Gelatin-Derived Graphene–Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

Abstract

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased the ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPARγ was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.

Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

“…Total RNA was isolated using TRIzol Reagents (Invitrogen, Carlsbad, CA, USA) as described. 54 , 55 , 56 The cDNA synthesis was carried out using hexamer and M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA). The cDNA products were diluted 10- to 50-fold and used as PCR templates.…”

Developing a Versatile Shotgun Cloning Strategy for Single-Vector-Based Multiplex Expression of Short Interfering RNAs (siRNAs) in Mammalian Cells

“…The image data were quantitatively analyzed by Amira 5.6 (Visage Imaging, Inc.), and 3-D volumetric data and bone density were determined as previously described. 84 − 87 …”

In vitro effect of graphene structures as an osteoinductive factor in bone tissue engineering: A systematic review

“…57 Polymers include poly(L-lactide) (PLLA), 46 poly(dopamine) (PDA), 68 poly(ɛ-caprolactone) (PCL), 14,36,203 poly(ethylenimine) (PEI), 37, 82 poly(lacticcoglycolic acid) (PLGA)-tussah, 61 silk fibroin (SF), 43 polyethylene terephthalate-based artificial ligament group (PET-ALs), 78 47,65,89 and inductively coupled plasma-optical emission spectroscopy (ICP-OES) 49,71 were employed to measure the release rate of ions, and inductively coupled plasma-mass spectroscopy (ICP-MS) 64 was utilized to assess the Ca/P ratio. Additional assays and measures used for sample characterization include thermal gravimetric analysis (TGA), 37,71,82,84,87 energy dispersive X-ray spectroscopy (EDX), 33 75 swelling study, 75 weight loss measurement, 60 diametral tensile strength (DTS), 60 nanoindentation measurements, 67, 76 photoluminescence (PL) spectra, 94 elemental analysis experiment, 25 magnetometry, 25 selected-area electron diffraction (SAED), 57, 91 wettability evaluation, 89 roughness evaluation 14,28,89 and PL spectrum. 94…”

“…The -NH2 and C=O functional groups of Ct were reported to contribute to bio-mineralization. 48 Zou et al 87 demonstrated that Gn-derived graphene/laponite (GL) powder enhanced osteodifferentiation due to the physical stresses induced on cells, as a result of the surface topographic, stiffness and roughness of graphene sheet .87 Furthermore, the GL-powder promoted matrix mineralization of the cells by increasing BMP9-induced osteogenesis; BMP9 is one of the most potent to induce MSC osteogenic differentiation.…”

“…CharacterizationSynthesized graphene, graphene derivatives and their compositions were chemically characterized using Fourier-transform infrared (FTIR)14, 21, 30, 36, 37, 42, 43, 45, 48, 61, 63, 65, 66, 68, 73 74, 75, 84-86, 91, 92 and raman spectroscopy.13, 17-19, 21, 22, 24-28 32-34, 36, 37, 40-42, 45, 47, 48, 54, 55, 57, 59, 61-65, 68, 69, 71, 72, 75-78, 86-89, 93 Also, morphology and topography of the samples were characterized by SEM,4, 13, 20-23, 25, 26, 29, 33-36, 38, 43, 45-56, 58, 60, 61, 63, 65, 66, 70, 71 73-85, 87, 91, 92 field emission scanning electron microscope (FESEM),17,40,42,44,72,79,86,90,91 transmission electron microscopy (TEM),4,18,30,31,43,44,50,53,61,64,66,71,77,80,82,86,87,[89][90][91][92][93][94] and atomic force microscope (AFM).14, 17-19, 21, 24-26, 36-40, 42, 45, 47, 49, 53, 54, 59, 62 69, 70, 77, 79, 80, 82, 89 Surface hydrophobicity of the composites and their chemical environments were evaluated by contact angle (CA) measurements14, 20, 28, 36-38, 41, 42, 46, , 70, 78, 81, 86, 88, 91, 92 were employed in the various studies to evaluate mechanical strength. Inductively coupled plasma atomic emission spectrometry (ICP-AES)…”