Surfactant-assisted photo-crosslinked silk fibroin sponges: A versatile platform for the design of bone scaffolds.

Critical size bone defects cannot heal without aid and current clinical approaches exhibit some limitations, underling the need for novel solutions. Silk fibroin, derived from silkworms, is widely utilized in tissue engineering and regenerative medicine due to its remarkable properties, making it a promising candidate for bone tissue regeneration in vitro and in vivo. However, the clinical translation of silk-based materials requires refinements in 3D architecture, stability, and biomechanical properties. In earlier research, improved mechanical resistance and stability of chemically crosslinked methacrylate silk fibroin (Sil-Ma) sponges over physically crosslinked counterparts were highlighted. Furthermore, the influence of photo-initiator and surfactant concentrations on silk properties was investigated. However, the characterization of sponges with Sil-Ma solution concentrations above 10 % (w/V) was hindered by production optimization challenges, with only cell viability assessed. This study focuses on the evaluation of methacrylate sponges' suitability as temporal bone tissue regeneration scaffolds. Sil-Ma sponge fabrication at a fixed concentration of 20 % (w/V) was optimized and the impact of photo-initiator (LAP) concentrations and surfactant (Tween 80) presence/absence was studied. Their effects on pore formation, silk secondary structure, mechanical properties, and osteogenic differentiation of hBM-MSCs were investigated. We demonstrated that, by tuning silk sponges' composition, the optimal combination boosted osteogenic gene expression, offering a strategy to tailor biomechanical properties for effective bone regeneration. Utilizing Design of Experiment (DoE), correlations between sponge composition, porosity, and mechanical properties are established, guiding tailored material outcomes. Additionally, correlation matrices elucidate the microstructure's influence on gene expressions, providing insights for personalized approaches in bone tissue regeneration.

A Gellan Gum, Polyethylene Glycol, Hydroxyapatite Composite Scaffold with the Addition of Ginseng Derived Compound K with Possible Applications in Bone Regeneration.

Engineered bone scaffolds should mimic the natural material to promote cell adhesion and regeneration. For this reason, natural biopolymers are becoming a gold standard in scaffold production. In this study, we proposed a hybrid scaffold produced using gellan gum, hydroxyapatite, and Poly (ethylene glycol) within the addition of the ginseng compound K (CK) as a candidate for bone regeneration. The fabricated scaffold was physiochemically characterized. The morphology studied by scanning electron microscopy (SEM) and image analysis revealed a pore distribution suitable for cells growth. The addition of CK further improved the biological activity of the hybrid scaffold as demonstrated by the MTT assay. The addition of CK influenced the scaffold morphology, decreasing the mean pore diameter. These findings can potentially help the development of a new generation of hybrid scaffolds to best mimic the natural tissue.

Modulation of Methacrylated Hyaluronic Acid Hydrogels Enables Their Use as 3D Cultured Model.

Bioengineered hydrogels represent physiologically relevant platforms for cell behaviour studies in the tissue engineering and regenerative medicine fields, as well as in in vitro disease models. Hyaluronic acid (HA) is an ideal platform since it is a natural biocompatible polymer that is widely used to study cellular crosstalk, cell adhesion and cell proliferation, and is one of the major components of the extracellular matrix (ECM). We synthesised chemically modified HA with photo-crosslinkable methacrylated groups (HA-MA) in aqueous solutions and in strictly monitored pH and temperature conditions to obtain hydrogels with controlled bulk properties. The physical and chemical properties of the different HA-MA hydrogels were investigated via rheological studies, mechanical testing and scanning electron microscopy (SEM) imaging, which allowed us to determine the optimal biomechanical properties and develop a biocompatible scaffold. The morphological evolution processes and proliferation rates of glioblastoma cells (U251-MG) cultured on HA-MA surfaces were evaluated by comparing 2D structures with 3D structures, showing that the change in dimensionality impacted cell functions and interactions. The cell viability assays and evaluation of mitochondrial metabolism showed that the hydrogels did not interfere with cell survival. In addition, morphological studies provided evidence of cell-matrix interactions that promoted cell budding from the spheroids and the invasiveness in the surrounding environment.

