Manipulating macrophage polarization with nanoparticles to control metastatic behavior in heterotypic breast cancer micro-tissues via exosome signaling.

This study aimed to investigate the effects of nanoparticles on macrophage polarization and their subsequent influence on post-tumorigenic behavior. Initially, seven different nanoparticles were applied to macrophages, and Zn-Ni-FeO (100 nm) and palladium nanoparticles (PdNPs, ∼25 nm) were found to induce M1-polarization in macrophages. A co-culture experiment was then conducted to examine the effects of macrophages on MCF-7 breast cancer micro-tissues. The M2-macrophages promoted tumor proliferation, while M1- and PdNPs-induced macrophages showed anti-tumor effects by suppressing cell proliferation. To reveal the mechanisms of effect, exosomes isolated from M1 (M1-Exo), M0 (M0-Exo), M2 (M2-Exo), and PdNPs-induced (PdNPs-Exo) macrophages were applied to the heterotypic tumor micro-tissues including MCF-7, human umbilical vein endothelial cells (HUVECs), and primary human dermal fibroblasts (phDFs). M2-Exo was seen to promote the migration of cancer cells and induce epithelial-mesenchymal transition (EMT), while M1-Exo suppressed these behaviors. PdNPs-Exo was effective in suppressing the aggressive nature of breast cancer cells similar to M1-Exo, moreover, the efficacy of 5-fluorouracil (5-FU) was increased in combination with PdNPs-Exo in both MCF-7 and heterotypic micro-tissues. In conclusion, PdNPs-Exo has potential anti-tumor effects, can be used as a combination therapy to enhance the efficacy of anti-cancer drugs, as well as innovative implants for breast cancer treatment.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Molecular docking, synthesis, anticancer activity, and metabolomics study of boronic acid ester-containing fingolimod derivatives.

In recent years, drugs that contain boronic acid groups, such as ixazomib (Ninlaro™) and bortezomib (Velcade™), have been used in the treatment of bone marrow cancer. The activity of compounds has been found to increase with the addition of boron atoms to the structure. In addition to these compounds, studies have found that fingolimod (FTY720) is more effective against breast cancer than cisplatin. Therefore, in this study, the first examples of boron-containing derivatives of fingolimod were designed and synthesized; in addition, their structures were confirmed by spectroscopic techniques. The synthesized boron-containing drug candidates were found to significantly inhibit cell proliferation and induce apoptosis-mediated cell death in HT-29 (colorectal cells), SaOs-2 (osteosarcoma cells), and U87-MG (glioblastoma cells). Moreover, we revealed that the anticancer effects of boron-containing fingolimod compounds were found to be significantly enhanced over boron-free control groups and, strikingly, over the widely used anticancer drug 5-fluorouracil. The metabolomic analysis confirmed that administration of the boron-containing drug candidates induces significant changes in the metabolite profiles in HT-29, SaOs-2, and U87-MG cells. Altogether, our results showed that boron-containing fingolimod compounds can be further examined to reveal their potential as anticancer drug candidates.

Development of plant-based biopolymer coatings for 3D cell culture: boron-silica-enriched quince seed mucilage nanocomposites.

Spheroid formation with spontaneous aggregation has captured interest in most cell culture studies due to its easy set-up and more reliable results. However, the economic and technical costs of the advanced systems and commercial ultra-low adhesive platforms have pushed researchers into pursuing alternatives. Nowadays, polymeric coatings, including poly-hydroxyethyl methacrylate and agar/agarose, are the commonly used polymers for non-adhesive plate fabrication, yet the costs and working solvent or heat-dependent preparation procedures maintain the need for the development of novel biomaterials. Here, we propose a greener and more economical approach for producing non-adherent surfaces and spheroid formation. For this, a plant waste-based biopolymer from quince fruit (Cydonia oblonga Miller, from Rosaceae family) seeds and boron-silica precursors were introduced. The unique water-holding capacity of quince seed mucilage (Q) was enriched with silanol and borate groups to form bioactive and hydrophilic nanocomposite overlays for spheroid studies. Moreover, 3D gel plates from the nanocomposite material were fabricated and tested in vitro as a proof-of-concept. The surface properties of coatings and the biochemical and mechanical properties of the nanocomposite materials were evaluated in-depth with techniques, and extra hydrophilic coatings were obtained. Three different cell lines were cultured on these nanocomposite surfaces, and spheroid formation with increased cellular viability was recorded on day 3 with a >200 µm spheroid size. Overall, Q-based nanocomposites are believed to be a fantastic alternative for non-adherent surface fabrication due to their low-cost, easy operation, and intrinsic hydration layer forming capacity with biocompatible nature in vitro.

