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Biomaterials

Biomaterials - Poster Session D

Poster B4 - Macroscopic Alignment of Biomimetic Self-Assembling Peptide Nanofibers

Friday, October 25, 2024
3:30 PM – 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  Materials mimicking the structure of the extracellular matrix (ECM) have been shown to facilitate cell proliferation, differentiation, and influence cell behavior. Synthetic peptide-based materials imitate the fibrous nature of the ECM, providing a scaffold for cellular growth. Nanofibrillar alignment of the peptide nanofiber K2(SL)6K2, or K2, has shown promise as a potential base for bottom-up tissue engineering. However, very little is known about the effect of biomimetic sequences on the desired nanoscale organization and how they influence cellular behavior on aligned hydrogels. The biomimetic sequence IKVAVG, derived from the extracellular matrix protein Laminin α1, was previously determined to enhance neurite outgrowth and regeneration after an injury. Here, we demonstrate the ability to produce anisotropic, nanofibrous hydrogels using a novel biomimetic self-assembling peptide, IKVAVGK2(SL)6K2, utilizing solid phase peptide synthesis, polarized light microscopy, and scanning electron microscopy.

Materials and

Methods:  Peptides were synthesized by solid phase peptide synthesis using a Rink-Amide resin. Unaligned gel formation was achieved by dissolving pure peptide into a 1X HBSS salt solution to create a 3 wt % hydrogel. Secondary structure analysis was achieved by ATR-FTIR using a Nicolet iS20 FT/IR spectrometer (Thermo Scientific, Waltham, MA), and Circular Dichroism using a J-810 spectropolarimeter (Jasco, Tokyo, Japan). Rheological and self-healing properties were determined through oscillatory rheology using an AR-G2 rheometer (TA Instruments, New Castle, DE). Aligned gels were created by first dissolving pure peptide into Milli-Q water and allowed to sit overnight to create a 3 wt % ink. 10 μL of the ink was pipetted into phosphate buffer solutions of various concentrations (1X PB, 3X PB, 5X PB, 7X, PB) out of a 0.1 – 10 μL pipette tip to form aligned noodles. Polarized light microscopy was used to determine anisotropy using an Eclipse E400 (Nikon Company, Tokyo, Japan) and pictures were taken on a Nikon D7000 (Nikon Company, Tokyo, Japan) using fixed exposure time and ISO. Noodles were placed on glass microscope slides to be imaged. Scanning electron microscopy was performed using a Helios NanoLab 660 Scanning Electron Microscope (FEI Company, Hillsboro, OR). Samples were prepared by ethanol dehydration (30%, 50%, 60%, 70%, 80%, 90%, 2 x 100%) followed by critical point dried using a Leica EM CPD300 (Leica Biosystems, Deer Park, IL). Dried samples were then sputter coated with 10 nm of gold using Denton Desk V Sputter System (Denton Vacuum, Moorestown, NJ)

Results, Conclusions, and Discussions: FTIR and CD results demonstrate the formation of anti-parallel β-sheet nanofibers. Rheological data shows that IKVAVG is a much weaker gel in comparison to K2, however, IKVAVG still shows the desired self-healing properties expected of self-assembling peptide hydrogels. Visualization of IKVAVG under polarized light microscopy demonstrated the desired anisotropic properties for noodles extruded into 3X, 5X, and 7X phosphate salt baths. 1X noodles showed no birefringent properties, indicating lack of anisotropy and alignment. This differs from K2 aligned noodles as 1X showed strong anisotropic properties. Scanning electron microscopy supported the results from polarized light microscopy. Images from 3X, 5X, and 7X noodles appeared aligned, especially in comparison to the unaligned 1X noodles.

This study suggests that alignment of biomimetic peptides is achievable although under different conditions than the original K2 alignment. In Vitro studies evaluating how nanofibrillar alignment affects cellular behavior are currently ongoing and seek to understand the utility of aligned biomimetic peptide hydrogels for tissue engineering. Future work includes assessing the 3D printability of IKVAVG hydrogels and In Vitro studies on 3D printed IKVAVG gel scaffolds.

Acknowledgements (Optional):