Enhancing influenza vaccine immunogenicity and efficacy through infection mimicry using silk microneedles.

Traditional bolus vaccine administration leads to rapid clearance of vaccine from lymphoid tissue. However, there is increasing evidence suggesting that the kinetics of antigen delivery can impact immune responses to vaccines, particularly when tailored to mimic natural infections. Here, we present the specific enhancements sustained release immunization confers to seasonal influenza vaccine, including the magnitude, durability, and breadth of humoral responses. To achieve sustained vaccine delivery kinetics, we have developed a microneedle array patch (MIMIX), with silk fibroin-formulated vaccine tips designed to embed in the dermis after a short application to the skin and release antigen over 1-2 weeks, mimicking the time course of a natural influenza infection. In a preclinical murine model, a single influenza vaccine administration via MIMIX led to faster seroconversion, response-equivalence to prime-boost bolus immunization, higher HAI titers against drifted influenza strains, and improved protective efficacy upon lethal influenza challenge when compared with intramuscular injection. These results highlight infection mimicry, achieved through sustained release silk microneedles, as a powerful approach to improve existing seasonal influenza vaccines, while also suggesting the broader potential of this platform technology to enable more efficacious next-generation vaccines and vaccine combinations.

Thin silk fibroin films as a dried format for temperature stabilization of inactivated polio vaccine.

Current inactivated polio vaccine (IPV) products are sensitive to both freezing and elevated temperatures and therefore must be shipped and stored between 2 °C and 8 °C, a requirement that imposes financial and logistical challenges for global distribution. As such, there is a critical need for a robust, thermally stable IPV to support global polio eradication and post-eradication immunization needs. Here, we present the development of air-dried thin films for temperature stabilization of IPV using the biomaterial silk fibroin. Thin-film product compositions were optimized for physical properties as well as poliovirus D-antigen recovery and were tested under accelerated and real-time stability storage conditions. Silk fibroin IPV films maintained 70% D-antigen potency after storage for nearly three years at room temperature, and greater than 50% potency for IPV-2 and IPV-3 serotypes at 45 °C for one year. The immunogenicity of silk fibroin IPV films after 2-week storage at 45 °C was assessed in Wistar rats and the stressed films generated equivalent neutralizing antibody responses to commercial vaccine for IPV-1 and IPV-2. However, the absence of IPV-3 responses warrants further investigation into the specificity of ELISA for intact IPV-3 D-antigen. By demonstrating immunogenicity post-storage, we offer the air-dried silk film format as a means to increase IPV vaccine access through innovative delivery systems such as microneedles.

Fabricating mechanically improved silk-based vascular grafts by solution control of the gel-spinning process.

There is a large unmet need for off-the-shelf biomaterial options to supplant venous autografts in bypass and reconstructive surgical procedures. Existing graft alternatives formed from non-degradable synthetic polymers are not capable of maintaining long-term patency and are thus not indicated for <6 mm inner diameter bypass procedures. To fill this void, degradable silk-based biomaterials have been proposed that can maintain their mechanical properties (i.e. compliance) while facilitating slow but progressive biomaterial remodeling and host integration mediated by cellular colonization. The goal of the present study was to enhance the porosity of gel-spun silk tubes, to facilitate faster degradation rates and improve cellularity, and thus improve host integration over time in vivo, while maintaining requisite mechanical functions. Silk solutions with a range of molecular weight distributions and, in turn, viscosities were used to generate tubes of varying porosities. A decrease in solution concentration correlated with an increase in mean pore size and overall porosity through a density-dependent mechanism. Tubes were mechanically analyzed, and these properties were the basis of an analytical model used to correlate tube formulations to structural compliance, which were shown to be similar to the saphenous vein. Tubes were also tested for suture retention to ensure surgical utility despite increased porosity. Tubes were implanted in the abdominal aorta of Sprague-Dawley rats via an end-to-end anastomosis model. Tubes with higher porosities showed early improvements in cell colonization that progressively increased over time; conversely, the dense architecture of less porous grafts (20MB) inhibited cell ingrowth and resulted in minimal biomaterial degradation at the 6-month time point. None of the highly porous tubes (5 MB and 10MB) remained patent at 6 months, likely due remodeling inducing bulk mechanical failure or a compromised blood-material interface.

Stabilization of RNA Encapsulated in Silk.

