Optimal Seeding Densities for In Vitro Chondrogenesis of Two- and Three-Dimensional-Isolated and -Expanded Bone Marrow-Derived Mesenchymal Stromal Stem Cells Within a Porous Collagen Scaffold

Abstract

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. The objective of this study was to assess the impact of cell seeding density within a collagen I scaffold on in vitro BMSC chondrogenesis following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments. It was hypothesized that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 × 10⁶ cells/cm³. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing an expansion medium, and seeded within collagen I scaffolds at densities of 50, 10, 5, 1, and 0.5 × 10⁶ BMSCs/cm³. For 3D isolation and expansion, aspirates containing known quantities of mononucleated cells (bone marrow-derived mononucleated cells [BMNCs]) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 × 10⁶ BMNCs/cm³ and cultured in the expansion medium for an equivalent duration to 2D expansion. Constructs were differentiated in vitro in the chondrogenic medium for 21 days and assessed with reverse-transcription quantitative polymerase chain reaction, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two-dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II messenger RNA (mRNA) relative to predifferentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 × 10⁶ BMSCs/cm³. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 × 10⁶ BMSCs/cm³ based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/deoxyribonucleic acid (DNA). For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 × 10⁶ BMNCs/cm³, while collagen II deposition occurred in scaffolds seeded at 10–50 × 10⁶ BMNCs/cm³. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 × 10⁶ BMNCs/cm³. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 × 10⁶ BMSCs/cm³ and 50 × 10⁶ BMNCs/cm³, respectively.