Developing Porous Fibrin Scaffolds with Tunable Anisotropic Features to Direct Myoblast Orientation.

Functional regeneration of anisotropically aligned tissues such as ligaments, microvascular networks, myocardium, or skeletal muscle requires a temporal and spatial series of biochemical and biophysical cues to direct cell functions that promote native tissue regeneration. When these cues are lost during traumatic injuries such as volumetric muscle loss (VML), scar formation occurs, limiting the regenerative capacity of the tissue. Currently, autologous tissue transfer is the gold standard for treating injuries such as VML but can result in adverse outcomes including graft failure, donor site morbidity, and excessive scarring. Tissue-engineered scaffolds composed of biomaterials, cells, or both have been investigated to promote functional tissue regeneration but are still limited by inadequate tissue ingrowth. These scaffolds should provide precisely tuned topographies and stiffnesses using proregenerative materials to encourage tissue-specific functions such as myoblast orientation, followed by aligned myotube formation and recovery of functional contraction. In this study, we describe the design and characterization of novel porous fibrin scaffolds with anisotropic microarchitectural features that recapitulate the native tissue microenvironment and offer a promising approach for regeneration of aligned tissues. We used directional freeze-casting with varied fibrin concentrations and freezing temperatures to produce scaffolds with tunable degrees of anisotropy and strut widths. Nanoindentation analyses showed that the moduli of our fibrin scaffolds varied as a function of fibrin concentration and were consistent with native skeletal muscle tissue. Quantitative morphometric analyses of myoblast cytoskeletons on scaffold microarchitectures demonstrated enhanced cell alignment as a function of microarchitectural morphology. The ability to precisely control the anisotropic features of fibrin scaffolds promises to provide a powerful tool for directing aligned tissue ingrowth and enhance functional regeneration of tissues such as skeletal muscle.

Functionalization of Bacterial Cellulose with the Antimicrobial Peptide KR-12 via Chimerical Cellulose-Binding Peptides.

Bacterial-derived cellulose (BC) has been studied as a promising material for biomedical applications, including wound care, due to its biocompatibility, water-holding capacity, liquid/gas permeability, and handleability properties. Although BC has been studied as a dressing material for cutaneous wounds, to date, BC inherently lacks antibacterial properties. The current research utilizes bifunctional chimeric peptides containing carbohydrate binding peptides (CBP; either a short version or a long version) and an antimicrobial peptide (AMP), KR-12. The secondary structure of the chimeric peptides was evaluated and confirmed that the α-helix structure of KR-12 was retained for both chimeric peptides evaluated (Long-CBP-KR12 and Short-CBP-KR12). Chimeric peptides and their individual components were assessed for cytotoxicity, where only higher concentrations of Short-CBP and longer timepoints of Short-CBP-KR12 exposure exhibited negative effects on metabolic activity, which was attributed to solubility issues. All KR-12-containing peptides exhibited antibacterial activity in solution against Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The lipopolysaccharide (LPS) binding capability of the peptides was evaluated and the Short-CBP-KR12 peptide exhibited enhanced LPS-binding capabilities compared to KR-12 alone. Both chimeric peptides were able to bind to BC and were observed to be retained on the surface over a 7-day period. All functionalized materials exhibited no adverse effects on the metabolic activity of both normal human dermal fibroblasts (NHDFs) and human epidermal keratinocyte (HaCaT) epithelial cells. Additionally, the BC tethered chimeric peptides exhibited antibacterial activity against E. coli. Overall, this research outlines the design and evaluation of chimeric CBP-KR12 peptides for developing antimicrobial BC membranes with potential applications in wound care.

Structural properties of optically clear bacterial cellulose produced by Komagataeibacter hansenii using arabitol.

Bacterial cellulose (BC) exhibits beneficial properties for use in biomedical applications but is limited by its lack of tunable transparency capabilities. To overcome this deficiency, a novel method to synthesize transparent BC materials using an alternative carbon source, namely arabitol, was developed. Characterization of the BC pellicles was performed for yield, transparency, surface morphology, and molecular assembly. Transparent BC was produced using mixtures of glucose and arabitol. Zero percent arabitol pellicles exhibited 25% light transmittance, which increased with increasing arabitol concentration through to 75% light transmittance. While transparency increased, overall BC yield was maintained indicating that the altered transparency may be induced on a micro-scale rather than a macro-scale. Significant differences in fiber diameter and the presence of aromatic signatures were observed. Overall, this research outlines methods for producing BC with tunable optical transparency, while also bringing new insight to insoluble components of exopolymers produced by Komagataeibacter hansenii.

