Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation and collagen production

Abstract

BACKGROUND:

Collagen production in fibroblasts is important for skin tissue repair. Cell-adhesive Arg-Gly-Asp (RGD) peptides immobilized on scaffolds stimulate fibroblast collagen production, but RGD peptides in solution exhibit opposite effects. Transgenic silkworm technology enables the design of fusion positions for RGD peptides in silk fibroin molecules. The effect of RGD-fused silk fibroin in solution on fibroblast cell activity remains unclear.

OBJECTIVE:

To clarify the effects of RGD peptides fused to silk fibroin heavy (H)-chain or light (L)-chain on fibroblast proliferation and collagen production when RGD-fused silk fibroin proteins were added to the culture medium.

METHODS:

Silk fibers with RGD-fused H-chains (H-RGD) or L-chains (L-RGD) were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions, respectively. These solutions were added to the fibroblast medium, and their proliferation and collagen production were quantified.

RESULTS:

Both L- and H-RGD stimulated fibroblast proliferation at a similar level, even in a solution format, but L-RGD promoted fibroblast collagen production significantly, indicating the synergistic effect of the native H-chain and RGD-fused L-chain.

CONCLUSION:

RGD-fused silk fibroin in solution stimulated fibroblast proliferation and collagen production, depending on the fusion position of the peptides.

Keywords

Silk fibroin solution format collagen production RGD transgenic silkworm

1. Introduction

Collagens are elongated fibrils in the extracellular matrix (ECM) and act as the principal tensile element of the skin [1]. During skin tissue repair, collagens are produced by fibroblasts [2], which are thereafter bundled to support the structural integration with other ECM components such as fibrin and fibronectin [3,4]. Thus, the stimulation of collagen production by fibroblasts is important for skin tissue repair. An increase in cell adhesion to a substrate is a way to stimulate fibroblast collagen production [5,6]. For instance, Graham et al. showed that fibronectin coating on a glass substrate promoted collagen type I production in fibroblasts [7].

An Arg-Gly-Asp (RGD) amino acid sequence is the minimum unit of the cell-adhesive activity of fibronectin [8], and this peptide is frequently used to modify scaffolds for tissue engineering to promote the cell-adhesive function of the scaffolds [8–11]. When immobilized on polymeric scaffolds, the RGD peptide stimulates fibroblast adhesion [12,13], proliferation [14,15], and collagen production [16]. In contrast, RGD peptides added to the cell culture medium in a solution format prevented these cell activities [17–20]. Brenner et al. [18] reported that RGD peptides in a solution format blocked transmembrane proteins (i.e., integrins) to inhibit tumor cell migration. Kambe et al. [19] showed that RGD peptides prevented chondrocyte adhesion to a scaffold, likely owing to the blocking of integrins on the chondrocyte surface when added to a culture medium in a solution format. In addition, Iwamoto et al. [20] revealed that RGD peptide stimulated collagenase expression in stellate cells, resulting in a significant reduction in collagen type I accumulation in cells in a solution format. Therefore, RGD peptides would affect cell activity depending on their format, position, and/or mobility.

Bombyx mori silk fibroin is a natural polymer that has a long history of use as a surgical suture material. This fibrous protein has been processed into versatile formats, such as solutions, sponges, and hydrogels, to be used as scaffolds for tissue engineering [19,21–24]. In addition, the establishment of transgenic silkworm technology has enabled the fusion of RGD peptides into silk fibroin proteins [25]. Silk fibroin protein consists of a disulfide-linked heavy (H)-chain (molecular weight, 360 kDa) and light (L)-chains (molecular weight, 27 kDa) [26], and each chain has been reported to be genetically modified with RGD peptides [20,27–30]. When processed into films, both silk fibroin with RGD-fused H-chain (H-RGD) and that with RGD-fused L-chain (L-RGD) promoted fibroblast adhesion [19,27,28]. However, the effects of RGD-fused silk fibroin in a solution format on cell activities remain unclear.

