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Reinforced chitosan membranes by microspheres for guided bone regeneration
Journal of the Mechanical Behavior of Biomedical Materials 81 (2018) 195–201
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Reinforced chitosan membranes by microspheres for guided bone regeneration
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Di Huanga,b,1, Lulu Niua,c,1, Jian Lia, Jingjing Dua, , Yan Weia, Yinchun Hua, Xiaojie Liana, ⁎ Weiyi Chenb, Kaiqun Wanga, a
Department of Biomedical Engineering, Research Center for Nano-biomaterials & Regenerative Medicine, College of Mechanics, Taiyuan University of Technology, Taiyuan 030024, PR China b Institute of Applied Mechanics & Biomedical Engineering, Shanxi Key Laboratory of Material Strength & Structural Impact, Taiyuan University of Technology, Taiyuan 030024, PR China c Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Guided bone regeneration Chitosan Nano-hydroxyapatite/chitosan microspheres Reinforce Cytocompatibility
In order to improve the osteogenic activity and mechanical strength of the guided bone regeneration (GBR) membrane for repairing bone defect, nano-hydroxyapatite/chitosan (nHA/CS) composite microspheres were prepared through in situ biomimetic method, then composite microspheres were incorporated into CS membrane. The morphologies and mechanical properties of the composite membranes were investigated through scanning electronic microscopy (SEM) and universal mechanical testing machine. The results show that the in situ biomimetic nHA/CS microspheres were embedded in CS membrane and were integrated tightly with CS matrix. The mechanical properties of GBR membranes containing in situ nHA/CS microspheres is significantly higher than that of membranes containing pure CS microspheres and blending nHA/CS microspheres. Its elongation rate at break reaches 5.61 ± 0.95%. The elastic modulus and strength of the GBR membranes can reach 766.27 ± 20.68 and 43.32 ± 0.95 MPa, respectively. Further, The work-of-fracture of the membranes with in situ microspheres approaches 2.71 ± 0.25 J/m2, which is about 3 times of the pure CS membrane. The cell culture results display that the GBR membranes containing in situ biomimetic nHA/CS microspheres exhibit good cytocompatibility.
1. Introduction The regeneration and repair of bone defect has become an important subject in the research of regenerative medicine at the present time (Vladescu et al., 2016). A large number of studies show that guided bone regeneration (GBR) membrane technology is an effective method to repair bone defects (Kharaziha et al., 2013; Xue et al., 2014; Al-Kattan et al., 2017). GBR membrane can give full play of space maintenance and bone guidance in the defect area. Biodegradable polymer materials (such as chitosan (CS), collagen, poly (lactic acid) (PLA)) have good biological effects and has become the main direction of GBR membrane research (Xue et al., 2014; Norowski et al., 2015; Ma et al., 2016; Hengjie et al., 2017). In the field of existing biodegradable GBR membrane materials, CS have attracted wide attention because of its good biocompatibility, controllable degradation rate and suitable structures (Ma et al., 2014, 2016; Norowski et al., 2015). Numerous studies have found that CS has
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excellent capacity to form microspheres, membranes and fibers, which make it significantly better than the other absorbable membrane materials for designing packaging structures (Sivakumar et al., 2002; Yang et al., 2009; Hengjie et al., 2017). Furthermore, CS is readily soluble in various acidic solvents and exhibits a positive charge, while it has similar structure to glycosaminoglycans, which provides the suitable environment for the cells to be able to effectively accomplish their biological functions such as inducing bone tissue regeneration and promoting drug absorption (Huang et al., 2011, 2012). However, the single CS membrane has poor mechanical properties and is insufficient to effectively support bone regeneration (Pourhaghgouy et al., 2016). CS membrane could not fully play the role of space maintaining and bone guiding in the bone defect area, thus limiting its application in bone regeneration (Mohammadi et al., 2016; Tamburaci and Tihminlioglu, 2017). It is viable to load hydroxyapatitebased microspheres in CS membranes to improve the mechanical and bone conductive properties of GBR membranes. Studies have shown
Corresponding authors. E-mail addresses: [email protected] (J. Du), [email protected] (K. Wang). These authors contribute equally to this work.
https://doi.org/10.1016/j.jmbbm.2018.03.006 Received 12 January 2018; Received in revised form 27 February 2018; Accepted 5 March 2018 Available online 07 March 2018 1751-6161/ © 2018 Elsevier Ltd. All rights reserved.
Journal of the Mechanical Behavior of Biomedical Materials 81 (2018) 195–201
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CS solution. 1.007 g Ca(NO3)2·4H2O was dissolved in the above polymer solution and stirred 0.5 h to obtain a homogeneous solution. The mixed solution was added slowly to 124.742 g Liquid paraffin containing 3.858 g span80 under constant stirring. The mixture was kept stirring at 37 °C for 3 h to obtain a homogeneous W/O emulsion. Afterwards, 0.5 mL 50% glutaraldehyde aqueous solution was gradually added to the emulsion and the stirring continued for a further 0.5 h at 37 °C for cross-linking process. Then, NaOH solution was added into the solution to adjust pH value to 8. Then, 0.972 g Na3PO4 solution was added slowly to the W/O emulsion, and kept stirring for 12 h. Finally, the stabilized composite microspheres were washed consecutively with petroleum ether and isopropyl alcohol and then were dried in an oven at 50 °C for 12 h.
that nano-hydroxyapatite/chitosan (nHA/CS) composite microspheres has excellent biological activity and osteoconduction (Sivakumar et al., 2002; Inanç et al., 2007; Li et al., 2014), which make it possible to enhance CS membrane and improve the bioactivity of the composite conducts to meet the clinical applications. According to the conducted studies, so far no work has been done on incorporating in situ nHA/CS composite microspheres into CS GBR membranes. In the current research, CS microspheres and both nHA/CS composite microspheres prepared by in situ biomimetic and blending method have been incorporated into CS membranes. The mechanical properties of GBR membranes are expected to be reinforced by loading in situ nHA/CS composite microspheres. Meanwhile, it is prospected that the cytocompatibility of GBR membranes could be improved by incorporation of in situ nHA/CS composite microspheres. Such a new type of GBR composite membrane with better biological function and mechanical properties will provide a good prospect for further research and development in GBR membrane.
2.5. Preparation of CS GBR membrane containing microspheres CS membrane containing microspheres was prepared through a solution casting method. 0.428 g CS power was dissolved in 2 wt% iceacetic acid to obtain a 4 wt% CS solution. Then, 0.043 g different microspheres were dispersed in the 4 wt% CS solution, and the mixture was stirred gently for 0.5 h. The mixed solution was set aside for about 4 h at room temperature to remove air bubbles, and then the mixture was casted it on the glass plate, dried at room temperature for 24 h until the solvent evaporated, resulting in a film containing microspheres. Afterward, the films were treated with 0.1 mol/L NaOH solution to neutralize the ice-acetic. Then, the samples were peeled off the glass mould and followed by washing repeatedly with deionized water to pH= 7.0. Finally, samples were set aside on filter paper to dry for 48 h at room temperature.
