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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 6930−6941
Homologous Sodium Alginate/Chitosan-Based Scaffolds, but Contrasting Effect on Stem Cell Shape and Osteogenesis Lili Zhang, Haowei Fang, Kunxi Zhang,* and Jingbo Yin* Department of Polymer Materials, Shanghai University, 99 Shangda Road, Shanghai 200444, P. R. China S Supporting Information *
ABSTRACT: Stem cell shape appears to be involved in the regulation of osteogenesis, which has been confirmed in two-dimensional surfaces and threedimensional hydrogels. The present study evaluated the effect of matrixcontrolled cellular shape on osteogenesis in three-dimensional porous scaffolds based on sodium alginate (ALG) and chitosan (CS). Three ALG/CS scaffolds, especially including a stiff one, were fabricated from different precursor matrices. Soft scaffold A was fabricated from the ALG/CS polyelectrolyte and further cross-linked by Ca2+ and glutaraldehyde to achieve soft scaffold B with alternative hydrophilicity. Stiff scaffold C with “hard-to-deform” feature was fabricated from “ALG/CS preformed gel”, which was an ALG gel network expanded by swelling force of the dissolving CS, and fixed using Ca2+ and glutaraldehyde. Scanning electron microscopy and F-actins staining showed rounded mesenchymal stem cells (MSCs) on the inner surfaces inside scaffold A with high swelling behavior, but spindlelike MSCs in scaffold B. Stiff scaffold C forced MSCs to adhere to polygonal shape. Fibronectin adsorption was found to be weakened in scaffold A. Integrin α5β1 expression, as well as osteogenesis-related genes (ALP, OCN) expression, was detected to be higher in the stiff scaffold C. Thus, the present study illustrated that the stiff scaffold C responded to cells with hard-to-deform information, leading to the amplification of focal adhesions and induction of high tension of cells, consequently enhancement of osteogenesis. KEYWORDS: sodium alginate, chitosan, cell shape, stiffness, osteogenesis
1. INTRODUCTION Toward stem cell based bone tissue engineering, it has been already demonstrated that mesenchymal stem cells (MSCs), which were allowed to adhere, flatten, and spread, underwent osteogenesis.1,2 According to a comparison between globally isotropic circular, square, triangular, and star cells, Ding and his colleagues found that optimal osteogenic differentiation happened in star cells.3 Thus, cellular shape appear to be integral to the commitment of stem cell fate. Properties of substrates, such as mechanical property, hydrophilicity and hydrophobicity, and surface topography, show significant effect on cell adhesion, cell shape, and spread.4−6 Researches have indicated that varying physical and chemical properties of substrates would modulate fibronectin (Fn) conformation and direct integrin binding and specificity, thus resulting in changes in cellular shape so to realize the regulation of cell differentiation.1,2 Matrix interfacial hydrophobicity showed the enhanced effect on osteogenesis of stem cell by harnessing the cytoskeletal organization of MSCs on the substrate.7 Besides, the elasticity and rigidity of threedimensional microenvironments regulated integrin binding as well as reorganization of adhesion ligands on the nanoscale, both of which were traction dependent and correlated with osteogenic commitment of mesenchymal stem cell populations.8 Thus, bone scaffolds should be designed not only to act as the temporary residence and delivery system for stem cells © 2018 American Chemical Society
