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Feasibility of SiAlON–Si3N4 composite ceramic as a potential bone repairing material
Journal Pre-proof Feasibility of SiAlON–Si3N4 composite ceramic as a potential bone repairing material Liguo Zhang, Xiaojie Liu, Miao Li, Enxia Xu, Fei Zhao, Huiyu Yuan, Xu Sun, Can Zhang, Lu Gao, Jinxing Gao PII:
S0272-8842(19)32681-1
DOI:
https://doi.org/10.1016/j.ceramint.2019.09.150
Reference:
CERI 22917
To appear in:
Ceramics International
Received Date: 15 July 2019 Revised Date:
31 August 2019
Accepted Date: 16 September 2019
Please cite this article as: L. Zhang, X. Liu, M. Li, E. Xu, F. Zhao, H. Yuan, X. Sun, C. Zhang, L. Gao, J. Gao, Feasibility of SiAlON–Si3N4 composite ceramic as a potential bone repairing material, Ceramics International (2019), doi: https://doi.org/10.1016/j.ceramint.2019.09.150. 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 Ltd.
Feasibility of SiAlON-Si3N4 Composite Ceramic as a Potential Bone Repairing Material Liguo Zhanga,b#; Xiaojie Liuc,b#; Miao Lid,e; Enxia Xud,e; Fei Zhaod,e; Huiyu Yuand,e; Xu Sunf; Can Zhanga,b; Lu Gaoa,b; Jinxing Gaod,e,* a
Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou University, Zheng zhou 450007, China
b
Henan Institute of Medical and Pharmaceutical Science, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, China
c
School of Basic Medical Sciences, Zhengzhou University, NO.100 Science Avenue, Zheng zhou 450001, China d
School of Material Science and Engineering, Zhengzhou University, Zhengzhou, NO.100 Science Avenue, Zheng zhou 450001, China e
Henan Key Laboratory of High Temperature Functional Ceramics, Zhengzhou University, 75 Daxue Road, Zhengzhou 450052, China
f
Henan Cancer Hospital,Zhengzhou University，Zhengzhou，No. 127 Dongming Road, Zhengzhou 450008, China #
These authors contributed equally to this work
*
Corresponding author. E-mail address: [email protected] (J. Gao)
Abstract In this study, SiAlON-Si3N4 composite ceramic are prepared by direct nitridation and investigated to overcome the limitations associated with ceramic Si3N4, which includes
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the difficulty in fabricating ceramic Si3N4 into shaped parts for use in the human body. Phase composition and microstructure of the SiAlON-Si3N4 composites were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively, and the porosity, bulk density, compressive strength, and ion release were also measured. The biological properties were evaluated by bone cell cultures on the ceramic surfaces. Results show that Si4Al2O2N6 is formed by the reaction of Al, Si, and Al2O3 with nitrogen at high temperature that forms Si3N4, thereby fabricating SiAlON-Si3N4 composite ceramics. Some α-Si3N4 grains underwent a phase transition from α- to β-Si3N4 fiber at high temperature. Porosity of the samples increases with increasing Si3N4 content, while the bulk density of the samples decreases. The compressive strength increases and then slightly decreases with increasing Si3N4 content. Water leaching experiments of the SiAlON-Si3N4 composite ceramics reveal that the composites exhibit outstanding chemical stability. Studies using bone cell culture indicate that the cells present a fusiform and extend two or three thin pseudopodia. The phenomena demonstrate that MC3T3-E1 cells have excellent growth activity and have the potential ability to proliferate to osteocytes on the surfaces of the samples, thus suggesting that SiAlON-Si3N4 based ceramics are biocompatible and could be implemented as a potential bone-repairing material. Keywords: SiAlON-Si3N4 based; Bone-repairing material; Composite ceramics; Biocompatible
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Introduction Silicon nitride based materials have outstanding biological properties that makes
them widely used in artificial bone substitute materials applications. In clinical applications, the high wear resistance, low friction, low density, nontoxicity, and good biocompatibility, are also especially important in high load-bearing bone replacement bioceramics [1-6]. This makes Si3N4 based ceramics an ideal biomaterial for orthopedics, such as in hip replacements, knee arthroplasty, and spinal fusion implants. Some research has shown that polished surfaces of Si3N4 have good ability to promote the growth of human osteoblast cells in vitro[2, 6]. In vivo, porous intramedullary Si3N4 rods implanted in rabbit femurs could guid bone regeneration[6]. Such reports clearly affirmed that, under certain conditions, Si3N4 presents favorable physico-chemical properties for osteoblast cell growth and metabolism. Hence, Si3N4 has been widely used clinically as a bone-repair and bone substitute material. Although Si3N4 exhibits outstanding performance as a biomaterial, the challenge to fabricate it obstructs its development and application. The Si3N4 sintering temperature is higher than 1800 ℃, which is not easy to achieve in many material preparation processes [7-11]. Modeling by hot pressing techniques is nearly the only way to fabricate high strength and dense Si3N4 [8]. However, human bone-repair and bone substitute materials are usually irregular shapes that cannot be achieved by using hot-pressed sintering. Therefore, exploring new ways to fabricate appropriately shaped, high strength, and dense Si3N4 is particularly important [9, 12]. In general, sintering aids
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are typically applied in the sintering process to reduce the sintering temperature; the added material reacts with Si3N4 to form a low-temperature melting phase, which facilities the formation of Si3N4. Commonly used materials are Y2O3, Al2O3, MgO, or a combination of thereof, of which Al2O3 is not only a sintering support but also a biomaterial[13-15]. When Al2O3 is add to the Si3N4 sintering process, it reacts with Si3N4, creating the SiAlON phase, which is a low-temperature melting phase [16-19]. If SiAlON phase is present in higher proportions during the sintering process, it can be used to fabricate high-strength and dense SiAlON-Si3N4 ceramics with appropriated shape at a relatively low sintering temperature. Bone-repairing material applications always require that the applied materials have good biological properties[14]. In a previous work[20], human umbilical vein endothelial cells (HUVECs) were cultured on the surface of SiAlON-Al2O3 ceramics. HUVECs grew and proliferated well for several days. Al2O3 has widely been demonstrated to be a biocompatible material and has been used clinically[21]. Although Si3N4 has been proven to be an excellent biomaterials, no reports have been found regarding the biological properties of SiAlON-Si3N4 composite ceramics. In clinical application, the artificial bone-repairing material is not only required to have good mechanical properties and be nontoxic[22, 23], but it must also be suitable for the growth and proliferation of osteocytes[24]. Only when cells continuously grow and proliferate well with the artificial materials, can it facilitate the fusion of implanted materials with human tissue and finally enable the implanted materials to possess