The purpose of this study is to investigate the effect of RGD-fused silk fibroin added to culture medium on fibroblast proliferation and collagen production in solution. Previous structural studies on silk fibroin in an aqueous solution format suggested its random coil conformation [31]. In contrast, recent studies on the primary sequence of silk fibroin H-chain have suggested the possibility of silk fibroin micellar structures in water [32]. This is because of the hydrophobic and hydrophilic block patterns of the H-chain [33]. These structural findings indicate that the effect of RGD peptides fused to silk fibroin would depend on the presence or absence of the hydrophobic–hydrophilic repeated structure. In this study, we prepared two types of RGD-fused silk fibroin proteins, H-RGD [27] and L-RGD [19], to determine the influence of the position of RGD peptides in silk fibroin molecules on fibroblast activity. In H-RGD, the hydrophobic and hydrophilic blocks in the H-chain were replaced with a repetitive hydrophilic peptide containing eight RGD sequences (Fig. 1A and 1B). This peptide is expected to form a random coil conformation in an aqueous solution. In L-RGD, two RGD sequences were fused at the carboxy-terminal of the hydrophilic L-chain (Fig. 1C and 1D), whereas the H-chain was not modified. Thus, L-RGD is expected to form a micellar structure in an aqueous solution similarly to native silk fibroin [32]. Cocoons of transgenic silkworms that produce H-RGD or L-RGD were degummed, dissolved, and dialyzed to prepare H-RGD or L-RGD aqueous solutions. These silk fibroin solutions were added to the fibroblast culture medium, and their proliferation and collagen production were quantified.

Fig. 1.

Amino acid sequences of (A) B. mori silk fibroin native H-chain, (B) RGD-fused H-chain, (C) native L-chain, and (D) RGD-fused L-chain. Numbers represent the positions of amino acid residues. Cell-adhesive RGD sequences are marked in red.

2. Materials and methods

2.1. Materials

Wild-type B. mori (silkworm strain, Gunma200) silk cocoons were provided by Dr. Masatoshi Iga (Silkworm Research Group, Division of Silk-Producing Insect Biotechnology, Institute of Agrobiological Sciences, NARO). Transgenic L-RGD and H-RGD B. mori (silkworm strain, LLL-RGD [19], and H6R-Gunma [27], respectively) silk cocoons were prepared at Adan Co. Ltd.

2.2. Preparation of silk fibroin aqueous solution

Wild-type, L-RGD, and H-RGD silk fibroin aqueous solutions were prepared as described previously [28,29,34]. Briefly, silkworm cocoons were degummed in boiled 0.02 M Na₂CO₃ aqueous solution. The degummed silk fibroin fibers were dissolved in 9 M LiBr aqueous solution and dialyzed against deionized water. The resultant silk fibroin aqueous solution was sterilized by autoclaving. The impurities were thereafter removed by centrifugation, and the concentration of the silk fibroin aqueous solution was determined by freeze-drying.

2.3. Evaluation of fibroblast proliferation

Cell culture experiments were conducted by Adan Co. Ltd. The effects of silk fibroin aqueous solutions on fibroblast proliferation were assessed as described previously [35,36] with minor modifications. Briefly, the mouse fibroblast-like cell line, NIH/3T3 (RCB2767), provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan, was seeded onto the wells of a 96-well tissue culture polystyrene (TCPS) plate (AGC Techno Glass, Japan) at 2 × 10³ cells/well and onto a 35 mm glass-based dish (AGC Techno Glass) at 4 × 10⁴ cells/dish. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, MA) containing 10% fetal bovine serum (FBS; Cytiva, MA) and 1% antibiotic mixture (final concentrations, 100 U/mL penicillin and 100 μg∕mL streptomycin; Gibco) at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ for 1 d. The medium was thereafter replaced with DMEM containing 1% FBS, 1% antibiotic mixture, and 0 or 100 μg∕mL silk fibroin. After 2 d of culture, the cells on the 35 mm dish were observed using a phase-contrast microscope (IX-70; Olympus, Japan) equipped with a ×10 objective. In contrast, the cells on the 96-well plate were incubated with WST-1 reagent (Roche Diagnostics, Germany) for 2 h. Absorbance at 450 nm was measured using a plate reader (Varioskan^TM LUX; Thermo Fisher Scientific, USA). Absorbance at 650 nm was used as a reference. Eight wells were used for each culture condition (n = 8).

2.4. Evaluation of fibroblast collagen production

2.4.1. Immunofluorescent staining of collagen type I

NIH/3T3 fibroblasts were seeded onto a 35 mm glass-based dish at a density of 4 × 10⁴ cells/dish and cultured in DMEM containing 10% FBS and 1% antibiotic mixture for 1 d. The medium was replaced with DMEM containing 1% FBS, 1% antibiotic mixture, and 0 or 100 μg∕mL silk fibroin. After culturing for 2 d, collagen type I and nuclei of the cells were immunostained as described previously [19,35] with minor modifications. A rabbit anti-collagen type I polyclonal antibody (ab34710; Abcam, UK) and Alexa Fluor^R488 conjugated goat anti-rabbit IgG H&L (ab150077; Abcam) were used as primary and secondary antibodies to stain collagen type I, respectively. Cell nuclei were stained with Hoechst 33342 (H342; Dojindo Laboratories, Japan). The stained cells were observed using a fluorescence microscope (BZ-X710; Keyence, Japan) equipped with ×10 and ×40 objectives.