2. Materials and methods 2.1. Materials Chitosan (CS) with a molecular weight of about 300,000 and a degree of N-deacetylation of 95% was purchased from Haidebei Biotechnology Co., Ltd. (Jinan, China). Ice-acetic acid was purchased from Qibang Chemical Co., Ltd. (Tianjin, China). Ca(NO3)2·4H2O, Na3PO4·12H2O, span 80, liquid paraffin, glutaraldehyde (50% aqueous solution, biological grade), petroleum ether (boiling point 30–60 °C), Isopropyl alcohol and NaOH were purchased from Guangfu Chemical Co., Ltd. (Tianjin, China). All reagents were of analytical grade.
2.6. Characterization 2.2. Preparation of CS microspheres 2.6.1. Morphologies The morphologies of pure CS microspheres, nHA/CS composite microspheres through blending method and in situ biomimetic method, CS GBR membranes containing different microspheres were observed by scanning electric microscopy (SEM, Tescan MIRA3, Czech; Jeol JSM7100F, Japan). Prior to examination, each specimen was coated with gold.
0.428 g CS powder was dissolved in 2 wt% ice-acetic acid to obtain a 4 wt% CS solution. 124.742 g liquid paraffin and 3.858 g span80 were added into a 250 mL conical flask and the mixture was stirred mechanically for 0.5 h to form an oil phase. Then CS solution was slowly added into the oil phase with constant stirring and the mixture was kept stirring at 37 °C for 3 h to obtain a homogeneous W/O emulsion. Then, 0.5 mL 50% glutaraldehyde aqueous solution was gradually incorporated to the emulsion and kept stirring for a further 3 h for crosslinking process. Finally, the products were separated by precipitation and washed consecutively with petroleum ether and isopropyl alcohol. CS microspheres were obtained after dried in an oven for 12 h at 50 °C.
2.6.2. Mechanical properties CS membranes containing different microspheres were tailored into dumbbell specimens of 30 mm in gauge length, 5 mm in width and 0.1 mm in thickness for the measurement of tensile strength. Mechanical properties were performed on a universal mechanical testing machine (500 N, Instron 5544, US) with a cross-head speed of 5 mm/min. The test conditions: the temperature is 24 °C and the relative humidity is 20%. After testing, the fracture surface of the specimens was analysed by SEM. The ultimate elastic modulus of the composite GBR membranes was calculated according the slope of stress-strain curve. And the tensile strength is the stress of fractures of samples. The elongation rate (δ) was calculated and recorded on the basis of the following formula: δ = Δl/l × 100%, where l is gauge length (mm), Δl is the variation of l. A three-point flexural test with a span of 10 mm was used to fracture the specimens at a cross-head speed of 0.5 mm/min on the universal mechanical testing machine (5 N, Instron 5544, US) (Xu et al., 2000). The specimens were cut into the size of 0.1 × 6 × 25 mm3. The workof-fracture (or the energy per volume) was evaluated from the loaddisplacement curve: Work-of-fracture (or the energy per volume): WOF = A/(bh) Where A is the area under the load-displacement curve, which is the work done by the applied load to deflect and fracture the specimen, b is the specimen width and h is the specimen thickness. For all the specimens, the test was stopped at a maximum cross-head displacement of 3 mm for a consistent calculation of the WOF values.
2.3. Preparation of nHA/CS composite microspheres through blending method nHA slurry was prepared through wet chemical precipitation as reference described (Huang et al., 2014a, 2014b). nHA slurry and 4 wt % CS ice-acetic solution were mixed together to prepare nHA/CS composite solution. 124.742 g Liquid paraffin and 3.858 g span80 were added into a flask and stirred mechanically for 0.5 h. Then nHA/CS composite solution was slowly added into the oil phase with constant stirring and the mixture was kept stirring at 37 °C for 3 h. Then, 0.5 mL 50% glutaraldehyde aqueous solution was gradually incorporated to the emulsion and kept stirring for a further 12 h at 37 °C for crosslinking process. Finally, the mixture of products was precipitated and the precipitation was washed consecutively with petroleum ether and isopropyl alcohol. The nHA/CS composite microspheres, thus, were obtained after dried in an oven for a 12 h at 50 °C. 2.4. Preparation of nHA/CS composite microspheres through in situ biomimetic method 0.428 g CS powder was dissolved in 2 wt% ice-acetic to give a 4 wt% 196
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microspheres prepared by blending method are shown in Fig. 1c. The shape of composite microspheres is irregular. The surface of composite microspheres exhibits rough, and some part of the nHA crystals display cluster-by-cluster aggregation. It may be caused by the high surface energy of nHA crystals in the slurry, resulting in spontaneous aggregation (Rodríguez-Clemente et al., 1998). Thereby, nHA crystals inhomogeneously disperse in CS matrix. Moreover, the aggregation of nHA will inevitably decrease the combining ability with the CS matrix and restraint the formation of CS hydrogen bonds. It may be why the composite microspheres are difficult to form a round structure.
2.7. Cytocompatibility Human osteosarcoma cell lines (MG63) were employed to evaluate the cytocompatibility of the GBR membranes containing different microspheres. The MG63 cells were routinely cultured in F12 medium (Cell-culture grade, Biowhittaker, Walkersville, MD, US) supplemented with 10% volume fraction of calf serum (Cell-culture grade, Gibco, Rockville, MD, US), 1% L-glutamine and 1% penicillin/streptomycin. The membranes containing different microspheres were cut into discs with diameter of 10 mm and thickness of 0.1 mm. The specimens were sterilized by ethylene oxide gas for 3 h and then put into 24-well plates and incubated in 1 mL MG63 cells suspension (about 2 × 104 cells per well). For observation of the MG63 cells on the surface of each sample, the seeded MG63 cells were pre-labelled with fluorescent 3,3′dioctadecyloxacarbocyanine perchlorate (DiO) dye (Moleccular Probes, China) at 37 °C for 20 min following the manufacture’ protocol. The cells grown on the discs were then observed using a fluorescence microscope (Nikon IS10, Melville, Japan) at day 4 and 7 after seeding. After culturing for 4 days, the cells were stained with 2% Alexa Fluor® 532 phalloidin (Invitrogen, US) at room temperature overnight and then stained with 0.2% blue fluorescent Hoechst 33342 (Invitrogen, US) for 5 min. The cytoskeletal actin and cell nuclei were observed through confocal laser scanning microscope（CLSM, A1R MP + , Nikon, Japan). The proliferation of MG63 cells with samples or blank control was determined using an MTT (3-{4,5-dimethylthiazol-2y}− 2,5-diphenyl2H-tetrazolium-bromide) assay. At each experimental interval (at day 1, 3 and 5), the medium was discarded, then 40 μL MTT solution was added into each well. After incubation for 4 h at 37 °C, the MTT solution was removed and the precipitated formazan was dissolved in DMSO (150 μL per well). The absorbance of solubilized formation at 570 nm was recorded with a microplate spectrophotometer (Biorad iMark, US).
