J. Mater. Sci. Technol. ›› 2020, Vol. 43: 52-63.DOI: 10.1016/j.jmst.2020.01.006
• Research Article • Previous Articles Next Articles
Paulina Kazimierczaka, Aleksandra Benkob, Krzysztof Palkac, Cristina Canalde, Dorota Kolodynskaf, Agata Przekoraa*()
Received:
2019-08-14
Accepted:
2019-10-01
Published:
2020-04-15
Online:
2020-04-26
Contact:
Przekora Agata
Paulina Kazimierczak, Aleksandra Benko, Krzysztof Palka, Cristina Canal, Dorota Kolodynska, Agata Przekora. Novel synthesis method combining a foaming agent with freeze-drying to obtain hybrid highly macroporous bone scaffolds[J]. J. Mater. Sci. Technol., 2020, 43: 52-63.
BET theory | MIP | MicroCT | |||
---|---|---|---|---|---|
Specific surface area (m2/g) | Total open porosity (%) | Total porosity (%) | Open porosity (%) | Closed porosity (%) | |
chit/aga/HA_L | 30.04 | 70.90 | 63.05 | 37.24 | 25.80 |
chit/aga/HA_H | 31.50 | 67.09 | 48.41 | 19.19 | 29.22 |
Table 1 Textural features of composite scaffolds: specific surface area and porosity of scaffolds.
BET theory | MIP | MicroCT | |||
---|---|---|---|---|---|
Specific surface area (m2/g) | Total open porosity (%) | Total porosity (%) | Open porosity (%) | Closed porosity (%) | |
chit/aga/HA_L | 30.04 | 70.90 | 63.05 | 37.24 | 25.80 |
chit/aga/HA_H | 31.50 | 67.09 | 48.41 | 19.19 | 29.22 |
Fig. 1. Porosity characterization of scaffolds: (a) Pore entrance size distribution determined by MIP; (b) MicroCT cross-section images of chit/aga/HA_L and chit/aga/HA_H (black colour—air voids; yellow/orange/green/blue—nanoHA, violet- polysaccharide matrix); (c) Pore share of different diameters in structure of produced biomaterials assessed by microCT; (d) Microstructure of scaffolds visualized by stereoscopic microscope and SEM.
Fig. 2. ATR-FTIR evaluation of biomaterials presented in two spectral regions: (a) Spectra of chitosan, agarose and mixture of these two; (b) Spectra of result of spectral subtractions of agarose spectrum from chit/aga spectrum, compared against spectrum of pure chitosan; (c) Spectra of result of spectral subtractions of HA spectrum from chit/aga/HA_H spectrum, compared against spectrum of chit/aga matrix and pure HA. All of spectra were maximized and offset for better clarity.
Fig. 3. Bioactivity test: (a) SEM images of biomaterials surfaces before (control) and after soaking in SBF for 14 and 28 days, along with Ca/P atomic ratio calculated for apatite crystals; (b) Changes in Ca2+ and HPO42- concentrations of SBF during incubation with biomaterials. *statistically significant results compared to control SBF (P < 0.05, unpaired t-test).
