions [34]. Above swelling study demonstrated that 2% chitosan scaffolds possess a higher swelling ratio than 3% chitosan scaffolds, in other words, 2% chitosan scaffolds could preserve a higher volume of media in the porous structure and could further enhance the penetration of cells into the inner area of the scaffolds more than 3% scaffolds. It would result in SHED and DPSC viabilities in 2% scaffold groups were significantly higher than those in 3% scaffold groups. These results suggest that the concentration of chitosan might affect the cell growth. However, cell responses are different for each cell type. Chatelet et al. studied the relationship between the cell type and adhesion by comparing between keratinocyte and fibroblasts. They concluded that the type of cell was a factor that also affected the adhesion, being more favorable for fibroblasts which exhibit a more negative surface charge [33]. It suggested that, besides DD, the cell types probably related to cytocompatibility. Notice from Fig. 8 and Table 3, SHED appeared better viability than DPSC, that may suggest surface of SHED possess more negative charge than DPSC. Different from DPSC viability of 3% chitosan group, SHED viability of 3% chitosan group had a decreasing trend from day 15 to day 21. We could assume that SHED began to differentiate on 3% chitosan scaffold. Accordingly, our results showed that 3% chitosan possibly induce SHED differentiation. However, further studies are needed to confirm this detection. The present study demonstrated that chitosan scaffolds prepared according to our novel method show a mild swelling in body fluid and degraded in lysozyme solution. The used scaffolds appear to simulate DPSC and SHED growth and are more suitable for SHED proliferation. The 3% chitosan may induce SHED differentiation. Further studies are needed to confirm that chitosan possesses the ability to induce differentiation of dental pulp derived cells.
Acknowledgements We would like to extend our gratitude to Prof. Vincent Everts for his critical reading of the manuscript. This study was supported by Thailand Research Fund and Prince of Songkla University, Hadyai, Songkla, Thailand.
S134
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Conflict of interest The authors have declared no conflict of interest.
References [1] S. Gronthos, M. Mankani, J. Brahim, P.G. Robey et al., Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo, Proc. Natl. Acad. Sci. USA 97 (2000), 13625–13630. [2] M. Miura, S. Gronthos, M. Zhao, B. Lu et al., SHED: stem cells from human exfoliated deciduous teeth, Proc. Natl. Acad. Sci. USA 100 (2003), 5807–5812. [3] P. Bianco and P.G. Robey, Stem cells in tissue engineering, Nature 414 (2001), 118–121. [4] G.T. Huang, T. Yamaza, L.D. Shea, F. Djouad et al., Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model, Tissue Eng. Part A 16 (2010), 605–615. [5] W. Zhang, X.F. Walboomers, T.H. van Kuppevelt, W.F. Daamen et al., The performance of human dental pulp stem cells on different three-dimensional scaffold materials, Biomaterials 27 (2006), 5658–5668. [6] K.W. Ng and D.W. Hutmacher, Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices, Biomaterials 27 (2006), 4591–4598. [7] G.T. Huang, W. Sonoyama, J. Chen and S.H. Park, In vitro characterization of human dental pulp cells: various isolation methods and culturing environments, Cell Tissue Res. 324 (2006), 225–236. [8] Q. Hu, B. Li, M. Wang and J. Shen, Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture, Biomaterials 25 (2004), 779–785. [9] M.V. Risbud and R.R. Bhonde, Polyacrylamide–chitosan hydrogels: in vitro biocompatibility and sustained antibiotic release studies, Drug Deliv. 7 (2000), 69–75. [10] A. Di Martino, M. Sittinger and M.V. Risbud, Chitosan: a versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005), 5983–5990. [11] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo et al., Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008), 1–21. [12] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed and A. Heras, Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009), 203–230. [13] S. Shi, P.M. Bartold, M. Miura, B.M. Seo et al., The efficacy of mesenchymal stem cells to regenerate and repair dental structures, Orthod. Craniofac. Res. 8 (2005), 191–199. [14] R. Seda Tigli, A. Karakecili and M. Gumusderelioglu, In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree, J. Mater. Sci. Mater. Med. 18 (2007), 1665–1674. [15] T. Freier, H.S. Koh, K. Kazazian and M.S. Shoichet, Controlling cell adhesion and degradation of chitosan films by N-acetylation, Biomaterials 26 (2005), 5872–5878. [16] O.S. Park L and J. Lee, Investigation of pore size effect on cell compatibility using pore size gradient chitosan scaffold, Key Engineering Materials 342–343 (2007), 285–288. [17] S.N. Park, H.J. Lee, K.H. Lee and H. Suh, Biological characterization of EDC-crosslinked collagen–hyaluronic acid matrix in dermal tissue restoration, Biomaterials 24 (2003), 1631–1641. [18] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi et al., Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W, J. Biomed. Mater. Res. 24 (1990), 721–734. [19] K.M. Varum, M.M. Myhr, R.J. Hjerde and O. Smidsrod, In vitro degradation rates of partially N-acetylated chitosans in human serum, Carbohydr. Res. 299 (1997), 99–101. [20] ISO/EN10993-5, Biological evaluation of medical devices-Part 5 tests for cytotoxicity. In vitro methods, 8.2 tests on extracts. [21] P. Ngamwongsatit, P.P. Banada, W. Panbangred and A.K. Bhunia, WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line, J. Microbiol. Methods 73 (2008), 211–215. [22] M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto et al., A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet, Biol. Pharm. Bull. 19 (1996), 1518–1520. [23] P. Arpornmaeklong, N. Suwatwirote, P. Pripatnanont and K. Oungbho, Growth and differentiation of mouse osteoblasts on chitosan–collagen sponges, Int. J. Oral Maxillofac. Surg. 36 (2007), 328–337. [24] G.T. Kose, H. Kenar, N. Hasirci and V. Hasirci, Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for bone tissue engineering, Biomaterials 24 (2003), 1949–1958.