2.4.2. Collagen quantification with Sirius red

NIH/3T3 fibroblasts were seeded onto a 6-well TCPS plate at a concentration of 4 × 10⁴ cells/well and incubated in DMEM containing 10% FBS and 1% antibiotic mixture at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. After 1 d of culture, the medium was changed to DMEM containing 1% FBS, 1% antibiotic, and 0 or 100 μg∕mL silk fibroin. The cells were cultured for 2 d, and the amount of deposited collagen was quantified using a Sirius red total collagen detection kit (9062; Chondrex, USA) following the manufacturer’s protocol, where pepsin (6011; Chondrex) was used for collagen solubilization. The absorbance at 530 nm was measured using a plate reader, and six different wells were used for each culture condition (n = 6).

Fig. 2.

Effects of the soluble wild-type, L-RGD, and H-RGD silk fibroin proteins on fibroblast proliferation. No silk fibroin protein was added to the medium of the blank group. (A) Fibroblast proliferation was assessed by WST-1 assay. Higher absorbance implies a larger cell number. Data are shown as mean ± SD (n = 8) and were analyzed by one-way ANOVA followed by Tukey’s post hoc comparison (^∗: p < 0.05, ^∗∗∗ p < 0.001, vs. the blank group). (B) Phase-contrast microscopic images of the cells cultured with/without soluble silk fibroin proteins. Scale bar = 100 μm. (C) Enlarged view of the red rectangles for each image in B. Fibrous precipitation (white arrows) was observed on the dish surface in the L- and H-RGD groups. Scale bar = 10 μm.

Fig. 3.

Effects of the soluble wild-type, L-RGD, and H-RGD silk fibroin proteins on fibroblast collagen production. No silk fibroin protein was added to the medium of the blank group. (A and B) Immunofluorescent staining for the detection of collagen type I. Scale bar = 50 μm (A) and 200 μm (B). (C) Amount of collagen produced by the cells. Data are shown as mean ± SD (n = 6) and were analyzed by one-way ANOVA followed by Tukey’s post hoc comparison (^∗: p < 0.05)

2.5. Statistical analysis

Quantitative data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc comparisons. Statistical significance was set at p < 0.05.

3. Results

3.1. Effects of RGD-fused silk fibroin in a solution format on fibroblast proliferation

Both the L-RGD and H-RGD groups exhibited statistically higher fibroblast proliferation than the blank group (Fig. 2A). This result was supported by microscopic observations; more cells were observed in the L- and H-RGD groups than in the blank and wild-type groups (Fig. 2B). In addition, fibrous substances precipitated on the surface of the cell culture dishes in the L- and H-RGD groups (Fig. 2C). However, no precipitation was observed in the blank or wild-type groups.

3.2. Effects of RGD-fused silk fibroin in a solution format on fibroblast collagen production

As shown in Fig. 3A, no remarkable difference in collagen type I stainability for individual cells was observed among the groups, and no visible collagen type I deposition was observed on the dish surface. However, according to the results of the cell proliferation assay (Fig. 2), more cells were observed in the L- and H-RGD groups (Fig. 3B). Both the L- and H-RGD groups tended to exhibit higher amounts of collagen compared with the blank and wild-type groups, but only the L-RGD group exhibited significantly increased collagen production (Fig. 3C).

Fig. 4.

Assumed mechanism for the formation of a 3-D fibrous network of L-RGD silk fibroin. (A) Schematic illustration of L-RGD silk fibroin, which consists of the native H-chain and RGD-fused L-chain. (B) Assumed molecular folding to form a micellar structure of L-RGD silk fibroin in water. (C) Assumed mechanism for the formation of a 3-D fibrous network of L-RGD silk fibroin when added to a cell culture medium.

Fig. 5.

Assumed mechanism for H-RGD silk fibroin to cover the dish surface two-dimensionally. (A) Schematic illustration of H-RGD silk fibroin, which consists of the RGD-fused H-chain and native L-chain. (B) Assumed molecular folding to form a random coil structure of H-RGD silk fibroin in water. (C) Assumed mechanism for H-RGD silk fibroin to cover the dish surface two-dimensionally when added to a cell culture medium.