% C | % H | % N | Reducing sugars [μg/mL] | ||
---|---|---|---|---|---|
Untreated samples | chit/aga/HA_L | 8.20 ± 0.01 | 1.07 ± 0.01 | 0.39 ± 0.01 | - |
chit/aga/HA_H | 4.27 ± 0.02 | 0.38 ± 0.01 | 0.19 ± 0.02 | - | |
Non-enzymatic solution 2nd week | chit/aga/HA_L control | 7.78 ± 0.02 | 0.8 ± 0.01 | 0.25 ± 0.02 | 0.00 |
chit/aga/HA_H control | 4.14 ± 0.01 | 0.44 ± 0.02 | 0.16 ± 0.01 | 0.00 | |
Enzymatic solution 2nd week | chit/aga/HA_L | 6.62 ± 0.21a, b, c | 0.81 ± 0.03b, c | 0.19 ± 0.00a, b | 35.87 ± 25.05a |
chit/aga/HA_H | 4.22 ± 0.22 | 0.44 ± 0.04b | 0.15 ± 0.05 | 36.13 ± 25.02a | |
Non-enzymatic solution 4th week | chit/aga/HA_L control | 7.73 ± 0.01 | 0.83 ± 0.01 | 0.27 ± 0.01 | 41.17 ± 4.04 |
chit/aga/HA_H control | 4.13 ± 0.01 | 0.43 ± 0.01 | 0.17 ± 0.02 | 44.12 ± 4.16 | |
Enzymatic solution 4th week | chit/aga/HA_L | 6.60 ± 0.21a, b, c | 0.78 ± 0.04b, c | 0.20 ± 0.00a, b | 72.07 ± 13.20a |
chit/aga/HA_H | 4.23 ± 0.20 | 0.44 ± 0.02b | 0.16 ± 0.05 | 69.92 ± 7.42a | |
Non-enzymatic solution 6th week | chit/aga/HA_L control | 7.73 ± 0.01 | 0.81 ± 0.02 | 0.27 ± 0.01 | 41.67 ± 3.53 |
chit/aga/HA_H control | 4.13 ± 0.01 | 0.44 ± 0.03 | 0.17 ± 0.01 | 44.93 ± 0.79 | |
Enzymatic solution 6th week | chit/aga/HA_L | 6.61 ± 0.22a, b, c | 0.80 ± 0.04b, c | 0.19 ± 0.00a, b | 76.19 ± 9.19a |
chit/aga/HA_H | 4.23 ± 0.22 | 0.44 ± 0.01b | 0.16 ± 0.06 | 72.17 ± 13.72a | |
Tris-HCl simulation solution | chit/aga/HA_L | ND | ND | ND | 46.23 ± 0.16 |
chit/aga/HA_H | ND | ND | ND | 46.76 ± 0.89 | |
Citric acid (C.A.) extreme solution | chit/aga/HA_L | ND | ND | 0.18 ± 0.01b, c | 433.72 ± 46.41c, d |
chit/aga/HA_H | ND | ND | 0.12 ± 0.01b | 347.33 ± 30.75d |
Table 2 Biodegradation of polysaccharide matrices of scaffolds assessed by CHN elemental analysis of retenate and detection of reducing sugars in filtrate.
% C | % H | % N | Reducing sugars [μg/mL] | ||
---|---|---|---|---|---|
Untreated samples | chit/aga/HA_L | 8.20 ± 0.01 | 1.07 ± 0.01 | 0.39 ± 0.01 | - |
chit/aga/HA_H | 4.27 ± 0.02 | 0.38 ± 0.01 | 0.19 ± 0.02 | - | |
Non-enzymatic solution 2nd week | chit/aga/HA_L control | 7.78 ± 0.02 | 0.8 ± 0.01 | 0.25 ± 0.02 | 0.00 |
chit/aga/HA_H control | 4.14 ± 0.01 | 0.44 ± 0.02 | 0.16 ± 0.01 | 0.00 | |
Enzymatic solution 2nd week | chit/aga/HA_L | 6.62 ± 0.21a, b, c | 0.81 ± 0.03b, c | 0.19 ± 0.00a, b | 35.87 ± 25.05a |
chit/aga/HA_H | 4.22 ± 0.22 | 0.44 ± 0.04b | 0.15 ± 0.05 | 36.13 ± 25.02a | |
Non-enzymatic solution 4th week | chit/aga/HA_L control | 7.73 ± 0.01 | 0.83 ± 0.01 | 0.27 ± 0.01 | 41.17 ± 4.04 |
chit/aga/HA_H control | 4.13 ± 0.01 | 0.43 ± 0.01 | 0.17 ± 0.02 | 44.12 ± 4.16 | |
Enzymatic solution 4th week | chit/aga/HA_L | 6.60 ± 0.21a, b, c | 0.78 ± 0.04b, c | 0.20 ± 0.00a, b | 72.07 ± 13.20a |
chit/aga/HA_H | 4.23 ± 0.20 | 0.44 ± 0.02b | 0.16 ± 0.05 | 69.92 ± 7.42a | |
Non-enzymatic solution 6th week | chit/aga/HA_L control | 7.73 ± 0.01 | 0.81 ± 0.02 | 0.27 ± 0.01 | 41.67 ± 3.53 |
