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Zi-Ran-Tong loaded brushite bone cement with enhanced osteoblast mineralization ability in vitro
Journal Pre-proofs Zi-Ran-Tong Loaded Brushite Bone Cement with Enhanced Osteoblast Mineralization Ability in vitro Zhengjun Pei, Kaili Zhang, Pengbin Li, Jiangbo Zhai, Guangda Li, Wenchao Shang PII: DOI: Reference:
S0167-577X(19)31540-X https://doi.org/10.1016/j.matlet.2019.126908 MLBLUE 126908
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
16 July 2019 12 September 2019 28 October 2019
Please cite this article as: Z. Pei, K. Zhang, P. Li, J. Zhai, G. Li, W. Shang, Zi-Ran-Tong Loaded Brushite Bone Cement with Enhanced Osteoblast Mineralization Ability in vitro, Materials Letters (2019), doi: https://doi.org/ 10.1016/j.matlet.2019.126908
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Zi-Ran-Tong Loaded Brushite Bone Cement with Enhanced Osteoblast Mineralization Ability in vitro Zhengjun Pei1, Kaili Zhang1, Pengbin Li2, Jiangbo Zhai2, Guangda Li1*, Wenchao Shang2*, 1 College of Medical Technology and Engineering, Henan University of Science and Technology, Luo-Yang 471023, Henan, China 2 Huanghe San-Men-Xia Hospital, San-Men-Xia, 472000, Henan, China * Corresponding author, E-mail address: [email protected], [email protected]
Abstract In this study, traditional Chinese medicine Zi-Ran-Tong (pyrite) extracted fluid rich-in-iron was used as cement liquid to prepare novel iron-loaded brushite bone cement. The microstructure and in vitro biological properties of the cement were investigated. The results showed that the phase composition of the novel cement was dominated by brushite while the surface morphology was significantly changed. Moreover, the cement showed no cytotoxicity to human osteosarcoma MG-63 cells. Additionally, the cement not only promoted the osteoblasts adhesion and spread but also enhanced the calcification ability. Therefore, Zi-Ran-Tong loaded brushite bone cement could be potentially used as bone repair material. Keywords: Brushite bone cement, Zi-Ran-Tong (Pyrite), Bone repair, Biomaterials, Bioceramics 1. Introduction Brushite bone cement (Bru) is popular as bone filling and graft material due to its similar chemical composition to bone, and to its good biocompatibility, excellent biodegradability and osteoconductivity [1, 2]. Recently, the addition of biological important metal ions to Bru to promote the biological performance
1
has attracted much attention [1-3]. Iron (Fe), one of the essential trace elements for human body, is very important for bone health [4]. Fe-loaded Bru, prepared from Fe-enriched calcium phosphate, exhibited osteoinductive activity [5]. In this study, Fe-loaded Bru was prepared using Fe-enriched liquid extracted from Zi-Ran-Tong. Zi-Ran-Tong, mainly composing of FeS2 (pyrite), is one of the most important traditional Chinese medicine and has been used by the Chinese people to treat bone fracture for over 1,000 years [6, 7]. Clinical applications show that Zi-Ran-Tong can promote bone reconstruction, relieve pain, and enrich blood. The experimental studies revealed that Zi-Ran-Tong could promote osteoblast differentiation and osteogenic function [8]. Recently, the incorporation of Zi-Ran-Tong to bone repair biomaterials is of interest. For example, the addition of Zi-Ran-Tong to PLLA could stimulate the growth of ROS17/2.8 osteoblasts [7]. The present research attempted to study the performance of Zi-Ran-Tong loaded Bru in vitro. 2. Materials and methods All chemicals used were of analytical grade (bought from Kemiou Lt. Co, China). The water used is thrice-distilled water. 2.1. Preparation of Zi-Ran-Tong extracted fluid The as-received Zi-Ran-Tong (Hebei ChuFeng Chinese Medicine CO., LTD, China) was prepared by calcining the raw Zi-Ran-Tong at 400 ℃ for 2 h and quenching in vinegar [6], and thus only 9.94 at% of sulfur was observed (Fig.1). To prepare the extract, 70 g of Zi-Ran-Tong was soaked in 140 ml water and decocted for 4 h to obtain 10 ml fluid with a pH value of 4 and Fe concentration of 10 mg/ml as measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (Fig.1).