Porous Thermoplastic Molded Regenerated Silk Crosslinked by the Addition of Citric Acid.

Thermoplastic molded regenerated silk fibroin was proposed as a structural material in tissue engineering applications, mainly for application in bone. The protocol allows us to obtain a compact non-porous material with a compression modulus in the order of a Giga Pascal in dry conditions (and in the order of tens of MPa in wet conditions). This material is produced by compressing a lyophilized silk fibroin powder or sponge into a mold temperature higher than the glass transition temperature. The main purpose of the produced resin was the osteofixation and other structural applications in which the lack of porosity was not an issue. In this work, we introduced the use of citric acid in the thermoplastic molding protocol of silk fibroin to obtain porosity inside the structural material. The citric acid powder during the compression acted as a template for the pore formation. The mean pore diameter achieved by the addition of the higher amount of citric acid was around 5 µm. In addition, citric acid could effectively crosslink the silk fibroin chain, improving its mechanical strength. This effect was proved both by evaluating the compression modulus (the highest value recorded was 77 MPa in wet conditions) and by studying the spectra obtained by Fourier transform infrared spectroscopy. This protocol may be applied in the near future to the production of structural bone scaffolds.

Modeling a Dynamic Printability Window on Polysaccharide Blend Inks for Extrusion Bioprinting.

Extrusion-based bioprinting is one of the most widespread technologies due to its affordability, wide range of processable materials, and ease of use. However, the formulation of new inks for this technique is based on time-consuming trial-and-error processes to establish the optimal ink composition and printing parameters. Here, a dynamic printability window was modeled for the assessment of the printability of polysaccharide blend inks of alginate and hyaluronic acid with the intent to build a versatile predictive tool to speed up the testing procedures. The model considers both the rheological properties of the blends (viscosity, shear thinning behavior, and viscoelasticity) and their printability (in terms of extrudability and the ability of forming a well-defined filament and detailed geometries). By imposing some conditions on the model equations, it was possible to define empirical bands in which the printability is ensured. The predictive capability of the built model was successfully verified on an untested blend of alginate and hyaluronic acid chosen to simultaneously optimize the printability index and minimize the size of the deposited filament.

Methacrylated Silk Fibroin Additive Manufacturing of Shape Memory Constructs with Possible Application in Bone Regeneration.

Methacrylated silk (Sil-MA) is a chemically modified silk fibroin specifically designed to be crosslinkable under UV light, which makes this material applicable in additive manufacturing techniques and allows the prototyping and development of patient-specific 2D or 3D constructs. In this study, we produced a thin grid structure based on crosslinked Sil-MA that can be withdrawn and ejected and that can recover its shape after rehydration. A complete chemical and physical characterization of Sil-MA was first conducted. Additionally, we tested Sil-MA biocompatibility according to the International Standard Organization protocols (ISO 10993) ensuring the possibility of using it in future trials. Sil-MA was also tested to verify its ability to support osteogenesis. Overall, Sil-MA was shown to be biocompatible and osteoconductive. Finally, two different additive manufacturing technologies, a Digital Light Processing (DLP) UV projector and a pneumatic extrusion technique, were used to develop a Sil-MA grid construct. A proof-of-concept of its shape-memory property was provided. Together, our data support the hypothesis that Sil-MA grid constructs can be injectable and applicable in bone regeneration applications.

Use of Bombyx mori silk fibroin in tissue engineering: From cocoons to medical devices, challenges, and future perspectives.

