Preparation and bioactivity of biodegradable β-tricalcium phosphate / calcium carbonate / phosphate bioactive glass composite porous ceramic

Abstract

Background:

At present, scaffold biomaterials with great biodegradation and biocompatibility are attracting more and more attention. Phosphate bioactive glass (PBG) without Si has been prepared successfully, with a glass transition temperature below 600°C. Calcium carbonate (CC)-based bioceramics with PBG as binder were sintered rapidly at a lower temperature. β-Tricalcium phosphate (β-TCP) has always been used to synthesize clinical ceramics due to its wonderful biocompatibility. Here, we combined the advantages of these raw materials to obtain a novel β-TCP/CC/PBG composite porous ceramic.

Method:

The preparation process of β-TCP/CC/PBG was optimized by controlling PBG content, NaCl ratio, sintering temperature, and holding time. Ceramic biodegradability was evaluated by soaking in a Tris-HCl buffer in vitro, and biocompatibility of the new material was indicated using CCK-8 tests and a live/dead fluorescence assay.

Results:

The best mechanical properties of β-TCP/CC/PBG composite porous ceramics were obtained with a PBG content of 60%, at which point the proportion of NaCl exerted the most significant influence on the density, porosity, and mechanical properties of the materials. The weight loss rate of the composite ceramics was 11.30%, which was much higher than that of β-TCP (1.41%) and hydroxyapatite (0.83%) ceramics. CCK-8 test and live/dead fluorescence assay indicated that the composite porous ceramics showed a biocompatibility similar to that of β-TCP ceramics.

Conclusion:

β-TCP/CC/PBG composite porous ceramics have potential applications in bone regeneration. It is hoped that the novel biomaterial developed in this study will prove useful for the repair of bone defects.

Keywords

porous ceramics phosphate bioactive glass biodegradation biocompatibility

Introduction

Bioceramics refers to a class of ceramic materials used for specific biological or physiological applications, that is, ceramic materials directly used in or around the human body for biological, medical, biochemical, and other purposes.¹

With the development of bone tissue engineering, there is now interest in biodegradable materials that can be used in vivo and have a degradation rate comparable to the growth rate of new bone. The properties of calcium phosphate porous ceramics are known to be similar to those of human tissues in terms of their composition, structure, and mechanical properties, and their ability of inducing stem cells to differentiate into osteoblasts.^2,3 In addition, β-tricalcium phosphate (β-TCP), hydroxyapatite (HA), and calcium sulphate (CS) with good biocompatibility, biodegradability, and bone conductivity, have been used widely in clinical applications.^4,5 In vivo, HAP ceramics do not degrade easily, and the degradation rate of β-TCP ceramics is slow; however, CS ceramics degrade too quickly.^6–8 In previous studies, researchers in our laboratory found that natural corals (mainly calcium carbonate (CC)) used as raw materials for orthopaedic and dental repair have good biocompatibility.^9–11 However, as CaCO₃ begins to decompose into CaO and CO₂ at about 650°C, the high temperature sintering used for ordinary ceramics cannot be used. In our previous research,¹² we prepared phosphate bioactive glass (PBG) with a glass conversion temperature below 600°C that was suitable for use as a binder for bioceramics to achieve low temperature sintering. On that basis, biphasic porous ceramics of CC/PBG were successfully prepared.¹² The results of in vitro and in vivo degradation experiments showed that the degradation rate of CC/PBG biphasic porous ceramics is between β-TCP and CS,¹³ CC/PBG biphasic porous ceramics have high bone guiding activity and fast degradation in the early stages, but the osteogenic effect is not comparable to that of β-TCP porous ceramics.¹⁴

In this study, composite porous ceramics of β-TCP/CC/PBG were prepared using β-TCP, CC, and PBG as raw materials, and NaCl as a pore-making agent. The final material benefitted from the respective characteristics of the raw materials. Compared with the previous study, the ceramic materials in this paper had lower sintering temperature and higher compressive strength.^12–14 Furthermore, the degradation rate of materials was improved; the effects of PBG and porosity on the properties of ceramics are discussed. This study provides a potential foundation for future research in the field of bone regeneration.

