Harvesting Silk Fibers from Plodia interpunctella: Role of Environmental Rearing Conditions in Fiber Production and Properties.

Silk fibers are produced by a wide variety of insects. The silkworm Bombyx mori (Bombyx) was domesticated because the physical properties of its silk fibers were amenable to the production of fine textiles. Subsequently, engineers have regenerated silk fibroin to form biomaterials. The monocular focus on Bombyx silk has underutilized the expanse of diverse silk proteins produced by more than 100,000 other arthropods. This vast array of silk fibers could be utilized for biomedical engineering challenges if sufficient rearing and purification processes are developed. Herein, we show that the moth, Plodia interpunctella (Plodia), represents an alternative silk source that is easily reared in highly regulated culture environments allowing for greater consistency in the silk produced. We controlled the temperature, resource availability (larvae/gram diet), and population density (larvae/mL) with the goal of increasing silk fiber production and improving homogeneity in Plodia silk proteins. We determined that higher temperatures accelerated insect growth and reduced life cycle length. Furthermore, we established initial protocols for the production of Plodia silk with optimal silk production occurring at 24 °C, with a resource availability of 10 larvae/gram and a population density of 0.72 larvae/mL. Population density was shown to be the most prominent driving force of Plodia silk mat formation among the three parameters assessed. Future work will need to link gene expression, protein production and purification, and resulting mechanical properties as a function of environmental cues to further transition Plodia silk into regenerated silk fibroin biomaterials.

Impact of crystalline domains on long-term stability and mechanical performance of anisotropic silk fibroin sponges.

Sponge-like materials made from regenerated silk fibroin biopolymers are a tunable and advantageous platform for in vitro engineered tissue culture and in vivo tissue regeneration. Anisotropic, three-dimensional (3D) silk fibroin sponge-like scaffolds can mimic the architecture of contractile muscle. Herein, we use silk fibroin solution isolated from the cocoons of Bombyx mori silkworms to form aligned sponges via directional ice templating in a custom mold with a slurry of dry ice and ethanol. Hydrated tensile mechanical properties of these aligned sponges were evaluated as a function of silk polymer concentration (3% or 5%), freezing time (50% or 100% ethanol), and post-lyophilization method for inducing crystallinity (autoclaving, water annealing). Hydrated static tensile tests were used to determine Young's modulus and ultimate tensile strength across sponge formulations at two strain rates to evaluate rate dependence in the calculated parameters. Results aligned with previous reports in the literature for isotropic silk fibroin sponge-like scaffolds, where the method by which beta-sheets were formed and level of beta-sheet content (crystallinity) had the greatest impact on static parameters, while polymer concentration and freezing rate did not significantly impact static mechanical properties. We estimated the crystalline organization using molecular dynamics simulations to show that larger crystalline regions may be responsible for strength at low strain amplitudes and brittleness at high strain amplitudes in the autoclaved sponges. Within the parameters evaluated, extensional Young's modulus is tunable in the range of 600-2800 kPa. Dynamic tensile testing revealed the linear viscoelastic region to be between 0% and 10% strain amplitude and 0.2-2 Hz frequencies. Long-term stability was evaluated by hysteresis and fatigue tests. Fatigue tests showed minimal change in the storage and loss modulus of 5% silk fibroin sponges for more than 6000 min of continuous mechanical stimulation in the linear regime at 10% strain amplitude and 1 Hz frequency. Furthermore, we confirmed that these mechanical properties hold when decellularized extracellular matrix is added to the sponges and when the mechanical property assessments were performed in cell culture media. We also used nano-computed tomography (nano-CT) and simulations to explore pore interconnectivity and tortuosity. Overall, these results highlight the potential of anisotropic, sponge-like silk fibroin scaffolds for long-term (>6 weeks) contractile muscle culture with an in vitro bioreactor system that provides routine mechanical stimulation.

Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs).