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S135
[25] F.J. O’Brien, B.A. Harley, I.V. Yannas and L.J. Gibson, The effect of pore size on cell adhesion in collagen-GAG scaffolds, Biomaterials 26 (2005), 433–441. [26] J. Zeltinger, J.K. Sherwood, D.A. Graham, R. Mueller et al., Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition, Tissue Eng. 7 (2001), 557–572. [27] L. Ma, C. Gao, Z. Mao, J. Zhou et al., Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering, Biomaterials 24 (2003), 4833–4841. [28] N. Shanmugasundaram, P. Ravichandran, P.N. Reddy, N. Ramamurty et al., Collagen–chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells, Biomaterials 22 (2001), 1943–1951. [29] W.C. Hsieh, C.P. Chang and S.M. Lin, Morphology and characterization of 3D micro-porous structured chitosan scaffolds for tissue engineering, Colloids Surf. B Biointerfaces 57 (2007), 250–255. [30] N.G. Schipper, K.M. Varum and P. Artursson, Chitosans as absorption enhancers for poorly absorbable drugs. 1: Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells, Pharm. Res. 13 (1996), 1686–1692. [31] G.I. Howling, P.W. Dettmar, P.A. Goddard, F.C. Hampson et al., The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro, Biomaterials 22 (2001), 2959–2966. [32] T. Mori, M. Okumura, M. Matsuura, K. Ueno et al., Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro, Biomaterials 18 (1997), 947–951. [33] C. Chatelet, O. Damour and A. Domard, Influence of the degree of acetylation on some biological properties of chitosan films, Biomaterials 22 (2001), 261–268. [34] L. Peng and R.-X. Zhuo, Biological evaluation of porous chitosan/collagen scaffolds for periodontal tissue engineering, in: The 2nd International Conference on Bioinformatics and Biomedical Engineering, 2008, pp. 897–900. [35] J.Z. Borysenko and W. Woods, Density, distribution and mobility of surface anions on a normal/transformed cell pair, Exp. Cell Res. 118 (1979), 215–227.
S121
Fabrication, characterization and cell cultures on a novel chitosan scaffold Zheng Guan a,b , Songtao Shi c , Buncha Samruajbenjakun a and Suttatip Kamolmatyakul a,∗ a
Prince of Songkla University, Preventive Dentistry, Hadyai, Songkhla, Thailand Biomedical Research Center, The First People’s Hospital of Kunming, Yunnan, China c University of Southern California, Center for Craniofacial Molecular Biology, Los Angeles, CA, USA b
Abstract. Chitosan has been used as scaffolds with various methods of fabrication including expensive commercial available ones for tissue engineering. The objective of this study is to assemble our novel method of chitosan scaffold fabrication in economical and uncomplicated way that suitable for dental pulp stem cell (DPSC) and stem cells of human exfoliated deciduous teeth (SHED). Chitosan scaffolds (2% and 3%) were fabricated in an uncomplicated procedure, including centrifugation and freeze-drying steps. The chitosan scaffolds were compared and the pore size, swelling and degradation were assessed. In addition, the cytocompatibility was assessed of chitosan scaffolds seeded with DPSC and SHED. The pore size of 2% and 3% chitosan scaffolds were similar being 188.71 ± 51.90 µm and 195.30 ± 67.21 µm, respectively. Swelling ratios of 3% chitosan scaffolds were significantly lower than those of 2% chitosan scaffolds. Dimension of scaffolds changed in first 5 minutes. After that, those scaffolds could maintain their dimension. Chitosan scaffolds degraded as from day 7. No differences were found between 2% and 3% chitosan scaffolds. The scaffolds were shown to be non-toxic and to promote DPSCs and SHED growth. The viability of DPSCs and SHED on 2% scaffolds proved to be higher than that of the 3% scaffold group. This study suggested that chitosan scaffolds fabricated with our novel method were suitable for the growth and survival of DPSC and SHED. Keywords: Biocompatibility, cell culture, chitosan scaffold, dental-derived stem cell
Abbreviations DPSC SHED DD Mw FBS PBS SBF
dental pulp stem cell; stem cells of human exfoliated deciduous teeth; deacetylation degree; molecular weight; fetal bovine serum; phosphate buffered saline; simulated body fluid.
1. Introduction Human dental pulp stem cell (DPSCs) [1] and stem cells of human exfoliated deciduous teeth (SHED) [2] are considered to facilitate pulp tissue engineering. Stem cells play an important role in regenerating * Address for correspondence: Associate Professor Suttatip Kamolmatyakul, D.Sc.D., Preventive Dentistry, Faculty of Dentistry, Prince of Songkla University, Hadyai, Songkhla, 90112, Thailand. Tel.: +66 74 287510, Fax: +66 74 212922; E-mail: [email protected].
