Vertical and uniform growth of MoS₂ nanosheets on GO nanosheets for efficient mechanical reinforcement in polymer scaffold

ABSTRACT

Graphene oxide (GO) and molybdenum disulphide (MoS₂), have drawn more and more attention as reinforcement in polymer. However, their large specific surface area and strong van der Waals forces result in serious aggregations in polymer matrix, which restrains the reinforcement effect. In this study, MoS₂ nanosheets were in situ synthesised vertically and uniformly on the surface of GO nanosheets with the help of a hexadecyl trimethyl ammonium bromide (CTAB) assisted hydrothermal process to facilitate their dispersion in polylactic acid (PLLA) scaffold manufactured via selective laser sintering (SLS). The experimental results revealed that the 1.5 wt% GO/MoS₂ nanocomposite exhibited better dispersion state compared with the 1.5 wt% GO and 1.5 wt% MoS₂ in PLLA matrix. Therefore, the PLLA scaffold loading 1.5 wt% GO/MoS₂ presented 10.2 and 1461 MPa of tensile strength and modulus with enhancement of 61% and 58%, respectively, compared with the pure PLLA scaffold. The reinforcement mechanisms mainly included crack deflection, crack bridging, crack pinning and pulling out of GO/MoS₂. What’s more, the GO/MoS₂-PLLA scaffold possessed benign cytocompatibility for cell behaviour, suggesting hopeful applications in bone defect repair.

KEYWORDS:

• Graphene oxide
• molybdenum disulphide
• scaffold
• in situ growth
• mechanical reinforcement

1. Introduction

Two-dimensional (2D) nanomaterials with outstanding properties, including unique flake structures, large surface areas and excellent mechanical strength, have attracted increasing attention in reinforcing polymer with the help of crack branching, crack bridging, pull-out and so on (Vatanpour et al. 2021; Dewapriya and Meguid 2018; Liu, Hui, and Lau 2022). As a typical kind of 2D nanomaterials, graphene oxide (GO), consisting of sp² bonded carbon atoms layers in hexagonal lattice, possesses desired Young’s modulus of 1 TPa and tensile strength of 130 GPa (Wang, Guan, Fischer, StefanWehner, Li, Wang, and Guoa, 2021; Dhinakaran et al. 2020; Yang, Shuai et al. 2022). Molybdenum disulphide (MoS₂), known as a novel graphene-like transition-metal dichalcogenides (TMDs), builds up of covalently bonded S-Mo-S atomic layers via forces, which exhibits outstanding mechanical properties including strong tensile strength of 23 GPa and Young’s modulus of 300 GPa (Rodriguez et al. 2021; Gupta, Chauhan, and Kumar 2020). GO and MoS₂ have been widely used as nanomaterials to enhance the mechanical properties of polymer scaffolds (Arslan et al. 2021; Sethulekshmia et al. 2021). Mollaqasem et al. (2021) introduced GO into the PCL/PHBV bone scaffold and enhanced the mechanical properties of the scaffold. Qi et al. (2021) added MoS₂ to the PVDF scaffold, which improved the mechanical properties of the scaffold. However, in spite of excellent mechanical properties, the most salient problem of GO and MoS₂ is that they are prone to generate irreversible agglomerations in polymer matrix because of high specific surface area and strong interaction forces, resulting in reduced reinforcement efficiency (Wu, Yin, Mu, Feng, Lu, and Shi, 2021; Gao, Liu, Zhang, Zhang, Li, and Han, 2022; Lin et al. 2021).

Surface modification has been widely applied in the field of improving dispersion of GO or MoS₂, which refers that modifiers can be attached or bonded on the surface of nanomaterials non-covalently or covalently, thereby contributing to arranging a thin layer as a physical barrier to weaken interaction force, and finally hinder their aggregations (Gnanasekar et al. 2021; Peng et al. 2021; Yan et al. 2021). For instance, Achagri et al. (2020) introduced octadecylamine (ODA) to modify GO nanosheets via covalent bonding and found that the dispersibility of GO in polyester fabrics (PET) was significantly improved. Peng, Wang, and Fu (2020) used Tannic acid (TA) to assist the exfoliation and functionalization of MoS₂ to prevent aggregations and found that TA was attached to the surface of MoS₂ to promote its dispersion in polyacrylonitrile (PAN). Besides, synergistic dispersion has been used to promote the dispersion of GO and MoS₂ simultaneously, which means GO and MoS₂ can construct a co-dispersion nanosystem with the help of their unique layered structures (Wu et al. 2022; Feng et al. 2022). Xu et al. (2019) used a simple salt assisted co-exfoliation method to realise the synergistic dispersion of GO and MoS₂ and found that GO/MoS₂ nanocomposite exhibited better dispersibility than pure GO and MoS₂. However, there are certain limitations in the field of surface modification and synergistic dispersion (Chen, Liu, Deng, Jiang, and Li, 2021; Osman et al. 2022). To be specific, for surface modification, it is relatively complex and difficult to control the process and condition of functionalization. What’s more, some treatments might destroy the structure of nanomaterials themselves, resulting in structural defects. Similarly, the process of synergistic dispersion is also difficult to control because it cannot realise thoroughly the mutual intercalation between nanomaterials.