Oxygen therapeutics have a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all-natural or semi-synthetic hemoglobin-based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products were removed from the market due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers, where immune responses and circulation times remain a challenge. This work looks to leverage the properties of silk fibroin, a cytocompatible and non-thrombogenic biopolymer, known to entrap protein-based cargo, to engineer a silk fibroin-hemoglobin-based oxygen carrier (sfHBOC). Herein, an all-aqueous solvent evaporation technique was used to form silk fibroin particles with and without hemoglobin to tailor the formulation for specific particle sizes. The encapsulation efficiency and ferrous state of hemoglobin were analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 µg/mL active hemoglobin in certain sfHBOC formulations. The system did not elicit a strong inflammation response in vitro, demonstrating the potential for this particle system to serve as an injectable HBOC.

Silk Fibroin Particles as Carriers in the Development of Hemoglobin-Based Oxygen Carriers.

Oxygen therapeutics have a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all-natural or semi-synthetic hemoglobin-based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products stalled in development due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers. This work leverages the properties of silk fibroin, a cytocompatible and non-thrombogenic biopolymer, known to entrap protein-based cargo, to engineer a fully protein-based oxygen carrier. Herein, an all-aqueous solvent evaporation technique was used to form silk particles via phase separation from a bulk polyvinyl alcohol phase (PVA). Particles size was tuned, and particles were formed with and without hemoglobin. The encapsulation efficiency and ferrous state of hemoglobin were analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 µg/mL active hemoglobin in certain sfHBOC formulations. The system did not elicit a strong inflammation response in vitro, demonstrating the potential for this particle system to serve as an injectable HBOC.

Extracellular matrix and cyclic stretch alter fetal cardiomyocyte proliferation and maturation in a rodent model of heart hypoplasia.

Introduction: Birth defects, particularly those that affect development of the heart, are a leading cause of morbidity and mortality in infants and young children. Babies born with heart hypoplasia (heart hypoplasia) disorders often have a poor prognosis. It remains unclear whether cardiomyocytes from hypoplastic hearts retain the potential to recover growth, although this knowledge would be beneficial for developing therapies for heart hypoplasia disorders. The objective of this study was to determine the proliferation and maturation potential of cardiomyocytes from hypoplastic hearts and whether these behaviors are influenced by biochemical signaling from the extracellular matrix (ECM) and cyclic mechanical stretch. Method: Congenital diaphragmatic hernia (CDH)-associated heart hypoplasia was induced in rat fetuses by maternal exposure to nitrofen. Hearts were isolated from embryonic day 21 nitrofen-treated fetuses positive for CDH (CDH+) and from fetuses without nitrofen administration during gestation. Results and discussion: CDH+ hearts were smaller and had decreased myocardial proliferation, along with evidence of decreased maturity compared to healthy hearts. In culture, CDH+ cardiomyocytes remained immature and demonstrated increased proliferative capacity compared to their healthy counterparts. Culture on ECM derived from CDH+ hearts led to a significant reduction in proliferation for both CDH+ and healthy cardiomyocytes. Healthy cardiomyocytes were dosed with exogenous nitrofen to examine whether nitrofen may have an aberrant effect on the proliferative ability of cardiomyocyte, yet no significant change in proliferation was observed. When subjected to stretch, CDH+ cardiomyocytes underwent lengthening of sarcomeres while healthy cardiomyocyte sarcomeres were unaffected. Taken together, our results suggest that alterations to environmental cues such as ECM and stretch may be important factors in the pathological progression of heart hypoplasia.

Right Ventricular Outflow Tract Surgical Resection in Young, Large Animal Model for the Study of Alternative Cardiovascular Patches.

Tetralogy of Fallot (ToF) is a severe congenital heart defect (CHD) that requires surgical reconstruction soon after birth. Reconstructive surgery involves the implantation of synthetic cardiovascular patches to widen the right ventricular outflow tract (RVOT) and repair defects in the septal wall. However, synthetic patches can cause complications for these patients later in life as they do not integrate or adapt in the tissue of a growing patient; a limitation that could be solved with the development of a patch fabricated from a degradable biomaterial. Unfortunately, the lack of appropriate pre-clinical models has hindered the development of novel patch materials. Currently, most studies use rodent models to study the efficacy of new patch materials; however, large animal models are necessary to develop realistically sized patches in a clinically relevant growing heart where gradients in diffusion and length scales for cell migration are more similar to the human. Here, we describe a novel method by which a Satinsky vascular clamp is used to isolate RVOT muscle for resection followed by implantation of a cardiovascular patch in an appropriately young, rapidly growing porcine model.

