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stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: Effect of degree of deacetylation
Colloids and Surfaces B: Biointerfaces 142 (2016) 105–113
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Relationships between surface roughness/stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: Effect of degree of deacetylation Shih-Feng Chou a,b , Jui-Yang Lai a,c,d,e,∗ , Ching-Hsien Cho a , Chih-Hung Lee a a
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA c Biomedical Engineering Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China d Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China e Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan 33305, Republic of China b
a r t i c l e
i n f o
Article history: Received 20 November 2015 Received in revised form 30 January 2016 Accepted 24 February 2016 Available online 27 February 2016 Keywords: Chitosan coating Degree of deacetylation Surface roughness/stiffness Corneal keratocyte Spheroid fabrication
a b s t r a c t Fabrication of the cell spheroids from corneal keratocytes has important implications to the advance in tissue engineering while stimulation from the interface of a biopolymer coating has the ability to modulate this event. This study aims to investigate the dependence of keratocyte migration, proliferation, and differentiation on the surface roughness/stiffness of the chitosan coatings through modifications by degree of deacetylation (DD). After a series of deacetylation process, chitosan coatings with increasing DD exhibited significantly decreased surface roughness and increased surface stiffness. Relationships between the behaviors of rabbit corneal keratocytes (RCKs) and biopolymer coatings with varying DDs (between 75% and 96%) were also found during in vitro cultivation. Both the surface roughness increase and stiffness decrease could lead to enhanced cell migration, which is the main driving force for the early stage spheroid formation on chitosan substrates (e.g., within 8 h). With these stimulations from the substrate interfaces, the size and morphology of RCK spheroids were greatly affected by the DD of chitosan. When fabricated on a lowered DD of chitosan material, the spheroids had a larger size with abundant extracellular matrix produced around the cells. At a later stage of spheroid cultivation (e.g., 5 days), significantly higher amount of RCKs on chitosan coatings was noted with increasing DD, indicating the substrate interface effects on cell proliferation. The keratocan expression of RCK spheroids grown on a lowered DD of chitosan was up-regulated, suggesting that both the surface roughness increase and stiffness decrease may facilitate the microenvironment for preservation of cellular phenotype. Overall, our work contributes to the scientific understanding of the keratocyte behaviors and spheroid fabrications in response to DD-mediated surface roughness/stiffness of chitosan coatings. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cell culture is a fundamental and important topic in tissue engineering since it simulates cell behaviors outside the native tissues with the possibility of engineering cells exhibiting desirable structure-function relationships. The underlying challenges in cell culture are to maintain the characteristic structures, behaviors, and metabolisms of the cells outside their complex tissues and organs [1]. Typically, formations of three-dimensional (3-D) spheroids and 2-D monolayers are the two most common cell configurations
∗ Corresponding author at: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China. E-mail address: [email protected] (J.-Y. Lai). http://dx.doi.org/10.1016/j.colsurfb.2016.02.051 0927-7765/© 2016 Elsevier B.V. All rights reserved.
during cultivation while the environmental conditions may determine the corresponding configurations in either 2-D or 3-D. Studies in fabrication of 3-D spheroids received many interests over the 2D monolayers given that the cells grown in a 3-D culture manner closely mimicked in vivo conditions and exhibited a higher similarity to the real tissues in many aspects. The unique 3-D structures are often accompanied with a high rate of proliferation and differentiation from cells, and their structure-function relationships have been reviewed by Lin and Chang [2]. It is generally believed that the most important aspect of spheroid cultivation is perhaps bridging in vitro cell assays and in vivo animal models. In addition, a 3-D spheroid culture technique has been used for purification and elimination of the undifferentiated cells as demonstrated by Hattori et al. [3]. Other biomedical applications that have been investigated by 3-D spheroids include the providing of ideal conditions for
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bioprinting and tissue regenerations [1,2]. Owing to these advantages, we seek for cultivation of keratocytes into 3-D spheroids using a biopolymer substrate while addressing the interfacial effects on the self-adjustment of spheroid configuration. Keratocytes, located in corneal stroma, are the major cellular components residing between the collagen lamellae. One of the important functions of keratocytes involves in the synthesis of extracellular matrix (ECM) that is essential to the strength and transparency of cornea. Upon injury or age-related damage to cornea, quiescent keratocytes initiate their cell cycles in proliferation, differentiation, and migration to the injury sites. However, depending on the types of injury and specific environmental conditions of the cornea, keratocytes may proliferate and differentiate into other cell types such as fibroblasts or myofibroblasts that may not function properly in native eyes and can sometimes reduce the vision [4]. Due to this issue, there is an unmet challenge in tissue engineering to allow the cultivation of corneal keratocytes in vitro while preserving their phenotype. To achieve this, cultivation of keratocytes into 3-D spheroids becomes an alternative route. In addition, several studies have suggested the formation of keratocyte spheroids while preserving the phenotypic expression. For example, Scott et al. showed the formation of keratocyte spheroids with specific phenotypic markers in a serum-free medium [5]. In another study, Li et al. demonstrated the formation of keratocyte spheroids in suspension with the addition of methylcellulose to promote spheroid formation [6]. Furthermore, Funderburgh et al. compared spheroid formation from the effect of matrix during cultivation [7]. Their results suggested that the expression of keratocan, a proteoglycan component found uniquely from keratocytes, was higher in unattached spheroids than those attached to the matrix during cultivation. In general, these researchers showed the possibility in the formation of keratocyte spheroid especially from an aqueous solution/suspension. Although keratocytes are anchorage-dependent cells, the formation of cell spheroids may become feasible with proper stimulations from the interface through cell-matrix crosstalks. Among all potential biopolymer substrates, chitosan coating has gained an increasing interest due to its excellent biocompatibility for cell culture and outstanding ability to promote spheroid formation. For example, Cheng et al. demonstrated the formation of spheroids from human adipose-derived stem cells (ASC) on chitosan coatings [8]. Their results suggested that chitosan materials enhanced spheroid formation of ASCs over 7 days of culture as compared to those on TCPS control. In another study, Lin et al. reported the formation of melanocyte spheroids on chitosan coatings and
compared the characteristics of the spheroids with different cell seeding densities [9]. These two examples clearly demonstrated the possibility of using chitosan coatings as substrate materials to fabricate spheroids during cell cultivation. However, research involving in the use of chitosan coatings for cell culture rarely considered the degree of deacetylation (DD) of chitosan as a crucial factor in spheroid formation, especially in keratocytes. DD is one of the most important physicochemical parameters affecting the surface properties of chitosan coatings as reported by Foster et al. [10]. In addition, the cell behaviors such as migration, aggregation, proliferation, and differentiation are known to be modulated by tuning the interface properties of the substrate materials. These considerations motivate us to address the gap in keratocyte spheroid fabrication on chitosan materials due to the effect of DD. As a promising ophthalmic biomaterial, chitosan is a polysaccharide consisting of randomly distributed units of (1–4)-linked d-glucosamine and N-acetyl-d-glucosamine [11]. It is usually obtained by alkaline N-deacetylation process of chitin, which may simultaneously lead to chemical depolymerization of biopolymer. Therefore, in this study, the deacetylated chitosan samples with varying DDs were prepared by heat-alkaline treatment under a nitrogen atmosphere to avoid the molecular weight modification due to oxidative mechanisms [12]. The molecular weight of biopolymer was controlled at the same level to ensure the clarification of the role of DD of chitosan in the bioengineering of corneal keratocyte spheroids. In the present study, we hypothesized that the DD-mediated surface roughness/stiffness of chitosan coatings plays important roles on the migration, proliferation, and differentiation of keratocytes. In addition, the cultivation of keratocyte spheroids and their corresponding morphology might be modulated by DD of the chitosan coatings. In a study by Chen et al., bovine corneal keratocyte spheroids were formed on a chitosan (DD value of 85%) coating [13]. Although it is promising to see that the keratocyte spheroids maintained phenotypes under serum-containing culture conditions, the effects of DD of chitosan on the fabrication of keratocyte spheroids and their corresponding cell behaviors remained unclear. Therefore, this work aims to investigate keratocyte spheroid fabrication on chitosan coatings of varying DD values. We found that chitosan deacetylation significantly affected the surface characteristics such as surface roughness and stiffness of the coating biomaterials. Furthermore, cell behaviors and spheroid fabrication were mediated through these stimulations from the substrate interface. For the first time, here, we demonstrated that the surface roughness/stiffness of chitosan coatings, as a result of DD,
Fig. 1. AFM measurements on surfaces of TCPS and samples coated with chitosan of varying DDs. (a) 3-D height images; (b) Rq (i.e., mean square roughness) and elastic modulus. Values are mean ± SD (n = 3). *P < 0.05 vs all groups.
