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poly(methyl vinyl ether-alt-maleic anhydride) composite with excellent biocompatibility
Materials Letters 261 (2020) 127102
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
A biomimetic nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) composite with excellent biocompatibility Pengfei Ren a, Faming Wang a, Tiantian Zhan b, Wanjun Hu a, Naizhen Zhou a, Tianzhu Zhang a,⇑, Jinhai Ye b,⇑ a b
State Key Laboratory of Bioelectronics, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing 210096, Jiangsu, PR China Nanjing Medical University, Nanjing 210029, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 9 August 2019 Received in revised form 20 November 2019 Accepted 1 December 2019 Available online 2 December 2019 Keywords: Nano-hydroxyapatite Chitosan Poly(methyl vinyl ether-alt-maleic anhydride) Electrostatic interaction Biomimetic Composite materials
a b s t r a c t Nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (NHA/CS/P(MVE-alt-MA)) composite was formed through electrostatic interaction. The results of characterization indicated that the nano-hydroxyapatite (NHA) in the composites was uniformly distributed in the polymer matrices (chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (CS/P(MVE-alt-MA)). With the increase of NHA content, the porosity and strength of the composites increase first and then decrease. The mechanical properties of composite were better than those of the three kinds of single component, the maximum compressive strength of composite was up to 8.48 MPa. Moreover, this composite showed excellent biocompatibility through SD rat bone marrow mesenchymal stem cells (SDBMSCs) culture. Therefore, this composite which combined the advantages of various materials exhibited good mechanical properties and biocompatibility, it was expected to be applied in bone tissue repair. Ó 2019 Published by Elsevier B.V.
1. Introduction Nano-hydroxyapatite (NHA) is the main inorganic component of human bone (60–70%) with good biocompatibility, bioactivity and osteoconductivity [1–4]. Chitosan (CS) is the only naturally occurring alkaline polysaccharide that differs from other commonly used polysaccharides. Since the amino group in the glucose unit has a cationic complexing ability, it can form a polyelectrolyte composite, making it a kind of biomaterial widely used in biomedical fields [5–7]. Poly(methyl vinyl ether-alt-maleic anhydride) (P(MVE-alt-MA)) is a non-toxic biomaterial for humans and animals with good hydrophilicity, chemical stability and biocompatibility. This bioadhesive polycarboxylic acid biomaterial has been widely applied in biotechnology, pharmacology and health care [8,9]. Some organisms in nature (such as animal bone tissue, etc.) are biocomposites with excellent mechanical properties formed by the peculiar interaction between inorganic and organic matter, in which the nanoinorganic substances are uniformly dispersed in the organic matter to enhance the strength of organic matter [10,11]. Inspired by this natural phenomenon, the NHA/CS/ ⇑ Corresponding authors. E-mail addresses: [email protected] (T. Zhang), [email protected] (J. Ye). https://doi.org/10.1016/j.matlet.2019.127102 0167-577X/Ó 2019 Published by Elsevier B.V.
P(MVE-alt-MA) composite prepared by the principle of bionics can better simulate the structure of artificial bone. Therefore, the synergy between the various materials give the composite excellent mechanical properties and biocompatibility to suit the complex physiological environment of body. Moreover, it’s expected to develop a novel bone tissue repair and replacement material [12,13], which is the original intention and goal of this paper.
2. Results and discussion Polyelectrolyte composite based on a polycation and a polyanion was formed from the oppositely charged polyelectrolytes [14]. CS was dissolved in acetic acid to form polycationic CS possessing the positive charged ammonium (–NH+3), P(MVE-alt-MA) was hydrolyzed to obtain polyanionic poly(methyl vinyl etheralt-maleic acid) possessing the negative charged carboxylate (–COO ). At room temperature, the polyelectrolyte composite (CS/P(MVE-alt-MA)) was formed through electrostatic interaction between ammoniums and carboxylates (–NH+3/–COO , ion crosslinking) after poly(methyl vinyl ether-alt-maleic acid) was added to CS in the aqueous solution. During the formation of the CS/P(MVE-alt-MA), NHA was deposited on surface or wrapped inside CS/P(MVE-alt-MA) to further form NHA/CS/P(MVE-alt-MA) composite, which was demonstrated in Fig. 1.
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P. Ren et al. / Materials Letters 261 (2020) 127102
Fig. 1. The synthesis procedure of NHA/CS /P(MVE-alt-MA) composite.