Jointly Optimized Spatial Histogram UNET Architecture (JOSHUA) for Adipose Tissue Segmentation.

Objective. We aim to develop a machine learning algorithm to quantify adipose tissue deposition at surgical sites as a function of biomaterial implantation. Impact Statement. To our knowledge, this study is the first investigation to apply convolutional neural network (CNN) models to identify and segment adipose tissue in histological images from silk fibroin biomaterial implants. Introduction. When designing biomaterials for the treatment of various soft tissue injuries and diseases, one must consider the extent of adipose tissue deposition. In this work, we analyzed adipose tissue accumulation in histological images of sectioned silk fibroin-based biomaterials excised from rodents following subcutaneous implantation for 1, 2, 4, or 8 weeks. Current strategies for quantifying adipose tissue after biomaterial implantation are often tedious and prone to human bias during analysis. Methods. We used CNN models with novel spatial histogram layer(s) that can more accurately identify and segment regions of adipose tissue in hematoxylin and eosin (H&E) and Masson's trichrome stained images, allowing for determination of the optimal biomaterial formulation. We compared the method, Jointly Optimized Spatial Histogram UNET Architecture (JOSHUA), to the baseline UNET model and an extension of the baseline model, attention UNET, as well as to versions of the models with a supplemental attention-inspired mechanism (JOSHUA+ and UNET+). Results. The inclusion of histogram layer(s) in our models shows improved performance through qualitative and quantitative evaluation. Conclusion. Our results demonstrate that the proposed methods, JOSHUA and JOSHUA+, are highly beneficial for adipose tissue identification and localization. The new histological dataset and code used in our experiments are publicly available.

Progress in silk and silk fiber-inspired polymeric nanomaterials for drug delivery.

The fields of drug and gene delivery have been revolutionized by the discovery and characterization of polymer-based materials. Polymeric nanomaterials have emerged as a strategy for targeted delivery because of features such as their impressive biocompatibility and improved availability. Use of naturally derived polymers in these nanomaterials is advantageous due to their biodegradability and bioresorption. Natural biopolymer-based particles composed of silk fibroins and other silk fiber-inspired proteins have been the focus of research in drug delivery systems due to their simple synthesis, tunable characteristics, and ability to respond to stimuli. Several silk and silk-inspired polymers contain a high proportion of reactive side groups, allowing for functionalization and addition of targeting moieties. In this review, we discuss the main classes of silk and silk-inspired polymers that are being used in the creation of nanomaterials. We also focus on the fabrication techniques used in generating a tunable design space of silk-based polymeric nanomaterials and detail how that translates into use for drug delivery to several distinct microenvironments.

Recent Advances in Natural Materials for Corneal Tissue Engineering.

Given the incidence of corneal dysfunctions and diseases worldwide and the limited availability of healthy, human donors, investigators are working to generate engineered cellular and acellular therapeutic approaches as alternatives to corneal transplants from human cadavers. These engineered strategies aim to address existing complications with human corneal transplants, including graft rejection, infection, and complications resulting from surgical methodologies. The main goals of these research endeavors are to (1) determine ideal mechanical properties, (2) devise methodologies to improve the efficacy of engineered corneal grafts and cell-based therapies, and (3) optimize transplantation of engineered tissue structures in the eye. Thus, recent innovations have sought to address these challenges through both in vitro and in vivo studies. This review covers recent work aimed at evaluating engineered materials, potential therapeutic cells, and the resulting cell-material interactions that lead to optimal corneal graft properties. Furthermore, we discuss promising strategies in corneal tissue engineering techniques and in vivo studies in animal models.

