Phospholipid‐induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration

Abstract

Silk fibroin (SF) hydrogels can be obtained via self-assembly, but this process takes several days or weeks, being unfeasible to produce cell carrier hydrogels. In this work, a phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DMPG), was used to induce and accelerate the gelation process of SF solutions. Due to the amphipathic nature and negative charge of DMPG, electrostatic and hydrophobic interactions between the phospholipids and SF chains will occur, inducing the structural transition of SF chains to the beta sheet and consequently a rapid gel formation is observed (less than 50 min). Moreover, the gelation time can be controlled by varying the lipid concentration. To assess the potential of the hydrogels as cell carriers, several mammalian cell lines, including L929, NIH/3T3, SaOS-2, and CaSki, were encapsulated into the hydrogel. The silk-based hydrogels supported the normal growth of fibroblasts, corroborating their cytocompatibility.Interestingly, an inhibition in the growth of cancer-derived cell lines was observed.Therefore, DMPG-induced SF hydrogels can be successfully used as a 3D platform for in situ cell encapsulation, opening promising opportunities in biomedical applications, such as in cell therapies and tissue regeneration.Bombyx mori "Nangnoi Srisaket 1" Thai silk cocoons were obtained from Queen Sirikit Sericulture Center, Srisaket province, Thailand.DMPG and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Lipids Polar, USA. Cell culture medium and all other reagents were purchased from Thermo Fisher Scientific.All reagents were of analytical grade. | SF solution preparation and its zeta potential determinationSF solution was prepared using the method adapted from Kim, Park, Joo Kim, Wada, and Kaplan (2005). In brief, silk cocoons were boiled in 0.02 M Na 2 CO 3 for 20 min, followed by their thoroughly washing with distilled water. The extracted silk fibre was then dissolved in 9.3 M LiBr at 60°C for 4 hr. The silk solution was dialysed against distilled water using a dialysis tube (MWCO 12-16 kDa; Sekisui, Japan) for 48 hr. The final concentration was approximately 6%, which was determined from dried solid weight.Analysis of zeta potential of the SF solution was conducted using laser doppler electrophoresis in a Zetasizer Nano ZS equipment (Malvern Instruments) at 25°C.

Exploring the Gelation Mechanisms and Cytocompatibility of Gold (III)-Mediated Regenerated and Thiolated Silk Fibroin Hydrogels

“…Indeed, a short gelation time is crucial for many applications, such as cell-loaded hydrogels or injectable controlled release systems. Crosslinking agents (e.g., glutaraldehyde, carbodiimides, or genipin), enzymes (e.g., tyrosinase or peroxidase), physical methods (e.g., vortexing or sonication), or chemical additives (e.g., alcohols [4], surfactants [5], or phospholipids [6]) have been applied to control the kinetics of SF gelation [7].…”

Comparative Study of Silk Fibroin-Based Hydrogels and Their Potential as Material for 3-Dimensional (3D) Printing

“…From our results, the 2SF/STS and 2SF/DMPG hydrogels showed better flow behavior, higher structure recovery and higher structure stability than 2SF/self-gelled hydrogel during printing and after post-treatment processes. In addition, the non-cytotoxicity of SF/STS and SF/DMPG hydrogel were shown in previous studies [ 26 , 37 ]. Therefore, these SF-based hydrogel formulations demonstrated the potential as material for 3D-printing and applied for several biomedical applications, such as tissue engineering.…”

“…This gelation processes takes over two weeks, which is not practicable for the applications as cell-encapsulated bioink or injectable hydrogels. To reduce the gelation time, some amphipathic chemicals, such as surfactants and phospholipids were added into SF solution [ 25 , 26 ]. It can enhance the formation of β-sheet structure due to electrostatic and hydrophobic interactions.…”

“…Three formulations of SF hydrogels including natural gelation (SF/self-gelled), STS-induced (SF/STS) and DMPG-induced (SF/DMPG) were prepared according to the methods reported in our previous researches with slight modifications ( Table 1 ) [ 25 , 26 ]. The SF solution was loaded in the syringe and the hydrogel was formed within the syringe used for 3D-printing.…”

“…Moreover, photopolymerization, and the chemical crosslinkers, such as carbodiimides, glutaraldehyde, or genipin were used. Our previous studies demonstrated various formulations of SF-based hydrogels that can be induced by different agents, such as alcohols [ 24 ], an anionic surfactant, sodium tetradecyl sulfate (STS) [ 25 ], and a phospholipid 1,2-dimyristoyl- sn -glycerol-3-phospho-(1′- rac -glycerol) (DMPG) [ 26 ]. STS is as anionic surfactant that have been shown to accelerate the gelation of SF rather than the cationic and nonionic surfactant.…”