Intrafibillarly-silicified Collagen Scaffolds for Tissue-Engineering. I. In-vitro Bioactivity, Osteogenic/Angiogenic Potential

Abstract

Objective: Highly-porous, biomimetic, intrafibrillarly-silicified collagen scaffolds (SCSs), inspired by diatom biosilicification, have been developed (Niu et al. Angew Chem Int Ed 2011;50:11688–91). This study examined the in-vitro bioactivity of SCSs, their biocompatibility and osteogenic/angiogenic potentials after exposure to murine mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). Method: SCS were prepared using choline-stabilized silicic acid to infiltrate poly(allylamine)-enriched, type I collagen sponges. Their in-vitro bioactivity, after immersion in simulated body fluid, was examined by TEM, selected-area electron diffraction and STEM-EDX. Viability/proliferation of murine MSCs and EPCs, after exposure to SCSs, was examined using MTT assay and flow cytometry. mRNA expressions of genes associated with osteogenesis and angiogenesis were examined with qRT-PCR. In-vitro osteogenesis of osteoblast-like cells differentiated from MSCs was examined by determining the calcium content of extracellular mineralization (Alizarin red S assay). In-vitro pro-angiogenesis was examined after culturing differentiated EPCs in ECMatrixTM and counting endothelial tube branch-points. Statistical analysis was performed for each parameter using one-way ANOVA and Holm-Sidak tests (α=0.05).

Result: Apatite was deposited along the SCS surface after 48 h. SCSs were as biocompatible as non-silicified CSs control (MTT-succinic dehydrogenase activity; flow cytometry-% apoptotic cells; P>0.05). Differentiated MSCs exposed to SCSs exhibit highly-significant (P<0.001) fold-increases in expressions of genes associated with alkaline phosphatase, Runt-related transcription factor 2, osteocalcin, and bone sialoprotein II, compared with those without SCS exposure, or exposed to non-silicified CSs. Differentiated EPCs exposed to SCSs exhibit highly-significant (P<0.001) fold increases in expressions of vascular endothelial growth factor-A and angiopoetin-2 genes. The results of mRNA expressions were further confirmed by significantly more extracellular calcium deposits by differentiated MSCs, and tube formation by differentiated EPCs in the presence of SCSs, compared with non-silicified CS (P<0.05).

Conclusion: The highly-desirable qualities of SCSs justify their loading with chemoattractants for stem/progenitor cell homing in hard tissue engineering.