Biomaterials
Victor Nguyen (he/him/his)
Undergraduate Researcher
University of Oklahoma Stephenson School of Biomedical Engineering
Oklahoma City, Oklahoma, United States
Handan Acar
Assistant Professor
University of Oklahoma Stephenson School of Biomedical Engineering, United States
The development of artificial scaffolds is a vital component for advancements in tissue engineering. Their consistent and modifiable properties provide reproducibility and controlled variability for tissue engineering research. A significant challenge in scaffold fabrication lies in effectively mimicking native tissue properties. While self-assembling peptides are used as scaffold materials composed of amino acids, the adjustment of their properties, (e.g., mechanical properties, side chain epitopes) specific to different tissues remains a challenge.
To overcome these challenges, we utilized our “co-assembly of oppositely charged peptides” (CoOP) strategy to identify peptide sequences that self-assemble into fibrillar structures and produce easily modifiable scaffolds. The CoOP strategy is a molecular framework that combines two oppositely charged hexapeptides which consist of three domains: charged, diphenylalanine (FF), and substitution domains ([XX]). The CoOP strategy encourages the fibrillar assembly of peptides and allows for the functionalization of peptide motifs for specific interactions, enabling the study of the structure-property relationship of self-assembling peptide structures (Figure 1A).1
To identify the most suitable CoOP sequence for tissue engineering applications, we studied the assembly kinetics and mechanical properties of various sequences in two-dimensional structures. Furthermore, we showed the modification of the identified sequence with RGD, a common cell binding motif, and demonstrated the effects on the differentiation of C2C12 mouse myoblast cells by comparing their myotube formation
CoOP assembly
We employed the CoOP strategy by combining oppositely charged peptides and substituting the amino acids in the substitution domain with divaline ([VV]), dileucine ([LL]), and dinorleucine ([nLnL]) (Figure 1B). Peptides were modified with RGD on C (cRGD) and N (nRGD) termini individually in a 1:10 ratio to positive peptide.
Assembly kinetics
To assess the assembly kinetics of CoOP scaffolds, we used thioflavin T (ThT), which fluoresces as it selectively binds to amyloid fibers, enabling the quantification of amyloid fiber formation in CoOPs with a BioTek Neo2SM microplate reader.
Differentiation
Cellular studies were done on C2C12 mouse myoblast cells, which differentiate and form myotubes for muscle repair.2 To identify myoblast differentiation into myotubes on CoOP scaffolds, we measured myosin heavy chain (MHC) expression through immunocytochemistry, imaged cells with Keyence bz-x710 microscope, and analyzed images with ImageJ/Fiji.
Ongoing studies
Ongoing studies are on measuring cytotoxicity of scaffolds on cells through Calcien AM and Ethidium Homodimer staining. Additionally, we are studying the mechanical properties of CoOP scaffolds through ViewSizer 3000 Particle Tracking Analyzer (Horiba).
Results
We compared the fibrilization of several CoOPs (Figure 1B) through ThT fluorescence intensity measurements (Figure 2A). [nLnL] showed greater fibril formation than [VV] and [LL], as shown by an increase in fluorescence intensity at 120 minutes by 2812 ± 690% and 18922 ± 831%, respectively (Figure 2A). As [nLnL] displayed superior fibrillar formation, further studies were done with the [nLnL] CoOP. Addition of RGD to [nLnL] reduced fibril formation by 25 ± 1% at 120 minutes (Figure 2A) but did not affect assembly time (Figure 2B) compared to [nLnL] scaffolds without RGD.
Differentiation of C2C12 myoblasts into myotubes on [nLnL] scaffolds was quantified by measuring MHC expression (Figure 3A). Differentiation was greater on 1 mM scaffolds compared to the control group and 0.5 mM scaffolds by 114 ± 19% and 171 ± 22%, respectively (Figure 3B). There was no significant difference in differentiation between the control group and 0.5 mM scaffolds. The addition of nRGD in 1 mM increased differentiation compared to [nLnL] without RGD by 39 ± 18% (Figure 3B).
Conclusion
Peptide-based scaffolds have shown properties that are suitable for tissue engineering applications. In this study, we employed the CoOP strategy to identify peptides that self-assemble into fibers that are suitable for tissue engineering applications. Furthermore, we demonstrated the ease of side chain modification by mixing peptides synthesized with RGD for enhanced cell binding. After measuring fibril structure assembly and cell differentiation in various CoOPs, we identified [nLnL] peptide as a promising candidate for tissue engineering applications, such as wound healing and repairing lost tissue function, to validate our CoOP method for use in peptide-based scaffold fabrication.
Discussion
Future directions for this research include studying the effects of CoOP scaffolds in three-dimensional structures to better emulate complex in-vivo conditions. Additionally, evaluating the stability of CoOP scaffolds against enzymes that may impede the assembly process could offer valuable insights into the interactions between CoOP scaffolds and native environments.
(1) Hamsici, S.; White, A. D.; Acar, H. Peptide Framework for Screening the Effects of Amino Acids on Assembly. Sci. Adv. 8 (3), eabj0305. https://doi.org/10.1126/sciadv.abj0305.
(2) Langlois, S.; Cowan, K. N. Regulation of Skeletal Muscle Myoblast Differentiation and Proliferation by Pannexins. Adv. Exp. Med. Biol. 2017, 925, 57–73. https://doi.org/10.1007/5584_2016_53.