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Fig. 2. Cell migration study after 8 h of RCK cultivation on surfaces of TCPS and samples coated with chitosan of varying DDs. The RCKs were further collected and placed on a porous membrane to allow migration to the opposite side of the membrane for 4 h. (a) Typical optical images of reverse side of the porous membrane stained with crystal violet. Scale bars indicate 50 m. (b) Quantitative analysis of cell migration level from the images shown in (a). Data in the experimental groups are percentages relative to that of TCPS groups. Values are mean ± SD (n = 4). *P < 0.05 vs all groups.
altered the migration, proliferation, and differentiation of keratocytes, thereby resulting in the various formations and architectures of the rabbit corneal keratocyte (RCK) spheroids.
2. Materials and methods 2.1. Materials Chitosan powder, derived from crab shell (Cat. No. 28191), was obtained from Fluka (Milwaukee, WI, USA). The DD and viscosityaverage molecular weight (Mv ) of as-received chitosan (DD75
group), as suggested by the manufacturer, were approximately 75–80% and 400 kDa, respectively. Deacetylated chitosan samples were prepared according to Tolaimate et al. [14]. In the DD85 group, the as-received chitosan was exposed to 60% (w/v) NaOH solution for 1 h at 100 ◦ C under a nitrogen atmosphere followed by extensive rinsing with deionized water until a neutral pH was reached. In addition, to obtain the highly deacetylated product (DD96 group), the as-received chitosan was treated twice by the same deacetylation procedure as described above for DD85 material, but with a minor modification. Each exposure time during the repeated alkaline treatment was set at 1.5 h. The DD and Mv of chitosan
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Fig. 3. (a) Representative light microscopic images of RCKs at 5 days after plating on surfaces of TCPS and samples coated with chitosan of varying DDs. Scale bars indicate 1000 m. (b) The diameter distribution histograms of RCK spheroids formed on various chitosan coatings and corresponding Gaussian fits.
samples were determined by ninhydrin assay and viscometric method, respectively (see Supporting information). Phosphatebuffered saline (PBS, pH 7.4) was purchased from Biochrom (Berlin, Germany). 24-well non-tissue culture polystyrene plates (Falcon 351147) and tissue culture polystyrene (TCPS) plates (Falcon 353047) were purchased from Becton Dickinson Labware (Franklin Lakes, NJ, USA). 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12) and TRIzol reagent were purchased from Gibco-BRL (Grand Island, NY, USA). Bovine serum albumin (BSA) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution (10,000 U/ml penicillin, 10 mg/ml streptomycin, and 25 g/ml amphotericin B) were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). All the other chemicals were of reagent grade and used as received without further purification.
2.2. Preparation of chitosan-coated culture substrates Chitosan solution was obtained by dissolving 1 g of as-received or deacetylated chitosan powder in 50 ml of acetic acid solution (1% v/v) with stirring until complete dissolution. The insoluble substances were removed by a filter paper (Tokyo Roshi Kaisha, Tokyo, Japan). To obtain chitosan coatings, 0.5 ml of the filtered chitosan solution was poured into the 24-well plate (Falcon 351147) and allowed to air-dry for 2 days at room temperature. The chitosan coatings were immersed in a 0.5 N NaOH solution for 1 h and rinsed extensively with deionized water until neutrality. After air-drying, the thickness of chitosan coatings was measured by a QuaNix 8500 thickness gauge (Automation Nix Gmbh, Koln, Germany). Ten measurements were done on different surface sites to calculate the thickness for each coating sample. Results were averaged on five
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Fig. 4. Representative scanning electron microscopic images of RCKs at 5 days after plating on surfaces of (a) TCPS and samples coated with chitosan of varying DDs (b) 75%, (c) 85%, and (d) 96%. Scale bars indicate 5 m.
Fig. 5. The OD value at 450 nm for RCKs cultured on TCPS and various chitosan coatings for 1, 3, and 5, days. An asterisk indicates statistically significant differences (*P < 0.05; n = 4) for the mean value of OD compared to value at previous time point. # P < 0.05 vs all groups; + P < 0.05 vs TCPS groups; ˆ P < 0.05 vs TCPS and DD75 groups (compared only within each time point group).
independent runs. Chitosan coatings had an average thickness of 12.3 ± 0.5, 11.9 ± 0.2, and 11.8 ± 0.5 m without statistical significance for DD75, DD85, and DD96, respectively (P > 0.05). 2.3. Atomic force microscopy An atomic force microscope (AFM) (NanoScope IV; Veeco Digital Instruments, Santa Barbara, CA, USA) was utilized to scan surface topography of the chitosan coatings [15]. The AFM samples were prepared as described in Section 2.2. The roughness of dry surfaces was analyzed in air. All measurements were made in tapping mode using a silicon cantilever at room temperature. AFM images were
recorded with a scan size of 5 m × 5 m. Five measurements were done on different surface sites to calculate the root mean square roughness (Rq) for each sample. Results were averaged on three independent runs. The nanoscale mechanical properties of the chitosan samples were characterized by force-volume spectroscopy using the same AFM. A silicon cantilever operated in contact mode was employed to observe the topography. During the measurements, a defined force was applied to various surface sites using a calibrated tip. The amount of cantilever deflection was monitored to determine the surface stiffness (elastic modulus) of the samples by collecting force–volume curves at four different regions. Results were averaged on three independent runs.
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Fig. 6. Gene expression level of keratocan in RCKs grown on surfaces of TCPS and samples coated with chitosan of varying DDs for 5 days by real-time RT-PCR. Data in the experimental groups are percentages relative to that of TCPS groups. Values are mean ± SD (n = 4). *P < 0.05 vs all groups.
2.4. Cell migration assays
2.6. Cell morphology studies
All animal procedures were approved by the Institutional Review Board and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-eight adult New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC) weighing 2.5–3.0 kg were used for this study. Primary rabbit corneal stromal cells were prepared according to our previously published methods [16]. The keratocytes were maintained in regular culture medium consisting of DMEM/F12, 10% FBS, and 1% A/A solution. In all experiments, third-passage rabbit corneal keratocytes (RCKs) were used. Cell migration assays were performed according to a reported method [17]. RCKs with a density of 5 × 104 cells/ml were seeded into 24-well TCPS (Falcon 353047) and various chitosan-coated plates by 1 ml/well. After incubation at 37 ◦ C for 8 h, the cultured cells were detached using trypsin-EDTA. A 100 l suspension containing 2 × 104 RCKs was plated into 24-well Millicell containing polycarbonate filters with 8-m pores (Millipore, Bedford, MA, USA). The cells were allowed to migrate for 4 h. Then, the migrated cells on the bottom surface of the filter were stained with 0.1% crystal violet and photographed using an optical microscope (Nikon, Melville, NY, USA) to capture cell migration images. The number of cells on the bottom side of the filter was counted at six different areas. All experiments were performed in quadruplicate, and the results were expressed as relative cell migration level normalized to the controls (TCPS groups).
After 5 days of cultivation of RCKs on various substrates, the cellular morphology was observed by scanning electron microscopy (SEM) [18]. The cell cultures were fixed with 2% glutaraldehyde in 0.1 M cacodylic acid buffer (pH 7.4) overnight at 4 ◦ C. After rinsing them with 0.1 M cacodylic acid buffer, the specimens were postfixed in 1% osmium tetroxide and dehydrated in a graded series of ethanol solutions. Then, the cell samples were dried with carbon dioxide in a critical point dryer (Balzers, Liechtenstein) and coated with gold in a sputter coater (Hitachi, Tokyo, Japan) before examination under a Hitachi S-3000N SEM with an accelerating voltage of 10 kV. 2.7. Cell proliferation assays After incubation for 1–5 days, the RCKs were counted by using the cell proliferation reagent WST-1 (Roche Diagnostics, Indianapolis, IN, USA) assay. The WST-1 assay is based on the cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) to a colored formazan by mitochondrial dehydrogenases in viable cells [19]. The amount of formazan product is proportional to the number of metabolically active cells. For staining, 100 l of WST-1 reagent was added to the cultures, and incubated for 4 h at 37 ◦ C in a CO2 incubator. The optical density (OD) value at 450 nm was recorded using the Multiskan Spectrum Microplate Spectrophotometer (ThermoLabsystems, Vantaa, Finland). All experiments were performed in quadruplicate.
