Graphene Oxide Hybridized nHAC/PLGA Scaffolds Facilitate the Proliferation of MC3T3-E1 Cells
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Biodegradable porous biomaterial scaffolds play a critical role in bone regeneration. In this study, the porous nano-hydroxyapatite/collagen/poly(lactic-co-glycolic acid)/graphene oxide (nHAC/PLGA/GO) composite scaffolds containing different amount of GO were fabricated by freeze-drying method. The results show that the synthesized scaffolds possess a three-dimensional porous structure. GO slightly improves the hydrophilicity of the scaffolds and reinforces their mechanical strength. Young’s modulus of the 1.5 wt% GO incorporated scaffold is greatly increased compared to the control sample. The in vitro experiments show that the nHAC/PLGA/GO (1.5 wt%) scaffolds significantly cell adhesion and proliferation of osteoblast cells (MC3T3-E1). This present study indicates that the nHAC/PLGA/GO scaffolds have excellent cytocompatibility and bone regeneration ability, thus it has high potential to be used as scaffolds in the field of bone tissue engineering.
KeywordsGraphene oxide Poly(lactic-co-glycolic acid) Collagen Nano-hydroxyapatite Biodegradable porous scaffold Bone tissue engineering
Atomic force microscopy
Cell counting kit-8
Computational topology design
Dulbecco’s modified Eagle media
Fetal bovine serum
Fourier transform infrared spectroscopy
Mesenchymal stem cells
Quantitative nano-mechanical atomic force microscope
Scanning electron microscope
Olid free-form fabrication
Bone tissue engineering combining three-dimensional porous scaffolds and bone cells has been widely studied as an attractive approach in the treatment of malfunctioning or lost tissue . Biodegradable scaffolds, which mimic the nature of the bone, play an important role for accommodating cells, controlling cell adhesion and proliferation, and facilitating bone regeneration . Till now, various methods including electrospinning, integration of computational topology design (CTD) and solid free-form fabrication (SFF), and freeze-drying have been applied to fabricate different three-dimensional (3D) porous structures [3, 4, 5, 6, 7]. Electrospinning is able to make nanofibrous or microfibrous scaffolds with complex structures (aligned, spring-like fiber) and compositions . However, the production efficiency of it is a bit low. The integration of CTD and SFF allows design of 3D anatomic scaffolds with porous architecture and better mechanical property. But this method requires strong professional knowledge . Compared to these two methods, freeze-drying method allows fabricating porous structures with a much simpler process by the sublimation of frozen liquid phase under vacuum to fabricate a porous structure .
Nature bones possess complex hierarchical architecture with two main components, collagen and hydroxyapatite [9, 10, 11]. In bone tissue engineering, fabricating an ideal biomimicry of the bone extracellular matrix accommodating for cell adhesion and proliferation for the treatment of malfunction is still a challenge . Nano-hydroxyapatite/collagen (nHAC)-based biodegradable scaffolds which are mimic of the natural bone could provide better biocompatibility, cell affinity, and bioresorbability . However, the drawbacks of collagen, including the poor mechanical and rapid degrading properties, remain an obstacle for its application in bone tissue engineering . Biodegradable aliphatic polymers, such as poly(lactic-co-glycolic) acid (PLGA), with high mechanical strength, outstanding biocompatibility, biodegradability, and good solubility in organic solvents, are ideal compensated material constructing 3D porous scaffolds for bone tissue engineering [15, 16]. A hybrid porous scaffold containing collagen and synthetic polymers combines the advantages of collagen and polymers and overcome their weaknesses, which is extensively used for bone repair and regeneration [17, 18, 19]. For instance, Liao et al. have developed a bone scaffold prepared by nHAC and poly(lactic acid) (PLA) to promote bone regeneration . Niu et al. have fabricated nHAC/poly(L-lactic acid)/chitosan microspheres composite scaffolds for enhancing osteoblast proliferation .
Recently, graphene oxide (GO), a novel carbon sheet with one-atom thickness [20, 21], have attracted great interest in biological field because it owns good biocompatibility. The GO hybridized scaffolds are able to enrich both the mechanical property of the scaffold and the cellular behaviors, such as cell spreading and proliferation [22, 23]. Luo et al. reported that the incorporation of GO into PLGA nanofibrous enhanced proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) . Jing et al. reported that the addition of 1.0 wt% GO into the thermoplastic polyurethane could facilitate the Swiss mouse fibroblasts cell proliferation . Compared with adding the chemical cross-linking agents (genipin, glutaraldehyde, carbodiimide, etc.) [25, 26], which have certain cytotoxicity, to improve the mechanical property of composite scaffolds, the small amount of GO hybridized scaffolds show good biocompatibility. Therefore, the hybridization of the GO and the nHAC/PLGA could be a novel artificial scaffold for bone tissues.