0959-2989/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved
S122
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
new tissue [3]. Frequently stem cells are seeded on a scaffold prior to their use. As a carrier, different types of scaffolds can be selected for tooth engineering. Several scaffolds have been tested for regeneration of bone, and these scaffolds are possibly also suitable for regeneration of dental tissue. However, regenerating dental tissue is not the same as regenerating bone. Pulp in the canal space has its specific locations; therefore, any scaffold system that is osteo-inductive such as hydroxyapatite and tricalcium phosphate is in fact not appropriate for pulp regeneration [4]. On the other hand, when pulp tissue engineering is used in vital pulp therapy, the material should possess preferably anti-microbial properties and should be shaped easily in order to fit the pulp space. Natural occurring materials such as collagen and chitosan have a good biocompatibility, and these materials can be shaped in any configuration. Collagen-based scaffolds have been studied with DPSC, and they were shown to support cell growth and differentiation in vivo [5]. Yet, the collagen scaffold used did not have anti-microbial properties, and in general collagen scaffolds often loose shape and size because of a fairly rapid degradation when contacted with body fluid [6]. Huang and coworkers reported a considerable contraction of the collagen scaffold by pulp cells; the scaffold decreased to half of its original size by 3–15 days [7]. In other words, three-dimensional space of collagen scaffold will decrease which may result in loss of space for pulp cells to proliferate. Therefore, a collagen matrix may not be a suitable scaffold for pulp tissue engineering [7]. There is, however, an alternative: chitosan. Chitosan is a natural polymer, is obtained from shell of shellfish, and the wastes of the seafood industry, and it is a deacetylated derivative of chitin. Chitosan scaffolds possess some special properties for application in tissue engineering. First, it can be easily molded in any form [8]. Second, it possesses excellent properties to form porous structures [9]. The microstructure of porous chitosan scaffolds allows swelling of the material, which stimulates cell attachment, easy nutrient supply and a proper 3-D structure. Third, the cationic nature of chitosan provides the means to bind cytokines and growth factors. This property is beneficial for tissue engineering purposes [10]. Another distinct property of chitosan is its antibacterial activity against a broad spectrum of bacteria [11]. Fourth, the degradation rate of the chitosan can be manipulated by the incorporation of deacetylation degree (DD) and by varying its molecular weight (Mw). Degradation has been shown to increase as DD decreases. Moreover, high Mw contribute to prolonged degradation [12]. Provided the many advantages of chitosan, scaffolds of this material were studied in various tissue engineering applications such as skin, bone, cartilage, liver, nerve and blood vessel [11]. We assume that such chitosan scaffolds are suitable also for vital pulp therapy and the restoration of dental structure. Therefore, we analyzed in the present study the cytocompatibility of dental pulp-derived stem cells seed on different types of chitosan scaffolds. 2. Materials and methods 2.1. Cell culture Primary and permanent teeth were collected under the approved guideline of the Ethical Committee of Prince of Songkla University. Human primary exfoliated teeth were collected from 6 to 12-year-old children (n = 6). Informed consent was obtained from the parents. Permanent teeth were obtained from adult donors (29 years old, n = 6). Impacted third molars and bicuspids were extracted due to orthodontic considerations. All of these teeth contained a normal healthy pulp and were confirmed by clinical and radiographic examination. SHED and DPSC were isolated as described by Gronthos et al. [1]. Tooth surfaces were cleaned by 70% alcohol and cut around the cementum–enamel junction by using sterilized dental fissure burs to
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S123
reveal the pulp chamber. The pulp tissue was gently separated and minced. The minced pulp tissues were digested in a mixture of 3 mg/ml collagenase type I and 4 mg/ml dispase (Dissolved in PBS, Sigma, St. Louis, Mo., USA) for 30–60 min at 37°C water-bath. Cell suspensions were obtained by passing the digested tissues through a 70 µm cell strainer (Becton/Dickinson, Franklin Lakes, NJ, USA). Single cell suspensions were seeded in 100 mm culture plates (Nunc, Denmark) containing DMEM (Life Technologies/GIBCO BRL) supplemented with 20% fetal bovine serum (FBS, Biochrom AG, Germany), 2 mM L-glutamine (Gibco Invitrogen, USA), 100 U/ml penicillin-G, 100 µg/ml streptomycin, 50 U/ml mycostatin and 100 µg/ml kanamycin and maintained under 5% CO2 at 37°C. Those cells were allowed to grow for 10 to 12 days, and then were collected by assessing their colony-forming efficiency (CFU-F) [13]. Cells aggregated in groups of less than 50 cells were scratched from the bottom of the plate and removed by phosphate buffered saline (PBS). The other colonies (50 cells) were transferred to T-75 cultural flasks (TPP, Switzerland) and were cultured up to 70–80% confluence, and then they were passaged at 1 : 3 ratios for experiment or storage. Cells at passage 3–5 were used in the experiments. 2.2. Fabrication of chitosan scaffolds The chitosan (Sea Fresh Chitosan (Lab) Co., Thailand) used had a DD of 85% and an Mw of 57,000 Dalton. To construct the scaffolds [14–16], chitosan was dissolved in 0.2 M acetic acid in final concentrations of 2 and 3% (w/v). Then they were injected with syringe into 1 M NaOH. Under these conditions a fibril-like chitosan was formed. The fibril-like chitosan was filtered though a sheet cloth, and placed in 15 ml centrifuge tubes and centrifuged at 3,000 rpm for 5 min, then kept at 4°C for 24 h, and subsequently frozen at −20°C. After 24 h the scaffolds were stabilized by immersing them in 96% alcohol for 1 h, 1 M NaOH for 5 min, and 70% alcohol for 12 h. The thus formed scaffolds were sectioned into slices with 5 mm diameter and 2 mm thickness (used for characterization of the scaffolds); or with 1 mm thickness (used for cell seeding). They were submerged into liquid nitrogen for a few seconds and then placed into 24-well plates and dried at 37°C for 2 days. 2.3. Analysis of swelling Swelling of the scaffolds was studied by analyzing water uptake [17] and the subsequent dimensional changes of the scaffolds. Simulated body fluid (SBF) was prepared according to the method described by Kokubo et al. [18]. Appropriate quantities of precursor chemicals were dissolved in deionized water, with final ion concentrations nearly equal to those of the inorganic constituents of human blood plasma. 2.3.1. Swelling ratio of scaffolds Chitosan scaffolds (n = 9 of both types of scaffold) were weighted using an electronic balance and placed in SBF for 5 minutes. After this period all excessive water was removed and the scaffolds were weighted again. Swelling ratios were determined by using the following equation: Swelling ratio = (W − W0 )/W0 , W0 represents initial dry weight and W denotes wet weight of scaffold. 2.3.2. Dimension changes of chitosan scaffolds Chitosan scaffolds (n = 9 of both types of scaffold) were immersed in SBF for a period of 21 days. After 5 min, 7, 14 and 21 days, of each specimen micrographs were taken using a converted microscope. The surface area of the scaffold was measured using Program Image Frame Work v.0.9.9.
