Biomimetic organization of collagen matrices to template bone-like microstructures

• A two-step process to create a lamellar bone-like composite is demonstrated.
• Step 1: molecular crowding to assemble collagen into cholesteric microstructure
• Step 2: PILP mineralization to attain intrafibrillar nanocrystals of hydroxyapatite
• Organized collagen matrices templated organized hydroxyapatite nanocrystals.
• A nano- to micro-scale hierarchical composite mimicking lamellar bone was formed.

The mineralized extracellular matrix (ECM) of bone is essential in vertebrates to provide structure, locomotion, and protect vital organs, while also acting as a calcium and phosphate reservoir to maintain homeostasis. Bone's structure comprises mainly structural collagen fibrils, hydroxyapatite nanocrystals and water, and it is the organization of the densely-packed collagen matrix that directs the organization of the mineral crystallites. Biogenic mineralization occurs when osteoblasts release “mineral bearing globules” which fuse into the preformed collagen matrix, and upon crystallization of this amorphous precursor, the fibrils become embedded with [001] oriented nanocrystals of hydroxyapatite. Our prior work has shown that this nanostructured organization of bone can be reproduced in vitro using the polymer-induced liquid-precursor (PILP) process. In this report, our focus is on using biomimetic processing to recreate both the nano- and micro-structure of lamellar bone. We first applied molecular crowding techniques to acidic, type-I collagen solutions to form dense, liquid crystalline collagen (LCC) scaffolds with cholesteric order. We subsequently mineralized these LCCs via the PILP process to achieve a high degree of intrafibrillar mineral, with compositions and organization similar to that of native bone and with a “lamellar” microstructure generated by the twisting LCC template. In depth characterization of the nano- and micro-structure was performed, including optical and electron microscopy, X-ray and electron diffraction, and thermogravimetric analyses. The results of this work lead us closer to our goal of developing hierarchically structured, collagen-hydroxyapatite composites which can serve as fully synthetic, bioresorbable, load-bearing bone substitutes that are remodeled by the native BRU.