Fabrication Methods and Form Factors of Gellan Gum-Based Materials for Drug Delivery and Anti-Cancer Applications.

Despite the success of cancer therapeutics, off target cell toxicity prevails as one of the main challenges of cancer treatment. Exploration of drug delivery methods is a growing field of research, which involves a variety of materials and processing techniques. A natural polymer, gellan gum presents physicochemical properties that enable drug loading for sustained release in a broad range of environmental conditions and anatomical locations. Gellan gum is an anionic exopolysaccharide, produced via fermentation by Sphingomonas elodea, which gels in the presence of cations. Additionally, it is biocompatible and nontoxic. Multiple physical and chemical gelation processes have been reported for the use of gellan gum in drug delivery applications to produced varying form factors, including hydrogels, nanohydrogels, beads, films, or patches, with tunable mechanical and physicochemical properties. The resulting formulations have shown promising outcomes for drug delivery including improving drug bioavailability, drug solubility, and drug release over time, without compromising biocompatibility or the introduction of adverse effects. This review presents studies in which gellan gum has been processed to enable the delivery of antibiotics, antiallergens, anti-inflammatory, or antifungal molecules with a special focus on drugs for anticancer applications.

HepaRG Maturation in Silk Fibroin Scaffolds: Toward Developing a 3D In Vitro Liver Model.

In vitro liver models are necessary tools for the development of new therapeutics. HepaRG cells are a commonly used cell line to produce hepatic progenitor cells and hepatocytes. This study demonstrates for the first time the suitability of 3% silk scaffolds to support HepaRG growth and differentiation. The modulus and pore size of 3% silk scaffolds were shown to be within the desired range for liver cell growth. The optimal seeding density for HepaRG cells on silk scaffolds was determined. The growth and maturation of scaffolded HepaRG cells was evaluated for 28 days, where the first 14 days of culture were a proliferation period and the last 14 days of culture were a differentiation period using dimethyl sulfoxide (DMSO) treatment. After the first 14 days of culture, the scaffolded HepaRG cells exhibited increased metabolic activity and albumin secretion compared to monolayer cultured controls and preserved these attributes through the duration of culture. Additionally, after the first 14 days of culture, the scaffolded HepaRG cells displayed a significantly reduced expression of genes associated with hepatocyte maturation. This difference in expression was no longer apparent after 28 days of culture, suggesting that the cells underwent rapid differentiation within the scaffold. The functionalization of silk scaffolds with extracellular matrix (ECM) components (type I collagen and/or an arginylglycylaspartic acid (RGD)-containing peptide) was investigated to determine the impact on HepaRG cell attachment and maturation. The inclusion of ECM components had no noticeable impact on cell attachment but did significantly influence CYP3A4 expression and albumin secretion. Finally, the matrix support provided by the 3% silk scaffolds could prime the HepaRG cells for steatosis liver model applications, as evidenced by lipid droplet accumulation and expression of steatosis-related genes after 24 h of exposure to oleic acid. Overall, our work demonstrates the utility of silk scaffolds in providing a modifiable platform for liver cell growth.

Influence of lyophilization primary drying time and temperature on porous silk scaffold fabrication for biomedical applications.