2.5. Quantification of cell spheroids RCKs with a density of 5 × 104 cells/ml were seeded into various chitosan-coated plates by 1 ml/well, and incubated in regular culture medium at 37 ◦ C for 1–5 days. The culture wells were photographed at six different fields by using an optical microscope. The RCK spheroid diameter was analyzed with the Adobe Photoshop software. The criterion for the formation of cell spheroids is considered to be that when the cell aggregate has a diameter > 50 m. All experiments were performed in quadruplicate. To quantitatively estimate the diameter distribution of the cell spheroids, the histogram and corresponding Gaussian fitting curve were obtained from the values for 30 cell spheroids.
2.8. Quantitative real-time reverse transcription polymerase chain reaction analyses After cultivation on TCPS and chitosan coatings for 5 days, the total RNA was isolated from cells with TRIzol reagent [20]. Reverse transcription of the extracted RNA (1 g) was performed using ImProm-II and Oligo(dT)15 primers (Promega, Madison, WI, USA). The primers used to amplify the rabbit keratocan complementary DNA (cDNA) were 5 -CTCACGTGGCTTTGATGTGT3 (sense) and 5 -GACCTTTGTGAGGCGATTGT-3 (antisense). The sequences of the primer pair used to amplify the internal control cDNA, glyceraldehyde-3-phosphate dehydrogenase
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Table 1 Physicochemical characteristics of chitosan samples. Sample code
Processing method
DDa (%)
Mv b (kDa)
DD75 DD85 DD96
As-received Deacetylation (60% NaOH, 100 ◦ C, 1 h) Deacetylation (60% NaOH, 100 ◦ C, 1.5 h*2 times)
75.6 ± 0.6 85.2 ± 0.4c 96.3 ± 0.4c
336.5 ± 2.4 339.2 ± 1.8 340.2 ± 2.5
a b c
Determination of deacetylation degree (DD) by ninhydrin assay. Data are expressed as mean ± standard deviation (n = 5). Determination of viscosity-average molecular weight (Mv ) by viscometric method. Data are expressed as mean ± standard deviation (n = 4). Significant difference as compared to the DD75 groups (P < 0.05).
(GAPDH), were 5 -TTGCCCTCAATGACCACTTTG-3 (sense) and 5 TTACTCCTTGGAGGCCATGTG-3 (antisense). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on a Light-Cycler instrument (Roche Diagnostics) with FastStart DNA Master SYBR Green I reagent. Each sample was determined in quadruplicate, and the gene expression results were normalized to the level of GAPDH mRNA. 2.9. Statistical analyses Results were expressed as mean ± standard deviation (SD). Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with P < 0.05.
Fig. 2a shows the optical images of RCKs on the reverse side of the transwell filter inserts stained with crystal violet. An obvious amount of RCKs can be seen, indicating that the cells pre-treated with chitosan can efficiently migrate through the transwell filter. In addition, Fig. 2b shows the quantitative analysis on the amount of the migrated RCKs, and the average cell migration, expressed in percentage of total cell numbers after normalizing to TCPS control (100%), was 234.7 ± 10.8%, 189.1 ± 12.4%, and 136.3 ± 7.0% for DD75, DD85, and DD96, respectively (P < 0.05). This suggested that RCK migration was facilitated due to the exposure of chitosan materials while cell migration ability decreased with increasing DD of the chitosan coatings.
3. Results 3.4. Quantification of cell spheroids 3.1. Preparation of chitosan-coated culture substrates Table 1 shows the DD and Mv of the as-received and deacetylated chitosan, after exposure to heat-alkaline treatment in a nitrogen environment. From the table, increasing NaOH treatment time increased DD. The findings from ninhydrin assays support the nuclear magnetic resonance (NMR) spectral data (Fig. S1). Further details are provided in the Supporting information. No significant modification to the Mv of the chitosan was found, suggesting that our deacetylation process of chitosan only modified the amide side groups on the N-acetyl-d-glucosamine units. The weight-average molecular weight of chitosan samples as determined by gel permeation chromatography (GPC) also showed no significant change between DD75, DD85, and DD96 groups (P > 0.05) (Fig. S2). Further details are provided in the Supporting information. Thus, changes in surface properties of the chitosan coatings were a dependent of DD instead of molecular weight of the chitosan. 3.2. Atomic force microscopy Surface roughness and stiffness (elastic modulus) of the chitosan coatings at various DDs were examined by AFM. Fig. 1a shows the 3D height images of DD75, DD85, and DD96 coatings where higher DD samples exhibited noticeable surface features such as humps and bumps. In addition, quantitative analyses of the AFM measurements shown in Fig. 1b indicated that the average Rq was 4.8 ± 0.5, 2.1 ± 0.2, and 0.9 ± 0.1 nm for DD75, DD85, and DD96, respectively (P < 0.05). The average elastic modulus of the chitosan coatings, also shown in Fig. 1b, was 6.5 ± 0.3, 7.8 ± 0.3, and 9.6 ± 0.6 MPa for DD75, DD85, and DD96, respectively (P < 0.05). Our results suggested a decreasing surface roughness and an increasing stiffness with increasing DD of the chitosan coatings. 3.3. Cell migration assays The surface characteristics of a biomaterial coating may influence cell behaviors such as the ability to migrate. Here, we performed studies on the migration ability of RCKs using transwell assays after 8 h of cultivation on various chitosan coatings.
According to our data, the formation of RCK spheroids could be observed following 1 day of direct contact with coating materials, irrespective of the DD of chitosan (Fig. S3). Further details are provided in the Supporting information. In addition, Fig. 3a shows representative light microscopic images of RCKs on TCPS control and chitosan coatings after a 5-day culture. It is interesting to note that the size and amount of the RCK spheroids appeared to be different on chitosan coatings of varying DD values. Quantitative analyses supported this finding where spheroids larger than 50 m in diameter were counted and categorized into Gaussian distribution plots shown in Fig. 3b. The RCK spheroids on DD75 exhibited the largest diameter distribution followed by DD85 and DD96. In addition, RCK spheroids with diameter over 150 m were found on DD75 with the majority of the spheroid diameter ranging from 100 to 150 m. Within DD85 and DD96, the diameter of the RCK spheroids decreased from a majority of 100–150 m to 50–100 m as increasing the DD in chitosan coatings. Therefore, DD of the chitosan seemed to have an effect in determination of the number of spheroids and their corresponding size.
3.5. Cell morphology studies Fig. 4 shows SEM images of representative RCK after 5 days of culture on TCPS control and various chitosan coatings. The morphology of RCKs on TCPS displayed a flat and confluent morphology without the evidence of spheroid formation. By contrast, the RCKs cultured on chitosan coatings typically showed the formation of spheroids with differences in morphology and architecture. In addition, RCK spheroids on DD75 coatings showed clear and distinct cell structures with features of ECM all over the surface of each individual cell. The surface features of the RCK spheroids were less noticeable on DD85 and DD96 coatings. Furthermore, RCK spheroids formed on DD75 coatings showed a better depth of field in the SEM image than those on DD85 and DD96. These observations were indicative of the dependence of the spheroid morphology and architecture on the DD of chitosan coatings.
