journal of the mechanical behavior of biomedical materials 116 (2021) 104320
Available online 21 January 2021
1751-6161/© 2021 Elsevier Ltd. All rights reserved.
CNT and rGO reinforced PMMA based bone cement for fixation of load bearing implants: Mechanical property and biological response
F. Pahlevanzadeh
a, H.R. Bakhsheshi-Rad
b,e,*, M. Kharaziha
c, M. Kasiri-Asgarani
b, M. Omidi
b, M. Razzaghi
b, Ahmad Fauzi Ismail
d, Safian Sharif
e, Seeram RamaKrishna
f, F. Berto
g,**aDepartment of Tissue Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran
bAdvanced Materials Research Center, Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran
cDepartment of Materials Engineering, Isfahan University of Technology, Isfahan, 84156-83111, Iran
dAdvanced Membrane Technology Research Center (AMTEC), Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia
eFaculty of Engineering, Universiti Teknologi Malaysia, 81310, Johor Bahru, Johor, Malaysia
fNanoscience and Nanotechnology Initiative, National University of Singapore, 9 Engineering Drive 1, Singapore, 1157, Singapore
gDepartment of Mechanical and Industrial Engineering, Norwegian University of Science and Technology, 7491, Trondheim, Norway
A R T I C L E I N F O Keywords:
Polymethyl methacrylate Carbon nanotube Reduced graphene oxide Mechanical properties Biocompatibility
A B S T R A C T
Polymethyl methacrylate (PMMA) bone cements (BCs) have some drawbacks, including limited bioactivity and bone formation, as well as inferior mechanical properties, which may result in failure of the BC. To deal with the mentioned issues, novel bioactive polymethyl methacrylate-hardystonite (PMMA-HT) bone cement (BC) rein- forced with 0.25 and 0.5 wt% of carbon nanotube (CNT) and reduced graphene oxide (rGO) was synthesized. In this context, the obtained bone cements were evaluated in terms of their mechanical and biological character- istics. The rGO reinforced bone cement exhibited better mechanical properties to the extent that the addition of 0.5 wt% of rGO where its compressive and tensile strength of bioactive PMMA-HT/rGO cement escalated from 92.07 ±0.72 MPa, and 40.02 ±0.71 MPa to 187.48 ±5.79 MPa and 64.92 ±0.75 MPa, respectively. Besides, the mechanisms of toughening, apatite formation, and cell interaction in CNT and rGO encapsulated PMMA have been studied. Results showed that the existence of CNT and rGO in BCs led to increase of MG63 osteoblast viability, and proliferation. However, rGO reinforced bone cement was more successful in supporting MG63 cell attachment compared to the CNT counterpart due to its wrinkled surface, which made a suitable substrate for cell adhesion. Based on the results, PMMA-HT/rGO can be a proper bone cement for the fixation of load-bearing implants.
1. Introduction
Acrylic bone cements have been mainly used as a cementing material for transferring the load between the bone and prosthetic implant in joint replacement surgeries (Pahlevanzadeh et al., 2018). This type of material offers many advantages like quick polymerization reaction, effortlessness in preparation and application, and fast patient recovery (Marrs et al., 2006). The failure rate after 16 years has been recorded as high as 67%, in patients younger than 45 years old (Marrs et al., 2006).
Two reasons for the mentioned failures are inferior mechanical prop- erties, and lack of bioactivity of PMMA (Pahlevanzadeh et al., 2019a).
The acrylic BCs do not directly stick to the bone. The indirect surface
adhesion increases the possibility of the formation of the gap between the bone/cement and cement/implant (Pahlevanzadeh et al., 2018, 2019a; Marrs et al., 2006), which can result in the loosening of the implant and can provide optimal sites for the colonization of bacteria (Cole et al., 2020). For improving the adhesion of cement to the bone, the approaches have mainly paid attention to using additional additives, particularly some bioactive ceramics like hydroxyapatite (Gonçalves et al., 2012), fluorapatite (Pahlevanzadeh et al., 2018), akermanite (Chen et al., 2015), baghdadite (Pahlevanzadeh et al., 2019b), and monticellite (Pahlevanzadeh et al., 2019a). Nevertheless, incorporating the additives can change the properties of the composite, particularly the mechanical properties, which are essential if the cement is applied for load-bearing usages (Cole et al., 2020; Yang et al., 2020).
* Corresponding author. Advanced Materials Research Center, Department of Materials Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran.
** Corresponding author.
E-mail addresses: [email protected], [email protected] (H.R. Bakhsheshi-Rad), [email protected] (F. Berto).
Contents lists available at ScienceDirect
Journal of the Mechanical Behavior of Biomedical Materials
journal homepage: http://www.elsevier.com/locate/jmbbm
https://doi.org/10.1016/j.jmbbm.2021.104320
Received 25 July 2020; Received in revised form 17 December 2020; Accepted 6 January 2021
Investigations show that for having a bioactive BC to exhibit osteo- conductivity after setting, the concentration of bioactive filler should be higher than 60 wt/wt.% (approximately 35 vol/vol%) (Tsukeoka et al., 2006).
Based on the previous research, hardystonite (Ca2ZnSi2O7), as a bioactive ceramic, has shown acceptable biological and mechanical properties. The mechanical characteristics of HT are close to that of natural bone, including Young’s modulus (37 GPa), fracture toughness (1.24 MPa m1/2) and the bending strength (136 MPa) (Pahlevanzadeh et al., 2019b; Tsukeoka et al., 2006). Besides, in a physiological envi- ronment, HT has superior chemical stability compared to calcium sili- cates. HT has shown anti-bacterial and anti-inflammatory properties due to the release of Zn from its network (Pahlevanzadeh et al., 2020; Wu et al., 2005). Hence, HT has been utilized to achieve a bioactive cement in this study, but it led to the decline of mechanical properties, pre- dictably. To deal with this problem, the incorporation of carbon-based materials (CBMs) such as graphene and its derivatives could be an effective method to compensate for the reduction of mechanical prop- erties (Pahlevanzadeh et al., 2020; Wu et al., 2005; Abdallah et al., 2020).