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suitable physiological function. In this research work, we first used Si3N4, Al, Si, and Al2O3 as raw materials, and fabricated composites of SiAlON-Si3N4 with a direct nitriding technique. We investigated the physical and chemical properties of the SiAlON-Si3N4 composites and finally, pre-osteoblastic, MC3T3-E1 cells, were seeded on the surface of SiAlON-Si3N4 to evaluate the biocompatibility of the SiAlON-Si3N4. 2
Material and Methods
2.1 Materials preparation Since Si3N4 had a higher melting point, making it very uneasy to sinter, the SiAlON-Si3N4 composites were prepared by forming the β-SiAlON phase with a low melting point such that Si3N4 particles would sinter together. The β-sialon (Si6−xAlxOxN8−x, where x may vary from 0 to 4.2) with x = 2 was prepared. The samples were produced by reaction-bonded sintering using Al, Si, Al2O3, and α-Si3N4 powder as raw materials, as shown in Fig. 1, the morphology of α-Si3N4 powder is made up of granules, as observed by SEM. The composition of the prepared composite samples is presented in Table 1. Chemically pure substances were used in sample preparation. Al powder (purity ≥99.9% and mean particle size of 5 µm), Si powder (purity ≥99.9% and mean particle size of 5 µm), α-Al2O3 powder (purity ≥99.9% and mean particle size of 5 µm), and α-Si3N4 powder (mean particle size of 2 µm, α-phase ≥95.0%) were mixed together with 5 wt.% polyvinyl alcohol, which acted as a binder, in ethanol to fabricate the sample slurry. After that, pellets were created using a pressure spraying pelletizer
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(YC-1000, Shanghai Yacheng Instrument Equipment Co., Ltd., China). Then, at 100 MPa, adopting hydraulic machine pressing technique to farbicate cylindrical samples which had 20 mm diameter and 2 mm thick. Ten cylindrical samples were farbicated for each kind of ceramic composite. The. Finally, the cylindrical samples were heated at 4 °C/min to 1500 °C and held isothermally for 3 h in N2 gas (1 atm) in a MoSi2 resistance furnace. 2.2 Materials characterization 2.2.1 Phase analysis and microstructure observation The phases of the samples resulting from nitridation were characterized by powder X-ray diffraction (XRD; X’Pert Pro MPD, PANalytical Co., Netherlands) with CuKα radiation; data were collected from 2θ = 10° to 90°. The microstructures were examined by slicing sections of the cyclinders, which were then ground and polished carefully. Pt was sputtered onto the cross sections, which were then observed using an SEM-EDAX (Nova 450 Nano, FEI, USA; EDAX, Energy Dispersive X-ray X-Max, OXFORD, UK). 2.2.2 Bulk density, porosity, and compressive strength test Sample bulk density and apparent porosity was measured using the Archimedes method, and water was used as the displacement liquid [25]. Samples were processed into prisms with dimension of 5 × 5 × 12.5 mm for the compressive strength test. Compressive strength was measured using a universal material testing machine (Instron 5567, Instron, USA) with a crosshead speed of 0.2 mm/min. The arithmetic average value of the three measurements taken on each sample is identified as the measured
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value. 2.2.3 Ion release studies of the SiAlON-Si3N4 composite ceramics Materials used as bone-repairing materials should have good chemical stability, which guarantees that ions in the composite material do not continually release into human tissue. The continual release of ions causes the materials not only to corrode, but also the increase in local ion concentration is not beneficial for reorganization. Therefore, ion release behavior of bone-repairing material becomes crucial. Soaking tests were used to investigate ion release in materials. Since the cell medium, as presented in section 2.2.4, contains a large quantity of ions and is easily contaminated, it is not suitable for soaking experiments. Therefore, deionized water was used in the experiment to soaking the ceramic samples. The chemical stability of the composites was estimated by measuring the total ion concentration in the deionized water over a 14-day soaking process. The composites were cut into disks (diameter 20 mm, height 10 mm). The weight ratio of the sample and deionized water was 1:7 at 37 ℃ during the soaking experiment. After soaking, the total ion concentration in the deionized water was measured once in every 24 h using Mettler Toledo S230-USP/EP-CN (Youyi Instrument Company, China) system. 2.2.4
Cell culture and proliferation on the composite ceramic surface
Mouse pre-osteoblastic cell line MC3T3-E1 was purchased from National Infrastructure of Cell Line Resource, China. A complete cell cuture medium containing α medium essential medium (Biological Industries, Israel) and 10% fetal bovine serum
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(Biological Industries, Israel) for cell culture was used. Cell culture was executed in 5% CO2 in an incubator (Thermo, USA) held at 37 ℃. Ceramic slices (thickness 2 mm) with a smooth surface were fabricated for cell culturing and biological properties evaluation. All the materials used for cell culture were sterilized in a sterilizer (Sanyo MLS-3751-PC, Japan) and dried in a dryer (Qilian power equipment Co.LTD, China). MC3T3-E1 cells were digested, centrifugated, and resuspended in a complete cell culture medium; 2 mL of the MC3T3-E1 cell suspension was added into a plate of six wells, each containing a composite ceramic slice at the bottom. After culturing (24 and 72 h), the cell culture medium was thrown away, and six well plates were cleaned with PBS twice. MC3T3-E1 cells on the ceramic surfaces were immersed in 2 µmol/L calcein-AM (Thermo Fisher, USA) for 40 min, after which the staining solution was thrown away and ceramic samples were cleaned twice with PBS. Calcein-AM is a cell-permeant fluorescent dye used to observe living cells without unfavorable effects. Ceramics samples with stained MC3T3-E1 cells on their surfaces were placed on a slide glass with surfaces contacting the glass. Laser scanning confocal microscopy (LSCM; Fluo1000, Olympus) is inverted microscopy which can observe the excited fluorescence of the opaque sample. Stained MC3T3-E1 cells on composite ceramic surfaces were also observed by LSCM. Transmitted light images captured by laser scanning confocal microscopy clearly show the 3D state of ceramic surface. When cells were stained by green fluorescent dye, the images captured by LSCM were referred to as confocal images. Merging the
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transmitted light images and confocal images enabled the clear observation of pseudopodia and shape of the cells on the ceramic surfaces. Cell pseudopodia were observed by imerging transmitted light images and confocal images captured by LSCM as well.
