Multi-spectral photoacoustic imaging combined with acoustic radiation force impulse imaging for applications in tissue engineering.

Tissue engineering is a dynamic field focusing on the creation of advanced scaffolds for tissue and organ regeneration. These scaffolds are customized to their specific applications and are often designed to be complex, large structures to mimic tissues and organs. This study addresses the critical challenge of effectively characterizing these thick, optically opaque scaffolds that traditional imaging methods fail to fully image due to their optical limitations. We introduce a novel multi-modal imaging approach combining ultrasound, photoacoustic, and acoustic radiation force impulse imaging. This combination leverages its acoustic-based detection to overcome the limitations posed by optical imaging techniques. Ultrasound imaging is employed to monitor the scaffold structure, photoacoustic imaging is employed to monitor cell proliferation, and acoustic radiation force impulse imaging is employed to evaluate the homogeneity of scaffold stiffness. We applied this integrated imaging system to analyze melanoma cell growth within silk fibroin protein scaffolds with varying pore sizes and therefore stiffness over different cell incubation periods. Among various materials, silk fibroin was chosen for its unique combination of features including biocompatibility, tunable mechanical properties, and structural porosity which supports extensive cell proliferation. The results provide a detailed mesoscale view of the scaffolds' internal structure, including cell penetration depth and biomechanical properties. Our findings demonstrate that the developed multimodal imaging technique offers comprehensive insights into the physical and biological dynamics of tissue-engineered scaffolds. As the field of tissue engineering continues to advance, the importance of non-ionizing and non-invasive imaging systems becomes increasingly evident, and by facilitating a deeper understanding and better characterization of scaffold architectures, such imaging systems are pivotal in driving the success of future tissue-engineering solutions.

Impact of Silk-Ionomer Encapsulation on Immune Cell Mechanical Properties and Viability.

Encapsulation of single cells is a powerful technique used in various fields, such as regenerative medicine, drug delivery, tissue regeneration, cell-based therapies, and biotechnology. It offers a method to protect cells by providing cytocompatible coatings to strengthen cells against mechanical and environmental perturbations. Silk fibroin, derived from the silkworm Bombyx mori, is a promising protein biomaterial for cell encapsulation due to the cytocompatibility and capacity to maintain cell functionality. Here, THP-1 cells, a human leukemia monocytic cell line, were encapsulated with chemically modified silk polyelectrolytes through electrostatic layer-by-layer deposition. The effectiveness of the silk nanocoating was assessed using scanning electron microscopy (SEM) and confocal microscopy and on cell viability and proliferation by Alamar Blue assay and live/dead staining. An analysis of the mechanical properties of the encapsulated cells was conducted using atomic force microscopy nanoindentation to measure elasticity maps and cellular stiffness. After the cells were encapsulated in silk, an increase in their stiffness was observed. Based on this observation, we developed a mechanical predictive model to estimate the variations in stiffness in relation to the thickness of the coating. By tuning the cellular assembly and biomechanics, these encapsulations promote systems that protect cells during biomaterial deposition or processing in general.

Redefining Surgical Materials: Applications of Silk Fibroin in Osteofixation and Fracture Repair.

Silk and silk derivatives have emerged as a possible alternative in surgical device development, offering mechanical strength, biocompatibility, and environmental sustainability. Through a systematic review following PRISMA guidelines, this study evaluated silk fibroin's application across pre-clinical and clinical settings, focusing on its role as screws and plates for osteofixation. A comprehensive search yielded 245 studies, with 33 subjected to full-text review and 15 ultimately included for qualitative analysis. The findings underscore silk fibroin's superior properties, including its tunable degradation rates and ability to be functionalized with therapeutic agents. In vivo and in vitro studies demonstrated its efficacy in enhancing bone healing, offering improved outcomes in osteofixation, particularly for craniofacial defects. Silk fibroin's remarkable attributes in biodegradation and drug release capabilities underscore its potential to enhance patient care. Ultimately, silk fibroin's integration into surgical practices promises a revolution in patient outcomes and environmental sustainability. Its versatility, coupled with the continuous progress in fabrication techniques, signals a promising horizon for its widespread acceptance in the medical field, potentially establishing a new benchmark in surgical treatment. Further research is expected to solidify the transition of silk products from basic science to patient care, paving the way for widespread use in various surgical applications.

Impact of Micro- and Nano-Plastics on Human Intestinal Organoid-Derived Epithelium.