Alternatively, in situ growth is a controllable choice to uniformly anchor one kind of nanomaterial on another nanomaterial surface to prepare nanocomposite materials from top to bottom (Wang, Wang, Zhu et al. 2021; Feng et al. 2022). That is to say, one can be directly grown on the surface of another one with the help of reducing agent and nucleating agent, which manifests that they can be bonded by chemical bonding. What’s more, in situ growth has the advantage of relatively easy operation and low cost. Above all, in situ growth can achieve selective distribution of newly grown nanomaterial and support the dispersion of matrix nanomaterial due to the formed steric hindrance effect (Wang, Yu et al. 2021; Rana et al. 2000; Yang et al. 2022; Wang et al. 2020). Considering that GO possesses plentiful oxygen-containing functional groups including carboxylic, epoxide and hydroxyl, which contribute to negative charge in aqueous solution, hexadecyl trimethyl ammonium bromide (CTAB) could be introduced as a typical cationic surface active agent due to existence of positively charged head groups (Abdelhalim et al. 2021; Krishnakumar and Elansezhian 2021; Qi, Gao, Shuai, Peng, Deng, Yang,Yang, and Shuai, 2022). Meanwhile, the precursor of MoS₂ reveals negative charge and thus CTAB plays a crucial part in improving compatibility of GO and negative ions for further in situ growth of MoS₂ and forming bonding between GO and MoS₂ (Niu et al. 2015; Rana, Mandal, and Bhattacharyya 1993; Chen et al. 2020). As far as we know, there were no reports of in situ growth of MoS₂ nanosheets on GO nanosheets to promote the dispersion of each other in polymer to improve the mechanical properties of the scaffold.

In this study, in-situ synthesis of MoS₂ nanosheets on GO nanosheets was controlled via a CTAB-assisted hydrothermal process to realise the co-dispersion of GO and MoS₂ in the poly-L-lactic acid (PLLA) bone scaffold manufactured via selective laser sintering (SLS). In our previous studies, PLLA nanocomposite scaffolds with Zn-doped mesoporous silica (Zn-MS) particles or GO nanosheets as reinforcements were fabricated to improve the osteogenic ability or hydrophilicity of the scaffold, respectively (Qian et al. 2021; Shuai et al. 2020). The main purpose of this paper was to facilitate the dispersion of GO nanosheets and MoS₂ nanosheets in the PLLA scaffold via MoS₂ nanosheets in situ synthesised on the surface of GO nanosheets, which could improve the mechanical properties of the scaffold. Dispersion states of GO, MoS₂, GO/MoS₂ nanocomposite within PLLA scaffold and their reinforcement effect on mechanical properties of PLLA scaffold were investigated. In addition, the cell behaviour of GO/MoS₂-PLLA scaffold was evaluated.

2. Materials and methods

2.1. Materials

GO was a nanosheet structure with a mean diameter of 8–15 μm and a thickness of 0.8–1.2 nm, which was supplied by Chengdu Organic Chemicals Co., Ltd. of China. PLLA (Mw: 1.7 × 10⁵ g/mol) was purchased from Shenzhen Polymtek Biomaterial Co., Ltd. of China. Hexadecyl trimethyl ammonium bromide (CTAB, 99% purity), Sodium molybdate dihydrate (Na₂MoO₄, 98% purity) and L-cysteine (99% purity) were provided by Aladdin Reagents Co., Ltd. of China.

2.2. Synthesis of GO/MoS₂ nanocomposite powder

GO/MoS₂ nanocomposite powder was prepared via a CTAB-assisted hydrothermal process (Huang et al. 2013). The specific preparation process of nanocomposite powder is shown in Figure 1(a). In a typical batch, 20 mg GO was suspended in deionised water and then under ultrasound for half an hour to obtain a homogeneous suspension. After that, 0.3 g of CTAB was added to the above suspension under stirring for another 24 h to ensure that CTAB was absorbed on the surface of GO. Subsequently, 0.3 g of Na₂MoO₄ was weighed and then mixed with the solution, followed by stirring for 3 h. And then the mixture solution and 1 g of L-cysteine were transferred into a sealed stainless steel autoclave to conduct hydrothermal reaction. After cooling naturally to indoor temperature, the product was taken out, then centrifuged with deionised water and washed with ethyl alcohol for three times. Finally, it was dried in a vacuum oven at 80°C for 12 h to obtain black powder and the GO/MoS₂ nanocomposite powder with 1:1 molar ratio of GO to MoS₂ was designated as GM-1. Similarly, other nanocomposite powder was also prepared via the same procedure as above except that the addition of GO was different. Then GO/MoS₂ with 2:1 (40 mg of GO) and 4:1 (80 mg of GO) molar ratio of GO to MoS₂ were named as GM-2 and GM-3, respectively. The reason for the selection of GO to MoS₂ with different molar ratios was that the synthesised GO/MoS₂ nanocomposite with different molar ratios might exhibit different spatial structures for the studying of dispersion state (Chang and Chen 2011; Thi et al. 2020; Sarwar et al. 2021). For comparison, the pure MoS₂ was synthesised under the similar hydrothermal process in the absence of GO and CTAB.

Figure 1. (a) The specific preparation process of GO-MoS₂ nanocomposite powder. (b) The dispersion states of GO, MoS₂, GM-1, GM-2 and GM-3 without PLLA in ethyl alcohol before and after 48 h at stationary conditions. (c) The micro morphologies of GO, MoS₂, GM-1, GM-2 and GM-3 powder under SEM.

2.3. Synthesis of GO/MoS₂-PLLA powder

The powder was prepared by means of adding 1.5 wt% GO, MoS₂, GM-1, GM-2 and GM-3 into PLLA, respectively. In a specific case, 0.045 g of GM-1 and 2.955 g PLLA were suspended uniformly in 100 ml ethyl alcohol under the action of ultrasonication for 30 min, respectively. And then the above solution was mixed together, followed by stirring for 60 min. Afterwards, the prepared solution was centrifugated at 6000 rpm for 10 min and then placed in a drying cabinet for collecting GM-1/PLLA composite powder. The preparation of other compound powder was the same method as above.

