Surface-Directed Mineralization of Fibrous Collagen Scaffolds in Simulated Body Fluid for Tissue Engineering Applications

Abstract

The use of polymer additives that stabilize fluidic amorphous calcium phosphate is key to obtaining intrafibrillar mineralization of collagen in vitro. On the other hand, this biomimetic approach inhibits the nucleation of mineral crystals in unconfined extrafibrillar spaces, that is, extrafibrillar mineralization. The extrafibrillar mineral content is a significant feature to replicate from hard connective tissues such as bone and dentin as it contributes to the final microarchitecture and mechanical stiffness of the biomineral composite. Herein, we report a straightforward route to produce densely mineralized collagenous composites via a surface-directed process devoid of the aid of polymer additives. Simulated body fluid (1×) is employed as a biomimetic crystallizing medium, following a preloading procedure on the collagen surface to quickly generate the amorphous precursor species required to initiate matrix mineralization. This approach consistently leads to the formation of extrafibrillar bioactive minerals in bulk collagen scaffolds, which may offer an advantage in the production of osteoconductive collagen-apatite materials for tissue engineering and repair purposes.

Keywords: amorphous precursor; apatite; mineralization; nanofibrous scaffolds; type-I collagen.

Figure 1.

As-synthesized collagen matrix seen through (A) 3–D laser scanning microscopy and (B,C) FESEM at different magnifications. The high resolution FESEM images reveal a dense network of type-I collagen fibrils with preserved interfibrillar spaces. Magnifications, ×9500 (B) and ×20,000 (C).

Figure 2.

FESEM morphologies of collagen fibrils subjected to different mineralization approaches: (A) incubation in 1× SBF solely; (B) alternate soaking in calcium- and phosphate-rich ionic solutions solely; (C) alternate soaking followed by incubation in 1× SBF. Remarkable mineralization occurred only when the fibrils were incubated in SFB after an initial mineral induction (i.e., soaking in calcium- and phosphate-rich solutions). Arrows, nucleation sites. Magnification, ×20,000.

Figure 3.

TEM images of type-I collagen fibrils mineralized in 1× SBF following the mineral induction step (preloading). (A) Longitudinal section of a banded collagen fibril completely encased by extrafibrillar apatite crystals in intimate contact with the collagen structure. (B) At lower magnification, it is possible to observe a continuous fibril strand surrounded by in-situ mineralized apatite crystals, which appear as dark needles due to the high electron density of crystalline CaP. Specimens lightly post-stained with uranyl acetate. Direct magnification, ×15,000 (A) and ×8000 (B).

Figure 4.

FESEM morphologies of collagen fibrils mineralized in SBF after incubation for (A,B) 24 h, (C,D) 48 h, (E,F) 72 h, and (G,H) 96 h. Yellow squares highlight the transitions between magnifications of ×9500 and ×20,000 for the same detail.

Figure 5.

Histograms showing the statistical distributions of diameter of (A) the control/as-synthesized collagen fibrils and (B–E) fibrils mineralized in SBF after incubation for 24–96 h. (F) Evolution of the average diameter with time as a consequence of gradual mineralization of apatite around the fibrils. Extended incubation in SBF for 96 h promoted an eightfold increase in diameter compared to the control collagen fibrils.

Figure 6.

(A) Concentration of Ca²⁺ ions in the mineralization medium after each 24-h incubation cycle. (B) Cumulative uptake of Ca²⁺ ions from the mineralization medium according to the total incubation time. Continuous extrafibrillar mineralization is verified by an increasing linear trend (dashed line) in the Ca²⁺ uptake (from the media to the fibrils' surface) during the incubation in SBF.

Figure 7.

(Left) Representative FESEM–EDS layered images and (right) EDS spectra of collagen fibrils mineralized in SBF for (A,B) 24 h, (C,D) 48 h, (E,F) 72 h, and (G,H) 96 h. Scale bars, 5 μm. Insets, relative concentration of the elements Calcium, Phosphorous, Oxygen, Carbon, Magnesium, and Sodium.

Figure 8.

Effect of incubation time on the Ca/P atomic ratio of the apatitic coatings mineralized around the collagen fibrils. Extended incubation in SBF enabled the transformation of precursor species (lower Ca/P ratios) into a carbonated apatite with Ca/P ratio of 1.61 ± 0.04. TCP, tricalcium phosphate.

Figure 9.

FTIR–ATR analysis. (A) Absorbance spectra of nonmineralized and apatite-mineralized collagen fibrils. (B) Normalized spectra of collagen fibrils mineralized after incubation in SBF for 24–96 h. Extended incubation in SBF led to a decrease in the peak intensity of the amide I band as the collagen structure became less accessible with the thickening of the external apatitic coating.