1. Introduction
Gelatin, a form of denatured collagen, is a versatile biomaterial that can be transformed into various forms such as hydrogels, films, and microparticles[1]. Due to its low immunogenicity and compatibility with different cell types, it is a desirable material for tissue engineering and regenerative medicine applications[2]. However, gelatin is biodegradable in vivo unless it is chemically crosslinked. To address this issue, gelatin has been used to modify synthetic polymers, including polycaprolactone scaffolds, electrospun poly(ε-caprolactone) fibers[3], and poly (L-lactic acid)[4], for various applications such as cartilage tissue engineering and drug delivery[5].
Fibroin is a structural protein of silk fiber that has been utilized for decades as a suture material and developed into biocompatible materials such as films, hydrogels, and coating materials[6]. Unlike gelatin, fibroin lacks the RGD peptide, which is a specific sequence of three amino acids that is commonly found in many extracellular matrix proteins and serves as a binding site for cell surface receptor called integrin[ 7]. Nevertheless, fibroin has superior mechanical strength and slower degradation rates than gelatin[8]. Therefore, integrating gelatin onto the surface of fibroin-based biomaterials has the potential to create advanced biofunctional materials. Both gelatin and fibroin are abundant and biocompatible substances[9], making them ideal candidates for this type of modification. Combining fibroin's impressive properties with the functional benefits of gelatin could lead to the development of innovative and highly desirable biofunctional materials.
To this end, we have aimed to modify the surface of fibroin microspheres with gelatin through three different techniques, including surface coating, incorporation, and covalent bonding. Given that the effectiveness of RGD peptides may differ based on the arrangement of gelatin molecules[10], we compared the three distinct methods of surface modification by assessing their impact on cell adhesion and migration. Our investigation would contribute to the advancement of biofunctional materials for tissue engineering and regenerative medicine applications.
2. Materials and methods
2.1. Materials
The freeze-dried fibroin was obtained from FineCo., Ltd. (Chuncheon, Korea), while papain (P3375), decane, propidium iodide (PI), Span 80, Tween 80, 4',6-diamidino-2-phenylindole (DAPI), gelatin type A (G1890) and other necessary reagents were sourced from Sigma-Aldrich (St. Louis, MO, USA). The BALB/c 3T3 cell line and human dermal fibroblast ATCC (HDF; PCS 201 012) was purchased from ATCC (American Type Culture Collection CCL163; Rockville, MD, USA).
2.2. Preparation and modification of Fibroin microspheres with gelatin
For the preparation of fibroin microspheres, an aqueous fibroin 2% solution (8 mL) was mixed with an organic phase consisting of Span 80 (2.2 mL), Tween 80 (1.8 mL), and decane (16 mL). The mixture was homogenized using a Polytron PT2100 homogenizer (Kinematica, Lucerne, Switzerland) at a speed of 30,000 rpm for 60 seconds. The resulting translucent emulsion was subsequently dried in a rotary vacuum evaporator (JP/N 1000S-W, Eyela, Tokyo, Japan) at 40 °C for 30 minutes. The microspheres formed in the emulsion were collected by centrifugation at 4,000 rpm for 5 minutes, and then washed several times with ethanol and air-dried at room temperature. The morphology of the microspheres was observed by field-emission scanning electron microscopy (FESEM; Hitachi S-4800; Hitachi, Japan) at an accelerating voltage of 5.0 kV.
To achieve gelatin coating, the fibroin microspheres were submerged in a warm 2% gelatin solution for 1 hour. Following this, the microspheres were collected and extensively washed with ethanol. Gelatin integration into fibroin was accomplished by solubilizing the gelatin at a concentration of 0.5% in the fibroin solution, followed by the emulsion dehydration process. To achieve covalent bonding of gelatin molecules on the surface of fibroin microspheres, an enzymatic peptide synthesis method under poor solvent conditions was employed, as previously described in the literature[11]. The quantity of gelatin on the surface of fibroin microspheres was assessed using Fluoroskan Ascent (ThermoLabsystems, Helsinki, Finland) and a confocal fluorescent microscope (LSM880, Carl Zeiss, Germany), with the aid of Fluorescein isothiocynate (FITC) labeled gelatin.
2.3. Cell culture and wound-healing assay with gelatin-modified microspheres
BALB/c 3T3 cells were cultured at 37 °C in a humidified atmosphere maintained at 5% CO2 using Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Walkersville, MD, USA) with 4.5 g/L glucose and l-glutamine supplemented with 10% bovine calf serum (BCS; Lonza, USA). Cells were dissociated using trypsin/EDTA (trypsin 0.25%, EDTA 1 mM in PBS; Lonza), counted and transferred. Fibroin microsphere suspension (1 mg/mL), which was sterilized by autoclaving, was added to the cells at the concentration of 0.05 mg/mL.
