Poster O19 - Fabricating Enzymatic Sensitive Anti-Inflammatory Scaffolds

Introduction: Osteoarthritis (OA) is a progressive musculoskeletal disease characterized by extracellular matrix (ECM) degradation. Cytokines secreted by immune cells trigger articular chondrocytes to produce catabolic enzymes that breakdown the ECM. Mesenchymal stromal cells (MSCs) are promising candidates for cartilage regeneration therapies due to their multipotent differentiation potential and capacity to produce cartilage-specific ECM. The harsh inflammatory microenvironment impedes effective differentiation and matrix deposition. To overcome this, synthetic corticosteroids, such as dexamethasone (DEX), have been used to inhibit gene expression of pro-inflammatory cytokines and induce chondrogenesis. However, high doses of DEX can lead to chondrocyte death and matrix degradation. Therefore, achieving DEX delivery in a tightly controlled manner is needed to avoid detrimental effects. To address this challenge, our approach involves tethering DEX to a 3D-printed poly(caprolactone) (PCL) scaffold via a peptide sensitive to pro-inflammatory enzymatic cues. The peptide will therefore be cleaved in the presence of inflammatory conditions, locally releasing DEX from the scaffold in a targeted and controlled manner. The aim of this study is to characterize DEX-peptide-PCL scaffolds and their potential as a viable tissue engineering model for OA therapies.

Materials and

Methods: DEX-peptide synthesis: DEX was modified with N,N′-disuccinimidyl carbonate and triethylamine in dry acetone under anhydrous conditions then concentrated and washed.1 A fast-degrading peptide KRVKRRLMETGGGC (Fast) was synthesized using solid-phase peptide synthesis techniques. DEX was coupled to the peptide on resin to produce DEX-Fast before cleaving and purifying using reversed-phase HPLC. A scrambled Fast peptide LTRLMEKRRKVGGGC (ScrFast) was also synthesized and modified with DEX to produce DEX-ScrFast as a non-degradable control to demonstrate sequence specificity. We also synthesized DEX modified with the cleaved peptide fragment (DEX-FastFragment) to confirm functionality after release. Syntheses were validated using 1H NMR spectroscopy and MALDI. Peptide-PCL conjugates: Conjugates were synthesized using methods previously described.2 Briefly, PCL diol and p-Maleimidophenyl isocyanate (PMPI) were dissolved in anhydrous 1-methyl-2-pyrrolidinone (NMP) and reacted overnight to synthesize PCL-maleimide. DEX-Fast-PCL and DEX-ScrFast-PCL conjugates were synthesized by reacting each peptide with PCL-mal in anhydrous NMP overnight. Each step was confirmed by 1H NMR. 3D Printing and Characterization: Peptide-PCL conjugate inks were prepared by dissolving 370 mg/mL unmodified PCL (80:25 kDa) and 5.3 mg/mL DEX-peptide-PCL conjugates in hexafluoroisopropanol and solvent-cast 3D-printed into scaffolds.2 DEX-peptide-PCL scaffolds were imaged using scanning electron microscopy to characterize scaffold architecture. In vitro DEX functionality: DEX-FastFragment was compared to DEX during MSC chondrogenesis to assess if the FastFragment inhibited DEX activity. MSCs were cultured as pellets in chondrogenic conditions for 3 weeks. Pellets were harvested and processed for biochemical analysis or fixed for histology to measure chondrogenic matrix deposition.

Results, Conclusions, and Discussions: NMR and MALDI confirmed DEX was successfully modified to the Fast, ScrFast, and FastFragment peptides. DEX-Fast and DEX-ScrFast peptides were successfully conjugated to PCL-mal to produce DEX-Fast-PCL and DEX-ScrFast-PCL conjugates. These peptide-PCL conjugates were incorporated into PCL inks and printed into scaffolds. No observable changes in scaffold architecture were noted between unmodified PCL and peptide-PCL scaffolds, confirming that the addition of the conjugates does not affect printing. In parallel, we confirmed that modifying DEX with the FastFragment peptide did not affect its chondrogenic activity. DEX-FastFragment added to chondrogenic media exhibited levels of DNA and glycosaminoglycans comparable to those of soluble DEX. This data indicates that DEX released from our scaffolds will enhance chondrogenesis. Current and ongoing work focuses on characterizing DEX release kinetics and DEX activity under pro-inflammatory enzymatic conditions.

References
1) Yang C, Mariner PD, Nahreini JN, et al. Cell-mediated delivery of glucocorticoids from thiol-ene hydrogels. Journal of Controlled Release 2012;162(3):612–618; doi: 10.1016/J.JCONREL.2012.08.002.
2) Camacho P, Busari H, Seims KB, et al. 3D printing with peptide–polymer conjugates for single-step fabrication of spatially functionalized scaffolds. Biomater Sci 2019;7(10):4237–4247; doi: 10.1039/C9BM00887J.

Acknowledgements (Optional): The authors acknowledge the generous support by Lehigh University Department of Bioengineering and the P.C. Rossin College of Engineering & Applied Science through the Rossin College Doctoral Dean’s fellowship and Maida’s fellowship (C.O.) and start-up funds (T.G.-F.). The authors acknowledge Lehigh University’s NMR Facility. The Bruker Neo 500 MHz NMR was acquired through NSF-MRI-1725883, with additional support from Lehigh University. This work was generously supported by National Science Foundation (NSF) through a Faculty Early Career Development (CAREER) award (DMR 1944914) and the National Institutes of Health (NIH) Grant (1R21AR079117) (L.W.C).