In this respect, CNTs as a category of CBMs are rolled-up graphene sheet layers in a tubular structure (Abdallah et al., 2020). CNTs show unique chemical and physical properties, which are highly dependent on different manufacturing routes. As the chemical bonding of CNTs is based upon the sp2 orbital bond, they have the hardest and strongest material documented in terms of modulus of elasticity and tensile strength, respectively (Abdallah et al., 2020). Applications of graphene and its nanomaterials family, like GO and rGO, the same as many novel materials, offer various technological opportunities because of outstanding thermal, electrical, optical, and mechanical properties of them (Lee et al., 2015). The questions about short-term and long-term cytotoxicity of graphene-based materials have raised because of growing biomedical applications of these materials (Abdallah et al., 2020; Lee et al., 2015). In this regard, an essential factor is the quantity of attached oxygen functional groups to the surface in which for a higher C/O levels, flakes are less cytotoxic, which can be attributed to the partially rGO structures (Tadyszak et al., 2018). So, rGO was used in this study to improve the BCs’ mechanical properties without sacrificing the biocompatibility. In recent years, both CNT (Halim et al., 2018; Qian et al., 2019) and rGO (Zhang and Gurunathan, 2016) have been utilized in various applications in the biomedical field, such as tissue engineer- ing (Liu et al., 2020; Li et al., 2020; Prakash et al., 2020), drug delivery (Abdallah et al., 2020; Lu et al., 2020), and properties improvement of BCs (Gonçalves et al., 2012; Ormsby et al., 2010a, 2010b). The ho- mogenous dispersion of graphene-based reinforcement into the poly- meric matrix is essential for having an appropriate interfacial bonding between the reinforcement and matrix and resulting in an optimal enhancement in mechanical properties (Ormsby et al., 2010a). For
example, Ormsby et al., 2010a, 2010b revealed that the most efficient method for producing PMMA containing CNT BCs is dispersing the MWCNT reinforcement in the liquid monomer ingredient utilizing ul- trasonic disintegrating before its introduction into the polymer powder.
Accordingly, they showed that acrylic BCs containing 0.1 to 0.25 wt% of MWCNT reinforcement resulted in a remarkable enhancement in both static and fatigue properties, while the levels of cytotoxic response were low (Ormsby et al., 2010a, 2010b).
Herein, we developed bioactive BC containing 60 wt% of HT, which was effective in achieving appropriate bioactivity and apatite-like layer formation on specimens’ surfaces. Furthermore, we aimed to create well-dispersed CNT, and rGO reinforced bioactive cements by ultraviolet dispersing in MMA liquid. There are many studies (Pahlevanzadeh et al., 2018, 2019a; Marrs et al., 2006; Cole et al., 2020; Gonçalves et al., 2012) for improvement of PMMA based cements features; however, a few of them have investigated incorporating carbon-based materials in PMMA-based cements. In addition to the effect of CNT and rGO incor- poration on the mechanical and cytotoxic properties of BC, comparing their effectiveness was examined in this study, which is scarcely studied before. The formation of the apatite-like layer, favorable compressive, tensile and bending strength, and high MG63 cell viability and prolif- eration demonstrated PMMA/HT/rGO BC could be an appropriate replacement for commercial PMMA.
2. Materials and methods 2.1. Preparation of the BCs
The powders of calcium carbonate (CaCO3, Merck, 98% purity), zinc oxide (ZnO, Merck, 99% purity), and silicon dioxide (SiO2, Aldrich, 99%
purity) were provided in order to the mechanochemical synthesis of HT.
The MMA liquid monomer (containing MMA: 84.4%, butyl methacry- late: 13.2%, N–N dimethyl-p-toluidine: 2.4%, and hydroquinone: 20 ppm), and PMMA powder (containing PMMA: 87.7%, benzoyl peroxide:
2.4% and barium sulfate: 10% as radio-opaque filler) were purchased from Teknimed, France (CEMFIX1 brand). The solid components of BCs were prepared, including powders of commercial CNT (purity >95%, lengths 1–5 mm, diameters 5–10 nm, Nanocyl-3150, Belgium), rGO (NanoInnova Technologies, Spain) and synthetic HT. Table 1 demon- strates the composition of the PMMA-based BCs. The different concen- trations of CNTs and rGO between 0.25 and 0.5 wt% were incorporated into the BC by dispersion of them into the MMA monomer utilizing an ultrasonic disintegrating instrument (MSE Ltd. UK) at an amplitude of 10 μA for 30 s. This method was employed to fabricate PMMA based cements because of the highest mechanical properties exhibited by specimens made by ultrasonic dispersing strategy in previous studies (Ormsby et al., 2010a).
The setting behavior of the BCs was assessed based on the ISO 5833-1 Abbreviations
BC Bone cement
CBM Carbon-based material CNT Carbon nanotube DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid
EDS Energy-dispersive X-ray spectroscopy FBS Fetal bovine serum
FTIR Fourier-transform infrared spectroscopy GO Graphene oxide
HA Hydroxyapatite HT Hardystonite MMA Methyl methacrylate
MMP Mitochondrial membrane potential
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MWCNT Multi-walled carbon nanotubes PBS Phosphate-buffered saline PMMA Polymethyl methacrylate rGO Reduced graphene oxide ROS Reactive oxygen species SBF Simulated body fluid SD Standard deviation
SEM Scanning electron microscope TEM Transmission electron microscope XRD X-ray diffraction
Table 1
Comparison of mechanical properties of different PMMA-based BCs containing carbon based derivatives.
Sample CSD (Mpa) CMD (MPa) CSW (Mpa) CMW (MPa) BS (MPa) BM (MPa) TS (MPa) TM (MPa) Ref.