4. Discussion

Peptides containing RGD sequence have been used to modify scaffolds for tissue engineering for stimulating cell adhesion [12,13], proliferation [14,15], and ECM production [16]. To exert this effect, RGD peptides need to be immobilized stably to the scaffold surface [35] because the formation of focal adhesions, which mediate transmembrane signaling, only occurs if the peptides on the surface resist the cell contractile forces [10]. Therefore, RGD peptide in a solution format that is added to the cell culture medium blocks transmembrane cell-adhesive molecules and inhibits cell adhesion [19] and collagen production [20]. However, as shown in Figs 2 and 3, L- and H-RGD in the solution format promoted fibroblast proliferation and/or collagen production. These specific effects of RGD-fused silk fibroin proteins added to the medium may stem from the deposition of proteins around the cells. As shown in Fig. 2C, fibrous substances were observed on the dish surfaces of the L- and H-RGD groups. These fibrous substances were considered RGD-fused silk fibroin proteins because they were not immunostained with the anti-collagen type I antibody (Fig. 3A). Thus, after attachment to the fibroblast surface, RGD-fused silk fibroin proteins might precipitate around the cells and act as a cell-adhesive substrate to stimulate cell adhesion and proliferation.

Although both L- and H-RGD exhibited a statistically significant stimulating effect on fibroblast proliferation (Fig. 2), only L-RGD induced significantly higher collagen production (Fig. 3). This might be explained by the synergistic effect of the native silk fibroin H-chain and the RGD-fused L-chain in the L-RGD silk fibroin. The H-chain consists of hydrophilic amino and carboxy-terminal regions and large intermediate hydrophobic blocks connected by short hydrophilic spacers [26]. Thus, the native H-chain is suggested to form a micellar structure [32] and the micellar structures assemble to construct a fibrous three-dimensional (3-D) network similar to a hydrogel [33]. In addition, the RGD peptides in the L-RGD silk fibroin would be the outer surface of the micelles because the RGD-fused L-chain is hydrophilic (the grand average of hydropathicity (GRAVY) value, −0.057, calculated using the ProtParam tool; http://web.expasy.org/protparam/) and is bound to the hydrophilic carboxy-terminal region of the native H-chain. Therefore, after binding to fibroblasts via easy access to the RGD present on the outer surface, L-RGD silk fibroin micelles can easily form a 3-D fibrous network (Fig. 4). In contrast, in the case of H-RGD silk fibroin, the hydrophobic blocks in the native H-chain were replaced with a hydrophilic repetitive peptide containing RGD sequences (GRAVY value, −0.789). This hydrophilic RGD-fused H-chain would not be able to form a micellar structure in an aqueous solvent; instead, it might remain in a random coil structure. Because silk fibroin with a random coil structure does not assemble into a 3-D fibrous network [38], H-RGD might precipitate to cover the dish surface two-dimensionally (Fig. 5), unlike L-RGD. Therefore, the precipitated L-RGD silk fibroin might assemble to act as an artificial fibronectin network to induce collagen production significantly, as fibronectin assembly/network is required for the deposition of collagen type I [39].

The results of this study suggest that both L- and H-RGD silk fibroin proteins in a solution format promote fibroblast proliferation, but the native silk fibroin H-chain with the RGD-fused L-chain is necessary for the stimulation of higher collagen production. Therefore, the application of the L-RGD aqueous solution to the skin would be useful for skin tissue repair.

5. Conclusions

In the present study, two evaluations were performed on L- and H-RGD silk fibroin proteins: fibroblast proliferation and collagen production. Both L- and H-RGD silk fibroin proteins in solution remarkably improved fibroblast proliferation to the same extent. However, the stimulating effect on fibroblast collagen production depended on the position of the fused RGD peptides in the silk fibroin molecules. L-RGD significantly promoted collagen production, indicating that the native H-chain and RGD-fused L-chain synergistic effect is important for activating collagen synthesis in individual fibroblasts.

Footnotes

Acknowledgements

This study was supported by Adan Co., Ltd. and NARO. The authors thank Drs. Tatsuya Iizuka and Masatoshi Iga, Silkworm Research Group, Institute of Agrobiological Sciences, NARO, for valuable advice about transgenic silkworm breading and for providing cocoons of Gunma200 silkworms, respectively.

Conflict of interest

This study was funded by Adan Co., Ltd.

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