2
2.2. Preparation of cement β-calcium phosphate (β-TCP) was synthesized using the method as a previous report except the coprecipitation process was at room temperature [9]. Cement powder was prepared by mixing β-TCP with Ca(H2PO4)·H2O at a molar ratio of 1:1. The Zi-Ran-Tong extract and 0.5 M citric acid solution was used as cement liquid to synthesize the cement loaded with Zi-Ran-Tong (Z-Bru) and the control sample (Bru), respectively. After mixing the powder with the liquid (3g/ml) for 30 s, the cement paste was immediately placed into modules and removed from the modules after setting. 2.3. Cement characterization After setting for 24 h, the cement was observed by scanning electron microscope (SEM, JSM-IT100) equipped with energy dispersion spectrum (EDS). Simultaneously, the cement was crushed and subjected to X-ray diffraction (XRD, PANalytical X’Pert-PRO MPD) test and Fourier transform infrared spectroscopy (FT-IR, Thermo Fisher Nicolet IS10) test, respectively. 2.4. MTT assay Cement disks (Φ 8×2 mm) were sterilized by soaking in alcohol and irradiating under UV light as a previous study [9]. Then, the cement extract for MTT test was prepared with a solid-liquid ratio of 1:5 [10]. Human osteosarcoma (MG-63) cells were grown in DMEM with 10% fetal bovine serum, supplemented with 1% Penicillin/Streptomycine Solution incubated at 37 ℃ and 5% CO2 [11]. MG-63 cells were seeded in 96-well plate at 1×103 cells/well and 200 μl of the cement extract was added in each well as culture medium. The medium was changed every 3 d. After 1, 3, and 7 d, MTT method was utilized to evaluate the OD values of the cells at 490 nm [10]. The test was repeated for five times.
3
2.5. Cell morphology and alizarin red staining Sterilized disks (Φ 5×2 mm) were pre-soaked in DMEM medium in 12-well plate for 30 min. Then, the medium was discarded and 1×104 MG-63 cells were seeded on each sample. On the 1st, 3rd, and 7th d, the discs were taken out, washed, fixed, dehydrated, and then observed by SEM [12]. On day 7, the 12-well plates were washed twice with PBS after the discs were removed. Then, the remained cells were fixed and stained with alizarin red solution [13]. Pictures around the sample were taken and the calcium nodes area percentage was analyzed using Photoshop in three typical pictures. 3. Results and discussion The phase composition of cements was dominated by brushite (JCPDS 009-0077) (Fig.2). The FI-IR spectra (Fig.2) further confirmed the XRD results as the characteristic bands of HPO4- group of brushite were detected at about 1213, 1140, 1064, 987, 874, 793, 578, and 527 cm-1 for both cements [14,15]. Compared the two spectra, the weak bands at 1121 cm-1 and 602 cm-1 (PO43- group) disappeared in Z-Bru [15], indicating the purer composition. Simultaneously, five blue shifts and two red shifts were found in Z-Bru, suggesting that the Fe ions with higher charge/radius ratio lead to lattice distortion in the cement. Fig. 3 shows the surface EDS and SEM images. Fe was observed on Z-Bru due to the addition of Zi-Ran-Tong. Moreover, flake-like particles growing in preferred orientation and a denser surface were observed for Z-Bru while thicker blade-like particles and more pores were found on Bru, which was quite different from the Fe-loaded Bru synthesized from Fe-loaded β-TCP showing much thicker and bigger crystal grains than the pure control [9]. The MTT results showed Z-Bru had no cytotoxicity to MG-63 cells (Fig. 4 A). Osteoblasts were observed on samples after co-culturing for different days (Fig.4 B1-C3). After 7 d, the cells almost