PMMA 128.73 ±
1.54 1182.82 ±
23.94 112.42 ±
1.49 1050.52 ±
21.50 92.09 ±
2.09 880.49 ±
15.76 54.01 ±
0.99 823.70 ±
13.81 This work
PMMA/HT 92.07 ±
0.72 2027.59 ±
36.86 69.03 ±
0.74 1821.51 ±
28.45 61.06 ±
0.68 1709.65 ±
25.56 40.02 ±
0.71 1662.50 ±
24.21 This work PMMA/HT/0.25CNT 146.67 ±
4.29 1496.75 ±
23.87 119.12 ±
1.56 1236.20 ±
23.94 79.05 ±
0.94 1082.58 ±
21.34 57.02 ±
1.12 996.61 ±
13.96 This work PMMA/HT/0.5CNT 173.10 ±
4.36 2003.39 ±
31.07 141.49 ±
3.00 1719.38 ±
25.42 82.07 ±
1.01 1298.53 ±
20.55 63.21 ±
1.70 1203.57 ±
15.76 This work PMMA/HT/0.25rGO 154.26 ±
2.90 1527.32 ±
25.49 121.72 ±
1.40 1241.60 ±
24.22 82.98 ±
1.18 1116.76 ±
21.21 58.06 ±
1.12 1067.71 ±
14.00 This work PMMA/HT/0.5rGO 187.48 ±
5.79 2058.50 ±
39.61 146.57 ±
2.46 1698.47 ±
25.20 86.03 ±
1.44 1403.27 ±
22.88 64.92 ±
0.75 1289.40 ±
16.46 This work
PMMA/HA/0.01GO - 9053 ±460 - - 23.01 ±
0.37 - - - Gonçalves et al. (2012)
PMMA/HA/0.1GO - 9084 ±470 - - 22.34 ±
0.74 - - - Gonçalves et al. (2012)
PMMA/HA/0.5GO - 9063 ±500 - - 24.89 ±
1.19 - - - Gonçalves et al. (2012)
PMMA/HA/1GO - 1022 ±670 - - 28.28 ±
1.09 - - - Gonçalves et al. (2012)
PMMA/HA/0.01CNT - 6089 ±680 - - 14.22 ±
0.63 - - - Gonçalves et al. (2012)
PMMA/HA/0.1CNT - 8047 ±380 - - 15.11 ±
0.90 - - - Gonçalves et al. (2012)
PMMA/HA/0.5CNT - 6093 ±650 - - 12.57 ±
0.63 - - - Gonçalves et al. (2012)
PMMA/HA/1CNT - 6080 ±680 - - 11.81 ±
1.69 - - - Gonçalves et al. (2012)
PMMA/0.1CNT (MS) 58.65 ±
4.85 2933 ±497 - - 46.46 ±
6.78 2770 ±181 - - Ormsby et al. (2010a)
PMMA/0.1CNT (DB) 61.14 ±
4.37 3152 ±348 - - 63.63 ±
8.99 2916 ±137 - - Ormsby et al. (2010a)
PMMA/0.1CNT
(Ultrasonic) 62.11 ±
3.83 3077 ±368 - - 57.23 ±
6.19 3109 ±292 - - Ormsby et al. (2010a)
PMMA/0.1cf-CNT
(MS) 52.26 ±
4.85 3043 ±497 - - 54.94 ±
7.28 3221 ±761 - - Ormsby et al. (2010a)
PMMA/0.1cf-CNT
(DB) 62.24 ±
3.60 3218 ±375 - - 68.48 ±
9.39 3261 ±118 - - Ormsby et al. (2010a)
PMMA/0.1cf-CNT
(Ultrasonic) 67.31 ±
3.83 3115 ±253 - - 59.61 ±
4.75 3480 ±181 - - Ormsby et al. (2010a)
PMMA/0.1CNT 109.0 ±
13.6 3804.0 ±
413.0 - - 56.5 ±
10.3 3573.9 ±
382.0 - - Ormsby et al. (2010b)
PMMA/0.25CNT 95.9 ±7.7 3499.0 ±
317.0 - - 54.1 ±5.2 3084.6 ±
239.0 - - Ormsby et al. (2010b)
PMMA/0.5CNT 80.2 ±4.9 3633.7 ±
962.0 - - 19.4 ±3.3 496.4 ±73.1 - - Ormsby et al. (2010b)
PMMA/1CNT 66.8 ±6.2 2812.0 ±
841.0 - - 41.0 ±4.4 2413.8 ±
158.0 - - Ormsby et al. (2010b)
PMMA/0.1Gr 95.7 ±12.8 - - - 48.3 ±7.9 2462 ±535 - - Paz et al. (2017)
PMMA/0.25Gr 98.8 ±11.4 - - - 50.6 ±9.6 2204 ±98 - - Paz et al. (2017)
PMMA/0.5Gr 98.9 ±15.2 - - - 52.4 ±
11.9 2510 ±452 - - Paz et al. (2017)
PMMA/1Gr 97.7 ±9.7 - - 46.6 ±7.1 2278 ±311 - - Paz et al. (2017)
PMMA/0.1GO 120.7 ±
16.1 - - - 66.4 ±6.5 3293 ±143 - - Paz et al. (2017)
PMMA/0.25 GO 106.3 ±
19.3 - - - 69.8 ±8.9 3375 ±169 - - Paz et al. (2017)
PMMA/0.5 GO 116.2 ±9.1 - - - 63.8 ±6.0 3226 ±98 - - Paz et al. (2017)
PMMA/1GO 103.3 ±
19.1 - - - 61.6 ±
10.2 3084 ±128 - - Paz et al. (2017)
PMMA/0.1CNT 130 ±4.16 - - - - - 48.03 ±
1.55 - Nien and Huang (2010)
PMMA/0.2CNT 130.16 ±
3.83 - - - - - 48.36 ±1 - Nien and Huang (2010)
PMMA/0.27CNT 127.25 ±
2.44 - - - - - 48.01 ±
3.09 - Nien and Huang (2010)
PMMA/0.43CNT 130.02 ±
7.41 - - - - - 45.55 ±
1.65 - Nien and Huang (2010)
PMMA/0.59CNT 129.06 ±
3.37 - - - - - 45.86 ±
4.23 - Nien and Huang (2010)
PMMA/0.75CNT 127.83 ±
3.54 - - - - - 46.58 ±
4.65 - Nien and Huang (2010)
PMMA/20Cs/GO 86.0 ±0.0 1044.4 ±
28.3 - - 75.2 ±0.9 - - - Bakhsheshi-Rad et al.
(2020)
(continued on next page)
standard. The setting temperature was measured by K-type thermo- couples connected to the data acquisition system linked to a computer with DEWESoft 7 (EPAD–TH8–K high-speed data acquisition). The maximum temperature (Tmax) in the profile is considered as the peak temperature within the polymerization of the PMMA-based BCs. The setting time was determined as the time elapsed from the start of the mixing until the hardening of the BC.
2.2. Mechanical tests of the BCs
The PMMA/HT powder component was mixed with MMA liquid containing CNT and rGO (40 g of powder in 14.4 mL of liquid) and injected to prepared moulds for evaluating the compressive strength based on the ASTM F451-08 standard, tensile strength based on the ASTM D638 standard, and bending strength based on the ISO 5833:2002 standard. The bending strength was calculated based on the equation: δf
=3Fabd2 , where: F is the fracture force in N, b is the sample width size in mm, d is the sample thickness in mm, and a is the distance between inner points of loading in mm (=20 mm).