Lyophilization of protein solutions, such as silk fibroin (silk), produces porous scaffolds useful for tissue engineering (TE). The impact of modifying lyophilization primary drying parameters on scaffold properties has not yet been explored previously. In this work, changes to primary drying duration and temperature were investigated using 3%, 6%, 9%, and 12% (w/v) silk solutions, via protocols labeled as Long Hold, Slow Ramp, and Standard. The 9% and 12% scaffolds were not successfully fabricated using the Standard protocol, while the Long Hold and Slow Ramp protocols resulted in scaffolds from all silk solution concentrations. Scaffolds fabricated using the Long Hold protocol had higher Young's moduli, smaller pore Feret diameters, and faster degradation. To investigate the utility of the different lyophilized scaffolds for in vitro cell culturing, the HepaRG liver cell line was cultured in the 3% to 12% scaffolds fabricated using the Long Hold protocol. The HepaRG cells grown in 3% scaffolds initially had greater lipid accumulation and metabolic activity than the other groups, although these differences were no longer apparent by Day 28. The deoxyribonucleic acid content of the HepaRG cells grown in 3% scaffold group was also initially significantly higher than the other groups. Significant differences in gene expression by 9% scaffolded HepaRG cells (CK19, HNFα) were seen on Day 14 while significant differences by 12% scaffolded HepaRG cells (ALB, APOA4) were seen on Day 28. Overall, modifying the primary drying parameters and silk concentration resulted in lyophilized scaffolds with tunable properties useful for TE applications.

Phomoxanthone A Targets ATP Synthase.

Phomoxanthone A is a naturally occurring molecule and a powerful anti-cancer agent, although its mechanism of action is unknown. To facilitate the determination of its biological target(s), we used affinity-based labelling using a phomoxanthone A probe. Labelled proteins were pulled down, subjected to chemoproteomics analysis using LC-MS/MS and ATP synthase was identified as a likely target. Mitochondrial ATP synthase was validated in cultured cells lysates and in live intact cells. Our studies show sixty percent inhibition of ATP synthase by 260 µM phomoxanthone A.

Silk Hydrogel-Mediated Delivery of Bone Morphogenetic Protein 7 Directly to Subcutaneous White Adipose Tissue Increases Browning and Energy Expenditure.

Objective: Increasing the mass and/or activity of brown adipose tissue (BAT) is one promising avenue for treating obesity and related metabolic conditions, given that BAT has a high potential for energy expenditure and is capable of improving glucose and lipid homeostasis. BAT occurs either in discrete "classical" depots, or interspersed in white adipose tissue (WAT), termed "inducible/recruitable" BAT, or 'beige/brite' adipocytes. We and others have demonstrated that bone morphogenetic protein 7 (BMP7) induces brown adipogenesis in committed and uncommitted progenitor cells, resulting in increased energy expenditure and reduced weight gain in mice. BMP7 is therefore a reliable growth factor to induce browning of WAT. Methods: In this study, we sought to deliver BMP7 specifically to subcutaneous (sc)WAT in order to induce tissue-resident progenitor cells to differentiate into energy-expending recruitable brown adipocytes, without off-target effects like bone formation, which can occur when BMPs are in the presence of bone progenitor cells (outside of WAT). BMP7 delivery directly to WAT may also promote tissue innervation, or directly activate mitochondrial activity in brown adipocytes, as we have demonstrated previously. We utilized silk protein in the form of an injectable hydrogel carrying BMP7. Silk scaffolds are useful for in vivo delivery of substances due to favorable material properties, including controlled release of therapeutic proteins in an active form, biocompatibility with minimal immunogenic response, and prior FDA approval for some medical materials. For this study, the silk was engineered to meet desirable release kinetics for BMP7 in order to mimic our prior in vitro brown adipocyte differentiation studies. Fluorescently-labeled silk hydrogel loaded with BMP7 was directly injected into WAT through the skin and monitored by non-invasive in vivo whole body imaging, including in UCP1-luciferase reporter mice, thereby enabling an approach that is translatable to humans. Results: Injection of the BMP7-loaded silk hydrogels into the subcutaneous WAT of mice resulted in "browning", including the development of multilocular, uncoupling protein 1 (UCP1)-positive brown adipocytes, and an increase in whole-body energy expenditure and skin temperature. In diet-induced obese mice, BMP7-loaded silk delivery to subcutaneous WAT resulted in less weight gain, reduced circulating glucose and lower respiratory exchange ratio (RER). Conclusions: In summary, BMP7 delivery via silk scaffolds directly into scWAT is a novel translational approach to increase browning and energy expenditure, and represents a potential therapeutic avenue for delivering substances directly to adipose depots in pursuit of metabolic treatments.

The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

Sunitinib-Loaded Chondroitin Sulfate Hydrogels as a Novel Drug-Delivery Mechanism for the Treatment of Pancreatic Neuroendocrine Tumors.