Silk fibroin has become a prominent material in tissue engineering (TE) over the last 20 years with almost 10,000 published works spanning in all the TE applications, from skeleton to neuronal regeneration. Fibroin is an extremely versatile biopolymer that, due to its ease of processing, has enabled the development of an entire plethora of materials whose properties and architectures can be tailored to suit target applications. Although the research and development of fibroin TE materials and devices is mature, apart from sutures, only a few medical products made of fibroin are used in the clinical routines. <40 clinical trials of Bombyx mori silk-related products have been reported by the FDA and few of them resulted in a commercialized device. In this review, after explaining the structure and properties of silk fibroin, we provide an overview of both fibroin constructs existing in the literature and fibroin devices used in clinic. Through the comparison of these two categories, we identified the burning issues faced by fibroin products during their translation to the market. Two main aspects will be considered. The first is the standardization of production processes, which leads both to the standardization of the characteristics of the issued device and the correct assessment of its failure. The second is the FDA regulations, which allow new devices to be marketed through the 510(k) clearance by demonstrating their equivalence to a commercialized medical product. The history of some fibroin medical devices will be taken as a case study. Finally, we will outline a roadmap outlining what actions we believe are needed to bring fibroin products to the market.

Novel hyphenation of DGT in-situ passive sampling with YES assay to ascertain the potency of emerging endocrine disruptors in water systems in New Zealand.

This study represents the first attempt to investigate selected estrogenic compounds that include 17α-ethynylestradiol (EE2), 17ß-estradiol (E2) bisphenol A (BPA), and bisphenol AF (BPAF) along the drinkable water, from river-to-tap, and wastewater, from effluent-to-treated wastewater, treatment processes of the Hamilton City Council and the monitoring of the freshwater, from source-to-outfall, of the Waikato River in New Zealand. This was accomplished by the adoption of a novel combination of diffusive gradients in thin films (DGTs) in-situ passive sampling coupled with high-performance liquid chromatography/mass spectrometry analysis (HPLC/MS) and the Yeast Estrogen Screen (YES). Estradiol equivalency quantities, integrated in time, were evaluated theoretically (cEEQ) by DGT-HPLC/MS and experimentally (EEQ) by DGT-YES assay. cEEQ and EEQ highlighted that primary treatments are not suitable for estrogens and bisphenolic plastics removal both at drinkable and wastewater treatment plants in Hamilton where they worsen the water quality in terms of estrogenicity making these pollutants more available in the water phase. All downstream sites monitored along the Waikato River showed higher cEEQ and EEQ, moreover the Waikato River water quality showed a moderate worsening moving from Taupo (source) to Tuakau (outfall). The most polluted sites were downstream of Hamilton city and Huntly township wastewater treatment plants that serve the main conurbations in the area. cEEQ and EEQ generally showed good agreement at low concentrations but differed substantially at more polluted sites where cEEQ consistently underestimated estrogenic potency, possibly due to DGT accumulation of estrogenic compounds not quantified by HPLC/MS.

Exploitation of response surface method for the optimization of RF-MEMS reconfigurable devices in view of future beyond-5G, 6G and super-IoT applications.

The emerging paradigms of the Beyond-5G, 6G and Super-IoT will demand for high-performance Radio Frequency (RF) passive components, and RF-MEMS technology, i.e. Microsystems-based RF passives, is a good candidate to meet such a challenge. As known, RF-MEMS have a complex behavior, that crosses different physical domains (mechanical; electrical; electromagnetic), making the whole design optimization and trimming phases particularly articulated and time consuming. In this work, we propose a novel design optimization approach based on the Response Surface Method (RSM) statistical methodology, focusing on a class of RF-MEMS-based programmable step power attenuators. The proposed method is validated both against physical simulations, performed with Finite Element Method (FEM) commercial software tools, as well as experimental measurements of physical devices. The case study here discussed features 3 DoFs (Degrees of Freedom), comprising both geometrical and material parameters, and aims to optimize the RF performances of the MEMS attenuator in terms of attenuation (S21 Scattering parameter) and reflection (VSWR-Voltage Standing Wave Ratio). When validated, the proposed RSM-based method allows avoiding physical FEM simulations, thus making the design optimization considerably faster and less complex, both in terms of time and computational load.

