(D-125) Controlling the Presentation of Growth Factor-Mimetic Peptides in 3D-Printed Scaffolds

Osteoarthritis is a debilitating disease characterized by progressive degeneration of the osteochondral (bone-cartilage) tissue interface in articulating joints. This project aims to develop biomaterial scaffolds to support functional osteochondral tissue regeneration. Growth factors (GFs), such as transforming growth factor beta-1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2), are commonly used to enhance osteochondral tissue repair and regeneration.[1] However, native GF delivery is limited by low GF stability, short half-lives, and poor  control of spatial and temporal presentation. GF-mimetic peptides are a promising alternative to native GFs because they are more stable, less costly, and can be modified with bioorthogonal groups for well-defined spatial and temporal delivery.[3] Our lab has successfully showed multi-peptide organization within  3D-printed polycaprolactone (PCL)-based scaffolds to direct local cell response.[4, 5] Here, we have used this platform to control the temporal presentation of a TGF-β1-mimetic peptide LIANAK, which has been shown to enhance chondrogenesis.[6] This approach will enable us to deliver bioactive cues at user-defined times during culture to guide cell behavior and matrix formation. 

he TGF-β1-mimetic peptide DBCO-CGGGLIANAKK(Cy3) (TGFpep) was synthesized using Fmoc solid phase peptide synthesis. The Cyanine 3 (Cy3) fluorophore was coupled to the epsilon amine on resin to detect peptide presentation. Dibenzocyclooctyne (DBCO) was added to the C-terminus to selectively react with azide on the scaffold surface. DBCO-maleimide was reacted with the thiol sidechain on the cysteine after the peptide was cleaved from the resin and purified using high performance liquid chromatography (HPLC). Peptide mass was verified using mass spectrometry. PCL (14 kDa) was modified with amine-poly(ethylene glycol)-azide to generate PCL-azide conjugates and confirmed using nuclear magnetic resonance (NMR) spectroscopy. PCL-azide scaffolds were 3Dprinted by co-dissolving 20 mg/mL PCL azide with 370 mg/mL unmodified PCL (80 kDa) in a  highly volatile solvent 1,1,1,3,3,3-hexafluoroisopropanol(HFIP). The resulting PCL-azide inks were solvent-cast 3D printed into scaffolds (14 layers, 260 µm programmed filament spacing) using 70psi print pressure and line speeds of 0.4mm/s for the first layer and 0.2mm/s for subsequent layers. PCL only and PCL-azide scaffolds were pretreated with a blocking solution containing 0.2% TWEEN and 0.2% Triton-X in phosphate-buffered saline (PBS) and washed with ultrapure water. DBCO-Cy3-peptide is diluted (1:100)  with PBS and 0.5% Bovine serum albumin (BSA) in PBS. The scaffolds were submerged in the diluted DBCO-Cy3 solution for 60 minutes and were washed with 1x PBS, ultrapure water, blocking solution, ultrapure water, 50% IPA, 100% IPA, 50% IPA, and ultrapure water respectively.The scaffolds were then left in ultrapure water overnight and imaged using a fluorescent microscope the next day

The TGF-β1 mimetic peptide was successfully synthesized with the Cy3 and DBCO  modifications.  The PCL-azide conjugate was successfully synthesized and printed into PCL-azide scaffolds. Fluorescence microscopy showed higher fluorescence intensity in the PCL-azide scaffolds compared to PCL only scaffolds, demonstrating that the DBCO-azide groups reacted to attach the TGFpep  to the scaffold surface (Figure 1).   

Figure 1: Representative fluorescence microscopy images of scaffolds 3D printed with (A) PCL only or (B) PCL with 20 mg/mL azide-PCL and labeled with DBCO-pep (red). (C) Quantification of mean fluorescence intensity using integrated density values (product of mean gray value by area).

 Our results show that we can successfully tether a GF-mimetic peptide to a 3D-printed scaffold using click chemistry. Our ongoing work focuses on demonstrating that the TGFpep can be attached to the scaffold surface in the presence of human mesenchymal stromal cells (hMSCs) during cell culture. This platform thus enables us to modify the cell’s extracellular environment over time. Future work will investigate hMSC differentiation response to temporal increases in TGFpep over time. 

Acknowledgements: This work was partially supported by NSF (CAREER DMR-1944914), NIH (R21AR079117-01A1), Lehigh University (LU) President’s Scholar Award (KBS), and start-up funds awarded to LWC from LU.  

References: [1] Di Luca+ Birth Defects Res. Part C 105(1): 34–52, 2015. [2] Occhetta+ PNAS 115(18): 4625–4630, 2018. [3] Seims+ Bioconj Chem 32(5): 861-878, 2021. [4] Camacho+ Biomater. Sci., 7: 4237, 2019. [5] Camacho+ Biomater Sci. 9: 6813-6829, 2021. [6] Zhang+ Adv. Funct. Mater. 25(3): 350–360, 2015.