2.4. Fabrication of composite scaffolds

The three-dimensional porous scaffold was fabricated via SLS system (Tino et al. 2020; Ge et al. 2020; Gu et al. 2020). Briefly, the composite powder was sintered selectively on the basis of predesigned model and turned into porous scaffolds with interconnected structure via layer by layer of construction, accompanying by 2.5 W of laser power, 120 mm/s of scanning speed and 0.6 mm of laser spot diameter, 0.3 mm of hatch distance and 0.2∼0.3 mm of powder layer thickness. These scaffolds loaded with 1.5 wt% GO, MoS₂, GM-1, GM-2 and GM-3 were designated as 1.5GO/PLLA, 1.5MoS₂/PLLA, 1.5GM-1/PLLA, 1.5GM-2/PLLA and 1.5GM-3/PLLA scaffolds, respectively.

2.5. Characterization

The microstructural morphologies and the dispersion states of GO and MoS₂ were investigated by a scanning electron microscopy (SEM, Phenom-World BV, Netherlands). The powder specimen was dispersed in ethanol, and then the suspension was dripped onto the specimen table with conductive tape and finally dried. The block specimen was fixed to the specimen table directly through conductive tape. All specimens were sprayed with a layer of gold before being put into SEM and the microstructural morphologies were observed at 20 kV. The morphologies of nanocomposite powder and the distribution of the elements were observed under a transmission electron microscope (TEM, FEI, USA). The nanocomposite powder was ultrasonically dispersed in ethanol and then dropped onto a carbon-supported membrane (Zhongjingkeji, China). The specimen was mounted on the specimen holder of TEM after drying and observed at an acceleration voltage of 200 kV. The zeta potential of GO, MoS₂, GM-1, GM-2 and GM-3 were determined in ethanol by a Zetasizer Nano (ZS90, Malvern, China) and all specimens were sonicated in ethanol for five minutes before testing. X-ray photoelectron spectroscopy (XPS, PHI-5000versaprobeIII, Japan) was used to characterise the elemental composition and atomic valence of specimens by using Al-Kα X-ray source in the scope of 0 eV to 1100 eV. All the peaks were fitted using CasaXPS program. Raman Spectrometer (Renishaw, UK) was used to study the chemical structure of specimens within the range of 250–1800 cm⁻¹. X-ray diffraction (XRD) was utilised for investigating the crystalline phase and structure of nanocomposite by using a XRD diffractometer (Ultima IV, Japan) under the condition of Cu-Kα radiation and scanning rate of 8°/min.

Tensile properties were investigated via a universal testing machine (Zhongluchang, China) according to the GB/T 528-2009/ISO 37:2005 standard with a crosshead movement rate of 0.5 mm/min (Chi et al. 2019). The dumbbell-shaped scaffold specimens (14 × 2 × 2.5 mm³) were prepared for tensile experiments and each test was conducted five times. And the results were extracted and analyzed using Origin software (OriginLab Corporation, USA). After tensile experiments, the fracture morphologies of specimens were investigated via SEM, and the specimens were carpeted with gold in advance. What’s more, the dispersion states of nanomaterials in polymer matrix were studied by using a polarised optical microscope (POM, Batuo, China). The specimens were sliced and then heated up from indoor temperature to 200°C and kept for 100 s. After that, they were cooled naturally and the dispersion states were observed through a camera of POM.

2.6. Cell culture

Human osteoblast-like (MG-63) cells were purchased from Institute of Reproductive and Stem Cell Engineering (Xiangya Medical College, China) in order to investigate the cytocompatibility (Shuai, Chen, He, Qian, Shuai, Peng, Deng, and Wenjing, 2022; Gao, Zeng et al. 2022; Shuai, Yuan, Shuai, Qian, Yao, Xu, Peng, and Yang, 2022). They were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Cellgro-Mediatech Inc., USA) equipped with 10% fetal bovine serum (FBS, Cellgro-Mediatech Inc., USA) and 1% penicillin/streptomycin antibiotics (Aladdin, China) under the circumstance of 37°C and 5% CO₂. And the MG-63 cells were isolated by the enzyme digestion method. For all cell tests, 4–6 passaged cells were used and the cell density in suspension was 1 × 10⁵ cells mL⁻¹. Before cell seeding, the cylindrical scaffolds (φ16 × 3 mm) were immersed in 70% ethanol for 30 min and then exposed to UV light for half an hour in order to achieve sterilization. Afterwards, the cells were seeded onto specimens in the culture medium at a density of 2 × 10⁵ cells/cm² for 1, 3 and 5 d, and the medium was refreshed every two days. Before the observation of morphologies, the specimens were fetched out and washed three times by using phosphate buffer saline (PBS, Cellgro-Mediatech Inc., USA). Subsequently, the cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich, USA), washed with PBS and then dehydrated with graded ethanol. The specimens were dried at a vacuum oven and then sprayed with a film of gold before investigating the morphologies of cell adhesion under SEM. After that, the area of cell adhesion was calculated by means of PhotoShop software (Adobe Systems Inc., USA). Followed by cell cultivation, these specimens were used to perform fluorescence staining assay. To be specific, the specimens were exposed to 2 mM calcein AM (Sigma-Aldrich, USA) and 4 mM propidium iodide (Sigma-Aldrich, USA) for one hour in order to investigate live cells and dead cells, respectively. The fluorescence images were obtained by using a microscope equipped with a digital camera (IX51, Olympus, Japan), where live cells were stained green. The cell density was obtained by calculating the ratio of the number of cells to the image area. Five specimens were prepared in each group.