3 Results and Discussion 3.1 Phase analysis and microstructure of the SiAlON-Si3N4 composite ceramics XRD patterns of the SiAlON-Si3N4 composite ceramics obtained by reactive sintering at 1500 ℃ for 4 h are presented in Fig. 2. The main phase in SL-1 was β-sialon, and the peak intensity of the detected α-Si3N4 increased with increasing Si3N4 content in the precursor material mix, thus confirming that the goal of the ceramic composition was achieved. As expected, Si, Al, and Al2O3 reacted with nitrogen at high temperature to generate the SiAlON phase in this experiment. The Si4Al2O2N6 formed in the samples holds Si3N4 together to form the SiAlON-Si3N4 composite ceramic. The presence of trace Al2O3 in the SL-1 and SLN-1 samples could be attributed to partial evaporation of Si and Al during heating process, which leads to a small quantity of excess Al2O3. The characteristic peak of Al2O3 disappears in the SLN-2, SLN-3, and SLN-4 samples which may be caused by Si3N4 reacting with Al2O3 to form the SiAlON phase; Al2O3 is often used as a sintering additive to create Si3N4 based ceramics [9]. The microstructure of the samples is shown in Fig. 3, showing the evolution of the microstructure of SiAlON-Si3N4. As can be seen in Fig. 3A and considering the corresponding XRD patterns in Fig. 2, the Si4Al2O2N6 grains reacted during sintering to
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form α-Si3N4. Overall, the grains become smaller with an increasing Si3N4 in the composites. Si4Al2O2N6 content also reduces with increasing Si3N4 content in the composite, which likely occurs because the Si4Al2O2N6 phase is formed by a direct reaction in SL-1. However, in the other samples (SLN-1–SLN-4), the synthesized Si4Al2O2N6 in the samples holds the α-Si3N4 grains together to form the SiAlON–Si3N4 composite ceramics, where α-Si3N4 grains inhibit the growth of the Si4Al2O2N6 phase during the nitriding sintering process. Fibrous crystals were also formed in the samples, except in SL-1. The elemental composition of the fibrous crystals was Si and N, as determined by EDAX. Therefore, it can be inferred that the fibrous crystals are β-Si3N4. Some grains of α-Si3N4 may undergo a phase transition from the α-Si3N4 phase to the β-Si3N4 phase at high temperature. Incorporation of fibers in ceramics often reinforces both the strength and toughness of the material, which may indicate that the ceramic properties may be improved in samples where fibrous β-Si3N4 phase is present. However, characteristic peaks of β-Si3N4 were not found by XRD, which may indicate that its trace amounts of β-Si3N4 could not be detected. 3.2 Porosity, bulk density, and compressive strength The change in the porosity and bulk density of the composites with the varying Si3N4 content is presented in Fig. 4. The porosity of samples is found to increase with increasing Si3N4 content, and the bulk density of samples is found to decrease. This is likely attributed to the Si3N4 having a higher melting point, which maks it difficult to
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sinter at the lower temperature of this experiment. The Si4Al2O2N6 formed in the samples and filled in the gap between the α-Si3N4 grains to form the SiAlON-Si3N4 composite ceramics. With decreasing Si4Al2O2N6 content and increasing Si3N4 content, the densification of the ceramics was reduced. The bulk density of the material prepared in this experiment is similar to that of the human skeleton [24]. It suggested that the use of SiAlON-Si3N4 as a bone-repairing material could not bring any adaptive problems. Otherwise, the porosity of material raises material surface roughness, which helps cell adhesion and promotes its combination with human tissue [26, 27]. Fig. 5 shows the measured compressive strength of the samples. The compressive strength first increases and then decreases with increasing Si3N4 content. This means that the addition of Si3N4 improved the compressive strength of the SiAlON-Si3N4 ceramics. In comparison with SLN-3, the compressive strength of SLN-4 is slightly decreased. This may be because the compressive strength decreased for with increasing porosity to a certain extent. However, the compressive strength of all samples met the needs of bone-repairing material, indicating that the SiAlON-Si3N4 composites have the feasibility to be used in the manufacture of novel load-bearing bone implnats[28]. The implementation of other sintering additives in the sample also could change the microstructure and improve the performance of SiAlON-Si3N4 composites. To avoid unexpected or undesirable effects on the biological properties, other sintering additives were not investigated in this study. The improvement in the properties of SiAlON-Si3N4 composites will be carried out in the future after confirming the materials are
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biocompatible. 3.3 Water leaching experiments of the SiAlON-Si3N4 composite ceramics Samples of the SiAlON-Si3N4 composite were immersed in deionized water for periods ranging from 1 to 15 days as shown in Fig. 6. The time-dependent profile of the ion concentration shows that ion release occurred mainly during the first five days after soaking. The ions concentration in deionized water tended to be stable after one week. There may be two reasons for this result. One is the impurities in the raw material; another is the ion release behavior of the composite materials. It is well understood that no material is 100% pure, which means that impurities may be present in the raw material. The impurity ions will also release from the formed SiAlON-Si3N4 composites. Pure Si3N4 and SiAlON have outstanding chemical stability, but they still undergo a slight ion release [29, 30]. Regardless of whether the ions are released from impurities or are leached from the composites, the maximum amount of ions released (<6 mg/L) arrived after two week of soaking. Based on this experiment, it is found that SiAlON-Si3N4 composite ceramics exhibit rather low ion release, thus being very chemically stable. 3.4 Viability and proliferation of MC3T3-E1 cells on SiAlON-Si3N4 MC3T3-E1 cells, as a preosteoblastic cell line, were used to investigate the properties of bone tissue materials since these cells can be induced to become osteoblastic cells [31]. During the calcein-AM incubation of the experiment, calcein-AM entered the viable cells and was inverted by intracellular esterases to calcein within the cells.