The use of mRNA and miRNA as diagnostic parameters and therapeutic agents has drawn wide interest both clinically and in research. However, RNA is a labile molecule, which requires strict storage conditions, often including cold temperatures or dry environments, in order to preserve RNA integrity. Achieving this requires huge costs for storage and added difficulty in transport. To address these issues, we introduce a system to encapsulate and store it long-term in dried silk fibroin matrices. At temperatures up to 45 °C, mRNA samples stored in lyophilized silk matrices showed good stability over 1 week, as measured by real-time PCR to assess transcript recovery. While the presence of the silk interfered with the generation of cDNA required for quantitation at roughly 1% w/v (400:1 silk:RNA mass), this phenomenon was resolved by the use of commercial RNA purification kits for silk concentrations up to 4% w/v. A higher concentration of silk correlated with increased thermal protection. As an alternative to lyophilization, RNA was simply air-dried in the presence of aqueous fibroin to create storage matrices. While air-dried matrices composed of low silk content were not protective, higher concentrations were protective and progressively lost additional water over time of storage because of the overall hydrophobic nature of the system. Taken together, these findings provide a new and potentially simpler method for preserving RNA samples for long-term storage and transportation, acting primarily on a water exclusion mechanism.

Enhanced Stabilization in Dried Silk Fibroin Matrices.

Preliminary studies have shown that silk fibroin can protect biomacromolecules from thermal degradation, but a deeper understanding of underlying mechanisms needed to fully leverage the stabilizing potential of this matrix has not been realized. In this study, we investigate stabilization of plasma C-reactive protein (CRP), a diagnostic indicator of infection or inflammation, to gain insight into stabilizing mechanisms of silk. We observed that the addition of antiplasticizing excipients that suppress ß-relaxation amplitudes in silk matrices resulted in enhanced stability of plasma CRP. These observations are consistent with those made in sugar-glass-based protein-stabilizing matrices and suggest fundamental insight into mechanisms as well as practical strategies to employ with silk protein matrices for enhanced stabilization utility.

Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly.

The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites.

Silk-based blood stabilization for diagnostics.

Advanced personalized medical diagnostics depend on the availability of high-quality biological samples. These are typically biofluids, such as blood, saliva, or urine; and their collection and storage is critical to obtain reliable results. Without proper temperature regulation, protein biomarkers in particular can degrade rapidly in blood samples, an effect that ultimately compromises the quality and reliability of laboratory tests. Here, we present the use of silk fibroin as a solid matrix to encapsulate blood analytes, protecting them from thermally induced damage that could be encountered during nonrefrigerated transportation or freeze-thaw cycles. Blood samples are recovered by simple dissolution of the silk matrix in water. This process is demonstrated to be compatible with a number of immunoassays and provides enhanced sample preservation in comparison with traditional air-drying paper approaches. Additional processing can remediate interactions with conformational structures of the silk protein to further enhance blood stabilization and recovery. This approach can provide expanded utility for remote collection of blood and other biospecimens empowering new modalities of temperature-independent remote diagnostics.

Optimizing Molecular Weight of Lyophilized Silk As a Shelf-Stable Source Material.

Storage of silk proteins in liquid form can lead to excessive waste from premature gelation, thus an alternative storage strategy is proposed using lyophilization to generate soluble and shelf-stable powder formats for on-demand use. Initial solution stability studies highlighted instabilities of higher-molecular-weight silks that could not be resolved by solution modifications such as autoclaving, pH increases, dilution, or combinations thereof. Conversely, shelf-stable lyophilized stock powders of silk fibroin of moderate to low molecular weights were developed that could be fully constituted even after 1 year of storage at elevated temperatures. Increasing dried silk powder loading in aqueous solution facilitated increased silk solution concentrations-here up to 80 mg/mL solubility was demonstrated across a range of formulations. Powders generated from silk solutions with higher-molecular-weight distributions were less soluble than moderate or lower-molecular-weight versions, despite no differences in their solution glass-transition temperatures. Instead, the aggregation and ß-sheet content of lyophilized higher molecular weight stock solutions were identified as the cause of the reduced powder solubility by circular dichroism and dynamic light scattering analyses. The solubility and molecular weight profiles of all formulations investigated were preserved after storing the lyophilized materials over 1 year, even at 37 °C. No long-term powder stability behaviors were influenced by the addition of a secondary drying step in the lyophilization procedure, suggesting that this protocol could be scaled without the burden of lengthy process times. Taken together, these findings provide a very flexible and potentially cost-saving approach to producing shelf-stable silk fibroin stock materials based on the use of moderate to lower-molecular-weight lyophilized preparations. This utility is demonstrated with the formation of silk material formats from the stored powders, including films, gels, and salt-leached porous scaffolds. In turn, a more efficient system allowing full resolubilization will enable stockpiling powder for on-demand usage and for deployment of dried silks for application demands in field settings.

