Assessing Collagen Assembly and Apatite Formation by Atomic Force Microscopy

OBJECTIVE: Use Atomic Force Microscopy (AFM) topography analysis to evaluate (1) the success of lab-processed collagen assembly to the biomimetic process seen and documented with MC3T3-E1 preosteoblast culture and (2) to compare the subsequent mineralization capacity, after exposure to simulated body fluid (SBF). METHODS: Acid-dissolved rat tail type-I collagen was diluted in Tris-HCl buffer (pH 9) containing 200mM KCl. This collagen solution (12ug/ml) was deposited onto a mica surface (10X10mm2) to develop the self-assembled fibrils (n=3), which were then characterized by AFM. Specimens of the multilayer collagen (MLCol; n=3) were prepared by repetitive depositions (up to 10 layers), followed by air-drying and immersion in SBF for 1 or 2 weeks. X-ray Diffraction (XRD) and AFM were used to detect the formation of hydroxyapatite in collagen. The AFM and picro-sirius red stain were used to assess collagen and apatite formation in MC3T3-E1 cell cultures (n=3) at day 21 following the differentiation assay. RESULTS: Most of the AFM spectra from the lab-processed assembled collagen fibrils showed parallel and unidirectional patterns, with minor branches running at 60° offset between the fibrils. Comparing with the MC3T3-E1 cell culture's AFM spectra, it was evident that both showed similar weaving-pattern collagen structure networks with ~67nm-periodicity. Moreover, XRD detected crystalline hydroxyapatite formed in both the 1- and the 2-week SBF immersion samples. AFM illustrated a mesh-like pattern of crystallization in the 2-week sample. The cell culture also revealed similar mesh-like apatite formation that was confirmed with Alizarin red staining. CONCLUSION: Unidirectional MLCol assembly can be produced and mineralized in the laboratory and is analogue to that of collagen synthesis and mineralization of the MC3T3-E1 cells. This finding is a promising direction for future improvement in biomaterials for bone regeneration. (Supported, in part, by NIDCR-K08DE018695, R43DE020971, AAOF, NC Biotech Center)