Materials and methods

Materials

As per methods described in our previous research,¹² PBG(50P₂O₅·18CaO·12MgO·20Na₂O) was prepared using CaCO₃, NH₄H₂PO₄, (MgCO₃)₄·Mg(OH)₂·5H₂O, NaCO₃ (analytical pure, Tianjin Fuchen, China). The β-TCP used in this study was prepared using Ca(OH)₂ and H₃PO₄ (analytical pure, Tianjin Fuchen, China) with a ratio of Ca/P = 1.5.¹⁵ The target material was obtained after filtration, ball-milling, and calcination in a muffle furnace at 850°C for 3 h.

Calcite (CaCO₃, 99%) powders were purchased from Shanghai Inorganic pigment Co. Ltd. (Shanghai, China) and had diameters less than 100 μm after grinding. The smaller size NaCl (< 100 μm) was purchased from Tianjin Zhiyuan Chemical Reagent co. Ltd. (Tianjin, China), while the larger size NaCl (300–600 μm) was prepared by milling and sifting with coarse salt purchased from Guangzhou Foundation Chemical Technology Co. Ltd. (Guangzhou, China).

Preparation of β-TCP/CC/PBG, PBG, and β-TCP porous ceramics

Equal quantities of β-TCP and CC were mixed with PBG in proportions of 20, 40, 50, 60, and 70% to fabricate ceramic starting powders. Equal amounts of two sizes of NaCl powders, which served as pore-forming agents, were blended with ceramics starting powders in proportions of 2:8, 3:7, 4:6, 5:5, and 6:4. The mixtures were poured into a mold and pressed at 10 MPa for 10 min with a pressing machine (FW-5, Tianjin Botianshengda, China), after which they were unmolded. The green bodies were placed in a muffle furnace and heated at a rate of 5°C/min up to one of several temperatures (500, 550, 600, 650, or 700°C), where they were held for 0, 20, 40, 60, or 80 min before being allowed to cool naturally. The sintered ceramics were immersed and soaked in deionized water for 24 h during which time the water was changed every 4 h. The ceramics were then dried at 80°C for 24 h to obtain different groups of β-TCP/CC/PBG composite porous ceramics. Detailed fabrication parameters for some ceramics are shown in Table 1.

Table 1.

Different group of β-TCP/CC/PBG composite porous ceramics.

Group	PBG content	NaCl radio	Temperature (°C)	Holding time (min)
7(60%PBG):3NaCl	60%	30%	600	40
6(60%PBG):4NaCl	60%	40%	600	40
5(60%PBG):5NaCl	60%	50%	600	40
6(40%PBG):4NaCl	40%	40%	600	40
6(50%PBG):4NaCl	50%	40%	600	40

PBG powders were mixed uniformly with 40% NaCl, and then compacted used a circular mold with a diameter of 22 mm under 10 MPa. The green bodies were heated to 600°C at a rate of 5°C/min, held at temperature for 20 min, and then cooled naturally, soaked in deionized water for 24 h, and dried for 24 h to obtain PBG porous ceramics.

The foaming agent was prepared by using rosin and a saturated NaOH solution, uniformly mixed with the β-TCP slurry, poured into a plaster mold, and dried after demolding. The raw embryos were placed in a muffle furnace at a rate of 5°C/min, sintered at 1100°C for 2 h, and cooled naturally to obtain porous β-TCP ceramics.¹⁶

Materials characterization

The broken section microstructure of the samples was observed using a scanning electron microscope (SEM, Phenom ProX, Netherlands).

The prepared β-TCP/CC/PBG composite porous ceramics were ground into powders, after which the ceramic and β-TCP, PBG, and calcite (CaCO₃) powders were analyzed using an X-ray diffractometer (XRD, D8 Advance, Bruker AXS, Germany).