The development of patient-derived intestinal organoids represents an invaluable model for simulating the native human intestinal epithelium. These stem cell-rich cultures outperform commonly used cell lines like Caco-2 and HT29-MTX in reflecting the cellular diversity of the native intestinal epithelium after differentiation. In our recent study examining the effects of polystyrene (PS), microplastics (MPs), and nanoplastics (NPs), widespread pollutants in our environment and food chain, on the human intestinal epithelium, these organoids have been instrumental in elucidating the absorption mechanisms and potential biological impacts of plastic particles. Building on previously established protocols in human intestinal organoid culture, we herein detail a streamlined protocol for the cultivation, differentiation, and generation of organoid-derived monolayers. This protocol is tailored to generate monolayers incorporating microfold cells (M cells), key for intestinal particle uptake but often absent in current in vitro models. We provide validated protocols for the characterization of MPs/NPs via scanning electron microscopy (SEM) for detailed imaging and their introduction to intestinal epithelial monolayer cells via confocal immunostaining. Additionally, protocols to test the impacts of MP/NP exposure on the functions of the intestinal barrier using transendothelial electrical resistance (TEER) measurements and assessing inflammatory responses using cytokine profiling are detailed. Overall, our protocols enable the generation of human intestinal organoid monolayers, complete with the option of including or excluding M cells, offering crucial techniques for observing particle uptake and identifying inflammatory responses in intestinal epithelial cells to advance our knowledge of the potential effects of plastic pollution on human gut health. These approaches are also amendable to the study of other gut-related chemical and biological exposures and physiological responses due to the robust nature of the systems. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Human intestinal organoid culture and generation of monolayers with and without M cells Support Protocol 1: Culture of L-WRN and production of WRN-conditioned medium Support Protocol 2: Neuronal cell culture and integration into intestinal epithelium Support Protocol 3: Immune cell culture and integration into intestinal epithelium Basic Protocol 2: Scanning electron microscopy: sample preparation and imaging Basic Protocol 3: Immunostaining and confocal imaging of MP/NP uptake in organoid-derived monolayers Basic Protocol 4: Assessment of intestinal barrier function via TEER measurements Basic Protocol 5: Cytokine profiling using ELISA post-MP/NP exposure.

Heterogeneous and Cooperative Rupture of Histidine-Ni2+ Metal-Coordination Bonds on Rationally Designed Protein Templates.

Metal-coordination bonds, a highly tunable class of dynamic noncovalent interactions, are pivotal to the function of a variety of protein-based natural materials and have emerged as binding motifs to produce strong, tough, and self-healing bioinspired materials. While natural proteins use clusters of metal-coordination bonds, synthetic materials frequently employ individual bonds, resulting in mechanically weak materials. To overcome this current limitation, we rationally designed a series of elastin-like polypeptide templates with the capability of forming an increasing number of intermolecular histidine-Ni2+ metal-coordination bonds. Using single-molecule force spectroscopy and steered molecular dynamics simulations, we show that templates with three histidine residues exhibit heterogeneous rupture pathways, including the simultaneous rupture of at least two bonds with more-than-additive rupture forces. The methodology and insights developed improve our understanding of the molecular interactions that stabilize metal-coordinated proteins and provide a general route for the design of new strong, metal-coordinated materials with a broad spectrum of dissipative time scales.

Tuning the Biodegradation Rate of Silk Materials via Embedded Enzymes.

Conventional thinking when designing biodegradable materials and devices is to tune the intrinsic properties and morphological features of the material to regulate their degradation rate, modulating traditional factors such as molecular weight and crystallinity. Since regenerated silk protein can be directly thermoplastically molded to generate robust dense silk plastic-like materials, this approach afforded a new tool to control silk degradation by enabling the mixing of a silk-degrading protease into bulk silk material prior to thermoplastic processing. Here we demonstrate the preparation of these silk-based devices with embedded silk-degrading protease to modulate the degradation based on the internal presence of the enzyme to support silk degradation, as opposed to the traditional surface degradation for silk materials. The degradability of these silk devices with and without embedded protease XIV was assessed both in vitro and in vivo. Ultimately, this new process approach provides direct control of the degradation lifetime of the devices, empowered through internal digestion via water-activated proteases entrained and stabilized during the thermoplastic process.

Recent advances in harnessing biological macromolecules for wound management: A review.

Wound dressings (WDs) are an essential component of wound management and serve as an artificial barrier to isolate the injured site from the external environment, thereby helping to prevent exogenous infections and supporting healing. However, maintaining a moist wound environment, providing protection from infection, good biocompatibility, and allowing for gas exchange, remain a challenge in device design. Functional wound dressings (FWDs) prepared from hybrid biological macromolecule-based materials can enhance efficacy of these systems for skin wound management. This review aims to provide an overview of the state-of-the-art FWDs within the field of wound management, with a specific focus on hybrid biomaterials, techniques, and applications developed over the past five years. In addition, we highlight the incorporation of biological macromolecules in WDs, the emergence of smart WDs, and discuss the existing challenges and future prospects for the development of advanced WDs.

