Hydrogels are considered useful scaffolds for encapsulation and delivery of cells and bioactive molecules, such as for tissue engineering and cell therapeutic applications, due to their high water content; usually >30% [1
]. Hydrogels used in these types of applications have mechanical and structural properties similar to some tissues and extracellular matrices (ECM), therefore, they can be implanted for tissue restoration or local release of therapeutic factors. To encapsulate and deliver cells, hydrogels must be formed without damaging cells, must be biocompatible, and must have suitable mass transport capability, sufficient mechanical integrity and strength, and controllable biodegradability [2
A variety of synthetic materials and naturally derived materials have been used to form hydrogels. Gelation occurs when the polymer chains crosslink either chemically or physically into networks, triggered by chemical reagents (cross-linkers) or physical stimulants (pH, temperature). Hydrogels formed from synthetic polymers offer the benefit of gelation and gel properties that are controllable and reproducible through the use of specific molecular weights, block structures, and modes of crosslinking. Generally, gelation of naturally derived polymers is reported to be less controllable, although the hydrogels formed are more compatible for hosting cell and bioactive molecules [3
]. Among naturally derived biomaterials, silk fibroin protein, the self-assembling structural protein in natural silkworm fibers, has been studied because of its excellent mechanical properties, biocompatibility, controllable degradation rates, and inducible formation of crystalline β-sheet structure networks [5
]. Silk fibroin has been fabricated into various material formats including films, three dimensional porous scaffolds, electrospun fibers and microspheres for tissue engineering and controlled drug release applications [10
]. In nature, silk fibroin aqueous solution is produced in the posterior section of the silkworm gland and then stored in the middle section at a concentration up to 30% (w/v) and contains a high content of random coil and alpha helical structure. During fiber spinning into air, shear forces and elongational flow-induced self-assembly result in a structural transition of the protein into the β-sheet structure, leading to the formation of solid fibers [15
]. The presence of metallic ions and pH changes in different sections of the gland influence this transition [16
]. In vitro,
purified silk fibroin aqueous solutions undergo self-assembly into β-sheet structures and form hydrogels. This sol-gel transition is influenced by temperature, pH, and ionic strength [20
]. The compressive strength and modulus of silk hydrogels increased with an increase in silk fibroin concentration and temperature [21
Silk fibroin hydrogels are of interest for many biomedical applications. Fini et al. used low-pH induced silk fibroin hydrogels as a bone-filling biomaterial to heal critical-size cancellous defects of rabbit distal femurs, and silk gels showed better bone healing than the control material, poly(D, L lactide-glycolide) [23
]. For many cell-based applications, gelation must be induced under mild conditions in a relatively short period of time (within hours). However, silk gelation time is prohibitively long unless nonphysiological treatments are considered (such as low pH, high temperature, additives) in the absence of chemical modifications to the native silk fibroin protein. For silk fibroin concentrations from 0.6 to 15 % (w/v), days to weeks were required for the sol-gel transition at room temperature or 37°C [21
]. Adding salts at concentrations above physiological levels did not significantly alter the gelation kinetics [21
]. Lowering pH (<5) or increasing temperature (>60°C) could reduce the gelation time to a few hours [21
]; conditions which could potentially alter cell function and affect cell viability.
In the present study, a new ultrasonication-based method was developed and then used to accelerate the sol-gel transition in a temporally controllable manner. Mechanistically, the process induces physical beta sheet crosslinks via alternations in hydrophobic hydration of the protein chains. This permitted cell additions post-sonication, followed by rapid gelation. Gelation time could be controlled from minutes to hours based on the sonication parameters used (energy output and duration time) and silk fibroin concentrations. The pH and salt concentration effects on gelation, the dynamic silk structural changes after gelation, and the behavior of encapsulated human bone marrow derived mesenchymal stem cells (hMSCs) in silk gels were studied.