Multiphoton imaging of myogenic differentiation in gelatin-based hydrogels as tissue engineering scaffolds
© Kim et al. 2016
Received: 4 November 2015
Accepted: 4 January 2016
Published: 18 January 2016
Hydrogels can serve as three-dimensional (3D) scaffolds for cell culture and be readily injected into the body. Recent advances in the image technology for 3D scaffolds like hydrogels have attracted considerable attention to overcome the drawbacks of ordinary imaging technologies such as optical and fluorescence microscopy. Multiphoton microscopy (MPM) is an effective method based on the excitation of two-photons. In the present study, C2C12 myoblasts differentiated in 3D gelatin hydroxyphenylpropionic acid (GHPA) hydrogels were imaged by using a custom-built multiphoton excitation fluorescence microscopy to compare the difference in the imaging capacity between conventional microscopy and MPM.
The physicochemical properties of GHPA hydrogels were characterized by using scanning electron microscopy and Fourier-transform infrared spectroscopy. In addition, the cell viability and proliferation of C2C12 myoblasts cultured in the GHPA hydrogels were analyzed by using Live/Dead Cell and CCK-8 assays, respectively. It was found that C2C12 cells were well grown and normally proliferated in the hydrogels. Furthermore, the hydrogels were shown to be suitable to facilitate the myogenic differentiation of C2C12 cells incubated in differentiation media, which had been corroborated by MPM. It was very hard to get clear images from a fluorescence microscope.
Our findings suggest that the gelatin-based hydrogels can be beneficially utilized as 3D scaffolds for skeletal muscle engineering and that MPM can be effectively applied to imaging technology for tissue regeneration.
Recently, the development of medical image technology for biomedical applications has attracted considerable interest . Conventional image technologies, including optical microscopy and confocal microscopy have contributed significantly to the development of biology and biomedicine [2–5]. On the other hand, the conventional image technologies have several disadvantages, such as light attenuation in tissue and limited light transparency. Multiphoton microscopy (MPM) is an effective method to overcome the limitations of conventional technologies .
MPM is a powerful tool that is based on the excitation of two-photons . Confocal microscope a useful tool to obtain three-dimensional (3D) image, is technically similar to MPM. However, it has limitation to observe thick specimens because the scattering of excitation and emission photons. Also, the absorption of the excitation light induced the photo-bleaching. On the other hand, MPM can measure the deeper layers of tissue due to a decrease in light attenuation and minimize the photo-damage site [8–11]. In addition, this technology improves the signal to background ratio as well as the sensitivity and spatial resolution. Therefore, MPM has attracted considerable attention as a superior image technology. This non-linear optical phenomenon allows one to obtain a three dimensional image of a cell and tissue [12–16]. The custom-built MPM used in this study consists of a femtosecond (fs) titanium:sapphire laser, polarizer, x-y scanner, beam expander, dichoic mirror, photomultiplier tube (PMT), discriminator, and etc. The nominal value of the laser pulse-width was approximately 120 fs and the in situ average power was between 5 to 50 mW.
On the other hand, the development of a biomimetic artificial scaffold for tissue engineering applications using a range of biomaterials has attracted interest. Among the many biomaterials, hydrogels are used widely to culture cells in 3D [17–19]. A hydrogel can be injected into the body to simultaneously synthesize a scaffold [20–22]. In particular, the hydrogel used in our study can be easily controlled the mechanical properties including stiffness by adjusting the concentration of hydrogen peroxide (H2O2) and horseradish peroxidase (HRP) [23, 24]. When the cells are cultured in hydrogel-based scaffolds, they are grown inside the hydrogel in 3D. Especially, gelatin-based hydrogels have excellent biocompatibility and non-immunogenicity, and also possess RGD sequences, which support the cell adhesion, proliferation and differentiation [25–30]. Although there are many systems for observing the cellular behaviors, there are limitations and restrictions to the observation of cellular behaviors using 2D imaging systems. Therefore, to address this limitation, MPM was used to observe cells growing inside a gelatin-based hydrogel in 3D.
In the present study, gelatin-based hydrogels were prepared and their physicochemical properties were characterized. The chemical composition of the hydrogels was characterized by Fourier-transform infrared (FT-IR) spectroscopy. The morphology of the hydrogels was observed by scanning electron microscopy (SEM). In addition, C2C12 myoblasts were cultured in the hydrogels and incubated in growth media (GM) and differentiation media (DM). Immunofluorescence analysis was conducted for the myosin heavy chain (MHC) of C2C12 myoblasts to examine the myogenic differentiation of myoblasts. The stained cells were imaged by custom-built multiphoton excitation fluorescence microscopy.
Gelatin hydroxyphenylpropionic acid (GHPA) conjugates were synthesized using the procedure described elsewhere (Fig. 2) [19, 21]. Briefly, gelatin was dissolved in 40 °C deionized water for 1 hour. Hydroxyphenylpropionic acid (HPA) was dissolved in water and dimethylformamide (DMF) then mixed with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After then, HPA solution was added to gelatin solution and stirred at 40 °C for 1 day. The resulting solution was dialyzed deionized water for 3 days and subjected to the dialysis in NaCl, water/ethanol and distilled water, subsequently. The purified solution was freeze-dried to obtain the GHPA conjugates. Peroxidase from horseradish (type VI, Sigma-Aldrich Co., St. Louis, MO) and H2O2 (30 wt. % in H2O, Junsei Chemical Co., Tokyo, Japan) were used for cross-linking the GHPA hydrogel.