The FTIR spectroscopy was used to analyze the interactions between the chemical bond of various substances in the composite formation process, as shown in Fig. 2(a). The stretching vibration absorption of –NH2 and –OH from CS was located at 3448 cm 1, –COOH from P(MVE-alt-MA) was located at 2956 cm 1 and 1714 cm 1. The above characteristic peak information appeared in CS/P(MVE-alt-MA) composite, demonstrating the successful synthesis of CS/P(MVE-alt-MA) complexes. The absorption peak of PO34 in NHA appeared at 567 cm 1, 604 cm 1, 961 cm 1 and 1037–1093 cm 1. The absorption band of water was located at 1634 and 3433 cm 1, and the weak absorption peak of –OH appeared at 3570 cm 1 and 630 cm 1. In the spectral curve of the formed NHA/CS/P(MVE-alt-MA) composite, a new peak appeared at 602 cm 1, the peak of 1032–1087 cm 1 broadened, and the stretching peaks of –OH and –NH2 near 3450 cm 1 widened and moved toward high wavenumber. All of the above information indicated a strong interaction between CS/P(MVE-alt-MA) and the NHA in the composite. As shown in Fig. 2(b), TG and DTG showed the mass loss and mass loss rate of composite with temperature, respectively. In the range of 50–150 °C, the mass loss of the sample was mainly
due to the loss of free water and crystal water, also included the dehydroxylation of NHA. The thermal stability of CS and P(MVE-alt-MA) was lower than NHA, which resulted from their high-water content and the destruction of hydrogen bonds. In the range of 150–600 °C, the mass loss of the sample was mainly caused by the decomposition of organic matter, so NHA thermal weight loss was small. The decomposition temperature range of CS was about 240–350 °C (the temperature corresponding to peak point of DTG is 305 °C), P(MVE-alt-MA) was about 100–500 °C (the temperature corresponding to peak point of DTG is 166 °C, 295 °C and 438 °C, respectively). Compared with NHA, CS and P(MVE-altMA), the decomposition temperature of CS/P(MVE-alt-MA) and NHA/CS/P(MVE-alt-MA) composite became higher, the decomposition rate becomes slower. The interaction between the substances led to a more stable composite. The decomposition amount and decomposition ratio of NHA/CS/P(MVE-alt-MA) were lower than CS/P(MVE-alt-MA), which mainly due to the unchanged weight of NHA. As shown in the Zeta Potential analysis (Fig. 2(c)), the zeta potential value of NHA/CS/P(MVE-alt-MA) composite was 17.36 mV. The negative charge of NHA/CS/P(MVE-alt-MA)
P. Ren et al. / Materials Letters 261 (2020) 127102
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Fig. 2. (a) The FTIR spectrogram and (b) TG-DTG graphs (TG is solid lines and DTG is dashed lines) of NHA, CS, P(MVE-alt-MA), CS/P(MVE-alt-MA) and NHA/CS/P(MVE-altMA); (c) the Zeta Potential analyzer of NHA/CS/P(MVE-alt-MA) in H2O; (d) the biocompatibility of NHA/CS/P(MVE-alt-MA) composite. Cell proliferation ability of SDBMSCs after culture in the presence of NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days (a normal complete medium was used as the positive control group).
composite probably resulted from the fact that the number of negatively charged –COOH was more than positively charged –NH2, which was beneficial for the bonding of positively charged particles to the surface of the material. The charge values inside the designated ‘‘unstable range” (from 30 to +30 mV), which meted the ideal range for biological applications [15]. The dense three-dimensional structure of composite network was shown in Fig. 3. From Fig. 3(a), it was found that the overall distribution of NHA was uniform with a size of 20–100 nm, although some agglomeration occurred. The CS/P(PVE-alt-MA) composite (b) has an irregular porous structure, its pore size was mainly distributed between 0.2 and 40 lm and there were a large number of internally interconnected pores. After binding with NHA (c and d), the pore walls of NHA/CS/P(PVE-alt-MA) composite became thicker and rougher without significant separation. The NHA was embedded in the CS/P(PVE-alt-MA) organic network, which made the structure of composite become more stable. These phenomena indicated that the two matrices were tightly bound together. To evaluate the biocompatibility of composite, SDBMSCs was cultured with NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days, the cell viability was assessed by a CCK-8 assay (Fig. 2(d)). When cultured with different composite, SDBMSCs showed a similar proliferation rate with the control group (a normal
complete medium without composite). This result indicated that the composite has no significant influence on cells viability. To better demonstrate the influence of NHA/CS/P(MVE-alt-MA) composite on SDBMSCs culture, the Live/Dead Staining Kit was employed to analyze the result of the cells after cultured for 1, 3, 5, and 7 days (Fig. 4). After staining, living cells were green and dead cells were red. The cell proliferation density of SDBMSCs exhibits consistent after cultured with composite extracts and normal complete medium (control group). Therefore, these results proved that the NHA/CS/P(MVE-alt-MA) composite had excellent biocompatibility.
