Despite bone's impressive ability to heal after traumatic injuries and fractures, a significant need still exists for developing strategies to promote healing of nonunion defects. To address this issue, we developed collagen-based hydrogels containing two-dimensional nanosilicates. Nanosilicates are ultrathin nanomaterials with a high degree of anisotropy and functionality that results in enhanced surface interactions with biological entities compared to their respective three-dimensional counterparts. The addition of nanosilicates resulted in a 4-fold increase in compressive modulus along with an increase in pore size compared to collagen-based hydrogels. In vitro evaluation indicated that the nanocomposite hydrogels are capable of promoting osteogenesis in the absence of any osteoinductive factors. A 3-fold increase in alkaline phosphatase activity and a 4-fold increase in the formation of a mineralized matrix were observed with the addition of the nanosilicates to the collagen-based hydrogels. Overall, these results demonstrate the multiple functions of nanosilicates conducive to the regeneration of bone in nonunion defects, including increased network stiffness and porosity, injectability, and enhanced mineralized matrix formation in a growth-factor-free microenvironment.

Although hydrogels are able to mimic native tissue microenvironments, their utility for biomedical applications is severely hampered due to limited mechanical stiffness and low toughness. Despite recent progress in designing stiff and tough hydrogels, it is still challenging to achieve a cell-friendly, high modulus construct. Here, we report a highly efficient method to reinforce collagen-based hydrogels using extremely low concentrations of a nanoparticulate-reinforcing agent that acts as a cross-link epicenter. Extraordinarily, the addition of these nanoparticles at a 10 000-fold lower concentration relative to polymer resulted in a more than 10-fold increase in mechanical stiffness and a 20-fold increase in toughness. We attribute the high stiffness of the nanocomposite network to the chemical functionality of the nanoparticles, which enabled the cross-linking of multiple polymeric chains to the nanoparticle surface. The mechanical stiffness of the nanoengineered hydrogel can be tailored between 0.2 and 200 kPa simply by manipulating the size of the nanoparticles (4, 8, and 12 nm), as well as the concentrations of the nanoparticles and polymer. Moreover, cells can be easily encapsulated within the nanoparticulate-reinforced hydrogel network, showing high viability. In addition, encapsulated cells were able to sense and respond to matrix stiffness. Overall, these results demonstrate a facile approach to modulate the mechanical stiffness of collagen-based hydrogels and may have broad utility for various biomedical applications, including use as tissue-engineered scaffolds and cell/protein delivery vehicles.

SignificanceWe demonstrate the use of next-generation sequencing technology (RNA-seq) to understand the effect of a two-dimensional nanomaterial on human stem cells at the whole-transcriptome level. Our results identify more than 4,000 genes that are significantly affected, and several biophysical and biochemical pathways are triggered by nanoparticle treatment. We expect that this systematic approach to understand widespread changes in gene expression due to nanomaterial exposure is key to develop new bioactive materials for biomedical applications.

Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell–cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ~100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.

Injectable hydrogels are investigated for cell encapsulation and delivery as they can shield cells from high shear forces. One of the approaches to obtain injectable hydrogels is to reinforce polymeric networks with high aspect ratio nanoparticles such as two-dimensional (2D) nanomaterials. 2D nanomaterials are an emerging class of ultrathin materials with a high degree of anisotropy and they strongly interact with polymers resulting in the formation of shear-thinning hydrogels. Here, we present 2D nanosilicate reinforced kappa-carrageenan (κCA) hydrogels for cellular delivery. κCA is a natural polysaccharide that resembles native glycosaminoglycans and can form brittle hydrogels via ionic crosslinking. The chemical modification of κCA with photocrosslinkable methacrylate groups renders the formation of a covalently crosslinked network (MκCA). Reinforcing the MκCA with 2D nanosilicates results in shear-thinning characteristics, and enhanced mechanical stiffness, elastomeric properties, and physiological stability. The shear-thinning characteristics of nanocomposite hydrogels are investigated for human mesenchymal stem cell (hMSC) delivery. The hMSCs showed high cell viability after injection and encapsulated cells showed a circular morphology. The proposed shear-thinning nanoengineered hydrogels can be used for cell delivery for cartilage tissue regeneration and 3D bioprinting.