The porous ceramic sample was cut into a cylindrical shape (diameter = 20 mm, height = 15 mm) and the dry, liquid, and wet weights of the porous ceramic samples were measured by the drainage method to calculate the porosity and density of the samples (n = 3).¹⁷

Both ends of the samples (diameter = 20 mm, height = 15 mm) were polished, and the compressive strength was measured at a crosshead speed of 0.5 mm/min with an electronic universal testing machine (UTM4304X, Shenzhen Sens, China) (n = 3).

In vitro degradation test

The cylindrical porous ceramic samples were dried and then weighed using an electronic balance. Tris-HCl buffer solution (pH 7.4) without inorganic metal ions was chosen for the degradation test, which does not affect the degradation and mineralization of ceramics. The samples were immersed in buffer solution at a ratio of 30 ml/g of solution to the weight of the sample, and were then oscillated in a constant temperature incubator shaker at the rate of 60 rpm at 37°C. The buffer solution was replaced after 3, 7, 14, 21, 28, and 35 days. The samples were cleaned using deionized water, and then dried and weighed. The weight loss rate of the porous ceramics was calculated as follows:

WL % = (W_{0} - W_{d}) / W_{0} \times 100 %,

(1)

where WL represents the weight loss rate of the porous ceramics, W₀ represents the starting weight of the dried samples, and W_d represents the weight of the dried samples after immersion (n = 3).

Evaluation of in vitro cell behaviors

Cell culture and implantation of materials

Mouse precranial bone cells (MC3T3-E1, Shanghai Department Biological Science, China) were cultured in alpha minimum essential medium (α-MEM, Gibco, Waltham, MA, USA) with 10% foetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Eallbio, China). Porous ceramic samples were cut into rectangular scaffolds of 6.5 mm × 6.5 mm × 1.5 mm. After being sterilized using an autoclave, each scaffold was soaked in 2 ml complete medium for 4 h (the pH rose from about 7.29 to 7.42). Then, the scaffolds were implanted in a 24-well plate to which 500 μL of a cell suspension (5 × 10⁴ cells) was added. The scaffolds were then cultured in a cell incubator at 37°C with 5% CO₂. The complete medium was replaced every 2 days.

Cell proliferation

A CCK-8 kit (Shanghai Dongren Technology, China) was used to evaluate the proliferation of the cells in the scaffolds after 1, 3, 5, and 7 days of culturing. The original medium was removed at the scheduled time and the cells were cleaned with PBS three times. Then, 200 μL of complete medium and 20 μL of CCK-8 reagent were added into each well under a dark light. After incubation of 2 h, 100 μL of supernatant was transferred into a 96-well plate. Finally, the absorbance was measured using a microplate reader at 450 nm. To reduce error and allow statistical evaluation, six holes were created at each time point in each test group.

Live/dead fluorescence detection

The activity of the cells on the materials was characterized using a live/dead double staining kit (Calcein-AM, Shanghai Yeasen, China). After culturing for 2 days, the cells were cleaned three times using PBS and then incubated in the Calcein-AM for 15 min at 37°C. Finally, the activity of the cells was observed using a 490 nm stimulated filter under a fluorescence microscope.

Statistical analysis

The n = 6 data points were expressed in the form of a mean ± standard deviation (SD) and a one-way analysis of variance (ANOVA) was used to compare the differences between the groups. In this case, the differences were considered to be statistically significant when P < 0.05 (*).

Results

Microstructure and composition

The appearance and broken section microstructure of the β-TCP/CC/PBG composite porous ceramics are shown in Figure 1. From Figure 1(a), it can be seen that the β-TCP/CC/PBG composite porous ceramics were white after sintering, and that the surface was covered with macroscopic pore structures; from Figure 1(b) it can be seen that the macropores had apertures ranging from 300 to 600 μm. Smaller interconnected pores and micropores can also be seen between the macropores (Figure 1(c,d)).

Figure 1.

(a) Appearance and (b), (c), (d) broken section microstructure of the β-TCP/CC/PBG composite porous ceramics.