S124
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
2.4. Degradation study To analyze the degradation of the scaffold, they were incubated in a solution containing lysozyme, an enzyme taken to be essential in the dissolvement of chitosan [19]. Scaffolds (n = 9 of both types of scaffold) were incubated for 7, 14 and 21 days in 1 × 104 U/ml lysozyme in PBS (pH 7.4) at 37°C. At the different time intervals, the scaffolds were washed with double distilled water and freeze-dried. The level of degradation was determined by assessing the weight loss by using the following formula: Percentage weight loss = (W0 − Wt )/W0 , W0 denotes the original weight, and Wt represents the weight at the different time intervals. 2.5. Cytotoxicity test The cytotoxicity of the scaffolds was evaluated by adding conditioned medium obtained from chitosan scaffolds to cultured cells (ISO/EN 109935 guidelines [20]). In brief, sterilized 2% and 3% chitosan scaffolds were immersed in culture medium consisting of DMED with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin-G, 100 µg/ml streptomycin, 50 U/ml mycostatin and 100 µg/ml kanamycin at 37°C. After 24 h the conditioned media were collected in sterilized tubes and kept at 4°C until use. SHED and DPSC were seeded at a density of 5 × 104 cells/well in 96-well-plates with the 150 µl of normal growth medium for 24 h (5% CO2 , 37°C) in order to establish an 80% confluent monolayer. After the removal of normal growth medium, the scaffold-conditioned medium was added to the different cell types. As a control cells were kept in non-conditioned medium. After a culture period of 24, 48, 72 h, the cells were evaluated by light microscopy, and after 72 h, the cell viability was performed assessed by an MTT assay. Culture medium was removed from 96-well-plate, and then the plate was washed by PBS twice. After 100 µl of medium and 10 µl of MTT solution (5 mg/ml, Sigma, USA) were added to each well, the plate was incubated for 4 h (5%, 37°C), then removed above solution and added 100 µl of DMSO (Dimethyl sulfoxide, Amresco, USA). The plate was incubated for 15 min at room temperature. Absorbance of the colored solution at wavelength 572 nm by plate reader (Biotrak II, Amersham Biosciences). 2.6. Morphology and viability of cells seeded in the chitosan scaffolds 2.6.1. Cell seeding in scaffolds Before cell seeding, chitosan scaffolds were immersed in 70% alcohol for 1 h, then thoroughly washed with sterilized distilled water several times, and then washed in PBS. The scaffolds were immersed in PBS and sterilized by an overnight ultraviolet light treatment. After removal of PBS, the sterilized scaffolds were placed in 48-well plates. 500 µl D-MEM was added supplemented with 10% FBS, glutamine and antibiotics (see above) for 24 h at 37°C. SHED and DPSC were counted by cell counter (Coutess, Invitrogen) and seeded with a density of 5 × 104 cells/scaffold and kept for 3 h (37°C, 5% CO2 ). After this period, the media were replaced by phenol red free DMEM with high glucose (Gibco) and the same additions as mentioned above. 2.6.2. Cell viability in the scaffolds The viability of the cells in the scaffolds was performed by using a WST-1 assay [21]. WST-1 assay is based on the finding that living cells are capable of cleaving slightly red colored WST salt to dark red colored water-soluble formazan by mitochondrial dehydrogenases [22]. Absorbance of water soluble
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S125
formazan was measured under 450 nm. Optical density (OD) is in proportion to cell number. The WST1 assay (Roche, Mannheim, Germany) was performed at day 8, 15 and 21 according to the protocol of the manufacturer. Absorbance of formazan was measured at 450 nm with 620 nm as reference and corrected to blank values (scaffolds without cells). 2.6.3. Scanning electron microscopy of cells and scaffolds Cell-loaded and non-cell-loaded scaffolds were prepared to observe the cell morphology on scaffold and microstructure of scaffold. Specimens were washed with phosphate buffer (PB) and fixed in 2.5% glutaraldehyde (Sigma) at room temperature for 2 h. After washing in PB, the cell-loaded scaffolds were dehydrated in ethanol and critically point dried and coated with gold. The specimens were observed by SEM (JSM-5800LV, JEOL). 2.7. Data analysis Statistical analyses were performed by using SPSS software (Version 16.0, Standard Software Package Inc., USA). The data was presented as mean ± SD. Differences among groups or differences among time intervals were analyzed using one-way analysis of variance (ANOVA). When a difference was statistically significant at P < 0.05, a multiple comparison test was performed. If the variances of the data were normal, the Scheffe method was used. If the variances of the data were not normal, the Dunnette T3 method was used. Significant differences were set at 95% confidence.
