Dual Peptide Functionalized Alginate Hydrogels to Control MSC Therapeutic Potency for Cell-mediated Matrix Repair

Introduction:: Age-related cell attrition is associated with progressive degeneration of cartilage in multiple tissues, including articular cartilage, meniscus, tendon and intervertebral disc. Cell supplementation to cartilaginous tissues has been widely explored to promote extracellular matrix synthesis and increase capacity for self-repair [1]. In particular, Mesenchymal Stem Cell (MSC) supplementation has been developed as a means of promoting matrix synthesis and for diminishing inflammation [2]. However, without a carrier, MSC survival and engraftment is poor [3]. Growth factors can promote transplanted cell survival, but side effects of growth factor leakage are concerning [4]. We hypothesized that short peptide mimetics of pro-survival growth factors, combined with cell adhesive peptides, could enhance MSC survival and secretion of cytokines to protect both transplanted cells from biochemical (i.e. interleukin-1β, IL-1β) induced inflammation. As insulin-like growth factor 1 (IGF-1) has been reported to promote cell survival and attenuate inflammatory signaling in MSC, we identified a candidate peptide reported to mimic the activity of IGF-1 and studied its influence on proliferation and modulation of cytokine production of bone marrow-derived MSCs.


Materials and Methods:: Bone marrow-derived MSCs were obtained from RoosterBio^TM. Alginate polymers were grafted with either cyclic-RGD (cRGD) alone or cRGD along with IGF-1 mimetic (IGM) peptides at varied peptide density using Strain Promoted Azide Alkyne Cycloaddition or maleimide-thiol click chemistry described previously [5][6]. cRGD-alginate (100 µM peptide density) and combination of cRGD-alginate and IGM-alginate (50 µM cRGD/ 50 µM IGM or 10 µM cRGD/ 50 µM IGM peptide density, respectively) were ionically crosslinked using divalent calcium ions to a final elastic modulus range of 12 kPa [6]. Bioactivity of the peptide coupled gels was assessed by measuring total and phosphorylated AKT using AlphaLISA kits (PerkinElmer). For studies of metabolic activity, MSCs were encapsulated into peptide modified gels either in RoosterBasal^TM alone or RoosterBasal^TM supplemented with soluble recombinant human IGF-1 (rhIGF1, 500 ng/mL). Hydrogels were cultured in RoosterBasal^TM media and RoosterBooster^TM. At 24h, the baseline metabolic activity of encapsulated MSCs was measured (Alamar Blue). Media was then replaced completely with RoosterBasal^TM media. Serial measures of metabolic activity were obtained at 1, 4, and 7 days of culture in serum-free media. For studies of cytokine secretion, MSCs encapsulated in each of the hydrogel condition were treated with IL-1β (1 ng/mL) for 4 days. Then, supernatant was collected, and concentration of cytokines was measured (Eve Technologies). Each hydrogel was defined as a single biological replicate. Data are presented as mean ± standard deviation of no less than 4 biological replicates for each hydrogel condition.


Results, Conclusions, and Discussions:: Growth factors, including IGF-1, have been investigated for their potential to promote cell-mediated repair following intradiscal injection into the cartilage [2][7]. IGF-1 has been shown to induce proliferation, promote cell survival, and block apoptosis in stem cells (i.e. MSCs) by activating the PI3K/AKT pathways. We observed that the combination of cell adhesive and IGF-1 mimetic peptides grafted to alginate hydrogels activated AKT signaling, elicited proliferation, and attenuated IL-1β induced inflammatory signaling in MSCs under serum-free conditions. Compared to MSCs seeded on cRGD alone, MSCs seeded on gels presented with cRGD peptide in combination with IGF-1 mimetic peptide exhibited enhanced Akt signaling (p< 0.05, Fig. 1B). Likewise, MSCs encapsulated in gels containing IGF-1 peptide mimetics demonstrated significantly enhanced metabolic activity compared to MSC in gels modified with cRGD alone (p< 0.05, Fig. 1C). Strikingly, cells encapsulated in gels dually modified with cRGD and IGM challenged with IL-1β demonstrated a significant reduction of proinflammatory cytokines (i.e. TNFα, p< 0.05; Fig. 1D) compared to cells encapsulated into gels modified with cRGD alone without diminishing IL-1β induced production of the anti-inflammatory cytokine IL-13 (data not shown). Altogether, these results demonstrate that alginate gels co-presenting cell-adhesive and IGF-1 mimetic peptides have the potential not only to promote survival of encapsulated MSC but also to directly reduce proinflammatory cytokine production in MSCs.

These results represent a promising step in using growth factor mimics tethered to a hydrogel vehicle, to create a localized environment that promotes cell survival and ameliorates pro-inflammatory signaling, while avoiding the risks associated with uncontrolled growth factor protein delivery (Fig. 1A). Our ongoing studies are focused on determining the receptors and signaling pathways through which IGM peptide exert their therapeutic effects and determine whether these receptors synergize with integrins to enhance therapeutic responses of MSC for in vivo transplantation.


Acknowledgements (Optional): : This work was supported by the NSF (Tan, DGE-1745038 and DGE-2139839), and the NIH (R01AR077678, AR07934 (R41)). 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 NSF.


References (Optional): : [1] Urlić I. and Ivković A. Cells. (2021). [2] Le H. J Tissue Eng. (2020). [3] Burdick J. Cell Stem Cell. (2016). [4] Masuda K. Eur Spine J. (2008). [5] Jain E. & Neal S. ACS Applied Bio Mater. (2021). [6] Tan X. Biomaterials. (2021). [7] Kennon J.C. BioMol Concepts. (2018).