chit/aga/HA_H control | 4.13 ± 0.01 | 0.44 ± 0.03 | 0.17 ± 0.01 | 44.93 ± 0.79 | |
Enzymatic solution 6th week | chit/aga/HA_L | 6.61 ± 0.22a, b, c | 0.80 ± 0.04b, c | 0.19 ± 0.00a, b | 76.19 ± 9.19a |
chit/aga/HA_H | 4.23 ± 0.22 | 0.44 ± 0.01b | 0.16 ± 0.06 | 72.17 ± 13.72a | |
Tris-HCl simulation solution | chit/aga/HA_L | ND | ND | ND | 46.23 ± 0.16 |
chit/aga/HA_H | ND | ND | ND | 46.76 ± 0.89 | |
Citric acid (C.A.) extreme solution | chit/aga/HA_L | ND | ND | 0.18 ± 0.01b, c | 433.72 ± 46.41c, d |
chit/aga/HA_H | ND | ND | 0.12 ± 0.01b | 347.33 ± 30.75d |
Fig. 4. Biodegradation of nanoHA component of scaffolds assessed by analysis of changes in Ca2+ and HPO42- amounts (μg) in filtrates: (a) Long-term test in PBS-based enzymatic solution (dotted lines) and non-enzymatic PBS solution; ion uptake (μg) was calculated by subtraction of ion amounts in PBS from ion amounts in filtrates (*statistically significant results compared to corresponding control solution before scaffolds immersion; $statistically significant results compared to chit/aga/HA_H + enzymes; &statistically significant results compared to chit/aga/HA_H; P < 0.05, One-way ANOVA followed by Tukey’s test); (b) Short-term test performed according to ISO 10993-14 in simulation solution (Tris-HCl, pH=7.4) and extreme solution (C.A., pH=3); ion release (μg) was calculated by subtraction of ion amounts in filtrates from ion amounts in Tris-HCl or C.A. (*statistically significant results compared to corresponding control solution before scaffolds immersion; #statistically significant results compared to the same sample incubated in C.A.; &statistically significant results compared to chit/aga/HA_H incubated in Tris-HCl; P < 0.05, One-way ANOVA followed by Tukey’s test).
Fig. 5. Liquid absorption ability test: (a) PBS absorption; (b) Human blood plasma absorption; absorption ability of biomaterials was expressed as a percentage of weight increase (Wi) with time calculated using the formula: Wi=(Wt - W0)/W0 × 100, where Wt is the weight of the sample at time t and W0 is the weight of the dry sample; *statistically significant results compared to the chit/aga/HA_H (P < 0.05, unpaired t-test).
Fig. 6. Cytotoxicity of scaffolds against MC3T3-E1 and hFOB 1.19 osteoblasts: (a) Indirect agar diffusion test performed according to ISO 10993-5 (latex material—positive control of cytotoxicity, polypropylene - negative control of cytotoxicity); (b) MTT test on extracts performed according to ISO 10993-5 (latex extract—positive control of cytotoxicity, polypropylene extract—negative control of cytotoxicity; *statistically significant results compared to negative control, #statistically significant results compared to positive control, P < 0.05, One-way ANOVA followed by Tukey′s test); (c) CLSM images presenting osteoblasts on the surface of scaffolds 48 h after seeding (viable cells—green fluorescence, dead cells—red fluorescence, magn. 100×scale bar = 100 μm).