3. Conclusion In this paper, NHA/CS/P(PVE-alt-MA) porous composite were successfully synthesized through electrostatic interaction, in which NHA nanoparticles were uniformly distributed in polymer matrices, and the strong interaction between the two phases in the composite offered a good mechanical property. The culture of SDBMSCs in composite extracts showed that the composite had excellent biocompatibility. Therefore, all of the above properties of composites will provide it a great application potential as a bone tissue repair material.
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P. Ren et al. / Materials Letters 261 (2020) 127102
Fig. 3. The SEM images of NHA (a), CS/P(PVE-alt-MA) (b), NHA/CS/P(PVE-alt-MA) (c), NHA/CS/P(PVE-alt-MA) under larger magnification (d).
Fig. 4. Live/dead fluorescence images of SDBMSCs after culture in the presence of NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
P. Ren et al. / Materials Letters 261 (2020) 127102
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CRediT authorship contribution statement
References
Pengfei Ren: Conceptualization, Methodology, Data curation, Visualization, Writing - original draft. Faming Wang: Methodology, Formal analysis, Writing - review & editing. Tiantian Zhan: Methodology, Resources. Wanjun Hu: Formal analysis, Writing review & editing. Naizhen Zhou: Software. Tianzhu Zhang: Conceptualization, Project administration, Funding acquisition. Jinhai Ye: Supervision, Funding acquisition.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by the Special Project on Development of National Key Scientific Instruments and Equipment of China (2011YQ03013403), National Natural Science Youth Foundation of China (81600407), National Natural Science Foundation of China (81371123), Priority Academic Program Development of Jiangsu Higher Education Institutions: PAPD, 2018-87, Qing-Lan Project (JX2161015067), and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.matlet.2019.127102.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/mlblue
A biomimetic nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) composite with excellent biocompatibility Pengfei Ren a, Faming Wang a, Tiantian Zhan b, Wanjun Hu a, Naizhen Zhou a, Tianzhu Zhang a,⇑, Jinhai Ye b,⇑ a b
State Key Laboratory of Bioelectronics, School of Biological Sciences & Medical Engineering, Southeast University, Nanjing 210096, Jiangsu, PR China Nanjing Medical University, Nanjing 210029, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 9 August 2019 Received in revised form 20 November 2019 Accepted 1 December 2019 Available online 2 December 2019 Keywords: Nano-hydroxyapatite Chitosan Poly(methyl vinyl ether-alt-maleic anhydride) Electrostatic interaction Biomimetic Composite materials
a b s t r a c t Nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (NHA/CS/P(MVE-alt-MA)) composite was formed through electrostatic interaction. The results of characterization indicated that the nano-hydroxyapatite (NHA) in the composites was uniformly distributed in the polymer matrices (chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (CS/P(MVE-alt-MA)). With the increase of NHA content, the porosity and strength of the composites increase first and then decrease. The mechanical properties of composite were better than those of the three kinds of single component, the maximum compressive strength of composite was up to 8.48 MPa. Moreover, this composite showed excellent biocompatibility through SD rat bone marrow mesenchymal stem cells (SDBMSCs) culture. Therefore, this composite which combined the advantages of various materials exhibited good mechanical properties and biocompatibility, it was expected to be applied in bone tissue repair. Ó 2019 Published by Elsevier B.V.