Nanosized Silk-Magnesium Complexes for Promotion of Angiogenic and Osteogenic Activities.

Injectable hydrogels with osteogenic and angiogenetic properties are of interest in bone tissue engineering. Since the bioactivity of ions is concentration-dependent, nanosized silk-magnesium (Mg) complexes were previously developed and assembled into hydrogels with angiogenic capabilities but failed to control both osteogenic and angiogenetic activities effectively. Here, nanosized silk particles with different sizes were obtained by using ultrasonic treatment to control silk-Mg coordination and particle formation, resulting in silk-Mg hydrogels with different types of bioactivity. Fourier transform infrared and X-ray diffraction results revealed that different coordination intensities were present in the different complexes as a basis for the differences in activities. Slow Mg ion release was controlled by these nanosized silk-Mg complexes through degradation. With the same amount of Mg ions, the different silk-Mg complexes exhibited different angiogenic and osteogenic capacities. Complexes with both angiogenic and osteogenic capacities were developed by optimizing the sizes of the silk particles, resulting in faster and improved quality of bone formed in vivo than complexes with the same composition of silk and Mg but only angiogenic or osteogenic capacities. The biological selectivity of silk-Mg complexes should facilitate applications in tissue regeneration.

Silk fibroin-based inks for in situ 3D printing using a double crosslinking process.

The shortage of tissues and organs for transplantation is an urgent clinical concern. In situ 3D printing is an advanced 3D printing technique aimed at printing the new tissue or organ directly in the patient. The ink for this process is central to the outcomes, and must meet specific requirements such as rapid gelation, shape integrity, stability over time, and adhesion to surrounding healthy tissues. Among natural materials, silk fibroin exhibits fascinating properties that have made it widely studied in tissue engineering and regenerative medicine. However, further improvements in silk fibroin inks are needed to match the requirements for in situ 3D printing. In the present study, silk fibroin-based inks were developed for in situ applications by exploiting covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing and in situ enzymatic crosslinking. Two different silk fibroin molecular weights were characterized and the synergistic effect of the covalent bonds with shear forces enhanced the shift in silk secondary structure toward ß-sheets, thus, rapid stabilization. These hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. Additionally, adhesion to tissues in vitro was demonstrated.

ForceGen: End-to-end de novo protein generation based on nonlinear mechanical unfolding responses using a language diffusion model.

Through evolution, nature has presented a set of remarkable protein materials, including elastins, silks, keratins and collagens with superior mechanical performances that play crucial roles in mechanobiology. However, going beyond natural designs to discover proteins that meet specified mechanical properties remains challenging. Here, we report a generative model that predicts protein designs to meet complex nonlinear mechanical property-design objectives. Our model leverages deep knowledge on protein sequences from a pretrained protein language model and maps mechanical unfolding responses to create proteins. Via full-atom molecular simulations for direct validation, we demonstrate that the designed proteins are de novo, and fulfill the targeted mechanical properties, including unfolding energy and mechanical strength, as well as the detailed unfolding force-separation curves. Our model offers rapid pathways to explore the enormous mechanobiological protein sequence space unconstrained by biological synthesis, using mechanical features as the target to enable the discovery of protein materials with superior mechanical properties.

Silk Nanofiber Carriers for Both Hydrophilic and Hydrophobic Drugs to Achieve Improved Combination Chemotherapy.

Combination chemotherapy is considered an effective strategy to inhibit tumor growth. Here, beta-sheet-rich silk nanofibers are co-loaded with hydrophilic doxorubicin (DOX) and hydrophobic paclitaxel (PTX) through a sequential physical blending-centrifugation-blending process. The ratio and amount of DOX and PTX on the nanofibers are regulated independently to optimize cooperative interaction. Both PTX and DOX are immobilized on the same nanofibers to avoid burst release problems. Besides the water-insoluble PTX, more than half of the DOX remained fixed on the nanofibers for more than 28 days, which facilitated the co-internalization of both DOX and PTX by tumor cells in vitro. Changing the ratio of co-loaded DOX and PTX achieved optimal combination therapy in vitro. The DOX-PTX co-loaded nanofibers are assembled into injectable hydrogels to facilitate in situ injection around tumor tissues in vivo. Long-term inhibition is achieved for tumors treated with DOX-PTX co-loaded hydrogels, superior to those treated with free DOX and PTX, and hydrogels loaded with only DOX or PTX. Considering the mild and controllable physical loading process and superior loading capacity for both hydrophilic and hydrophobic ingredients, these injectable silk nanofiber hydrogels are promising carriers to deliver multiple drug types simultaneously in situ, enhancing combination chemotherapies towards clinical applications.