2.7. Statistical analysis

If not specified, all experiments in this study were performed three times. Statistical Package for the Social Science (SPSS, IBM Corporation, USA) software was used to analyze experimental data, which was indicated as mean ± standard error. p < 0.05 (*) and p < 0.01 (**) were considered significant difference and very significant difference in statistics, respectively.

3. Results and discussion

3.1. GO, MoS₂ and GO/MoS₂ nanocomposite characterization and analysis

The dispersion states of GO, MoS₂ and GO/MoS₂ powder suspended in ethyl alcohol after 48 h were investigated, and the results were exhibited in Figure 1(b). All solutions exhibited a uniform and stable state immediately after ultrasound for 30 min while these solutions presented different dispersion states after standing for 48 h. For GO and MoS₂ suspension, the GO and MoS₂ finally settled down on the bottom and the limpid supernatant was observed obviously, which was ascribed to the aggregations of GO and MoS₂. It could be observed that GM-1, GM-2 and GM-3 solutions presented relatively improved dispersibility compared with the pure GO and MoS₂ solutions. What’s more, some sediments could be both observed in the GM-1 and GM-3 solutions but not in the GM-2 solution. And the colours of the GM-1 and GM-3 solution changed from dark to shallow because the excess MoS₂ and GO resulted in poor dispersibility, respectively. By contrast, it could be observed that the GM-2 suspension still maintained a relatively uniform and stable state and few precipitates appeared at the bottom of the solution, which indicated a good dispersion state. The zeta potential could be used as an index to characterise the repulsive effect between nanoparticles, and the higher positive potential or the lower negative potential, which meant the better dispersion stability of the nanoparticles (Krishnakumar and Elansezhian 2022; Gupta et al. 2021; Abedi, Fangueiro, and Correia 2020). As shown in Figure 1(c–d), the zeta potential of GO, MoS₂, GM-1, GM-2 and GM-3 were 1.8 mV, −0.5 mV, −10.1 mV, −18.7 mV and −11.6 mV, respectively. It could be inferred that GM-1, GM-2 and GM-3 were better dispersed than GO and MoS₂, and GM-2 had the best dispersion. It meant that the in-situ growth of MoS₂ on GO could improve dispersion states, and the ratio of GO to MoS₂ in GM-2 was relatively appropriate compared with other ratios of GO to MoS₂, which was in favour of better dispersibility.

The micromorphologies of GO, MoS₂ and GO/MoS₂ powder were observed, as presented in Figure 1(e). The pristine GO exhibited a typical laminated structure with curled and corrugated morphologies and stacked together. And from high magnification image, the smooth surface could be observed. While as-synthesised MoS₂ consisted of spherical particles whose average diameter was about 2 μm, and a large number of agglomerations were significantly observed. When GO and CTAB were introduced into the synthetic process of MoS₂, it could be found that spherical structure of MoS₂ was transformed into nanosheet structure. For GM-1 powder, there were large-scale MoS₂ nanosheets which tightly stacked together due to the excess precursor of Mo. It could be observed from GM-2 powder that MoS₂ nanosheets were uniformly arranged on the surface of GO, and the phenomenon of agglomeration of MoS₂ was significantly improved on account of the appropriate ratio of GO to MoS₂. What’s more, for GM-3 powder, it was also found that MoS₂ could be dispersed well while there existed some aggregations due to the addition of excess GO. From the above results, it could be concluded that the phenomena of agglomerations were significantly restrained, and the morphologies of MoS₂ were greatly influenced through controlling the ratios of GO to MoS₂ (Chen et al. 2021).

The TEM images and EDS mapping images of GO, MoS₂ and GO/MoS₂ powder were provided for further investigation of microstructure, as shown in Figure 2. It could be observed from Figure 2(a) that the pure GO presented typical lamellar structure and smooth surface accompanied with wrinkle morphology, and there existed some GO nanosheets stacked together. The lattice spacing of adjacent nanosheets was approximately 0.84 nm, which was corresponding to the (002) plane of GO (Mohamed et al. 2019), and the C and O elements were observed to disperse uniformly. From Figure 2(b), the pure MoS₂ exhibited spherical appearance, which was corresponding to the results of SEM, and MoS₂ was proved to stack together. For the GO/MoS₂ nanocomposite powder, it could be found that MoS₂ nanosheets exhibited lamellar structure, which were in situ grown vertically on the surface of GO, and lattice spacing of adjacent MoS₂ nanosheets was approximately 0.94 nm, which was corresponding to (002) plane (Zhang and Liang 2018). For GM-1, excess MoS₂ was observed to generate some agglomerations, which was shown as large areas of black images in Figure 2(c). And the Mo and S elements were observed to disperse ununiformly. From Figure 2(d), MoS₂ nanosheets were grown uniformly on the surface of GO without significant aggregations under the appropriate ratio of MoS₂ to GO. And the C, Mo and S elements could be observed to distribute uniformly from EDS mapping results. In comparison, from Figure 2(e), it could be found that the number and area of MoS₂ decreased relatively and the area of GO increased, which produced some agglomerations due to the excess amount. From the above results, it could be concluded that MoS₂ nanosheets were in situ grown vertically onto GO nanosheets. Besides, the appropriate ratio of MoS₂ to GO played an important role in the uniform dispersion of MoS₂/GO because excess MoS₂ and GO both resulted in the generation of agglomerations (Chang and Chen 2011), respectively, which was corresponding to the results of SEM.

Figure 2. TEM images at different magnifications, HERTEM images and EDS mapping images of pure GO (a1-a4), pure MoS₂ (b1-b4), GM-1 (c1-c4), GM-2 (d1-d4) and GM-3 (e1-e4) powder.

