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. 2018 May 16;8(32):17860-17877.
doi: 10.1039/c8ra00383a. eCollection 2018 May 14.

Enhanced bioactivity and osteoinductivity of carboxymethyl chitosan/nanohydroxyapatite/graphene oxide nanocomposites

Zhang Yu  1 , Caiwen Xiao  1 , Yazhuo Huang  1 , Mingjiao Chen  1 , Wei Wei  1 , Xiaoxuan Yang  1 , Huifang Zhou  1 , Xiaoping Bi  1 , Linna Lu  1 , Jing Ruan  1 , Xianqun Fan  1
Affiliations

Enhanced bioactivity and osteoinductivity of carboxymethyl chitosan/nanohydroxyapatite/graphene oxide nanocomposites

Zhang Yu et al. RSC Adv. .

Abstract

Tissue engineering approaches combine a bioscaffold with stem cells to provide biological substitutes that can repair bone defects and eventually improve tissue functions. The prospective bioscaffold should have good osteoinductivity. Surface chemical and roughness modifications are regarded as valuable strategies for developing bioscaffolds because of their positive effects on enhancing osteogenic differentiation. However, the synergistic combination of the two strategies is currently poorly studied. In this work, a nanoengineered scaffold with surface chemistry (oxygen-containing groups) and roughness (R q = 74.1 nm) modifications was fabricated by doping nanohydroxyapatite (nHA), chemically crosslinked graphene oxide (GO) and carboxymethyl chitosan (CMC). The biocompatibility and osteoinductivity of the nanoengineered CMC/nHA/GO scaffold was evaluated in vitro and in vivo, and the osteogenic differentiation mechanism of the nanoengineered scaffold was preliminarily investigated. Our data demonstrated that the enhanced osteoinductivity of CMC/nHA/GO may profit from the surface chemistry and roughness, which benefit the β1 integrin interactions with the extracellular matrix and activate the FAK-ERK signaling pathway to upregulate the expression of osteogenic special proteins. This study indicates that the nanocomposite scaffold with surface chemistry and roughness modifications could serve as a novel and promising bone substitute for tissue engineering.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Scheme 1
Scheme 1. Introduction of CMC/nHA/GO scaffolds modified surface properties, including, topographies and roughness, ions releasing and chemical composition. The synergistic effects promote the osteogenic differentiation of hADSCs and the formation of new bone.
Fig. 1
Fig. 1. Characterization of the scaffolds. (a) SEM images of the CMC, CMC/GO and CMC/nHA/GO scaffolds at low (Mag = 50, scale bars: 1 mm), middle (Mag = 200, scale bars: 200 μm) and high (Mag = 2000, scale bars: 20 μm) magnifications and optical images of the CMC, CMC/GO and CMC/nHA/GO scaffolds; (b) FTIR spectra of the scaffolds; (c) XRD spectra of the scaffolds; (d) XPS spectra of the scaffolds; (e) Raman spectra of the scaffolds.
Fig. 2
Fig. 2. Mechanical properties of the scaffolds. (a) AFM of the scaffolds; (b) hardness of the scaffolds as determined by nanoindenter testing (**P < 0.01); (c) stress–strain curve of the scaffolds; (d) Young modulus, compress strength and porosity of the scaffolds (*P < 0.05; **P < 0.01; ***P < 0.001).
Fig. 3
Fig. 3. Effects of the CMC/nHA/GO scaffold on cell adhension and proliferation. (a) Growth and morphology of hADSCs on the CMC, CMC/GO and CMC/nHA/GO scaffolds detected by SEM at low (Mag = 1000, scale bars: 100 μm) and high (Mag = 5000, scale bars: 30 μm) magnification; (b) adhesion rate of hADSCs on substrates for 8 h normalized to the CMC 1 h; (c) proliferation of hADSCs on substrates for 72 h; (d) Ki-67 immunofluorescence staining of cells on substrates for 3 d (scale bars: 20 μm); (e) positive cell ratios of Ki-67 were determined by dividing the number of immune-positive cells to the number of nuclei stained with Hoechst (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 4
Fig. 4. Evaluation of the expression of osteogenesis-related genes of hADSCs on CMC/nHA/GO substrates. The expression of OSX (a), BSP (b), OPN (c), CON (d) and ALP (e) by qPCR after hADSCs were incubated in PM for 7 d or 14 d; the expression of OSX (f), BSP (g), OPN (h), CON (i) and ALP (j) by qPCR after hADSCs were cultured in DM for 7 d or 14 d (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 5
Fig. 5. Evaluation of the osteogenic differentiation of hADSCs on CMC/nHA/GO substrates. (a) The protein expression of osteogenic markers of hADSCs incubated in PM or DM for 7 d; (b) BSP immunofluorescence staining of hADSCs on substrates incubated in PM for 7 d (scale bars: 50 μm); (c) positive cell ratios of BSP were determined by dividing the number of immune-positive cells to the number of nuclei stained with Hoechst (*P < 0.05, **P < 0.01, ***P < 0.001); (d) light microscopy and optical images of ALP staining of hADSCs on substrates incubated in PM or DM for 7 d and ARS staining of hADSCs on substrates incubated in PM or DM for 14 d (scale bars: 200 μm).
Fig. 6
Fig. 6. The molecular mechanism by which CMC/nHA/GO scaffolds promoted osteogenesis. (a) Schematic illustration of the mechanism of CMC/nHA/GO scaffolds promoting osteogenesis; (b) immunoblots showing the up-regulation of β1 integrin in hADSCs on CMC/nHA/GO substrates after incubated in PM and DM for 3 days; (c) immunoblots displaying the phosphorylation of Erk1/2 and FAK in hADSCs on CMC/nHA/GO substrates after incubated in PM for 24 h or 48 h.
Fig. 7
Fig. 7. Histological analysis and immunohistochemical analysis of CMC/nHA/GO scaffolds postimplantation. (a) HE staining images of hADSCs/scaffold complexes after implantation for 8 weeks; Von Kossa staining of cells/scaffold complexes at 8 weeks postimplantation; dark deposits indicated the calcium deposition (scale bars: 100 μm); (b) immunohistochemical analysis of osteogenic differentiation in cells/scaffold complexes after being implanted for 8 weeks. BSP and OPN staining demonstrated positive brown staining in the tissue (scale bars: 200 μm); positive expression ratios of BSP (c) and OPN (d) in CMC/nHA/GO, CMC/GO and CMC groups at 8 weeks postimplantation (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig. 8
Fig. 8. Micro-CT imaging analysis. (a) Representative coronal and sagittal images of calvarial bone defects after 8 weeks implantation; (b) bone volume to tissue volume (BV/TV); (c) bone mineral density (BMD) morphometric analysis (*P < 0.05, **P < 0.01, ***P < 0.001).
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