Mineralized DNA-collagen complex-based biomaterials for bone tissue engineering

Abstract

DNA is a highly polyanionic biomolecule that complexes with both collagen and hydroxyapatite. By combining these complexes, we synthesized nucleic-acid collagen complexes (NACC) mineralized with hydroxyapatite. The composite complexes were made using a short, monodisperse single-stranded DNA, type I collagen, and mineralizing medium. They rapidly self-assembled into both mineralized NACC microfibers and 3D NACC gels. At the nanoscale, these complexes are hierarchical, interwoven, curly nanofibrils resembling native extracellular matrix, which mineralized an interpenetrating nanocrystalline hydroxyapatite phase. Mineralization was able to be done either before or after NACC formation enabling temporal control of the process. In response to the NACC material, primary human osteoblasts took on an osteocyte-like morphology. Moreover, the cells agglomerated and remodeled the NACC gels into densified, tissue-like structures within 3 days. NACC fibers and gels have promise not only as osteoconductive coatings and scaffolds, but as coatings and scaffolds for any tissue using this new form of naturally-derived biomaterials.

Keywords: DNA-collagen complex; Self-assembly; Tissue engineering.

Figure 1.

A representative image of NACC fibers formed at ~14% mass fraction DNA in solution stained for DNA (a). ssDNA-collagen bindings on a mole per mass basis (b) as a function of mass fraction DNA in solution. A representative image of mineralized immobilized NACC fibers formed at ~14% mass fraction DNA in solution stained with alizarin red (c). Quantified alizarin red stain bound to the mineralized immobilized NACC fibers as a function of bound ssDNA/collagen on a mole per mass basis (d). Each data point in (b) and (d) was measured in triplicate and error bars are one standard deviation.

Figure 2.

ssDNA fluorescently stained using SYBR Safe DNA stain (Invitrogen) to visualize the NACC gel after formation in 0.6 mL microcentrifuge tubes.

Figure 3.

TEM micrographs of 1% phosphotungstic acid stained NACC fibrils (a) and high resolution TEM micrographs of those fibrils with an inset representative line profile of an individual fibril (b).

Figure 4.

Characterization of mineralized NACCs. XRD patterns for two-step mineralized NACCgels after 1, 3, and 6 days in mineralization solution (a). A representative TEM micrograph of two-step mineralized NACC fibers after 6 days in mineralization solution (b) with a representative SAED pattern (inset).

Figure 5.

X-ray diffraction pattern of one-step mineralized NACC gel (a). Transmission electron micrograph of one-step mineralized incubated in mineralizing solution for 1 day (b) and unmineralized (c) NACC fibers with their associated small area electron diffraction patterns (b-c insets).

Figure 6.

Representative SEM micrographs of the surface of a random ssDNA two-step mineralized gel after 6 days in mineralization solution (a) with higher magnification micrographs of central region designated (b).

Figure 7.

Representative phase contrast microscope images of HObs taken over 2 days of culture on tissue culture polystyrene (a, d), unmineralized (b, e), and mineralized (c, f) immobilized NACC fibers.

Figure 8.

Representative immunocytochemistry fluorescence microscope images of HObs taken after 3 days of culture stained for cytoskeletal F-actin (a, b) and osteopontin (c, d) on unmineralized and mineralized immobilized NACC fibers.

Figure 9.

HObs aggregated the dispersed NACC gel into a dense tissue over a 2 day period.

Figure 10.

A representative image of live HObs stained with calcein AM (Invitrogen) following the manufacturer’s instructions after 24 hours of culture in the NACC gel (a). Microscopy image of the fixed unmineralized cell densified NACC gel (b) with associated composite images of the embedded cell nuclei stained with Hoescht 33342 (c). Scale bar in (b-c) is 3000 μm.

Figure 11.

Microscopy image of the fixed mineralized cell densified random NACC gel (a) with associated composite images of the embedded cell nuclei stained with Hoescht 33342 (b). Scale bar in (a-b) is 3000 μm.