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Calcein produces a green signal with laser stimulation and can be used as a living cell tracer[32]. Living cells on the composite ceramic surfaces were observed after 24 and 72 hour cultures, as shown in Fig. 7. After the 24 h culture, MC3T3-E1 cells adhered well on all ceramic sample surfaces. Most of the cells were presented as a fusiform and extended two or three thin pseudopodia. These phenomena indicate that MC3T3-E1 cells had the growth ability and potential to migrate, as that introduced in Klemke’s article[33]. When the culture time was increased to 72 h, MC3T3-E1 cells proliferated on all ceramic sample surfaces with time and reached 90% confluence, as shown in Fig. 7. MC3T3-E1 cells had a larger cell body, extended more and wider pseudopodia than those cultured for 24 h (Fig. 8A). The pseudopodia around the cells cross linked with each other (Fig. 8B) as culture time increased to 72 h. Due to cytoplasmic streaming during pseudopodia extension and withdrawal, the growth and cross linking of pseudopodia generated intracellular movement [34]. The changes in the pseudopodia and cell shape with culture time evidences that MC3T3-E1 cells have excellent growth activity and potential ability to proliferate to osteocytes. The increase in the number of living cells on the composite ceramic sample surfaces shows that these ceramics are biocompatible and have no toxity. SiAlON-Si3N4 ceramics have a good biological environment and are suitable for MC3T3-E1 cell growth and proliferation. Previous water leaching experiments revealed that SiAlON-Si3N4 composite ceramics exhibit a slight ion release. The maximum amount of ions released was <6 mg/L, which
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is less than the ion concentration of Hanks balanced salt solution [35]. The good growth status of cells cultured with SiAlON-Si3N4 composite ceramics illustrates that ions released from SiAlON-Si3N4 based ceramics did not influcece cell growth. When considering the results of this work as well as those from previous research, the Si4Al2O2N6 phase can not only be used as soft-tissue compatible materials but can also be readily combined with MC3T3-E1 cells (bone cells, hard tissue). The Si4Al2O2N6 phase can also be combined with materials (such as Al2O3 and Si3N4) to form compound and composite to improve its performance. Therefore, the developed SiAlON-Si3N4 composite is expected to be applicable in biomaterials, such as bone-repairing material and biological scaffolds. However, more biological properties of the composites must further be evaluated for clinical applications. 4
Conclusion Si4Al2O2N6 phase is prepared by reacting Si, Al, and Al2O3 with nitrogen at high
temperature; during sintering, it combines with α-Si3N4 to form SiAlON-Si3N4 composite ceramics. The porosity of the samples is found to increase with increasing Si3N4 content and the bulk density is found to decrease. The compressive strength increases first and then slightly decreases with increasing Si3N4 content. Water leaching experiments of the SiAlON-Si3N4 composites show that they exhibit outstanding chemical stability. The results of bone cell cultures show that the cells present a fusiform and extend two or three thin pseudopodia. These phenomena indicate that MC3T3-E1 cells have excellent growth activity and have the potential ability to
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proliferate to osteocytes on the surfaces of SiAlON-Si3N4 samples. Therefore, we conclude that SiAlON-Si3N4 based ceramics exhibit excellent biocompatibility and could be implemented as a potential bone-repairing Material.
Financial support and sponsorship: This work was funded by the National Natural Science Foundation of China (3160067), Key Scientific Research Project of Colleges and Universities in Henan Province (17210231004, 19A430027, 16A350011), Major Science and Technology Project of Henan Province (161100311400) and China Postdoctoral Science Foundation (2017M612416).
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3-4.
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Table captions Table 1. Chemical composition of all samples in wt.%
Figure captions Fig.1 The morphology of α-Si3N4 powder Fig.2 XRD patterns of the SiAlON-Si3N4 composite ceramics Fig.3 The microstructure of the samples: A: SL-1; B: SLN-1; C: SLN-2; D: SLN-3; and D: SLN-4 Fig.4 The change in the porosity and bulk density of the composites with the varying Si3N4 content Fig.5 The compressive strength of the samples Fig.6 The ion concentration of the SiAlON-Si3N4 composite Fig.7 Cell culture results of the SiAlON–Si3N4 composite ceramics Fig.8 The pseudopodia around the cells
21
Table
Table 1. Chemical composition of all samples in wt.% Sample
Si
Al
Al2O3
Si3N4
Total
SL-1
56.57
9.09
34.34
0.00
100.00
SLN-1
45.26
7.27
27.47
20.00
100.00
SLN-2
33.94
5.45
20.60
40.00
100.00
SLN-3
22.63
3.64
13.74
60.00
100.00
SLN-4
11.31
1.82
6.87
80.00
100.00
22
Figure
Fig.1 The morphology of α-Si3N4 powder
Fig.2 XRD patterns of the SiAlON-Si3N4 composite ceramics
23
Fig.3 The microstructure of the samples: A: SL-1; B: SLN-1; C: SLN-2; D: SLN-3; D: SLN-4
Fig.4 The change in the porosity and bulk density of the composites with the varying Si3N4 content
24
Fig.5 The compressive strength of the samples
Fig.6 The ion concentration of the SiAlON-Si3N4 composite
25
Fig.7 Cell culture results of the SiAlON-Si3N4 composite ceramics
Fig.8 The pseudopodia around the cells
26
S0272-8842(19)32681-1
DOI:
https://doi.org/10.1016/j.ceramint.2019.09.150
Reference:
CERI 22917
To appear in:
Ceramics International
Received Date: 15 July 2019 Revised Date:
31 August 2019
Accepted Date: 16 September 2019
Please cite this article as: L. Zhang, X. Liu, M. Li, E. Xu, F. Zhao, H. Yuan, X. Sun, C. Zhang, L. Gao, J. Gao, Feasibility of SiAlON–Si3N4 composite ceramic as a potential bone repairing material, Ceramics International (2019), doi: https://doi.org/10.1016/j.ceramint.2019.09.150. 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 Ltd.