BACKGROUND: Pancreatic neuroendocrine tumors (PanNETs) are increasingly common. Experts debate whether small tumors should be resected. Tumor destruction via injection of cytotoxic agents could offer a minimal invasive approach to this controversy. We hypothesize that a new drug delivery system comprising chondroitin sulfate (CS) hydrogels loaded with sunitinib (SUN) suppresses tumor growth in PanNET cells. METHODS: Injectable hydrogels composed of CS modified with methacrylate groups (MA) were fabricated and loaded with SUN. Loading target was either 200 µg (SUN200-G) or 500 µg (SUN500-G) as well as sham hydrogel with no drug loading (SUN0-G). SUN release from hydrogels was monitored in vitro over time and cytotoxicity induced by the released SUN was evaluated using QGP-1 and BON1 PanNET cell lines. QGP-1 xenografts were developed in 35 mice and directly injected with 25 µL of either SUN200-G, SUN500-G, SUN0-G, 100 µL of Sunitinib Malate (SUN-inj), or given 40 mg/kg/day oral sunitinib (SUN-oral). RESULTS: SUN-loaded CSMA hydrogel retained complete in vitro cytotoxicity toward the QGP-1 PanNET and BON-1 PanNET cell lines for 21 days. Mouse xenograft models with QGP-1 PanNETs showed a significant delay in tumor growth in the SUN200/500-G, SUN-inj and SUN-oral groups compared with SUN0-G (p = 0.0014). SUN500-G hydrogels induced significantly more tumor necrosis than SUN0-G (p = 0.04). There was no difference in tumor growth delay between SUN200/500G, SUN-inj, and SUN-oral. CONCLUSIONS: This study demonstrates that CSMA hydrogels loaded with SUN suppress PanNETs growth. This drug delivery could approach represents a novel way to treat PanNETs and other neoplasms via intratumoral injection.

Development of a mechanically matched silk scaffolded 3D clear cell renal cell carcinoma model.

Development of a 3D, biomaterials-based model for clear cell renal cell carcinoma (ccRCC) would be advantageous for understanding disease progression in vitro. This study demonstrated the development of lyophilized silk scaffolds that mechanically match the experimentally determined Young's modulus for ex vivo ccRCC samples and normal kidney tissue. Scaffolds fabricated from silk solutions ranging from 3 to 12% (w/v) were evaluated through mechanical testing. Following mechanical characterization of ccRCC samples, it was demonstrated that 6% silk scaffolds mechanically matched ccRCC samples. No impact of pathological grade and stage on the calculated ccRCC modulus was observed and all tumors evaluated mechanically matched the 6% silk scaffold formulation. Stratifying tissue specimens based upon histological observations (e.g. evidence of high levels of collagen deposition) resulted in no significant differences between groups. To investigate the impact of a mechanically matched culturing environment on in vitro ccRCC disease characteristics a model ccRCC cell line, 786-O, was utilized. Scaffolded 786-O cells demonstrated increased lipid droplet accumulation, a hallmark of ccRCC, compared to standard two-dimensional (2D) culture conditions. Additionally, scaffolded 786-O cells demonstrated increased expression of genes associated with ccRCC aggressiveness (ex. VEGFA, TNF, and IL-6) or immune markers under investigation as therapeutic targets (ex. PDL1, CTLA4). Comparison with 786-O cells grown on non-mechanically matched scaffolds demonstrated that these improved ccRCC characteristics were driven by scaffold modulus. Overall, our findings support the use of silk scaffolds in replicating physiologic tumor behavior for clear cell renal cell carcinoma and provide a platform for investigating disease progression.

Development of a stacked, porous silk scaffold neuroblastoma model for investigating spatial differences in cell and drug responsiveness.