3.2. Morphologies of the surface of GBR membranes As shown in Fig. 2a, CS membrane containing pure CS microspheres is closely smooth with small amount protrusions of microspheres, higher magnification SEM images (Fig. 2b) shows that microspheres are encapsulated into membrane and no microspheres can be found on the surface of membrane. Fig. 2c shows a lot of in situ nHA/CS microspheres protrusions uniformly appear on the surface of membrane. The protrusions present microparticles with uniform sizes. Further amplification (Fig. 2d) illustrates that the surface of the composite membrane presents macroporous architecture similarly to sponge structure. The macroporous architecture of membrane is favorable to the adhesion of osteoblasts, and may directly bond with surrounding bony tissues (Olszta et al., 2007; Joughehdoust et al., 2013; Huang et al., 2014a, 2014b). As shown in Fig. 2e, the protrusions of nHA/CS microspheres prepared by blending method are clearly observed on the external surface of the membrane. As mentioned above, nHA crystals aggregation and non-uniformly distribution in nHA/CS composite microspheres by blending method would lead to efficiently increase of irregular morphology and particle size, which may have negative impact on cell adhesion and growth (Sivakumar et al., 2002).
2.8. Statistical analysis
3.3. Morphologies of the cross section of GBR membranes
All experiments were performed in triplicate. The data were expressed as the mean ± standard deviation. Statistical significance was assessed using the Student's t-test, values were considered significant at p < 0.05, highly significant at p < 0.01.
The morphologies of cross section of CS membrane containing microspheres are shown in Fig. 3. The SEM images exhibit presence of microspheres with different morphologies in CS membrane. As shown in Fig. 3a-b, there are obvious gaps between CS membrane matrix and pure CS microspheres or blending nHA/CS microspheres. This phenomenon can be attributed to the reason that the surface of pure CS microspheres after cross-linking by glutaraldehyde keeps stable. It becomes difficult to form chemical bonds with CS matrix. For blending nHA/CS microspheres, then nHA crystals aggregation and non-uniformly distribution results the decrease of effective combination with the CS matrix. Differently from the both groups, there are no obvious gaps between in situ nHA/CS microspheres and membrane matrix (Fig. 3c). Higher magnification SEM image shows that in situ nHA/CS microspheres are tightly attached with membrane matrix (Fig. 3d). On the other hand, there are layers of honeycomb distributed in membrane, and the outer layer presents macroporous similarly to sponge
3. Results and discussion 3.1. Characterization of different microspheres Fig. 1a shows the SEM photographs of pure CS microspheres. CS microspheres were of spherical regular structure with smooth and compact surface. nHA/CS microspheres prepared through in situ biomimetic method were shown in Fig. 1b. It shows that the shape of composite microspheres was close to round, a large number of inorganic nanoparticles were dispersed well on the surface of microspheres, and no obvious aggregation phenomenon is found. nHA/CS
Fig. 1. SEM photographs of pure CS microspheres (a), in situ nHA/CS microspheres (b) and blending nHA/CS microspheres (c).
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Fig. 2. SEM images of pure CS microspheres membranes(a) and higher magnification corresponding to the yellow rectangle region in a (b); in situ nHA/CS microspheres membranes(c) and higher magnification corresponding to the yellow rectangle region in c (d); blending nHA/CS microspheres membranes (e) and higher magnification corresponding to the yellow rectangle region in e (f).
Moreover, the composite membrane is ruptured completely at the strain value of about 0.057 (Fig. 4d). As shown in Table 1, the pure CS membrane exhibits excellent mechanical properties. The elongation rate, elastic modulus and strength of the pure CS membrane are higher than those of CS microspheres embedded membranes. It can be illustrated by the reason that the interfacial gap between microspheres and CS matrix may decrease the strength of composite membrane (Zhang and Ma, 2010). The elastic modulus and strength of in situ nHA/CS microspheres embedded membrane reaches 766.27 ± 20.68 and 43.32 ± 0.95 MPa, respectively, which is the highest of all other membranes. Moreover, its elongation rate (about 5.61 ± 0.95%) is higher than the other microspheres embedded membrane. The in situ microspheres embedded membrane exhibit better toughness. nHA particles can evenly disperse on the surface of CS microspheres and effectively reinforce the composite membrane by constructing bonding interactions among microspheres and CS matrix. It can effectively improve the behavior of plastic deformation, which is why the elastic modulus, tensile strength and elongation rate is much larger than those of the other two composite membranes. Fig. 5a shows the typical three-point bending curves of the pure CS and microspheres-embedded membranes. It clearly indicates that the pure CS membrane exhibits higher flexible. It doesn’t fail until maximum cross-head displacement of 3 mm. The curve of in situ microspheres embedded membrane exhibits a highest load and a higher deformation behavior than that of the other both microspheres embedded membranes. Even after reaching the highest load, the in situ microspheres membrane could still bear the bending load due to the in situ nHA/CS microspheres bridging through the crack. Fig. 5b shows the effects of the microspheres on the mechanical properties of the membranes. The WOF of the membranes with in situ microspheres (2.71 ± 0.25 J/m2) is about 3 times of the pure membrane (p < 0.01).
and coheres with central layer. Such membrane with porous structure is beneficial to the adhesion, migration and proliferation of osteoblasts (Huang et al., 2014a, 2014b).
3.4. Mechanical properties of GBR membranes The stress-strain curves of the membranes are shown in Fig. 4. Fig. 4a shows the stress-strain curve of pure CS membrane. It appears a platform when the strain value is about 0.04. It may be caused by that CS molecular chain changes from random to axial orientation with the increase of strain in the tensile processing. Further, the stress decreases suddenly when the strain value is about 0.057 because of the cracks growth during tensile processing. Finally, the stress gradually reduces to 0 when the strain value is about 0.07, which indicates that CS membrane fracture occurs entirely. However, after incorporation of microspheres, the stress-strain behavior of composite membranes would be determined by interfacial conditions between microspheres and membrane matrix (Zhang et al., 2010). There is a clear gap between pure CS microspheres and CS matrix. Therefore, the binding force is so small that the composite membrane is broken completely when the strain value reaches about 0.036 (Fig. 4b). After incorporation of blending nHA/CS microspheres, there is a clear gap between composite microspheres and CS matrix. Even so, as mechanical enhanced phase, nHA crystals are introduced into the microspheres system. Therefore, the elastic modulus and strength of the composite membranes are higher than those of pure CS microspheres embedded membranes. Furthermore, the composite membrane is broken entirely when the strain value is about 0.046 (Fig. 4c). In the in situ nHA/CS microspheres embedded membrane, in situ nHA crystals link the microspheres with CS matrix together and can be severed as the main stress point to effectively disperse or absorb the external load. It gives the composites membrane highest elastic modulus and strength of all groups. 198
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Fig. 3. SEM images of the cross-section of pure CS microspheres membranes (a); blending nHA/CS microspheres membranes (b); in situ nHA/CS microspheres membranes (c) and higher magnification corresponding to the yellow rectangle region in c (d).
