Silk Hydrogels Research Articles

Characterization and deposition behavior of silk hydrogels soaked in simulated body fluid

This paper reports the mineralization ability of semi-interpenetrating networks composed of regenerated silk fibroin and polyacrylamide hydrogels soaked in simulated body fluid (SBF1x). Hydrogels were prepared by polymerization of acrylamide and N,N′-methylenebisacrylamide in silk fibroin solution with a redox pair as initiator. The incorporation of the fibroin within the polyacrylamide matrix was proved by FTIR–ATR spectroscopy. Swelling measurements in saline solution were performed to evaluate the behavior of these hydrogels having various compositions. Mineralization assays in SBF1x solution revealed the presence of apatite-like crystals onto the surface of the silk fibroin/polyacrylamide hydrogels. Cytotoxicity test by extract method revealed that these hydrogels with various contents in silk fibroin have not developed cytotoxic effects on human fibroblast cultures which increases the possibility of their use in biomedical applications. Mechanical compressive tests revealed good strengths for silk fibroin/polyacrylamide hydrogels. In this way, organic–inorganic hybrids analogous to bone structure can be produced under biomimetic conditions and could be further used in biomedical applications.

抗菌丝素蛋白水凝胶敷料的制备<br>Preparation of Antimicrobial Silk Hydrogels for Surgical Dressings

季铵盐类抗菌剂作为凝胶促进剂，诱导丝素蛋白水溶液快速原位凝胶化从而制备抗菌性丝素水凝胶敷料。利用X射线衍射、红外光谱、扫描电镜、zeta电位分析、差热分析等手段对所制丝素水凝胶的结构、zeta电位、热稳定、降解性及其抗菌性能进行了表征。研究结果表明，季铵盐会引起丝素颗粒负电位值下降，由此促发形成的丝素水凝胶为β-折叠片层结构，凝胶内部形貌为多孔的三维网状结构，且对革兰氏阳性菌和阴性菌都有很明显的抗菌效果。同时由体外降解、差热分析结果可知，季铵盐引发丝素蛋白溶液快速形成水凝胶，蛋白酶可以在体外降解水凝胶。季铵盐在水凝胶中主要以无定形或亚稳态晶体状态存在，容易扩散释放，达到较好的抗菌效果。抗菌性原位丝素蛋白水凝胶预计可以用于外科伤口敷料。 Using quaternary ammonium salts (QAS) as initiating agents, silk fibroin aqueous solutions were induced to gel rapidly. The structure, zeta-potential change and thermal stability properties of QAS-induced silk gel were charac- terized by means of X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), zeta- potential measurements and differential thermal analysis (DTA). The results show that QAS-induced silk gelation is accompanied by the formation of β-sheets and less negative zeta-potential values. With the observation of SEM, the internal morphology of gel exhibited a porous three-dimensional network structure. In vitro degradation and DTA analysis revealed that QAS-induced fibroin gel was not quite stable and disintegrated more easily in the enzyme solu- tion. Also, QAS is amorphous or metastable crystal state in silk gel and thus easy to release and diffuse. The antibacte- rial test in vitro suggested that silk-QAS gel showed distinctly antibacterial activities against both Gram-positive and Gram-negative bacteria. In situ antimicrobial silk hydrogel is expected to be used for a surgical dressing.

抗菌丝素蛋白水凝胶敷料的制备

季铵盐类抗菌剂作为凝胶促进剂，诱导丝素蛋白水溶液快速原位凝胶化从而制备抗菌性丝素水凝胶敷料。利用X射线衍射、红外光谱、扫描电镜、zeta电位分析、差热分析等手段对所制丝素水凝胶的结构、zeta电位、热稳定、降解性及其抗菌性能进行了表征。研究结果表明，季铵盐会引起丝素颗粒负电位值下降，由此促发形成的丝素水凝胶为β-折叠片层结构，凝胶内部形貌为多孔的三维网状结构，且对革兰氏阳性菌和阴性菌都有很明显的抗菌效果。同时由体外降解、差热分析结果可知，季铵盐引发丝素蛋白溶液快速形成水凝胶，蛋白酶可以在体外降解水凝胶。季铵盐在水凝胶中主要以无定形或亚稳态晶体状态存在，容易扩散释放，达到较好的抗菌效果。抗菌性原位丝素蛋白水凝胶预计可以用于外科伤口敷料。 Using quaternary ammonium salts (QAS) as initiating agents, silk fibroin aqueous solutions were induced to gel rapidly. The structure, zeta-potential change and thermal stability properties of QAS-induced silk gel were charac- terized by means of X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), zeta- potential measurements and differential thermal analysis (DTA). The results show that QAS-induced silk gelation is accompanied by the formation of β-sheets and less negative zeta-potential values. With the observation of SEM, the internal morphology of gel exhibited a porous three-dimensional network structure. In vitro degradation and DTA analysis revealed that QAS-induced fibroin gel was not quite stable and disintegrated more easily in the enzyme solu- tion. Also, QAS is amorphous or metastable crystal state in silk gel and thus easy to release and diffuse. The antibacte- rial test in vitro suggested that silk-QAS gel showed distinctly antibacterial activities against both Gram-positive and Gram-negative bacteria. In situ antimicrobial silk hydrogel is expected to be used for a surgical dressing.