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3.6. Cell proliferation assays The mitochondrial activity of RCK cultures was measured by means of WST-1 assays. Fig. 5 shows the average optical density (OD) of RCKs on TCPS control and various chitosan coatings after cultivation for 1–5 days. The amount of RCKs cultured on TCPS increased significantly from 1 to 3 days (P < 0.05) but no significance from 3 to 5 days (P > 0.05) perhaps due to a minimal space for cells to proliferate. For RCKs cultured on various chitosan coatings, a similar trend in the increase of the OD but less pronounced than that of the TCPS control was found over 5 days of cultivation. Furthermore, considering substrate interface effects, increasing DD of the chitosan coatings increased the OD at 5 days of culture. Based on these, it appeared that surface properties of the chitosan coatings significantly affected the growth of the RCKs. Our results suggested that proliferation of the RCKs on DD75 was less active than DD85 and DD96. 3.7. Quantitative real-time reverse transcription polymerase chain reaction analyses Fig. 6 shows the average keratocan mRNA expression level of RCKs on various chitosan coatings in percentage after normalizing to TCPS control (100%). After cultivation for 5 days, keratocan expression of RCKs on chitosan substrates was significantly higher than that of the TCPS control (P < 0.05). This suggested that the keratocytes maintained their phenotype on all chitosan coatings. In addition to the above observation, we also noticed a significant decrease in keratocan expression with increasing DD of the chitosan materials. Keratocan expression from RCKs on DD75, DD85 and DD96 was found at 493.6 ± 20.8%, 360.5 ± 12.2% and 291.0 ± 10.4%, respectively (P < 0.05). This finding suggested that the surface properties of the chitosan coatings, mediated through DD, were responsible for the phenotypic modulation of keratocytes. 4. Discussion In this study, we reported RCK behaviors and spheroid fabrication due to the substrate interface effects of chitosan coatings modulated through DD. The Rq of underlying TCPS substrates determined by AFM was 3.5 ± 0.4 nm [21]. After coating with chitosan materials of varying DDs, the culture surfaces showed different roughness levels. The influence of increasing DD of chitosan on the reduction of surface roughness was reported by others [22]. In addition, the decrease in surface roughness was due to the removal of fibrillar aggregates in chitosan [23]. More specifically, besides processing conditions of chitosan, other factors such as charge density and molecular chain length of the biopolymer were correlated with chemical heterogeneity-mediated formation of the fibrillar aggregates [24]. From our results, increasing DD decreased the chemical heterogeneity (on the basis of no significant molecular weight changes) and prevented the formation of fibrillar aggregates, and thus, leading to a smoother surface of the chitosan coating. By contrast, decreasing chemical heterogeneity was accompanied by a higher stiffness. Majd et al. have used nanoindentation to obtain surface stiffness of various chitosan films having a DD from 76% to 96% and have found that the surface stiffness increased at a higher DD due to the increase in film crystallinity [25]. Several studies have also correlated stiffness and DD of the chitosan coatings [26–28]. The dependence of stiffness on DD of chitosan may be explained by a much closer packing of the biopolymer chains due to deacetylation where the substituted amino groups attract the neighboring biopolymer chains resulting in a much stronger and stiffer films. Furthermore, the chitosan with a higher DD exhibits a more flexible chain, which may facilitate hydrogen bonding through amino
groups on glucosamine units of the deacetylated chitosan. Overall, our findings in surface characteristics of chitosan coatings were in agreement with these earlier findings, which depended on the DD of the chitosan. Studies have shown that surface properties of a biomaterial coating may greatly influence cell migration ability [29–32]. These studies suggested that cell migration was positively correlated to surface roughness and inversely correlated to matrix stiffness. More specifically, cell migration ability was enhanced due to cellmatrix crosstalks mediated through focal adhesion where short focal adhesions were associated with the highest migration speed [32]. Similar to these literature findings, our data suggested a strong dependence on cell migration due to important physical cues such as matrix stiffness and surface roughness. A higher migration ability of the RCK cells was correlated to the dependence on the size and amount of the spheroids (Fig. S3). For example, RCKs on DD75 may receive a longer time to migrate since no cell attachment was observed up to day 3 as compared to some attachments on DD85 and DD96 after day 1. This resulted in a wider size distribution of RCK spheroids on DD75 than DD85 and DD96. Moreover, an excess of ECM molecules on the surface of cells indicated higher cell migration ability since a constant formation and dissociation of the focal adhesions was required through ECM during migration [32]. In a similar study, the formation of spheroids from mesenchymal stem cells was reported in porous scaffolds with excess deposition of ECM on the spheroids after 2 days of culture [33]. Of particular importance, the study suggested the correlations between anchorages of the cells to the walls of the scaffold as a function of the spheroid formation as well as how fast the spheroids were formed [33]. In general, our observations demonstrated that RCK migration was the main driving force on spheroid formation while the number and size of the spheroids strongly depended on the surface roughness/stiffness of chitosan coatings. According to our results, RCK proliferation on all chitosan coatings significantly increased from 1 to 3 days whereas significant increases in RCK proliferation were only observed on DD85 and DD96 from 3 days to 5 days (P < 0.05). In addition, no statistical significance of RCK proliferation was found within chitosan coatings at 1 day (P > 0.05) whereas significance differences in RCK proliferation were found in all chitosan coatings after 5 days (P < 0.05). These findings suggested that the surface properties of chitosan coatings affected cell proliferation and were in agreement with others who suggested that cell proliferation decreased with increasing surface roughness [34–37]. More importantly, it had been suggested that surface roughness of a biopolymer coating significantly affected the adsorption of fibronectin, a pre-requisite for cell attachment and growth [38]. By contrast, studies showed that stimulations from a stiffer matrix increased cell proliferation [39–43]. In addition, the differences in stiffness-induced cell proliferation were due to increase in integrin signaling through focal adhesion complexes [40]. In general, these earlier findings supported our present proliferation results where the RCKs on DD96 coatings exhibited the highest increase in cell proliferation during 5 days of cultivation. There are at least three phenotypes associated with corneal keratocytes including keratocytes, fibroblast, and myofibroblast [4]. Keratocan, a protein that belongs to one of the keratan sulfate proteoglycans, is the phenotypic marker when keratocytes are being mass-produced during cell proliferation and differentiation [44]. Studies have shown that keratocan expression was significantly up-regulated during cultivation of keratocytes using biomaterial coatings after several weeks [13,45]. In addition, our results in the phenotypic expression of keratocytes suggested that the keratocan levels were not only up-regulated by chitosan coatings but also were mediated through differences in surface characteristics of the substrate materials. The dependence of phenotypic expression of cells on surface roughness of a biomaterial was reported previously
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[46–49]. According to these earlier findings, increasing surface roughness increased the phenotypic expression of cells. In addition, cells with high phenotypic expression found on rougher surfaces produced noticeable ECM in cell morphology [46]. This observation was similar to our results (Fig. 4) where the surface of RCK spheroids on DD75 exhibited a significant amount of ECM as compared to DD85 and DD96. On the other hand, surface stiffness is another important surface property that has been demonstrated to significantly affect the phenotypic expression of cells. Studies suggested that cellular phenotypic expression was inversely proportional to the stiffness of the biomaterial coatings [49–51]. Supported by these examples, we reported a strong dependence of phenotypic expression in keratocyte spheroids with respect to roughness and stiffness of the chitosan materials modulated through deacetylation. 5. Conclusions In summary, this work demonstrated the feasibility of the fabrication of 3-D keratocyte spheroids using chitosan substrates at various DDs without significant modification to their corresponding Mv . In particular, increasing DD from 75 to 96% decreased surface roughness and increased surface stiffness of the chitosan materials. Studies in RCK behaviors suggested the substrate interface effects of biopolymer coatings. Both the surface roughness increase and stiffness decrease could lead to enhancement of cell migration, spheroid formation, and phenotypic maintenance. In addition, the proliferation of RCKs and size of cell spheroids were greatly affected by the surface roughness/stiffness of coating materials. This experimental work confirmed the dependence of the spheroid morphology and architecture on the DD of chitosan. Overall, our findings contributed to the scientific understanding of the keratocyte cultivation and spheroid fabrication in response to DDmediated surface roughness/stiffness of chitosan substrates. Acknowledgements This work was supported by grants CMRPD1B0451 and CMRPD1C0111 from Chang Gung Memorial Hospital and grant NSC99-2221-E-182-008 from the National Science Council of Republic of China. The authors are grateful to Dr. David Hui-Kang Ma (Department of Ophthalmology, Chang Gung Memorial Hospital) and Miss Hsiao-Yun Cheng (Molecular Medicine Research Center, Chang Gung University) for technical assistance.