Development of in vitro, preclinical cancer models that contain cell-driven microenvironments remains a challenge. Engineering of millimeter-scale, in vitro tumor models with spatially distinct regions that can be independently assessed to study tumor microenvironments has been limited. Here, we report the use of porous silk scaffolds to generate a high cell density neuroblastoma (NB) model that can spatially recapitulate changes resulting from cell and diffusion driven changes. Using COMSOL modeling, a scaffold holder design that facilitates stacking of thin, 200 µm silk scaffolds into a thick, bulk millimeter-scale tumor model (2, 4, 6, and 8 stacked scaffolds) and supports cell-driven oxygen gradients was developed. Cell-driven oxygen gradients were confirmed through pimonidazole staining. Post-culture, the stacked scaffolds were separated for analysis on a layer-by-layer basis. The analysis of each scaffold layer demonstrated decreasing DNA and increasing expression of hypoxia related genes (VEGF, CAIX, and GLUT1) from the exterior scaffolds to the interior scaffolds. Furthermore, the expression of hypoxia related genes at the interior of the stacks was comparable to that of a single scaffold cultured under 1% O2 and at the exterior of the stacks was comparable to that of a single scaffold cultured under 21% O2. The four-stack scaffold model underwent further evaluation to determine if a hypoxia activated drug, tirapazamine, induced reduced cell viability within the internal stacks (region of reduced oxygen) as compared with the external stacks. Decreased DNA content was observed in the internal stacks as compared to the external stacks when treated with tirapazamine, which suggests the internal scaffold stacks had higher levels of hypoxia than the external scaffolds. This stacked silk scaffold system presents a method for creating a single culture model capable of generating controllable cell-driven microenvironments through different stacks that can be individually assessed and used for drug screening.

Development of a dinutuximab delivery system using silk foams for GD2 targeted neuroblastoma cell death.

Neuroblastoma is the most common extracranial solid tumor of childhood and is associated with poor survival in high risk patients. Recently, dinutuximab (DNX) has emerged as an effective immunotherapy to treat patients with high risk neuroblastoma. DNX works through the induction of cell lysis via complement-dependent cytotoxicity (CDC) or antibody dependent cellular cytotoxicity (ADCC). However, one third of patients who undergo DNX treatment exhibit tumor relapse and the therapy is dose limited by side effects such as severe pain. To overcome delivery challenges of DNX, including large size and dose limiting side effects, we fabricated a delivery system capable of sustained local delivery of bioactive DNX utilizing silk fibroin. We evaluated the impact of silk properties (MW, crystallinity, and concentration) on release properties and confirmed the bioactivity of the release product. Additionally, we observed that the effectiveness of CDC induction by DNX could be correlated to the GD2 expression level of the target cells, with both the intravenous DNX formulation and the released DNX. Collectively, these data highlights a strategy to overcome delivery challenges and potentially improve therapeutic efficacy in cells expressing heterogenous levels of GD2.

Horseradish Peroxidase-Catalyzed Crosslinking of Fibrin Microthread Scaffolds.

Horseradish peroxidase (HRP) has been investigated as a catalyst to crosslink tissue-engineered hydrogels because of its mild reaction conditions and ability to modulate the mechanical properties of the matrix. Here, we report the results of the first study investigating the use of HRP to crosslink fibrin scaffolds. We examined the effect of varying HRP and hydrogen peroxide (H2O2) incorporation strategies on the resulting crosslink density and structural properties of fibrin in a microthread scaffold format. Primary (1°) and secondary (2°) scaffold modification techniques were evaluated to crosslink fibrin microthread scaffolds. A primary scaffold modification technique was defined as incorporating crosslinking agents into the microthread precursor solutions during extrusion. A secondary scaffold modification technique was defined as incubating the microthreads in a postprocessing crosslinker bath. Fibrin microthreads were enzymatically crosslinked through primary, secondary, or a combination of both approaches. All fibrin microthread scaffolds crosslinked with HRP and H2O2 via primary and/or secondary methods exhibited an increase in dityrosine crosslink density compared with uncrosslinked control microthreads, demonstrated by scaffold fluorescence. Fourier transform infrared spectroscopy indicated the formation of isodityrosine bonds in 1° HRP crosslinked microthreads. Characterization of tensile mechanical properties revealed that all HRP crosslinked microthreads were significantly stronger than control microthreads. Primary (1°) HRP crosslinked microthreads also demonstrated significantly slower degradation than control microthreads, suggesting that incorporating HRP and H2O2 during extrusion yields scaffolds with increased resistance to proteolytic degradation. Finally, cells seeded on HRP crosslinked microthreads retained a high degree of viability, demonstrating that HRP crosslinking yields biocompatible scaffolds that are suitable for tissue engineering. The goal of this work was to facilitate the logical design of enzymatically crosslinked fibrin microthreads with tunable structural properties, enabling their application for engineered tissue constructs with varied mechanical and structural properties.

