Abstract
This work explores in depth the simultaneous self-assembly and mineralization of type I collagen by a base-acid neutralization technique to prepare biomimetic collagen-apatite fibrils with varying mineralization extent and doped with luminescent bactericidal Tb3+ ions. Two variants of the method are tested: base-acid titration, a solution of Ca(OH)2 is added dropwise to a stirred solution containing type I collagen dispersed in H3 PO4 ; and direct mixing, the Ca(OH)2 solution is added by fast dripping onto the acidic solution. Only the direct mixing variant yielded an effective control of calcium phosphate polymorphism. Luminescence spectroscopy reveals the long luminescence lifetime and high relative luminescence intensity of the Tb3+ -doped materials, while two-photon confocal fluorescence microscopy shows the characteristic green fluorescence light when using excitation wavelength of 458nm, which is not harmful to bone tissue. Cytotoxicity/viability tests reveal that direct mixing samples show higher cell proliferation than titration samples. Additionally, osteogenic differentiation essays show that all mineralized fibrils promote the osteogenic differentiation, but the effect is more pronounced when using samples prepared by direct mixing, and more notably when using the Tb3+ -doped mineralized fibrils. Based on these findings it is concluded that the new nanocomposite is an ideal candidate for bone regenerative therapy.
Highlights
Type-I collagen (Col) is the most abundant structural protein in mammals
We studied the cytocompatibility of the samples and their ability to induce osteogenic differentiation of human mesenchymal stem cells
The X-ray diffraction (XRD) patterns of samples prepared by the base-acid titration variant at reagent concentrations C1, C2, C3 and Col/calcium phosphate (CaP) ratios 50:50, 70:30 and 90:10 are shown in Figure 1 (a-c)
Summary
Type-I collagen (Col) is the most abundant structural protein in mammals. It can be found in tendon and epithelial tissues, as well as in bone and teeth, where plays a structural role as an extracellular protein, and determine their mechanical and elastic behaviour. [1,2]Bone is a mineralized tissue with particular hierarchical architecture, mechanical properties and remodeling capabilities.[2]. Type-I collagen (Col) is the most abundant structural protein in mammals. It can be found in tendon and epithelial tissues, as well as in bone and teeth, where plays a structural role as an extracellular protein, and determine their mechanical and elastic behaviour. Bone is a mineralized tissue with particular hierarchical architecture, mechanical properties and remodeling capabilities.[2] The basic building unit of the intimate structure of the bone is a self–assembled collagen fibril, mainly type I collagen, mineralized with apatite nanocrystals at both intrafibrillar and interfibrillar zones.[3,4,5,6] Type I collagen is a trimeric molecule that consist of two 1 and one 2 peptide chains, formed by a repetitive sequence of glycine–X–Y, with X and Y being normally proline and hydroxyproline residues. The interaction between tropocollagen units leads to the formation of fibrillar structures with a regular array of gaps and overlap spaces, observed in transmission electron microscopy (TEM) as a periodic banding pattern of 67 nm (D-spacing).[3,4,5,6] The mineral component is a poorly crystalline, non-stoichiometric Ca2+– and OH–deficient apatite -the stoichiometric hydroxyapatite (HA) is Ca10(PO4)6(OH)2-, coated with citrate and doped with 4– 6 wt % of carbonate, 0.9 wt% Na, 0.5 wt% Mg and others minor elements.[7,8]
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