3. Results 3.1. Characterization of chitosan scaffolds 3.1.1. Morphology of chitosan scaffolds We measured the pore size in different position of the 15 ml tube after centrifugal step (Fig. 1). Specimens of 2–2.5 cm and 3–3.5 cm from 2% (Fig. 1(a)) and 3% (Fig. 1(b)) chitosan scaffolds were chosen for these measurements, and their pore size were 188.71 ± 51.90 µm and 195.30 ± 67.21 µm, respec-
Fig. 1. Pore size of chitosan scaffolds in different positions of 15 ml tube (a) 2% chitosan scaffolds; (b) 3% chitosan scaffolds. (Colors are visible in the online version of the article; http://dx.doi.org/10.3233/BME-141231.)
S126
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Fig. 2. SEM micrographs showed the microstructures of chitosan scaffolds: (a) Cross-section of 2% chitosan scaffolds; (b) Longitudinal-section of 2% chitosan scaffolds; (c) Cross-section of 3% chitosan scaffolds; (d) Longitudinal-section of 3% chitosan scaffolds.
tively (Fig. 2, NS, analyzed by non-parametric analysis, Mann–Whitney U test, n = 9). Microstructures of scaffolds revealed the presence of interconnected micropores. 3.1.2. Swelling study As shown in Fig. 3, swelling ratio of 3% chitosan scaffolds was significantly lower than that of 2% scaffolds (738.47 ± 18.27% and 883.89 ± 20.92%, respectively; P < 0.05, Student’s t-test, n = 9). For a further investigation on the dimensional changes of chitosan scaffolds, the specimens were immersed in the SBF buffer for 21 days. The longitudinal (Fig. 4(a)) and cross (Fig. 4(b)) sectional sizes of the scaffolds only significantly changed in the first 5 minutes. Scaffolds maintained their dimension after that and there were no significant differences between the 2% and 3% scaffolds. 3.1.3. Degradation of chitosan scaffolds Compared to day 0, 2% chitosan scaffolds had a 2.12 ± 1.25% weight loss and 3% scaffolds had a 2.85 ± 2.38% weight loss on day 7. The weight loss of the scaffolds was significantly increased during day 7, day 14 and day 21 (P < 0.05, one-way ANOVA, multiple comparisons, n = 9) (Table 1). However, there were no significant differences in degradation between 2% and 3% chitosan scaffolds at any measured time point (P > 0.05, Student’s t-test, n = 9) (Table 1, Fig. 5).
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S127
Fig. 3. Swelling ratios (738.47 ± 18.27) of 3% chitosan scaffolds were significantly lower than that (883.89 ± 20.92) of 2% chitosan scaffolds (P < 0.05, analyzed by Student t-test, n = 9).
Fig. 4. Dimensional changes of 2% and 3% chitosan scaffolds in the longitudinal-section (a) and in cross-section (b).
S128
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold Table 1 In vitro degradation (%) of chitosan scaffolds 2% 3%
Day 7 2.88 ± 1.75 2.85 ± 0.79
Day 14 20.86 ± 2.32 14.67 ± 3.44
Day 21 30.42 ± 3.20 24.15 ± 1.23
Notes: Between different time point, P < 0.05, analyzed by Mann–Whitney U test, n = 9.
Fig. 5. In vitro degradation of chitosan scaffolds, Error bars represent means ± SD. Comparisons among time points: P > 0.05, data was analyzed by Student t-test, n = 9; comparisons between 2% and 3% chitosan scaffolds P < 0.05, were analyzed by one-way ANOVA, multiple comparisons, n = 9.
3.2. Cytocompatibility 3.2.1. Cytotoxicity test After 24, 48 and 72 h, the response of the cells to scaffold-conditioned medium was evaluated microscopically, and no obvious differences in cellular morphology were observed. Table 2 and Fig. 6 demonstrates the cell viability after cells exposed to scaffold-conditioned medium for 72 h (one-way ANOVA, multiple comparisons, n = 6). The viability of DPSC incubated with scaffold-conditioned medium of 2% and 3% groups were not significantly different, but they were significantly higher than cell viability of the normal media control group (P < 0.01). For SHED, cell viabilities of 2% groups were significantly higher than control groups (P < 0.01), but cell viability of 3% group was not significantly different from those of normal media control group and 2% group. 3.2.2. Morphology of cells seeded in scaffolds Cells were found to attach to the bottom (Fig. 7(c)) and wall (Fig. 7(d)) of the pores in scaffolds. They attached on the wall, and the pseudopods could be seen clearly (Fig. 7(a)). When the cells were confluent, they could cross the border of the pores (Fig. 7(b)) and were shown to have contact with each other. 3.2.3. Cell viability on scaffolds The cell viability as analyzed by the WST-1 assay of DPSC and SHED in chitosan scaffolds is shown in Table 3. The optical densities of DPSC in the 2% and 3% scaffold groups were significantly higher than those of the controls on day 8, 15 and 21. Particularly on day 21, the optical density of 2% scaffold group was not only higher than that of the control group but also higher than that of 3% scaffold group
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S129
Table 2 Cytotoxicity test by MTT assay DPSC SHED
Control 0.430 ± 0.044 0.366 ± 0.021
2% chitosan 0.680 ± 0.055∗ 0.454 ± 0.022∗
3% chitosan 0.627 ± 0.043∗ 0.417 ± 0.016
Notes: Means ± SD of optical density in different cell groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.01).
Fig. 6. Cytotoxicity test by MTT assay: OD (A = 572 nm) was expressed as a measure of cell viability after exposure to the 2% and 3% extraction media after 72 h, and the cells cultured with normal media were set as a control, n = 6. Error bars represent means ± SD. Data was analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.01).