Local delivery of dinutuximab from lyophilized silk fibroin foams for treatment of an orthotopic neuroblastoma model.

Immunotherapy targeting GD2 is a primary treatment for patients with high-risk neuroblastoma. Dinutuximab is a monoclonal antibody with great clinical promise but is limited by side effects such as severe pain. Local delivery has emerged as a potential mechanism to deliver higher doses of therapeutics into the tumor bed, while limiting systemic toxicity. We aim to deliver dinutuximab locally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Dinutuximab-loaded silk fibroin foams were fabricated through lyophilization. In vitro release profile and bioactivity of the release through complement-dependent cytotoxicity were characterized. MYCN-amplified neuroblastoma cells (KELLY) were injected into the left gland of mice to generate an orthotopic neuroblastoma model. Once the tumor volume reached 100 mm3 , dinutuximab-, human IgG-, or buffer-loaded foams were implanted into the tumor and growth was monitored using high-resolution ultrasound. Post-resection histology was performed on tumors. Dinutuximab-loaded silk fibroin foams exhibited a burst release, with slow release thereafter in vitro with maintenance of bioactivity. The dinutuximab-loaded foam significantly inhibited xenograft tumor growth compared to IgG- and buffer-loaded foams. Histological analysis revealed the presence of dinutuximab within the tumor and neutrophils and macrophages infiltrating into dinutuximab-loaded silk foam. Tumors treated with local dinutuximab had decreased MYCN expression on histology compared to control or IgG-treated tumors. Silk fibroin foams offer a mechanism for local release of dinutuximab within the neuroblastoma tumor. This local delivery achieved a significant decrease in tumor growth rate in a mouse orthotopic tumor model.

Silk Particle Production Based on silk/PVA Phase Separation Using a Microfabricated Co-flow Device.

Polymeric particles are ideal drug delivery systems due to their cellular uptake-relevant size. Microparticles could be developed for direct injection of drug formulations into a diseased site, such as a tumor, allowing for drug retention and slow drug exposure over time through sustained release mechanisms. Bombyx mori silk fibroin has shown promise as a biocompatible biomaterial both in research and the clinic. Silk has been previously used to make particles using an emulsion-based method with poly(vinyl alcohol) (PVA). In this study, polydimethylsiloxane-based microfluidic devices were designed, fabricated, and characterized to produce silk particles through self-association of silk when exposed to PVA. Three main variables resulted in differences in particle size and size distribution, or polydispersity index (PDI). Utilizing a co-flow microfluidic device decreased the PDI of the silk particles as compared to an emulsion-based method (0.13 versus 0.65, respectively). With a flow-focusing microfluidics device, lowering the silk flow rate from 0.80 to 0.06 mL/h resulted in a decrease in the median particle size from 6.8 to 3.0 µm and the PDI from 0.12 to 0.05, respectively. Lastly, decreasing the silk concentration from 12% to 2% resulted in a decrease in the median particle size from 5.6 to 2.8 µm and the PDI from 0.81 to 0.25, respectively. Binding and release of doxorubicin, a cytotoxic drug commonly used for cancer treatment, with the fabricated silk particles was evaluated. Doxorubicin loading in the silk particles was approximately 41 µg/mg; sustained doxorubicin release occurred over 23 days. When the cytotoxicity of the released doxorubicin was tested on KELLY neuroblastoma cells, significant cell death was observed. To demonstrate the potential for internalization of the silk particles, both KELLY and THP-1-derived macrophages were exposed to fluorescently labelled silk particles for up to 24 h. With the macrophages, internalization of the silk particles was observed. Additionally, THP-1 derived macrophages exposure to silk particles increased TNF-α secretion. Overall, this microfluidics-based approach for fabricating silk particles utilizing PVA as a means to induce phase separation and silk self-assembly is a promising approach to control particle size and size distribution. These silk particles may be utilized for a variety of biomedical applications including drug delivery to multiple cell types within a tumor microenvironment.