Silk-based stabilization of biomacromolecules.

Silk fibroin is a high molecular weight amphiphilic protein that self-assembles into robust biomaterials with remarkable properties including stabilization of biologicals and tunable release kinetics correlated to processing conditions. Cells, antibiotics,monoclonal antibodies and peptides, among other biologics, have been encapsulated in silk using various processing approaches and material formats. The mechanistic basis for the entrapment and stabilization features, along with insights into the modulation of release of the entrained compounds from silks will be reviewed with a focus on stabilization of bioactive molecules.

Static and cyclic mechanical loading of mesenchymal stem cells on elastomeric, electrospun polyurethane meshes.

Biomaterial substrates composed of semi-aligned electrospun fibers are attractive supports for the regeneration of connective tissues because the fibers are durable under cyclic tensile loads and can guide cell adhesion, orientation, and gene expression. Previous studies on supported electrospun substrates have shown that both fiber diameter and mechanical deformation can independently influence cell morphology and gene expression. However, no studies have examined the effect of mechanical deformation and fiber diameter on unsupported meshes. Semi-aligned large (1.75 µm) and small (0.60 µm) diameter fiber meshes were prepared from degradable elastomeric poly(esterurethane urea) (PEUUR) meshes and characterized by tensile testing and scanning electron microscopy (SEM). Next, unsupported meshes were aligned between custom grips (with the stretch axis oriented parallel to axis of fiber alignment), seeded with C3H10T1/2 cells, and subjected to a static load (50 mN, adjusted daily), a cyclic load (4% strain at 0.25 Hz for 30 min, followed by a static tensile loading of 50 mN, daily), or no load. After 3 days of mechanical stimulation, confocal imaging was used to characterize cell shape, while measurements of deoxyribonucleic acid (DNA) content and messenger ribonucleic acid (mRNA) expression were used to characterize cell retention on unsupported meshes and expression of the connective tissue phenotype. Mechanical testing confirmed that these materials deform elastically to at least 10%. Cells adhered to unsupported meshes under all conditions and aligned with the direction of fiber orientation. Application of static and cyclic loads increased cell alignment. Cell density and mRNA expression of connective tissue proteins were not statistically different between experimental groups. However, on large diameter fiber meshes, static loading slightly elevated tenomodulin expression relative to the no load group, and tenascin-C and tenomodulin expression relative to the cyclic load group. These results demonstrate the feasibility of maintaining cell adhesion and alignment on semi-aligned fibrous elastomeric substrates under different mechanical conditions. The study confirms that cell morphology is sensitive to the mechanical environment and suggests that expression of select connective tissue genes may be enhanced on large diameter fiber meshes under static tensile loads.

Rapid prototyped sutureless anastomosis device from self-curing silk bio-ink.

Sutureless anastomosis devices are designed to reduce surgical time and difficulty, which may lead to quicker and less invasive cardiovascular anastomosis. The implant uses a barb-and-seat compression fitting composed of one male and two female components. The implant body is resorbable and capable of eluting heparin. Custom robotic deposition equipment was designed to fabricate the implants from a self-curing silk solution. Curing did not require deleterious processing steps but devices demonstrated high crush resistance, retention strength, and leak resistance. Radial crush resistance is in the range of metal vascular implants. Insertion force and retention strength of the anastomosis was dependent on fit sizing of the male and female components and subsequent vessel wall compression. Anastomotic burst strength was dependent on the amount of vessel wall compression, and capable of maintaining higher than physiological pressures. In initial screening using a porcine implant, the devices remained intact for 28 days (the length of study). Histological sections revealed cellular infiltration within the laminar structure of the male component, as well as at the interface between the male and female components. Initial degradation and absorption of the implant wall were observed. The speed per anastomosis using this new device was much faster than current systems, providing significant clinical improvement.

Characteristics of platelet gels combined with silk.

Platelet gel, a fibrin network containing activated platelets, is widely used in regenerative medicine due the capacity of platelet-derived growth factors to accelerate and direct healing processes. However, limitations to this approach include poor mechanical properties, relatively rapid degradation, and the lack of control of release of growth factors at the site of injection. These issues compromise the ability of platelet gels for sustained function in regenerative medicine. In the present study, a combination of platelet gels with silk fibroin gel was studied to address the above limitations. Mixing sonicated silk gels with platelet gels extended the release of growth factors without inhibiting gel-forming ability. The released growth factors were biologically active and their delivery was modified further by manipulation of the charge of the silk protein. Moreover, the silk gel augmented both the rheological properties and compressive stiffness of the platelet gel, tuned by the silk concentration and/or silk/platelet gel ratio. Silk-platelet gel injections in nude rats supported enhanced cell infiltration and blood vessel formation representing a step towards new platelet gel formulations with enhanced therapeutic impact.