To perform the wound-healing assay[12], the cells were seeded into 12-well plates and grown to confluency. A sterile 200 μL pipette tip was used to create a scratch/wound in the cell monolayer. The cells were then washed with phosphate-buffered saline (PBS) to remove any debris, and fresh medium with or without the microspheres was added to the wells. After 24 hours of incubation, images of the cells were captured and the distance between the edges of the scratch/wound was measured using ImageJ (NIH, Bethesda, MD, USA)[13]. Furthermore, cell proliferation was evaluated using 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay for detecting DNA synthesis in cells[14].
2.4. Measurement of individual cell migration speed
Individual cell migration speed was measured by culturing 10,000 cells, allowing them to adhere for 3 hours, and then capturing time-lapse images using a remote camera control program every 5 minutes for 4 hours. A total of 43 images with a resolution of 5456 × 3632 pixels were obtained and then reduced to 200 × 400 pixels using software. The iTrack4U software was used to measure the cell migration speed[15].
3. Results and discussion
3.1. Preparation of gelatin-modified microspheres
Gelatin-modified fibroin microspheres were prepared using three methods: coating, incorporation, and covalent bonding. Microsphere size and morphology were characterized using scanning electron microscopy (Figure 1). Gelatin-coated microspheres had a slightly increased diameter of 5%, while the other modified microspheres had similar shapes and size distributions to unmodified fibroin microspheres. The modified microspheres' precipitation behavior was investigated, and gelatin-modified microspheres showed a delayed precipitation pattern compared to unmodified fibroin microspheres as shown in Figure 2(a). Settling continued for several days, suggesting that collagen had altered the surface interaction among the modified microspheres.
The utilization of FITC-labeled gelatin allowed confirmation of the surface modification of fibroin microspheres. Confocal micrographs of the modified microspheres (Figure 2(b)) showed that FITC-gelatin molecules were associated with fibroin microspheres. However, the fluorescent patterns were different between the three modification methods. The microspheres modified with gelatin coating displayed blurry fluorescent regions, whereas the gelatin-incorporated and gelatin-coupled microspheres exhibited fluorescent spots of similar size to that of microspheres. The intensity of fluorescence was measured to be 182 ± 9.1, 11 ± 1.2, and 54 ± 1.2 U/mg for FITC-gelatin-coated, FITC-gelatin- incorporated, and FITC-gelatin-coupled microspheres, respectively (as shown in Figure 2(c)). These results indicated that the level of FITC-gelatin coverage on fibroin microspheres was dependent on the modification method used.
Therefore, the surface of fibroin microspheres was successfully modified using three different methods, although with differing surface coverage. Prior to optimizing the modification methods, this study aimed to determine whether gelatin modification could enhance the biofunctionality of fibroin microspheres.
3.2. Enhanced 3T3 cell migration with gelatin-modified microspheres
To evaluate the impact of fibroin microspheres modified with gelatin, a wound-healing assay was performed. Micrographs of mechanically scratched gaps of confluent monolayers of 3T3 cells in the presence of microspheres and gelatin-modified microspheres (concentration of 0.05 mg/L) at 0 and 24 h are presented in Figure 3(a). The migration of the monolayer edge was quantified and found to be significantly higher in the presence of gelatin-coated, gelatin-incorporated, and gelatin-coupled microspheres, with distances of 328 ± 15.6, 316.4 ± 38, and 259.8 ± 18.7 μm, respectively (Fig. 3(b)), compared to the unmodified microspheres at 145.3 ± 6.2 μm. Furthermore, after 24 hours, the gap between monolayers was filled by cells and became sub-confluent only in the presence of gelatin-modified microspheres.
The cells migrated and filled the gap of the monolayers were stained with DAPI and quantified using a fluorescent microscope. The cell densities at the center of the monolayer gaps for gelatin-coated, gelatin- incorporated, and gelatin-coupled microspheres were 1.0 × 103, 4.2 × 102, 3.4 × 102 cells/mm2, respectively, which were significantly higher than the density of 1.8 × 102 cells/mm2 when unmodified fibroin microspheres were supplemented in the wound-healing assay. The highest cell migration distance was observed with gelatin-coated microspheres, followed by gelatin-incorporated and gelatin-coupled microspheres, which were significantly higher than unmodified fibroin microspheres. These findings indicate that the presence of gelatin on the surface of fibroin microspheres promotes cell migration.
It should be noted that the gap between the microspheres could have been filled by cell proliferation stimulated by gelatin molecules in addition to cell migration[13]. To determine if 3T3 cells were proliferating or not, a nucleoside incorporation assay using EdU was conducted. Figure 4 displays the percentage of EdU-incorporated 3T3 cells at 2 and 12 hours. The background percentages were approximately 7%, suggesting that the migration of 3T3 cell monolayers was due to cell migration rather than cell proliferation. However, the percentage of EdU-positive 3T3 cells increased to 13% in DMEM with 10% serum. These results clearly indicate that gelatin-modified microspheres have a significant impact on 3T3 cell migration.