Surgical method for critical sized cranial defects in rat cranium.

Cranial tissue models are a widely used model to show the bone repair and the regeneration ability of candidate biomaterials for tissue engineering purposes. Until now, efficacy studies of different biomaterials for calvarial defect bone regeneration have been reported, generally in small animal models. This paper offers a versatile, reliable, and reproducible surgical method for creating a critical-sized cranial defect in rats including critical steps and tried-and-tested tips. The method proposed here,•Shows a general procedure for in vivo cranial models.•Provide an insight to restore bone tissue repair that may be used in combination with several tissue engineering strategies•Is a crucial technique that may guide in vivo bone tissue engineering.

3D Printing of Extracellular Matrix-Based Multicomponent, All-Natural, Highly Elastic, and Functional Materials toward Vascular Tissue Engineering.

3D printing offers an exciting opportunity to fabricate biological constructs with specific geometries, clinically relevant sizes, and functions for biomedical applications. However, successful application of 3D printing is limited by the narrow range of printable and bio-instructive materials. Multicomponent hydrogel bioinks present unique opportunities to create bio-instructive materials able to display high structural fidelity and fulfill the mechanical and functional requirements for in situ tissue engineering. Herein, 3D printable and perfusable multicomponent hydrogel constructs with high elasticity, self-recovery properties, excellent hydrodynamic performance, and improved bioactivity are reported. The materials' design strategy integrates fast gelation kinetics of sodium alginate (Alg), in situ crosslinking of tyramine-modified hyaluronic acid (HAT), and temperature-dependent self-assembly and biological functions of decellularized aorta (dAECM). Using extrusion-based printing approach, the capability to print the multicomponent hydrogel bioinks with high precision into a well-defined vascular constructs able to withstand flow and repetitive cyclic compressive loading, is demonstrated. Both in vitro and pre-clinical models are used to show the pro-angiogenic and anti-inflammatory properties of the multicomponent vascular constructs. This study presents a strategy to create new bioink whose functional properties are greater than the sum of their components and with potential applications in vascular tissue engineering and regenerative medicine.

Bacterial Membrane Vesicles as Smart Drug Delivery and Carrier Systems: A New Nanosystems Tool for Current Anticancer and Antimicrobial Therapy.

Bacterial membrane vesicles (BMVs) are known to be critical communication tools in several pathophysiological processes between bacteria and host cells. Given this situation, BMVs for transporting and delivering exogenous therapeutic cargoes have been inspiring as promising platforms for developing smart drug delivery systems (SDDSs). In the first section of this review paper, starting with an introduction to pharmaceutical technology and nanotechnology, we delve into the design and classification of SDDSs. We discuss the characteristics of BMVs including their size, shape, charge, effective production and purification techniques, and the different methods used for cargo loading and drug encapsulation. We also shed light on the drug release mechanism, the design of BMVs as smart carriers, and recent remarkable findings on the potential of BMVs for anticancer and antimicrobial therapy. Furthermore, this review covers the safety of BMVs and the challenges that need to be overcome for clinical use. Finally, we discuss the recent advancements and prospects for BMVs as SDDSs and highlight their potential in revolutionizing the fields of nanomedicine and drug delivery. In conclusion, this review paper aims to provide a comprehensive overview of the state-of-the-art field of BMVs as SDDSs, encompassing their design, composition, fabrication, purification, and characterization, as well as the various strategies used for targeted delivery. Considering this information, the aim of this review is to provide researchers in the field with a comprehensive understanding of the current state of BMVs as SDDSs, enabling them to identify critical gaps and formulate new hypotheses to accelerate the progress of the field.

