The Osteoinductive Potential of Biomimetic Mineralized Collagen Scaffolds for Bone Regeneration

Luo, Lucy (1), Uludag, Hasan (2), Viswanathan, Sowmya (1, 3), Sone, Eli (1)

(1) Institute of Biomaterials and Biomedical Engineering, University of Toronto

(2) Chemical and Materials Engineering, University of Alberta

(3) Cell Therapy Program, University Health Network

INTRODUCTION: Bone comprises fibrillar collagen with aligned apatitic mineral in embedded within collagen fibrils (intrafibrillar mineral) and surrounding fibrils (extrafibrillar mineral). Bone tissue engineering scaffolds could serve as alternatives to autografts when bone healing fails. This project aims to assess the effect of mineral location and alignment in collagen scaffolds on osteoinduction in MSCs in vitro. Biomimetic scaffolds are expected to induce greater expression of mature osteoblast markers in MSCs compared to non-biomimetic scaffolds.

METHODS: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)-fixed collagen scaffolds were mineralized for 2, 3, 4, and 6 days with a supersaturated Ca and P solution stabilized by poly-Aspartic acid (pAsp). This process permits biomimetic, intrafibrillar mineralization seen in native bone as opposed to mineralization with a supersaturated Ca and P solution without pAsp.1,2 Scanning electron microscopy (SEM) was used to assess mineral alignment and location, and backscattered electrons detected heavier elements such as Ca and P. Transmission electron microscopy (TEM) was used to identify the mineral phase as apatite and mineral alignment was determined using 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 (e.g. type I collagen) vs. late osteoblastic markers (e.g. osteocalcin).

RESULTS: Scaffolds mineralized with pAsp for 6 days exhibited more unaligned surface mineral compared to those mineralized for 3 or 4 days. SAED confirmed aligned, intrafibrillar mineral. Scaffolds mineralized for 2 days exhibited no detectable mineralization with SEM and backscatter SEM. Non-biomimetic scaffolds mineralized without pAsp demonstrated extrafibrillar spherulites of mineral.

DISCUSSION & CONCLUSIONS: Mineralization for 3 vs. 6 days will allow for comparison between scaffolds with aligned, intrafibrillar mineral compared to scaffolds with less aligned surface mineral for future experiments regarding osteogenic differentiation in MSCs. Scaffolds mineralized without pAsp will be used as non-biomimetic controls. Ultimately, this bone tissue-engineering approach is expected to enhance the osteoinductivity of bone scaffolds as alternatives to autografts in the treatment of non-union fractures.