2.3. Hardystonite synthesize process
The mechanochemical synthesis method was utilized for preparing the hardystonite powder. The powders of zinc oxide, calcium carbonate, and silicon dioxide, with a molar ratio of 1:2:2 correspondingly, were blended applying a planetary ball mill instrument (Retsch, PM 100) in a zirconia vial having five zirconia balls with the diameter of 20 mm. The mass ratio of ball to powder was chosen 10:1, and the speeds of vial and disc rotations were adjusted at 500 and 250 rpm, correspondingly. The time of 20 h was considered for the milling, and samples were sintered at 900 ◦C after milling, which was reported as the best condition for HT synthesis in Ref. (Sadeghzade et al., 2016).
2.4. Characterization of PMMA-based BCs
The X-ray diffractometer (D5000, Siemens) was applied to determine the HT powder XRD patterns via Cu- Kα radiation (45 kV, 40 mA), in the diffraction angles (2θ) between 10 and 60◦at a scanning rate of 4◦/min.
The FTIR spectrum of HT was recorded in a spectral range of 4000 to 400 cm−1. The PMMA-based BCs microstructure was observed applying SEM (JEOL JSM 6380LA) equipped with EDS analysis (Oxford) with an operating voltage of 20 kV and TEM (Hitachi HT7700).
2.5. Evaluation of bioactivity
In the present research, the formation of the layer of calcium phos- phate (Ca–P) on the surface of the specimens in SBF solution with an ionic concentration similar to human blood plasma based on the Kokubo formulation and method was considered as an indicator for bioactivity (Kokubo and Takadama, 2006). The BC samples in tablet shape with 6 mm diameter and 3 mm thickness were prepared for evaluation of the bioactivity. The samples were immersed in the SBF solution at 37 ◦C temperature for the duration of 7, 14, 21, and 28 days. After the im- mersion, the specimens were washed using distilled water after
removing the SBF solution and finally were dried in an oven for 4 h at 60 ◦C. Thereafter, SEM studies were performed to observe the specimens surface morphology and investigating the formation of Ca–P layer, to measure the amount of Ca and P elements in the deposition of the sur- face of the sample.
2.6. MTT assay
The activity of the cell mitochondrial dehydrogenase enzyme and subsequently, the rate of tetrazolium salt reduction and the formation of formazan crystals, were measured by MTT assay test as an indicator of living MG63 cells activity. The evaluation of cytotoxicity was performed after 1, 4, and 7 days after starting the MTT assay test 104 cells/ml were seeded on each BC disc. For each evaluation, three samples were considered, and a monolayer culture in a polystyrene container with three replications was used as control. At the end of each day, the culture medium in wells was removed and replaced with DMEM and MTT at 10:1 ratio. The plate was held in the incubator for 4 h. The wells were then evacuated, and the formed formazan crystals were dissolved in 350 μL of DMSO, and the plates were put in an incubator again for 30 min. In the next step, a uniform solution was prepared by pipetting, and every 350 μL of DMSO in each well was transferred into 2 wells of another plate. Finally, the light absorption at 570 nm was measured by the ELISA reader. The percentage of the cell viability was calculated based on the following equation:
Relative cell viability(%) =Asmple−Ab
Ac−Ab (1)
where Asample indicates the absorption of the specimen, Ab is the ab- sorption of DMSO solution, and Ac is the absorption of the control sample.
2.7. Cell adhesion
The evaluation of cell adhesion was performed after 4 days of cul- ture, and for each sample, 2 replications were considered. In this regard, 3% glutaraldehyde diluted with PBS was prepared. At the first step, the culture medium was extracted, and after rinsing the samples by PBS, 350 μL of 3% glutaraldehyde was poured into each well, and the plate was covered with foil and placed in a refrigerator at 4 ◦C for 2 h. Then, glutaraldehyde was extracted, and the specimens were dehydrated using alcohol at the concentrations of 50%, 70%, 90%, and 100%, corre- spondingly, and for each percentage, the duration of 15 min was considered. SEM images were prepared for evaluating the cell adhesion on the specimens.
2.8. Statistical analysis
All tests repeated at least 3 times to have more accurate results and performing statistical analysis. The results were presented as the means
±standard deviation (SD). For the p-values lower than 0.05 (p <0.05), the results were considered as significant statistically. The GraphPad Prism Software (V.6) was applied to compare the results of the different samples.
Table 1 (continued)
Sample CSD (Mpa) CMD (MPa) CSW (Mpa) CMW (MPa) BS (MPa) BM (MPa) TS (MPa) TM (MPa) Ref.
PMMA/25Cs/GO 93.0 ±4.0 1216.9 ±
82.7 - - 79.9 ±1.8 - - - Bakhsheshi-Rad et al.
(2020) PMMA/30Cs/GO 88.6 ±1.6 1106.2 ±
98.7 - - 76.1 ±1.3 - - - Bakhsheshi-Rad et al.
(2020)
Standard* 70<
Compressive strength in dry condition (CSD); Compressive modulus in dry condition (CMD); Compressive strength in wet condition (CSW); Compressive modulus in wet condition (CMW); Bending strength (BS); Bending modulus (BM); Tensile strength (TS); Tensile modulus (TM); Magnetic stirring (MS); Dry blending (DB);
carboxyl functionalized CNT (cf-CNT).
3. Results and discussion 3.1. Characterizations of HT
Fig. 1a depicts the XRD pattern of the HT after synthesis. As can be seen, only characteristic peaks of HT (XRD JCPDS data file No.
1219075–0916) were detected, and no impurities peak was observed.
Hence, the process, including 20 h milling of the ingredients and raising the temperature to 900 ◦C, is sufficient for the synthesis of HT powder (Ormsby et al., 2010b). According to the Williamson–Hall method, the crystallite size of HT powder was measured equal to 85 ±4 nm. The TEM images in the frame depicted that the HT powder presented a particulate shape-like with size of about 95 nm. The FTIR spectrum of HT is shown in Fig. 1b. As can be observed, the bands of intensity at about 1668 cm−1 and 3457 cm−1 could be related with stretching and bending vibrational
water because of the powders’ exposure to the atmosphere (Mohammadi et al., 2015). Moreover, the band of intense at around 840 cm−1 could be related to the Si–O bond (Manso et al., 2003), and the intense bands at about 974 cm−1, and 919 cm−1 are related to the asymmetric stretching vibration of Si–O–Si bond (Mohammadi et al., 2015; Agathopoulos et al., 2006). The Zn–O and Ca–O was detected at around 521 cm−1 and 511 cm−1 respectively, which is further, confirmed formation of HT. The peaks detected in 1066, 991, and 839 cm−1 were attributed to PMMA, while characteristic peaks of O–CH3 stretching, CH3 stretching, C––O stretching, and C–H asymmetric stretching vibrations were detected at 1163, 1439, 1719, and 2949 cm−1, respectively (Pahlevanzadeh et al., 2018). The BCs that contained HT in its composition had some more peaks, including at around 841 cm−1, which can be related to the Si–O bond and two other peaks at 973, and 922 cm−1, which can be attributed to the asymmetric stretching vibration of Si–O–Si bond. A slight
Fig. 1. (a) x-ray diffraction pattern, (b) FTIR absorption spectra of hardystonite powder and (c) FTIR absorption spectra of PMMA, HT, CNT, rGO, PMMA/HT, PMMA/HT/0.5rGO, PMMA/HT/0.5CNT. Note: In frame is TEM micrograph of hardystonite powder.