Annulus fibrosus tissue engineering using lamellar silk scaffolds.

Degeneration of the intervertebral disc (IVD) represents a significant muscular skeletal disease. Recently, scaffolds composed of synthetic, natural and hybrid biomaterials have been investigated as options to restore the IVD; however, they lack the hallmark lamellar morphological features of annulus fibrosus (AF) tissue. The goal of regenerating the disc is to achieve anatomical morphology as well as restoration of mechanical and biological function. In this study, two types of scaffold morphology formed from silk fibroin were investigated towards the goal of AF tissue restoration. The first design mimics the lamellar features of the IVD that are associated with the AF region. The second is a porous spongy scaffold that serves as a control. Toroidal scaffolds were formed from the lamellar and porous silk material systems to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm and a height of 3 mm. The inter-lamellar spacing in the lamellar scaffold was 150-250 µm and the average pore sizes in the porous scaffolds were 100-250 µm. The scaffolds were seeded with porcine AF cells and, after growth over defined time frames in vitro, histology, biochemical assays, mechanical testing and gene expression indicated that the lamellar scaffold generated results that were more favourable in terms of ECM expression and tissue function than the porous scaffold for AF tissue. Further, the seeded porcine AF cells supported the native shape of AF tissue in the lamellar silk scaffolds. The lamellar silk scaffolds were effective in the formation of AF-like tissue in vitro.

Mechanical improvements to reinforced porous silk scaffolds.

Load-bearing porous biodegradable scaffolds are required to engineer functional tissues such as bone. Mechanical improvements to porogen leached scaffolds prepared from silk proteins were systematically studied through the addition of silk particles in combination with silk solution concentration, exploiting interfacial compatibility between the two components. Solvent solutions of silk up to 32 w/v % were successfully prepared in hexafluoroisopropanol (HFIP) for the study. The mechanical properties of the reinforced silk scaffolds correlated to the material density and matched by a power law relationship, independent of the ratio of silk particles to matrix. These results were similar to the relationships previously shown for cancellous bone. From these data we conclude that the increased mechanical properties were due to a densification effect and not due to the inclusion of stiffer silk particles into the softer silk matrix. A continuous interface between the silk matrix and the silk particles, as well as homogeneous distribution of the silk particles within the matrix was observed. Furthermore, we note that the roughness of the pore walls was controllable by varying the ratio of the particles matrix, providing a route to control topography. The rate of proteolytic hydrolysis of the scaffolds decreased with increase in mass of silk used in the matrix and with increasing silk particle content.

Bioreactor system using noninvasive imaging and mechanical stretch for biomaterial screening.

Screening of biomaterial and tissue systems in vitro, for guidance of performance in vivo, remains a major requirement in the field of tissue engineering. It is critical to understand how culture stimulation affects both tissue construct maturation and function, with the goal of eliminating resource-intensive trial-and-error screening and better matching specifications for various in vivo needs. In this article a multifunctional and robust bioreactor design that addresses this need is presented. The design enables a range of mechanical inputs, durations, and frequencies to be applied in coordination with noninvasive optical assessments. A variety of biomaterial systems, including micro- and nano-fiber and porous sponge biomaterials, as well as cell-laden tissue engineering constructs were used in validation studies to demonstrate the versatility and utility of this new bioreactor design. The silk-based biomaterials highlighted in these studies offered several unique optical signatures for use in label-free nondestructive imaging that allowed for sequential profiling. Both short- and long-term culture studies were conducted to evaluate several practical scenarios of usage: on a short-term basis, the authors demonstrate that construct cellularity can be monitored by usage of nonpermanent dyes; on a more long-term basis, the authors show that cell ingrowth can be monitored by green-fluorescent protein (GFP)-labeling, and construct integrity probed with concurrent load/displacement data. The ability to nondestructively track cells, biomaterials, and new matrix formation without harvesting designated samples at each time point will lead to less resource-intensive studies and should enhance our understanding and the discovery of biomaterial designs related to functional tissue engineering.

Ingrowth of human mesenchymal stem cells into porous silk particle reinforced silk composite scaffolds: An in vitro study.