Tidy dataset of the experimental design of the optimization of the alkali degumming process of Bombyx mori silk.

Silk fibroin is the structural fiber of the silk filament and it is usually separated from the external protein, named sericine, by a chemical process called degumming. This process consists of an alkali bath in which the silk cocoons are boiled for a determined time. It is also known that the degumming process impacts the property of the outcoming silk fibroin fibers. In this work, we described the dataset obtained from a Design of Experiment (DoE) screening made on the alkali degumming. Four process factors were considered: the number of degumming baths, the process time, the process temperature, and the salt concentration. The data on the properties of the silk fibroin fibers were collected. In particular, the molecular weight was obtained by gel permeation chromatography (GPC), the mechanical data by tensile test and the secondary structure by Fourier Infrared Transform Spectroscopy (FTIR).

Molecularly Imprinted Silk Fibroin Nanoparticles.

Nanosized biomimetics prepared by the strategy of molecular imprinting, that is, the stamping of recognition sites by means of a template-assisted synthesis, are demonstrating potential as plastic antibodies in medicine, proving effective for cell imaging and targeted therapies. Most molecularly imprinted nanoparticles (MIP-NPs) are currently made of soft matter, such as polyacrylamide and derivatives. Yet, MIP-NPs biocompatibility is crucial for their effective translation into clinical uses. Here, we propose the original idea to synthesize fully biocompatible molecularly imprinted nanoparticles starting from the natural polymer silk fibroin (MIP SF-NPs), which is nontoxic and highly biocompatible. The conditions to produce MIP SF-NPs of different sizes (dmean ∼ 50 nm; dmean ∼ 100 nm) were set using the response surface method. The stamping of a single, high affinity (KD = 57 × 10-9 M), and selective recognition site per silk fibroin nanoparticle was demonstrated, together with the confirmation of nontoxicity. Additionally, MIP SF-NPs were used to decorate silk microfibers and silk nanofibers, providing a general means to add entailed biofunctionalities to materials.

Preparation and characterization of a soluble eggshell membrane/agarose composite scaffold with possible applications in cartilage regeneration.

Articular hyaline cartilage is an extremely hydrated, not vascularized tissue with a low-cell density. The damage of this tissue can occur after injuries or gradual stress and tears (osteoarthritis), minor damages can be self-healed in several weeks, but major injuries may eventually require surgery. In fact, in this case, because of nature of the cartilage (the absence of cells and vascularization) it is difficult to expect its natural regeneration in a reasonable amount of time. In recent years, cell therapy, in which cells are directly transplanted, has attracted attention. In this study, a scaffold for implanting chondrocytes was prepared. The scaffold was made as a sponge using the eggshell membrane and agarose. The eggshell membrane is structurally similar to the extracellular matrix and nontoxic due to its many collagen components and has good biocompatibility and biodegradability. However, scaffolds made of collagen only has poor mechanical properties. For this reason, the disulfide bond of collagen extracted from the insoluble eggshell membrane was cut, converted into water-soluble, and then mixed with agarose to prepare a scaffold. Agarose is capable of controlling mechanical properties, has excellent biocompatibility, and is suitable for forming a hydrogel having a three-dimensional porosity. The scaffold was examined for Fourier-transform infrared, mechanical properties, biodegradability, and biocompatibility. In in vitro experiment, cytotoxicity, cell proliferation, and messenger RNA expression were investigated. The study demonstrated that the agarose/eggshell membrane scaffold can be used for chondrocyte transplantation.

A Design of Experiment Rational Optimization of the Degumming Process and Its Impact on the Silk Fibroin Properties.