[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]
Appendix A. Supplementary data
[45]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02. 051.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Relationships between surface roughness/stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: Effect of degree of deacetylation Shih-Feng Chou a,b , Jui-Yang Lai a,c,d,e,∗ , Ching-Hsien Cho a , Chih-Hung Lee a a
Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA c Biomedical Engineering Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China d Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China e Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan 33305, Republic of China b
a r t i c l e
i n f o
Article history: Received 20 November 2015 Received in revised form 30 January 2016 Accepted 24 February 2016 Available online 27 February 2016 Keywords: Chitosan coating Degree of deacetylation Surface roughness/stiffness Corneal keratocyte Spheroid fabrication
a b s t r a c t Fabrication of the cell spheroids from corneal keratocytes has important implications to the advance in tissue engineering while stimulation from the interface of a biopolymer coating has the ability to modulate this event. This study aims to investigate the dependence of keratocyte migration, proliferation, and differentiation on the surface roughness/stiffness of the chitosan coatings through modifications by degree of deacetylation (DD). After a series of deacetylation process, chitosan coatings with increasing DD exhibited significantly decreased surface roughness and increased surface stiffness. Relationships between the behaviors of rabbit corneal keratocytes (RCKs) and biopolymer coatings with varying DDs (between 75% and 96%) were also found during in vitro cultivation. Both the surface roughness increase and stiffness decrease could lead to enhanced cell migration, which is the main driving force for the early stage spheroid formation on chitosan substrates (e.g., within 8 h). With these stimulations from the substrate interfaces, the size and morphology of RCK spheroids were greatly affected by the DD of chitosan. When fabricated on a lowered DD of chitosan material, the spheroids had a larger size with abundant extracellular matrix produced around the cells. At a later stage of spheroid cultivation (e.g., 5 days), significantly higher amount of RCKs on chitosan coatings was noted with increasing DD, indicating the substrate interface effects on cell proliferation. The keratocan expression of RCK spheroids grown on a lowered DD of chitosan was up-regulated, suggesting that both the surface roughness increase and stiffness decrease may facilitate the microenvironment for preservation of cellular phenotype. Overall, our work contributes to the scientific understanding of the keratocyte behaviors and spheroid fabrications in response to DD-mediated surface roughness/stiffness of chitosan coatings. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cell culture is a fundamental and important topic in tissue engineering since it simulates cell behaviors outside the native tissues with the possibility of engineering cells exhibiting desirable structure-function relationships. The underlying challenges in cell culture are to maintain the characteristic structures, behaviors, and metabolisms of the cells outside their complex tissues and organs [1]. Typically, formations of three-dimensional (3-D) spheroids and 2-D monolayers are the two most common cell configurations
∗ Corresponding author at: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China. E-mail address: [email protected] (J.-Y. Lai). http://dx.doi.org/10.1016/j.colsurfb.2016.02.051 0927-7765/© 2016 Elsevier B.V. All rights reserved.
during cultivation while the environmental conditions may determine the corresponding configurations in either 2-D or 3-D. Studies in fabrication of 3-D spheroids received many interests over the 2D monolayers given that the cells grown in a 3-D culture manner closely mimicked in vivo conditions and exhibited a higher similarity to the real tissues in many aspects. The unique 3-D structures are often accompanied with a high rate of proliferation and differentiation from cells, and their structure-function relationships have been reviewed by Lin and Chang [2]. It is generally believed that the most important aspect of spheroid cultivation is perhaps bridging in vitro cell assays and in vivo animal models. In addition, a 3-D spheroid culture technique has been used for purification and elimination of the undifferentiated cells as demonstrated by Hattori et al. [3]. Other biomedical applications that have been investigated by 3-D spheroids include the providing of ideal conditions for
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bioprinting and tissue regenerations [1,2]. Owing to these advantages, we seek for cultivation of keratocytes into 3-D spheroids using a biopolymer substrate while addressing the interfacial effects on the self-adjustment of spheroid configuration. Keratocytes, located in corneal stroma, are the major cellular components residing between the collagen lamellae. One of the important functions of keratocytes involves in the synthesis of extracellular matrix (ECM) that is essential to the strength and transparency of cornea. Upon injury or age-related damage to cornea, quiescent keratocytes initiate their cell cycles in proliferation, differentiation, and migration to the injury sites. However, depending on the types of injury and specific environmental conditions of the cornea, keratocytes may proliferate and differentiate into other cell types such as fibroblasts or myofibroblasts that may not function properly in native eyes and can sometimes reduce the vision [4]. Due to this issue, there is an unmet challenge in tissue engineering to allow the cultivation of corneal keratocytes in vitro while preserving their phenotype. To achieve this, cultivation of keratocytes into 3-D spheroids becomes an alternative route. In addition, several studies have suggested the formation of keratocyte spheroids while preserving the phenotypic expression. For example, Scott et al. showed the formation of keratocyte spheroids with specific phenotypic markers in a serum-free medium [5]. In another study, Li et al. demonstrated the formation of keratocyte spheroids in suspension with the addition of methylcellulose to promote spheroid formation [6]. Furthermore, Funderburgh et al. compared spheroid formation from the effect of matrix during cultivation [7]. Their results suggested that the expression of keratocan, a proteoglycan component found uniquely from keratocytes, was higher in unattached spheroids than those attached to the matrix during cultivation. In general, these researchers showed the possibility in the formation of keratocyte spheroid especially from an aqueous solution/suspension. Although keratocytes are anchorage-dependent cells, the formation of cell spheroids may become feasible with proper stimulations from the interface through cell-matrix crosstalks. Among all potential biopolymer substrates, chitosan coating has gained an increasing interest due to its excellent biocompatibility for cell culture and outstanding ability to promote spheroid formation. For example, Cheng et al. demonstrated the formation of spheroids from human adipose-derived stem cells (ASC) on chitosan coatings [8]. Their results suggested that chitosan materials enhanced spheroid formation of ASCs over 7 days of culture as compared to those on TCPS control. In another study, Lin et al. reported the formation of melanocyte spheroids on chitosan coatings and
compared the characteristics of the spheroids with different cell seeding densities [9]. These two examples clearly demonstrated the possibility of using chitosan coatings as substrate materials to fabricate spheroids during cell cultivation. However, research involving in the use of chitosan coatings for cell culture rarely considered the degree of deacetylation (DD) of chitosan as a crucial factor in spheroid formation, especially in keratocytes. DD is one of the most important physicochemical parameters affecting the surface properties of chitosan coatings as reported by Foster et al. [10]. In addition, the cell behaviors such as migration, aggregation, proliferation, and differentiation are known to be modulated by tuning the interface properties of the substrate materials. These considerations motivate us to address the gap in keratocyte spheroid fabrication on chitosan materials due to the effect of DD. As a promising ophthalmic biomaterial, chitosan is a polysaccharide consisting of randomly distributed units of (1–4)-linked d-glucosamine and N-acetyl-d-glucosamine [11]. It is usually obtained by alkaline N-deacetylation process of chitin, which may simultaneously lead to chemical depolymerization of biopolymer. Therefore, in this study, the deacetylated chitosan samples with varying DDs were prepared by heat-alkaline treatment under a nitrogen atmosphere to avoid the molecular weight modification due to oxidative mechanisms [12]. The molecular weight of biopolymer was controlled at the same level to ensure the clarification of the role of DD of chitosan in the bioengineering of corneal keratocyte spheroids. In the present study, we hypothesized that the DD-mediated surface roughness/stiffness of chitosan coatings plays important roles on the migration, proliferation, and differentiation of keratocytes. In addition, the cultivation of keratocyte spheroids and their corresponding morphology might be modulated by DD of the chitosan coatings. In a study by Chen et al., bovine corneal keratocyte spheroids were formed on a chitosan (DD value of 85%) coating [13]. Although it is promising to see that the keratocyte spheroids maintained phenotypes under serum-containing culture conditions, the effects of DD of chitosan on the fabrication of keratocyte spheroids and their corresponding cell behaviors remained unclear. Therefore, this work aims to investigate keratocyte spheroid fabrication on chitosan coatings of varying DD values. We found that chitosan deacetylation significantly affected the surface characteristics such as surface roughness and stiffness of the coating biomaterials. Furthermore, cell behaviors and spheroid fabrication were mediated through these stimulations from the substrate interface. For the first time, here, we demonstrated that the surface roughness/stiffness of chitosan coatings, as a result of DD,
Fig. 1. AFM measurements on surfaces of TCPS and samples coated with chitosan of varying DDs. (a) 3-D height images; (b) Rq (i.e., mean square roughness) and elastic modulus. Values are mean ± SD (n = 3). *P < 0.05 vs all groups.
