Fabrication, Modification, and Evaluation of Modular, Gelatin-Based Microcarriers for the Delivery of Progenitor Cells in Bone Tissue Engineering Applications

Abstract

Complete regeneration of large bone defects is constrained by metabolite diffusion and spatiotemporal limits to osteogenic cue distribution. Macroscale bone filler osseointegration is relatedly hampered by limited cytocompatibility and infill of large voids. In this project, cellularizable, injectable microcarriers were thus developed with the goal of creating a mechanobiologically osteogenic bone filler material. Gelatin-based microcarriers (GmC) crosslinked with genipin were emulsified and the effect of impeller geometry and stirring rate on microparticle morphology were examined. GmC were seeded at varying densities with mesenchymal stromal cells (MSC) and were evaluated physically and biologically. The effect of GmC matrix composition was examined by varying gelatin concentration and exogenous addition of hydroxyapatite (HA) and/or solution-mineralization. Suspension culture of GmC in a fibrin carrier gel was examined to determine the effect on MSC localization and phenotype, and as a model of GmC delivery. In a first study, GmC size was modulated to produce microparticles suitable for injectable delivery. Seeding density of MSC was modulated to maximize osteogenic response. Particle size was negatively related with impeller rate. Increasing the rotation rate of an axial impeller (1.0 mm blade diameter) from 250 rpm to 500 rpm decreased particle diameter from 341.7±17.0 μm to 177.3±13.0 μm. A corresponding experiment with 0.3 mm diameter impeller resulted in a diameter decrease from 173.2±9.4 μm to 86.2±14.2 μm. MSC-seeded GmC were cultured in packed beds, loosely simulating a void-filler environment. All conditions exhibited increased DNA-normalized calcium deposition over three weeks of culture, though the effect was most pronounced in osteogenic medium. In a follow-on study, GmC were modified to enhance stiffness-induced osteogenesis. Size and shear modulus (G’) were positively related with increasing gelatin and hydroxyapatite content. The diameter of 6.0% gelatin GmC increased from 86.2±14.20 μm to 149.6±6.2 μm in 14.0% GmC. Hydroxyapatite addition increased the diameter of 6.0% GmC from 80.3±7.1 μm to 149.5±4.3 μm with 5.0% HA. Rheometric mechanical characterization revealed increasing gelatin and hydroxyapatite concentration to be related with matrix stiffness. G' of 6.0% gelatin matrix was increased from 34.7±3.7 kPa to 47.5±3.7 kPa in 14.0% gelatin. HA addition increased G' of 6.0% gelatin to 70.8±2.9 kPa in 10.0% HA. Cytocompatibility related with gelatin:HA ratio and all seeding efficiencies exceeded 92%. Viability and DNA were negatively related with calcium content and peaked after one week of culture. DNA-normalized ALP and calcium deposition were upregulated in all conditions, indicating osteogenicity even in the absence of supplements. Lastly, MSC were pre-seeded onto or simultaneously mixed with GmC in a fibrin matrix to suspend and culture cells in carrier gel. Both seeding methods resulted in osteogenic activity on 6.0% and 14.0% gelatin GmC. Notably, DNA content was essentially maintained over three weeks in culture, which had been a limitation in previous studies, particularly when calcium was present in the GmC. Calcium deposition was markedly increased in osteogenic medium in both GmC types and seeding models, suggesting that a protein carrier gel may allow improved osteogenic development of GmC. Overall, this research illuminates processing-structure-function relationships in mechanobiologically osteoinductive, non-toxically genipin-crosslinked GmC designed for longterm bone-filling applications. Microcarriers can be used to culture and deliver progenitor cells, and to potentiate their osteogenic ability. Packed-bed culture and delivery in a protein matrix can create an environment to enhance formation of new bone tissue, and may have utility for treatment of large bone defects.