The WOF value indicates that the reinforced effect is achieved by in situ nHA/CS microspheres. The in situ nHA crystals linking the microspheres with CS matrix together allow the membranes to consume more energy. The interaction between CS matrix and the microspheres introduces frictional sliding of microspheres during deformation, which consumes energy as well (Zuo et al., 2010).
3.5. Cytocompatibility of GBR membranes To understand the cells growth and proliferation on the surface of GBR membranes containing different microspheres, the MG63 cells were observed y DiO labeling at 4 days after seeding. As shown in Fig. 6a, the cells spread well on all samples and exhibit a similar spindle-like appearance. However, the cell population attached to nHA/ CS microspheres embedded membranes is higher than that of pure CS microspheres membranes (Fig. 6b-c). The result determines that nHA/ CS microspheres embedded samples have excellent cytocompatibility. Fig. 6a1-c1 shows MG63 cells cultured on different membranes for 7 days. It can be seen that all specimens has excellent cellular response. MG63 cells connect with each other. The cells spread and cover almost all the sample surface. CLSM was used to observe cytoskeleton of MG63 cells grown onto
Fig. 4. Stress-strain curves of pure CS membrane (a); pure CS microspheres membrane (b); blending nHA/CS microspheres membrane (c); in situ nHA/CS microspheres membrane (d).
Table 1 The elastic modulus, tensile strength and elongation rate of CS microspheres, blending microspheres and in situ microspheres embedded membranes, compared with pure CS membrane.
Elastic modulus (MPa) Tensile strength (MPa) Elongation rate (%)
Pure CS membrane
CS microspheres membrane
Blending microspheres membrane
In situ microspheres membrane
615.82 ± 13.67 20.92 ± 0.74 7.23 ± 1.25
546.32 ± 19.84 17.57 ± 0.421 3.47 ± 0.64
742.56 ± 16.47 32.25 ± 0.84 4.37 ± 0.75
766.27 ± 20.68 43.32 ± 0.951 5.61 ± 0.95
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Fig. 5. Typical three-point bending load-displacement curves (a) and WOF (b) of the pure CS and microspheres-embedded membranes. Significantly different at *p < 0.05 and **p < 0.01.
crystals has been considered to be the ideal material to improve bone regeneration due to its osteoconductivity and bioactivity (Huang et al., 2011, 2012). The results demonstrate that the nHA/CS microspheres embedded membranes have better cytocompatibility.
the different membranes for 4 days and the results were shown in Fig. 7a-c. MG63 cells cultured onto both nHA/CS microspheres embedded specimens exhibit remarkable filopodia extensions and F-action expressions when compared with those grown onto pure CS microspheres membranes, indicating that MG63 cells seeded onto nHA/CS microspheres embedded substrates have felt the chemical stimulus (nHA crystal) for their initial adhesion and spreading. After 1, 3 and 5 day(s) of culturing, the cell proliferation in each group was determined using MTT test. As shown in Fig. 8, during a period of 5 days of culture, the cells proliferate obviously with the culture time in all groups. At the early stage (day 1), there is no significant difference between the samples. After 3 days co-culturing, the proliferation of cells cultured on both nHA/CS microspheres embedded membranes shows a significant difference from the blank control (p < 0.05). At day 5, the cell proliferation cultured on both nHA/CS microspheres embedded membranes increases significantly compared with CS microspheres and blank control (p < 0.01), which may be caused by the incorporation of nHA crystal because of its similarity to the inorganic component of human hard tissues. As reported, nHA
4. Conclusions In this article, CS guided bone regeneration membrane containing nHA/CS composite microspheres were prepared through in situ biomimetic method and solution casting method. The mechanical properties of composite GBR membranes were successfully carried out. Its elongation rate at break reaches 5.61 ± 0.95%. While, the elasticity modulus and ultimate strength of the membranes can reach 766.272 ± 20.675 and 43.318 ± 0.951 MPa, respectively. The WOF of the membranes with in situ microspheres approaches 2.71 ± 0.25 J/ m2, which is about 3 times of the pure CS membrane. It demonstrates better mechanical properties than containing pure CS microspheres or blending nHA/CS microspheres. GBR membrane containing microspheres is a sort of biomaterials with strong space maintaining ability
Fig. 6. Fluorescent micrographs of DiO-labelled MG63 cells cultured for 4 and 7 days on: pure CS microspheres membranes (a, a1); in situ microspheres membranes (b, b1) and blending microspheres membranes (c, c1).
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Fig. 7. Representative confocal images of MG63 cells cultured for 4 days with staining of actin filament (red) and nuclei (blue) on: pure CS microspheres membranes (a); in situ microspheres membranes (b) and blending microspheres membranes (c).
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Fig. 8. MTT assay for proliferation of MG63 cells cultured on pure CS microspheres, blending microspheres and in situ microspheres membranes for 1, 3 and 5 day(s), compared with the blank control (tissue culture plastic) under the same culture conditions. Significantly different at *p < 0.05 and **p < 0.01.