Feasibility of SiAlON-Si3N4 Composite Ceramic as a Potential Bone Repairing Material Liguo Zhanga,b#; Xiaojie Liuc,b#; Miao Lid,e; Enxia Xud,e; Fei Zhaod,e; Huiyu Yuand,e; Xu Sunf; Can Zhanga,b; Lu Gaoa,b; Jinxing Gaod,e,* a
Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou University, Zheng zhou 450007, China
b
Henan Institute of Medical and Pharmaceutical Science, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, China
c
School of Basic Medical Sciences, Zhengzhou University, NO.100 Science Avenue, Zheng zhou 450001, China d
School of Material Science and Engineering, Zhengzhou University, Zhengzhou, NO.100 Science Avenue, Zheng zhou 450001, China e
Henan Key Laboratory of High Temperature Functional Ceramics, Zhengzhou University, 75 Daxue Road, Zhengzhou 450052, China
f
Henan Cancer Hospital,Zhengzhou University，Zhengzhou，No. 127 Dongming Road, Zhengzhou 450008, China #
These authors contributed equally to this work
*
Corresponding author. E-mail address: [email protected] (J. Gao)
Abstract In this study, SiAlON-Si3N4 composite ceramic are prepared by direct nitridation and investigated to overcome the limitations associated with ceramic Si3N4, which includes
1
the difficulty in fabricating ceramic Si3N4 into shaped parts for use in the human body. Phase composition and microstructure of the SiAlON-Si3N4 composites were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM), respectively, and the porosity, bulk density, compressive strength, and ion release were also measured. The biological properties were evaluated by bone cell cultures on the ceramic surfaces. Results show that Si4Al2O2N6 is formed by the reaction of Al, Si, and Al2O3 with nitrogen at high temperature that forms Si3N4, thereby fabricating SiAlON-Si3N4 composite ceramics. Some α-Si3N4 grains underwent a phase transition from α- to β-Si3N4 fiber at high temperature. Porosity of the samples increases with increasing Si3N4 content, while the bulk density of the samples decreases. The compressive strength increases and then slightly decreases with increasing Si3N4 content. Water leaching experiments of the SiAlON-Si3N4 composite ceramics reveal that the composites exhibit outstanding chemical stability. Studies using bone cell culture indicate that the cells present a fusiform and extend two or three thin pseudopodia. The phenomena demonstrate that MC3T3-E1 cells have excellent growth activity and have the potential ability to proliferate to osteocytes on the surfaces of the samples, thus suggesting that SiAlON-Si3N4 based ceramics are biocompatible and could be implemented as a potential bone-repairing material. Keywords: SiAlON-Si3N4 based; Bone-repairing material; Composite ceramics; Biocompatible
2
1
Introduction Silicon nitride based materials have outstanding biological properties that makes
them widely used in artificial bone substitute materials applications. In clinical applications, the high wear resistance, low friction, low density, nontoxicity, and good biocompatibility, are also especially important in high load-bearing bone replacement bioceramics [1-6]. This makes Si3N4 based ceramics an ideal biomaterial for orthopedics, such as in hip replacements, knee arthroplasty, and spinal fusion implants. Some research has shown that polished surfaces of Si3N4 have good ability to promote the growth of human osteoblast cells in vitro[2, 6]. In vivo, porous intramedullary Si3N4 rods implanted in rabbit femurs could guid bone regeneration[6]. Such reports clearly affirmed that, under certain conditions, Si3N4 presents favorable physico-chemical properties for osteoblast cell growth and metabolism. Hence, Si3N4 has been widely used clinically as a bone-repair and bone substitute material. Although Si3N4 exhibits outstanding performance as a biomaterial, the challenge to fabricate it obstructs its development and application. The Si3N4 sintering temperature is higher than 1800 ℃, which is not easy to achieve in many material preparation processes [7-11]. Modeling by hot pressing techniques is nearly the only way to fabricate high strength and dense Si3N4 [8]. However, human bone-repair and bone substitute materials are usually irregular shapes that cannot be achieved by using hot-pressed sintering. Therefore, exploring new ways to fabricate appropriately shaped, high strength, and dense Si3N4 is particularly important [9, 12]. In general, sintering aids
3
are typically applied in the sintering process to reduce the sintering temperature; the added material reacts with Si3N4 to form a low-temperature melting phase, which facilities the formation of Si3N4. Commonly used materials are Y2O3, Al2O3, MgO, or a combination of thereof, of which Al2O3 is not only a sintering support but also a biomaterial[13-15]. When Al2O3 is add to the Si3N4 sintering process, it reacts with Si3N4, creating the SiAlON phase, which is a low-temperature melting phase [16-19]. If SiAlON phase is present in higher proportions during the sintering process, it can be used to fabricate high-strength and dense SiAlON-Si3N4 ceramics with appropriated shape at a relatively low sintering temperature. Bone-repairing material applications always require that the applied materials have good biological properties[14]. In a previous work[20], human umbilical vein endothelial cells (HUVECs) were cultured on the surface of SiAlON-Al2O3 ceramics. HUVECs grew and proliferated well for several days. Al2O3 has widely been demonstrated to be a biocompatible material and has been used clinically[21]. Although Si3N4 has been proven to be an excellent biomaterials, no reports have been found regarding the biological properties of SiAlON-Si3N4 composite ceramics. In clinical application, the artificial bone-repairing material is not only required to have good mechanical properties and be nontoxic[22, 23], but it must also be suitable for the growth and proliferation of osteocytes[24]. Only when cells continuously grow and proliferate well with the artificial materials, can it facilitate the fusion of implanted materials with human tissue and finally enable the implanted materials to possess
4
suitable physiological function. In this research work, we first used Si3N4, Al, Si, and Al2O3 as raw materials, and fabricated composites of SiAlON-Si3N4 with a direct nitriding technique. We investigated the physical and chemical properties of the SiAlON-Si3N4 composites and finally, pre-osteoblastic, MC3T3-E1 cells, were seeded on the surface of SiAlON-Si3N4 to evaluate the biocompatibility of the SiAlON-Si3N4. 2