C2C12 myoblasts (derived from thigh muscle of C3H mice) were obtained from the American Type Culture Collection (Rockville, MD) and maintained routinely in GM, Dulbecco’s modified Eagle’s Medium (DMEM, Welgene, Daegu, Korea) complemented with 10 % fetal bovine serum (Welgene) and a 1 % antibiotic-antimycotic solution (including 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B per mL, Sigma-Aldrich Co.). DM for myogenic differentiation is a low-serum media [DMEM containing 2 % of horse serum (Welgene) and 1 % antibiotic-antimycotic solution]. Live and dead cell assay was conducted by using the Live/Dead Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR). A cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was used to evaluate the proliferation of C2C12 myoblasts in GHPA hydrogels.
Physicochemical characterizations of GHPA hydrogels
The morphology of the GHPA hydrogels was examined by SEM (S-800, Hitachi, Tokyo, Japan). The GHPA hydrogels were prepared in a cell culture slide (SPL life science, Gyeonggi-do, Korea) by mixing 100 μL of 3 % GHPA containing HRP (0.005 mg/mL) and H2O2 (0.01 wt. %). The cross-linked hydrogels were freeze-dried over a 2 day period. The dehydrated hydrogels were sputter coated with an ultrathin layer of gold/platinum by ion sputtering (E1010, Hitach, Tokyo, Japan) prior to the SEM observations.
Compositional analysis of the GHPA hydrogel was performed by FT-IR spectroscopy (Nicolet 560, Nicolet Co., Madison, WI). The GHPA hydrogel was prepared on a slide glass covered with cover glass.
Live and dead cell assay of C2C12 cells in GHPA hydrogels
A live and dead cell assay was implemented to measure the viability of the C2C12 myoblasts in the GHPA hydrogel. The C2C12 myoblasts were seeded in GHPA hydrogel at a density of 1 × 105 cells/mL and incubated in GM. The GM was changed every 2-3 days. After 1 and 7 days of culture, the live and dead cell assay solution (2 μM calcein AM and 4 μM ethidium homodimer-1) was added to each cell-seeded hydrogel and incubated for 30 min at 37 °C. The cells were then observed using Olympus IX81 inverted fluorescence microscope (Olympus Corp, Osaka, Japan).
Proliferation of C2C12 cells in GHPA hydrogels
To evaluate the proliferation of the C2C12 myoblasts in GHPA hydrogel, the C2C12 myoblasts were seeded in GHPA hydrogels at a density of 1 × 105 cells/mL and each cell-seeded hydrogel was incubated with a CCK-8 solution in the last 4 hours of the culture periods (1, 3, 5, and 7 days) at 37 °C in the dark. The absorbance was measured at 450 nm using an ELISA reader (SpectraMax® 340, Molecular Device Co, Sunnyvale, CA).
Immunostaining of C2C12 cells in GHPA hydrogels
To examine the myogenic differentiation, C2C12 myoblasts were seeded in GHPA hydrogels at a density of 1 × 105 cells/mL and incubated in GM for 4 days. The cells were then incubated for an additional 3 days in GM and DM, respectively. The cells were fixed with a 3.7 % formaldehyde solution (Sigma-Aldrich Co.) for 10 min and permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich Co.) for 5 min. Subsequently, the cells were blocked with a 2 % bovine serum albumin (BSA, GenDEPOT, Barker, TX) solution in Dulbecco’s phosphate-buffered saline (DPBS, Gibco BRL, Rockville, MD) for 1 hour and incubated with Alexa Fluor 488-conjugated anti-MHC monoclonal antibody (1:200 in 1 % BSA solution in DPBS, eBioscienceInc., San Diego, CA) overnight at 4 °C. Subsequently, the cells were incubated with TRITC-labeled phalloidin (200 units/mL in methanol, 1:40 in 1 % BSA solution in DPBS, Molecular Probes, Eugene, OR) for 1 hour at room temperature. The stained cells were imaged using an Olympus IX81 inverted fluorescence microscope and a custom-built MPM. The fluorescence images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD).
Custom-built multiphoton microscopy
All variables were tested in three independent cultures for each experiment, which was repeated twice (n = 6). All the quantitative data is expressed as the mean ± standard deviation (SD). Statistical comparisons were carried out by a one-way analysis of variance (ANOVA), followed by a Bonferroni test for multiple comparisons. A value of p < 0.05 was considered statistically significant.
Results and discussion
Characterizations of GHPA hydrogels
Live and dead cell assay and proliferation of C2C12 cells in GHPA hydrogels
Immunofluorescence staining analysis of C2C12 cells in GHPA hydrogels
In this study, MPM, which is based on the excitation of two-photons, was customized and the C2C12 myoblasts in GHPA hydrogels were observed. These results showed that SEM and 2D fluorescence microscopy have a high resolution, but the images provide only morphological information. On the other hand, MPM images can provide both morphological and functional information. In addition, MPM has outstanding resolution with a spatial resolution of several hundreds of nanometers. Moreover, MPM images of cells, cultured even inside the GHPA hydrogels could be obtained. Therefore, MPM is a suitable imaging system for observing and analyzing cells and 3D tissues. Overall, MPM can be applied effectively to biomedical imaging technology for tissue engineering applications.
This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (No. 2015M3A9E2028643) and by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2013R1A2A1A09013980).
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