|
[1] | Mengyang Wang, Shichao Bi, Jianhui Pang, Zhongzheng Zhou, Di Qin, Honglei Wang, Xiaojie Cheng, Xiguang Chen. Precise quantification of the antibacterial activity of chitosan by NB medium neutralizer [J]. J. Mater. Sci. Technol., 2021, 70(0): 224-232. |
[2] | Xiaoxue Yuan, Ran Liu, Wenchang Zhang, Xiaoqiang Song, Lei Xu, Yan Zhao, Lei Shang, Jingsong Zhang. Preparation of carboxylmethylchitosan and alginate blend membrane for diffusion-controlled release of diclofenac diethylamine [J]. J. Mater. Sci. Technol., 2021, 63(0): 210-215. |
[3] | Kai Chen, Changci Tong, Jinge Yang, Peifang Cong, Ying Liu, Xiuyun Shi, Xu Liu, Jun Zhang, Rufei Zou, Keshen Xiao, Yuyang Ni, Lei Xu, Mingxiao Hou, Hongxu Jin, Yunen Liu. Injectable melatonin-loaded carboxymethyl chitosan (CMCS)-based hydrogel accelerates wound healing by reducing inflammation and promoting angiogenesis and collagen deposition [J]. J. Mater. Sci. Technol., 2021, 63(0): 236-245. |
[4] | Lin Lu, Qianqian Liu. Synergetic effects of photo-oxidation and biodegradation on failure behavior of polyester coating in tropical rain forest atmosphere [J]. J. Mater. Sci. Technol., 2021, 64(0): 195-202. |
[5] | Yongyong Xue, Na Wang, Zhi Zeng, Jinpeng Huang, Zhiming Xiang, Yan-Qing Guan. Neuroprotective effect of chitosan nanoparticle gene delivery system grafted with acteoside (ACT) in Parkinson’s disease models [J]. J. Mater. Sci. Technol., 2020, 43(0): 197-207. |
[6] | Mingli Lin, Huanhuan Liu, Jingjing Deng, Ran An, Minjuan Shen, Yanqiu Li, Xu Zhang. Carboxymethyl chitosan as a polyampholyte mediating intrafibrillar mineralization of collagen via collagen/ACP self-assembly [J]. J. Mater. Sci. Technol., 2019, 35(9): 1894-1905. |
[7] | Wen Zhang, Lili Tan, Dingrui Ni, Junxiu Chen, Ying-Chao Zhao, Long Liu, Cijun Shuai, Ke Yang, Andrej Atrens, Ming-Chun Zhao. Effect of grain refinement and crystallographic texture produced by friction stir processing on the biodegradation behavior of a Mg-Nd-Zn alloy [J]. J. Mater. Sci. Technol., 2019, 35(5): 777-783. |
[8] | Junxiu Chen, Lili Tan, Xiaoming Yu, Ke Yang. Effect of minor content of Gd on the mechanical and degradable properties of as-cast Mg-2Zn-xGd-0.5Zr alloys [J]. J. Mater. Sci. Technol., 2019, 35(4): 503-511. |
[9] | Ming-Shi Song, Rong-Chang Zeng, Yun-Fei Ding, Rachel W. Li, Mark Easton, Ivan Cole, Nick Birbilis, Xiao-Bo Chen. Recent advances in biodegradation controls over Mg alloys for bone fracture management: A review [J]. J. Mater. Sci. Technol., 2019, 35(4): 535-544. |
[10] | Peng Chen, Yunliang Zhao, Tianxing Chen, Tingting Zhang, Shaoxian Song. Synthesis of montmorillonite-chitosan hollow and hierarchical mesoporous spheres with single-template layer-by-layer assembly [J]. J. Mater. Sci. Technol., 2019, 35(10): 2325-2330. |
[11] | Baihao You, Qingtao Li, Hua Dong, Tao Huang, Xiaodong Cao, Hua Liao. Bilayered HA/CS/PEGDA hydrogel with good biocompatibility and self-healing property for potential application in osteochondral defect repair [J]. J. Mater. Sci. Technol., 2018, 34(6): 1016-1025. |
[12] | Zhong Haoxiang, Lu Jidian, He Aiqin, Sun Minghao, He Jiarong, Zhang Lingzhi. Carboxymethyl chitosan/poly(ethylene oxide) water soluble binder: Challenging application for 5 V LiNi0.5Mn1.5O4 cathode [J]. J. Mater. Sci. Technol., 2017, 33(8): 763-767. |
[13] | Baojie Wang, Daokui Xu, Junhua Dong, Wei Ke. Effect of Texture on Biodegradable Behavior of an As-Extruded Mg-3%Al-1%Zn Alloy in Phosphate Buffer Saline Medium [J]. J. Mater. Sci. Technol., 2016, 32(7): 646-652. |
[14] | Weiping Ding, Cheng Liang, Sijie Sun, Liqun He, Dayong Gao. On-Chip Fabrication of Carbon Nanoparticle-Chitosan Composite Membrane [J]. J. Mater. Sci. Technol., 2015, 31(11): 1087-1093. |
[15] | Mei Li, Ling Ren, LiHua Li, Peng He, GuoBo Lan, Yu Zhang, Ke Yang. Cytotoxic Effect on Osteosarcoma MG-63 Cells by Degradation of Magnesium [J]. J. Mater. Sci. Technol., 2014, 30(9): 888-893. |
Viewed | ||||||
Full text |
|
|||||
Abstract |
|
|||||