Material and Methods
2.1 Materials preparation Since Si3N4 had a higher melting point, making it very uneasy to sinter, the SiAlON-Si3N4 composites were prepared by forming the β-SiAlON phase with a low melting point such that Si3N4 particles would sinter together. The β-sialon (Si6−xAlxOxN8−x, where x may vary from 0 to 4.2) with x = 2 was prepared. The samples were produced by reaction-bonded sintering using Al, Si, Al2O3, and α-Si3N4 powder as raw materials, as shown in Fig. 1, the morphology of α-Si3N4 powder is made up of granules, as observed by SEM. The composition of the prepared composite samples is presented in Table 1. Chemically pure substances were used in sample preparation. Al powder (purity ≥99.9% and mean particle size of 5 µm), Si powder (purity ≥99.9% and mean particle size of 5 µm), α-Al2O3 powder (purity ≥99.9% and mean particle size of 5 µm), and α-Si3N4 powder (mean particle size of 2 µm, α-phase ≥95.0%) were mixed together with 5 wt.% polyvinyl alcohol, which acted as a binder, in ethanol to fabricate the sample slurry. After that, pellets were created using a pressure spraying pelletizer
5
(YC-1000, Shanghai Yacheng Instrument Equipment Co., Ltd., China). Then, at 100 MPa, adopting hydraulic machine pressing technique to farbicate cylindrical samples which had 20 mm diameter and 2 mm thick. Ten cylindrical samples were farbicated for each kind of ceramic composite. The. Finally, the cylindrical samples were heated at 4 °C/min to 1500 °C and held isothermally for 3 h in N2 gas (1 atm) in a MoSi2 resistance furnace. 2.2 Materials characterization 2.2.1 Phase analysis and microstructure observation The phases of the samples resulting from nitridation were characterized by powder X-ray diffraction (XRD; X’Pert Pro MPD, PANalytical Co., Netherlands) with CuKα radiation; data were collected from 2θ = 10° to 90°. The microstructures were examined by slicing sections of the cyclinders, which were then ground and polished carefully. Pt was sputtered onto the cross sections, which were then observed using an SEM-EDAX (Nova 450 Nano, FEI, USA; EDAX, Energy Dispersive X-ray X-Max, OXFORD, UK). 2.2.2 Bulk density, porosity, and compressive strength test Sample bulk density and apparent porosity was measured using the Archimedes method, and water was used as the displacement liquid [25]. Samples were processed into prisms with dimension of 5 × 5 × 12.5 mm for the compressive strength test. Compressive strength was measured using a universal material testing machine (Instron 5567, Instron, USA) with a crosshead speed of 0.2 mm/min. The arithmetic average value of the three measurements taken on each sample is identified as the measured
6
value. 2.2.3 Ion release studies of the SiAlON-Si3N4 composite ceramics Materials used as bone-repairing materials should have good chemical stability, which guarantees that ions in the composite material do not continually release into human tissue. The continual release of ions causes the materials not only to corrode, but also the increase in local ion concentration is not beneficial for reorganization. Therefore, ion release behavior of bone-repairing material becomes crucial. Soaking tests were used to investigate ion release in materials. Since the cell medium, as presented in section 2.2.4, contains a large quantity of ions and is easily contaminated, it is not suitable for soaking experiments. Therefore, deionized water was used in the experiment to soaking the ceramic samples. The chemical stability of the composites was estimated by measuring the total ion concentration in the deionized water over a 14-day soaking process. The composites were cut into disks (diameter 20 mm, height 10 mm). The weight ratio of the sample and deionized water was 1:7 at 37 ℃ during the soaking experiment. After soaking, the total ion concentration in the deionized water was measured once in every 24 h using Mettler Toledo S230-USP/EP-CN (Youyi Instrument Company, China) system. 2.2.4
Cell culture and proliferation on the composite ceramic surface
Mouse pre-osteoblastic cell line MC3T3-E1 was purchased from National Infrastructure of Cell Line Resource, China. A complete cell cuture medium containing α medium essential medium (Biological Industries, Israel) and 10% fetal bovine serum
7
(Biological Industries, Israel) for cell culture was used. Cell culture was executed in 5% CO2 in an incubator (Thermo, USA) held at 37 ℃. Ceramic slices (thickness 2 mm) with a smooth surface were fabricated for cell culturing and biological properties evaluation. All the materials used for cell culture were sterilized in a sterilizer (Sanyo MLS-3751-PC, Japan) and dried in a dryer (Qilian power equipment Co.LTD, China). MC3T3-E1 cells were digested, centrifugated, and resuspended in a complete cell culture medium; 2 mL of the MC3T3-E1 cell suspension was added into a plate of six wells, each containing a composite ceramic slice at the bottom. After culturing (24 and 72 h), the cell culture medium was thrown away, and six well plates were cleaned with PBS twice. MC3T3-E1 cells on the ceramic surfaces were immersed in 2 µmol/L calcein-AM (Thermo Fisher, USA) for 40 min, after which the staining solution was thrown away and ceramic samples were cleaned twice with PBS. Calcein-AM is a cell-permeant fluorescent dye used to observe living cells without unfavorable effects. Ceramics samples with stained MC3T3-E1 cells on their surfaces were placed on a slide glass with surfaces contacting the glass. Laser scanning confocal microscopy (LSCM; Fluo1000, Olympus) is inverted microscopy which can observe the excited fluorescence of the opaque sample. Stained MC3T3-E1 cells on composite ceramic surfaces were also observed by LSCM. Transmitted light images captured by laser scanning confocal microscopy clearly show the 3D state of ceramic surface. When cells were stained by green fluorescent dye, the images captured by LSCM were referred to as confocal images. Merging the
8
transmitted light images and confocal images enabled the clear observation of pseudopodia and shape of the cells on the ceramic surfaces. Cell pseudopodia were observed by imerging transmitted light images and confocal images captured by LSCM as well.
