CELL ADHESIVE AND GROWTH FACTOR MIMICKING PEPTIDES WORK SYNERGYSTICALLY TO INFLUENCE OSTEOGENIC ACTIVITY IN MESENCHYMAL STEM CELLS

Introduction:: Matrix immobilized growth factor mimicking peptides have been studied as potential substitutes for soluble growth factors, such as rhBMP-2, to provide for local, safe delivery. Prior studies in drosophila and mice strongly suggest that bone growth and repair both require co-occupancy of growth factor receptors and integrins^1,2. For this reason, co-presentation of integrin-mediated cell adhesive peptides with growth factor mimicking peptides may be optimal for promoting growth and regeneration in target sites³. Co-localization of αβ integrins and BMP receptors in osteogenic cells treated with rhBMP-2 has been observed by several groups⁴. There is also evidence that materials in which these ligands are co-localized enhances osteogenic signaling⁵. Here, we tested the hypothesis that nanometer scale co-localization adhesive ligands and growth factor mimicking peptides can enhance osteogenic activity of soft gel materials. Using orthogonal click chemistries, we conjugated integrin-binding peptides (cRGD) and short chain mimetics of the rhBMP-2 knuckle epitope (KE) to alginate hydrogels.


Materials and Methods:: Alginate was modified with either BCN-amine (N-[(1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane; Sigma) or BMPH (N-β-maleimidopropionic acid hydrazide, ThermoFisher)^2,3. We grafted cell adhesive cyclo-RGD (cRGD, Peptides International) and growth factor mimetic BMP-2 knuckle epitope (KE, Peptides 2.0) via two orthogonal click chemistries: strain promoted azide-alkyne cycloaddition (SPAAC) for cRGD and maleimide-thiol for KE onto alginate polymers either in separate reactions (homovalent) (Figure 1,2) or within the same polymer chain (heterovalent) (Figure 3). Conjugation was verified with fluorophore surrogates and ¹H NMR. Clonally derived D1 mouse mesenchymal stem cells (mMSC) were encapsulated in alginate hydrogels with or without cRGD and KE with a targeted peptide density of 5-18 µM of each peptide. Hydrogels were crosslinked with either 50 mM-100 mM calcium sulfate slurry or varying concentrations of (22.5 mM-150 mM) CaCO₃ and glucono-delta-lactone (GDL; 2:1 molar ratio to CaCO₃) to achieve a range of elastic moduli from 12 kPa -90 kPa³. Cell-gel constructs were cultured in 10% FBS media containing osteogenic supplements, with or without rhBMP-2 for 5 or 15 days. Cell-gel constructs were fixed in 4% paraformaldehyde for RUNX2 staining or cells were extracted via alginate lyase for early osteogenesis marker assays. Cell lysates were separated from DNA and assessed for alkaline phosphatase activity (ALP) and osteocalcin (OCN) production via OCN ELISA. ALP and OCN content were normalized to total DNA quantified via Picogreen assay.

Results, Conclusions, and Discussions:: Differential nanopatterning of peptides on alginate via homovalent or heterovalent conjugation was verified by FRET (Figure 4A-B). Fluorophores conjugated to the same chain of alginate resulted in loss of donor signal to acceptor, indicating the distance between peptides is 7 ± 0.3nm – mimicking the separation for integrin-growth factor receptor co-localization, while in the homovalent condition, fluorophore separation was ≥ 10nm. D1 mMSC encapsulated in hydrogels with either homovalent or heterovalent cRGD and KE exhibited a significantly increased osteocalcin (OCN) expression over 15 days compared to unmodified alginate control, over all three stiffnesses (12kPa-90kPa) as demonstrated in Figure 4C-E. There were no significant differences in cells encapsulated in homovalent or heterovalent conditions across any stiffness; however, we did observe a trend toward higher OCN expression in MSC cultured within stiffer gels (50 or 90kPa) with heterovalent peptide presentation.

Analysis of cellular osteocalcin (OCN) expression, consistent with other assays (ALP activity and RUNX2 staining) demonstrated the effectiveness of short chain mimetic peptides and display a similar potency in osteogenic potential compared to soluble rhBMP-2. However, there was no trend toward an improvement in osteogenesis when cRGD and KE were nanopatterned within soft (12kPa) gels, and the trends observed in stiffer (50 and 90kPa) gels toward improvement in MSC osteogenesis when these ligands were nanopatterned together was not statistically significant. It is possible that at lower stiffness (12kPa), MSC may mechanically reorganize their environment in the homovalent hydrogels to force the peptides closer together⁶. Trends toward increased OCN expression at higher stiffnesses in heterovalent gels suggests MSC may respond favorably to nano-scale co-presentation of integrin binding and growth factor mimicking peptides only from materials that resist cell-mediated reorganization of peptides^3,6.

Nanopatterning of peptides is of special interest for many current studies on cell adhesive peptide and growth factor-based biomaterials. However, our results suggest that whilst on rigid surfaces, it may be beneficial to nanopattern peptides within receptor-scale difference of one another, the impact of nanopatterning on soft materials may be much less extensive. This may stem from cells' ability to mechanically reorganize soft materials, especially those with viscoelastic creep like alginate gels.


Acknowledgements (Optional): : This work was performed with support from NIH R01AR077678, Grant No. DGE-1745038 and DGE-2139839, and DoD Discovery Award W81XWH-22-1-0020. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation

References (Optional): : ¹Sawala et al., Cell Reports. (2015); ²Jain, Neal et al. ACS Applied Biomaterials. (2021); ³Tan et al. Biomaterials. (2021); ⁴Lai, et al. JBMR. (2005); ⁵Hubbell, et al. Sci. Transl. Med. (2011); ⁶Huebsch, et al., Nature Materials. (2010).

¹Sawala et al., Cell Reports. (2015); ²Jain, Neal et al. ACS Applied Biomaterials. (2021); ³Tan et al. Biomaterials. (2021); ⁴Lai, et al. JBMR. (2005); ⁵Hubbell, et al. Sci. Transl. Med. (2011); ⁶Huebsch, et al., Nature Materials. (2010).