In this study, the porous nano-hydroxyapatite/collagen/poly(lactic-co-glycolic acid) /graphene oxide (nHAC/PLGA/GO) scaffolds, which contains different weight ratio of GO (0.0, 0.5, 1.0, and 1.5 wt%) have been fabricated and characterized. The hybridization scaffolds show porous structures. The addition of GO modifies the hydrophilic property and the mechanical property of the hybridization scaffolds. To investigate the effect of the nHAC/PLGA/GO scaffold on bone tissue engineering, the MC3T3-E1 cells were cultured on the porous hybridization scaffolds. The results show that the 1.5 wt% GO-doped hybridization scaffolds facilitate cell adhesion, growth, and proliferation, further indicating nHAC/PLGA/GO scaffold can be considered as a promising candidate in bone tissue engineering.
Results and Discussion
Structure of nHAC/PLGA/GO Composite Scaffolds
Physicochemical and Mechanical Characterizations of nHAC/PLGA/GO Composite Scaffolds
The hydrophilicity of the scaffolds plays a key role in interacting with cells. The addition of GO not only increases the mechanical property of the composite scaffolds, but also changes the hydrophobicity of four kinds of scaffolds. Figure 4f shows the contact angles of different nHAC/PLGA/GO scaffolds. The contact angles of the nHAC/PLGA scaffolds was ~ 125.1° while for nHAC/PLGA/GO with different GO amount (0.5. 1.0, and 1.5 wt%) are ~ 113.4°, ~ 103.4°, and ~ 101.4°, respectively. As the increasing of the GO amount, the contact angles of the composite scaffolds decrease slightly because of both the hydroxyl groups and the negatively charged groups, such as carboxylic acid groups on the GO surface . Thus, GO can provide remarkable bioactivity to 3D nHAC/PLGA scaffolds.
In general, scaffolds for tissue engineering not only require exhibition of biocompatible morphology and properties, but also porous structure and physical strength . The freeze-dried 3D nHAC/PLGA/GO scaffolds possess porous structure because of the sublimation of solvent. The functional groups, including hydroxyl (OH), epoxy (C-O-C), and carboxyl (COOH) species on scaffolds surfaces  induces good hydrophilicity. The addition of PLGA and GO provides sufficient physical strength. Thus, the 3D nHAC/PLGA/GO scaffolds could be a promising candidate for tissue engineering.
Young’s modulus and OD values of different PLGA/nHAC/GO scaffolds
Young’s modulus and OD values
PLGA/nHAC/GO (0.5 wt%)
PLGA/nHAC/GO (1.0 wt%)
PLGA/nHAC/GO (1.5 wt%)
7.53 ± 1.25 (GPa)
8.34 ± 1.00 (GPa)
9.25 ± 0.85 (GPa)
10.20 ± 1.28 (GPa)
OD (1 day)
0.294 ± 0.006
0.268 ± 0.014
0.269 ± 0.007
0.300 ± 0.008
OD (3 days)
0.433 ± 0.033
0.406 ± 0.018
0.458 ± 0.028
0.505 ± 0.021
OD (5 days)
0.470 ± 0.015
0.450 ± 0.017
0.470 ± 0.013
0.534 ± 0.010
OD (7 days)
0.674 ± 0.039
0.645 ± 0.025
0.704 ± 0.026
0.843 ± 0.071
The cytotoxicity of GO is an essential concern for its application in biology field. Till now, two arguments have been arisen. One claim the GO would induce cytotoxicity and its effect is concentration dependent. For example, Chatterjee, et al. reported the toxic response with differential dose dependency for GO . Pinto, et al. reported that only low concentrations of GO may be incorporated safety in PLA to facilitate cell adhesion and proliferation . The others state that even higher amount of GO would have good biocompatibility and enhance both mechanical properties of the substrates and the cellular behaviors. Shin, et al. studied the C2C12 skeletal myoblasts were enhanced on PLGA-GO-collagen hybrid matrices than PLGA, PLGA-collagen matrices . And Luo, et al. reported the GO-doped PLGA nanofiber scaffolds can enhance the osteogenic differentiation of MSCs . In this study, the GO was selected based on the first argument. The limited amount is added into the composite scaffolds for non-cytotoxicity and enhanced mechanical property. The conjugation of GO into nHAC/PLGA scaffolds significantly enhanced cell growth, proliferation. Although the cell number on both nHAC/PLGA and nHAC/PLGA with small amount of GO, for example, nHAC/PLGA/GO (0.5 wt%), is more or less the same, the number of cells on nHAC/PLGA/GO (1.5 wt%) scaffold was higher than that on the nHAC/PLGA scaffolds. These results indicate the nHAC/PLGA/GO scaffolds are biofunctional with the ability of enhancing the growth and proliferation of MC3T3-E1 cells. Therefore, the excellent biocompatibility and biofunctionality allows nHAC/PLGA/GO to be employed as effective scaffolds for bone regeneration.
