Abstract
Background & Aim In bone healing, osteoblasts derived from periosteal mesenchymal stromal cells (MSCs) form new bone. Osteoblastic differentiation from MSCs (osteoinduction), is partly driven by the components and mechanics of the extracellular matrix (ECM). Bone ECM comprises fibrillar collagen with apatitic mineral in gap zones between collagen molecules (intrafibrillar) and surrounding fibrils (extrafibrillar). This project aims to optimize mineralization conditions for fabricating bone biomimetic hydroxyapatite (HA)-collagen scaffolds. These biomimetic scaffolds will later be tested for their osteoinductive potential in mesenchymal stromal cells (MSCs). Methods, Results & Conclusion Methods EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed fibrillar collagen scaffolds were mineralized with a metastable, supersaturated Ca and P solution for 2, 4, 6, and 8 days. This process permits biomimetic, intrafibrillar mineralization seen in native bone. Non-biomimetic scaffolds were mineralized in an unstable, supersaturated Ca and P solution. Scanning electron microscopy (SEM) assessed mineral morphology and location, and backscattered electrons detected heavier elements such as Ca and P. Transmission electron microscopy (TEM) identified HA and its alignment with selected area electron diffraction (SAED). To compare osteoinduction by biomimetic scaffolds vs. non-biomimetic scaffolds, qRT-PCR and immunofluorescent staining will be used to quantify gene and protein expression of early (ie. type I collagen and fibronectin) vs. late osteoblastic markers (ie. alkaline phosphatase and osteocalcin). Biomimetic scaffolds are expected to induce greater expression of later osteoblast markers in MSCs compared to non-biomimetic scaffolds. Results Biomimetic scaffolds mineralized for 6 and 8 days exhibited unaligned, extrafibrillar mineral compared to those mineralized for 4 days (Fig.1). SAED confirmed aligned, intrafibrillar mineral. Scaffolds mineralized for 2 days exhibited no detectable mineralization with SEM and backscatter SEM. Non-biomimetic scaffolds demonstrated extrafibrillar amorphous and crystalline mineral phases. Conclusion Mineralization for 4 days with pAsp is the optimal time point for biomimetic mineralized collagen scaffolds for future studies regarding osteogenic differentiation in MSCs. Ultimately, this biomimetic approach is expected to enhance the osteoinductivity of scaffolds as alternatives to autografts in the treatment of non-union fractures.
Published Version
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