Remodeling of Three-Dimensional Collagen I Matrices by Human Bone Marrow Stromal Cells during Osteogenic Differentiation In Vitro

Abstract

Cell fate is triggered by the characteristics of the surrounding extracellular matrix (ECM) including its composition and topological and mechanical properties. Human bone marrow stromal cells (hBMSC) are known to reside in a niche environment where they are maintained in a quiescent, multipotent state, also controlled by the ECM characteristics. In this in vitro study, three-dimensional (3D) fibrillary collagen I (Col)-based matrices with defined topological and mechanical characteristics were used (pore size of 3-4 μm, fibril diameter of ∼0.7 μm, ∼90 Pa (non-cross-linked), and ∼160 Pa (cross-linked)), mimicking conditions of the environment in the bone marrow. The performance of non-cross-linked and cross-linked scaffolds during osteogenic differentiation of hBMSC in terms of matrix stiffness and proteolytic degradability was investigated. Cell adhesion, morphology, and invasion as well as matrix remodeling were investigated on cross-linked and non-cross-linked Col matrices over 22 days. About 25% of the cells invaded the matrices and showed a spread morphology independent of cross-linking. Cellular proteolytic matrix degradation in terms of a decreased matrix layer thickness was only found for non-cross-linked matrices at constant pore size and fibril diameter. Osteogenic differentiation of hBMSC was examined by alkaline phosphatase staining and enzyme activity (early marker) and calcium phosphate deposition (late marker) and was similarly supported in both scaffolds. Furthermore, both matrices were strongly stiffened by about 10-fold because of high mineralization under osteogenic conditions. In summary, these results emphasize that fibrillary 3D Col matrices are a suitable model to study primary hBMSC behavior in terms of ECM remodeling during osteogenesis at defined in vitro conditions.