but also to regulate the stem cell differentiation of certain lineages. However, although the effect of matrix-controlled cellular shape on osteogenesis has been confirmed in twodimensional surfaces and three-dimensional hydrogels, whether the three-dimensional porous scaffolds perform the similar effect of matrix-controlled cellular shape on osteogenesis with two-dimensional surfaces and three-dimensional hydrogels remains elusive. Sodium alginate (ALG) and chitosan (CS) are two kinds of natural macromolecules that have been widely used in bone tissue engineering.9 There were various methods to fabricate scaffolds based on ALG and CS to act as scaffolds. However, due to the hydrophilic nature of ALG and CS, the ALG/CSbased scaffolds generally exhibited a soft character when wetted in water and culture medium. These spongelike scaffolds performed well in absorbing water and deformation. It was a challenge to fabricate an ALG/CS-based scaffold that was stiff and hard to deform, so to reinforce the traction forces of stem cells, promoting osteogenesis. Toward bone tissue engineering, it is worth to construct a stiffer ALG/CS-based scaffold for bone tissue engineering and to evaluate the effect of the stiff feature of the ALG/CS-based scaffolds on the regulation of Received: December 11, 2017 Accepted: February 2, 2018 Published: February 2, 2018 6930
DOI: 10.1021/acsami.7b18859 ACS Appl. Mater. Interfaces 2018, 10, 6930−6941
Research Article
ACS Applied Materials & Interfaces
Japan). Pore diameters of scaffolds were obtained by analyzing the SEM images from sectioned microspheres. 2.2.2. Swelling Behavior of Scaffolds. Each sample was measured immediately after vacuum-drying as dry weights (Wd); next, these samples were immersed in phosphate buffered saline (PBS) solution at a constant temperature of 37 °C and weighed at given time points to conform the swollen weights (Ws); the completely swollen samples were centrifuged at 500 rpm for 3 min and then weighed straightway as Ws′. The swelling ratio of each simple was calculated by the following equation: swelling ratio = (Ws − Wd)/Wd × 100%. The water retention was calculated by the following equation: ratio of water retention = (Ws′ − Wd)/Wd × 100%. 2.2.3. Mechanical Properties of Scaffolds. The compressive performance of the scaffolds was evaluated using a Dynamic Mechanical Analyzer (TA Q800 instrument, TA Instruments Waters LLC) in 0.1 M PBS at 37 °C, fitted with a custom compression clamp. A cubic sample (4 mm × 4 mm × 4 mm) was compressed with a constant strain rate of 10% min−1. 2.3. Evaluation of Cellular Shape and Proliferation. 2.3.1. Cell Seeding and in Vitro Culture. MSCs, derived from femurs of neonatal Sprague-Dawley rats, were purchased from Servicebio (China). MSCs were cultured in a low-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 μg mL−1 ascorbic acid, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin in 100 mm diameter culture dishes at 37 °C in a 5% CO2 atmosphere. MSCs before passage three were collected and resuspended in a culture medium at the concentration of 1 × 105 cells per mL, followed by seeding into ALG/CS-based scaffolds. After incubation for 3 h to allow cell attachment, osteogenic-induced medium, which was composed of high-glucose DMEM, 10% FBS, 50 μM ascorbic acid-2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone, was added for in vitro induction. 2.3.2. Observation of Cellular Shape. SEM was used to observe the cellular shape inside the scaffolds. At 3 and 24 h after seeding, the scaffolds/MSCs were collected and washed with PBS for three times, followed by immersion in 4% paraformaldehyde. After gradually replacing H2O by ethanol, hexamethyldisilazane was used to replace ethanol. The samples, collected after the evaporation of hexamethyldisilazane, were treated with gold sputtering and observed with SEM. Rhodamine phalloidin was also used to stain F-actins to further identify the cellular shape. The scaffolds/MSCs samples were collected and washed with PBS for three times, followed by immersion in 4% paraformaldehyde. 0.5% Triton was used to treat the samples and then washed by PBS for 20 min. After immersion in staining solution for 30 min, the samples were washed with PBS and observed with confocal laser microscope (FV-1000, Japan, Instrumental Analysis and Research Center, Shanghai University). 2.3.3. Adsorption of Fn on Scaffolds. The scaffolds immersed in culture medium for 4 h were washed with PBS for 2 h, followed by determination by immunofluorescent staining. Briefly, the samples were blocked with bovine serum albumin and reacted with polyclonal to Fn antibody at 4 °C, followed by immersion in Alexa Fluor 568 labeled secondary antibody at 37 °C and observed using confocal laser scanning microscopy. 