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The goal of this study was to determine the impact of silk biomaterial structure (e.g., solution, hydrogel, film) on proteolytic susceptibility. In vitro enzymatic degradation of silk fibroin hydrogels and films was studied using a variety of proteases, including proteinase K, protease XIV, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2. Hydrogels were used to assess bulk degradation while films were used to assess surface degradation. Weight loss, secondary structure determined by Fourier Transform Infrared (FT-IR) spectroscopy and degradation products analyzed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to evaluate degradation through five days. Silk films were significantly degraded by proteinase K, while silk hydrogels were degraded more extensively by protease XIV and proteinase K. Collagenase preferentially degraded the β-sheet content in hydrogels while protease XIV and α-chymotrypsin degraded the amorphous structures. MMP-1 and MMP-2 degraded silk fibroin in solution resulting in a decrease in peptide fragment sizes over time. The link between primary sequence mapping with protease susceptibility provides insight into the role of secondary structure in impacting proteolytic access by comparing solution vs. solid state proteolytic susceptibility.
In the field of soft tissue reconstruction, custom implants could address the need for materials that can fill complex geometries. Our aim was to develop a material system with optimal rheology for material extrusion, that can be processed in physiological and non-toxic conditions and provide structural support for soft tissue reconstruction. To meet this need we developed silk based bioinks using gelatin as a bulking agent and glycerol as a non-toxic additive to induce physical crosslinking. We developed these inks optimizing printing efficacy and resolution for patient-specific geometries that can be used for soft tissue reconstruction. We demonstrated in vitro that the material was stable under physiological conditions and could be tuned to match soft tissue mechanical properties. We demonstrated in vivo that the material was biocompatible and could be tuned to maintain shape and volume up to three months while promoting cellular infiltration and tissue integration.
Silk-based bioinks were developed for 2D and 3D printing. By incorporating nontoxic polyols into silk solutions, two-part formulations with selfcuring features at room temperature were generated. By varying the formulations the crystallinity of the silk polymer matrix could be controlled to support printing in 2D and 3D formats interfaced with CAD geometry and with good feature resolution. The self-curing phenomenon was tuned and exploited in order to demonstrate the formation of both structural and support materials. Biocompatible aqueous protein inks for printing that avoid the need for chemical or photo initiators and that form aqueous-stable structures with good resolution at ambient temperatures provide useful options for biofunctionalization and a broad range of applications.
Porous silk protein scaffolds were designed to display shape memory characteristics and volumetric recovery following compression. Two strategies were utilized to realize shape recovery: the addition of hygroscopic plasticizers like glycerol and tyrosine modifications with hydrophilic sulfonic acid chemistries. Silk sponges were evaluated for recovery following 80% compressive strain, total porosity and pore size distribution, secondary structure development, in vivo volume retention, cell infiltration, and inflammatory responses. Glycerol-modified sponges recovered up to 98.3% of their original dimensions following compression, while sulfonic acid / glycerol modified sponges swelled in water up to 71 times their compressed volume, well in excess of their original size. Longer silk extraction times (giving lower silk molecular weights) and higher concentrations of glycerol gave sponges with greater flexibility and shape fidelity, with no loss in modulus following compression. Sponges were over 95% porous, with secondary structure analysis indicating glycerol-induced β-sheet physical crosslinking. Tyrosine modifications with sulfonic acid interfered with β-sheet formation. Glycerol-modified sponges exhibited improved rates of cellular infiltration at subcutaneous implant sites with minimal immune response in mice. They also degraded more rapidly than unmodified sponges, a result posited to be cell-mediated. Overall, this work suggests that silk sponge systems may be useful for minimally invasive deployment in soft tissue augmentation procedures.
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.
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