RESUMEN
The de novo design of self-assembling peptides has garnered significant attention in scientific research. While alpha-helical assemblies have been extensively studied, exploration of polyproline type II (PPII) helices, such as those found in collagen, remains relatively limited. In this study, we focused on understanding the sequence-structure relationship in hierarchical assemblies of collagen-like peptides, using defense collagen SP-A as a model. By dissecting the sequence derived from SP-A and synthesizing short collagen-like peptides, we successfully constructed a discrete bundle of hollow triple helices. Mutation studies pinpointed amino acid sequences, including hydrophobic and charged residues that are critical for oligomer formation. These insights guided the de novo design of collagen-like peptides, resulting in the formation of diverse quaternary structures, including discrete and heterogenous bundled oligomers, 2D nanosheets, and pH-responsive nanoribbons. Our study represents a significant advancement in the understanding and harnessing of collagen higher-order assemblies beyond the triple helix.
RESUMEN
Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.
Asunto(s)
Hidrogeles , Nanofibras , Péptidos , Nanofibras/química , Péptidos/química , Hidrogeles/química , Humanos , Materiales Biocompatibles/química , Materiales Biocompatibles/síntesis química , Anisotropía , AnimalesRESUMEN
Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.
RESUMEN
3D printing has become one of the primary fabrication strategies used in biomedical research. Recent efforts have focused on the 3D printing of hydrogels to create structures that better replicate the mechanical properties of biological tissues. These pose a unique challenge, as soft materials are difficult to pattern in three dimensions with high fidelity. Currently, a small number of biologically derived polymers that form hydrogels are frequently reused for 3D printing applications. Thus, there exists a need for novel hydrogels with desirable biological properties that can be used as 3D printable inks. In this work, the printability of multidomain peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations, is established. MDPs with different charge functionalities are optimized as distinct inks and are used to create complex 3D structures, including multi-MDP prints. Additionally, printed MDP constructs are used to demonstrate charge-dependent differences in cellular behavior in vitro. This work presents the first time that self-assembling peptides have been used to print layered structures with overhangs and internal porosity. Overall, MDPs are a promising new class of 3D printable inks that are uniquely peptide-based and rely solely on supramolecular mechanisms for assembly.
Asunto(s)
Hidrogeles , Nanofibras , Hidrogeles/química , Nanofibras/química , Porosidad , Impresión Tridimensional , Péptidos/química , Ingeniería de Tejidos/métodosRESUMEN
Adjuvants play a critical role in enhancing vaccine efficacy; however, there is a need to develop new immunomodulatory compounds to address emerging pathogens and to expand the use of immunotherapies. Multidomain peptides (MDPs) are materials composed of canonical amino acids that form injectable supramolecular hydrogels under physiological salt and pH conditions. MDP hydrogels are rapidly infiltrated by immune cells in vivo and have previously been shown to influence cytokine production. Therefore, we hypothesized that these immunostimulatory characteristics would allow MDPs to function as vaccine adjuvants. Herein, we demonstrate that loading antigen into MDP hydrogels does not interfere with their rheological properties and that positively charged MDPs can act as antigen depots, as demonstrated by their ability to release ovalbumin (OVA) over a period of 7-9 days in vivo. Mice vaccinated with MDP-adjuvanted antigen generated significantly higher IgG titers than mice treated with the unadjuvanted control, suggesting that these hydrogels potentiate humoral immunity. Interestingly, MDP hydrogels did not elicit a robust cellular immune response, as indicated by the lower production of IgG2c and smaller populations of tetramer-positive CD8+ T splenocytes compared to mice vaccinated alum-adjuvanted OVA. Together, the data suggest that MDP hydrogel adjuvants strongly bias the immune response towards humoral immunity while evoking a very limited cellular immune response. As a result, MDPs may have the potential to serve as adjuvants for applications that benefit exclusively from humoral immunity.
Asunto(s)
Hidrogeles , Inmunidad Humoral , Ratones , Animales , Ovalbúmina , Adyuvantes Inmunológicos/química , Antígenos , Péptidos , Adyuvantes Farmacéuticos , Inmunoglobulina G , Aminoácidos , CitocinasRESUMEN
Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.