2.3.4. Evaluation of Cellular Proliferation in Scaffolds. Cell numbers in the scaffold at 1, 7, and 14 days after seeding were quantified by DNA assay using Hoechst 33258 dye (Sigma-Aldrich). Briefly, the scaffolds/MSCs samples were collected and washed with PBS for three times and crushed, followed by treatment with 0.5 mg mL−1 proteinase K at 56 °C for 12 h. After centrifugation, the DNA content in the supernatant was quantified spectrophotometrically using Hoechst 33258 dye by correlating with a standard curve (SpectraMax M2, Molecular Devices). To visualize the growth and distribution of MSCs in scaffolds, the MSCs were prelabeled with fluorescent 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) dye (Molecular Probes) for 15 min at 37 °C before seeding. The cells grown in scaffolds were observed by confocal laser microscope. The relative cytotoxicity was evaluated by Cell Counting Kit-8 (CCK-8). The scaffolds extracts were collected by immersing 200 mg
cellular shape, as well as the effect of shape regulation on promoting osteogenesis of stem cells in three-dimensional porous scaffold. Here, in the present study, soft and stiff scaffolds were all fabricated based on ALG and CS. The soft scaffolds were prepared from the ALG/CS polyelectrolyte matrices. At the same time, cross-linking components were employed to alter the hydrophilicity of the scaffold matrix. A new strategy that used swelling pressure of CS during dissolution to expend the ALG gel network was proposed to prepare the stiff scaffold with the “hard-to-deform” property. These ALG/CS scaffolds were created for the purpose of evaluating the effect of scaffold on promoting osteogenic differentiation of stem cells through forcing the adhesive stem cells to present different cellular shape on the inner surfaces inside the scaffolds. The structure, swelling property, and mechanical property of the ALG/CS scaffolds were studied. The MSCs were seeded into these scaffolds. The shape and proliferation of adherent MSCs was observed. Furthermore, the osteogenic differentiation of MSCs in different ALG/CS scaffolds was monitored to evaluate the effect of scaffolds-controlled cellular shape on osteogenesis in three-dimensional porous scaffold.
2. EXPERIMENTAL SECTION 2.1. Preparation of ALG/CS-Based Scaffolds. All of the scaffolds in this work were obtained by mixing chitosan (Mv = 4 × 104, DA ≤ 5%, purchased from Jinan Haidebei Marine Bioengineering Corp.) and sodium alginate (Mv: 7.8 × 105, purchased from Sinopharm Chemical Reagent Co., Ltd.). The mole ratio of −COOH in ALG and −NH2 of CS was 1:1. The solid content (the sum of mass fraction of ALG and CS) was 3%. The fabrication of three types of ALG/CS-based scaffolds, named scaffold A, scaffold B, and scaffold C, will be listed in later sections. To study the viscoelastic behavior of the ALG/CS precursor matrices during the preparation of different types of scaffolds, rheological experiments were performed on an AR2000 rheometer from TA Instruments with 25 mm diameter parallel plates at 37 °C in the oscillatory mode. The frequency sweep test was performed, which covered a range of frequencies from 1 to 100 rad s−1 at the regular strain of γ = 0.01. The storage modulus G′, loss modulus G″, and dynamic viscosity η were obtained with respect to frequency. Fourier transform infrared (FT-IR) measurements were performed with an AVATAR 370 FTIR spectrophotometer from Nicolet. The KBr used for pellet preparation, each matrix were detected after drying. 2.1.1. Preparation of Soft Scaffold A. Sodium alginate was dissolved in deionized water to form a homogeneous solution. The CS powder (3 wt %) that evenly dispersed in the ALG solution was dissolved by slowly adding dilute acids until the pH value reached 4−5. Then, the emulsion was sped up with a FA25 High Shear Emulsifier (FA25, FLUKO, Shanghai) and continued to stir for 12 h to form a homogeneous polyelectrolyte suspension. After lyophilization at −20 °C, scaffold A was obtained. 