1. Introduction Nano-hydroxyapatite (NHA) is the main inorganic component of human bone (60–70%) with good biocompatibility, bioactivity and osteoconductivity [1–4]. Chitosan (CS) is the only naturally occurring alkaline polysaccharide that differs from other commonly used polysaccharides. Since the amino group in the glucose unit has a cationic complexing ability, it can form a polyelectrolyte composite, making it a kind of biomaterial widely used in biomedical fields [5–7]. Poly(methyl vinyl ether-alt-maleic anhydride) (P(MVE-alt-MA)) is a non-toxic biomaterial for humans and animals with good hydrophilicity, chemical stability and biocompatibility. This bioadhesive polycarboxylic acid biomaterial has been widely applied in biotechnology, pharmacology and health care [8,9]. Some organisms in nature (such as animal bone tissue, etc.) are biocomposites with excellent mechanical properties formed by the peculiar interaction between inorganic and organic matter, in which the nanoinorganic substances are uniformly dispersed in the organic matter to enhance the strength of organic matter [10,11]. Inspired by this natural phenomenon, the NHA/CS/ ⇑ Corresponding authors. E-mail addresses: [email protected] (T. Zhang), [email protected] (J. Ye). https://doi.org/10.1016/j.matlet.2019.127102 0167-577X/Ó 2019 Published by Elsevier B.V.
P(MVE-alt-MA) composite prepared by the principle of bionics can better simulate the structure of artificial bone. Therefore, the synergy between the various materials give the composite excellent mechanical properties and biocompatibility to suit the complex physiological environment of body. Moreover, it’s expected to develop a novel bone tissue repair and replacement material [12,13], which is the original intention and goal of this paper.
2. Results and discussion Polyelectrolyte composite based on a polycation and a polyanion was formed from the oppositely charged polyelectrolytes [14]. CS was dissolved in acetic acid to form polycationic CS possessing the positive charged ammonium (–NH+3), P(MVE-alt-MA) was hydrolyzed to obtain polyanionic poly(methyl vinyl etheralt-maleic acid) possessing the negative charged carboxylate (–COO ). At room temperature, the polyelectrolyte composite (CS/P(MVE-alt-MA)) was formed through electrostatic interaction between ammoniums and carboxylates (–NH+3/–COO , ion crosslinking) after poly(methyl vinyl ether-alt-maleic acid) was added to CS in the aqueous solution. During the formation of the CS/P(MVE-alt-MA), NHA was deposited on surface or wrapped inside CS/P(MVE-alt-MA) to further form NHA/CS/P(MVE-alt-MA) composite, which was demonstrated in Fig. 1.
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P. Ren et al. / Materials Letters 261 (2020) 127102
Fig. 1. The synthesis procedure of NHA/CS /P(MVE-alt-MA) composite.
The FTIR spectroscopy was used to analyze the interactions between the chemical bond of various substances in the composite formation process, as shown in Fig. 2(a). The stretching vibration absorption of –NH2 and –OH from CS was located at 3448 cm 1, –COOH from P(MVE-alt-MA) was located at 2956 cm 1 and 1714 cm 1. The above characteristic peak information appeared in CS/P(MVE-alt-MA) composite, demonstrating the successful synthesis of CS/P(MVE-alt-MA) complexes. The absorption peak of PO34 in NHA appeared at 567 cm 1, 604 cm 1, 961 cm 1 and 1037–1093 cm 1. The absorption band of water was located at 1634 and 3433 cm 1, and the weak absorption peak of –OH appeared at 3570 cm 1 and 630 cm 1. In the spectral curve of the formed NHA/CS/P(MVE-alt-MA) composite, a new peak appeared at 602 cm 1, the peak of 1032–1087 cm 1 broadened, and the stretching peaks of –OH and –NH2 near 3450 cm 1 widened and moved toward high wavenumber. All of the above information indicated a strong interaction between CS/P(MVE-alt-MA) and the NHA in the composite. As shown in Fig. 2(b), TG and DTG showed the mass loss and mass loss rate of composite with temperature, respectively. In the range of 50–150 °C, the mass loss of the sample was mainly
due to the loss of free water and crystal water, also included the dehydroxylation of NHA. The thermal stability of CS and P(MVE-alt-MA) was lower than NHA, which resulted from their high-water content and the destruction of hydrogen bonds. In the range of 150–600 °C, the mass loss of the sample was mainly caused by the decomposition of organic matter, so NHA thermal weight loss was small. The decomposition temperature range of CS was about 240–350 °C (the temperature corresponding to peak point of DTG is 305 °C), P(MVE-alt-MA) was about 100–500 °C (the temperature corresponding to peak point of DTG is 166 °C, 295 °C and 438 °C, respectively). Compared with NHA, CS and P(MVE-altMA), the decomposition temperature of CS/P(MVE-alt-MA) and NHA/CS/P(MVE-alt-MA) composite became higher, the decomposition rate becomes slower. The interaction between the substances led to a more stable composite. The decomposition amount and decomposition ratio of NHA/CS/P(MVE-alt-MA) were lower than CS/P(MVE-alt-MA), which mainly due to the unchanged weight of NHA. As shown in the Zeta Potential analysis (Fig. 2(c)), the zeta potential value of NHA/CS/P(MVE-alt-MA) composite was 17.36 mV. The negative charge of NHA/CS/P(MVE-alt-MA)
P. Ren et al. / Materials Letters 261 (2020) 127102
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Fig. 2. (a) The FTIR spectrogram and (b) TG-DTG graphs (TG is solid lines and DTG is dashed lines) of NHA, CS, P(MVE-alt-MA), CS/P(MVE-alt-MA) and NHA/CS/P(MVE-altMA); (c) the Zeta Potential analyzer of NHA/CS/P(MVE-alt-MA) in H2O; (d) the biocompatibility of NHA/CS/P(MVE-alt-MA) composite. Cell proliferation ability of SDBMSCs after culture in the presence of NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days (a normal complete medium was used as the positive control group).
