Mechanical properties, and in vitro biocompatibility assessment of biomimetic dual layered keratin/ hydroxyapatite scaffolds

Abstract

A novel biomimetic dual layered keratin/hydroxyapatite (keratin/HA) scaffold was designed using iterative freeze-drying technique. The prepared scaffolds were studied using several analytical techniques to better understand the biological, structural, and mechanical properties. The developed multilayered, interconnected, porous keratin scaffold with different hydroxyapatite (HA) content in the outer and inner layer, mimics the inherent gradient structure of alveolar bone. SEM studies showed an interconnected porous architecture of the prepared scaffolds with seamless integration between the upper and lower layers. The incorporation of HA improved the mechanical properties keratin/HA scaffolds. The keratin/HA scaffolds exhibited superior mechanical properties in terms of Young's modulus and compressive strength in comparison to pure keratin scaffolds. The biocompatibility studies suggested that both keratin and keratin/HA scaffolds were cyto-compatible, in terms of cell proliferation. Furthermore, it showed that both the tested materials can served as an ideal substrate for the differentiation of Saos-2 cells, leading to mineralization of the extracellular matrix. In summary, ionic liquid based green technique was employed for keratin extraction to fabricate keratin/HA scaffolds and our detailed in vitro investigations suggest the great potential for these composite scaffolds for bone tissue engineering in future.

Keywords: alveolar bone; biomimetic; bone tissue engineering; dental implants; keratin.

FIGURE 1

Preparation of Keratin/hydroxyapatite bi-layered scaffold using HPMC as a cross-linking agent.

FIGURE 2

Multilayered Keratin-HA scaffold general appearance (diameter: 18 mm, height 9 mm) (A,B). Vertical section of multilayered scaffold (C). Scale Bar = 1 mm.

FIGURE 3

(A): Keratin hydroxyapatite scaffold upper layer containing 60% of HA and the lower layer with HA content of 40% (B). Scale Bars 100 µm.

FIGURE 4

Compressive strength (MPa) of keratin and keratin/hydroxyapatite composite scaffolds (n = 10 per scaffold, Error bars represent ±SE of the mean, t-test, **** P ˂ 0.0001).

FIGURE 5

Graphical representation of the Young’s modulus of keratin and keratin/Hydroxyapatite scaffolds (n = 10, Error bars represents ± SE of the mean, t-test, **** P˂ 0.0001).

FIGURE 6

Graphical representation showing Saos-2 cell proliferation on keratin and keratin/Hydroxyapatite scaffolds at three tested time points of 24, 48 and 72 h (Error bars are presented as the standard error of the mean, n = 3, * P ˂ 0.05).

FIGURE 7

(A,C) SEM micrographs of Saos-2 cells on keratin scaffolds showing the cell attachment and the formation of mesh like network on the surface and within the porous structure, (B,D) Saos-2 cell adhesion on keratin/HA scaffolds facilitated by long cytoplasmic extensions within the porous 3-dimensional matrices. (Scale bars represents 10 µm).

FIGURE 8

ALP activity of Saos-2 cells on keratin and keratin/Hydroxyapatite scaffolds after three predefined time points of 3,7 and 14 days (n = 3, Error bars present ± SE of the mean).

FIGURE 9

Optical images of the Alizarin Red S-stained keratin (A) and keratin/HA scaffolds (B). Phase contrast images of Alizarin Red S-stained mineralized nodules formed by Saos-2 cells on keratin (C) and keratin/Hydroxyapatite scaffolds (D) over a culture period of 14 days. (Original magnification: ×200; Scale bars = 20 µm).

FIGURE 10

Optical images of the Alizarin Red S-stained keratin (A) and keratin/Hydroxyaptite scaffolds (B). Phase contrast images of Alizarin Red S-stained mineralized nodules formed by Saos-2 cells on keratin (C) and keratin/Hydroxyapatite scaffolds (D) over a culture period of 21 days. (Original magnification: ×200; Scale bars = 20 µm).

FIGURE 11

Quantitative analysis of calcium deposits by measuring the absorbance of the extracted Alizarin Red dye at day 14 and 21 (n = 3, *** p = 0.0003, ****P˂0.0001, error bars represent ± SE of the mean).

FIGURE 12

Osteocalcin content of keratin and keratin/Hydroxyapatite scaffolds at different cultured time (n = 3, error bars represent ± SE of the mean, **** P˂0.0001, ** p = 0.0012).