In Vitro Biocompatibility of Decellularized Cultured Plant Cell-Derived Matrices.

There has been a recent increase in exploring the use of decellularized plant tissue as a novel "green" material for biomedical applications. As part of this effort, we have developed a technique to decellularize cultured plant cells (tobacco BY-2 cells and rice cells) and tissue (tobacco hairy roots) that uses deoxyribonuclease I (DNase I)). As a proof of concept, all cultured plant cells and tissue were transformed to express recombinant enhanced green fluorescent protein (EGFP) to show that the proteins of interest could be retained within the matrices. Decellularization of lyophilized tobacco BY-2 cells with DNase for 30 min depleted the DNA content from 1503 ± 459 to 31 ± 5 ng/sample. The decellularization procedure resulted in approximately 36% total protein retention (154 ± 60 vs 424 ± 70 µg/sample) and 33% EGFP retention. Similar results for DNA removal and protein retention were observed with the rice cells and tobacco hairy root matrices. When exposed to decellularized BY-2 cell-derived matrices, monolayer cultures of human foreskin fibroblasts (hFFs) maintained or increased metabolic activity, which is an indicator of cell viability. Furthermore, hFFs were able to attach, spread, and proliferate when cultured with the decellularized BY-2 cell-derived matrices in an aggregate model. Overall, these studies demonstrate that cultured plant cells and tissue can be effectively decellularized with DNase I with substantial protein retention. The resulting material has a positive impact on hFF metabolic activity and could be employed to create a three-dimensional environment for cell growth. These results thus show the promise of using naturally derived cellulose matrices from cultured plant cells and tissues for biomedical applications.

Silk Reservoirs for Local Delivery of Cisplatin for Neuroblastoma Treatment: In Vitro and In Vivo Evaluations.

Neuroblastoma is the most common extracranial childhood tumor, and current treatment requires surgical resection and multidrug chemotherapy. Local, perioperative delivery of chemotherapeutics is a promising treatment method for solid tumors that require surgical removal. In this study, we have aimed to develop a controlled-release implant system to deliver cisplatin in tumor or tumor resection area. Silk fibroin, a biodegradable, nonimmunogenic biopolymer was used to encapsulate different doses of cisplatin in a reservoir system. The physical integrity of the reservoirs was characterized by evaluating the crystalline structure of silk secondary structure using FTIR spectroscopy. The in vitro release of cisplatin was evaluated in phosphate-buffered saline at 37°C, and the reservoirs were able to release the drug up to 30 days. The cytotoxicity of cisplatin and cisplatin reservoirs were tested on KELLY cells. Cytotoxicity data showed 3.2 µg/mL cisplatin was required to kill 50% of the cell population, and the released cisplatin from the silk reservoirs showed significant cytotoxicity up to 21 days. Intratumoral implantation of silk reservoirs into an orthotopic neuroblastoma mouse model decreased tumor growth significantly when compared with control subjects. These results suggest that silk reservoirs are promising carriers for cisplatin delivery to the tumor site.

Scaffolding kidney organoids on silk.

End stage kidney disease affects hundreds of thousands of patients in the United States. The therapy of choice is kidney replacement, but availability of organs is limited, and alternative sources of tissue are needed. Generation of new kidney tissue in the laboratory has been made possible through pluripotent cell reprogramming and directed differentiation. In current procedures, aggregates of cells known as organoids are grown either submerged or at the air-liquid interface. These studies have demonstrated that kidney tissue can be generated from pluripotent stem cells, but they also identify limitations. The first is that perfusion of cell aggregates is limited, restricting the size to which they can be grown. The second is that aggregates lack the structural integrity required for convenient engraftment and suturing or adhesion to regions of kidney injury. In this study, we evaluated the capacity of silk to serve as a support for the growth and differentiation of kidney tissue from primary cells and from human induced pluripotent stem cells. We find that cells can differentiate to epithelia characteristic of the developing kidney on this material and that these structures are maintained following engraftment under the capsule of the adult kidney. Blood vessel investment can be promoted by the addition of vascular endothelial growth factor to the scaffold, but the proliferation of stromal cells within the graft presents a challenge, which will require some readjustment of cell growth and differentiation conditions. In summary, we find that silk can be used to support growth of stem cell derived kidney tissue.