The XRD pattern of the β-TCP/CC/PBG composite porous ceramics for CC, β-TCP, and PBG is shown in Figure 2. The characteristic peaks of calcite (CaCO₃) and β-TCP can be observed in the XRD pattern of the ceramic materials. The PBG was a glass material with an amorphous structure with no sharp crystal diffraction peak.¹⁹

Figure 2.

XRD patterns of the β-TCP/CC/PBG composite porous ceramics, CC, β-TCP, and PBG.

Porosity and density

The effects of different PBG content, NaCl ratios, sintering temperature, and holding time on the porosity and density of the composite porous ceramics is shown in Figure 3, where it can be seen that the trends of the porosity and density were approximately opposite. With an increase in the PBG content, the porosity of the ceramic materials decreased and the density increased. The radio of the pore-making agent NaCl had the greatest influence on the porosity and density of the ceramic materials. When the NaCl ratio was 60%, porous ceramics with a maximum porosity of 76.7 ± 1.4% were obtained. On the contrary, sintering temperature and holding time had little effect on the porosity and density of the resulting ceramics.

Figure 3.

Effect of different amounts of PBG content, NaCl ratios, sintering temperatures, and holding times on the porosity and density of the composite porous ceramics.

Compressive strength

The effect of different PBG content, NaCl ratios, sintering temperatures, and holding times on the compressive strength of composite porous ceramics are shown in Figure 4. When the PBG content was below 60%, increasing the PBG content improved the compressive strength of the ceramic materials. In contrast, the compressive strength of the ceramics decreased as the PBG content increased when the PBG content was higher than 60%. When the ratio of NaCl was 20%, the compressive strength of the composite porous ceramics was as high as 12.94 ± 2.08 MPa. A sintering temperature below 600°C had little influence on the compressive strength of the ceramics. Ceramics with the highest compressive strength were obtained at a holding time of 40 min, which was therefore deemed to be optimal.

Figure 4.

(a) Effects of the PBG content, (b) NaCl ratio, (c) sintering temperature, and (d) holding time on the compression strength of the composite porous ceramics.

Degradation

As can be seen from Figure 5, as the soaking time was prolonged, each group of ceramics degraded gradually in the Tris-HCl buffer. The weight loss rate of the PBG porous ceramics was highest, and the weight loss after immersion for 35 day was 26.98 ± 0.11%. The second highest weight loss rate was for the β-TCP/CC/PBG composite porous ceramics, while the least weight was lost for the β-TCP and HA porous ceramics (the weight loss ratios after immersion for 35 days were 1.41 ± 0.74% and 0.83 ± 0.05%, respectively). Comparing the five groups of composite ceramics, it can be seen that the degradation rate was: 5(60%PBG):5NaCl > 6(60%PBG):4NaCl > 7(60%PBG):3NaCl > 6(50%PBG):4NaCl > 6(40%PBG):4NaCl. Among these, the group of 5(60%PBG):5NaCl composite porous ceramics had the highest degradation ratio, and the weight loss ratio after immersion for 35 days was 11.30 ± 0.61%. In addition, the effect of the NaCl ratio on the degradation rate of the composite porous ceramics was greater than the PBG content.

Figure 5.

Weight loss rate of HA porous ceramics, β-TCP porous ceramics, PBG porous ceramics, and composite porous ceramics with different PBG content / NaCl ratios after immersion in Tris-HCl for different lengths of time.

Cell proliferation

As depicted in Figure 6, the cells proliferated gradually in different materials and blank groups as the culturing time was extended. During the culturing process, no significant difference was observed in the proliferation of the cells on the β-TCP/CC/PBG composite porous ceramics with different PBG content / NaCl ratios (P > 0.05). The cell proliferation of five groups of β-TCP/CC/PBG composite porous ceramics and β-TCP porous ceramics were significantly different (P < 0.05) after 3, 5, and 7 days. When the cell proliferation of the composite ceramics and blank group were compared, no significant differences were observed at 1 day. However, on the 3rd and 5th days, the proliferation of cells in the blank group was noticeably higher than that in the composite porous ceramic group (P < 0.05). In contrast, the OD value of the porous ceramic group was close to that of the blank group on the 7th day and there was no significant difference (P > 0.05).