The relationship between bacterial outer membrane vesicles and halophilic adaptation.

Many cells are known to actively release nano-sized outer membrane vesicles (OMVs) that contain bioactive proteins, lipids, and nucleic acids into the extracellular environment. These vesicles have been associated with adaptation to environmental stress in other species, but their role in halophilic salt stress adaptation is not known. This study aimed to isolate and characterize the OMVs of Halomonas caseinilytica KB2 at various salt concentrations [6% (KB2-6), 12% (KB2-12), and 18% (KB2-18)] and to identify the patterns of adaptations to increasing salinity in its structure, protein composition, and expression. Also, a comparison with the composition of OMVs of E. coli, a mesophilic bacterium, was performed. Bioinformatics and statistical analysis were carried out to elucidate the underlying proteome differences that may exist as a result of increasing salinity. The results show that OMV production in H. caseinilytica KB2 is promoted by a decrease in salinity. OMVs also revealed possible structural and metabolic changes happening in the cells which led to the deduction that cells become more stable with increasing salt concentrations. Cell wall integrity, protein expression and folding are important. Although H. caseinilytica KB2 OMVs show cellular changes with changing salt concentration, they may not play a direct role in adaptation to changing salinity.

Tuning the Cell-Adhesive Properties of Two-Component Hybrid Hydrogels to Modulate Cancer Cell Behavior, Metastasis, and Death Pathways.

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material's adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

Decellularized Bone Extracellular Matrix-Coated Electrospun PBAT Microfibrous Membranes with Cell Instructive Ability and Improved Bone Tissue Forming Capacity.

Current approaches to develop bone tissue engineering scaffolds have some limitations and shortcomings. They mainly suffer from combining mechanical stability and bioactivity on the same platform. Synthetic polymers are able to produce mechanically stable sturctures with fibrous morphology when they are electrospun, however, they cannot exhibit bioactivity, which is crucial for tissue engineering and regenerative medicine. One current strategy to bring bioactivity in synthetic materials is to combine extracellular matrix (ECM)-sourced materials with biologically inert synthetic materials. ECM-sourced materials without any modifications are mechanically unstable; therefore, reinforcing them with mechanically stable platforms is indispensable. In order to overcome this bifacial problem, we have demonstrated that poly(butylene adipate-co-terephthalate) (PBAT) electrospun microfibrous membranes can be successfully modified with decellularized bone ECM to endow fibers with bioactive hydrogel and mimic natural micro-features of the native bone tissue. The developed structures have been shown to support osteogenesis, confirmed by histochemical staining and gene expression studies. Furthermore, ECM-coated PBAT fibers, when they were aligned, supplied an improved level of osteogenesis. The strategy demonstrated can be adapted to any other tissues, and the emerging microfibrous, mechanically stable, and bioactive materials can find implications in the specific fields of tissue engineering and regenerative medicine.

Omics technologies for high-throughput-screening of cell-biomaterial interactions.

Recent research effort in biomaterial development has largely focused on engineering bio-instructive materials to stimulate specific cell signaling. Assessing the biological performance of these materials using time-consuming and trial-and-error traditional low-throughput screening techniques remains a critical challenge in the field. In contrast, the use of increasingly sophisticated omics technologies to facilitate high-throughput screening of unbiased global understanding of cell-biomaterial interactions at gene, epigenetic, mRNA, protein, metabolite, and lipid levels holds great potential to predict the therapeutic outcome of biomaterials with specific properties. In this review, we highlight the potential use of omics technologies - namely transcriptomics, proteomics, metabolomics and lipidomics - in biomaterial design and deciphering of the fundamental cell behaviors (e.g., adhesion, migration, differentiation) in response to cell-biomaterial interactions. Moreover, the potential challenges and prospects of high-throughput analysis platforms are discussed rationally, providing an insight into the developing field and its use in biomaterials science.

Xenogenic Neural Stem Cell-Derived Extracellular Nanovesicles Modulate Human Mesenchymal Stem Cell Fate and Reconstruct Metabolomic Structure.