and biomimetic function, and can promote the bone defects recovery effectively. It may have great potential in curative applications for bone defects. Acknowledgements This work has been supported by the Natural Science Foundation of China (Grant no.: 11502158, 11632013, 51503140, 11502156, 11572213, 51502192). The financial support from China Scholarship Council (Grant no.: 201706935057), Natural Science Foundation of Shanxi Province (Grant no.: 2015021195), International Cooperation Project Foundation of Shanxi Province (Grant no.: 201603D421037) is also acknowledged with gratitude. References Al-Kattan, R., Retzepi, M., Calciolari, E., Donos, N., 2017. Microarray gene expression during early healing of GBR-treated calvarial critical size defects. Clin. Oral. Implants Res. 28, 1248–1257. Hengjie, S., Kwei-Yu, L., Anastasios, K., Daniel, G.A., Chaoxi, W., Kenneth, M.A., Najib, G., Pradeep, A., Tomoko, F., Joel, D.B., 2017. In vitro and in vivo evaluations of a novel post-electrospinning treatment to improve the fibrous structure of chitosan membranes for guided bone regeneration. Biomed. Mater. 12, 015003. Huang, D., Niu, L., Wei, Y., Guo, M., Zuo, Y., Zou, Q., Hu, Y., Chen, W., Li, Y., 2014a. Interfacial and biological properties of the gradient coating on polyamide substrate for bone substitute. J. R. Soc. Interface 11. Huang, D., Xi, S., Zuo, Y., Wei, Y., Guo, M., Wang, H., Chen, W., Li, Y., 2014b. Facile fabrication of gradient bioactive coating with hierarchically porous structures and superior cell response. Mater. Lett. 133, 105–108. Huang, D., Zuo, Y., Zou, Q., Wang, Y., Gao, S., Wang, X., Liu, H., Li, Y., 2012. Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue
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Journal of the Mechanical Behavior of Biomedical Materials journal homepage: www.elsevier.com/locate/jmbbm
Reinforced chitosan membranes by microspheres for guided bone regeneration
T
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Di Huanga,b,1, Lulu Niua,c,1, Jian Lia, Jingjing Dua, , Yan Weia, Yinchun Hua, Xiaojie Liana, ⁎ Weiyi Chenb, Kaiqun Wanga, a
Department of Biomedical Engineering, Research Center for Nano-biomaterials & Regenerative Medicine, College of Mechanics, Taiyuan University of Technology, Taiyuan 030024, PR China b Institute of Applied Mechanics & Biomedical Engineering, Shanxi Key Laboratory of Material Strength & Structural Impact, Taiyuan University of Technology, Taiyuan 030024, PR China c Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610064, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Guided bone regeneration Chitosan Nano-hydroxyapatite/chitosan microspheres Reinforce Cytocompatibility
In order to improve the osteogenic activity and mechanical strength of the guided bone regeneration (GBR) membrane for repairing bone defect, nano-hydroxyapatite/chitosan (nHA/CS) composite microspheres were prepared through in situ biomimetic method, then composite microspheres were incorporated into CS membrane. The morphologies and mechanical properties of the composite membranes were investigated through scanning electronic microscopy (SEM) and universal mechanical testing machine. The results show that the in situ biomimetic nHA/CS microspheres were embedded in CS membrane and were integrated tightly with CS matrix. The mechanical properties of GBR membranes containing in situ nHA/CS microspheres is significantly higher than that of membranes containing pure CS microspheres and blending nHA/CS microspheres. Its elongation rate at break reaches 5.61 ± 0.95%. The elastic modulus and strength of the GBR membranes can reach 766.27 ± 20.68 and 43.32 ± 0.95 MPa, respectively. Further, The work-of-fracture of the membranes with in situ microspheres approaches 2.71 ± 0.25 J/m2, which is about 3 times of the pure CS membrane. The cell culture results display that the GBR membranes containing in situ biomimetic nHA/CS microspheres exhibit good cytocompatibility.
1. Introduction The regeneration and repair of bone defect has become an important subject in the research of regenerative medicine at the present time (Vladescu et al., 2016). A large number of studies show that guided bone regeneration (GBR) membrane technology is an effective method to repair bone defects (Kharaziha et al., 2013; Xue et al., 2014; Al-Kattan et al., 2017). GBR membrane can give full play of space maintenance and bone guidance in the defect area. Biodegradable polymer materials (such as chitosan (CS), collagen, poly (lactic acid) (PLA)) have good biological effects and has become the main direction of GBR membrane research (Xue et al., 2014; Norowski et al., 2015; Ma et al., 2016; Hengjie et al., 2017). In the field of existing biodegradable GBR membrane materials, CS have attracted wide attention because of its good biocompatibility, controllable degradation rate and suitable structures (Ma et al., 2014, 2016; Norowski et al., 2015). Numerous studies have found that CS has
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excellent capacity to form microspheres, membranes and fibers, which make it significantly better than the other absorbable membrane materials for designing packaging structures (Sivakumar et al., 2002; Yang et al., 2009; Hengjie et al., 2017). Furthermore, CS is readily soluble in various acidic solvents and exhibits a positive charge, while it has similar structure to glycosaminoglycans, which provides the suitable environment for the cells to be able to effectively accomplish their biological functions such as inducing bone tissue regeneration and promoting drug absorption (Huang et al., 2011, 2012). However, the single CS membrane has poor mechanical properties and is insufficient to effectively support bone regeneration (Pourhaghgouy et al., 2016). CS membrane could not fully play the role of space maintaining and bone guiding in the bone defect area, thus limiting its application in bone regeneration (Mohammadi et al., 2016; Tamburaci and Tihminlioglu, 2017). It is viable to load hydroxyapatitebased microspheres in CS membranes to improve the mechanical and bone conductive properties of GBR membranes. Studies have shown
Corresponding authors. E-mail addresses: [email protected] (J. Du), [email protected] (K. Wang). These authors contribute equally to this work.
https://doi.org/10.1016/j.jmbbm.2018.03.006 Received 12 January 2018; Received in revised form 27 February 2018; Accepted 5 March 2018 Available online 07 March 2018 1751-6161/ © 2018 Elsevier Ltd. All rights reserved.
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CS solution. 1.007 g Ca(NO3)2·4H2O was dissolved in the above polymer solution and stirred 0.5 h to obtain a homogeneous solution. The mixed solution was added slowly to 124.742 g Liquid paraffin containing 3.858 g span80 under constant stirring. The mixture was kept stirring at 37 °C for 3 h to obtain a homogeneous W/O emulsion. Afterwards, 0.5 mL 50% glutaraldehyde aqueous solution was gradually added to the emulsion and the stirring continued for a further 0.5 h at 37 °C for cross-linking process. Then, NaOH solution was added into the solution to adjust pH value to 8. Then, 0.972 g Na3PO4 solution was added slowly to the W/O emulsion, and kept stirring for 12 h. Finally, the stabilized composite microspheres were washed consecutively with petroleum ether and isopropyl alcohol and then were dried in an oven at 50 °C for 12 h.
that nano-hydroxyapatite/chitosan (nHA/CS) composite microspheres has excellent biological activity and osteoconduction (Sivakumar et al., 2002; Inanç et al., 2007; Li et al., 2014), which make it possible to enhance CS membrane and improve the bioactivity of the composite conducts to meet the clinical applications. According to the conducted studies, so far no work has been done on incorporating in situ nHA/CS composite microspheres into CS GBR membranes. In the current research, CS microspheres and both nHA/CS composite microspheres prepared by in situ biomimetic and blending method have been incorporated into CS membranes. The mechanical properties of GBR membranes are expected to be reinforced by loading in situ nHA/CS composite microspheres. Meanwhile, it is prospected that the cytocompatibility of GBR membranes could be improved by incorporation of in situ nHA/CS composite microspheres. Such a new type of GBR composite membrane with better biological function and mechanical properties will provide a good prospect for further research and development in GBR membrane.
2.5. Preparation of CS GBR membrane containing microspheres CS membrane containing microspheres was prepared through a solution casting method. 0.428 g CS power was dissolved in 2 wt% iceacetic acid to obtain a 4 wt% CS solution. Then, 0.043 g different microspheres were dispersed in the 4 wt% CS solution, and the mixture was stirred gently for 0.5 h. The mixed solution was set aside for about 4 h at room temperature to remove air bubbles, and then the mixture was casted it on the glass plate, dried at room temperature for 24 h until the solvent evaporated, resulting in a film containing microspheres. Afterward, the films were treated with 0.1 mol/L NaOH solution to neutralize the ice-acetic. Then, the samples were peeled off the glass mould and followed by washing repeatedly with deionized water to pH= 7.0. Finally, samples were set aside on filter paper to dry for 48 h at room temperature.