Silk fibroin protein is biodegradable and biocompatible, exhibiting excellent mechanical properties for various biomedical applications. However, porous three-dimensional (3-D) silk fibroin scaffolds, or silk sponges, usually fall short in matching the initial mechanical requirements for bone tissue engineering. In the present study, silk sponge matrices were reinforced with silk microparticles to generate protein-protein composite scaffolds with desirable mechanical properties for in vitro osteogenic tissue formation. It was found that increasing the silk microparticle loading led to a substantial increase in the scaffold compressive modulus from 0.3 MPa (non-reinforced) to 1.9 MPa for 1:2 (matrix:particle) reinforcement loading by dry mass. Biochemical, gene expression, and histological assays were employed to study the possible effects of increasing composite scaffold stiffness, due to microparticle reinforcement, on in vitro osteogenic differentiation of human mesenchymal stem cells (hMSCs). Increasing silk microparticle loading increased the osteogenic capability of hMSCs in the presence of bone morphogenic protein-2 (BMP-2) and other osteogenic factors in static culture for up to 6 weeks. The calcium adsorption increased dramatically with increasing loading, as observed from biochemical assays, histological staining, and microcomputer tomography (µCT) analysis. Specifically, calcium content in the scaffolds increased by 0.57, 0.71, and 1.27 mg (per µg of DNA) from 3 to 6 weeks for matrix to particle dry mass loading ratios of 1:0, 1:1, and 1:2, respectively. In addition, µCT imaging revealed that at 6 weeks, bone volume fraction increased from 0.78% for non-reinforced to 7.1% and 6.7% for 1:1 and 1:2 loading, respectively. Our results support the hypothesis that scaffold stiffness may strongly influence the 3-D in vitro differentiation capabilities of hMSCs, providing a means to improve osteogenic outcomes.

A model for the stretch-mediated enzymatic degradation of silk fibers.

To restore physiological function through regenerative medicine, biomaterials introduced into the body must degrade at a rate that matches new tissue formation. For effective therapies, it is essential that we understand the interaction between physiological factors, such as routine mechanical loading specific to sites of implantation, and the resultant rate of material degradation. These relationships are poorly characterized at this time. We hypothesize that mechanical forces alter the rates of remodeling of biomaterials, and this impact is modulated by the concentration of enzymes and the duration of the mechanical loads encountered in situ. To test this hypothesis we subjected silk fibroin fibers to repeated cyclic loading in the presence of enzymatic degradation (either alpha-chymotrypsin or Protease XIV) and recorded the stress-strain response. Data were collected daily for a duration of 2 weeks and compared to the control cases of stretched fibers in the presence of phosphate buffered saline or non-stretched samples in the presence of enzyme alone. We observed that incubation with proteases in the absence of mechanical loads causes a reduction of the ultimate tensile strength but no change in stiffness. However, cyclic loading caused the accumulation of residual strain and softening in the material's properties. We utilize these data to formulate a mathematical model to account for residual strain and reduction of mechanical properties during silk fiber degradation. Numerical predictions are in fair agreement with experimental data. The improved understanding of the degradation phenomenon will be significant in many clinical repair cases and may be synergistic to decrease silk's mechanical properties after in vivo implantation.

Modular elastic patches: mechanical and biological effects.

A modular approach to engineering cross-linked elastic biomaterials is presented for fine-tuning of material mechanical and biological properties. The three components, soluble elastin, hyaluronic acid, and silk fibroin, contribute with different features to the overall properties of the final material system. The elastic biomaterial is chemically cross-linked via interaction between primary amine groups naturally present on the two proteins, silk and elastin, or chemically introduced on hyaluronan and N-succinimide functionalities of the cross-linker. The materials obtained by cross-linking the three components in different ratios have Young's moduli ranging from ∼ 100 to 230 kPa, strain to failure between ∼ 15-40% and ultimate tensile strengths of ∼ 30 kPa. The biological effects and enzymatic degradation rates of the different composites are also different based on material composition. These findings further underline the strength of modular, multicomponent systems in creating a range of biomaterials, targeted tissue engineering, and regenerative medicine applications, with application-tailored mechanical and biological properties.

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.

Effect of hydration on silk film material properties.

Effects of hydration on silk fibroin film properties were investigated for water-annealed and MeOH-treated samples. Hydration increased thickness by 60% for MeOH-immersed films, while water-annealed samples remained constant. MeOH-immersed films showed an 80% mass loss due to water, while water-annealed lost only 40%. O(2) permeability was higher in MeOH-immersed films with Dk values of 10(-10) (mL O(2) x cm) x (cm(-1) x s(-1) x mmHg(-1)), while those of water-annealed films reached only one fifth of this value. All films showed a decrease in Young's modulus and increased plastic deformation by two orders of magnitude when submerged in saline solution. FT-IR showed that beta-sheet content in water-annealed films increased with increasing water vapor pressure, while MeOH-immersed films showed no change.