XPS was conducted to characterise the chemical composition and atomic valence of the GO, MoS₂ and GO/MoS₂ powder, and the results were presented in Figure 3(a–c). The full-scan spectra of the GO, MoS₂ and GO/MoS₂ powder were exhibited in Figure 3(a). GO possessed two typical peaks of C1s and O1s, and MoS₂ possessed corresponding peaks of Mo3d and S2p. The survey spectrum of the GO/MoS₂ powder showed the presence of C, O, Mo and S elements, which reached a preliminary conclusion that MoS₂ was successfully synthesised on the surface of GO. Besides, the narrow scans of Mo3d and S2p in the GO/MoS₂ powder were investigated for further proof. In the high-resolution spectra of Mo3d, there existed two strong characteristic peaks assigned to Mo3d_3/2 (232.4 eV) and Mo3d_5/2 (229.2 eV), respectively, which revealed the characteristic +4 oxidation state of Mo. What’s more, two weak peaks at 233.6 eV and 226.7 eV were attributed to Mo⁶⁺ and S2s, respectively. Similarly, the two feature peaks of S2p_1/2 and S2p_3/2 were observed at 163.3 and 162.2 eV, respectively, as presented in Figure 3(c), corresponding to binding energy of S²⁻ (Wang et al. 2019; Zou et al. 2019; Ye et al. 2016). Therefore, the above results of XPS manifested that MoS₂ was successfully grown on the surface of GO.

Figure 3. (a) The full-scan XPS spectra of GO, MoS₂ and GO/MoS₂ powder. (b) The narrow scan spectra of Mo3d in GO/MoS₂ powder. (c) The narrow scan spectra of S2p in GO/MoS₂ powder. (d–e) Raman spectra of GO, MoS₂ and GO/MoS₂ powder. (f) XRD spectra of GO, MoS₂, GM-1, GM-2 and GM-3 powder.

Raman spectra related to the GO, MoS₂ and GO/MoS₂ powder were investigated, as presented in Figure 3(d-e). For pure MoS₂ powder, there were two peaks at 374 cm⁻¹ and 404 cm⁻¹, corresponding to E¹_2g and A_1g vibration modes, respectively, which were ascribed to the in-plane vibration of Mo-S atoms and the out-of-plane vibration of Mo-S atoms, respectively (Wang et al. 2016). What’s more, it could be observed that there existed two typical characteristic peaks of GO, which were attributed to the D band and G band at 1345 cm⁻¹ and 1589 cm⁻¹, respectively (Lee et al. 2021). The D band was related to vibration of sp³-bonded C atoms, while G band arose from the vibration of sp² carbon atoms, representing the structure ordering. For the GO/MoS₂ nanocomposite powder, it could be found that there existed two peaks at 374 and 404 cm⁻¹ attributing to MoS₂ while the other two typical peaks at 1346 and 1578 cm⁻¹ belonged to GO, respectively, which demonstrated that MoS₂ was successfully in situ grown on the surface of GO. In addition, G band was shifted from 1589 cm⁻¹ to 1578 cm⁻¹, revealing the chemical doping of carbon material and the formation of dyadic bonding between GO and MoS₂ (Thangappan et al. 2016). It could be noted that the value of I_D/I_G (∼1.10) of the GO/MoS₂ powder increased compared with that of pure GO powder (∼1.01), which indicated there emerged some defects and disordered structures (Xie et al. 2016). In general, it could be concluded from the above results that MoS₂ was successfully grown onto GO under the condition of hydrothermal reaction.

XRD spectroscopy was used to investigate the crystalline phase and structure of the GO, MoS₂ and GO/MoS₂ powder with different ratios of MoS₂ to GO, as shown in Figure 3(f). It could be observed that there existed two diffraction peaks of GO at 11.8° and 26.9°, which were corresponding to (001) and (002) planes, respectively, and the lattice spacing of adjacent nanosheets was approximately 0.84 nm corresponding to (002) plane. For the pure MoS₂ powder, the typical characteristic peaks at 8.8°, 17.4°, 33.2° and 57.0° were ascribed to (002), (004), (100) and (110) planes, respectively. GO/MoS₂ nanocomposites powder possessed similar diffraction peaks to pure MoS₂, which indicated that they possessed similar crystalline structure to MoS₂. What’s more, the diffraction peak of them corresponding to (002) plane at 8.8° indicated a d-spacing of 0.94 nm, which corresponded with the results of TEM. And the intensity of relevant diffraction peaks was weakened monotonously accompanied by reduced proportions of MoS₂. Besides, the newly appeared diffraction peak of GM-3 at 27.4° was ascribed to GO, which suggested that the content of GO was excessive, resulting in some agglomerations (Qin et al. 2015; Wang et al. 2018; Lin et al. 2020). The above results of XRD demonstrated that the synthetic MoS₂ possessed good crystal structure, and MoS₂ was successfully in situ grown on GO. Besides, the ratio of MoS₂ to GO played a crucial part in the dispersion of GO/MoS₂ nanocomposite.

The mechanism diagram of the in-situ growth of MoS₂ nanosheets onto GO nanosheets was illustrated in Figure 4. It was well known that GO carried negative charge in aqueous solution due to plentiful oxygenated groups including carboxyl and hydroxyl. Therefore, CTAB served as a typical cationic surfactant, containing positively charged head groups, which could be absorbed on the surface of GO through electrostatic interaction. It endowed GO with positive charge in order to overcome charge incompatibility between GO and anion. Afterwards, molybdate ion (MoO₄^2-) could be attracted on CTA⁺-GO via electrostatic action and then gradually nucleated and grew into MoS₂ nanosheets in the presence of L-cysteine, which acted as sulphur source and reducing agent. Finally, MoS₂ nanosheets were vertically, selectively and uniformly arranged onto the surface of GO nanosheets.