3 Results and Discussion 3.1 Phase analysis and microstructure of the SiAlON-Si3N4 composite ceramics XRD patterns of the SiAlON-Si3N4 composite ceramics obtained by reactive sintering at 1500 ℃ for 4 h are presented in Fig. 2. The main phase in SL-1 was β-sialon, and the peak intensity of the detected α-Si3N4 increased with increasing Si3N4 content in the precursor material mix, thus confirming that the goal of the ceramic composition was achieved. As expected, Si, Al, and Al2O3 reacted with nitrogen at high temperature to generate the SiAlON phase in this experiment. The Si4Al2O2N6 formed in the samples holds Si3N4 together to form the SiAlON-Si3N4 composite ceramic. The presence of trace Al2O3 in the SL-1 and SLN-1 samples could be attributed to partial evaporation of Si and Al during heating process, which leads to a small quantity of excess Al2O3. The characteristic peak of Al2O3 disappears in the SLN-2, SLN-3, and SLN-4 samples which may be caused by Si3N4 reacting with Al2O3 to form the SiAlON phase; Al2O3 is often used as a sintering additive to create Si3N4 based ceramics [9]. The microstructure of the samples is shown in Fig. 3, showing the evolution of the microstructure of SiAlON-Si3N4. As can be seen in Fig. 3A and considering the corresponding XRD patterns in Fig. 2, the Si4Al2O2N6 grains reacted during sintering to
9
form α-Si3N4. Overall, the grains become smaller with an increasing Si3N4 in the composites. Si4Al2O2N6 content also reduces with increasing Si3N4 content in the composite, which likely occurs because the Si4Al2O2N6 phase is formed by a direct reaction in SL-1. However, in the other samples (SLN-1–SLN-4), the synthesized Si4Al2O2N6 in the samples holds the α-Si3N4 grains together to form the SiAlON–Si3N4 composite ceramics, where α-Si3N4 grains inhibit the growth of the Si4Al2O2N6 phase during the nitriding sintering process. Fibrous crystals were also formed in the samples, except in SL-1. The elemental composition of the fibrous crystals was Si and N, as determined by EDAX. Therefore, it can be inferred that the fibrous crystals are β-Si3N4. Some grains of α-Si3N4 may undergo a phase transition from the α-Si3N4 phase to the β-Si3N4 phase at high temperature. Incorporation of fibers in ceramics often reinforces both the strength and toughness of the material, which may indicate that the ceramic properties may be improved in samples where fibrous β-Si3N4 phase is present. However, characteristic peaks of β-Si3N4 were not found by XRD, which may indicate that its trace amounts of β-Si3N4 could not be detected. 3.2 Porosity, bulk density, and compressive strength The change in the porosity and bulk density of the composites with the varying Si3N4 content is presented in Fig. 4. The porosity of samples is found to increase with increasing Si3N4 content, and the bulk density of samples is found to decrease. This is likely attributed to the Si3N4 having a higher melting point, which maks it difficult to
10
sinter at the lower temperature of this experiment. The Si4Al2O2N6 formed in the samples and filled in the gap between the α-Si3N4 grains to form the SiAlON-Si3N4 composite ceramics. With decreasing Si4Al2O2N6 content and increasing Si3N4 content, the densification of the ceramics was reduced. The bulk density of the material prepared in this experiment is similar to that of the human skeleton [24]. It suggested that the use of SiAlON-Si3N4 as a bone-repairing material could not bring any adaptive problems. Otherwise, the porosity of material raises material surface roughness, which helps cell adhesion and promotes its combination with human tissue [26, 27]. Fig. 5 shows the measured compressive strength of the samples. The compressive strength first increases and then decreases with increasing Si3N4 content. This means that the addition of Si3N4 improved the compressive strength of the SiAlON-Si3N4 ceramics. In comparison with SLN-3, the compressive strength of SLN-4 is slightly decreased. This may be because the compressive strength decreased for with increasing porosity to a certain extent. However, the compressive strength of all samples met the needs of bone-repairing material, indicating that the SiAlON-Si3N4 composites have the feasibility to be used in the manufacture of novel load-bearing bone implnats[28]. The implementation of other sintering additives in the sample also could change the microstructure and improve the performance of SiAlON-Si3N4 composites. To avoid unexpected or undesirable effects on the biological properties, other sintering additives were not investigated in this study. The improvement in the properties of SiAlON-Si3N4 composites will be carried out in the future after confirming the materials are
11
biocompatible. 3.3 Water leaching experiments of the SiAlON-Si3N4 composite ceramics Samples of the SiAlON-Si3N4 composite were immersed in deionized water for periods ranging from 1 to 15 days as shown in Fig. 6. The time-dependent profile of the ion concentration shows that ion release occurred mainly during the first five days after soaking. The ions concentration in deionized water tended to be stable after one week. There may be two reasons for this result. One is the impurities in the raw material; another is the ion release behavior of the composite materials. It is well understood that no material is 100% pure, which means that impurities may be present in the raw material. The impurity ions will also release from the formed SiAlON-Si3N4 composites. Pure Si3N4 and SiAlON have outstanding chemical stability, but they still undergo a slight ion release [29, 30]. Regardless of whether the ions are released from impurities or are leached from the composites, the maximum amount of ions released (<6 mg/L) arrived after two week of soaking. Based on this experiment, it is found that SiAlON-Si3N4 composite ceramics exhibit rather low ion release, thus being very chemically stable. 3.4 Viability and proliferation of MC3T3-E1 cells on SiAlON-Si3N4 MC3T3-E1 cells, as a preosteoblastic cell line, were used to investigate the properties of bone tissue materials since these cells can be induced to become osteoblastic cells [31]. During the calcein-AM incubation of the experiment, calcein-AM entered the viable cells and was inverted by intracellular esterases to calcein within the cells.