shoulder was seen in the CNT’s spectra between 827 and 846 cm−1. The peaks at 1539 and 2411 cm−1 were related to the carbon skeleton and CO2 asymmetric stretching, correspondingly (Pahlevanzadeh et al., 2019a). As far as the rGO is concerned, the peak at 1591 cm−1 was attributed to C––C, and the absorption peak at 1166 cm−1, which could be related to C–OH was still existed (Gong et al., 2015). Based on the results, the FTIR spectra of the PMMA-HT-xCNT and PMMA-HT-xrGO BCs revealed peaks characteristic of PMMA, HT, CNT, and rGO.
3.2. Mechanical properties
PMMA-based BCs typically have a mechanical ability to distribute the loaded mechanical stresses equally. So, as a result, the loaded stresses to the implant can be transferred to the bone by PMMA-based BCs. The transfer of mechanical forces is attained through the cement interface, which makes a significant surface area, resulting in reducing the stress concentrations over the bone (Gao et al., 2017; Jing et al., 2015; Wang et al., 2015). The results of mechanical properties, including compressive, bending, and tensile strengths, are summarized in Table 1 and all stress-strain curves presented in supportive informa- tion (Fig. S1-Fig. S4). The results are related to the PMMA cement and the PMMA cement containing 60 wt% of HT as the control samples, the PMMA/HT cement incorporating 0.25 and 0.5 wt% of both rGO and CNT reinforcements, as well as the outcomes of the other studies related to PMMA based cements containing graphene and its derivatives in order to the comparison. As reported in the previous studies (Soleymani
Eil Bakhtiari et al., 2020a; Abazari et al., 2020a; Munir et al., 2019), small loadings of rGO and CNTs can result in remarkable improvement in mechanical properties due to providing high surface area, the small concentrations of 0.25 and 0.5 wt% was chosen as reinforcement in this study. Moreover, lower levels of CNT loadings can reduce the possibility of reinforcement agglomeration and prevent chemical or physical interference during the polymerization reaction of the PMMA cement (Ormsby et al., 2010a). The concentration of CNTs was related to the dispersion of functional groups and loading on CNTs into the cement microstructure (Munir et al., 2019). Besides, Zanello et al. presented that the implantation of CNTs in the bone can enhance the mechanical properties of injured bone tissue (Zanello et al., 2006). In this perspec- tive, the homogenous distribution of CNTs into the BCs caused a delay in propagation of cracks through the BC mantle during dynamic and static loadings. The outcomes showed that incorporating rGO and CNT to the PMMA-based BCs could enhance both the bending and tensile strengths of the resulting nanocomposite (Gao et al., 2017; Wang et al., 2015;
Munir et al., 2019). Based on the outcomes, it could be summarized that improvement in mechanical properties is due to the MWCNTs arresting and delaying of the cracks propagation in the BCs using a bridging effect, and hindering crack propagation resulted in properly uniform dispersion of rGO and CNT in the PMMA-based BCs, which can attract and delay the crack propagation in the BC. Other studies (Broda et al., 2014; Ashcroft et al., 2008; Schiavone et al., 2016; Zhang et al., 2019; Gao et al., 2013;
Mi et al., 2016; Pu´ertolas and Kurtz, 2014; Abdel-Mottaleb et al., 2019;
Fathyunes et al., 2019; Yadav et al., 2018; Thomas et al., 2020; Canal
Fig. 2.Compressive modulus and strength of PMMA-based cements containing rGO and CNT reinforcement agents in (a,b) dry and (c,d) wet condition.
and Ginebra, 2011) showed that uniform dispersion of reinforcement agents is able to reduce the acceleration rate of crack propagation within the matrix. However, rGO reinforcement had more effect on the bending and tensile strengths of the cements in comparison with CNTs rein- forcement due to the happening of CNTs clustering in the cement microstructure. Attaining a sufficiently uniform dispersion is crucial for an acceptable bonding between the carbon-based reinforcement agent and the matrix, which can better transfer the loads and delays the propagation of the crack.
As it is shown in Fig. 2 the incorporation of HT powders to PMMA cements led to reduction of compressive strength from 128.73 ±1.54 MPa and 112.42 ±1.49 MPa for PMMA control specimens to 92.07 ± 0.72 and 69.03 ±0.74 for PMMA/HT cements in dry (Fig. 2a and b) and wet condition (Fig. 2c and d) tests results, respectively. Fig. 2 depicts the modulus of elasticity and compressive strength of the BCs after 7 days of immersion in the SBF solution. The mechanical properties of all BCs in wet conditions have the same trend as the ones before immersion but with a significant decrease in value. In wet conditions, agglomerated HA particles can cause segregation of phase and lack of uniformity in the composite, as well as weak bonding to the matrix, which can result in a decline in the mechanical properties. This reduction in wet condition was also reported by Puskas et al. (Puska et al., 2003) study, which revealed the lower compressive strength for oligomer-modified cements after one week remaining in wet condition. Generally, similar trends in Goncalves et al. (Gonçalves et al., 2012), Chen et al. (2015), and Zamarron et al. (Rentería-Zamarr´on et al., 2009) study results regarding the reduction of mechanical properties due to the existence of bioactive
ceramics in cement composition which was in agreement with this study results. In fact, the poor interaction between PMMA and HT, as well as agglomeration of the HT, may result in increasing cement brittleness and faster break of BC subsequently. The addition of rGO at 0.5 wt% could raise the compressive modulus and strength of PMMA/HT cements to 187.48 ±5.79 MPa and 2058.50 ±39.61 MPa, respectively, which was more successful in the betterment of mechanical features than CNT.