Figure 4. The mechanism diagram of the MoS₂ nanosheets in situ grown on both sides of GO nanosheets via CTAB-assisted hydrothermal process.

3.2. Dispersion of GO, MoS₂ and GO/MoS₂ in the matrix

The morphologies of PLLA, GM/PLLA powder and GM/PLLA scaffold were investigated through SEM. The PLLA powder possessed irregular shape with average particle size of ∼150 μm as shown in Figure 5(a), and it could be found from high magnified image that the surface had a smooth condition (Figure 5(b)). For the GM/PLLA powder, the surface of PLLA became relatively rough, and there existed some GO/MoS₂ nanosheets that adhered to the surface of PLLA in Figure 5(c–d). The fabrication process of GM/PLLA scaffold was presented in Figure 5(e), and the porous scaffold was obtained via sintering layer by layer according to the given model. From the top view of the scaffold as shown in Figure 5(f), the scaffold appeared black in appearance with a cylindrical shape and the diameter was approximately 16 mm. It was observed under SEM from Figure 5(f–g) that the interior of the scaffold was an interconnected pore structure, which could facilitate nutrient exchange and promote cell proliferation (Shuai et al. 2022). So, the scaffold was prospective to apply in bone tissue engineering. The EDS results were exhibited in Figure 5(i–l). The results indicated that the C, O, Mo and S elements were uniformly distributed, which suggested the good dispersion of GO/MoS₂ nanosheets on the PLLA scaffold.

Figure 5. The morphologies of the PLLA (a), GM/PLLA powder (c) and their corresponding high magnified images (b, d). (e) The fabrication process of the GM/PLLA scaffold via SLS. (f) The top view of the scaffold. (g) The optical microscope (OM) image of the scaffold. (h) The SEM images of the surface of scaffold. (i-l) The EDS elemental mappings of C, O, Mo and S signals.

The dispersibility of nanomaterials played a crucial part in the properties of composite materials (Teng et al. 2021), so the dispersion states of nanomaterials in PLLA matrix were investigated, and the results were presented in Figure 6. There were a number of big dark masses stacked together in the 1.5GO/PLLA scaffold under POM, which indicated that GO nanosheets formed large-scale agglomerations because of the high content of GO. Similarly, there also existed numerous agglomerations of MoS₂, which were observed to cover most of the area from the high magnification image of the 1.5MoS₂/PLLA scaffold. It could be concluded that the GO and MoS₂ were prone to generate irreversible aggregations in PLLA matrix, respectively, under the circumstance of high content. After the in-situ growth of MoS₂ onto GO, the dispersibility of GO/MoS₂ nanocomposite was also conducted. From the 1.5GM-1/PLLA scaffold, it could be observed that the phenomenon of aggregation was alleviated while the MoS₂ still formed some continuous agglomerations due to the relatively high proportion. Compared with that, it could be observed that the dispersibility was significantly improved after increasing ratio of GO and GM-2 dispersed uniformly in the PLLA matrix. What’s more, for the 1.5GM-3/PLLA scaffold, there existed large chunks of aggregations reappeared, which was ascribed to excess GO stacked together. From the above results, it could be demonstrated that the dispersion states of GO and MoS₂ could be improved significantly by means of in-situ growth of MoS₂ onto GO. Besides, the ratio of GO to MoS₂ played a crucial part in their dispersibility in PLLA matrix. For GO/MoS₂ nanocomposite, the excessive GO and MoS₂ both resulted in poor dispersion and serious aggregations and only appropriate proportion of GO and MoS₂ could contribute to good dispersibility in the PLLA matrix.

Figure 6. The dispersion states of the 1.5GO, 1.5MoS₂, 1.5GM-1, 1.5GM-2 and 1.5GM-3 powder in the PLLA matrix under POM and SEM, respectively.

The dispersibility could also be investigated from the results of SEM, and it could be observed that the surface of PLLA had a relatively smooth condition. For the 1.5GO/PLLA scaffold, there were some large GO nanosheets which were stacked together, forming serious agglomerations. Similarly, it could be significantly observed that the agglomerations of MoS₂ were formed in PLLA matrix. In contrast, the phenomenon of the aggregations of them could be improved by introducing MoS₂ on GO. From the 1.5GM-1/PLLA scaffold, there existed a few agglomerations of GM in the PLLA matrix due to excess MoS₂ although it showed better dispersion compared with the 1.5GO/PLLA and 1.5MoS₂/PLLA scaffolds. Besides, for the 1.5GM-2/PLLA scaffold, GM presented a uniform dispersion state and no obvious aggregations were observed because of the appropriate ratio of MoS₂ to GO. There were also some excess GO nanosheets stacked together in the 1.5GM-3/PLLA scaffold, indicating relatively decreased dispersibility. Therefore, it could be concluded that the introduction of MoS₂ on GO and the appropriate ratio of MoS₂ to GO both had positive effects on their dispersion in PLLA matrix (Song et al. 2017).