composite probably resulted from the fact that the number of negatively charged –COOH was more than positively charged –NH2, which was beneficial for the bonding of positively charged particles to the surface of the material. The charge values inside the designated ‘‘unstable range” (from 30 to +30 mV), which meted the ideal range for biological applications [15]. The dense three-dimensional structure of composite network was shown in Fig. 3. From Fig. 3(a), it was found that the overall distribution of NHA was uniform with a size of 20–100 nm, although some agglomeration occurred. The CS/P(PVE-alt-MA) composite (b) has an irregular porous structure, its pore size was mainly distributed between 0.2 and 40 lm and there were a large number of internally interconnected pores. After binding with NHA (c and d), the pore walls of NHA/CS/P(PVE-alt-MA) composite became thicker and rougher without significant separation. The NHA was embedded in the CS/P(PVE-alt-MA) organic network, which made the structure of composite become more stable. These phenomena indicated that the two matrices were tightly bound together. To evaluate the biocompatibility of composite, SDBMSCs was cultured with NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days, the cell viability was assessed by a CCK-8 assay (Fig. 2(d)). When cultured with different composite, SDBMSCs showed a similar proliferation rate with the control group (a normal
complete medium without composite). This result indicated that the composite has no significant influence on cells viability. To better demonstrate the influence of NHA/CS/P(MVE-alt-MA) composite on SDBMSCs culture, the Live/Dead Staining Kit was employed to analyze the result of the cells after cultured for 1, 3, 5, and 7 days (Fig. 4). After staining, living cells were green and dead cells were red. The cell proliferation density of SDBMSCs exhibits consistent after cultured with composite extracts and normal complete medium (control group). Therefore, these results proved that the NHA/CS/P(MVE-alt-MA) composite had excellent biocompatibility.
3. Conclusion In this paper, NHA/CS/P(PVE-alt-MA) porous composite were successfully synthesized through electrostatic interaction, in which NHA nanoparticles were uniformly distributed in polymer matrices, and the strong interaction between the two phases in the composite offered a good mechanical property. The culture of SDBMSCs in composite extracts showed that the composite had excellent biocompatibility. Therefore, all of the above properties of composites will provide it a great application potential as a bone tissue repair material.
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Fig. 3. The SEM images of NHA (a), CS/P(PVE-alt-MA) (b), NHA/CS/P(PVE-alt-MA) (c), NHA/CS/P(PVE-alt-MA) under larger magnification (d).
Fig. 4. Live/dead fluorescence images of SDBMSCs after culture in the presence of NHA/CS/P(MVE-alt-MA) composite extracts for 1, 3, 5, and 7 days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
P. Ren et al. / Materials Letters 261 (2020) 127102
5
CRediT authorship contribution statement
References
Pengfei Ren: Conceptualization, Methodology, Data curation, Visualization, Writing - original draft. Faming Wang: Methodology, Formal analysis, Writing - review & editing. Tiantian Zhan: Methodology, Resources. Wanjun Hu: Formal analysis, Writing review & editing. Naizhen Zhou: Software. Tianzhu Zhang: Conceptualization, Project administration, Funding acquisition. Jinhai Ye: Supervision, Funding acquisition.
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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The work was supported by the Special Project on Development of National Key Scientific Instruments and Equipment of China (2011YQ03013403), National Natural Science Youth Foundation of China (81600407), National Natural Science Foundation of China (81371123), Priority Academic Program Development of Jiangsu Higher Education Institutions: PAPD, 2018-87, Qing-Lan Project (JX2161015067), and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.matlet.2019.127102.