12
Calcein produces a green signal with laser stimulation and can be used as a living cell tracer[32]. Living cells on the composite ceramic surfaces were observed after 24 and 72 hour cultures, as shown in Fig. 7. After the 24 h culture, MC3T3-E1 cells adhered well on all ceramic sample surfaces. Most of the cells were presented as a fusiform and extended two or three thin pseudopodia. These phenomena indicate that MC3T3-E1 cells had the growth ability and potential to migrate, as that introduced in Klemke’s article[33]. When the culture time was increased to 72 h, MC3T3-E1 cells proliferated on all ceramic sample surfaces with time and reached 90% confluence, as shown in Fig. 7. MC3T3-E1 cells had a larger cell body, extended more and wider pseudopodia than those cultured for 24 h (Fig. 8A). The pseudopodia around the cells cross linked with each other (Fig. 8B) as culture time increased to 72 h. Due to cytoplasmic streaming during pseudopodia extension and withdrawal, the growth and cross linking of pseudopodia generated intracellular movement [34]. The changes in the pseudopodia and cell shape with culture time evidences that MC3T3-E1 cells have excellent growth activity and potential ability to proliferate to osteocytes. The increase in the number of living cells on the composite ceramic sample surfaces shows that these ceramics are biocompatible and have no toxity. SiAlON-Si3N4 ceramics have a good biological environment and are suitable for MC3T3-E1 cell growth and proliferation. Previous water leaching experiments revealed that SiAlON-Si3N4 composite ceramics exhibit a slight ion release. The maximum amount of ions released was <6 mg/L, which
13
is less than the ion concentration of Hanks balanced salt solution [35]. The good growth status of cells cultured with SiAlON-Si3N4 composite ceramics illustrates that ions released from SiAlON-Si3N4 based ceramics did not influcece cell growth. When considering the results of this work as well as those from previous research, the Si4Al2O2N6 phase can not only be used as soft-tissue compatible materials but can also be readily combined with MC3T3-E1 cells (bone cells, hard tissue). The Si4Al2O2N6 phase can also be combined with materials (such as Al2O3 and Si3N4) to form compound and composite to improve its performance. Therefore, the developed SiAlON-Si3N4 composite is expected to be applicable in biomaterials, such as bone-repairing material and biological scaffolds. However, more biological properties of the composites must further be evaluated for clinical applications. 4
Conclusion Si4Al2O2N6 phase is prepared by reacting Si, Al, and Al2O3 with nitrogen at high
temperature; during sintering, it combines with α-Si3N4 to form SiAlON-Si3N4 composite ceramics. The porosity of the samples is found to increase with increasing Si3N4 content and the bulk density is found to decrease. The compressive strength increases first and then slightly decreases with increasing Si3N4 content. Water leaching experiments of the SiAlON-Si3N4 composites show that they exhibit outstanding chemical stability. The results of bone cell cultures show that the cells present a fusiform and extend two or three thin pseudopodia. These phenomena indicate that MC3T3-E1 cells have excellent growth activity and have the potential ability to
14
proliferate to osteocytes on the surfaces of SiAlON-Si3N4 samples. Therefore, we conclude that SiAlON-Si3N4 based ceramics exhibit excellent biocompatibility and could be implemented as a potential bone-repairing Material.
Financial support and sponsorship: This work was funded by the National Natural Science Foundation of China (3160067), Key Scientific Research Project of Colleges and Universities in Henan Province (17210231004, 19A430027, 16A350011), Major Science and Technology Project of Henan Province (161100311400) and China Postdoctoral Science Foundation (2017M612416).
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3-4.
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Table captions Table 1. Chemical composition of all samples in wt.%
Figure captions Fig.1 The morphology of α-Si3N4 powder Fig.2 XRD patterns of the SiAlON-Si3N4 composite ceramics Fig.3 The microstructure of the samples: A: SL-1; B: SLN-1; C: SLN-2; D: SLN-3; and D: SLN-4 Fig.4 The change in the porosity and bulk density of the composites with the varying Si3N4 content Fig.5 The compressive strength of the samples Fig.6 The ion concentration of the SiAlON-Si3N4 composite Fig.7 Cell culture results of the SiAlON–Si3N4 composite ceramics Fig.8 The pseudopodia around the cells
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Table
Table 1. Chemical composition of all samples in wt.% Sample
Si
Al
Al2O3
Si3N4
Total
SL-1
56.57
9.09
34.34
0.00
100.00
SLN-1
45.26
7.27
27.47
20.00
100.00
SLN-2
33.94
5.45
20.60
40.00
100.00
SLN-3
22.63
3.64
13.74
60.00
100.00
SLN-4
11.31
1.82
6.87
80.00
100.00
22
Figure
Fig.1 The morphology of α-Si3N4 powder
Fig.2 XRD patterns of the SiAlON-Si3N4 composite ceramics
23
Fig.3 The microstructure of the samples: A: SL-1; B: SLN-1; C: SLN-2; D: SLN-3; D: SLN-4
Fig.4 The change in the porosity and bulk density of the composites with the varying Si3N4 content
24
Fig.5 The compressive strength of the samples
Fig.6 The ion concentration of the SiAlON-Si3N4 composite
25
Fig.7 Cell culture results of the SiAlON-Si3N4 composite ceramics
Fig.8 The pseudopodia around the cells
26