3.3. Mechanical properties

The tensile properties of scaffolds were investigated, as shown in Figure 7. It could be observed that the dumbbell-shaped specimens were broken after tensile experiments. For the pure PLLA scaffold, it could present strength of 6.32 MPa and modulus of 921 MPa, respectively, which showed the lowest tensile properties. The strength and modulus of the 1.5GO/PLLA and 1.5MoS₂/PLLA scaffolds were slightly improved due to the addition of GO and MoS₂, while the improvement was inconspicuous because of the excessive contents, compared with pure PLLA scaffold. After in-situ growth of MoS₂ onto GO, tensile properties of the scaffold were significantly improved. It could be observed that the tensile strength and modulus of the 1.5GM-1/PLLA scaffold showed 8.35 MPa and 1163 MPa, respectively, which were considerably higher than that of the PLLA scaffold (p < 0.05). And the tensile strength and modulus of the 1.5GM-1/PLLA scaffold were increased by 32% and 26%, respectively, compared with pure PLLA scaffold. What’s more, 1.5GM-2/PLLA scaffold presented the best tensile properties, whose strength and modulus were considerably higher than that of the PLLA scaffold (p < 0.01). The strength and modulus of the 1.5GM-2/PLLA scaffold was 10.2 and 1461 MPa, respectively, increasing by 61% and 58%, respectively, compared with pure PLLA scaffold. For 1.5GM-3/PLLA scaffold, the tensile properties were declined compared with the 1.5GM-2/PLLA scaffold, due to the addition of excess GO. Table 1 presented a comparison of the tensile strength of our work with that of others on the PLLA and PLLA composite scaffolds (Prabhakaran, Venugopal, and Ramakrishna 2009; Zhou et al. 2017; Jing et al. 2020; Öztatlı and Ege 2017; Liao, Li, and Tjong 2019; Adithya et al. 2020). It could be observed that the tensile strength of the GO/PLLA, MoS₂/PLLA and GM-2/PLLA scaffolds were significantly higher than that of the PLLA scaffold. Previous studies have indicated that the addition of appropriate amount of GO or MoS₂ could improve the mechanical properties of the scaffold due to the unique flake structures, large surface areas and excellent mechanical properties of GO and MoS₂ (Guo et al. 2022). Furthermore, the tensile strength of the GM-2/PLLA scaffold was higher than that of the GO/PLLA and MoS₂/PLLA scaffolds. The reason was that GO or MoS₂ had a high specific surface area and strong interaction force, which were prone to agglomeration in the polymer matrix, resulting in reduced enhancement efficiency (Wu, Yin et al. 2021; Lin et al. 2021). In the present study, MoS₂ nanosheets were in situ synthesised vertically and uniformly on the surface of GO nanosheets to obtain GM-2, which formed steric hindrance effect to support the dispersion of GO. It allowed GM-2 to have better dispersion properties than GO and MoS₂ in the matrix, which could fully exert the mechanical strengthening effect of GO and MoS₂ to enhance the mechanical properties of the scaffold. Through the above tensile results, tensile properties of the scaffold were significantly enhanced by means of in-situ growth of MoS₂ onto GO, and the appropriate ratio of GO to MoS₂ also played a crucial part in the tensile properties of the scaffold. Because the 1.5GM-2/PLLA scaffold had the best mechanical properties in all of the above scaffolds, and subsequent experiments were only conducted to test and analyze the 1.5GM-2/PLLA scaffold.

Figure 7. (a) The tensile specimens clamped with tongs of the universal testing machine. (b) The tensile specimens before and after tensile experiment. (c–d) The stress-strain curves, tensile strength and modulus of the PLLA, 1.5GO/PLLA, 1.5MoS₂/PLLA, 1.5GM-1/PLLA, 1.5GM-2/PLLA and 1.5GM-3/PLLA scaffold specimens. * represents statistical difference (*p < 0.05, **p < 0.01) compared with the PLLA scaffold.

Table 1. Tensile strength of PLLA, GO/PLLA, MoS₂/PLLA and GM-2/PLLA.

Materials	Tensile strength (MPa)	References
PLLA	4.7	Prabhakaran et al.
PLLA	4.0	Zhou et al.
PLLA	4.9	Jing et al.
GO/PLLA	8.1	Öztatlı et al.
GO/PLLA	8.7	Liao et al.
MoS2/PLLA	9.5	Adithya et al.
GM-2/PLLA	10.2	Our work

The crack propagation, which was generated via the Vickers indentation method at the load of 9.8 N, was used to investigate the influence of GM-2 on the scaffold, and the morphologies were presented in Figure 8. GM-2 was broken under the condition of crack propagation, which required a lot of energy. For the morphology of pull out, GM-2 was pulled out of the PLLA matrix, which enhanced the interfacial connection between them (Florek et al. 2021). It was observed that there existed some GM-2 at the destination of the crack because the energy was insufficient for crack propagation, which was referred as crack pinning (Fan et al. 2021). What’s more, there existed GM-2 bridges on the surface to connect the crack, restricting the interface slip and delaying further propagation (Ghazanlou, Ghazanlou, and Ashraf 2021). Crack was generally extended in a monotonous direction with no deflection while the crack would be obstructed and then deviate from the original direction under the condition of encountering GM-2, which resulted in consuming more energy for crack propagation. Similarly, the extended crack branched in the way of river-delta when encountering GM-2 at the end of crack tip (Abazari, Shamsipur, and RezaBakhsheshi-Rad 2021). The corresponding crack propagation model was presented in Figure 8(g). It could be concluded that crack propagation caused by the above results would consume more fracture energy in order to effectively enhance the tensile properties and fracture toughness.

Figure 8. (a–f) SEM images of crack propagation in the 1.5GM-2/PLLA scaffold. (g) The corresponding crack propagation model.