Estimating Kinetic Rate Parameters for Enzymatic Degradation of Lyophilized Silk Fibroin Sponges.

Sponge-like biomaterials formed from silk fibroin are promising as degradable materials in clinical applications due to their controllable breakdown into simple amino acids or small peptides in vivo. Silk fibroin, isolated from Bombyx mori silkworm cocoons, can be used to form sponge-like materials with a variety of tunable parameters including the elastic modulus, porosity and pore size, and level of nanocrystalline domains. These parameters can be independently tuned during formulation resulting in a wide parameter space and set of final materials. Determining the mechanism and rate constants for biomaterial degradation of these tunable silk materials would allow scientists to evaluate and predict the biomaterial performance for the large array of tissue engineering applications and patient ailments a priori. We first measured in vitro degradation rates of silk sponges using common protein-degrading enzymes such as Proteinase K and Protease XIV. The concentration of the enzyme in solution was varied (1, 0.1, 0.01 U/mL) along with one silk sponge formulation parameter: the level of crystallinity within the sponge. Additionally, two experimental degradation methods were evaluated, termed continuous and discrete degradation methods. Silk concentration, polymer chain length and scaffold pore size were held constant during experimentation and kinetic parameter estimation. Experimentally, we observed that the enzyme itself, enzyme concentration within the bulk solution, and the sponge fabrication water annealing time were the major experimental parameters dictating silk sponge degradation in our experimental design. We fit the experimental data to two models, a Michaelis-Menten kinetic model and a modified first order kinetic model. Weighted, non-linear least squares analysis was used to determine the parameters from the data sets and Monte-Carlo simulations were utilized to obtain estimates of the error. We found that modified first order reaction kinetics fit the time-dependent degradation of lyophilized silk sponges and we obtained first order-like rate constants. These results represent the first investigations into determining kinetic parameters to predict lyophilized silk sponge degradation rates and can be a tool for future mathematical representations of silk biomaterial degradation.

The ties that bind: functional clusters in limb-girdle muscular dystrophy.

The limb-girdle muscular dystrophies (LGMDs) are a genetically pleiomorphic class of inherited muscle diseases that are known to share phenotypic features. Selected LGMD genetic subtypes have been studied extensively in affected humans and various animal models. In some cases, these investigations have led to human clinical trials of potential disease-modifying therapies, including gene replacement strategies for individual subtypes using adeno-associated virus (AAV) vectors. The cellular localizations of most proteins associated with LGMD have been determined. However, the functions of these proteins are less uniformly characterized, thus limiting our knowledge of potential common disease mechanisms across subtype boundaries. Correspondingly, broad therapeutic strategies that could each target multiple LGMD subtypes remain less developed. We believe that three major "functional clusters" of subcellular activities relevant to LGMD merit further investigation. The best known of these is the glycosylation modifications associated with the dystroglycan complex. The other two, mechanical signaling and mitochondrial dysfunction, have been studied less systematically but are just as promising with respect to the identification of significant mechanistic subgroups of LGMD. A deeper understanding of these disease pathways could yield a new generation of precision therapies that would each be expected to treat a broader range of LGMD patients than a single subtype, thus expanding the scope of the molecular medicines that may be developed for this complex array of muscular dystrophies.

Assessing the compatibility of primary human hepatocyte culture within porous silk sponges.

Donor organ shortages have prompted the development of alternative implantable human liver tissues for patients suffering from end-stage liver failure. Purified silk proteins provide desirable features for generating implantable tissues, including sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation. While different cell types were previously cultured for weeks within silk-based scaffolds, it remains unclear whether such scaffolds can be used to culture primary human hepatocytes (PHH) isolated from livers. Therefore, here we assessed the compatibility of PHH culture within porous silk scaffolds that enable diffusion of oxygen/nutrients through the pores. We found that incorporation of type I collagen during the fabrication and/or autoclaving of porous silk scaffolds, as opposed to simple adsorption of collagen onto pre-fabricated silk scaffolds, was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 ± 25 µm) promoted larger PHH spheroids and consequently higher PHH functions than large pores (235 ± 84 µm) for at least 1 month in culture. Further incorporation of supportive fibroblasts into scaffolds enhanced PHH functions up to 5-fold relative to scaffolds with PHHs alone and 2D co-cultures on plastic. Lastly, encapsulating PHHs within protein hydrogels while housed in the silk scaffold led to higher functions than protein hydrogel-only or silk-only controls. In conclusion, porous silk scaffolds containing extracellular matrix proteins can be used for the culture of PHHs ± supportive non-parenchymal cells, which can be further built on in the future to create optimized silk-based liver tissue surrogates for cell-based therapy.