Silk fibroin is a protein with a unique combination of properties and is widely studied for biomedical applications. The extraction of fibroin (degumming) from the silk filament impacts the properties of the outcoming material. The degumming can be conducted with different procedures. Among them, the most used and studied procedure in the research field is the alkali degumming with sodium carbonate (Na2CO3). In this study, by the use of a statistical method, namely, design of experiment (DOE), we characterized the Na2CO3 degumming, taking into consideration the main process factors involved and changing them within a selected range of values. We considered the process temperature and time, the salt concentration, and the number of baths used, testing the impact of these variables on the fibroin properties by building empirical models. These models not only took into consideration the direct effect of the process factors but also their combined effect, which are not conventionally detectable with other methods. The weight loss and the amount of sericin removed in the process were determined and used as a measure of the effectiveness of the process. The secondary structure, the molecular weight, the diameter of fibers, and their morphology and mechanical properties were studied with the intent to correlate the macroscopical properties with the structural changes. We report, for the first time, the possibility to effectively remove all sericin from the silk fibroin using Na2CO3, using a process that requires less salt, water, and energy, in comparison with the standard alkali protocol, making this technique overall more environmentally sustainable; in addition, we have demonstrated the possibility to tune the material properties by varying the degumming conditions and even to optimize them with empirical statistically based equations that allow one to directly set the optimal process parameters. The major effect on the macroscopical properties (such as the ultimate strength and Young's modulus) has been proved to be correlated with the removal of sericin instead of the microstructural variations. Finally, a ready-to-use table with a set of optimized degumming procedures to maximize or minimize the studied properties was provided.

Optimization of a Low-Power Chemoresistive Gas Sensor: Predictive Thermal Modelling and Mechanical Failure Analysis.

The substrate plays a key role in chemoresistive gas sensors. It acts as mechanical support for the sensing material, hosts the heating element and, also, aids the sensing material in signal transduction. In recent years, a significant improvement in the substrate production process has been achieved, thanks to the advances in micro- and nanofabrication for micro-electro-mechanical system (MEMS) technologies. In addition, the use of innovative materials and smaller low-power consumption silicon microheaters led to the development of high-performance gas sensors. Various heater layouts were investigated to optimize the temperature distribution on the membrane, and a suspended membrane configuration was exploited to avoid heat loss by conduction through the silicon bulk. However, there is a lack of comprehensive studies focused on predictive models for the optimization of the thermal and mechanical properties of a microheater. In this work, three microheater layouts in three membrane sizes were developed using the microfabrication process. The performance of these devices was evaluated to predict their thermal and mechanical behaviors by using both experimental and theoretical approaches. Finally, a statistical method was employed to cross-correlate the thermal predictive model and the mechanical failure analysis, aiming at microheater design optimization for gas-sensing applications.

Micropatterning of Substrates for the Culture of Cell Networks by Stencil-Assisted Additive Nanofabrication.

The fabrication of in vitro neuronal cell networks where cells are chemically or electrically connected to form functional circuits with useful properties is of great interest. Standard cell culture substrates provide ensembles of cells that scarcely reproduce physiological structures since their spatial organization and connectivity cannot be controlled. Supersonic Cluster Beam Deposition (SCBD) has been used as an effective additive method for the large-scale fabrication of interfaces with extracellular matrix-mimicking surface nanotopography and reproducible morphological properties for cell culture. Due to the high collimation of SCBD, it is possible to exploit stencil masks for the fabrication of patterned films and reproduce features as small as tens of micrometers. Here, we present a protocol to fabricate micropatterned cell culture substrates based on the deposition of nanostructured cluster-assembled zirconia films by stencil-assisted SCBD. The effectiveness of this approach is demonstrated by the fabrication of micrometric patterns able to confine primary astrocytes. Calcium waves propagating in the astrocyte networks are shown.

Comparative Study on the Effect of the Different Harvesting Sources of Demineralized Bone Particles on the Bone Regeneration of a Composite Gellan Gum Scaffold for Bone Tissue Engineering Applications.