3.4. Cytocompatibility

The results of fluorescence images could be used to elucidate the cell behaviour of 1.5GM-2/PLLA scaffold (the PLLA scaffold as control). As shown in Figure 9(a), it could be seen that the number of living cells on both scaffolds gradually increased with the increase of culture time, and only few dead cells appeared at each time point. From Figure 9(b–c), the PLLA scaffold and 1.5GM-2/PLLA scaffold exhibited cell density of 819 cells/mm² and 926 cells/mm² at 5 d, respectively, which was considerably much higher than that of the PLLA scaffold and 1.5GM-2/PLLA scaffold at 1 d (p < 0.01), suggesting that both scaffolds promoted cell proliferation (Deng et al. 2022; Qi, Liao, Shuai, Pan, Qian, Peng, and Shuai, 2022). In addition, after the same time of culture, there were slightly more live cells on the 1.5GM-2/PLLA scaffold than on the pure PLLA scaffold, which might be due to a small amount of GO contained in the 1.5GM-2/PLLA scaffold. In previous studies, it was found that adding a small amount of GO to the scaffold could promote cell proliferation. The MG-63 cells were cultivated on the 1.5GM-2/PLLA scaffold for 1, 3 and 5 d to investigate morphological features. As shown in Figure 10(a), it could be obviously observed that there existed many individual spherical cells adhered on the scaffold after 1 d of culture. For 3 d, cells gradually expanded and then grew into fusiform shape, and the filopodia was newly appeared. After culturing for 5 d, the cells continued expanding widely and eventually reached confluence to form a fused cell layer. The shape of cells was transformed into polygonal and few spherical cells could be observed. What’s more, the cell relative area gradually increased with increasing incubation time and reached a maximum of 92% after 5 d of culture as shown in Figure 10(b), which was considerably much higher than that of the 1.5GM-2/PLLA scaffold at 1 d (p < 0.01). It could be concluded that the cells on the 1.5GM-2/PLLA scaffold possessed benign adhesion and growth. It could be demonstrated that the 1.5GM-2/PLLA scaffold possessed favourable cytocompatibility, which was expected to be applied in the field of bone implants (Sharma et al. 2021; Wu, Zou et al. 2021; Qi, Wang, Shuai, Peng, and Shuai. 2022).

Figure 9. (a) Fluorescence images of MG-63 cells cultivated on the PLLA scaffold and 1.5GM-2/PLLA scaffold for 1, 3 and 5 d. (b) Live cells density of MG-63 cells cultivated on the PLLA scaffold for 1, 3 and 5 d. (c) Live cells density of MG-63 cells cultivated on the 1.5GM-2/PLLA scaffold for 1, 3 and 5 d. * represents statistical difference (*p < 0.05, **p < 0.01) compared with the PLLA scaffold or 1.5GM-2/PLLA scaffold at 1 d.

Figure 10. (a) SEM images of MG-63 cells cultivated on the 1.5GM-2/PLLA scaffold for 1, 3 and 5 d. (b) Cell relative area of MG-63 cells cultivated on the 1.5GM-2/PLLA scaffold for 1, 3 and 5 d. * represents statistical difference (*p < 0.05, **p < 0.01) compared with the 1.5GM-2/PLLA scaffold at 1 d.

4. Conclusions

In this study, MoS₂ nanosheets were in-situ grown onto the surface of GO nanosheets to construct a co-dispersion nanosheets/nanosheets system via CTAB-assisted hydrothermal process for improving their dispersion. And then they were added into PLLA matrix to fabricate composite scaffold by means of SLS. In the GO-MoS₂ nanostructure, MoS₂ nanosheets were selectively grown on GO to achieve uniform dispersion, and then the growth of MoS₂ nanosheets could form steric hindrance effect to hinder the aggregations of GO nanosheets, leading to better dispersion states and reinforcement efficiency. According to tensile experimental results, the GO/MoS₂-PLLA scaffold exhibited the best tensile strength and modulus, whose values were 10.2 and 1461 MPa, respectively, which enhanced significantly compared with the GO/PLLA and MoS₂/PLLA scaffolds, owing to improved dispersibility of nanomaterials in the PLLA matrix. What’s more, the reinforcement mechanisms of tensile properties involved crack deflection, crack bridging, crack pinning and pulling out of GO/MoS₂. In addition, the GO/MoS₂-PLLA scaffold possessed benign cytocompatibility, indicating hopeful applications for bone implants.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the following funds: (1) The Natural Science Foundation of China [51905553, 51935014, 82072084, 81871498]; (2) Hunan Provincial Natural Science Foundation of China [2021JJ20061, 2020JJ3047, 2019JJ50588]; (3) The Provincial Key R & D Projects of Jiangxi [20201BBE51012]; (4) The Project of State Key Laboratory of High Performance Complex Manufacturing ; (5) Support by the Open Sharing Fund for the Large-scale Instruments and Equipments of Central South University; (6) The Fundamental Research Funds for the Central Universities of Central South University [2022ZZTS0729].

Notes on contributors

Pei Feng

Pei Feng is an associate professor in the School of Mechanical and Electrical Engineering at Central South University. He is engaged in the research of additive manufacturing, and bone repair materials. He has published more than 30 Sci papers in Adv Sci, Bioact Mater, etc.

Saipu Shen

Saipu Shen is a postgraduate of the School of Mechanical and Electrical Engineering at Central South University. His current research focuses on laser additive manufacturing and bone repair materials.

Liuyimei Yang

Liuyimei Yang is currently the assistant research fellow at the Ganjiang Innovation Academy of Chinese Academy of Science in China. Her research interest mainly focuses on granular materials, numerical simulation, powder mixing, the addictive manufacturing and the multi-material 3D printing.

Ye Kong

Ye Kong is a postgraduate of the School of Mechanical and Electrical Engineering at Central South University. His current research focuses on laser additive manufacturing and bone repair materials.

Sheng Yang

Sheng Yang is a professor currently working at Shenzhen University. His research centres on the differentiation mechanism of human totipotent stem cells into oocytes and the mechanism of stem cells repairing endometrium to improve clinical pregnancy outcomes.

Cijun Shuai

Cijun Shuai is a professor in Central South University, and Jiangxi University of Science and technology. He has been engaged in the research of biological additive manufacturing. Moreover, he has published over 200 research papers in journals including Nano Energy, Adv Sci, etc.