Comparison of Uncultured Marrow Mononuclear Cells and Culture-Expanded Mesenchymal Stem Cells in 3D Collagen-Chitosan Microbeads for Orthopedic Tissue Engineering

Abstract

Stem cell-based therapies have shown promise in enhancing repair of bone and cartilage. Marrow-derived mesenchymal stem cells (MSC) are typically expanded in vitro to increase cell number, but this process is lengthy, costly, and there is a risk of contamination and altered cellular properties. Potential advantages of using fresh uncultured bone marrow mononuclear cells (BMMC) include heterotypic cell and paracrine interactions between MSC and other marrow-derived cells including hematopoietic, endothelial, and other progenitor cells. In the present study, we compared the osteogenic and chondrogenic potential of freshly isolated BMMC to that of cultured-expanded MSC, when encapsulated in three-dimensional (3D) collagen-chitosan microbeads. The effect of low and high oxygen tension on cell function and differentiation into orthopedic lineages was also examined. Freshly isolated rat BMMC (25 · 10 6 cells/mL, containing an estimated 5 · 10 4 MSC/mL) or purified and culture-expanded rat bone marrow-derived MSC (2 · 10 5 cells/mL) were added to a 65-35 wt% collagenchitosan hydrogel mixture and fabricated into 3D microbeads by emulsification and thermal gelation. Microbeads were cultured in control MSC growth media in either 20% O 2 (normoxia) or 5% O 2 (hypoxia) for an initial 3 days, and then in control, osteogenic, or chondrogenic media for an additional 21 days. Microbead preparations were evaluated for viability, total DNA content, calcium deposition, and osteocalcin and sulfated glycosaminoglycan expression, and they were examined histologically. Hypoxia enhanced initial progenitor cell survival in fresh BMMC-microbeads, but it did not enhance osteogenic potential. Fresh uncultured BMMCmicrobeads showed a similar degree of osteogenesis as culture-expanded MSC-microbeads, even though they initially contained only 1/10th the number of MSC. Chondrogenic differentiation was not strongly supported in any of the microbead formulations. This study demonstrates the microbead-based approach to culturing and delivering cells for tissue regeneration, and suggests that fresh BMMC may be an alternative to using cultureexpanded MSC for bone tissue engineering.

Chitosan-based scaffolds for bone tissue engineering

“…113 Recently, a chitosan-collagen hydrogel microbead was reported as a vehicle for culture and delivery of cultured-expanded mesenchymal stem cells or fresh bone marrow mononuclear cells. 117 The microbeads were fabricated via a two-step process. Chitosan, collagen and β-glycerophosphate were mixed in solution and bead formation was achieved using a water-in-oil emulsion system followed by thermally induced gelation at physiological temperature.…”

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

“…19,20 Cell collections containing bone marrow-derived mononucleated cells (BMNCs)-a small fraction of which are BMSCs-are seeded within biomaterials for 3D isolation, expansion, and subsequent differentiation. 20,21 Cell seeding density is a transplantation variable that has not been evaluated in detail to date for either 2D-or 3D-expanded BMSCs. Healthy articular cartilage naturally contains 9.6 × 10 6 chondrocytes/cm 3 .…”

Distributed vasculogenesis from modular agarose-hydroxyapatite-fibrinogen microbeads

“…A two-paddle impeller stirred the mixture at 700 rpm for 6 min at 37 °C and then for 30 min on ice to gel the resulting microbeads. Previous work has shown that use of FBS acts as a surfactant that facilitates the separation of the beads from the oil phase during production, and also aids in maintaining high cell viability [17, 21]. …”