2. Materials and methods 2.1. Materials Chitosan (CS) with a molecular weight of about 300,000 and a degree of N-deacetylation of 95% was purchased from Haidebei Biotechnology Co., Ltd. (Jinan, China). Ice-acetic acid was purchased from Qibang Chemical Co., Ltd. (Tianjin, China). Ca(NO3)2·4H2O, Na3PO4·12H2O, span 80, liquid paraffin, glutaraldehyde (50% aqueous solution, biological grade), petroleum ether (boiling point 30–60 °C), Isopropyl alcohol and NaOH were purchased from Guangfu Chemical Co., Ltd. (Tianjin, China). All reagents were of analytical grade.
2.6. Characterization 2.2. Preparation of CS microspheres 2.6.1. Morphologies The morphologies of pure CS microspheres, nHA/CS composite microspheres through blending method and in situ biomimetic method, CS GBR membranes containing different microspheres were observed by scanning electric microscopy (SEM, Tescan MIRA3, Czech; Jeol JSM7100F, Japan). Prior to examination, each specimen was coated with gold.
0.428 g CS powder was dissolved in 2 wt% ice-acetic acid to obtain a 4 wt% CS solution. 124.742 g liquid paraffin and 3.858 g span80 were added into a 250 mL conical flask and the mixture was stirred mechanically for 0.5 h to form an oil phase. Then CS solution was slowly added into the oil phase with constant stirring and the mixture was kept stirring at 37 °C for 3 h to obtain a homogeneous W/O emulsion. Then, 0.5 mL 50% glutaraldehyde aqueous solution was gradually incorporated to the emulsion and kept stirring for a further 3 h for crosslinking process. Finally, the products were separated by precipitation and washed consecutively with petroleum ether and isopropyl alcohol. CS microspheres were obtained after dried in an oven for 12 h at 50 °C.
2.6.2. Mechanical properties CS membranes containing different microspheres were tailored into dumbbell specimens of 30 mm in gauge length, 5 mm in width and 0.1 mm in thickness for the measurement of tensile strength. Mechanical properties were performed on a universal mechanical testing machine (500 N, Instron 5544, US) with a cross-head speed of 5 mm/min. The test conditions: the temperature is 24 °C and the relative humidity is 20%. After testing, the fracture surface of the specimens was analysed by SEM. The ultimate elastic modulus of the composite GBR membranes was calculated according the slope of stress-strain curve. And the tensile strength is the stress of fractures of samples. The elongation rate (δ) was calculated and recorded on the basis of the following formula: δ = Δl/l × 100%, where l is gauge length (mm), Δl is the variation of l. A three-point flexural test with a span of 10 mm was used to fracture the specimens at a cross-head speed of 0.5 mm/min on the universal mechanical testing machine (5 N, Instron 5544, US) (Xu et al., 2000). The specimens were cut into the size of 0.1 × 6 × 25 mm3. The workof-fracture (or the energy per volume) was evaluated from the loaddisplacement curve: Work-of-fracture (or the energy per volume): WOF = A/(bh) Where A is the area under the load-displacement curve, which is the work done by the applied load to deflect and fracture the specimen, b is the specimen width and h is the specimen thickness. For all the specimens, the test was stopped at a maximum cross-head displacement of 3 mm for a consistent calculation of the WOF values.
2.3. Preparation of nHA/CS composite microspheres through blending method nHA slurry was prepared through wet chemical precipitation as reference described (Huang et al., 2014a, 2014b). nHA slurry and 4 wt % CS ice-acetic solution were mixed together to prepare nHA/CS composite solution. 124.742 g Liquid paraffin and 3.858 g span80 were added into a flask and stirred mechanically for 0.5 h. Then nHA/CS composite solution was slowly added into the oil phase with constant stirring and the mixture was kept stirring at 37 °C for 3 h. Then, 0.5 mL 50% glutaraldehyde aqueous solution was gradually incorporated to the emulsion and kept stirring for a further 12 h at 37 °C for crosslinking process. Finally, the mixture of products was precipitated and the precipitation was washed consecutively with petroleum ether and isopropyl alcohol. The nHA/CS composite microspheres, thus, were obtained after dried in an oven for a 12 h at 50 °C. 2.4. Preparation of nHA/CS composite microspheres through in situ biomimetic method 0.428 g CS powder was dissolved in 2 wt% ice-acetic to give a 4 wt% 196
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microspheres prepared by blending method are shown in Fig. 1c. The shape of composite microspheres is irregular. The surface of composite microspheres exhibits rough, and some part of the nHA crystals display cluster-by-cluster aggregation. It may be caused by the high surface energy of nHA crystals in the slurry, resulting in spontaneous aggregation (Rodríguez-Clemente et al., 1998). Thereby, nHA crystals inhomogeneously disperse in CS matrix. Moreover, the aggregation of nHA will inevitably decrease the combining ability with the CS matrix and restraint the formation of CS hydrogen bonds. It may be why the composite microspheres are difficult to form a round structure.
2.7. Cytocompatibility Human osteosarcoma cell lines (MG63) were employed to evaluate the cytocompatibility of the GBR membranes containing different microspheres. The MG63 cells were routinely cultured in F12 medium (Cell-culture grade, Biowhittaker, Walkersville, MD, US) supplemented with 10% volume fraction of calf serum (Cell-culture grade, Gibco, Rockville, MD, US), 1% L-glutamine and 1% penicillin/streptomycin. The membranes containing different microspheres were cut into discs with diameter of 10 mm and thickness of 0.1 mm. The specimens were sterilized by ethylene oxide gas for 3 h and then put into 24-well plates and incubated in 1 mL MG63 cells suspension (about 2 × 104 cells per well). For observation of the MG63 cells on the surface of each sample, the seeded MG63 cells were pre-labelled with fluorescent 3,3′dioctadecyloxacarbocyanine perchlorate (DiO) dye (Moleccular Probes, China) at 37 °C for 20 min following the manufacture’ protocol. The cells grown on the discs were then observed using a fluorescence microscope (Nikon IS10, Melville, Japan) at day 4 and 7 after seeding. After culturing for 4 days, the cells were stained with 2% Alexa Fluor® 532 phalloidin (Invitrogen, US) at room temperature overnight and then stained with 0.2% blue fluorescent Hoechst 33342 (Invitrogen, US) for 5 min. The cytoskeletal actin and cell nuclei were observed through confocal laser scanning microscope（CLSM, A1R MP + , Nikon, Japan). The proliferation of MG63 cells with samples or blank control was determined using an MTT (3-{4,5-dimethylthiazol-2y}− 2,5-diphenyl2H-tetrazolium-bromide) assay. At each experimental interval (at day 1, 3 and 5), the medium was discarded, then 40 μL MTT solution was added into each well. After incubation for 4 h at 37 °C, the MTT solution was removed and the precipitated formazan was dissolved in DMSO (150 μL per well). The absorbance of solubilized formation at 570 nm was recorded with a microplate spectrophotometer (Biorad iMark, US).