(Fig. 8(a)). SHED showed similar results as DPSC except that between day 15 and day 21, the optical density of the 3% group was somewhat lower (Fig. 8(b)). 4. Discussion The procedure used in the present study for preparation of chitosan scaffold is easy and the used materials are not expensive due to locally availability. We have food grade chitosan produce by a local factory using law material from the sea. We introduced a centrifuge step before the material was freezedried according to the method used by Park et al. [16], and then we obtained 2% chitosan scaffolds with the pore size of 188.71 ± 51.90 µm and 3% scaffolds with the pore size of 195.30 ± 67.21 µm. Viable cell sizes of DPSC and SHED can be read from cell counter that were at the range of 10–40 µm. Mean size range of osteoblasts is 10–30 µm [23]. Kose et al. suggested that the average pore size should be at least three times larger than the size of cells so that a single cell could establish contact with others cells [24]. Fibroblasts have been demonstrated to bind to a wide range of pore sizes from 95 to 150 µm and the viability of the cells would increase with decreasing pore size until no cells could fit into the pores
S130
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Fig. 7. Scanning electron micrographs of cells cultured in chitosan scaffolds for 16 days. (a) On the scaffolds; (b) across the border of the pores; (c) on the bottom of the pores; (d) on the wall of the pores.
Table 3 Cell viability on scaffolds detected by WST-1 assay Control Day 8 Day 15 Day 21
0.777 ± 0.063 1.414 ± 0.275 1.534 ± 0.018
Day 8 Day 15 Day 21
0.787 ± 0.098 1.214 ± 0.129 1.453 ± 0.012
2% chitosan DPSC 1.142 ± 0.112∗ 2.286 ± 0.049∗ 2.854 ± 0.103*,# SHED 1.365 ± 0.067∗ 2.470 ± 0.099∗ 2.905 ± 0.066*,#
3% chitosan 1.105 ± 0.095∗ 2.062 ± 0.101∗ 2.343 ± 0.212∗,# 1.603 ± 0.018∗ 2.150 ± 0.076∗ 2.121 ± 0.178*,#
Notes: Means ± SD of optical density in different cell and chitosan scaffold groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level (∗ P < 0.05 between control and test groups, # P < 0.05 within 2% and 3% scaffold groups).
[25]. Another study suggested that porous scaffold microstructure with minimal pore size ranging from 100 to 150 µm was usually required to allow tissue in growth [26].
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S131
Fig. 8. OD (A = 450 nm) was expressed as a measure of cell ((a) DPSC; (b) SHED) viability in scaffolds on day 8, 15 and 21, n = 6. Error bars represent means ± SD. Means ± SD of optical density in different cell and chitosan scaffold groups analyzed by one-way ANOVA, and the statistical significance was accepted at the 0.05 confidence level.
Pore size of used scaffolds in our study was approximately three times the size of SHED and DPSC. Therefore, it is reasonable to choose scaffolds with the pore size as found in our study. Figure 7 confirmed that the cells were able to maintain their normal morphology and could get into the pore of scaffolds.
S132
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Next, we examined the swelling ratio of those chitosan scaffolds. Both chitosan concentrations of 2% and 3% scaffolds possess an equally high swelling ratio. It could preserve a high volume of liquid within the porous structure, maintained their dimensions, and further enhance the penetration of cells into the inner area of the scaffold [17,27,28]. Seda et al. found [14] that swelling ratios of 2% chitosan scaffolds were significantly higher than that of 3% scaffolds since in their study pore size of 2% scaffold was larger than 3% scaffolds, and a larger pore was assumed to contain more liquid. However, when we used the scaffolds with almost equal pore size, we got the same result as theirs. Hsieh et al. suggested that the mechanical property of scaffold increased when the chitosan concentration increased from 1 to 3% [29]. It resulted in 3% scaffold possess stronger compressive strength. When liquid got into scaffolds, the pores of 2% scaffolds would expand more than those of 3% scaffolds even though their sizes were equal. Degradation occurred of both 2% and 3% scaffolds. Chitosan is mainly degraded by lysozyme [19] which is present in various human body fluids and tissues [24]. β-(1-4)-glycosidic bonds between polysaccharide chains are related to the functional activity of lysozyme, and after all β-(1-4)-glycosidic bonds are broken, the degradation will decelerate [29]. Chitosan with more β-(1-4)-glycosidic bonds would decelerate degradation rate. The 2% chitosan scaffold contains about 6% less β-(1-4)-glycosidic bonds than the 3% scaffold. Therefore, the degradation rate of 2% chitosan scaffold was only about 6% higher than those of 3% chitosan scaffold. Moreover, we agree with a previous study, slight increase of chitosan concentration could decelerate the degradation [29], but would not significant impact on the scaffold degradation. The rate of scaffold degradation should mirror the rate of new tissue formation or be adequate for the controlled release of bioactive molecules [12]. According to above, we can easily control the rate of scaffold degradation by adjusting the chitosan concentration. The chitosan scaffold appeared to be non-cytotoxic. After 24, 48 and 72 h, no changes in cellular morphology were detected microscopically in both test and control groups. Toxicity of chitosan is reported to depend on degree of deacetylation (DD). Chitosan with DD higher than 35% showed low toxicity [30]. Chitosan DD in our study was 85%, and the cytotoxicity experiments showed that the viability of SHED and DPSC was