Also, similar to Paz et al. (2017) study, which reported increasing graphene-based materials up to 0.5 wt% leads to mechanical features betterment, the more amounts of CNT and rGO (0.5 wt% against 0.25 wt
%) led to higher compressive, bending and tensile strengths. Also, similar trends were observed for bending and tensile strengths so that bending strength of PMMA/HT cements reached from 61.06 ±0.68 to 82.07 ±1.01 MPa, 86.03 ±1.44 MPa and tensile strength raised from 40.02 ±0.71 MPa to 63.21 ±1.70 MPa and 64.92 ±0.75 MPa caused by incorporating 0.5 wt% CNT and rGO, respectively (Fig. 3). Although the trend of bending strength in this study is similar to Goncalves et al.
(Gonçalves et al., 2012), this value is much more than their PMMA/HT/GO cements bending results, and it can be due to a different method of CNT and rGO incorporation to PMMA based cements. In another research (Paz et al., 2019) it was shown that silanes function- alized GO could improve the mechanical performance of PMMA-based cements because of silanization enhancing the long-term dispersion of the graphene in the MMA, reducing the formation of clusters. Based on the results, the silanization approach enhances the covalent bond for- mation at the interface of graphene and the matrix and, resulting in a more vigorous bonding between the matrix and graphene following
Fig. 3. (a,b) Bending modulus and strength and (c,d) tensile modulus and strength of PMMA-based cements containing rGO and CNT reinforcement agents.
polymerization. In this study the main reason for exhibiting superior mechanical properties by rGO reinforced bone cement compared to the CNT reinforced sample in equal reinforcement incorporation can be attributed to the higher specific area and wrinkled surface provided by rGO sheets, which can contribute in the improvement of integration between the reinforcement and BC matrix and better adhesio- n/interlocking. In contrast, the CNTs with a cylindrical shape do not provide the identical interlocking with the micron-sized PMMA beads and HT.
As presented in Fig. 4, the mechanism of the inhabitation of crack propagation for CNT and rGO could be described as follows: (a) crack bridging: rGO acts as a bridge of two sides of the crack and provides closure stress to counteract the applied stress (Pahlevanzadeh et al., 2020), resulting in a delay for further crack propagation (b) pullout: rGO pulls out the matrix and slows down the propagation of the crack by the interfacial friction between graphene and matrix. (c) crack deflection:
crack deflects into a different plane when it encounters graphene, resulting in a tortuous path and more energy dissipation for crack propagation. (d) crack tip shielding: the crack tip is restricted in the vicinity of graphene because of the low energy needed for the deboning of the interface (Pahlevanzadeh et al., 2020; Gao et al., 2014). It should be mentioned that among the mechanisms mentioned above, bridging is the highlighted strategy of CNTs to inhibit crack propagation. The mentioned improvements could have clinical benefits for the usage of PMMA/rGO or PMMA/CNT nanocomposite cement in total joint replacement surgery because of a decrease in the rate of crack propagation.
As can be seen in Fig. 5 the trends in mechanical properties are also consistent with the morphology of the fractured surfaces of the tensile
test specimens. Pores exist in all carbon nanostructure-reinforced ce- ments, despite the uniform dispersion of the nanofiller in the BCs matrix (Fig. 5c and d). In comparison with the PMMA/HT BCs, the bone ce- ments containing the CNT or rGO powders presented a more undulated and wrinkled surface, which is typical for the material and more ductile.
In the rGO-reinforced cements the pores were smaller and fewer, irre- spective of the loading (e.g., Fig. 5e and f), while a porous structure was seen in the cements reinforced with CNTs. The origin of these pores may be related to the release of the unreacted volatile MMA monomer during the polymerization of the cement as well as the degradation of HT particles. The similar result demonstrated in Goncalves et al. (Gonçalves et al., 2012) study, which reported the same results regarding the better capability of rGO based sheets than functionalized CNTs in PMMA/HA BCs.
It should be noted that CNTs can be agglomerated very quickly due to the existence of van der Waals forces in each tube. Nevertheless, rGO favored its single-layer, two-dimensional (2D) hexagonal crystalline structure, with oxygen groups on its surface, could provide an improved bonding with the matrix comparing with the CNTs (Munir et al., 2019), which leads to an increase in the mechanical characteristics of the BCs.
Based on all mechanical test results, PMMA/HT composite cements containing 0.5 wt% CNT and rGO exhibited higher compressive, bending, and tensile tests. Hence bioactivity, cell viability, and adhesion tests were performed on PMMA cement (as control specimens), PMMA/HT without carbon-based reinforcement, and PMMA/HT ce- ments containing 0.5 wt% CNT and rGO as mechanically optimized specimens.
The temperature rising during the polymerization occurs for all PMMA based cement so that temperature reaches its maximum point
Fig. 4.Scheme illustration for inhibition mechanism of crack propagation by PMMA-based cements containing (a) CNT and (b) rGO reinforcement agents.
and then followed by a decrease (listed in Table 2). The peak of tem- perature declined from 90.06 ±1.93 to 75.49 ±1.25 due to the incor- poration of HT and 0.5 wt% of rGO. Generally, the addition of HT, CNT, and rGO led to a reduction of polymerization temperature peak. On the other hand, setting time was increased in the existence of HT, CNT and rGO, which may be due to the less exposure to PMMA in the polymer- ization process. The higher loading of CNT and rGO can significantly reduce the exothermic polymerization reaction, because the carbon- based materials act similar to a heat sink in the BC matrix. Similar re- ports (Chen et al., 2015) demonstrated that ceramic-based particles could absorb heat slightly, which is suitable for declining necrosis of surrounding tissue. The outcomes of the mentioned research revealed
that the polymerization reaction of the BC was influenced by the incorporation of the CNT and rGO; in this respect, the maximum curing temperature reduced, and the reaction was delayed, increasing the setting time. The higher loadings of MWCNTs have more remarkable effects.
3.3. Bioactivity evaluation
Fig. 6a–d shows the SEM images of PMMA, PMMA/HT, PMMA/HT/
0.5CNT, and PMMA/HT/0.5rGO BCs surface before immersion in SBF.