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Fig. 2. Cell migration study after 8 h of RCK cultivation on surfaces of TCPS and samples coated with chitosan of varying DDs. The RCKs were further collected and placed on a porous membrane to allow migration to the opposite side of the membrane for 4 h. (a) Typical optical images of reverse side of the porous membrane stained with crystal violet. Scale bars indicate 50 m. (b) Quantitative analysis of cell migration level from the images shown in (a). Data in the experimental groups are percentages relative to that of TCPS groups. Values are mean ± SD (n = 4). *P < 0.05 vs all groups.
altered the migration, proliferation, and differentiation of keratocytes, thereby resulting in the various formations and architectures of the rabbit corneal keratocyte (RCK) spheroids.
2. Materials and methods 2.1. Materials Chitosan powder, derived from crab shell (Cat. No. 28191), was obtained from Fluka (Milwaukee, WI, USA). The DD and viscosityaverage molecular weight (Mv ) of as-received chitosan (DD75
group), as suggested by the manufacturer, were approximately 75–80% and 400 kDa, respectively. Deacetylated chitosan samples were prepared according to Tolaimate et al. [14]. In the DD85 group, the as-received chitosan was exposed to 60% (w/v) NaOH solution for 1 h at 100 ◦ C under a nitrogen atmosphere followed by extensive rinsing with deionized water until a neutral pH was reached. In addition, to obtain the highly deacetylated product (DD96 group), the as-received chitosan was treated twice by the same deacetylation procedure as described above for DD85 material, but with a minor modification. Each exposure time during the repeated alkaline treatment was set at 1.5 h. The DD and Mv of chitosan
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Fig. 3. (a) Representative light microscopic images of RCKs at 5 days after plating on surfaces of TCPS and samples coated with chitosan of varying DDs. Scale bars indicate 1000 m. (b) The diameter distribution histograms of RCK spheroids formed on various chitosan coatings and corresponding Gaussian fits.
samples were determined by ninhydrin assay and viscometric method, respectively (see Supporting information). Phosphatebuffered saline (PBS, pH 7.4) was purchased from Biochrom (Berlin, Germany). 24-well non-tissue culture polystyrene plates (Falcon 351147) and tissue culture polystyrene (TCPS) plates (Falcon 353047) were purchased from Becton Dickinson Labware (Franklin Lakes, NJ, USA). 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F12) and TRIzol reagent were purchased from Gibco-BRL (Grand Island, NY, USA). Bovine serum albumin (BSA) was acquired from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution (10,000 U/ml penicillin, 10 mg/ml streptomycin, and 25 g/ml amphotericin B) were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). All the other chemicals were of reagent grade and used as received without further purification.
2.2. Preparation of chitosan-coated culture substrates Chitosan solution was obtained by dissolving 1 g of as-received or deacetylated chitosan powder in 50 ml of acetic acid solution (1% v/v) with stirring until complete dissolution. The insoluble substances were removed by a filter paper (Tokyo Roshi Kaisha, Tokyo, Japan). To obtain chitosan coatings, 0.5 ml of the filtered chitosan solution was poured into the 24-well plate (Falcon 351147) and allowed to air-dry for 2 days at room temperature. The chitosan coatings were immersed in a 0.5 N NaOH solution for 1 h and rinsed extensively with deionized water until neutrality. After air-drying, the thickness of chitosan coatings was measured by a QuaNix 8500 thickness gauge (Automation Nix Gmbh, Koln, Germany). Ten measurements were done on different surface sites to calculate the thickness for each coating sample. Results were averaged on five
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Fig. 4. Representative scanning electron microscopic images of RCKs at 5 days after plating on surfaces of (a) TCPS and samples coated with chitosan of varying DDs (b) 75%, (c) 85%, and (d) 96%. Scale bars indicate 5 m.
Fig. 5. The OD value at 450 nm for RCKs cultured on TCPS and various chitosan coatings for 1, 3, and 5, days. An asterisk indicates statistically significant differences (*P < 0.05; n = 4) for the mean value of OD compared to value at previous time point. # P < 0.05 vs all groups; + P < 0.05 vs TCPS groups; ˆ P < 0.05 vs TCPS and DD75 groups (compared only within each time point group).
independent runs. Chitosan coatings had an average thickness of 12.3 ± 0.5, 11.9 ± 0.2, and 11.8 ± 0.5 m without statistical significance for DD75, DD85, and DD96, respectively (P > 0.05). 2.3. Atomic force microscopy An atomic force microscope (AFM) (NanoScope IV; Veeco Digital Instruments, Santa Barbara, CA, USA) was utilized to scan surface topography of the chitosan coatings [15]. The AFM samples were prepared as described in Section 2.2. The roughness of dry surfaces was analyzed in air. All measurements were made in tapping mode using a silicon cantilever at room temperature. AFM images were
recorded with a scan size of 5 m × 5 m. Five measurements were done on different surface sites to calculate the root mean square roughness (Rq) for each sample. Results were averaged on three independent runs. The nanoscale mechanical properties of the chitosan samples were characterized by force-volume spectroscopy using the same AFM. A silicon cantilever operated in contact mode was employed to observe the topography. During the measurements, a defined force was applied to various surface sites using a calibrated tip. The amount of cantilever deflection was monitored to determine the surface stiffness (elastic modulus) of the samples by collecting force–volume curves at four different regions. Results were averaged on three independent runs.
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Fig. 6. Gene expression level of keratocan in RCKs grown on surfaces of TCPS and samples coated with chitosan of varying DDs for 5 days by real-time RT-PCR. Data in the experimental groups are percentages relative to that of TCPS groups. Values are mean ± SD (n = 4). *P < 0.05 vs all groups.
2.4. Cell migration assays
2.6. Cell morphology studies
All animal procedures were approved by the Institutional Review Board and were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-eight adult New Zealand white rabbits (National Laboratory Animal Breeding and Research Center, Taipei, Taiwan, ROC) weighing 2.5–3.0 kg were used for this study. Primary rabbit corneal stromal cells were prepared according to our previously published methods [16]. The keratocytes were maintained in regular culture medium consisting of DMEM/F12, 10% FBS, and 1% A/A solution. In all experiments, third-passage rabbit corneal keratocytes (RCKs) were used. Cell migration assays were performed according to a reported method [17]. RCKs with a density of 5 × 104 cells/ml were seeded into 24-well TCPS (Falcon 353047) and various chitosan-coated plates by 1 ml/well. After incubation at 37 ◦ C for 8 h, the cultured cells were detached using trypsin-EDTA. A 100 l suspension containing 2 × 104 RCKs was plated into 24-well Millicell containing polycarbonate filters with 8-m pores (Millipore, Bedford, MA, USA). The cells were allowed to migrate for 4 h. Then, the migrated cells on the bottom surface of the filter were stained with 0.1% crystal violet and photographed using an optical microscope (Nikon, Melville, NY, USA) to capture cell migration images. The number of cells on the bottom side of the filter was counted at six different areas. All experiments were performed in quadruplicate, and the results were expressed as relative cell migration level normalized to the controls (TCPS groups).
After 5 days of cultivation of RCKs on various substrates, the cellular morphology was observed by scanning electron microscopy (SEM) [18]. The cell cultures were fixed with 2% glutaraldehyde in 0.1 M cacodylic acid buffer (pH 7.4) overnight at 4 ◦ C. After rinsing them with 0.1 M cacodylic acid buffer, the specimens were postfixed in 1% osmium tetroxide and dehydrated in a graded series of ethanol solutions. Then, the cell samples were dried with carbon dioxide in a critical point dryer (Balzers, Liechtenstein) and coated with gold in a sputter coater (Hitachi, Tokyo, Japan) before examination under a Hitachi S-3000N SEM with an accelerating voltage of 10 kV. 2.7. Cell proliferation assays After incubation for 1–5 days, the RCKs were counted by using the cell proliferation reagent WST-1 (Roche Diagnostics, Indianapolis, IN, USA) assay. The WST-1 assay is based on the cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) to a colored formazan by mitochondrial dehydrogenases in viable cells [19]. The amount of formazan product is proportional to the number of metabolically active cells. For staining, 100 l of WST-1 reagent was added to the cultures, and incubated for 4 h at 37 ◦ C in a CO2 incubator. The optical density (OD) value at 450 nm was recorded using the Multiskan Spectrum Microplate Spectrophotometer (ThermoLabsystems, Vantaa, Finland). All experiments were performed in quadruplicate.