A silk hydrogel-based delivery system of bone morphogenetic protein for the treatment of large bone defects

The use of tissue grafting for the repair of large bone defects has numerous limitations including donor site morbidity and the risk of disease transmission. These limitations have prompted research efforts to investigate the effects of combining biomaterial scaffolds with biochemical cues to augment bone repair. The goal of this study was to use a critically-sized rat femoral segmental defect model to investigate the efficacy of a delivery system consisting of an electrospun polycaprolactone (PCL) nanofiber mesh tube with a silk fibroin hydrogel for local recombinant bone morphogenetic protein 2 (BMP-2) delivery. Bilateral 8 mm segmental femoral defects were formed in 13-week-old Sprague Dawley rats. Perforated electrospun PCL nanofiber mesh tubes were fitted into the adjacent native bone such that the lumen of the tubes contained the defect (Kolambkar et al., 2011b). Silk hydrogels with or without BMP-2 were injected into the defect. Bone regeneration was longitudinally assessed using 2D X-ray radiography and 3D microcomputed topography (μCT). Following sacrifice at 12 weeks after surgery, the extracted femurs were either subjected to biomechanical testing or assigned for histology. The results demonstrated that silk was an effective carrier for BMP-2. Compared to the delivery system without BMP-2, the delivery system that contained BMP-2 resulted in more bone formation (p<0.05) at 4, 8, 12 weeks after surgery. Biomechanical properties were also significantly improved in the presence of BMP-2 (p<0.05) and were comparable to age-matched intact femurs. Histological evaluation of the defect region indicated that the silk hydrogel has been completely degraded by the end of the study. Based on these results, we conclude that a BMP-2 delivery system consisting of an electrospun PCL nanofiber mesh tube with a silk hydrogel presents an effective strategy for functional repair of large bone defects.

Silk–hyaluronan-based composite hydrogels: A novel, securable vehicle for drug delivery

A new, biocompatible hyaluronic acid (HA)-silk hydrogel composite was fabricated and tested for use as a securable drug delivery vehicle. The composite consisted of a hydrogel formed by cross-linking thiol-modified HA with poly(ethylene glycol)-diacrylate, within which was embedded a reinforcing mat composed of electrospun silk fibroin protein. Both HA and silk are biocompatible, selectively degradable biomaterials with independently controllable material properties. Mechanical characterization showed the composite tensile strength as fabricated to be 4.43 ± 2.87 kPa, two orders of magnitude above estimated tensions found around potential target organs. In the presence of hyaluronidase (HAse) in vitro, the rate of gel degradation increased with enzyme concentration although the reinforcing silk mesh was not digested. Composite gels demonstrated the ability to store and sustainably deliver therapeutic agents. Time constants for in vitro release of selected representative antibacterial and anti-inflammatory drugs varied from 46.7 min for cortisone to 418 min for hydrocortisone. This biocomposite showed promising mechanical characteristics for direct fastening to tissue and organs, as well as controllable degradation properties suitable for storage and release of therapeutically relevant drugs.

The use of injectable sonication-induced silk hydrogel for VEGF 165 and BMP-2 delivery for elevation of the maxillary sinus floor