In vitro and in vivo analysis of visible light crosslinkable gelatin methacryloyl (GelMA) hydrogels.

Photocrosslinkable materials have been frequently used for constructing soft and biomimetic hydrogels for tissue engineering. Although ultraviolet (UV) light is commonly used for photocrosslinking such materials, its use has been associated with several biosafety concerns such as DNA damage, accelerated aging of tissues, and cancer. Here we report an injectable visible light crosslinked gelatin-based hydrogel for myocardium regeneration. Mechanical characterization revealed that the compressive moduli of the engineered hydrogels could be tuned in the range of 5-56 kPa by changing the concentrations of the initiator, co-initiator and co-monomer in the precursor formulation. In addition, the average pore sizes (26-103 µm) and swelling ratios (7-13%) were also shown to be tunable by varying the hydrogel formulation. In vitro studies showed that visible light crosslinked GelMA hydrogels supported the growth and function of primary cardiomyocytes (CMs). In addition, the engineered materials were shown to be biocompatible in vivo, and could be successfully delivered to the heart after myocardial infarction in an animal model to promote tissue healing. The developed visible light crosslinked hydrogel could be used for the repair of various soft tissues such as the myocardium and for the treatment of cardiovascular diseases with enhanced therapeutic functionality.

Elastic, silk-cardiac extracellular matrix hydrogels exhibit time-dependent stiffening that modulates cardiac fibroblast response.

Heart failure is the leading cause of death in the United States and rapidly becoming the leading cause of death worldwide. While pharmacological treatments can reduce progression to heart failure following myocardial infarction, there still exists a need for new therapies that promote better healing postinjury for a more functional cardiac repair and methods to understand how the changes to tissue mechanical properties influence cell phenotype and function following injury. To address this need, we have optimized a silk-based hydrogel platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM hydrogels have tunable mechanical properties, as well as rate-controllable hydrogel stiffening over time. In vitro, silk-cECM scaffolds led to enhanced cardiac fibroblast (CF) cell growth and viability with culture time. cECM incorporation improved expression of integrin an focal adhesion proteins, suggesting that CFs were able to interact with the cECM in the hydrogel. Subcutaneous injection of silk hydrogels in rats demonstrated that addition of the cECM led to endogenous cell infiltration and promoted endothelial cell ingrowth after 4 weeks in vivo. This naturally derived silk fibroin platform is applicable to the development of more physiologically relevant constructs that replicate healthy and diseased tissue in vitro and has the potential to be used as an injectable therapeutic for cardiac repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3058-3072, 2016.

Electrical and mechanical stimulation of cardiac cells and tissue constructs.

The field of cardiac tissue engineering has made significant strides over the last few decades, highlighted by the development of human cell derived constructs that have shown increasing functional maturity over time, particularly using bioreactor systems to stimulate the constructs. However, the functionality of these tissues is still unable to match that of native cardiac tissue and many of the stem-cell derived cardiomyocytes display an immature, fetal like phenotype. In this review, we seek to elucidate the biological underpinnings of both mechanical and electrical signaling, as identified via studies related to cardiac development and those related to an evaluation of cardiac disease progression. Next, we review the different types of bioreactors developed to individually deliver electrical and mechanical stimulation to cardiomyocytes in vitro in both two and three-dimensional tissue platforms. Reactors and culture conditions that promote functional cardiomyogenesis in vitro are also highlighted. We then cover the more recent work in the development of bioreactors that combine electrical and mechanical stimulation in order to mimic the complex signaling environment present in vivo. We conclude by offering our impressions on the important next steps for physiologically relevant mechanical and electrical stimulation of cardiac cells and engineered tissue in vitro.