Figure 6.

Cell proliferation in β-TCP/CC/PBG composite porous ceramics with different PBG content/NaCl ratios, β-TCP porous ceramics, and blank groups; *significant difference (P < 0.05).

Fluorescence detection

The fluorescence images of cells cultured on β-TCP/CC/PBG composite porous ceramics with different PBG content / NaCl ratios and β-TCP porous ceramics for 2 days using a live/dead kit are shown in Figure 7. As shown, the living cells exhibited green fluorescence while the dead cells displayed red fluorescence. A large number of living cells and sporadic numbers of dead cells could be observed on the surface of all materials. Moreover, many cells grew in the porous structure of the ceramics.

Figure 7.

Fluorescence images of cells on porous ceramics: (a) 7(60%PBG):3NaCl; (b) 6(60%PBG):4NaCl; (c) 5(60%PBG):5NaCl; (d) 6(40%PBG):4NaCl; and (e) 6(50%PBG):4NaCl; (f) β-TCP.

Discussion

The macropores of β-TCP/CC/PBG composite porous ceramics were the same diameters as those of the pore-making agent NaCl. The micropores were distributed widely throughout the porous ceramics and made the internal connectivity of the entire ceramic structure favorable. This phenomenon was caused by the use of two different sizes of NaCl as pore formers, in line with experimental expectations.

In the low-temperature sintering process, CaCO₃ was well controlled and did not decompose into CaO and CO₂. The diffraction peaks in ceramics were derived mainly from CaCO₃ and β-TCP. These all indicate that the temperature program for sintering was suitable.

The PBG melted sealed the pores inside the ceramic at high temperature, thereby reducing the porosity and increasing the density.^18,19 According to Mocciaro, the pore-making agent can be removed by soaking in deionized water and dissolving, leaving the position vacant to create pores.²⁰ For this reason, the higher the NaCl ratio, the higher the porosity and the lower the density.

The PGB used as the binder and main component of the composite ceramics was melted at high temperature during sintering, which caused the bonding between the raw material particles in the ceramics to become tighter, thereby increasing the corresponding compressive strength. However, when the PBG content was too high, the melting state of the PBG generated stress during the cooling process, which compromised the mechanical properties of the ceramic materials. Wan et al. prepared calcium polyphosphate bioceramics with different porosities by controlling the concentration of pore-making agent, and their results showed that the porosity of the ceramics increased as the concentration of the pore-making agent increased, although the compressive strength of the ceramic materials decreased.²¹ The CaCO₃ begins to decompose at 650°C to produce CaO and CO₂, which reduces the compressive strength of the ceramics. When the holding time was too short, the PBG failed to melt completely into the glass state and the ceramics were not completely sintered. However, when the holding time was too long, the PBG melted excessively, which then generated stress during the cooling process and caused partial fracture of ceramic. Note that both factors may negatively affect the compressive strength of ceramics.

He et al. found that PBG had good biodegradability and the degradation rate of PBG was faster than that of other ingredients.¹³ Thus, increasing the PBG content may improve the degradability of ceramics. A study by Fu et al. showed that the degradation rate of glass ceramics was rapid due to the fast ion-exchange in the early days, but the degradation rate at later stage was slow due to the high dense mineral layer.²² In order to improve this undesirable degradability, Shuai et al. added β-TCP to regulate the degradation rate of early and late glass ceramics in the process of preparing 58S glass.²³ The NaCl ratio determines the porosity of the ceramics, which can accelerate the degradation rate by increasing the contact area between the ceramics and buffer solution.²⁴ For these reasons, the degradation rate of the composite porous ceramics with large NaCl ratios was relatively high.

