IGF-1- and IGFBP-5-Loaded Collagen Sponges for Skeletal Muscle Regeneration

Introduction: Volumetric muscle loss (VML) is a condition in which trauma to a skeletal muscle induces loss of muscle mass. This loss of mass overcomes the muscle’s ability to regenerate, and thus results in a permanent loss of muscle functionality.1 Autologous tissue transfer is the standard of care to treat VML; however, it often results in graft failure and does not fully restore muscle functionality.2 To address these limitations, biomaterial scaffolds are being studied to regenerate muscle tissue and restore functionality. These scaffolds are limited in their ability to regenerate muscle in part because of a lack of sufficient satellite cell recruitment and myofiber formation.1 Insulin-like growth factor-1 (IGF-1) enhances both myoblast proliferation and differentiation,3 and thus we expect that its release from a biomaterial scaffold should overcome this limitation. Additionally, the complex formed between IGF-1 and an IGF-binding protein (IGFBP-5) enhances myoblast differentiation and may aid in the delivery of IGF-1 from scaffolds.4 Thus, our goal is to add both IGF-1 and IGFBP-5 to our scaffold to enhance the proliferation and differentiation of myoblasts.

Materials and

Methods: Type I collagen hydrogels were formed in cylindrical PTFE molds and frozen to form anisotropic porous sponges, as outlined in our previous work.1 Sponges were crosslinked for 24 hours with 12.9 mM EDC, 10.4 mM NHS, and 34 mM heparin. IGFBP-5 was then incubated on the sponges overnight at 4°C, followed an overnight incubation of IGF-1 (37°C and 5% CO2). Six groups of sponges were fabricated: 1) heparin/IGFBP-5/IGF-1, 2) heparin/IGF-1, 3) heparin, 4) IGFBP-5/IGF-1, 5) IGF-1, and 6) none (control). To measure IGF-1 release, conditioned myoblast growth medium from each sponge was collected every 24 hours, and replaced with either fresh growth or differentiation medium, which was immediately transferred to myoblasts pre-plated in a 96-well plate. A cell metabolism assay (CCK8) was used to analyze cell proliferation on days 0 and 3. Conditioned differentiation medium was similarly collected to analyze the effect of released IGF-1 on myofiber formation. Cells were fixed after 4 days of culture in conditioned differentiation medium, immunostained against myosin, and counterstained with DAPI. The fusion index was calculated to quantify myofiber formation. This process of alternating between proliferation and differentiation medium was conducted four times to quantify the release of IGF-1 over a 1 month period (Fig 1A). Statistical analysis was conducted using one-way ANOVA with Tukey’s post-hoc analysis (p < 0.05).

Results, Conclusions, and Discussions:

Results: To quantify cell proliferation, we performed CCK8 assays and found an increase in cell proliferation between days 0 to 3 for all groups. At day 3, all sponge groups except heparin-only increased proliferation as compared to the controls. At days 10, 17, and 24, all of the heparin-loaded sponges did not enhance proliferation, although notably they did not decrease proliferation with respect to controls. To quantify differentiation, fusion index was calculated after 4 days in culture with conditioned medium from IGF-1/IGFBP-5 loaded sponges. At day 7, heparin-containing sponges supported significantly higher fusion indices than sponges without heparin, suggesting that heparin-loaded sponges induce greater amounts of myofiber differentiation than heparin unloaded sponges (Fig 1B). At day 14, heparin/IGFBP-5/IGF-1 sponges induced more differentiation than controls (Fig 1C), and at day 21, this same group induces more differentiation than all of the heparin-free sponges (Fig 1D). At day 28, heparin/IGFBP-5/IGF-1 sponges qualitatively enhanced the fusion index compared to all other treatment groups (Fig 1E).

Conclusions: These studies confirm the ability of our sponges to induce myoblast proliferation and differentiation in vitro through the release of IGFBP-5/IGF-1 over a one-month period. At later time points, collagen sponges containing heparin supported less proliferation than other groups, but they instead induced the most differentiation. These data suggest that heparin/IGFBP-5/IGF-1 scaffolds initially release enough IGF-1 to induce a proliferative response, which is then modulated at later time points to enhance myogenic differentiation while maintaining proliferation levels similar to those of control sponges. This ability to support the most myofiber formation supports our hypothesis that heparin-loaded sponges will be ideal for muscle regeneration. Further, the time-course for this release fits previous literature that describes satellite cell infiltration occurring within the first week of regeneration 5. Heparin-loaded sponges incubated with IGF-1/IGFBP-5 elevated myoblast proliferation levels, and as such may be advantageous for implantation to promote skeletal muscle tissue regeneration. Future work includes testing the ability of these sponges to facilitate muscle regeneration in vivo by implanting them into a murine model of VML.

Acknowledgements (Optional):

References: 1. Kozan, NG et al. J. Fuct. Biomater. 2023; 11:p.1245897. 2. Mulbauer, G.D. et al. Discoveries (Craiova). 2019;7(1):e90. 3. Yang, SY et al. FEBS Lett. 2002;522(1-3):p.156-60. 4. Duan, C et al. Gen Comp Endocrinol. 2010;167(3):p.344-51. 5. Oyaizu, T., et al., Scientific Reports. 2018;8(1).