Bone is the rigid tissue that constitutes the skeleton. The material for bone regeneration has to provide the mechanical stability by maintaining the mechanical loads both in the rest conditions and during the body movements. Bone is dynamic tissue constantly reshaped by the action of cells (osteoblasts and osteoclasts). This activity is normally enough to heal bone injuries; however, in several conditions, when bone is subjected to fatal damages, self-renewal is difficult, if not even impossible, and a medical treatment is required. The transplantation of a biomaterial is one of the common surgical procedures to overcome critical injuries. In this study, we exploited the effect of the use of different sources of demineralized bone powder (DBP) in combination with gellan gum (GG) to form a GG-DBP hydrogel scaffold with the purpose of regenerating the bone tissue. DBP was extracted from the femurs of two typologies of Gallus gallus domesticus (the Yeonsan Ogye, a traditional and rare black chicken from Korea, and the Cornish cross, the most common breeds for industrial meat production) and the Pekin duck. The composite scaffold has been tested both in vitro and in vivo. In vitro studies using rat bone marrow-derived mesenchymal stem cells (rBMSCs) confirmed the cellular suitability of bone-specific gene expression for seeded GG-DBP scaffolds, differentiation capacity, and marked upregulation. The scaffold containing a DBP derived from the Yeonsan Ogye (YO) bone showed higher levels of cell proliferation and osteogenic differentiation in comparison with the scaffold with the DBP obtained from the other studied sources. These results have been related with the higher amount of melanin, studied by fluorescence, of the YO DBP compared to Cornish cross and Pekin duck. Overall, this study clearly shows the use of YO DBP as a promising material in bone tissue regeneration.

Natural Sources and Applications of Demineralized Bone Matrix in the Field of Bone and Cartilage Tissue Engineering.

Demineralized bone matrix (DBM) is one of the most widely used materials for bone repair. Recently, different strategies in tissue engineering have been used to improve preparation of biomaterials from natural sources suitable for the use in bone regeneration. However, the application of DBM in tissue engineering is still a challenge, because the mechanical properties which are essential to bear tensile and load and the risk of transmission of disease by donor are still a matter of homework. A solution to this problem is to blend natural and synthetic polymers to complement defects and make them ideal biomaterials. An ideal biomaterial improves survival, adhesion, proliferation, induction, and differentiation of cells in the biomaterial after in vivo transplantation. In this review, we will look at the study of DBM made of natural and synthetic materials giving a direction for future research.

Preparation and Statistical Characterization of Tunable Porous Sponge Scaffolds using UV Cross-linking of Methacrylate-Modified Silk Fibroin.

Silk fibroin sponges have been widely studied and reported in literature for tissue engineering applications. Several fabrication methods have been proposed during the years to cover most of the demands in terms of properties, which should be adapted to the considered tissue. Most of these procedures are based on the secondary structure transition of the protein to the stable ß crystalline form. This transition, known as physical cross-linking, makes the sponge resistant to dissolution in water, and, in general, increases the sponge stiffness. In our work, we propose an alternative method to ensure the stability of the sponge based on chemical cross-linking of a methacrylated version of silk fibroin (Sil-MA) obtained via chemical modification. The Sil-MA water solution with the addition of a photoinitiator (LAP) allows the opening, under UV radiation, of a double carbon-carbon bond and radical polymerization. The incorporation of air bubbles (that serves as a template for the pores) was accomplished by a mixer; then, the foam was stabilized under UV light and the excess water was removed by freeze-drying. Because of the cytotoxicity of the photoinitiator (found when used at a high concentration), an additional washing step in water has been introduced to eliminate the residues and improve the cells' viability. Fourier transform infrared (FTIR) analysis confirmed the functionalization of the protein. To evaluate the effect of the composition on the sponge properties, a 23 full factorial design of the experiment has been adopted. FTIR analysis revealed that the sponge composition did not affect the protein's secondary structure. The analysis of images obtained by SEM allowed some statistical measures of the porosity curves to be studied and modeled. The same modeling procedure was applied to the dissolution test in a simulated body fluid, to the water absorption, and to the cell viability (tested by the MTT and LDH assays). An empirical model for each property was built, showing how by changing the composition it is possible to tune the sponge properties.