2.5. Quantification of cell spheroids RCKs with a density of 5 × 104 cells/ml were seeded into various chitosan-coated plates by 1 ml/well, and incubated in regular culture medium at 37 ◦ C for 1–5 days. The culture wells were photographed at six different fields by using an optical microscope. The RCK spheroid diameter was analyzed with the Adobe Photoshop software. The criterion for the formation of cell spheroids is considered to be that when the cell aggregate has a diameter > 50 m. All experiments were performed in quadruplicate. To quantitatively estimate the diameter distribution of the cell spheroids, the histogram and corresponding Gaussian fitting curve were obtained from the values for 30 cell spheroids.
2.8. Quantitative real-time reverse transcription polymerase chain reaction analyses After cultivation on TCPS and chitosan coatings for 5 days, the total RNA was isolated from cells with TRIzol reagent [20]. Reverse transcription of the extracted RNA (1 g) was performed using ImProm-II and Oligo(dT)15 primers (Promega, Madison, WI, USA). The primers used to amplify the rabbit keratocan complementary DNA (cDNA) were 5 -CTCACGTGGCTTTGATGTGT3 (sense) and 5 -GACCTTTGTGAGGCGATTGT-3 (antisense). The sequences of the primer pair used to amplify the internal control cDNA, glyceraldehyde-3-phosphate dehydrogenase
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Table 1 Physicochemical characteristics of chitosan samples. Sample code
Processing method
DDa (%)
Mv b (kDa)
DD75 DD85 DD96
As-received Deacetylation (60% NaOH, 100 ◦ C, 1 h) Deacetylation (60% NaOH, 100 ◦ C, 1.5 h*2 times)
75.6 ± 0.6 85.2 ± 0.4c 96.3 ± 0.4c
336.5 ± 2.4 339.2 ± 1.8 340.2 ± 2.5
a b c
Determination of deacetylation degree (DD) by ninhydrin assay. Data are expressed as mean ± standard deviation (n = 5). Determination of viscosity-average molecular weight (Mv ) by viscometric method. Data are expressed as mean ± standard deviation (n = 4). Significant difference as compared to the DD75 groups (P < 0.05).
(GAPDH), were 5 -TTGCCCTCAATGACCACTTTG-3 (sense) and 5 TTACTCCTTGGAGGCCATGTG-3 (antisense). Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on a Light-Cycler instrument (Roche Diagnostics) with FastStart DNA Master SYBR Green I reagent. Each sample was determined in quadruplicate, and the gene expression results were normalized to the level of GAPDH mRNA. 2.9. Statistical analyses Results were expressed as mean ± standard deviation (SD). Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with P < 0.05.
Fig. 2a shows the optical images of RCKs on the reverse side of the transwell filter inserts stained with crystal violet. An obvious amount of RCKs can be seen, indicating that the cells pre-treated with chitosan can efficiently migrate through the transwell filter. In addition, Fig. 2b shows the quantitative analysis on the amount of the migrated RCKs, and the average cell migration, expressed in percentage of total cell numbers after normalizing to TCPS control (100%), was 234.7 ± 10.8%, 189.1 ± 12.4%, and 136.3 ± 7.0% for DD75, DD85, and DD96, respectively (P < 0.05). This suggested that RCK migration was facilitated due to the exposure of chitosan materials while cell migration ability decreased with increasing DD of the chitosan coatings.
3. Results 3.4. Quantification of cell spheroids 3.1. Preparation of chitosan-coated culture substrates Table 1 shows the DD and Mv of the as-received and deacetylated chitosan, after exposure to heat-alkaline treatment in a nitrogen environment. From the table, increasing NaOH treatment time increased DD. The findings from ninhydrin assays support the nuclear magnetic resonance (NMR) spectral data (Fig. S1). Further details are provided in the Supporting information. No significant modification to the Mv of the chitosan was found, suggesting that our deacetylation process of chitosan only modified the amide side groups on the N-acetyl-d-glucosamine units. The weight-average molecular weight of chitosan samples as determined by gel permeation chromatography (GPC) also showed no significant change between DD75, DD85, and DD96 groups (P > 0.05) (Fig. S2). Further details are provided in the Supporting information. Thus, changes in surface properties of the chitosan coatings were a dependent of DD instead of molecular weight of the chitosan. 3.2. Atomic force microscopy Surface roughness and stiffness (elastic modulus) of the chitosan coatings at various DDs were examined by AFM. Fig. 1a shows the 3D height images of DD75, DD85, and DD96 coatings where higher DD samples exhibited noticeable surface features such as humps and bumps. In addition, quantitative analyses of the AFM measurements shown in Fig. 1b indicated that the average Rq was 4.8 ± 0.5, 2.1 ± 0.2, and 0.9 ± 0.1 nm for DD75, DD85, and DD96, respectively (P < 0.05). The average elastic modulus of the chitosan coatings, also shown in Fig. 1b, was 6.5 ± 0.3, 7.8 ± 0.3, and 9.6 ± 0.6 MPa for DD75, DD85, and DD96, respectively (P < 0.05). Our results suggested a decreasing surface roughness and an increasing stiffness with increasing DD of the chitosan coatings. 3.3. Cell migration assays The surface characteristics of a biomaterial coating may influence cell behaviors such as the ability to migrate. Here, we performed studies on the migration ability of RCKs using transwell assays after 8 h of cultivation on various chitosan coatings.
According to our data, the formation of RCK spheroids could be observed following 1 day of direct contact with coating materials, irrespective of the DD of chitosan (Fig. S3). Further details are provided in the Supporting information. In addition, Fig. 3a shows representative light microscopic images of RCKs on TCPS control and chitosan coatings after a 5-day culture. It is interesting to note that the size and amount of the RCK spheroids appeared to be different on chitosan coatings of varying DD values. Quantitative analyses supported this finding where spheroids larger than 50 m in diameter were counted and categorized into Gaussian distribution plots shown in Fig. 3b. The RCK spheroids on DD75 exhibited the largest diameter distribution followed by DD85 and DD96. In addition, RCK spheroids with diameter over 150 m were found on DD75 with the majority of the spheroid diameter ranging from 100 to 150 m. Within DD85 and DD96, the diameter of the RCK spheroids decreased from a majority of 100–150 m to 50–100 m as increasing the DD in chitosan coatings. Therefore, DD of the chitosan seemed to have an effect in determination of the number of spheroids and their corresponding size.
3.5. Cell morphology studies Fig. 4 shows SEM images of representative RCK after 5 days of culture on TCPS control and various chitosan coatings. The morphology of RCKs on TCPS displayed a flat and confluent morphology without the evidence of spheroid formation. By contrast, the RCKs cultured on chitosan coatings typically showed the formation of spheroids with differences in morphology and architecture. In addition, RCK spheroids on DD75 coatings showed clear and distinct cell structures with features of ECM all over the surface of each individual cell. The surface features of the RCK spheroids were less noticeable on DD85 and DD96 coatings. Furthermore, RCK spheroids formed on DD75 coatings showed a better depth of field in the SEM image than those on DD85 and DD96. These observations were indicative of the dependence of the spheroid morphology and architecture on the DD of chitosan coatings.