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration.

Bacterial membrane vesicle functions, laboratory methods, and applications.

Bacterial membrane vesicles (BMVs) are cupped-shaped structures formed by bacteria in response to environmental stress, genetic alteration, antibiotic exposure, and others. Due to the structural similarities shared with the producer organism, they can retain certain characteristics like stimulating immune responses. They are also able to carry molecules for long distances, without changes in the concentration and integrity of the molecule. Bacteria originally secrete membrane vesicles for gene transfer, excretion, cell to cell interaction, pathogenesis, and protection against phages. These functions are unique and have several innovative applications in the pharmaceutical industry that have attracted both scientific and commercial interest.This led to the development of efficient methods to artificially stimulate vesicle production, purification, and manipulation in the lab at nanoscales. Also, for specific applications, engineering methods to impart pathogen antigens against specific diseases or customization as cargo vehicles to deliver payloads to specific cells have been reported. Many applications of BMVs are in cancer drugs, vaccines, and adjuvant development with several candidates in clinical trials showing promising results. Despite this, applications in therapy and commercialization stay timid probably due to some challenges one of which is the poor understanding of biogenesis mechanisms. Nevertheless, so far, BMVs seem to be a reliable and cost-efficient technology with several therapeutic applications. Research toward characterizing more membrane vesicles, genetic engineering, and nanotechnology will enable the scope of applications to widen. This might include solutions to other currently faced medical and healthcare-related challenges.

Mechanically robust hybrid hydrogels of photo-crosslinkable gelatin and laminin-mimetic peptide amphiphiles for neural induction.

Self-assembling bio-instructive materials that can provide a biomimetic tissue microenvironment with the capability to regulate cellular behaviors represent an attractive platform in regenerative medicine. Herein, we develop a hybrid neuro-instructive hydrogel that combines the properties of a photo-crosslinkable gelatin methacrylate (GelMA) and self-assembling peptide amphiphiles (PAs) bearing a laminin-derived neuro-inductive epitope (PA-GSR). Electrostatic interaction and ultraviolet light crosslinking mechanisms were combined to create dual-crosslinked hybrid hydrogels with tunable stiffness. Spectroscopic, microscopic and theoretical techniques show that the cationic PA-GSR(+) electrostatically co-assembles with the negatively charged GelMA to create weak hydrogels with hierarchically ordered microstructures, which were further photo-crosslinked to create mechanically robust hydrogels. Dynamic oscillatory rheology and micromechanical testing show that photo-crosslinking of the co-assembled GelMA and PA-GSR(+) hydrogel results in robust hydrogels displaying improved stiffness. Gene expression analysis was used to show that GelMA/PA-GSR(+) hydrogels can induce human mesenchymal stem cells (hMSCs) into neural-lineage cells and supports neural-lineage specification of neuroblast-like cells (SH-SY5Y) in a growth-factor-free manner. Also, metabolomics analysis suggests that the hydrogel alters the metabolite profiles in the cells by affecting multiple molecular pathways. This work highlights a new approach for the design of PA-based hybrid hydrogels with robust mechanical properties and biological functionalities for nerve tissue regeneration.

Decellularized spinal cord meninges extracellular matrix hydrogel that supports neurogenic differentiation and vascular structure formation.

Decellularization of extracellular matrices offers an alternative source of regenerative biomaterials that preserve biochemical structure and matrix components of native tissues. In this study, decellularized bovine spinal cord meninges (dSCM)-derived extracellular matrix hydrogel (MeninGEL) is fabricated by employing a protocol that involves physical, chemical, and enzymatic processing of spinal meninges tissue and preserves the biochemical structure of meninges. The success of decellularization is characterized by measuring the contents of residual DNA, glycosaminoglycans, and hydroxyproline, while a proteomics analysis is applied to reveal the composition of MeninGEL. Frequency and temperature sweep rheometry show that dSCM forms self-supporting hydrogel at physiological temperature. The MeninGEL possesses excellent cytocompatibility. Moreover, it is evidenced with immuno/histochemistry and gene expression studies that the hydrogel induces growth-factor free differentiation of human mesenchymal stem cells into neural-lineage cells. Furthermore, MeninGEL instructs human umbilical vein endothelial cells to form vascular branching. With its innate bioactivity and low batch-to-batch variation property, the MeninGEL has the potential to be an off-the-shelf product in nerve tissue regeneration and restoration.

