Hierarchical Assembly by Peptide Amphiphiles and Their Use in Cartilage Repair

Lewis, Jacob Alexander

doi:https://doi.org/10.21985/n2-yf3c-4z77

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Hierarchical Assembly by Peptide Amphiphiles and Their Use in Cartilage Repair

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In nature, materials with complex architectures are formed through hierarchical self-assembly. Therefore, the study and design of hierarchically assembling materials is important in producing materials that mimic biological structures and is a key challenge in biomaterials science and engineering. In articular cartilage, hierarchical assembly of extracellular matrix (ECM) components provides mechanical resilience to this musculoskeletal tissue. However, following injury or degradation of adult human cartilage, this critical tissue in joints lacks the intrinsic ability to repair the damaged ECM and regenerate functional tissue. Therefore osteoarthritis, or cartilage degradation, is a leading cause of morbidity, affecting a quarter of a billion people worldwide. This work describes the development of several hierarchically assembling peptide amphiphile (PA) systems to study how these structures form and to apply bioactive superstructures for regeneration of cartilage tissue. PA molecules, comprising a peptide sequence conjugated to a hydrophobic alkyl tail, self-assemble in aqueous media to form filament networks that mimic the topology of ECM, making PAs a valuable tool in producing scaffolds for regeneration. To study how hierarchical assemblies form in peptide systems, the first project explored the formation of supramolecular superstructures by oppositely charged short PAs. In previous work on PA molecules covalently conjugated to DNA segments (DNA-PA), co-assembled PA molecules demonstrated formation of superstructures containing many nanoscale filaments formed due to dynamic exchange of monomers in the supramolecular assemblies. Superstructure formation was triggered by mixing of supramolecular structures containing complementary DNA segments followed by dynamic exchange of molecules to enhance Watson-Crick pairing in the bundled regions. While this assembly process relied on pairing of complementary sequences to overcome strong cohesive interactions among molecules within the assemblies, it remained unknown if PA superstructures could form from simpler building blocks. Upon mixing aggregates of oppositely charged short PA molecules, the system formed filament bundles over the course of several days as a result of molecular redistribution among the oppositely charged assemblies, producing mixed charge primary assemblies that could form hierarchical bundles. Comparing the short PA supramolecular assemblies to oppositely charged PA mixtures with stronger or with weaker intermolecular cohesion showed that more cohesive structures had a decreased tendency to redistribute and form superstructures, while less cohesive structures were better able to redistribute and quickly formed robust superstructures. To better understand the role that charged groups play in determining how supramolecular systems assemble, the second study focused on a nonionic PA molecule with a decaethylene glycol group replacing the charged residues like glutamic acid and lysine needed for solubility in aqueous conditions. These nonionic PA molecules formed high aspect ratio filaments, but unlike charged PA molecules their assembly was largely independent of buffer pH. Mixing charged and uncharged PA molecules revealed that if only a minority of the PA molecules within the assemblies were charged, the co-assembled filaments had similar morphologies to the completely charged system. X-ray scattering and spectroscopic analysis showed mixing charged and uncharged PA molecules resulted in assemblies with high intermolecular order by decreasing repulsion among assemblies. Previous work indicated that nonionic hydrophilic functional groups in nanomaterials may improve osteogenic differentiation of progenitor cells. Therefore, the study concluded with in vitro experiments that nonionic PA filaments increased osteogenesis of human mesenchymal stem cells (hMSCs), suggesting tuning filament charge may be an important tool for improving musculoskeletal regeneration. The third project aimed to develop PA filaments for cartilage repair and to study the way in which their hierarchical assembly affects their bioactivity. Deamidation of hydrogel forming PA sequences displaying a peptide epitope programmed to bind the chondrogenic cytokine transforming growth factor β-1 was shown to improve retention of the growth factor and chondrogenesis of encapsulated cells. Studying the self-assembly of these systems revealed that without deamidation, PA filaments formed bundled structures which likely masked the growth factor binding peptide sequence. This work established the deamidated sequence as the preferred PA design for chondrogenic applications, while highlighting the implications of superstructure formation on PA bioactivity. In the final study, these improved chondrogenic PA filaments were used for cartilage regeneration in a sheep model. To produce a more resilient material that could withstand the mechanical environment in a large-animal joint, the supramolecular PA assemblies were mixed with particles of the covalent biopolymer hyaluronic acid (HA). Combining the two components produced PA filament bundles with regular order, which was attributed to swelling by the HA component packing the PA filaments into a confined volume. The supramolecular-covalent hybrid was found to support chondrogenesis by encapsulated hMSCs in vitro. After implantation in cartilage defects in sheep stifles, the material was well retained and improved the macroscopic appearance of healing cartilage in both the load-bearing femoral condyle and in the less mechanically active trochlear groove at early times, indicating the material had the physical properties needed to remain present in defects long enough to affect healing. After 12 weeks, condyle defects continued to show improved macroscopic appearance due to treatment, while histological staining of trochlear groove defects showed improved regeneration of hyaline-like tissue, demonstrating the PA/HA hybrid superstructures had the biological efficacy to direct cartilage repair. While treated condyle defects may have developed hyaline-like tissue if evaluated at later times, further improvements in the PA/HA hybrids mechanical properties may still be necessary for comparable healing of the high load-bearing condyle defects to the observed regeneration in the less mechanically active trochlear groove. Together, these studies help explain how superstructures form in natural systems and how their hierarchical assembly can affect their biological performance. Successful regeneration of hyaline-like tissue in the clinically relevant sheep model suggests that through further optimization of hybrid formulation and of bioactive signal presentation, hybrid PA superstructures have the potential to immensely improve the lives of many millions of patients suffering from damaged cartilage.

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