17. Bioink With Induced Myoprogenitor and Humoral Factors Promotes Muscle Regeneration

Background: In settings of muscular trauma that leads to significant volumetric muscle loss (VML), fibrosis and aberrant healing replaces normal myofiber regeneration. This scarring phenotype leads to poor muscle function and patient morbidity. Previous studies have shown muscle regeneration requires the coordination of progenitor cells, supportive extracellular matrix, and a promyogenic niche. We hypothesized printing a bioink replacing these components will facilitate muscle repair following VML. Platelet-rich plasma (PRP), was selected to stimulate cellular integration and growth, as it contains many myogenic and vasculogenic growth factors. METHODS: Patient blood samples (pooled) were centrifuged to isolate PRP. Commercially available gelatin and methacrylic anhydride were used to synthesize GelMA (Sigma). These constructs were impregnated with PRP isolates. For the cellular fraction, commercially available CD34+ umbilical cord blood derived (NCRM1, NIH) hiPSCs were differentiated via primary and secondary conditioned media to generate muscular progenitor cells (hiPSC-MPCs) over 30 days. These terminally differentiated cells were then incubated with the PRP-gel matrix to generate a high-fidelity promyogenic bioink. Using a murine VML model, 4 mm punch biopsies were performed in the posterior compartment of 10wk old immunocompromised mice (Rag knockout) and assigned into one of four groups: Sham, VML-only, VML+GelMA+PRP, and VML+GelMA+PRP+cells. 3D scaffolds were implanted onto the wounded muscles and secured with photo-crosslinking. Functional muscle measured torque at the ankle and in situ gastrocnemius force generated. Fibrosis, CD31+, and iPSC transplantation quantifications were performed with standard histology techniques. RESULTS: Primary differentiation induced a robust transition from OCT4+ hiPSCs to PAX3+ muscular stem cells to PAX7+ mature satellite cells. Secondary differentiation exhibit PAX7+ satellite cells with robust deposition of actinin and formation of myofibrils. In a titration curve examining optimal cell density (3-7%) and PRP fraction of ink media (0-50%), the 3% gel matrix generated the most robust actinin (F-Actin) and actin (ACTN2) deposition by day 3 of culture, further improved with 25% PRP. Ultra-performance liquid chromatography identified glycerophospholipids, glycerolipids, and sphingolipids as the primary constituents of the PRP enrichments. On in vitro experiments, printed myofibers with gel matrix with PRP to be enriched in Pax7 satellite cell and Myh3 myosin heavy chain expression, with increased F-actin and ACTN2 immunolabeling suggesting more robust myofibril formation. Subsequent in vivo experiments demonstrated marked reduction in generated force and tetanic strength within VML cohorts with dose dependent increase in generated force with the addition of GelMA+PRP and most pronounced with the GelMA+PRP+Cells bioink. These muscles also exhibited 50% of the total fibrosis as seen in VML only groups, suggesting the development of an anti-fibrotic niche as well as concurrent statistically significant 6-fold increases in CD31+ vascular staining in the bioink vs. VML. CONCLUSION: A bioink consisting of induced myoprogenitor cells and humoral factors coupled to a scaffold can be printed into a muscular defect to significantly improve muscular regeneration and function.