4
formed a layer to cover the surface of Z-Bru while separated cells, stretching out lamellipodia and filopodia to connect with each other, were observed on Bru. Simultaneously, significantly more mineral nodes were observed for Z-Bru (Fig. 4 D1-F). These results suggested that Zi-Ran-Tong was beneficial for the growth and calcification of osteoblasts, which was very important for the bone reconstruction process. Feng et al reported that Zi-Ran-Tong itself could enhance the calcification ability of osteoblasts and promote the expression of IGF [16]. Zhang et al found that the incorporation of Zi-Ran-Tong to PLLA could improve the ALP activity of osteoblast [7]. The Fe ions in Zi-Ran-Tong may be of great importance to the enhancement of osteoblast performance. As is known, iron is crucial for bone reconstruction [4]. Recent studies showed that the addition of Fe to β-TCP not only improved the osteoblast response in vitro but also enhanced bone formation in vivo [17, 18]. However, for present study, further studies need to be done to reveal how Zi-Ran-Tong helps Bru promote the cellular behavior as well as how it affects the crystal forming process. Moreover, Fe-loaded brushite cement was proven to be antibacterial [5, 9].Whether the Zi-Ran-Tong loaded cement rich-in-Fe has antibacterial ability is on researching in our lab. 4 Conclusion Fe-loaded brushite cement was prepared by utilizing the Zi-Ran-Tong extracted fluid rich-in-Fe as cement liquid. The novel cement exhibited brushite as the dominant phase and showed significantly different surface morphology from the contrast. Moreover, the novel cement displayed no cytotoxicity to MG-63 cells and promoted the spread process as well as the calcifying ability of the osteoblasts. Therefore, Zi-Ran-Tong loaded brushite cement could be potentially used as bone repair material.
5
Acknowledgements This research was funded by Natural Science Foundation of China [grant number 81402225, U1304805 and 21461009]; Young Backbone Teachers Project in Henan Higher Education Institution [2018GGJS050], China; Huanghe San-Men-Xia Hospital, China; Scientific and Technological Research Key Projects of Henan Province [182102311114], China; Innovation Scientists and Technicians Troop Construction Projects of Henan Province, China. References [1] F. Tamimi, Z. Sheikh, J. Barralet, Acta Biomater. 8 (2012) 474–487. [2] J. Cabrejos-Azama, M. H. Alkhraisat, C. Rueda, et al, Mater. Sci. Eng. C. 43 (2014) 403-410. [3] M. H. Alkhraisat, C. Moseke, L. Blanco, Biomaterials. 29 (35) (2008) 4691-4697 [4] E. Balogh, G. Paragh, V. Jeney, Pharmaceuticals. 11 (4) (2018) 107. [5] V. Uskoković, V. Graziani, V. M. Wu, et al, Mater. Sci. Eng., C. 94 (2019) 798-810. [6] National pharmacopoeia commission, Chinese Pharmacopeia, 2015ed, China Medical Science and Technology Press, Beijing, 2015. [7] L. F. Zhang, Y. Y. Zheng, C. D. Xiong, Bull. Mater. Sci. 38 (3) (2015): 811-816. [8] J. Zhang, K. M. Chen, D. J. Zhi, et al, Arch. Pharm. Res. 38 (2015): 2228–2240. [9] G. D. Li, N. Zhang, S. T. Zhao, et al, Mater. Lett. 215, (2018) 27-30. [10] S. M. H. Dabiri, A. Lagazzo, F. Barberis, et al, Mater. Sci. Eng. C. 67 (2016) 502-510. [11] G. F Liang, S. Kan, Y. L. Zhu, et al, Int. J. Nanomed. 13 (2018) 585-599. [12] K. V. Nishad, S. Sureshbabu, M. Komath, et al, Mater. Lett. 209 (2017) 19-22. [13] Y. F. Su, C. C. Lin, T. H. Huang, et al, Mater. Sci. Eng. C. 42 (2014) 672-680.