Sonication-induced silk hydrogels were previously prepared as an injectable bone replacement biomaterial, with a need to improve osteogenic features. Vascular endothelial growth factor (VEGF 165) and bone morphogenic protein-2 (BMP-2) are key regulators of angiogenesis and osteogenesis, respectively, during bone regeneration. Therefore, the present study aimed at evaluating in situ forming silk hydrogels as a vehicle to encapsulate dual factors for rabbit maxillary sinus floor augmentation. Sonication-induced silk hydrogels were prepared in vitro and the slow release of VEGF 165 and BMP-2 from these silk gels was evaluated by ELISA. For in vivo studies for each time point (4 and 12 weeks), 24 sinus floors elevation surgeries were made bilaterally in 12 rabbits for the following four treatment groups: silk gel (group Silk gel), silk gel/VEGF 165 (group VEGF), silk gel/BMP-2 (group BMP-2), silk gel/VEGF 165/BMP-2 (group V + B) ( n = 6 per group). Sequential florescent labeling and radiographic observations were used to record new bone formation and mineralization, along with histological and histomorphometric analysis. At week 4, VEGF 165 promoted more tissue infiltration into the gel and accelerated the degradation of the gel material. At this time point, the bone area in group V + B was significantly larger than those in the other three groups. At week 12, elevated sinus floor heights of groups BMP-2 and V + B were larger than those of the Silk gel and VEGF groups, and the V + B group had the largest new bone area among all groups. In addition, a larger blood vessel area formed in the remaining gel areas in groups VEGF and V + B. In conclusion, VEGF 165 and BMP-2 released from injectable and biodegradable silk gels promoted angiogenesis and new bone formation, with the two factors demonstrating an additive effect on bone regeneration. These results indicate that silk hydrogels can be used as an injectable vehicle to deliver multiple growth factors in a minimally invasive approach to regenerate irregular bony cavities.

Controlled Hydrogel Formation of a Recombinant Spider Silk Protein

Due to their biocompatibility, biodegradability, and low immunogenicity, recombinant spider silk proteins have a high potential for a variety of applications when processed into morphologies such as films, capsules, beads, or hydrogels. Here, hydrogels made of the engineered and recombinantly produced spider silk protein eADF4(C16) were analyzed in detail. It has previously been shown that eADF4(C16) nanofibrils self-assemble by a mechanism of nucleation-aggregation, providing the basis of silk hydrogels. We focused on establishing a reproducible gelation process by employing different protein concentrations, chemical crosslinking, and functionalization of eADF4(C16) with fluorescein. Fluorescein strongly influenced assembly as well as the properties of the hydrogels, such as pore sizes and mechanical behavior, possibly due to its interference with packing of silk nanofibrils during hydrogel formation.

State of Water, Molecular Structure, and Cytotoxicity of Silk Hydrogels

A novel technique was developed to regulate the bulk water content of silk hydrogels by adjusting the concentrations of silk proteins, which is helpful to investigate the effects of the state of water in polymeric hydrogel on its biological functions, such as cytotoxicity. Gelation of the silk hydrogel was induced with ethanol and its gelation behavior was analyzed by rheometry. The silk hydrogels prepared at various silk concentrations were characterized with respect to their water content, molecular and network structures, state of water, mechanical properties, and cytotoxicity to human mesenchymal stem cells. The network structure of silk hydrogel was heterogeneous with β-sheet and fibrillar structures. The influence of the state of water in the silk hydrogel on the cytotoxicity was recognized by means of differential scanning calorimetry and cell proliferation assay, which revealed that the bound water will support cell-adhesion proteins in the cellular matrix to interact with the surface of the silk hydrogels.

Silk Fibroin Sol-Gel Transitions in Different Solutions

Silk hydrogels have mechanical properties and structural features that are similar to load-bearing soft tissues, and can be implanted for tissue restoration. In the present study, we investigate silk fibroin sol-gel transition processes in detail. The effect of different surfactants and high temperature pretreatment were studied detailedly. Based on the study, a mechanism was summarized. The result showed that the surfactant accelerated sol-gel transitions while heat pretreatment decelerated sol-gel transitions. Gelation time was decreased to several hours when added surfactant with negative charge. On the contrary, gelation time was increased to dozens of days when extended the time of high-temperature processing. Upon gelation, a random coil structure of the silk fibroin was transformed into a β-sheet structure.

Silk Fibroin Hydrogels Coupled with the n16N−β-Chitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization

Previous results have shown that the nacre specific peptide, n16N, from the Japanese pearl oyster Pinctada fucata has a binding affinity for β-chitin. As a result, the n16N-chitin assembly is able to selectivity nucleate aragonite. Here, we have added silk fibroin hydrogels to the in vitro assay to more fully represent the in vivo matrix. Crystallization, with a silk fibroin hydrogel and n16N on β-chitin, results in metastable vaterite and amorphous calcium carbonate, which form as flat deposits with hemispherical centers. Acidic peptide controls (p-Asp/p-Glu) were also tested in the silk-chitin assay and result in flat calcite that grows into the β-chitin substrate. Fluorescence imaging of that matrix, made with labeled n16N, shows that n16N binds to β-chitin in the presence of silk gel. These results demonstrate that the addition of a silk hydrogel to the n16N−β-chitin assembly changes the microenvironment for mineralization. This work contributes to our understanding of the roles of individual nacre ma...