De Novo Design of Functional Coassembling Organic-Inorganic Hydrogels for Hierarchical Mineralization and Neovascularization.

Synthetic nanostructured materials incorporating both organic and inorganic components offer a unique, powerful, and versatile class of materials for widespread applications due to the distinct, yet complementary, nature of the intrinsic properties of the different constituents. We report a supramolecular system based on synthetic nanoclay (Laponite, Lap) and peptide amphiphiles (PAs, PAH3) rationally designed to coassemble into nanostructured hydrogels with high structural integrity and a spectrum of bioactivities. Spectroscopic and scattering techniques and molecular dynamic simulation approaches were harnessed to confirm that PAH3 nanofibers electrostatically adsorbed and conformed to the surface of Lap nanodisks. Electron and atomic force microscopies also confirmed an increase in diameter and surface area of PAH3 nanofibers after coassembly with Lap. Dynamic oscillatory rheology revealed that the coassembled PAH3-Lap hydrogels displayed high stiffness and robust self-healing behavior while gas adsorption analysis confirmed a hierarchical and heterogeneous porosity. Furthermore, this distinctive structure within the three-dimensional (3D) matrix provided spatial confinement for the nucleation and hierarchical organization of high-aspect ratio hydroxyapatite nanorods into well-defined spherical clusters within the 3D matrix. Applicability of the organic-inorganic PAH3-Lap hydrogels was assessed in vitro using human bone marrow-derived stromal cells (hBMSCs) and ex vivo using a chick chorioallantoic membrane (CAM) assay. The results demonstrated that the organic-inorganic PAH3-Lap hydrogels promote human skeletal cell proliferation and, upon mineralization, integrate with the CAM, are infiltrated by blood vessels, stimulate extracellular matrix production, and facilitate extensive mineral deposition relative to the controls.

Human cardiomyocyte-derived exosomes induce cardiac gene expressions in mesenchymal stromal cells within 3D hyaluronic acid hydrogels and in dose-dependent manner.

Accomplishing a reliable lineage-specific differentiation of stem cells is vital in tissue engineering applications, however, this need remained unmet. Extracellular nanovesicles (particularly exosomes) have previously been shown to have this potential owing to their rich biochemical content including proteins, nucleic acids and metabolites. In this work, the potential of human cardiomyocytes-derived exosomes to induce in vitro cardiac gene expressions in human mesenchymal stem cells (hMSCs) was evaluated. Cardiac exosomes (CExo) were integrated with hyaluronic acid (HA) hydrogel, which was functionalized with tyramine (HA-Tyr) to enable the development of 3D (three dimensional), robust and bioactive hybrid cell culture construct through oxidative coupling. In HA-Tyr/CExo 3D hybrid hydrogels, hMSCs exhibited good viability and proliferation behaviours. Real time quantitative polymerase chain reaction (RT-qPCR) results demonstrated that cells incubated within HA-Tyr/CExo expressed early cardiac progenitor cell markers (GATA4, Nkx2.5 and Tbx5), but not cTnT, which is expressed in the late stages of cardiac differentiation and development. The expressions of cardiac genes were remarkably increased with increasing CExo concentration, signifying a dose-dependent induction of hMSCs. This report, to some extent, explains the potential of tissue-specific exosomes to induce lineage-specific differentiation. However, the strategy requires further mechanistic explanations so that it can be utilized in translational medicine.

Ultrasonics-Assisted Effective Isolation and Characterization of Exosomes from Whole Organs.