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[14] A. T. Saleh, L. S. Ling, R. Hussain, J. Mater. Sci. 51 (2016) 7427-7439. [15] A. Laskus, A. Zgadzaj, J. Kolmas, Int. J. Mol. Sci. 19 (2018) 12. [16] W. Feng, W Fu, Y. P. Zhu, et al, ZheJiang Coll. Tradit. Chin. Med. 28 (2) (2004) 65-67. [17] S. Vahabzadeh, S. Bose, Ann. Biomed. Eng. 45 (3) (2017) 819-828. [18] A. Manchón, M. H. Alkhraisat, C. Rueda-Rodriguez, et al. Biomed. Mater. 10 (5) (2015) 055012. Figures Fig1.
Fig2.
7
Fig3.
Fig4.
Figure captions Fig1. Preparation of Zi-Ran-Tong extracted fluid (A) and EDS of the as-received Zi-Ran-Tong (B) Fig.2. XRD patterns (A1 Bru, A2 Z-Bru) and FT-IR spectra (B1, B2) of cements Fig.3.SEM images (Bru: A1, A2; Z-Bru: B1, B2) and EDS results (Bru: A3; Z-Bru: A3) of cements Fig.4 In vitro biological behavior of Bru and Z-Bru: MTT results (A); Typical SEM images of MG-63 cells spread on Bru (B1,1d; B2,3d; B3,7d) and Z-Bru (C1, 1d; C2, 3d; C3, 7d); Typical staining pictures and the corresponding treated pictures of Bru (D1, D2) and Z-Bru (E1, E2); Area percentage of calcium nodules in the staining pictures (F). (*p=0.727＞0.05, **p=0.104＞0.05, ***p =0.007<0.01）
8
Fe-loaded brushite bone cement was prepared by using Zi-Ran-Tong (pyrite) extracted fluid as cement liquid. The cement phase composition was dominated by brushite. The cement exhibited no cytotoxicity to MG-63 cells and enhanced osteoblasts spreading and calcifying ability.
Declaration of interests ☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
9
We have no conflicts of interest to declare.
10
11
12
S0167-577X(19)31540-X https://doi.org/10.1016/j.matlet.2019.126908 MLBLUE 126908
To appear in:
Materials Letters
Received Date: Revised Date: Accepted Date:
16 July 2019 12 September 2019 28 October 2019
Please cite this article as: Z. Pei, K. Zhang, P. Li, J. Zhai, G. Li, W. Shang, Zi-Ran-Tong Loaded Brushite Bone Cement with Enhanced Osteoblast Mineralization Ability in vitro, Materials Letters (2019), doi: https://doi.org/ 10.1016/j.matlet.2019.126908
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Zi-Ran-Tong Loaded Brushite Bone Cement with Enhanced Osteoblast Mineralization Ability in vitro Zhengjun Pei1, Kaili Zhang1, Pengbin Li2, Jiangbo Zhai2, Guangda Li1*, Wenchao Shang2*, 1 College of Medical Technology and Engineering, Henan University of Science and Technology, Luo-Yang 471023, Henan, China 2 Huanghe San-Men-Xia Hospital, San-Men-Xia, 472000, Henan, China * Corresponding author, E-mail address: [email protected], [email protected]
Abstract In this study, traditional Chinese medicine Zi-Ran-Tong (pyrite) extracted fluid rich-in-iron was used as cement liquid to prepare novel iron-loaded brushite bone cement. The microstructure and in vitro biological properties of the cement were investigated. The results showed that the phase composition of the novel cement was dominated by brushite while the surface morphology was significantly changed. Moreover, the cement showed no cytotoxicity to human osteosarcoma MG-63 cells. Additionally, the cement not only promoted the osteoblasts adhesion and spread but also enhanced the calcification ability. Therefore, Zi-Ran-Tong loaded brushite bone cement could be potentially used as bone repair material. Keywords: Brushite bone cement, Zi-Ran-Tong (Pyrite), Bone repair, Biomaterials, Bioceramics 1. Introduction Brushite bone cement (Bru) is popular as bone filling and graft material due to its similar chemical composition to bone, and to its good biocompatibility, excellent biodegradability and osteoconductivity [1, 2]. Recently, the addition of biological important metal ions to Bru to promote the biological performance