3.2. Morphologies of the surface of GBR membranes As shown in Fig. 2a, CS membrane containing pure CS microspheres is closely smooth with small amount protrusions of microspheres, higher magnification SEM images (Fig. 2b) shows that microspheres are encapsulated into membrane and no microspheres can be found on the surface of membrane. Fig. 2c shows a lot of in situ nHA/CS microspheres protrusions uniformly appear on the surface of membrane. The protrusions present microparticles with uniform sizes. Further amplification (Fig. 2d) illustrates that the surface of the composite membrane presents macroporous architecture similarly to sponge structure. The macroporous architecture of membrane is favorable to the adhesion of osteoblasts, and may directly bond with surrounding bony tissues (Olszta et al., 2007; Joughehdoust et al., 2013; Huang et al., 2014a, 2014b). As shown in Fig. 2e, the protrusions of nHA/CS microspheres prepared by blending method are clearly observed on the external surface of the membrane. As mentioned above, nHA crystals aggregation and non-uniformly distribution in nHA/CS composite microspheres by blending method would lead to efficiently increase of irregular morphology and particle size, which may have negative impact on cell adhesion and growth (Sivakumar et al., 2002).
2.8. Statistical analysis
3.3. Morphologies of the cross section of GBR membranes
All experiments were performed in triplicate. The data were expressed as the mean ± standard deviation. Statistical significance was assessed using the Student's t-test, values were considered significant at p < 0.05, highly significant at p < 0.01.
The morphologies of cross section of CS membrane containing microspheres are shown in Fig. 3. The SEM images exhibit presence of microspheres with different morphologies in CS membrane. As shown in Fig. 3a-b, there are obvious gaps between CS membrane matrix and pure CS microspheres or blending nHA/CS microspheres. This phenomenon can be attributed to the reason that the surface of pure CS microspheres after cross-linking by glutaraldehyde keeps stable. It becomes difficult to form chemical bonds with CS matrix. For blending nHA/CS microspheres, then nHA crystals aggregation and non-uniformly distribution results the decrease of effective combination with the CS matrix. Differently from the both groups, there are no obvious gaps between in situ nHA/CS microspheres and membrane matrix (Fig. 3c). Higher magnification SEM image shows that in situ nHA/CS microspheres are tightly attached with membrane matrix (Fig. 3d). On the other hand, there are layers of honeycomb distributed in membrane, and the outer layer presents macroporous similarly to sponge
3. Results and discussion 3.1. Characterization of different microspheres Fig. 1a shows the SEM photographs of pure CS microspheres. CS microspheres were of spherical regular structure with smooth and compact surface. nHA/CS microspheres prepared through in situ biomimetic method were shown in Fig. 1b. It shows that the shape of composite microspheres was close to round, a large number of inorganic nanoparticles were dispersed well on the surface of microspheres, and no obvious aggregation phenomenon is found. nHA/CS
Fig. 1. SEM photographs of pure CS microspheres (a), in situ nHA/CS microspheres (b) and blending nHA/CS microspheres (c).
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Fig. 2. SEM images of pure CS microspheres membranes(a) and higher magnification corresponding to the yellow rectangle region in a (b); in situ nHA/CS microspheres membranes(c) and higher magnification corresponding to the yellow rectangle region in c (d); blending nHA/CS microspheres membranes (e) and higher magnification corresponding to the yellow rectangle region in e (f).
Moreover, the composite membrane is ruptured completely at the strain value of about 0.057 (Fig. 4d). As shown in Table 1, the pure CS membrane exhibits excellent mechanical properties. The elongation rate, elastic modulus and strength of the pure CS membrane are higher than those of CS microspheres embedded membranes. It can be illustrated by the reason that the interfacial gap between microspheres and CS matrix may decrease the strength of composite membrane (Zhang and Ma, 2010). The elastic modulus and strength of in situ nHA/CS microspheres embedded membrane reaches 766.27 ± 20.68 and 43.32 ± 0.95 MPa, respectively, which is the highest of all other membranes. Moreover, its elongation rate (about 5.61 ± 0.95%) is higher than the other microspheres embedded membrane. The in situ microspheres embedded membrane exhibit better toughness. nHA particles can evenly disperse on the surface of CS microspheres and effectively reinforce the composite membrane by constructing bonding interactions among microspheres and CS matrix. It can effectively improve the behavior of plastic deformation, which is why the elastic modulus, tensile strength and elongation rate is much larger than those of the other two composite membranes. Fig. 5a shows the typical three-point bending curves of the pure CS and microspheres-embedded membranes. It clearly indicates that the pure CS membrane exhibits higher flexible. It doesn’t fail until maximum cross-head displacement of 3 mm. The curve of in situ microspheres embedded membrane exhibits a highest load and a higher deformation behavior than that of the other both microspheres embedded membranes. Even after reaching the highest load, the in situ microspheres membrane could still bear the bending load due to the in situ nHA/CS microspheres bridging through the crack. Fig. 5b shows the effects of the microspheres on the mechanical properties of the membranes. The WOF of the membranes with in situ microspheres (2.71 ± 0.25 J/m2) is about 3 times of the pure membrane (p < 0.01).
and coheres with central layer. Such membrane with porous structure is beneficial to the adhesion, migration and proliferation of osteoblasts (Huang et al., 2014a, 2014b).
3.4. Mechanical properties of GBR membranes The stress-strain curves of the membranes are shown in Fig. 4. Fig. 4a shows the stress-strain curve of pure CS membrane. It appears a platform when the strain value is about 0.04. It may be caused by that CS molecular chain changes from random to axial orientation with the increase of strain in the tensile processing. Further, the stress decreases suddenly when the strain value is about 0.057 because of the cracks growth during tensile processing. Finally, the stress gradually reduces to 0 when the strain value is about 0.07, which indicates that CS membrane fracture occurs entirely. However, after incorporation of microspheres, the stress-strain behavior of composite membranes would be determined by interfacial conditions between microspheres and membrane matrix (Zhang et al., 2010). There is a clear gap between pure CS microspheres and CS matrix. Therefore, the binding force is so small that the composite membrane is broken completely when the strain value reaches about 0.036 (Fig. 4b). After incorporation of blending nHA/CS microspheres, there is a clear gap between composite microspheres and CS matrix. Even so, as mechanical enhanced phase, nHA crystals are introduced into the microspheres system. Therefore, the elastic modulus and strength of the composite membranes are higher than those of pure CS microspheres embedded membranes. Furthermore, the composite membrane is broken entirely when the strain value is about 0.046 (Fig. 4c). In the in situ nHA/CS microspheres embedded membrane, in situ nHA crystals link the microspheres with CS matrix together and can be severed as the main stress point to effectively disperse or absorb the external load. It gives the composites membrane highest elastic modulus and strength of all groups. 198
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Fig. 3. SEM images of the cross-section of pure CS microspheres membranes (a); blending nHA/CS microspheres membranes (b); in situ nHA/CS microspheres membranes (c) and higher magnification corresponding to the yellow rectangle region in c (d).