Exosomes, natural and nanovesicular structures surrounded by a lipid membrane, tend to be secreted toward extracellular environments by almost all cell types. Late studies have shown them to be effective in several complex biological processes like cancer development and metastasis, immune system regulation, cellular signal transduction, stem cell differentiation, and regeneration of damaged tissues. Although there are many studies dealing with the role of exosomes in the aforementioned fields, the mechanisms remained largely unknown. There is therefore a need for further study on exosome isolation from different sources. While researchers mostly use serum, plasma, urine, and cell culture media as a source for exosome isolation, there are no studies dealing with direct isolation of exosomes from whole organs in literature. In this study, we propose a protocol for effective isolation of exosomes from whole organs. Mouse brain, heart, and liver were chosen as the sources of exosomes in this study. Isolated exosomes were successfully characterized with BCA test, western blot, transmission electron microscopy and ELISA.

Synthesis of Silica-Based Boron-Incorporated Collagen/Human Hair Keratin Hybrid Cryogels with the Potential Bone Formation Capability.

Tissue engineering and regenerative medicine have evolved into a different concept, the so-called clinical tissue engineering. Within this context, the synthesis of next-generation inorganic-organic hybrid constructs without the use of chemical crosslinkers emerges with a great potential for treating bone defects. Here, we propose a sophisticated approach for synthesizing cost-effective boron (B)- and silicon (Si)-incorporated collagen/hair keratin (B-Si-Col-HK) cryogels with the help of sol-gel reactions. In this approach, collagen and hair keratin were engaged with a B-Si network using tetraethyl orthosilicate as a silica precursor, and the obtained cryogels were characterized in depth with attenuated total reflectance-Fourier transform infrared spectroscopy, solid-state NMR, X-ray diffraction, thermogravimetric analysis, porosity and swelling tests, Brunauer-Emmett-Teller and Barrett-Joyner-Halenda analyses, frequency sweep and temperature-dependent rheology, contact angle analysis, micromechanical tests, and scanning electron microscopy with energy dispersive X-ray analysis. In addition, the cell survival and osteogenic features of the cryogels were evaluated by the MTS test, live/dead assay, immuno/histochemistry, and quantitative real-time polymerase chain reaction analyses. We conclude that the B-Si-networked Col-HK cryogels having good mechanical durability and osteoinductive features would have the potential bone formation capability.

Multicomponent hydrogels for the formation of vascularized bone-like constructs in vitro.

The native extracellular matrix (ECM) is a complex gel-like system with a broad range of structural features and biomolecular signals. Hydrogel platforms that can recapitulate the complexity and signaling properties of this ECM would have enormous impact in fields ranging from tissue engineering to drug discovery. Here, we report on the design, synthesis, and proof-of-concept validation of a microporous and nanofibrous hydrogel exhibiting multiple bioactive epitopes designed to recreate key features of the bone ECM. The material platform integrates self-assembly with orthogonal enzymatic cross-linking to create a supramolecular environment comprising hyaluronic acid modified with tyramine (HA-Tyr) and peptides amphiphiles (PAs) designed to promote cell adhesion (RGDS-PA), osteogenesis (Osteo-PA), and angiogenesis (Angio-PA). Through individual and co-cultures of human adipose derived mesenchymal stem cells (hAMSCs) and human umbilical vascular endothelial cells (HUVECs), we confirmed the capacity of the HA-Tyr/RGDS-PA/Osteo-PA/Angio-PA hydrogel to promote cell adhesion as well as osteogenic and angiogenic differentiation in both 2D and 3D setups. Furthermore, using immunofluorescent staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we demonstrated co-differentiation and organization of hAMSCs and HUVECs into 3D aggregates resembling vascularized bone-like constructs. STATEMENT OF SIGNIFICANCE: This body of work presents a new approach to develop more complex, yet functional, in vitro environments for cell culture while enabling a high level of control, tuneability, and reproducibility. The multicomponent self-assembling bioactive 2D and 3D hydrogels with nanofibrous architecture designed to recreate key molecular and macromolecular features of the native bone ECM and promote both osteogenesis and angiogenesis. The materials induce endothelial cells towards large vascular lumens and MSCs into bone cells on/within the same platform and form vascularized-bone like construct in vitro. This strategy looks encouraging for lifelike bone tissue engineering in vitro and bone tissue regeneration in vivo.