Rapid Volumetric Bioprinting of Decellularized Extracellular Matrix Bioinks.

Decellularized extracellular matrix (dECM)-based hydrogels are widely applied to additive biomanufacturing strategies for relevant applications. The extracellular matrix components and growth factors of dECM play crucial roles in cell adhesion, growth, and differentiation. However, the generally poor mechanical properties and printability have remained as major limitations for dECM-based materials. In this study, heart-derived dECM (h-dECM) and meniscus-derived dECM (Ms-dECM) bioinks in their pristine, unmodified state supplemented with the photoinitiator system of tris(2,2-bipyridyl) dichlororuthenium(II) hexahydrate and sodium persulfate, demonstrate cytocompatibility with volumetric bioprinting processes. This recently developed bioprinting modality illuminates a dynamically evolving light pattern into a rotating volume of the bioink, and thus decouples the requirement of mechanical strengths of bioprinted hydrogel constructs with printability, allowing for the fabrication of sophisticated shapes and architectures with low-concentration dECM materials that set within tens of seconds. As exemplary applications, cardiac tissues are volumetrically bioprinted using the cardiomyocyte-laden h-dECM bioink showing favorable cell proliferation, expansion, spreading, biomarker expressions, and synchronized contractions; whereas the volumetrically bioprinted Ms-dECM meniscus structures embedded with human mesenchymal stem cells present appropriate chondrogenic differentiation outcomes. This study supplies expanded bioink libraries for volumetric bioprinting and broadens utilities of dECM toward tissue engineering and regenerative medicine.

Cultivated Meat from Aligned Muscle Layers and Adipose Layers Formed from Glutenin Films.

Cultivated meat production is a promising technology to generate meat while reducing the reliance on traditional animal farming. Biomaterial scaffolds are critical components in cultivated meat production, enabling cell adhesion, proliferation, differentiation, and orientation. In the present work, naturally derived glutenin was fabricated into films with and without surface patterning and in the absence of toxic cross-linking or stabilizing agents for cell culture related to cultivated meat goals. The films were stable in culture media for at least 28 days, and the surface patterns induced cell alignment and guided myoblast organization (C2C12s) and served as a substrate for 3T3-L1 adipose cells. The films supported adhesion, proliferation, and differentiation with mass balance considerations (films, cells, and matrix production). Freeze-thaw cycles were applied to remove cells from glutenin films and monitor changes in glutenin mass with respect to culture duration. Extracellular matrix (ECM) extraction was utilized to quantify matrix deposition and changes in the original biomaterial mass over time during cell cultivation. Glutenin films with C2C12s showed mass increases with time due to cell growth and new collagen-based ECM expression during proliferation and differentiation. All mass balances were compared among cell and noncell systems as controls, along with gelatin control films, with time-dependent changes in the relative content of film, matrix deposition, and cell biomass. These data provide a foundation for cell/biomaterial/matrix ratios related to time in culture as well as nutritional and textural features.

Biomedical Applications of Electro-Initiated Polymerisation on Ti6Al4†V Titanium Alloy using Silk Fibroin Coatings for Antibiotic Delivery and Improved Cell Metabolism.

Silk fibroin interactions with metallic surfaces can provide utility for medical materials and devices. Toward this goal, titanium alloy (Ti6Al4 V) was covalently grafted with polyacrylamide via electrochemically reducing 4-nitrobenzene diazonium salt in the presence of acrylamide. Analysis of the modified surfaces with FT-IR spectra, SEM and AFM were consistent with surface grafting. Functionalised titanium samples with a silk fibroin membrane, with and without impregnated therapeutics, were used to assess cytocompatibility and drug delivery. Initial cytocompatibility experiments using fibroblasts showed that the functionalised samples, both with and without silk fibroin coatings, supported significant increases between 72-136 % in cell metabolism, compared to the controls after 7 days. A 7-days release profiling showed consistent bacterial inhibition through gentamicin release with average inhibition zones of 239 mm2 . Over a 5-week period, silk fibroin coated samples, both with and without growth factors, supported better human mesenchymal stem cell metabolism with increases reaching 1031 % and 388 %, respectively, compared to samples without the silk fibroin coating with.

Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

ForceGen: End-to-end de novo protein generation based on nonlinear mechanical unfolding responses using a protein language diffusion model.

Through evolution, nature has presented a set of remarkable protein materials, including elastins, silks, keratins and collagens with superior mechanical performances that play crucial roles in mechanobiology. However, going beyond natural designs to discover proteins that meet specified mechanical properties remains challenging. Here we report a generative model that predicts protein designs to meet complex nonlinear mechanical property-design objectives. Our model leverages deep knowledge on protein sequences from a pre-trained protein language model and maps mechanical unfolding responses to create novel proteins. Via full-atom molecular simulations for direct validation, we demonstrate that the designed proteins are novel, and fulfill the targeted mechanical properties, including unfolding energy and mechanical strength, as well as the detailed unfolding force-separation curves. Our model offers rapid pathways to explore the enormous mechanobiological protein sequence space unconstrained by biological synthesis, using mechanical features as target to enable the discovery of protein materials with superior mechanical properties.

Silk Nanofiber Scaffolds with Multiple Angiogenic Cues to Accelerate Wound Regeneration.

Niches with multiple physical and chemical cues can influence the fate of cells and tissues in vivo. Simulating the in vivo niche in the design of bioactive materials is a challenge, particularly to tune multiple cues simultaneously in the same system. Here, an assembly strategy was developed to regulate multiple cues in the same scaffold based on the use of two silk nanofiber components that respond differently during the fabrication processes. An aqueous solution containing the two components, amorphous silk nanofibers (ASNFs) and ß-sheet-rich silk nanofibers (BSNFs), was sequentially treated with an electrical field and freeze-drying processes where the BSNFs oriented to the electrical field, while the ASNFs formed stable porous structures during the lyophilization process to impact the mechanical properties. Bioactive cargo, such as deferoxamine (DFO), was loaded on the BSNFs to enrich cell responses with the scaffolds. The in vitro results revealed that the loaded DFO and the anisotropic structures with improved mechanical properties resulted in better vascularization than those of the scaffolds without the anisotropic features. The multiple cues in the scaffolds provided angiogenic niches to accelerate wound healing.

Concentric-mineralized hybrid silk-based scaffolds for bone tissue engineering in vitro models.

There are many challenges in the development of 3D-tissue models for studying bone physiology and disease. Silk fibroin (SF), a natural fibrous protein used in biomedical applications has been studied for bone tissue engineering (TE) due to its mechanical properties, biocompatibility and biodegradability. However, low osteogenic capacity as well as the necessity to reinforce the protein mechanically for some orthopedic applications prompts the need for further designs for SF-based materials for TE bone. Concentric mineralized porous SF-based scaffolds were developed to improve mechanics and mineralization towards osteoregeneration. Hybrid SF silica microparticles (MP) or calcium carbonate nano-structured microparticles (NMP) were seeded with hMSCs co-cultured under osteogenic and osteoclastic conditions with THP-1 human monocytes up to 10 weeks to simulate and recapitulate bone regeneration. Scaffolds with appropriate pore size for cell infiltration, resulted in improved compressive strength, increased cell attachment and higher levels of expression of osteogenic markers and mineralization after adding the NMPs, compared to controls systems without these particles. These hybrid SF-based 3D-structures can provide improved scaffold designs for in vitro bone TE.

Simulation of Cortical and Cancellous Bone to Accelerate Tissue Regeneration.

Different tissues have complex anisotropic structures to support biological functions. Mimicking these complex structures in vitro remains a challenge in biomaterials designs in support of tissue regeneration. Here, inspired by different types of silk nanofibers, a composite materials strategy was pursued towards this challenge. A combination of fabrication methods was utilized to achieve separate control of amorphous and beta-sheet rich silk nanofibers in the same solution. Aqueous solutions containing these two structural types of silk nanofibers were then simultaneously treated with an electric field and with ethylene glycol diglycidyl ether (EGDE). Under these conditions, the beta-sheet rich silk nanofibers in the mixture responded to the electric field while the amorphous nanofibers were active in the crosslinking process with the EGDE. As a result, cryogels with anisotropic structures were prepared, including mimics for cortical- and cancellous-like bone biomaterials as a complex osteoinductive niche. In vitro studies revealed that mechanical cues of the cryogels induced osteodifferentiation of stem cells while the anisotropy inside the cryogels influenced immune reactions of macrophages. These bioactive cryogels also stimulated improved bone regeneration in vivo through modulation of inflammation, angiogenesis and osteogenesis responses, suggesting an effective strategy to develop bioactive matrices with complex anisotropic structures beneficial to tissue regeneration.