The WOF value indicates that the reinforced effect is achieved by in situ nHA/CS microspheres. The in situ nHA crystals linking the microspheres with CS matrix together allow the membranes to consume more energy. The interaction between CS matrix and the microspheres introduces frictional sliding of microspheres during deformation, which consumes energy as well (Zuo et al., 2010).
3.5. Cytocompatibility of GBR membranes To understand the cells growth and proliferation on the surface of GBR membranes containing different microspheres, the MG63 cells were observed y DiO labeling at 4 days after seeding. As shown in Fig. 6a, the cells spread well on all samples and exhibit a similar spindle-like appearance. However, the cell population attached to nHA/ CS microspheres embedded membranes is higher than that of pure CS microspheres membranes (Fig. 6b-c). The result determines that nHA/ CS microspheres embedded samples have excellent cytocompatibility. Fig. 6a1-c1 shows MG63 cells cultured on different membranes for 7 days. It can be seen that all specimens has excellent cellular response. MG63 cells connect with each other. The cells spread and cover almost all the sample surface. CLSM was used to observe cytoskeleton of MG63 cells grown onto
Fig. 4. Stress-strain curves of pure CS membrane (a); pure CS microspheres membrane (b); blending nHA/CS microspheres membrane (c); in situ nHA/CS microspheres membrane (d).
Table 1 The elastic modulus, tensile strength and elongation rate of CS microspheres, blending microspheres and in situ microspheres embedded membranes, compared with pure CS membrane.
Elastic modulus (MPa) Tensile strength (MPa) Elongation rate (%)
Pure CS membrane
CS microspheres membrane
Blending microspheres membrane
In situ microspheres membrane
615.82 ± 13.67 20.92 ± 0.74 7.23 ± 1.25
546.32 ± 19.84 17.57 ± 0.421 3.47 ± 0.64
742.56 ± 16.47 32.25 ± 0.84 4.37 ± 0.75
766.27 ± 20.68 43.32 ± 0.951 5.61 ± 0.95
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Fig. 5. Typical three-point bending load-displacement curves (a) and WOF (b) of the pure CS and microspheres-embedded membranes. Significantly different at *p < 0.05 and **p < 0.01.
crystals has been considered to be the ideal material to improve bone regeneration due to its osteoconductivity and bioactivity (Huang et al., 2011, 2012). The results demonstrate that the nHA/CS microspheres embedded membranes have better cytocompatibility.
the different membranes for 4 days and the results were shown in Fig. 7a-c. MG63 cells cultured onto both nHA/CS microspheres embedded specimens exhibit remarkable filopodia extensions and F-action expressions when compared with those grown onto pure CS microspheres membranes, indicating that MG63 cells seeded onto nHA/CS microspheres embedded substrates have felt the chemical stimulus (nHA crystal) for their initial adhesion and spreading. After 1, 3 and 5 day(s) of culturing, the cell proliferation in each group was determined using MTT test. As shown in Fig. 8, during a period of 5 days of culture, the cells proliferate obviously with the culture time in all groups. At the early stage (day 1), there is no significant difference between the samples. After 3 days co-culturing, the proliferation of cells cultured on both nHA/CS microspheres embedded membranes shows a significant difference from the blank control (p < 0.05). At day 5, the cell proliferation cultured on both nHA/CS microspheres embedded membranes increases significantly compared with CS microspheres and blank control (p < 0.01), which may be caused by the incorporation of nHA crystal because of its similarity to the inorganic component of human hard tissues. As reported, nHA
4. Conclusions In this article, CS guided bone regeneration membrane containing nHA/CS composite microspheres were prepared through in situ biomimetic method and solution casting method. The mechanical properties of composite GBR membranes were successfully carried out. Its elongation rate at break reaches 5.61 ± 0.95%. While, the elasticity modulus and ultimate strength of the membranes can reach 766.272 ± 20.675 and 43.318 ± 0.951 MPa, respectively. The WOF of the membranes with in situ microspheres approaches 2.71 ± 0.25 J/ m2, which is about 3 times of the pure CS membrane. It demonstrates better mechanical properties than containing pure CS microspheres or blending nHA/CS microspheres. GBR membrane containing microspheres is a sort of biomaterials with strong space maintaining ability
Fig. 6. Fluorescent micrographs of DiO-labelled MG63 cells cultured for 4 and 7 days on: pure CS microspheres membranes (a, a1); in situ microspheres membranes (b, b1) and blending microspheres membranes (c, c1).
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Fig. 7. Representative confocal images of MG63 cells cultured for 4 days with staining of actin filament (red) and nuclei (blue) on: pure CS microspheres membranes (a); in situ microspheres membranes (b) and blending microspheres membranes (c).
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Fig. 8. MTT assay for proliferation of MG63 cells cultured on pure CS microspheres, blending microspheres and in situ microspheres membranes for 1, 3 and 5 day(s), compared with the blank control (tissue culture plastic) under the same culture conditions. Significantly different at *p < 0.05 and **p < 0.01.
and biomimetic function, and can promote the bone defects recovery effectively. It may have great potential in curative applications for bone defects. Acknowledgements This work has been supported by the Natural Science Foundation of China (Grant no.: 11502158, 11632013, 51503140, 11502156, 11572213, 51502192). The financial support from China Scholarship Council (Grant no.: 201706935057), Natural Science Foundation of Shanxi Province (Grant no.: 2015021195), International Cooperation Project Foundation of Shanxi Province (Grant no.: 201603D421037) is also acknowledged with gratitude. References Al-Kattan, R., Retzepi, M., Calciolari, E., Donos, N., 2017. Microarray gene expression during early healing of GBR-treated calvarial critical size defects. Clin. Oral. Implants Res. 28, 1248–1257. Hengjie, S., Kwei-Yu, L., Anastasios, K., Daniel, G.A., Chaoxi, W., Kenneth, M.A., Najib, G., Pradeep, A., Tomoko, F., Joel, D.B., 2017. In vitro and in vivo evaluations of a novel post-electrospinning treatment to improve the fibrous structure of chitosan membranes for guided bone regeneration. Biomed. Mater. 12, 015003. Huang, D., Niu, L., Wei, Y., Guo, M., Zuo, Y., Zou, Q., Hu, Y., Chen, W., Li, Y., 2014a. Interfacial and biological properties of the gradient coating on polyamide substrate for bone substitute. J. R. Soc. Interface 11. Huang, D., Xi, S., Zuo, Y., Wei, Y., Guo, M., Wang, H., Chen, W., Li, Y., 2014b. Facile fabrication of gradient bioactive coating with hierarchically porous structures and superior cell response. Mater. Lett. 133, 105–108. Huang, D., Zuo, Y., Zou, Q., Wang, Y., Gao, S., Wang, X., Liu, H., Li, Y., 2012. Reinforced nanohydroxyapatite/polyamide66 scaffolds by chitosan coating for bone tissue
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