A shape-controlled tunable microgel cell delivery platform for low-dose delivery of primed stem cells for in vivo therapeutic neovascularization

摘要

Introduction: Hydrogel-based cell delivery platforms fabricated from synthetic or natural polymers have evolved from a cytoprotective design to a functional biomaterial design, which not only offers better cell survival but also modulates cellular behaviour via intrinsic biophysical or biochemical cues. Tissue regenerative processes are driven by reparative mesenchymal stem cells (MSCs) under the influence of biochemical cues dictated by the microenvironment. Prolonged ischemia and lack of blood supply drive cellular apoptosis and tissue death. However, delivery of primed stem cells via a tunable microenvironment triggers an 'angiocrine' response, driving therapeutic angiogenesis. Hence, in this study, it is hypothesised that delivery of primed human mesenchymal stem cells on a shape-controlled microgel platform at a low-cell dose promotes angiogenesis in a murine model of hind-limb ischemia (HLI). The specific objectives of the study were to investigate the cellular behaviour on tunable parameters such as matrix concentration, cross-linker concentration and cell densities and therapeutic efficacy of the optimized group in a hind-limb ischemia model. Materials and Methods: Collagen-based microgels of concentrations 1,2 and 3mg/ml were fabricated by neutralizing type-Ⅰ collagen and dispensing a mixture composed of cells and cross-linker 4S-Star-PEG Mw 10000 KDa (Jenkem Tech, U.S.A) on to a hydrophobic surface. hMSCs at an optimized cell density of 0.8×106 were embedded within the microgels. Cell embedded microgels were tested for changes in morphology by confocal microscopy; protein and gene expression quantitation by multiplex ELISA and gene arrays; and changes in matrix stiffness with atomic force microscopy and confocal microscopy. In vivo experiments were performed in a Balb/c nude hind-limb ischemia mouse model. Animal groups (n=12/group) were PBS; microgels alone; microgels with 50,000 hMSCs; 50,000 and 1,000,000 cells alone. Laser-Doppler perfusion and clinical signs of disease severity were assessed along with molecular evaluation using; multiplex ELISA, gene expression arrays and MALDI-imaging mass spectrometry. Statistical analysis was performed using two-way ANOVA with p＜0.05. Results and Discussion: The use of 4S-StarPEG combined with the high surface hydrophobicity allowed gelation after 40 minutes, conferring a spherical shape to the microgels. Over 80% viability and low apoptosis was observed using flow-cytometry. 2mg/ml microgels at 0.8×10~6 cell density showed up-regulation of paracrine factors involved in angiogenesis and distinguishing integrin expression profile (Figure1). AFM analysis revealed insignificant changes in matrix stiffness and unique cell morphology in 2mg/ml microgels compared to 1 and 3mg/ml microgels. Results from the in vivo hind-limb ischemia studies (Figure 2) showed increased blood perfusion, up-regulation of protein and gene markers related to angiogenesis, and changes in glycan environment between the treatment and control groups. Conclusion: Tunable shape-controlled collagen based microgel platform can be used to deliver hMSCs at a low cell dose for angiogenesis in vivo.