Silk hydrogel for cartilage tissue engineering

Cartilage tissue engineering based on cultivation of immature chondrocytes in agarose hydrogel can yield tissue constructs with biomechanical properties comparable to native cartilage. However, agarose is immunogenic and nondegradable, and our capability to modify the structure, composition, and mechanical properties of this material is rather limited. In contrast, silk hydrogel is biocompatible and biodegradable, and it can be produced using a water-based method without organic solvents that enables precise control of structural and mechanical properties in a range of interest for cartilage tissue engineering. We observed that one particular preparation of silk hydrogel yielded cartilaginous constructs with biochemical content and mechanical properties matching constructs based on agarose. This finding and the possibility to vary the properties of silk hydrogel motivated this study of the factors underlying the suitability of hydrogels for cartilage tissue engineering. We present data resulting from a systematic variation of silk hydrogel properties, silk extraction method, gel concentration, and gel structure. Data suggest that silk hydrogel can be used as a tool for studies of the hydrogel-related factors and mechanisms involved in cartilage formation, as well as a tailorable and fully degradable scaffold for cartilage tissue engineering.

Matrix Interactions in Biomineralization: Aragonite Nucleation by an Intrinsically Disordered Nacre Polypeptide, n16N, Associated with a β-Chitin Substrate

Previous literature by Falini et al. suggests that the cooperation between β-chitin, proteins, and a silk fibroin-like hydrogel determines polymorph selectivity within the nacreous layer of mollusk shells (favoring aragonite over calcite formation). Here we present an in vitro assay in which we combine functionalized organic surfaces with soluble peptides to probe the role of surface−peptide interactions in calcium carbonate polymorph selectivity. Specifically, we combined n16N (a 30 amino acid peptide from the Japanese pearl oyster Pinctada fucata) and its sequence variants, n16Ns (randomly scrambled) and n16NN (global Asp → Asn, Glu → Gln substitution), with different forms of chitin (α and β). We found that the combination of n16N adsorbed onto β-chitin leads to the formation of aragonite in vitro as well as demonstrated chitin binding ability. Negative controls, including sequence modified versions of n16N (n16Ns and n16NN), exhibit variation in β-chitin binding and the ability to nucleate aragonite. ...

Structure and gelation mechanism of silk hydrogels

Silk fibroin was regenerated from cocoons produced by the silkworm Bombyx Mori. Light scattering showed that an aqueous solution of the regenerated silk fibroin (RSF) was made of individual proteins with a weight average molar mass of about 4 x 10(5) g mol(-1) and a hydrodynamic radius of about 10 nm. Gel formation of RSF in acidic solutions was investigated as a function of the pH (2-4), concentration (0.5-10 g L(-1)) and temperature (5-70 degrees C). The structure of the gels was studied using light scattering and confocal laser scanning microscopy. The structure was found to be self-similar from length scales of less than 15 nm up to length scales of about 1 microm, and characterized by a correlation length of a few microns. Gel formation was tracked using turbidity, rheology, light scattering and circular dichroism. Gelation involves the formation of self-similar aggregates with a growth rate that increases exponentially. The protein aggregation is correlated to, and perhaps caused by, the formation of beta-sheets, the fraction of which also increases exponentially with time.

Non-equilibrium silk fibroin adhesives

Regenerated silkworm silk solutions formed metastable, soft-solid-like materials (e-gels) under weak electric fields, displaying interesting mechanical characteristics such as dynamic adhesion and strain stiffening. Raman spectroscopy, in situ electric field dynamic oscillatory rheology and polarized optical microscopy indicated that silk fibroin electrogelation involved intermolecular self-assembly of silk molecules into amorphous, micron-scale, micellar structures and the formation of relatively long lifetime, intermicellar entanglement crosslinks. Overall, the electrogelation process did not require significant intramolecular beta-strand or intermolecular beta-sheet formation, unlike silk hydrogels. The kinetics of e-gel formation could be tuned by changing the field strength and assembly conditions, such as silk concentration and solution pH, while e-gel stiffness was partially reversible by removal of the applied field. Transient adhesion testing indicated that the adhesive characteristics of e-gels could at least partially be attributed to a local increase in proton concentration around the positive electrode due to the applied field and surface effects. A working model of electrogelation was described en route to understanding the origins of the adhesive characteristics.