The nature of the biomaterial and the fabrication process are very important to scaffold properties . So far, the biomaterials have been extensively studied, including metals , ceramics , glass , chemically synthesized polymers , natural polymers , and combinations of these materials to form composites . Changing components of composite scaffolds will induce the scaffolds property. For instance, to fabricate the biomimic scaffold of natural bone, the type I collagen is been used in this study. Currently, the collagen family includes more than 20 different types of collagen existing in the skin, bone, cartilage, etc. Replacing type I collagen with other types, it is possible to fabricate different composite scaffolds for different purpose. For example, collagen type II is one of the fibril-forming collagens and the predominant type of collagen in cartilage. Coordinating collagen type II into the scaffolds may be able to facilitate cartilage bone regeneration . In addition, the collagen with proper annealing may further strength the scaffolds, which may induce a new composite material with functional structures. Besides the nature of biomaterials, the processing also determines the functional of scaffolds, such as different processing methods. Material chemistry and processing determines the maximum functional properties as well as how cells interact with the scaffold. Scaffolds of properties and requirements in bone tissue engineering have been extensively investigated, including degradation , mechanical properties , cytokine delivery , and combinations of scaffolds and cells .
In summary, nHAC/PLGA/GO scaffolds with different amount of GO (0.0, 0.5, 1.0, and 1.5 wt%) were fabricated by freeze-drying method. The fabricated nHAC/PLGA/GO scaffolds show porous structure. Furthermore, the mechanical property and the hydrophilicity of the scaffolds are enhanced because of the addition of PLGA and GO. The in vitro study shows the porous scaffolds facilitate the cell adsorption, growth, and proliferation. These nHAC/PLGA/GO scaffolds could be a promising candidate for bone tissue applications.
The purified lyophilized type 1 collagen was obtained from Tianjin Saining Biological Engineering Technology Co., Ltd. PLGA with lactide:glycolide ratio of 75:25 and Mw of 95,000 g/mol was purchased from Shandong Medical Appliance Factory (China). GO was purchased from Shanghai Aladdin biochemical Polytron Technologies Inc. MC3T3-E1 osteoblast cells were provided by cell bank of Shanghai Chinese Academy of Sciences. Fetal bovine serum (FBS), antibiotic-antimycotic, CCK-8, and Dulbecco’s modified Eagle media (DMEM) were accessed from Tianjin Nobuo Letter Technology Co., Ltd. 1,4-dioxane, phosphate buffered saline (PBS, 0.1 M, PH 7.4), and all other chemicals were analytical grade and used as received with no further purification.
Preparation of the nHAC Power and nHAC/PLGA/GO Composite Scaffolds
The method of compositing nHAC powder has been reported previously [66, 67, 68]. Briefly, collagen was dissolved in acetic acid (0.5 mol/L) forming a solution with the concentration of 4 g/L. The CaCl2 and H3PO4 (Ca/P = 1.66) solutions were then added separately by drops. The dropping rate is 100 drops per minute. The solution was gently stirred and titrated at 37 °C with ammonia solution to pH 9. After 24 h, the nHAC deposition was harvested by centrifugation and freeze-drying. For the preparation of nHAC/PLGA/GO composite scaffolds, GO was evenly dispersed in dioxane by using an ultrasonic cell crusher, forming a final concentration of 0.0, 0.5, 1.0, and 1.5 g/L, respectively. The PLGA was then added into GO solutions, forming a final concentration of 10% (m/v). The GO/PLGA solutions were then stirred gently at room temperature for 12 h. The final solution was formed by adding the nHAC power into the GO/PLGA solution at a 1:1 nHAC:PLGA weight ratio. The formed nHAC/PLGA/GO solution was then stirred and ultrasonicated for 4 h. After frozen at − 20 °C overnight, the nHAC/PLGA/GO composite scaffolds were obtained by lyophilizing to remove dioxane.