1
has attracted much attention [1-3]. Iron (Fe), one of the essential trace elements for human body, is very important for bone health [4]. Fe-loaded Bru, prepared from Fe-enriched calcium phosphate, exhibited osteoinductive activity [5]. In this study, Fe-loaded Bru was prepared using Fe-enriched liquid extracted from Zi-Ran-Tong. Zi-Ran-Tong, mainly composing of FeS2 (pyrite), is one of the most important traditional Chinese medicine and has been used by the Chinese people to treat bone fracture for over 1,000 years [6, 7]. Clinical applications show that Zi-Ran-Tong can promote bone reconstruction, relieve pain, and enrich blood. The experimental studies revealed that Zi-Ran-Tong could promote osteoblast differentiation and osteogenic function [8]. Recently, the incorporation of Zi-Ran-Tong to bone repair biomaterials is of interest. For example, the addition of Zi-Ran-Tong to PLLA could stimulate the growth of ROS17/2.8 osteoblasts [7]. The present research attempted to study the performance of Zi-Ran-Tong loaded Bru in vitro. 2. Materials and methods All chemicals used were of analytical grade (bought from Kemiou Lt. Co, China). The water used is thrice-distilled water. 2.1. Preparation of Zi-Ran-Tong extracted fluid The as-received Zi-Ran-Tong (Hebei ChuFeng Chinese Medicine CO., LTD, China) was prepared by calcining the raw Zi-Ran-Tong at 400 ℃ for 2 h and quenching in vinegar [6], and thus only 9.94 at% of sulfur was observed (Fig.1). To prepare the extract, 70 g of Zi-Ran-Tong was soaked in 140 ml water and decocted for 4 h to obtain 10 ml fluid with a pH value of 4 and Fe concentration of 10 mg/ml as measured by Inductively Coupled Plasma-Atomic Emission Spectrometry (Fig.1).
2
2.2. Preparation of cement β-calcium phosphate (β-TCP) was synthesized using the method as a previous report except the coprecipitation process was at room temperature [9]. Cement powder was prepared by mixing β-TCP with Ca(H2PO4)·H2O at a molar ratio of 1:1. The Zi-Ran-Tong extract and 0.5 M citric acid solution was used as cement liquid to synthesize the cement loaded with Zi-Ran-Tong (Z-Bru) and the control sample (Bru), respectively. After mixing the powder with the liquid (3g/ml) for 30 s, the cement paste was immediately placed into modules and removed from the modules after setting. 2.3. Cement characterization After setting for 24 h, the cement was observed by scanning electron microscope (SEM, JSM-IT100) equipped with energy dispersion spectrum (EDS). Simultaneously, the cement was crushed and subjected to X-ray diffraction (XRD, PANalytical X’Pert-PRO MPD) test and Fourier transform infrared spectroscopy (FT-IR, Thermo Fisher Nicolet IS10) test, respectively. 2.4. MTT assay Cement disks (Φ 8×2 mm) were sterilized by soaking in alcohol and irradiating under UV light as a previous study [9]. Then, the cement extract for MTT test was prepared with a solid-liquid ratio of 1:5 [10]. Human osteosarcoma (MG-63) cells were grown in DMEM with 10% fetal bovine serum, supplemented with 1% Penicillin/Streptomycine Solution incubated at 37 ℃ and 5% CO2 [11]. MG-63 cells were seeded in 96-well plate at 1×103 cells/well and 200 μl of the cement extract was added in each well as culture medium. The medium was changed every 3 d. After 1, 3, and 7 d, MTT method was utilized to evaluate the OD values of the cells at 490 nm [10]. The test was repeated for five times.
