Supermagnetic Sugarcane Bagasse Hydrochar for Enhanced Osteoconduction in Human Adipose Tissue-Derived Mesenchymal Stem Cells

Abstract

Hydrothermally carbonized sugarcane bagasse (SCB) has exceptional surface properties. Looking at the huge amount of SCB produced, its biocompatible nature, cheap-cost for carbonization, and its easy functionalization can give impeccable nano-biomaterials for tissue engineering applications. Herein, sugarcane bagasse was converted into hydrochar (SCB-H) by hydrothermal carbonation. The SCB-H produced was further modified with iron oxide (Fe₃O₄) nanoparticles (denoted as SCB-H@Fe₃O₄). Facile synthesized nano-bio-composites were characterized by SEM, HR-TEM, XRD, FT-IR, XPS, TGA, and VSM analysis. Bare Fe₃O₄ nanoparticles (NPs), SCB-H, and SCB-H@Fe₃O₄ were tested for cytocompatibility and osteoconduction enhancement of human adipose tissue-derived mesenchymal stem cells (hADMSCs). The results confirmed the cytocompatible and nontoxic nature of SCB-H@Fe₃O₄. SCB-H did not show enhancement in osteoconduction, whilst on the other hand, Fe₃O₄ NPs exhibited a 0.5-fold increase in the osteoconduction of hADMSCs. However, SCB-H@Fe₃O₄ demonstrated an excellent enhancement in osteoconduction of a 3-fold increase over the control, and a 2.5-fold increase over the bare Fe₃O₄ NPs. Correspondingly, the expression patterns assessment of osteoconduction marker genes (ALP, OCN, and RUNX2) confirmed the osteoconductive enhancement by SCB-H@Fe₃O₄. In the proposed mechanism, the surface of SCB-H@Fe₃O₄ might provide a unique topology, and anchoring to receptors of hADMSCs leads to accelerated osteogenesis. In conclusion, agriculture waste-derived sustainable materials like "SCB-H@Fe₃O₄₄" can be potentially applied in highly valued medicinal applications of stem cell differentiation.

Keywords: hADMSCs; hydrochar; hydrothermal carbonation; osteoconduction enhancement; sugarcane bagasse.

Scheme 1

Flow chart of sugarcane bagasse (SCB) source and synthesis process of SCB-H@Fe₃O₄.

Figure 1

SEM image of (A) SCB hydrochar (SCB-H) and (B) SCB-H@Fe₃O₄, (C) the zoomed (100 nm) surface of SCB-H@Fe₃O₄, (D) HR-TEM image of the SCB-H@Fe₃O₄ surface, (E) HAADF-STEM image of SCB-H@Fe₃O₄, and EDS elemental mapping images of (F) carbon (C), (G) iron (Fe) and (H) oxygen (O).

Figure 2

XRD (A) and FT-IR (B) analysis of SCB-H and SCB-H@Fe₃O₄, (C) XPS analysis of SCB-H@Fe₃O₄, (D) high-resolution spectrum of Fe 2p, TGA analysis of SCB-H and SCB-H@Fe₃O₄ (E), and VSM analysis of Fe₃O₄ and SCB-H@Fe₃O₄ (F).

Figure 3

Cytotoxicity of studied materials against the hADMSCs. (A–C) hADMSCs viability measured after 24 h, and (D–F) hADMSCs viability measured after 14 d. Nano-scaffolds; (A,D) Fe₃O₄ (B,E) SCB-H, and (C,F) SCB-H@Fe₃O₄. *** p < 0.001.

Figure 4

Analysis of calcium deposition by ARS staining. hADMSCs were differentiated to osteoblasts for 14 days in osteogenic induction media with or without nano-scaffolds. After differentiation, the calcium deposition was determined with ARS staining. Intracellular and extracellular calcium was stained to red by ARS. C: vehicle, 1: 25 μg /mL, 2: 50 μg /mL 3: 100 μg/mL.

Figure 5

Quantification of osteogenesis. hADMSCs were differentiated to osteoblasts for 14 days in osteogenic induction media with or without nano-scaffolds. After differentiation, the osteogenic markers were quantified by using ARS staining and real-time RT PCR. (A) Quantification of ARS staining. (B–D) mRNA level of osteogenic marker. C: vehicle, nano-scaffolds concentration from 25, 50 to 100 µg/mL for every sample of bare Fe₃O₄, SCB-H, and SCB-H@Fe₃O₄. * p < 0.05, ** p < 0.01 *** p < 0.001 in comparison with untreated cells.

Figure 6

The protein levels of OCN and ALP. hADMSCs were differentiated to osteoblasts for 14 days in osteogenic induction media with or without nano-scaffolds. After differentiation, the protein levels of osteogenic markers (A) ALP and (B) OCN were analyzed by immunocytochemistry. All results were quantified using ImageJ Software (C) ALP and (D) OCN. All materials are treated with 100 µg/mL for every cell. C: vehicle, * p < 0.05.