3.3. Migration velocity of individual 3T3 cells
We also investigated the effect of gelatin-modified microspheres on the individual cell migration speed of 3T3 cells in the absence of contact inhibition. 3T3 cells were inoculated into a culture dish without contact with each other and observed using a phase-contrast microscope at room temperature for approximately 9 hours. Digital images of 40x magnification were captured every 5 minutes and analyzed using an automated cell image tracking software, iTrack4U. The software tracked the movement paths and measured the migration speeds of 100 individual cells from each starting point. 3T3 cells moved more actively in gelatin-modified microspheres supplemented with serum-free DMEM medium than in the control group, and most of them moved far away from the starting point.
The mean migration speeds of cells with microspheres modified by gelatin coating, incorporation, and coupled by enzyme were 1.99, 1.86, and 2.08 μm/min, respectively. While the cells showed the highest activity in microspheres enzyme-modified with gelatin, there was no significant difference among the modified microspheres. The velocity was significantly increased to 1.69 μm/min from 0.87 μm/min for serumfree DMEM medium and 1.16 μm/min for 10% serum-containing DMEM medium by unmodified fibroin microspheres. The findings suggest that microspheres are more efficient than serum in stimulating cell migration under non-contact inhibition conditions.
3.4 Enhancing HDF cell migration by gelatin-modified microspheres
To evaluate the effect of fibroin microspheres modified with gelatin in wound-healing assays, HDFs were used instead of the commonly employed 3T3 cells. HDF cells are primary human cells that are derived from skin tissue and have a similar phenotype to fibroblast found in vivo[12]. As shown in Figure 5(a), when gelatin-coated, gelatin-incorporated, and gelatin-coupled microspheres were supplemented, the migration distances of HDF monolayers were 410.6 ± 26.2, 298.4 ± 28.3, and 381.7 ± 21.7 μm, respectively. These values were significantly higher than those observed with unmodified microspheres and without microsphere supplementation. Gelatin-coating and gelatin- coupling demonstrated a similar impact on HDF migration. However, the addition of serum to the culture medium showed an acceleration in HDF migration at 12 hours, reaching the highest migration distance of 538.2 ± 36.3 μm at 24 hours.
The gelatin-modified microspheres exhibited a significant improvement in both 3T3 and HDF cell migration in vitro. Wound healing assays revealed that the cell migration was increased from 50% to 200% compared to the unmodified microspheres. However, no significant differences were found among the gelatin-modified microspheres based on the different modification methods used. Although the gelatin- coated microspheres exhibited the highest level of FITC-gelatin, the cell migration enhancement was comparable to the gelatin-incorporated microspheres for 3T3 cells and to the gelatin-coupled microspheres for HDFs. Among the presented methods of surface modification with gelatin, gelatin-coating and gelatin-incorporation are relatively simple and can be scaled up easily compared to enzymatic gelatin-coupling on fibroin microspheres. These results suggest that further optimization of gelatin modification is necessary to maximize the biofunctionality of fibroin microspheres.
These findings agree with previous studies that demonstrated recombinant fibroins carrying RGD to have superior properties for promoting rapid cell adhesion, spreading, and proliferation of L929 cells [14]. Moreover, surface modification with gelatin has been shown to enhance the biofunctionality of synthetic polymers such as polyurethane vascular grafts[15] and polycaprolactone scaffolds[16]. Fibroin microspheres are particularly attractive for surface modification with gelatin due to their proteinaceous surface, which can facilitate the folding of gelatin molecules. These results suggest that gelatin modification of fibroin microspheres may offer a promising strategy for developing functional biomaterials with enhanced biological properties.
4. Conclusions
In conclusion, this study has successfully demonstrated the modification of the surface of fibroin microspheres with gelatin using three different methods: coating, incorporation, and covalent bonding. Despite the differences in FITC-gelatin coverage depending on the method of modification, this study aimed to determine whether the modification of fibroin microspheres with gelatin could enhance their biofunctionality. Wound-healing assays were conducted to evaluate the effect of the gelatin-modified microspheres on cell migration, and the results revealed that gelatin modification of fibroin microspheres enhances cell migration. There was any significant difference among the modified microspheres showing the highest cell migration distance. Furthermore, the gelatin-modified microspheres were also found to increase the migration velocity of individual 3T3 cells under non-contact inhibition conditions.
This study has demonstrated that gelatin-modified fibroin microspheres can promote wound healing by stimulating cell migration possibly through enhanced cell-microsphere interactions via the RGD peptide sequence. This has important implications for various biomedical applications, including scaffolds for cell culture, injectable dermal fillers, and cell tracking. These findings suggest that gelatin-modified fibroin microspheres could be a versatile tool for regenerative medicine and tissue engineering research. It is worth noting that further optimization of the gelatin modification process is necessary to maximize the biofunctionality of fibroin microspheres.