3
2.5. Cell morphology and alizarin red staining Sterilized disks (Φ 5×2 mm) were pre-soaked in DMEM medium in 12-well plate for 30 min. Then, the medium was discarded and 1×104 MG-63 cells were seeded on each sample. On the 1st, 3rd, and 7th d, the discs were taken out, washed, fixed, dehydrated, and then observed by SEM [12]. On day 7, the 12-well plates were washed twice with PBS after the discs were removed. Then, the remained cells were fixed and stained with alizarin red solution [13]. Pictures around the sample were taken and the calcium nodes area percentage was analyzed using Photoshop in three typical pictures. 3. Results and discussion The phase composition of cements was dominated by brushite (JCPDS 009-0077) (Fig.2). The FI-IR spectra (Fig.2) further confirmed the XRD results as the characteristic bands of HPO4- group of brushite were detected at about 1213, 1140, 1064, 987, 874, 793, 578, and 527 cm-1 for both cements [14,15]. Compared the two spectra, the weak bands at 1121 cm-1 and 602 cm-1 (PO43- group) disappeared in Z-Bru [15], indicating the purer composition. Simultaneously, five blue shifts and two red shifts were found in Z-Bru, suggesting that the Fe ions with higher charge/radius ratio lead to lattice distortion in the cement. Fig. 3 shows the surface EDS and SEM images. Fe was observed on Z-Bru due to the addition of Zi-Ran-Tong. Moreover, flake-like particles growing in preferred orientation and a denser surface were observed for Z-Bru while thicker blade-like particles and more pores were found on Bru, which was quite different from the Fe-loaded Bru synthesized from Fe-loaded β-TCP showing much thicker and bigger crystal grains than the pure control [9]. The MTT results showed Z-Bru had no cytotoxicity to MG-63 cells (Fig. 4 A). Osteoblasts were observed on samples after co-culturing for different days (Fig.4 B1-C3). After 7 d, the cells almost
4
formed a layer to cover the surface of Z-Bru while separated cells, stretching out lamellipodia and filopodia to connect with each other, were observed on Bru. Simultaneously, significantly more mineral nodes were observed for Z-Bru (Fig. 4 D1-F). These results suggested that Zi-Ran-Tong was beneficial for the growth and calcification of osteoblasts, which was very important for the bone reconstruction process. Feng et al reported that Zi-Ran-Tong itself could enhance the calcification ability of osteoblasts and promote the expression of IGF [16]. Zhang et al found that the incorporation of Zi-Ran-Tong to PLLA could improve the ALP activity of osteoblast [7]. The Fe ions in Zi-Ran-Tong may be of great importance to the enhancement of osteoblast performance. As is known, iron is crucial for bone reconstruction [4]. Recent studies showed that the addition of Fe to β-TCP not only improved the osteoblast response in vitro but also enhanced bone formation in vivo [17, 18]. However, for present study, further studies need to be done to reveal how Zi-Ran-Tong helps Bru promote the cellular behavior as well as how it affects the crystal forming process. Moreover, Fe-loaded brushite cement was proven to be antibacterial [5, 9].Whether the Zi-Ran-Tong loaded cement rich-in-Fe has antibacterial ability is on researching in our lab. 4 Conclusion Fe-loaded brushite cement was prepared by utilizing the Zi-Ran-Tong extracted fluid rich-in-Fe as cement liquid. The novel cement exhibited brushite as the dominant phase and showed significantly different surface morphology from the contrast. Moreover, the novel cement displayed no cytotoxicity to MG-63 cells and promoted the spread process as well as the calcifying ability of the osteoblasts. Therefore, Zi-Ran-Tong loaded brushite cement could be potentially used as bone repair material.