not influenced by scaffold-conditioned medium. Hence, those scaffolds were considered to be non-toxic. During scaffold-conditioned medium preparation, some chitosan oligosaccharides may be released into the medium. Those oligosaccharides had been demonstrated to possess biological activities [31]. In line with this possibility, DPSC viability in scaffold-conditioned medium groups and SHED viability in 2% scaffold-conditioned medium group were significantly higher than those of normal medium control group. In addition, the 3% chitosan scaffold possess stronger mechanical force than 2% chitosan scaffold [29], therefore oligosaccharides released from 3% scaffold-conditioned medium were possibly less than those from 2% scaffold-conditioned medium. For this reason, SHED and DPSC viabilities in 2% scaffold-conditioned medium group were higher than those in 3% scaffold-conditioned medium group. On the other hand, a small number of growth factor or components from fetal bovine serum, for example insulin, may bond to chitosan [32] when scaffold-conditioned medium was prepared. Insulin could promote glucose taken up by cells to provide enough energy for cell growth. The 2% chitosan scaffold bond less such components than 3% chitosan scaffold during scaffold-conditioned medium preparation, thus 2% scaffold-conditioned medium contain more such components result in higher cell viabilities in 2% scaffold-conditioned medium. Moreover, Howling et al. found chitosan could stimulate, no effect or inhibit cell proliferation when it cultured with different fibroblast strains or cell lines [31]. In our study,
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S133
DPSC viability of 3% scaffold-conditioned medium group was significantly higher than control group whereas SHED viability of 3% scaffold-conditioned medium group was not significantly different from control group. It suggested that chitosan also effected on stimulation of DPSC and SHED proliferation differently. The cytocompatibility of chitosan has been proved in vitro with myocardial, endothelial and epithellial cells, fibroblasts, hepatocytes, condrocytes, keratinocytes [33] and periodontal ligament cells [34]. Chitosan contains a large amount of amino groups, which give it high positive charges. It has been demonstrated that all vertebrate cells possess unevenly distributed negative surface charges [35]. Cells could bind tightly within chitosan via electrostatic interaction. Figure 7 shows that the cells were able to maintain their normal morphology in the scaffolds. These results indicate that chitosan scaffolds were suitable for DPSC and SHED to adhere to and proliferate. General recommendations for chitosan application in tissue engineering are summarized by Inmaculada et al.: (1) A DD of around 85% is good for cell proliferation and scaffold structure maintenance; (2) A high Mw assists in prolonging biodegradation of scaffolds [12]. We chose the commercial chitosan product of 85% as mentioned above, which is good for cell proliferation. Results showed that SHED and DPSC viabilities in scaffold groups were significant higher than those in control groups, since there was much more culture space in the three-dimensional scaffolds, and cells could continue growing without contacting inhibitions [34]. Above swelling study demonstrated that 2% chitosan scaffolds possess a higher swelling ratio than 3% chitosan scaffolds, in other words, 2% chitosan scaffolds could preserve a higher volume of media in the porous structure and could further enhance the penetration of cells into the inner area of the scaffolds more than 3% scaffolds. It would result in SHED and DPSC viabilities in 2% scaffold groups were significantly higher than those in 3% scaffold groups. These results suggest that the concentration of chitosan might affect the cell growth. However, cell responses are different for each cell type. Chatelet et al. studied the relationship between the cell type and adhesion by comparing between keratinocyte and fibroblasts. They concluded that the type of cell was a factor that also affected the adhesion, being more favorable for fibroblasts which exhibit a more negative surface charge [33]. It suggested that, besides DD, the cell types probably related to cytocompatibility. Notice from Fig. 8 and Table 3, SHED appeared better viability than DPSC, that may suggest surface of SHED possess more negative charge than DPSC. Different from DPSC viability of 3% chitosan group, SHED viability of 3% chitosan group had a decreasing trend from day 15 to day 21. We could assume that SHED began to differentiate on 3% chitosan scaffold. Accordingly, our results showed that 3% chitosan possibly induce SHED differentiation. However, further studies are needed to confirm this detection. The present study demonstrated that chitosan scaffolds prepared according to our novel method show a mild swelling in body fluid and degraded in lysozyme solution. The used scaffolds appear to simulate DPSC and SHED growth and are more suitable for SHED proliferation. The 3% chitosan may induce SHED differentiation. Further studies are needed to confirm that chitosan possesses the ability to induce differentiation of dental pulp derived cells.
Acknowledgements We would like to extend our gratitude to Prof. Vincent Everts for his critical reading of the manuscript. This study was supported by Thailand Research Fund and Prince of Songkla University, Hadyai, Songkla, Thailand.
S134
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
Conflict of interest The authors have declared no conflict of interest.