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3.6. Cell proliferation assays The mitochondrial activity of RCK cultures was measured by means of WST-1 assays. Fig. 5 shows the average optical density (OD) of RCKs on TCPS control and various chitosan coatings after cultivation for 1–5 days. The amount of RCKs cultured on TCPS increased significantly from 1 to 3 days (P < 0.05) but no significance from 3 to 5 days (P > 0.05) perhaps due to a minimal space for cells to proliferate. For RCKs cultured on various chitosan coatings, a similar trend in the increase of the OD but less pronounced than that of the TCPS control was found over 5 days of cultivation. Furthermore, considering substrate interface effects, increasing DD of the chitosan coatings increased the OD at 5 days of culture. Based on these, it appeared that surface properties of the chitosan coatings significantly affected the growth of the RCKs. Our results suggested that proliferation of the RCKs on DD75 was less active than DD85 and DD96. 3.7. Quantitative real-time reverse transcription polymerase chain reaction analyses Fig. 6 shows the average keratocan mRNA expression level of RCKs on various chitosan coatings in percentage after normalizing to TCPS control (100%). After cultivation for 5 days, keratocan expression of RCKs on chitosan substrates was significantly higher than that of the TCPS control (P < 0.05). This suggested that the keratocytes maintained their phenotype on all chitosan coatings. In addition to the above observation, we also noticed a significant decrease in keratocan expression with increasing DD of the chitosan materials. Keratocan expression from RCKs on DD75, DD85 and DD96 was found at 493.6 ± 20.8%, 360.5 ± 12.2% and 291.0 ± 10.4%, respectively (P < 0.05). This finding suggested that the surface properties of the chitosan coatings, mediated through DD, were responsible for the phenotypic modulation of keratocytes. 4. Discussion In this study, we reported RCK behaviors and spheroid fabrication due to the substrate interface effects of chitosan coatings modulated through DD. The Rq of underlying TCPS substrates determined by AFM was 3.5 ± 0.4 nm [21]. After coating with chitosan materials of varying DDs, the culture surfaces showed different roughness levels. The influence of increasing DD of chitosan on the reduction of surface roughness was reported by others [22]. In addition, the decrease in surface roughness was due to the removal of fibrillar aggregates in chitosan [23]. More specifically, besides processing conditions of chitosan, other factors such as charge density and molecular chain length of the biopolymer were correlated with chemical heterogeneity-mediated formation of the fibrillar aggregates [24]. From our results, increasing DD decreased the chemical heterogeneity (on the basis of no significant molecular weight changes) and prevented the formation of fibrillar aggregates, and thus, leading to a smoother surface of the chitosan coating. By contrast, decreasing chemical heterogeneity was accompanied by a higher stiffness. Majd et al. have used nanoindentation to obtain surface stiffness of various chitosan films having a DD from 76% to 96% and have found that the surface stiffness increased at a higher DD due to the increase in film crystallinity [25]. Several studies have also correlated stiffness and DD of the chitosan coatings [26–28]. The dependence of stiffness on DD of chitosan may be explained by a much closer packing of the biopolymer chains due to deacetylation where the substituted amino groups attract the neighboring biopolymer chains resulting in a much stronger and stiffer films. Furthermore, the chitosan with a higher DD exhibits a more flexible chain, which may facilitate hydrogen bonding through amino
groups on glucosamine units of the deacetylated chitosan. Overall, our findings in surface characteristics of chitosan coatings were in agreement with these earlier findings, which depended on the DD of the chitosan. Studies have shown that surface properties of a biomaterial coating may greatly influence cell migration ability [29–32]. These studies suggested that cell migration was positively correlated to surface roughness and inversely correlated to matrix stiffness. More specifically, cell migration ability was enhanced due to cellmatrix crosstalks mediated through focal adhesion where short focal adhesions were associated with the highest migration speed [32]. Similar to these literature findings, our data suggested a strong dependence on cell migration due to important physical cues such as matrix stiffness and surface roughness. A higher migration ability of the RCK cells was correlated to the dependence on the size and amount of the spheroids (Fig. S3). For example, RCKs on DD75 may receive a longer time to migrate since no cell attachment was observed up to day 3 as compared to some attachments on DD85 and DD96 after day 1. This resulted in a wider size distribution of RCK spheroids on DD75 than DD85 and DD96. Moreover, an excess of ECM molecules on the surface of cells indicated higher cell migration ability since a constant formation and dissociation of the focal adhesions was required through ECM during migration [32]. In a similar study, the formation of spheroids from mesenchymal stem cells was reported in porous scaffolds with excess deposition of ECM on the spheroids after 2 days of culture [33]. Of particular importance, the study suggested the correlations between anchorages of the cells to the walls of the scaffold as a function of the spheroid formation as well as how fast the spheroids were formed [33]. In general, our observations demonstrated that RCK migration was the main driving force on spheroid formation while the number and size of the spheroids strongly depended on the surface roughness/stiffness of chitosan coatings. According to our results, RCK proliferation on all chitosan coatings significantly increased from 1 to 3 days whereas significant increases in RCK proliferation were only observed on DD85 and DD96 from 3 days to 5 days (P < 0.05). In addition, no statistical significance of RCK proliferation was found within chitosan coatings at 1 day (P > 0.05) whereas significance differences in RCK proliferation were found in all chitosan coatings after 5 days (P < 0.05). These findings suggested that the surface properties of chitosan coatings affected cell proliferation and were in agreement with others who suggested that cell proliferation decreased with increasing surface roughness [34–37]. More importantly, it had been suggested that surface roughness of a biopolymer coating significantly affected the adsorption of fibronectin, a pre-requisite for cell attachment and growth [38]. By contrast, studies showed that stimulations from a stiffer matrix increased cell proliferation [39–43]. In addition, the differences in stiffness-induced cell proliferation were due to increase in integrin signaling through focal adhesion complexes [40]. In general, these earlier findings supported our present proliferation results where the RCKs on DD96 coatings exhibited the highest increase in cell proliferation during 5 days of cultivation. There are at least three phenotypes associated with corneal keratocytes including keratocytes, fibroblast, and myofibroblast [4]. Keratocan, a protein that belongs to one of the keratan sulfate proteoglycans, is the phenotypic marker when keratocytes are being mass-produced during cell proliferation and differentiation [44]. Studies have shown that keratocan expression was significantly up-regulated during cultivation of keratocytes using biomaterial coatings after several weeks [13,45]. In addition, our results in the phenotypic expression of keratocytes suggested that the keratocan levels were not only up-regulated by chitosan coatings but also were mediated through differences in surface characteristics of the substrate materials. The dependence of phenotypic expression of cells on surface roughness of a biomaterial was reported previously
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[46–49]. According to these earlier findings, increasing surface roughness increased the phenotypic expression of cells. In addition, cells with high phenotypic expression found on rougher surfaces produced noticeable ECM in cell morphology [46]. This observation was similar to our results (Fig. 4) where the surface of RCK spheroids on DD75 exhibited a significant amount of ECM as compared to DD85 and DD96. On the other hand, surface stiffness is another important surface property that has been demonstrated to significantly affect the phenotypic expression of cells. Studies suggested that cellular phenotypic expression was inversely proportional to the stiffness of the biomaterial coatings [49–51]. Supported by these examples, we reported a strong dependence of phenotypic expression in keratocyte spheroids with respect to roughness and stiffness of the chitosan materials modulated through deacetylation. 5. Conclusions In summary, this work demonstrated the feasibility of the fabrication of 3-D keratocyte spheroids using chitosan substrates at various DDs without significant modification to their corresponding Mv . In particular, increasing DD from 75 to 96% decreased surface roughness and increased surface stiffness of the chitosan materials. Studies in RCK behaviors suggested the substrate interface effects of biopolymer coatings. Both the surface roughness increase and stiffness decrease could lead to enhancement of cell migration, spheroid formation, and phenotypic maintenance. In addition, the proliferation of RCKs and size of cell spheroids were greatly affected by the surface roughness/stiffness of coating materials. This experimental work confirmed the dependence of the spheroid morphology and architecture on the DD of chitosan. Overall, our findings contributed to the scientific understanding of the keratocyte cultivation and spheroid fabrication in response to DDmediated surface roughness/stiffness of chitosan substrates. Acknowledgements This work was supported by grants CMRPD1B0451 and CMRPD1C0111 from Chang Gung Memorial Hospital and grant NSC99-2221-E-182-008 from the National Science Council of Republic of China. The authors are grateful to Dr. David Hui-Kang Ma (Department of Ophthalmology, Chang Gung Memorial Hospital) and Miss Hsiao-Yun Cheng (Molecular Medicine Research Center, Chang Gung University) for technical assistance.
[4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]
Appendix A. Supplementary data
[45]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.02. 051.
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