Neat PMMA does not have any microstructure and exhibits a relatively smooth surface. The SEM images of PMMA-based BCs reinforced with HT, CNT, and rGO showed that the reinforcements have uniformly distributed in the polymeric BC matrix, which has a remarkable influ- ence on the biological and mechanical characteristics of the BC. The observation of the surface of PMMA cements revealed that there is no sign of apatite-like layer deposition (Fig. 6e). Fig. 6 f-h show the SEM images of the formed apatite layer on the PMMA/HT BCs with and without carbon-based additives. SEM images of PMMA cements con- taining HT showed that a compacted apatite-layer has formed on the entire surface of the specimens. On the other hand, SEM images of specimen surfaces indicated the existence of CNT, and rGO led to increasing bioactivity features of BCs. The EDS analysis from the surface of PMMA-based BCs before immersion in SBF showed that the presence Fig. 5. SEM images of fracture surfaces for (a) PMMA, (b) PMMA/HT, (c) PMMA/HT/0.25CNT, (d) PMMA/HT/0.5CNT, (e) PMMA/HT/0.25rGO, and (f) PMMA/HT/
0.5rGO bone cements.
Table 2
The setting time and highest temperature within polymerization of PMMA-based bone cements containing rGO and CNT reinforcement agents.
Specimen Setting time (min) Maximum temperature (◦C)
PMMA 9.80 ±0.25 90.06 ±1.93
PMMA/HT 13.6 ±0.34 78.81 ±1.87
PMMA/HT/0.25CNT 13.78 ±0.38 77.02 ±1.85 PMMA/HT/0.5CNT 14.29 ±0.39 76.92 ±1.43 PMMA/HT/0.25rGO 13.85 ±0.41 76.53 ±1.39 PMMA/HT/0.25rGO 14.36 ±0.43 75.49 ±1.25
Standard 5–15 90˂
of S and Ba elements were observed in the PMMA bone cement, which is attributed to the presence of BaSO4 (Area A). However, PMMA/HT/CNT and PMMA/HT/rGO depicted the existence of Mg, Zn, Si, and O repre- senting the incorporation of HT in PMMA-based BCs. Moreover, all characteristic of CNT and rGO incorporation such as carbon Kα peak at 0.277 eV was found on the EDS pattern of PMMA-based containing CNT or rGO (Area B and C) as shown in Fig. 6i.
As can be seen in Fig. 6j, it was recommended that the existence of the Ca2+ions in HT could be exchanged with the H+ions existed in the medium and generate a hydrated silica film, which in turn can present suitable locations for the nucleation of PO43− ion on the cements (Hamvar et al., 2020). These locations stimulate the formation of an apatite layer on the surface of PMMA-based cements in the physiological medium. When the specimens are immersed into the SBF solution, the dissolution of the HT happens, it leads to the exchange and release of Ca2+ion with H3O+or H+ions in the solution (Bakhsheshi-Rad et al.,
2019). After the dissolution of HT, the decreasing the SiO2 content in the solution causes some changes in the Si–O–Si bonds and the formation of Si–OH silane groups. On the other hand, the presence of rGO and CNTs besides HT in the polymer-based cement also positively charged the Ca2+ions, which are adsorbed onto the negatively charged rGO and CNTs surface, and it led to the escalation of bioactivity of PMMA/HT cements. Even though HT’s incorporation could improve the bioactivity, the synergistic influence of the simultaneous addition of HT and rGO was more remarkable. Similar results presented in a study by Kanayama et al. (2014a) revealed that the addition of rGO could enormously improve the calcium absorption and, subsequently, enhance the for- mation of the apatite layer. Based on the results of another investigation conducted by Wan et al. (2011), the addition of GO nanosheets enhanced gelatin’s bioactivity by inducing more calcium phosphate nanocrystals on the gelatin–graphene oxide composites. In the same way, it was stated that (Munir et al., 2019) because of their nanoscale Fig. 6. SEM images of PMMA based BCs surfaces before soaking in SBF for (a) PMMA, (b) PMMA/HT, (c) PMMA/HT/0.5CNT, (d) PMMA/HT/0.5rGO and after 28 days soaking for (e) PMMA, (f) PMMA/HT, (g) PMMA/HT/0.5CNT, (h) PMMA/HT/0.5rGO, (i) EDS analysis of PMMA-based cements before immersion in SBF and (j) schematic illustration of bioactivity mechanism for PMMA/HT/CNT and PMMA/HT/rGO bone cements.
dimensions of graphene and CNTs, they have the potential for entering into different cellular compartments, including the nucleus, which makes nano-carbon based as a promising candidate for delivery of bioactive molecules.
3.4. Cell viability and adhesion
Fig. 7a shows the results of the MTT assay test conducted on PMMA specimens, PMMA/HT cements without and with 0.5 wt% CNT and rGO, after 1, 4, and 7 days of culture times. As can be observed in the figure, cell viability was improved for all specimens with increasing the culture time from 1 day to 7 days, which demonstrated cell-friendly behavior and no toxicity of all cements for the viability of MG63 cells. Incorpo- ration of HT to cement enhanced MG63 cell viability at day 7 of culture to 219.71 ±9.09 versus 162.55 ±7.01 for PMMA, which is associated with the release of Ca and Si ions from HT that helps to the formation of apatite-like layer. Similar result (Diba et al., 2014) revealed silicate-based ceramics additives are significantly improved cell response of the matrix. On the other hand, the presence of 0.5 wt% CNT and rGO in PMMA/HT cements led to the betterment of MG63 cell viability and reached the percentage of viability to 241.74 ±9.85 and 249.53 ±10.04 at day 7 respectively. Other studies have confirmed that rGO nanostructures can improve cell proliferation (Crisan et al., 2015;
Bai et al., 2016). For instance, in the case of using graphene-based materials, the outcomes of a study by Sumathra et al. (2018) confirmed that the addition of GO into hydroxyapatite had no toxicity effect on osteoblasts and augmented propagation and osteogenic discrimination. Also, Li et al. (2014) study revealed that the existence of rGO in the substrate could improve the MG63 cell proliferation, which was in agreement with MTT assay results of PMMA/HT specimens containing CNT and rGO. In this perspective, Porter et al. (2007) more outlined the alteration of CNT and rGO through the nuclear membrane layer and their presence in the nucleus. This stimulated the use of CNTs as an appealing additive phase for the delivery of bioactive molecules, including DNA and RNA, regarding their healing rate, particularly within the nucleus of the cell. Based on the results, it was recommended that (Soleymani Eil Bakhtiari et al., 2020a) the nanoscale dimensions and rod-shaped morphology of CNTs are helpful as they show morphological biomimicry of the principal structural proteins of the body, e.g., fibrillar proteins in the extracellular matrix.