2.1.2. Preparation of Soft Scaffold B. Scaffold A was first soaked in 3% calcium chloride (CaCl2) solution for 15 min and then in 1% glutaraldehyde aqueous solution for 15 min. After lyophilization, scaffold B was achieved. 2.1.3. Preparation of Stiff Scaffold C. After dissolving ALG in deionized water, the CS powder (3 wt %) that evenly dispersed in ALG solution was dissolved by adding dilute acids to immediately reach the pH value lower than 3. Then, a colorless transparent gel-like mixture was obtained. After lyophilization at −20 °C, the matrix was first soaked in 3% calcium chloride (CaCl2) solution for 15 min, followed by soaking in 1% glutaraldehyde aqueous solution for 15 min. After lyophilization at −20 °C, scaffold C was obtained. 2.2. Characterization of ALG/CS-Based Scaffolds. 2.2.1. Porous Structure of Scaffolds. The microstructures of scaffolds were observed by scanning electron microscopy (SEM) (JXA-840, JEOL, 6931
DOI: 10.1021/acsami.7b18859 ACS Appl. Mater. Interfaces 2018, 10, 6930−6941
Research Article
ACS Applied Materials & Interfaces
Figure 1. Schematic representation for the preparation of the ALG/CS precursor matrices for the soft scaffolds and stiff scaffold. For soft scaffold, the pH was adjusted slowly to 4−5 to support the ALG/CS polyelectrolyte suspension formation. For stiff scaffold, a new preparation strategy was created. CS dissolution was designed to take place inside the ALG gel, so to provide swelling pressure to expand the ALG network to an expansion state. After freeze-drying, the networks were fixed by Ca2+ and glutaraldehyde to yield the stiff scaffold. 2.5. Statistical Analysis. All of the data were reported as mean ± standard deviation. Single-factor analysis of variance was used to assess the statistical significance of the results. Values of p < 0.05 were indicative of significant differences.
scaffolds in 1 mL DMEM and 10% FBS for 1, 2, 4, 6, and 8 weeks. DMEM and 10% FBS and dimethyl sulfoxide were employed as control groups. MSCs were seeded in 96-well plates (10 000 cells per well) for 24 h, followed by changing growth medium with 110 μL extract to culture for another 48 h. Then, 10 μL of CCK-8 solution was added to each well of the plate to be incubated for 4 h, followed by measuring the absorbance (optical density (OD) value) at 450 nm using a microplate reader (SpectraMax M2, Molecular Devices). Cell viability% = (ODsample − ODDMEM)/ODDMEM × 100%. 2.4. Evaluation of in Vitro Osteogenic Differentiation. Scaffolds/MSCs samples were fixed with 4% paraformaldehyde and embedded in paraffin, sectioned (5 μm thick). von Kossa and alkaline phosphatase (ALP) staining were carried out to show the osteogenesis of MSCs in the three scaffolds. For von Kossa staining, the sections were incubated in 5% silver nitrate solution for 1 h and washed. The ALP staining was performed according to the manufacturer’s protocol of Leukocyte Alkaline Phosphatase Kit (Sigma; MO). Total RNA was isolated using Trizol and determined from the optical absorbance at 260 nm. Then, cDNA was synthesized from RNA using a PrimeScript 1st strand cDNA synthesis Kit (TaKaRa). Real-time polymerase chain reaction (RT-PCR) was performed using SYBRGreen PCR MasterMix in each reaction. Expression of osteogenesis-related genes, including ALP, OCN, and OPG were detected. Relative expression levels for each gene were calculated by normalizing the quantified cDNA transcript level to that of the GAPDH. Also, the relative gene amount of the noninduced group was set as 1. GAPDH: 5′-tgaacaggaagctcactgg-3′ and 5′-tccaccaccctgttgctgta-3′; OCN: 5′-tctctctgctcactctgctg-3′ and 5′-attttggagcagctgtgccg-3′; ALP: 5′-acgtggctaagaatgtcatc-3′ and 5′-acgtggctaagaatgtcatc-3′; OPG: 5′-caggagagtgaggcaggctatt-3′ and 5′-cttcaggggttggagatttagc-3′. Besides, expression of cell adhesion molecules, integrin α5 and β1, was also monitored by RT-PCR. Relative expression levels for these two genes were calculated by normalizing the quantified cDNA transcript level to that of the GAPDH. Integrin α5: 5′-acttagggcaagtcgggaatt-3′ and 5′gggcagtgagtggggtttat-3′; integrin β1: 5′-tctgtggaggaaatggtgtgtg-3′ and 5′-tttagtgaggttgaaatgggagc-3′.
