Biomimetic mineralized collagen scaffolds and their effect on osteogenic differentiation

Background & Aim During bone healing, MSCs differentiate into bone-forming osteoblasts. This process of osteogenic differentiation is partly driven by the main noncellular constituents of bone: collagen and hydroxyapatite (HA). In terms of ultrastructure, aligned HA crystals are inside collagen fibrils (intrafibrillar) and surround fibrils (extrafibrillar). Mimicking this ultrastructure could enhance autograft alternatives in bone healing. This project aims to test ultrastructurally biomimetic mineralized collagen substrates with two extremes of extrafibrillar mineralization (Fig. 1, conditions A and B) for their effect on gene and protein expression during osteogenic differentiation in comparison to a conventional, nonbiomimetic control (Fig. 1, condition C). Methods, Results & Conclusion Methods EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed collagen suspensions were mineralized with a supersaturated Ca and P solution stabilized with poly-aspartic acid (pAsp), which permits intrafibrillar mineralization. Non-biomimetic substrates were mineralized without pAsp. Scanning electron microscopy (SEM) assessed surface mineral morphology, whereas transmission electron microscopy (TEM) assessed intrafibrillar mineral morphology and location. 2D substrates were generated by drying mineralized suspensions on tissue culture surfaces. Future work will involve testing osteogenic differentiation in 3D using similar mineralization conditions in scaffolds. For testing osteogenic differentiation of human bone marrow MSCs in different mineralization conditions, RT-PCR and immunofluorescent staining are used to quantify gene and protein expression of early (ie. runx2 and osteopontin) vs. later osteoblastic markers (ie. osteocalcin). During bone healing, MSCs differentiate into bone-forming osteoblasts. This process of osteogenic differentiation is partly driven by the main noncellular constituents of bone: collagen and hydroxyapatite (HA). In terms of ultrastructure, aligned HA crystals are inside collagen fibrils (intrafibrillar) and surround fibrils (extrafibrillar). Mimicking this ultrastructure could enhance autograft alternatives in bone healing. This project aims to test ultrastructurally biomimetic mineralized collagen substrates with two extremes of extrafibrillar mineralization (Fig. 1, conditions A and B) for their effect on gene and protein expression during osteogenic differentiation in comparison to a conventional, nonbiomimetic control (Fig. 1, condition C). EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed collagen suspensions were mineralized with a supersaturated Ca and P solution stabilized with poly-aspartic acid (pAsp), which permits intrafibrillar mineralization. Non-biomimetic substrates were mineralized without pAsp. Scanning electron microscopy (SEM) assessed surface mineral morphology, whereas transmission electron microscopy (TEM) assessed intrafibrillar mineral morphology and location. 2D substrates were generated by drying mineralized suspensions on tissue culture surfaces. Future work will involve testing osteogenic differentiation in 3D using similar mineralization conditions in scaffolds.