Material properties and osteogenic differentiation of marrow stromal cells on fiber-reinforced laminated hydrogel nanocomposites

Abstract

The fibrils in the bone matrix are glued together by extracellular matrix proteins to form laminated structures (osteons) to provide elasticity and a supportive substrate for osteogenesis. The objective of this work was to investigate material properties and osteogenic differentiation of bone marrow stromal (BMS) cells seeded on osteon-mimetic fiber-reinforced hydrogel/apatite composites. Layers of electrospun poly( l-lactide) fiber mesh coated with a poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogel precursor solution were stacked and pressed together, and crosslinked to produce a laminated fiber-reinforced composite. Hydroxyapatite (HA) nanocrystals were added to the precursor solution to produce an osteoconductive matrix for BMS cells. Acrylamide-terminated Arg–Gly–Asp (RGD) peptide (Ac-GRGD) was conjugated to the PLEOF/HA hydrogel phase to promote focal point adhesion of BMS cells. Laminates were characterized with respect to the Young’s modulus, degradation kinetics and osteogenic differentiation of BMS cells. The moduli of the laminates under dry and wet conditions were significantly higher than those of the fiber mesh and PLEOF/HA hydrogel, and within the range of values reported for wet human cancellous bone. At days 14 and 21, alkaline phosphatase (ALPase) activity of the laminates was significantly higher than those of the fiber mesh and hydrogel. Lamination significantly increased the extent of mineralization of BMS cells and laminates with HA and conjugated with RGD (Lam-RGD-HA) had 2.7-, 3.5- and 2.8-fold higher calcium content (compared to laminates without HA or RGD) after 7, 14 and 21 days, respectively. The Lam-RGD-HA group had significantly higher expression of osteopontin and osteocalcin compared to the hydrogel or laminates without HA or RGD, consistent with the higher ALPase activity and calcium content of Lam-RGD-HA. Laminated osteon-mimetic structures have the potential to provide mechanical strength to the regenerating region as well as supporting the differentiation of progenitor cells to the osteogenic lineage.