Anisotropic silk biomaterials containing cardiac extracellular matrix for cardiac tissue engineering.

Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults, respectively. In both of these cases, a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue, wound healing complications, and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need, we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures, degradation rates, and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro, silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling.

Clinical applications of naturally derived biopolymer-based scaffolds for regenerative medicine.

Naturally derived polymeric biomaterials, such as collagens, silks, elastins, alginates, and fibrins are utilized in tissue engineering due to their biocompatibility, bioactivity, and tunable mechanical and degradation kinetics. The use of these natural biopolymers in biomedical applications is advantageous because they do not release cytotoxic degradation products, are often processed using environmentally-friendly aqueous-based methods, and their degradation rates within biological systems can be manipulated by modifying the starting formulation or processing conditions. For these reasons, many recent in vivo investigations and FDA-approval of new biomaterials for clinical use have utilized natural biopolymers as matrices for cell delivery and as scaffolds for cell-free support of native tissues. This review highlights biopolymer-based scaffolds used in clinical applications for the regeneration and repair of native tissues, with a focus on bone, skeletal muscle, peripheral nerve, cardiac muscle, and cornea substitutes.

Cardiac extracellular matrix-fibrin hybrid scaffolds with tunable properties for cardiovascular tissue engineering.

Solubilized cardiac extracellular matrix (ECM) is being developed as an injectable therapeutic that offers promise for promoting cardiac repair. However, the ECM alone forms a hydrogel that is very soft compared to the native myocardium. As both the stiffness and composition of the ECM are important in regulating cell behavior and can have complex synergistic effects, we sought to develop an ECM-based scaffold with tunable biochemical and mechanical properties. We used solubilized rat cardiac ECM from two developmental stages (neonatal, adult) combined with fibrin hydrogels that were cross-linked with transglutaminase. We show that ECM was retained within the gels and that the Young's modulus could be tuned to span the range of the developing and mature heart. C-kit+ cardiovascular progenitor cells from pediatric patients with congenital heart defects were seeded into the hybrid gels. Both the elastic modulus and composition of the scaffolds impacted the expression of endothelial and smooth muscle cell genes. Furthermore, we demonstrate that the hybrid gels are injectable, and thus have potential for minimally invasive therapies. ECM-fibrin hybrid scaffolds offer new opportunities for exploiting the effects of both composition and mechanical properties in directing cell behavior for tissue engineering.

Terminal sterilization of alginate hydrogels: efficacy and impact on mechanical properties.

Terminal, or postprocessing, sterilization of composite biomaterials is crucial for their use in wound healing and tissue-engineered devices. Recent research has focused on optimizing traditional biomaterial formulations to create better products for commercial and academic use which incorporate hydrophobic compounds or secondary gel networks. To use a hydrogel in a clinical setting, terminal sterilization is necessary to ensure patient safety. Lyophilization, gamma-irradiation, and ethylene oxide treatment all have negative consequences when applied to alginate scaffolds for clinical use. Here, we aim to find alternative terminal sterilization methods for alginate and alginate-based composite hydrogels which maintain the structure of composite alginate networks for use in biomedical applications. A thorough investigation of the effect of common sterilization methods on swollen alginate-based hydrogels has not been reported and therefore, this work examines autoclaving, ethanol washing, and ultraviolet light as sterilization techniques for alginate and alginate/Pluronic® F68 composite hydrogels. Preservation of structural integrity is evaluated using shear rheology and analysis of water retention, and efficacy of sterilization is determined via bacterial persistence within the hydrogel. Results indicate that ethanol sterilization is the best method of those investigated because ethanol washing results in minimal effects on mechanical properties and water retention and eliminates bacterial persistence. Furthermore, this study suggests that ethanol treatment is an efficacious method for terminally sterilizing interpenetrating networks or other composite hydrogel systems.