3. RESULTS AND DISCUSSION 3.1. Construction of ALG/CS Scaffolds Using Different Methods. Thanks to numerous carboxyl groups of ALG and amino groups of CS, various methods are reported to fabricate scaffolds based on ALG and CS.9−12 ALG is a water-soluble polymer, whose network has to be fixed by other components such as Ca2+ to yield a three-dimensional structure. CS is well known to be a semicrystalline polymer. The stable, crystalline structure makes CS normally insoluble in aqueous solutions with pH above 7 but fully soluble in dilute acids, thus providing a convenient mechanism for processing under mild conditions. The viscous CS solutions can be gelled in high-pH solutions or baths of solvents. Thus, CS possesses excellent ability to be processed into porous scaffold.13 Here, in the present study, three types of scaffold based on ALG and CS are fabricated from different ALG/CS precursor matrices. 3.1.1. Preparation of ALG/CS Precursor Matrices for Soft Scaffolds. Theoretically, ALG is a polyelectrolyte carrying negative charges, which could interact with CS, a polyelectrolyte carrying positive charges. In the present study, the electronic interaction between ALG and CS is found to significantly depend on the pH value. ALG aqueous solution (3 wt %) shows the pH of 7. CS power (3 wt %) that evenly dispersed in ALG solution is dissolved by slowly adding dilute acids until the pH value reaches 4−5. Along with the protonation of amino groups on CS, the electronic interaction between CS and ALG is formed strongly enough to overcome their solvation effect, leading to the precipitation of the ALG/ 6932
DOI: 10.1021/acsami.7b18859 ACS Appl. Mater. Interfaces 2018, 10, 6930−6941
Research Article
ACS Applied Materials & Interfaces
Figure 2. Evaluation of the different ALG/CS precursor matrices during the preparation of different types of scaffolds. (a) General view of the ALG/ CS polyelectrolyte, ALG gel, and ALG/CS preformed gel. (b) FT-IR spectra of CS, ALG, ALG/CS polyelectrolyte, and ALG/CS preformed gel. (c) Frequency dependence of complex viscosity |η*| of ALG solution, ALG gel, ALG/CS polyelectrolyte suspension, ALG solution/CS power, and ALG/CS preformed gel. (d) Storage modulus/loss modulus (G′/G″) of ALG/CS preformed gel and ALG/CS polyelectrolyte suspension. (e) Storage modulus/loss modulus (G′/G″) of ALG solution and ALG gel. 6933
DOI: 10.1021/acsami.7b18859 ACS Appl. Mater. Interfaces 2018, 10, 6930−6941
Research Article
ACS Applied Materials & Interfaces
Figure 3. Features of the three scaffolds. The SEM images of scaffold A (a), scaffold B (b), and scaffold C (c); the swelling ratio (d); and water retention ratio (e) of the three scaffolds. (f) CCK-8 assay for evaluation of the relative cytotoxicity of scaffolds extracts. (g) Scaffolds bearing 0.5 kg weight. Compression tests for the wetted scaffolds at 25 °C: (h) the stress−strain curves and (i) the compressive modulus (*p < 0.05, **p < 0.01).
is 2400 Pa s. As shown in Figure 2d, G″ is higher than G′, indicating that ALG/CS polyelectrolyte suspension possesses a typical flow behavior. 3.1.2. Preparation of ALG/CS Precursor Matrices for Stiff Scaffold. A new strategy, based on the gelation characteristics of ALG and dissolution characteristics of CS, is introduced to create a stiff ALG/CS scaffold. Briefly, the CS dissolution is designed to take place inside the ALG gel, so to provide a swelling pressure to expand the ALG network to an expansion state. After freeze-drying, the networks are fixed by Ca2+ and glutaraldehyde to yield the stiff scaffold (Figures 1 and 2a). Along with the protonation of carboxyl groups in ALG, ALG can be gelled through hydrogen bonds and hydrophobic effects. As shown in Figure 2c, the η* of ALG solution is 43 Pa s. When the pH value is lower than 3, the η* dramatically reaches 4900 Pa s. Also, the G′ is higher than G″, indicating the gel formation (Figure 2e). At the same time, CS is dissolved along with the decrease in the pH value from 7 to