5
Acknowledgements This research was funded by Natural Science Foundation of China [grant number 81402225, U1304805 and 21461009]; Young Backbone Teachers Project in Henan Higher Education Institution [2018GGJS050], China; Huanghe San-Men-Xia Hospital, China; Scientific and Technological Research Key Projects of Henan Province [182102311114], China; Innovation Scientists and Technicians Troop Construction Projects of Henan Province, China. References [1] F. Tamimi, Z. Sheikh, J. Barralet, Acta Biomater. 8 (2012) 474–487. [2] J. Cabrejos-Azama, M. H. Alkhraisat, C. Rueda, et al, Mater. Sci. Eng. C. 43 (2014) 403-410. [3] M. H. Alkhraisat, C. Moseke, L. Blanco, Biomaterials. 29 (35) (2008) 4691-4697 [4] E. Balogh, G. Paragh, V. Jeney, Pharmaceuticals. 11 (4) (2018) 107. [5] V. Uskoković, V. Graziani, V. M. Wu, et al, Mater. Sci. Eng., C. 94 (2019) 798-810. [6] National pharmacopoeia commission, Chinese Pharmacopeia, 2015ed, China Medical Science and Technology Press, Beijing, 2015. [7] L. F. Zhang, Y. Y. Zheng, C. D. Xiong, Bull. Mater. Sci. 38 (3) (2015): 811-816. [8] J. Zhang, K. M. Chen, D. J. Zhi, et al, Arch. Pharm. Res. 38 (2015): 2228–2240. [9] G. D. Li, N. Zhang, S. T. Zhao, et al, Mater. Lett. 215, (2018) 27-30. [10] S. M. H. Dabiri, A. Lagazzo, F. Barberis, et al, Mater. Sci. Eng. C. 67 (2016) 502-510. [11] G. F Liang, S. Kan, Y. L. Zhu, et al, Int. J. Nanomed. 13 (2018) 585-599. [12] K. V. Nishad, S. Sureshbabu, M. Komath, et al, Mater. Lett. 209 (2017) 19-22. [13] Y. F. Su, C. C. Lin, T. H. Huang, et al, Mater. Sci. Eng. C. 42 (2014) 672-680.
6
[14] A. T. Saleh, L. S. Ling, R. Hussain, J. Mater. Sci. 51 (2016) 7427-7439. [15] A. Laskus, A. Zgadzaj, J. Kolmas, Int. J. Mol. Sci. 19 (2018) 12. [16] W. Feng, W Fu, Y. P. Zhu, et al, ZheJiang Coll. Tradit. Chin. Med. 28 (2) (2004) 65-67. [17] S. Vahabzadeh, S. Bose, Ann. Biomed. Eng. 45 (3) (2017) 819-828. [18] A. Manchón, M. H. Alkhraisat, C. Rueda-Rodriguez, et al. Biomed. Mater. 10 (5) (2015) 055012. Figures Fig1.
Fig2.
7
Fig3.
Fig4.
Figure captions Fig1. Preparation of Zi-Ran-Tong extracted fluid (A) and EDS of the as-received Zi-Ran-Tong (B) Fig.2. XRD patterns (A1 Bru, A2 Z-Bru) and FT-IR spectra (B1, B2) of cements Fig.3.SEM images (Bru: A1, A2; Z-Bru: B1, B2) and EDS results (Bru: A3; Z-Bru: A3) of cements Fig.4 In vitro biological behavior of Bru and Z-Bru: MTT results (A); Typical SEM images of MG-63 cells spread on Bru (B1,1d; B2,3d; B3,7d) and Z-Bru (C1, 1d; C2, 3d; C3, 7d); Typical staining pictures and the corresponding treated pictures of Bru (D1, D2) and Z-Bru (E1, E2); Area percentage of calcium nodules in the staining pictures (F). (*p=0.727＞0.05, **p=0.104＞0.05, ***p =0.007<0.01）
8
Fe-loaded brushite bone cement was prepared by using Zi-Ran-Tong (pyrite) extracted fluid as cement liquid. The cement phase composition was dominated by brushite. The cement exhibited no cytotoxicity to MG-63 cells and enhanced osteoblasts spreading and calcifying ability.
Declaration of interests ☒ 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.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
9
We have no conflicts of interest to declare.
10
11
12