Biomimetic mineralization of collagen
Diana Zeiger, Yajun Cheng, Ryan Koseski, John Howarter, Chris Stafford, and Sheng Lin-Gibson
Mineralized tissues such as bones and teeth are composed of an organic network (primarily type I collagen) infiltrated with carbonated hydroxyapatite. Current understanding of the process by which the mineral nucleates on and in the collagen is limited, although it has been established that acidic proteins act as “chaperone” molecules facilitating calcium phosphate nucleation and orderly mineralization of the collagen fibrils. The objective of this study was to use a polyelectrolyte to direct calcium phosphate mineralization on thin films of type I collagen in a biomimetic manner. Glass cover slips were treated with chlorodimethyl-n-octadecylsilane, and collagen films were permitted to self-assemble on the hydrophobized surface. The collagen was characterized using laser scanning confocal microscopy and atomic force microscopy (AFM). The films were soaked 4 h to 5 d in Tris-buffered saline containing 5 mM CaCl2 and 2.5 mM K2HPO4 (TBS) and various concentrations (0 to 100 µg/mL) of poly(acrylic acid) (pAA). After mineralization, the films were evaluated using AFM, scanning electron microscopy (SEM), electron backscattered diffraction (EBSD), and X-ray photoelectron spectroscopy (XPS). XPS spectra were collected at low take-off angles, sampling 7 nm to 10 nm into the collagen. The diameter of the hydrated collagen fibrils was consistent with that of native type I collagen (~ 300 nm), and the fibrils were observed to assemble in a mesh-like layer. Mineral nucleation on collagen films incubated in pAA-containing TBS was observed as early as 4 h after immersion. AFM images revealed high phase contrast between cover slips and collagen fibrils that were incubated in TBS with pAA; this was not evident in images of films incubated in pAA-free TBS. SEM images showed crystalline material in intimate association with fibrils on samples incubated in mineralizing solution with pAA. This was not observed on samples incubated in pAA-free mineralizing solution. EBSD confirmed the presence of crystalline material in collagen films incubated in TBS with pAA. XPS studies detected substantial C, O, N, Ca, and P. The ratio of calcium to phosphate varied with the concentration of pAA in the mineralizing solution, and also varied over time, with a Ca:P ratio that most closely resembled that of biological apatite achieved using a pAA concentration of 80 µg/mL for an incubation period of 5 d. From this study, it can be concluded that mineralization of collagen fibrils occurred via pAA-directed nucleation. XPS data indicated the presence of CaP, while phase contrasts in AFM images indicated changes in fibril hardness. Support: NIDCR/NIST Interagency Agreement Y1-DE-7005-01.