As shown from the results, β-TCP had better biocompatibility than the composite ceramics in vitro. This may be due to the high degradation of composite ceramics, which caused a localized high ionic conductivity and weakened cell proliferation. Findings in the study by Gao et al. were similar.²⁵ Note that the opposite may be the case in vivo. De Aza et al. implanted calcium phosphate ceramics into rabbits and found that the ceramics were continuously degraded in vivo.²⁶ The ceramics did not degrade significantly when soaked in complete medium for 1 day. On the 3rd and 5th days, the powders was dropped, affecting cell growth. This resulted in similar cell growth in all groups on the 1st day, while on the 3rd and 5th day, the porous ceramic group differed from the blank group. After changing the liquid to empty the powders, calcium ions and phosphate ions were gradually degraded in the culture medium to promote cell growth.²⁶ Therefore, there was no difference between the ceramic group and the blank group on the 7th day. In addition, Makhaniok’s research demonstrates that the presence of porous structures increased the space for cell growth, and also could facilitate differentiation and osteogenesis.²⁷ Based on the results of the cell proliferation experiment, the β-TCP/CC/PBG composite porous ceramics were found to have no noticeably detrimental effect on cell growth and had good biocompatibility.

The results of cells fluorescence images were similar to those of the experiment by Tang et al. in which they prepared porous BCP ceramics with good connectivity and found that a large number of cells grew into the interior of porous ceramics.²⁸ These results indicated that the cells exhibited good bioactivity on the materials. Both β-TCP/CC/PBG composite porous ceramics and β-TCP porous ceramics had good biocompatibility.

Conclusion

In this study, PBG was employed as a binder and the main component and was mixed with β-TCP and CC. Then, NaCl was used as a pore-making agent to prepare β-TCP/CC/PBG composite porous ceramics. The NaCl ratio was found to have the greatest effect on the porosity, density, and compressive strength of the prepared composite porous ceramics. Ceramic material with the best comprehensive properties was obtained for a sintering temperature of 600°C and a holding time of 40 min. The β-TCP/CC/PBG composite porous ceramics were shown to have good degradability in vitro. And among all the properties, the PBG content was the most important factor. Cells were found to grow well on the ceramic materials, and there was no significant difference with the blank group at 7 days. The prepared β-TCP/CC/PBG composite porous ceramics exhibited excellent biocompatibility, and the content and proportion of the ceramic components were found to have no significant effect on the bioactivity of the ceramic material. It is anticipated that the results of this study of composite porous ceramics will provide solid foundation for clinical application of bone repair materials.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Guangzhou “121 talents” project [grant number 158010], the Natural Science Foundation of Guangdong Province of China [grant number 2018A0303130051], and the National Natural Science Foundation of China [grant number 81470724].

ORCID iD

Xiaoming Chen

References

Saiz

Boccaccini

Chevalier

et al . Editorial on bioceramics for bone repair. J Eur Ceram Soc 2018; 38(3): 821–822.

Yuan

De Bruijn

et al . Bone formation induced by calcium phosphate ceramics in soft tissue of dogs: a comparative study between porous alpha-TCP and beta-TCP. J Mater Sci Mater Med 2001; 12(1): 7–13.

Palmer

Nelson

Schatton

et al . Biocompatibility of calcium phosphate bone cement with optimised mechanical properties: an in vivo study. J Mater Sci Mater Med 2016; 27(12): 191.

Szponder

Mytnik

Jaegermann

Use of calcium sulfate as a biomaterial in the treatment of bone fractures in rabbits—Preliminary studies. Bull Vet Inst Pulawy 2013; 57(1): 119–122.

Bui

XV.

Synthesis and characterization of HA/β-TCP bioceramic powder. Vietnam J Chem 2018; 56(2): 152–155.

Grondahl

Hui

In vitro degradation study of polyanhydride copolymers/surface grafted hydroxyapatite composites for bone tissue application. Polym Degrad Stab 2017; 140: 136–146.

Kuang

Mao

Enhanced degradation and osteogenesis of β-tricalcium phosphate/calcium sulfate composite bioceramics: The effects of phase ratio. J Biomater Tissue Eng 2014; 4(5): 389–398.