The consolidation behavior of silk hydrogels

Hydrogels have mechanical properties and structural features that are similar to load-bearing soft tissues including intervertebral disc and articular cartilage, and can be implanted for tissue restoration or for local release of therapeutic factors. To help predict their performance, mechanical characterization and mathematical modeling are the available methods for use in tissue engineering and drug delivery settings. In this study, confined compression creep tests were performed on silk hydrogels, over a range of concentrations, to examine the phenomenological behavior of the gels under a physiological loading scenario. Based on the observed behavior, we show that the time-dependent response can be explained by a consolidation mechanism, and modeled using Biot’s poroelasticity theory. Two observations are in strong support of this modeling framework, namely, the excellent numerical agreement between increasing load step creep data and the linear Terzaghi theory, and the similar values obtained from numerical simulations and direct measurements of the permeability coefficient. The higher concentration gels (8% and 12% w/v) clearly show a strain-stiffening response to creep loading with increasing loads, while the lower concentration gel (4% w/v) does not. A nonlinear elastic constitutive formulation is employed to account for the stiffening. Furthermore, an empirical formulation is used to represent the deformation-dependent permeability.

Vortex-Induced Injectable Silk Fibroin Hydrogels

A novel, to our knowledge, technique was developed to control the rate of β-sheet formation and resulting hydrogelation kinetics of aqueous, native silk solutions. Circular dichroism spectroscopy indicated that vortexing aqueous solutions of silkworm silk lead to a transition from an overall protein structure that is initially rich in random coil to one that is rich in β-sheet content. Dynamic oscillatory rheology experiments collected under the same assembly conditions as the circular dichroism experiments indicated that the increase in β-sheet content due to intramolecular conformational changes and intermolecular self-assembly of the silk fibroin was directly correlated with the subsequent changes in viscoelastic properties due to hydrogelation. Vortexing low-viscosity silk solutions lead to orders-of-magnitude increase in the complex shear modulus, G ∗ , and formation of rigid hydrogels ( G ∗ ≈ 70 kPa for 5.2 wt % protein concentration). Vortex-induced, β-sheet-rich silk hydrogels consisted of permanent, physical, intermolecular crosslinks. The hydrogelation kinetics could be controlled easily (from minutes to hours) by changing the vortex time, assembly temperature and/or protein concentration, providing a useful timeframe for cell encapsulation. The stiffness of preformed hydrogels recovered quickly, immediately after injection through a needle, enabling the potential use of these systems for injectable cell delivery scaffolds.

Sonication-induced gelation of silk fibroin for cell encapsulation

Purified native silk fibroin forms β-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of β-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K + at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca 2+. The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5–2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol–gel transitions, such as for cell encapsulation.

Rheological characterization of hydrogels formed by recombinantly produced spider silk

Many fibrous proteins such as spider silks exhibit impressive mechanical properties and are highly biocompatible leading to many potential biomaterial applications. For applications such as tissue engineering, polymer hydrogels have been proposed as an effective means of producing porous but stable scaffolds. Here, nanofiber-based hydrogels were produced from engineered and recombinantly produced spider silk proteins. The silk nanofibers are stable semi-flexible polymers which assemble into hydrogel networks. We studied the hydrogel rheology and determined the concentration dependence of the elastic modulus. AFM images indicate that the nanofibers might assemble into branch-like structures, which would also be consistent with the measured rheological behavior. Since the developed spider silk hydrogels are stable over weeks and show a high elastic modulus at low volume fractions, they are well suited for a broad variety of applications.

Structure and Properties of Silk Hydrogels

Control of silk fibroin concentration in aqueous solutions via osmotic stress was studied to assess relationships to gel formation and structural, morphological, and functional (mechanical) changes associated with this process. Environmental factors potentially important in the in vivo processing of aqueous silk fibroin were also studied to determine their contributions to this process. Gelation of silk fibroin aqueous solutions was affected by temperature, Ca(2+), pH, and poly(ethylene oxide) (PEO). Gelation time decreased with increase in protein concentration, decrease in pH, increase in temperature, addition of Ca(2+), and addition of PEO. No change of gelation time was observed with the addition of K(+). Upon gelation, a random coil structure of the silk fibroin was transformed into a beta-sheet structure. Hydrogels with fibroin concentrations >4 wt % exhibited network and spongelike structures on the basis of scanning electron microscopy. Pore sizes of the freeze-dried hydrogels were smaller as the silk fibroin concentration or gelation temperature was increased. Freeze-dried hydrogels formed in the presence of Ca(2+) exhibited larger pores as the concentration of this ion was increased. Mechanical compressive strength and modulus of the hydrogels increased with increase in protein concentration and gelation temperature. The results of these studies provide insight into the sol-gel transitions that silk fibroin undergoes in glands during aqueous processing while also providing important insight in the in vitro processing of these proteins into useful new materials.