However, it must be taken into account that the toxicity versus biocompatibility is dependent on several physicochemical properties,
including stiffness, surface properties, concentration, exposure time to the cells, and the dispersibility (Zhang and Gurunathan, 2016; Liao et al., 2011). In this context, it has beenreported that incorporation of a high amount of CNT and rGO may attach the membranes of the cell and accumulate in the cytoplasm with revealing toxicity to cells. Neverthe- less, rGO can absorb calcium strongly, suggesting that the encapsulation with a low amount of rGO is effective for osteoblasts viability and pro- liferation, which was reported in Kanayama et al. (2014b) study that revealed films of GO and rGO demonstrated high adsorption of calcium in culture medium with FBS. They speculate that calcium absorption on rGO films was improved by graphite intercalation (i.e., insertion of calcium between layers of graphite) (Kanayama et al., 2014b). So, the low amount of graphene-based materials not only did not have any destructive effect but also enhanced osteoblast cell proliferation (Gurunathan et al., 2019). Also, the same results demonstrated for CNTs considering to promote regeneration of the bone (Nien and Huang, 2010) that occur just at a low concentration which regarding dose-dependent effects of carbon nanostructures on cell viability. In higher dosages, graphene-based materials have a toxic effect on cell viability due to the generation of ROS (Bai et al., 2016). Fig. 7b indicates the MG63 cell morphology in PMMA based cements with and without CNT and rGO structures. As can be observed in Fig. 7b, the existence of rGO and CNT has resulted in changing the morphology to a more round shape. Regarding this issue, Zanello et al. (2006) investigated the use of CNTs as appropriate scaffold materials for the growth and proliferation of osteoblasts and, subsequently, bone formation. They revealed that CNTs could maintain the proliferation of osteoblast and result in the formation of the bone. Besides, they showed that cell shape could be controlled by using SWNTs or MWNTs. The SEM micrographs and sub- sequent approval by MTT assay showed live osteoblast cells, but glob- ular morphology was confusing. Regarding this issue, the below reference confirmed that CNT’s existence in the substrate leads to a round morphology of osteoblast. Zhang et al. (2007) presented that the cell morphology can be changed through treatment with CNTs, in which the majority of the cells treated with CNTs were round, and fewer were elongated, tended to grow separately, and detached from the cell pop- ulations compared to the control cells. Well attachment of MG63 cells on all specimens can be observed clearly. However, cells attachment in PMMA/HT cements containing CNT and rGO was better in compare with specimens without the existence of carbon-based nanostructures which was associated to the creation of osteointegration substrate caused by HT and improvement of protein absorption and cells
Fig. 7.(a) MTT assay and (b) SEM images of MG63 cells attached on PMMA-based BCs containing CNT and rGO.
attachment consequently due to the presence of CNTs and rGO (Tadyszak et al., 2018). Nevertheless, biological examinations showed that the in-vitro biocompatibility of rGO was less influenced compared to that of CNTs and provided a direct relationship between adherence and proliferation of osteoblast and Mesenchymal stem cells (MSCs) type cells (Soleymani Eil Bakhtiari et al., 2020a; Abazari et al., 2020a). So, the formation of granuloma inside the mice lungs when they were dosed with different concentrations of CNTs was observed. This investigation revealed that CNTs show cytotoxicity in the lungs primarily because of the existence of metal catalysts, e.g., Ni. In another study (Munir et al., 2019), it was shown that CNT and graphene cytotoxicity problems are attributed to their nanotoxicological potential effects on human macrophage cells and phagocytes may trigger inflammatory reactions on the implants and scaffolds surfaces, resulting in their loosening and the metal ions release into the surrounding tissues. In the same way, Xu et al. (JMeng et al., 2011) also reported the relocation of CNTs from implants to surrounding organs in association with inflammatory cyto- kine alterations, in which rGO presented better biological properties because of promising desired surface properties like reducing the func- tional oxygen-containing groups.
For a better understanding of CNTs and rGO positive and negative effects on the cell interaction with the BCs surface, Fig. 8 presented schematic illustrations. For discussing carbon-based nanostructured helping effect for cell attachment and proliferation pathway 1, express protein absorption and better cell adsorption in following due to ripples and wrinkles on the surface of the graphene-based material. Similar results reported by Gao et al. (Wang et al., 2015) study that confirm the positive effect of graphene-based materials surfaces in protein absorp- tion. On the other hand, the destructive effect of CNTs and rGO (in high dosages) indicated in pathway 2: mitochondrial dysfunction is also associated with the overproduction of ROS. Li et al. (2012) showed that graphene-based materials cause a reduction in mitochondrial membrane potential (MMP) and, consequently, increase the levels of intracellular ROS, which activate the mitochondria-dependent apoptotic pathway
(Nien and Huang, 2010; Li et al., 2012). Also, ROS induced DNA dam- age, which both can lead to a decline in cell viability (Abazari et al., 2020b; Soleymani Eil Bakhtiari et al., 2020b; Bakhsheshi-Rad et al., 2020). It should be mentioned that because of the significant potential of CNTs and graphene for utilizing in tissue engineering applications due to their purposeful interactions with proteins, osteoblast-like human cells, and nucleic acids, exhibiting the promising cytocompatibility of these nanomaterials. In another research, it was revealed that (Wojtoniszak et al., 2012) the graphene particles are nontoxic at the loadings lower than 50 μg mL−1. In this respect, lately, in a research conducted by Munir et al. (2019) it was reported that adsorption and binding of blood proteins to CNTs are helpful for decreasing their cytotoxicity. Their outcomes from this study shine a light on the design and development of novel and safe PMMA-based bone cements encapsulated with a low concentration of CNT and rGo.
4. Conclusion
In this research, the effects of two nanostructured carbon re- inforcements (rGO and CNTs), when incorporated into the PMMA/HT bone cement have been compared on an extensive group of properties of the cements. Both CNT and rGO in PMMA-based BC display the capa- bility to tailor their compressive, tensile, and bending strength of the cement. Nevertheless, for the rGO-reinforced bone cement, some me- chanical properties were higher compared to the corresponding values for the non-reinforced cement. This could be because of the high degree of surface functionalization, high wrinkled surface and specific area, available in rGO reinforcement which all of them lead to high adhesion and interlocking of the nanosized reinforcement with the bone cement matrix. Regarding the biocompatibility of PMMA-based cement, a low amount of CNT and rGO (0.5 wt%) has been recommended as the highest allowable concentration can be entered into the human body.
However, the cements with 0.5 wt% rGO indicated higher MG63 viability and proliferation support. In fact, PMMA/HT/0.5rGO is
Fig. 8. Schematic illustration of PMMA-based BCs containing CNTs and rGO positive (pathway 1) and negative effect (pathway 2) on cells attachment.
