Casein-Coated Molybdenum Disulfide Nanosheets Augment the Bioactivity of Alginate Microspheres for Orthopedic Applications

Abstract

Defects and disorders of the bone due to disease, trauma, or abnormalities substantially affect a person's life quality. Research in bone tissue engineering is motivated to address these clinical needs. The present study demonstrates casein-mediated liquid exfoliation of molybdenum disulfide (MoS₂) and its coupling with alginate to create microspheres to engineer bone graft substitutes. Casein-exfoliated nano-MoS₂ was chemically characterized using different analytical techniques. The UV-visible spectrum of nano-MoS₂-2 displayed strong absorption peaks at 610 and 668 nm. In addition, the XPS spectra confirmed the presence of the molybdenum (Mo, 3d), sulfur (S, 2p), carbon (C, 1s), oxygen (O, 1s), and nitrogen (N, 1s) elements. The exfoliated MoS₂ nanosheets were biocompatible with the MG-63, MC3T3-E1, and C2C12 cells at 250 μg/mL concentration. Further, microspheres were created using alginate, and they were characterized physiochemically and biologically. Stereomicroscopic images showed that the microspheres were spherical with an average diameter of 1 ± 0.2 mm. The dispersion of MoS₂ in the alginate matrix was uniform. The alginate-MoS₂ microspheres promoted apatite formation in the SBF (simulated body fluid) solution. Moreover, the alginate-MoS₂ was biocompatible with MG-63 cells and promoted cell proliferation. Higher alkaline phosphatase activity and mineralization were observed on the alginate-MoS₂ with the MG-63 cells. Hence, the developed alginate-MoS₂ microsphere could be a potential candidate for a bone graft substitute.

Figure 1

(A) Schematic depiction of the exfoliation of MoS₂ with casein. (B) Visual observations of the exfoliated nano-MoS₂ solution at different time intervals. (C) UV–vis spectra of the exfoliated nano-MoS₂ at different time intervals. (D) Absorbance of the exfoliated nano-MoS₂ at 660 nm at different time intervals.

Figure 2

(A) Bulk production of nano-MoS₂ with casein (100 mL batch). (B) FT-IR spectrum of (a) casein, (b) commercial MoS₂, (c) casein nano-MoS₂-1, and (d) casein nano-MoS₂-2. (C) XRD spectrum of exfoliated nano-MoS₂-2. (D) Raman spectra of exfoliated nano-MoS₂-2. (E) HR-TEM images of exfoliated nano-MoS₂-2. (F) AFM images of exfoliated nano-MoS₂-2.

Figure 3

XPS analysis of the developed nano-MoS₂-2 and the individual elemental image: (A) nano-MoS₂-2; (B) Mo, 3d; (C) S, 2p; (D) C, 1s; (E) O, 1s; and (F) N, 1s.

Figure 4

(A) Schematic representation of the development of microspheres. (B) Photographs of solution of (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2. (C) Photographs of the microspheres of (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2. (D) stereomicrographs of (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2.

Figure 5

(A) FT-IR spectrum of (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2. (B) XRD patterns of (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2. (C) TGA graph of (a) alginate and (b) alginate–MoS₂-1. (D) Mechanical strength of the developed microspheres (alginate–MoS₂-2). (E) High- and low-magnification FE-SEM images and corresponding EDS spectra of alginate (a, b, and c), alginate–MoS₂-1 (d, e, and f), and alginate–MoS₂-2 (g, h, and i).

Figure 6

(A) Water uptake and retention of the developed microspheres containing 3% alginate, alginate–MoS₂-1, and alginate–MoS₂-2. (B) Biodegradation results of 3% alginate, 3% alginate–MoS₂-1, and alginate–MoS₂-2 biocomposite microspheres. (C) Protein adsorption studies of the alginate–MoS₂-1 and alginate–MoS₂-2 composite microspheres.

Figure 7

(A) Stereo-microscopic images of the microspheres after immersion in SBF: (a) alginate, (b) alginate–MoS₂-1, and (c) alginate–MoS₂-2. (B) FT-IR spectrum of microspheres after immersion in SBF: graph (a) alginate, (b) alginate–MoS₂-1. (c) alginate–MoS₂-2; and (C) MTT assay results with MG-63 osteoblast-like cells at different concentrations of the microspheres, measured using three independent values.

Figure 8

FE-SEM-EDX images (A, B) for 3% alginate at 500 and 10 μm magnifications, respectively; images (D, E) for alginate-nano-MoS₂-1 at 500 and 10 μm magnifications, respectively; images (G, H) for alginate-nano-MoS₂-2 at 500 and 10 μm magnifications, respectively; images (C, F) and (I) EDX images of 3% alginate, alginate-nano-MoS₂-1, and alginate-nano-MoS₂-2, respectively.

Figure 9

Optical micrographs of (A) C2C12 cells, (B) MC3T3-E1 cells, and (C) MG-63 cells after treatment with (a) control, (b) 50 μg/mL of exfoliated nano-MoS₂-2, and (c) 250 μg/mL of exfoliated nano-MoS₂-2.

Figure 10

Fluorescent micrographs showing AO/EB-stained MG-63 cells with microspheres. (A) Green channel images for (a₁) alginate, (b₁) alginate–MoS₂-1, and (c₁) alginate–MoS₂-2; (B) Red channel images for (a₂) alginate, (b₂) alginate–MoS₂-1, and (c₂) alginate–MoS₂-2; (C) Merged images of green and red channels for (a₃) alginate, (b₃) alginate–MoS₂-1, and (c₃) alginate–MoS₂-2; (D) Hoechst 33342 staining images for (a₄) alginate, (b₄) alginate–MoS₂-1, and (c₄) alginate–MoS₂-2. Scale bar = 100 μm.

Figure 11

Alkaline phosphatase (ALP) activity of alginate, alginate–MoS₂-1, and alginate–MoS₂-2 microspheres. ALP activity of the microspheres after (A) 7 days and (B) 14 days of incubation. The data are presented as the mean ± standard deviation (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant (compared with 3% alginate microspheres).

Figure 12

Mineralization potential of the developed microspheres on the MG-63 cell lines. In image A, images (a₁, b₁, and c₁) are the optical microscopic images of alginate, alginate–MoS₂-1, and alginate–MoS₂-2, respectively, taken after 7 days, and images (a₂, b₂, c₂) after 14 days (scale bar = 50 μm). Image B represents the quantitative mineralization measured at 562 nm after 14 days. The data are presented as the mean ± standard deviation (n = 3).