The presented research highlights a novel approach using fmoc-protected peptide hydrogels for the encapsulation and stretching of mesenchymal stem cells (MSCs). This study utilized a custom mechanical stretching device with a PDMS chamber to stretch human MSCs encapsulated in Fmoc hydrogels. The study assessed the influence of various solvents on the self-assembly and mechanical properties of the hydrogels, and MSC viability and alignment. Particularly we focused on fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) prepared in dimethyl sulfoxide (DMSO), hexafluoro-2-propanol (HFP), and deionized water (DiH₂O). Through molecular self-assembly of the peptide sequence into β-sheets connected by π-π aromatic stacking of F-F groups, the peptide hydrogel was found to form a stiff, hydrated gel with nanofiber morphology and a compressive modulus ranging from 174 to 277 Pa. Therefore, this hydrogel can mimic certain critical features of the extracellular matrix and collagen. Evaluations of MSCs cultured on the peptide hydrogels, including viability, morphology, and alignment assessments using various staining techniques, demonstrated that 3D-cultured MSCs in Fmoc-FF/HFP and Fmoc-FF/DMSO, followed by mechanical stretching, exhibited elongated morphology with distinct microfilament fibers compared to the control cells, which maintained a round and spherical F-actin shape. Notably, peptide gels with a concentration of 5 mM maintained 100 % MSC viability. The findings indicate the potential and specific conditions for successful cell encapsulation and alignment within peptide hydrogels, highlighting a promising tissue engineering platform through the encapsulation of MSCs in peptide nanofibers followed by a stretching process. By enhancing our understanding of MSC-peptide hydrogel interactions, this research contributes to the development of biomaterials tailored for regenerative medicine.

There is a pressing need for new cell-laden, printable bioinks to mimic stiffer tissues such as cartilage, fibrotic tissue and bone. PEGDA monomers are bioinks that crosslink with light to form a viscoelastic solid, however, they lack cell adhesion properties. Here, we utilized a hybrid bioink by combining self-assembled peptide nanofibers with PEGDA for 3D printing lumens. Adult human dermal fibroblast (aHDF) cells were first seeded in peptide-laden in 2D and 3D layers and cell behavior were studied. The cell's morphology remained spheres when they were infused in the 3D hydrogel and highly aligned with 2D overlay hydrogels. HDF cells did not adhere to unmodified PEGDA lumens, however, they successfully attached and proliferated on PEGDA/peptide lumens. Moreover, HDF cells seeded on the hybrid PEGDA/peptide lumens displayed a distinct spread F-actin morphology. The results showcase the potential of peptide hydrogels in facilitating interaction of anchorage dependent cells with PEGDA structures.

Mesenchymal stem cell (MSC) osteogenic differentiation requires scaffolds to support multiple stages of growth and differentiation signals. Fluorenyl-9-methoxycarbonyl diphenylalanine (Fmoc-FF) peptides self-assemble to create 3D nanofibers. Here, we cultured MSC in 2D and 3D Fmoc-FF layers to support their osteogenic differentiation. The stiffness of the hydrogels was tunable between 100 and 10,000 Pa which allows precise modulation of the cellular microenvironment. Scaffold stiffness impacted cell viability which softer scaffolds (100 Pa) favored higher viability. MSC formed spheroids in 3D hydrogel and showed spread morphology in 2D overlayers. Our results demonstrate that the Fmoc-FF 3D cultures significantly enhanced osteogenic differentiation, as evidenced by increased calcium deposition, elevated phosphatase activity, and the secretion of osteocalcin. We propose that the peptides provide integrin-binding sites that activate a cytoplasmic feedback loop essential for differentiation. These findings suggest that self-assembled Fmoc-FF peptide hydrogels, is a promising platform for bone tissue engineering applications.

Enzyme-induced self-assembly (EISA) is a recently developed nanotechnology technique in which small molecules are induced by cellular enzymes self-assembling into nanostructures inside cancer cells. This technique can boost the efficacy of chemotherapy drugs by avoiding drug efflux, inhibiting the cells’ DNA repair mechanisms, and targeting the mitochondria. In this work, we study the self-assembly of a short peptide and its fluorescence analogue induced by Eyes absent (EYA) tyrosine phosphatases to boost the efficacy of doxorubicin (DOX) therapy in drug-resistant types of breast cancer cells, MDA-MB-231 and MCF-7. The peptides Fmoc–FF–YP and NBD–FF–YP were synthesized with the solid-phase peptide synthesis (SPPS) method and analyzed with HPLC and MALDI-TOF. Dynamic light scattering was used to determine the size distribution of peptides exposed to the EYA enzyme in vitro. The presence of EYA enzymes in breast cancer cells was confirmed using the western blotting assay. The intracellular location of the peptide self-assembly was studied by imaging fluorescence NBD-tagged peptides. The efficacy of the peptide alone and with DOX was determined against MCF-7 and MDA-MB-231 using MTT and LIVE-DEAD assays. Nucleus and cytoplasm F-actin (Phalloidin) staining was used to determine cell morphology changes in response to the combination therapy of peptides/DOX. At an optimal concentration, the peptides are not toxic to the cells; however, they boost the efficacy of DOX against drug-resistant breast cancer cells. We used state-of-the-art computer-aided techniques to predict the molecular structure of peptides and their interactions with EYA. This study demonstrates an approach for incorporating non-cytotoxic components into DOX combination therapy, thereby avoiding increased systemic burden or adverse effects.