References [1] S. Gronthos, M. Mankani, J. Brahim, P.G. Robey et al., Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo, Proc. Natl. Acad. Sci. USA 97 (2000), 13625–13630. [2] M. Miura, S. Gronthos, M. Zhao, B. Lu et al., SHED: stem cells from human exfoliated deciduous teeth, Proc. Natl. Acad. Sci. USA 100 (2003), 5807–5812. [3] P. Bianco and P.G. Robey, Stem cells in tissue engineering, Nature 414 (2001), 118–121. [4] G.T. Huang, T. Yamaza, L.D. Shea, F. Djouad et al., Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model, Tissue Eng. Part A 16 (2010), 605–615. [5] W. Zhang, X.F. Walboomers, T.H. van Kuppevelt, W.F. Daamen et al., The performance of human dental pulp stem cells on different three-dimensional scaffold materials, Biomaterials 27 (2006), 5658–5668. [6] K.W. Ng and D.W. Hutmacher, Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices, Biomaterials 27 (2006), 4591–4598. [7] G.T. Huang, W. Sonoyama, J. Chen and S.H. Park, In vitro characterization of human dental pulp cells: various isolation methods and culturing environments, Cell Tissue Res. 324 (2006), 225–236. [8] Q. Hu, B. Li, M. Wang and J. Shen, Preparation and characterization of biodegradable chitosan/hydroxyapatite nanocomposite rods via in situ hybridization: a potential material as internal fixation of bone fracture, Biomaterials 25 (2004), 779–785. [9] M.V. Risbud and R.R. Bhonde, Polyacrylamide–chitosan hydrogels: in vitro biocompatibility and sustained antibiotic release studies, Drug Deliv. 7 (2000), 69–75. [10] A. Di Martino, M. Sittinger and M.V. Risbud, Chitosan: a versatile biopolymer for orthopaedic tissue-engineering, Biomaterials 26 (2005), 5983–5990. [11] I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo et al., Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008), 1–21. [12] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed and A. Heras, Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009), 203–230. [13] S. Shi, P.M. Bartold, M. Miura, B.M. Seo et al., The efficacy of mesenchymal stem cells to regenerate and repair dental structures, Orthod. Craniofac. Res. 8 (2005), 191–199. [14] R. Seda Tigli, A. Karakecili and M. Gumusderelioglu, In vitro characterization of chitosan scaffolds: influence of composition and deacetylation degree, J. Mater. Sci. Mater. Med. 18 (2007), 1665–1674. [15] T. Freier, H.S. Koh, K. Kazazian and M.S. Shoichet, Controlling cell adhesion and degradation of chitosan films by N-acetylation, Biomaterials 26 (2005), 5872–5878. [16] O.S. Park L and J. Lee, Investigation of pore size effect on cell compatibility using pore size gradient chitosan scaffold, Key Engineering Materials 342–343 (2007), 285–288. [17] S.N. Park, H.J. Lee, K.H. Lee and H. Suh, Biological characterization of EDC-crosslinked collagen–hyaluronic acid matrix in dermal tissue restoration, Biomaterials 24 (2003), 1631–1641. [18] T. Kokubo, H. Kushitani, S. Sakka, T. Kitsugi et al., Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W, J. Biomed. Mater. Res. 24 (1990), 721–734. [19] K.M. Varum, M.M. Myhr, R.J. Hjerde and O. Smidsrod, In vitro degradation rates of partially N-acetylated chitosans in human serum, Carbohydr. Res. 299 (1997), 99–101. [20] ISO/EN10993-5, Biological evaluation of medical devices-Part 5 tests for cytotoxicity. In vitro methods, 8.2 tests on extracts. [21] P. Ngamwongsatit, P.P. Banada, W. Panbangred and A.K. Bhunia, WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line, J. Microbiol. Methods 73 (2008), 211–215. [22] M. Ishiyama, H. Tominaga, M. Shiga, K. Sasamoto et al., A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet, Biol. Pharm. Bull. 19 (1996), 1518–1520. [23] P. Arpornmaeklong, N. Suwatwirote, P. Pripatnanont and K. Oungbho, Growth and differentiation of mouse osteoblasts on chitosan–collagen sponges, Int. J. Oral Maxillofac. Surg. 36 (2007), 328–337. [24] G.T. Kose, H. Kenar, N. Hasirci and V. Hasirci, Macroporous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) matrices for bone tissue engineering, Biomaterials 24 (2003), 1949–1958.
Z. Guan et al. / Fabrication, characterization and cell cultures on a novel chitosan scaffold
S135
[25] F.J. O’Brien, B.A. Harley, I.V. Yannas and L.J. Gibson, The effect of pore size on cell adhesion in collagen-GAG scaffolds, Biomaterials 26 (2005), 433–441. [26] J. Zeltinger, J.K. Sherwood, D.A. Graham, R. Mueller et al., Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition, Tissue Eng. 7 (2001), 557–572. [27] L. Ma, C. Gao, Z. Mao, J. Zhou et al., Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering, Biomaterials 24 (2003), 4833–4841. [28] N. Shanmugasundaram, P. Ravichandran, P.N. Reddy, N. Ramamurty et al., Collagen–chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells, Biomaterials 22 (2001), 1943–1951. [29] W.C. Hsieh, C.P. Chang and S.M. Lin, Morphology and characterization of 3D micro-porous structured chitosan scaffolds for tissue engineering, Colloids Surf. B Biointerfaces 57 (2007), 250–255. [30] N.G. Schipper, K.M. Varum and P. Artursson, Chitosans as absorption enhancers for poorly absorbable drugs. 1: Influence of molecular weight and degree of acetylation on drug transport across human intestinal epithelial (Caco-2) cells, Pharm. Res. 13 (1996), 1686–1692. [31] G.I. Howling, P.W. Dettmar, P.A. Goddard, F.C. Hampson et al., The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro, Biomaterials 22 (2001), 2959–2966. [32] T. Mori, M. Okumura, M. Matsuura, K. Ueno et al., Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro, Biomaterials 18 (1997), 947–951. [33] C. Chatelet, O. Damour and A. Domard, Influence of the degree of acetylation on some biological properties of chitosan films, Biomaterials 22 (2001), 261–268. [34] L. Peng and R.-X. Zhuo, Biological evaluation of porous chitosan/collagen scaffolds for periodontal tissue engineering, in: The 2nd International Conference on Bioinformatics and Biomedical Engineering, 2008, pp. 897–900. [35] J.Z. Borysenko and W. Woods, Density, distribution and mobility of surface anions on a normal/transformed cell pair, Exp. Cell Res. 118 (1979), 215–227.