The composite scaffolds were coated with gold and were observed under a SEM (JSM-7100F). We spray gold for 20 s for preparation of electron microscopy samples. The topography and the mechanical properties of the matrices were characterized by atomic force microscopy (AFM, Multimode VIII, Bruker, Germany) in air. Image analysis was performed using Gwyddion and Nanoscope Analysis Software. Compositional analysis of the nHAC/PLGA/GO composite scaffolds was performed by a FT-IR spectrophotometer (VECTOR22, Bruker, Germany). All spectra were recorded in absorption mode in the wavelength range of 1000–2200 cm−1 with a resolution of 4.0 cm−1 and 16-times scanning. The contact angles of the samples were measured using a contact angle measurement system by the sessile drop method (EasyDrop, model DAS30, kruss, Germany). The XRD patterns were measured using the X-ray diffractometer (D8 DISCOVER). The Cu-Kα radiation (λ = 0.154 nm) is 40 kV and 30 mA. The scan rate of the measurements is 8°min−1 over the 2θ range of 5–80° at RT. The porosity of the scaffolds were measured by an automatic surface area and porosity analyzer (ASAP 2460, Micromeritics, GA, USA).
MC3T3-E1 osteoblast cells were incubated in DMEM supplemented with 10% FBS and 3% antibiotic-antimycotic solution at 37 °C and 5% CO2 in a cell incubator. The initial attachment and proliferation were tested by using CCK-8 according to the manufacturer’s instruction, in which the number of viable cells was directly proportional to the metabolic reaction products obtained in the CCK-8 assay . Briefly, the MC3T3-E1 osteoblast cells were seeded at a density of 2.5 × 104 cells per well on the nHAC/PLGA, nHAC/PLGA/GO (0.5 wt%), nHAC/PLGA/GO (1.0 wt%), and nHAC/PLGA/GO (1.5 wt%) matrices embedded in 48-well cell culture plate. The cells were incubated with the CCK-8 solution in the last 2 h of the culture periods (1, 3, 5, and 7 days) for the proliferation at 37 °C in the dark. The absorbance was measured at the wavelength of 450 nm using an ELISA reader (DNM-9602).
The cell samples for SEM measurement were fixed with formaldehyde, and the specimens were then dehydrated through a graded series of ethanol (30, 50, 75, 95, and 100%) for 15 min at each concentration. Then, the samples were drying by critical point drying was allowed to occur with a carbon dioxide analyzer (Hitachi, HCP-2). Finally, the samples with gold coating were observed by SEM.
Where A (experiment) represents absorbance of wells with cells, CCK-8 solution and power samples solution; A (blank) represents absorbance of wells with medium and CCK-8 solution without cells and A (control) represents absorbance of wells with cells, CCK-8 solution without power samples solution.
Quantitative results were expressed as the mean value from at least triplicate samples ± standard deviation (SD). Student’s t test was used to the statistical analysis. A value of p < 0.05 was considered statistically significant. Data are marked ** to indicate p < <0.01.
The authors gratefully acknowledge the support from the National Natural Science Foundation of China (No. 31600753), from the Natural Science Foundation of Tianjin (No. 16JCYBJC43400) and from the National Science Foundation of Hebei (No. C2017202206) and from Hebei Province Foundation for Returned Overseas Chinese Scholars (CL201711). CL acknowledges the financial support by the National Natural Science Foundation of China (Project Nos. 51201056, 51171058, 51401146), Natural Science Foundation of Hebei Province of China (Project Nos. E2013202021, E2013202022, and E2015202037), Outstanding Youth Foundation of Hebei Province of China (Project No. E2015202282), and Science and Technology Correspondent Project of Tianjin (Nos. 14JCTPJC00496, 15JCYBJC29900).
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YL, GY, and LL carried out the experiments and analysis. XZ, HW, and DX supervised the writing of the manuscript and created the figures. YL supervised the whole work. All the authors have read and approve the final manuscript.
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