Hao

Qin

Liu

et al . Assessment of calcium sulfate hemihydrate-tricalcium silicate composite for bone healing in a rabbit femoral condyle model. Mater Sci Eng C 2018; 88: 53–60.

Guillemin

Patat

Fournie

et al . The use of coral as a bone graft substitute. J Biomed Mater Res 1987; 21(5): 557–567.

10.

Martina

Subramanyam

Weaver

et al . Developing macroporous bicontinuous materials as scaffolds for tissue engineering. Biomaterials 2005; 26(28): 5609–5616.

11.

Cooper

Hirabayashi

Strychar

et al . Corals and their potential applications to integrative medicine. Evid Based Complement Alternat Med 2014; 2014: 1–9.

12.

Yang

Zhu

et al . Fabrication of a novel calcium carbonate composite ceramic as bone substitute. J Am Ceram Soc 2015; 98(1): 223–228.

13.

Zhang

Yang

et al . In vitro degradation and cell response of calcium carbonate composite ceramic in comparison with other synthetic bone substitute materials. Mater Sci Eng C 2015; 50: 257–265.

14.

Ren

Tian

et al . Comparative study on in vivo response of porous calcium carbonate composite ceramic and biphasic calcium phosphate ceramic. Mater Sci Eng C 2016; 64: 117–123.

15.

Liu

Yang

et al . Preparation of bioactive β-tricalcium phosphate microspheres as bone graft substitute materials. Mater Sci Eng C 2017; 70: 1200–1205.

16.

Studart

Gonzenbach

Tervoort

et al . Processing routes to macroporous ceramics: a review. J Am Ceram Soc 2006; 89(6): 1771–1789.

17.

Lin

Tadai

Takahashi

et al . An experimental study on measurement methods of bulk density and porosity of rock samples. J Geosci Environ Protect 2015; 3(5): 72–79.

18.

Iatsenko

Sych

Tomila

Effect of sintering temperature on structure and properties of highly porous glass-ceramics. Process Appl Ceram 2015; 9(2): 99–105.

19.

Dorokhova

Zhernovaya

Bessmertnyi

et al . Control of the structure of porous glass-ceramic material. Glass Ceram 2017; 74: 95–98.

20.

Mocciaro

Lombardi

Scian

AN.

Ceramic material porous structure prepared using pore-forming additives. Refract Ind Ceram 2017; 58(1): 65–68.

21.

Wan

Wang

The effect of porosity on the structure and properties of calcium polyphosphate bioceramics. Ceramics-Silikáty 2011; 55(1): 43–48.

22.

Rahaman

et al . Silicate, borosilicate, and borate bioactive glass scaffolds with controllable degradation rate for bone tissue engineering applications. I. Preparation and in vitro degradation. J Biomed Mater Res 2010; 1(95A); 164–171.

23.

Shuai

Cao

Dan

et al . Improvement in degradability of 58s glass scaffolds by ZnO and beta-TCP modification. Bioengineered 2016; 5(7); 342–351.

24.

Tanaka

Kumagae

Saito

et al . Bone formation and resorption in patients after implantation of β-tricalcium phosphate blocks with 60% and 75% porosity in opening-wedge high tibial osteotomy. J Biomed Mater Res B Appl Biomater 2010; 86B(2): 453–459.

25.

Gao

Peng

Feng

et al . Bone biomaterials and interactions with stem cells. Bone Res 2017; 5: 17059.

26.

De Aza

Peña

Luklinska

et al . Bioeutectic® ceramics for biomedical application obtained by laser floating zone method. In vivo Evaluation. Materials 2014; 7(4): 2395–2410.

27.

Makhaniok

Haranava

Goranov

et al . In silico prediction of the cell proliferation in porous scaffold using model of effective pore. Biosystems 2013; 114(3): 227–237.

28.

Tang

Mao

Liu

et al . Fabrication, characterization and cellular biocompatibility of porous biphasic calcium phosphate bioceramic scaffolds with different pore sizes. Ceram Int 2016; 42(14): 15311–15318.