Introduction: Alginate hydrogels are biocompatible, easy to use, and optically clear allowing cell growth monitoring, making them a promising biomaterial to support not only cell growth, stem cell differentiation, and tissue formation, but also cell delivery and implantation. In particular, alginate hydrogels can recapitulate not only the elasticity but also the viscoelasticity of soft tissue extracellular matrix (ECM). Mesenchymal stem/stromal cells (MSCs) have potential to restore tissue function due to their regenerative, anti-fibrotic and anti-inflammatory properties. The long-term goal of this study is to demonstrate the feasibility of using two alginate hydrogel systems, cryoelectrospun scaffolds and microfibers for stromal cell delivery.
Materials and Methods: To mimic the soft tissue ECM, porous sponge-like scaffolds were fabricated by cryoelectrospinning of an alginate-elastin-PEG solution. To facilitate delivery of high density cells, alginate hydrogel microfibers were fabricated using microfluidic synthesis. The morphology of both alginate hydrogel scaffolds was characterized by optical microscopy and scanning electron microscopy. The scaffold viscoelasticity was measured using a CellScale MicroTester. Mouse NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary gland mesenchyme cells were used as model mesenchymal and MSC cells. Cell loading efficiency in cryoelectrospun scaffolds and microfibers was evaluated. Cell viability was assessed by trypan blue exclusion and calcein AM/ethidium homodimer-1 LIVE/DEAD staining. Cell proliferation in cryoelectrospun scaffolds and microfibers was examined by Cell-Titer Glo® 3D Viability Assay and alamarBlue Assay, respectively. Expression of mesenchymal and fibrotic markers was evaluated by immunocytochemistry analysis. Furthermore, proteomic analysis was performed to evaluate ECM protein produced by primary E16 salivary gland cells grown on alginate hydrogel scaffolds.
Results, Conclusions, and Discussions: Cryoelectrospun alginate scaffolds exhibited honeycomb-like morphology, and high compliance and viscoelasticity, thereby mimicking not only soft-tissue ECM topography but also its biomechanical properties. Cryoelectrospun scaffolds allowed deep cell penetration into the scaffold with optimal cell-seeding density of ~50,000 cells/scaffold. Alginate microfibers could also mimic soft tissue viscoelasticity, while allowing high cell loading efficiency and high-density cell growth (0.25-20 × 106 cells/scaffold). Cells grown in both alginate hydrogel systems maintained a viability above 90% and retained characteristic expression of fibroblast markers such as platelet-derived growth factor α (PDFGα) and vimentin, with minimal expression of fibrotic markers such as α-smooth muscle actin (α-SMA). Proteomic analysis confirmed expression of ECM proteins and anti-fibrotic molecules in cells grown in alginate hydrogel scaffolds, highlighting the potential of alginate scaffolds to maintain mesenchymal cell homeostatic phenotype and anti-fibrotic properties. For future cell delivery and implantation in vivo, cryoelectrospun scaffold can be used to deliver 50,000 cells or combined to deliver larger cell numbers. Alginate microfibers that accommodate high cell density also hold promise for in vivo cell delivery. In summary, soft-tissue-inspired alginate hydrogels are versatile systems for stem cell maintenance, growth and delivery, and may promote functional tissue regeneration.
