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Macroporous bioceramic scaffolds based on tricalcium phosphates reinforced with silica: microstructural, mechanical, and biological evaluation Macroporous bioceramic scaffolds based on tricalcium phosphates reinforced with silica:...
Novotna, Lenka; Chlup, Zdenek; Jaros, Josef; Castkova, Klara; Drdlik, Daniel; Pospisil, Jakub; Hampl, Ales; Koutna, Irena; Cihlar, Jaroslav
2022-04-03 00:00:00
JOURNAL OF ASIAN CERAMIC SOCIETIES 2022, VOL. 10, NO. 2, 356–369 https://doi.org/10.1080/21870764.2022.2053278 FULL LENGTH ARTICLE Macroporous bioceramic scaffolds based on tricalcium phosphates reinforced with silica: microstructural, mechanical, and biological evaluation a b c,d a,e a,e c Lenka Novotna , Zdenek Chlup , Josef Jaros , Klara Castkova , Daniel Drdlik , Jakub Pospisil , c,d c,f a,e Ales Hampl , Irena Koutna and Jaroslav Cihlar a b CEITEC - Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; CEITEC - IPM, Academy of Sciences of the Czech Republic, Brno, Czech Republic; Faculty of Medicine, Department of Histology and Embryology, Masaryk University, Brno, Czech Republic; International Clinical Research Center - Center of Biomolecular and Cellular Engineering, St. Anne’s University Hospital Brno, Brno, Czech Republic; Faculty of mechanical engineering, Institute of Materials Science and Engineering, Brno University of Technology, Brno, Czech Republic; International Clinical Research Center – Cell and Tissue Engineering facility, St. Anne’s University Hospital Brno, Brno, Czech Republic ABSTRACT ARTICLE HISTORY Received 21 December 2021 The positive effect of silica on microstructural, mechanical and biological properties of calcium Accepted 11 March 2022 phosphate scaffolds was investigated in this study. Scaffolds containing 3D interconnected spherical macropores with diameters in the range of 300–770 µm were prepared by the KEYWORDS polymer replica technique. Reinforcement was achieved by incorporating 5 to 20 wt % of Bioceramics; scaffold; colloidal silica into the initial hydroxyapatite (HA) powder. The HA was fully decomposed into calcium phosphate; silica; alpha and beta-tricalcium phosphate, and silica was transformed into cristobalite at 1200°C. compressive strength Silica reinforced scaffolds exhibited compressive strength in the range of 0.3 to 30 MPa at the total porosity of 98–40%. At a nominal porosity of 75%, the compressive strength was doubled compared to scaffolds without silica. When immersed into a cultivation medium, the formation of an apatite layer on the surfaces of scaffolds indicated their bioactivity. The supportive effect of the silicon enriched scaffolds was examined using three different types of cells (human adipose-derived stromal cells, L929, and ARPE-19 cells). The cells firmly adhered to the surfaces of composite scaffolds with no sign of induced cell death. Scaffolds were non-cytotoxic and had good biocompatibility in vitro. They are promising candidates for therapeutic applications in regenerative medicine. 1. Introduction biomaterial scaffold and support vascularization of the Nowadays, many people face problems related to ingrown tissue. Pores must be interconnected, with bone disorders. Bone tissue is able to completely a pore size of minimally 100 µm in diameter (ideally regenerate on its own if the damaged part is small >300 µm) [7,8]. Besides such macropores, the micro- enough. If not, it is necessary to heal such trauma, porosity (<10 µm) of the struts is desirable because it e.g. by using bone grafts. Autografts, i.e. parts of provides a larger surface area, which is critical for bone harvested from the patient’s body, naturally protein adsorption, and adhesion and growth of cells have the most suitable properties, but some pro- [7,9]. Within few months the scaffold should resorb in blems, such as lack of available tissue material and the body environment. The resorption kinetics should the necessity of multiple surgical procedures, were ideally be equal to the bone turnover rate in order to reported [1]. Nonetheless, because the bone is facilitate load transfer directly to the newly developing the second most common transplanted tissue, the bone. The by-products of the body-scaffold interaction demand for bone grafts is huge – several million must not be toxic and should be easy to eliminate via people need them every year [2]. Hence, the devel- relevant body systems [10]. Also, mechanical proper- opment of a new type of synthetic graft, further ties should be similar to those of replaced bone, i.e. referred to as a scaffold, seems to be a promising compressive strength of cancellous bone is in the 11–13 choice [3,4]. range of approx. from 1 to 38 MPa [ ], and the The requirements on the synthetic scaffolds are scaffold must not collapse during handling and manifold [5,6]; the ideal scaffold must be biocompati- in vivo during normal physical activities. Scaffolds ble, i.e. must not elicit any inflammatory response and/ should be easy to manufacture in shapes, which accu- or demonstrate immunogenicity or cytotoxicity. It rately fit the defects in the bone. Hence, the intrinsic should support tissue formation by 3D structures structure, as well as the composition, play crucial roles with pores allowing cells to migrate throughout the in the clinical success of the scaffold. CONTACT Daniel Drdlik daniel.drdlik@ceitec.vutbr.cz CEITEC - Central European Institute of Technology, Brno University of Technology, Purkynova 123, Brno 612 00, Czech Republic © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The Korean Ceramic Society and The Ceramic Society of Japan. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. JOURNAL OF ASIAN CERAMIC SOCIETIES 357 Bioceramic materials based on calcium phosphates slurries were prepared. A silica-free slurry (as exhibit the greatest chemical similarity to the bone a reference) was prepared from HA powder (purity mineral component [14]. Their wide expansion into >90%, Fluka, Switzerland) bonded by 5 wt % polyvinyl clinical practice is, however, limited by insufficient alcohol (PVA, Mowiol 10–98, Sigma Aldrich, Germany), mechanical properties if they are prepared syntheti- deionized water, 0.2 wt % glycerol (Onex, Czech cally. The objective of this work was to develop a new Republic) and 0.1 wt % n-octanol (Lachema, Czech composite biomaterial with biological characteristics Republic). The second type was prepared by mixing and compressive strength similar to highly porous HA powder (purity > 90%, Fluka, Switzerland), colloidal hard tissues. Silica was chosen as the reinforcing silica solution LUDOX® SK-R (Grace, US) and deionized 4+ phase because silicon (as Si ion) is considered to be water. The weight fraction of HA in the slurry was in the one of the essential trace elements required for the range of 0.45 to 0.5. The coating process was repeated development of healthy bones. It acts as a biological if a lower porosity of the scaffolds was required. Slurry cross-linking agent in the extracellular matrix. residues were then gently removed from the surface of Moreover, it enhances osteoblast proliferation, differ - impregnated PU templates by compressed air to entiation, and collagen production [15,16]. Calcium achieve the desired calculated porosity. The scaffolds phosphate ceramics substituted by silicate ions exhibit prepared were dried at 25°C for 24 h. To burnout the superior biological properties compared to their stoi- PU template and achieve a sufficient manipulation chiometric counterparts [17]. Up to now, a great deal strength the scaffolds were calcined at 1000°C with of material research was focused on bioceramics con- a heating rate of 1°C/min. The scaffolds were finally 18– taining amorphous silica such as bioactive glasses [ pressureless sintered in air at 1200°C for 3 h with 21 22–25 ] (pseudo) wollastonite [ ], dicalcium silicate [26] a heating rate of 5°C/min and a cooling rate of 27–29 and Si-doped CaP [17, ]. 10°C/min. On the other hand, materials composed of crystal- line silica in the form of quartz or cristobalite for med- 2.2. Thermal, physical and structural ical applications were poorly studied so far. There are characterization of scaffolds only a few studies concerning bioactive composites composed of cristobalite and calcium phosphate Thermal analysis of the as-coated PU template was matrix such as dicalcium phosphates [30,31], tetracal- performed using a 6300 Seiko Instruments TG-DTA cium phosphate [32] or HA (reinforced with biogenic (Seiko Instruments, Japan). The specimen was mea- silica) [33]. Therefore, here we aimed to extend the sured at temperatures between 35 and 1000°C with knowledge on bioactive material composition based a heating rate of 2°C/min in a mixture of air and argon on silica – tricalcium phosphate (TCP/SiO ), where the (1:1); the flow rate was set to 400 ml/min. crystalline silica, in the form of cristobalite formed after The phase composition of HA and composites (5– sintering, plays a crucial role. 20 wt % SiO ) was determined via an X-ray powder In this study, the TCP/ SiO composite scaffolds diffractometer SmartLab 3 kW (XRD, Rigaku, Japan). were fabricated by the polymer replica technique. The diffraction patterns were measured from 15° to The silica content varied from 0 to 20 wt % and the 90° (2θ) with Cu Kα radiation. For this purpose, the effect of cristobalite, overall phase composition, sinter- sintered scaffolds were crushed into a fine powder ing temperature, pore size, and total porosity on which was subsequently analyzed. The phase content microstructural, mechanical and biological properties was quantified using the Rietveld analysis. The evalua- of tricalcium phosphate scaffolds were investigated. tion of the crystallographic structures and quantitative analyses were realized using the PDXL2 software. The morphology of sintered scaffolds was observed 2. Materials and methods using a scanning electron microscope (SEM, ZEISS Ultra Plus, Germany) equipped with an EDX analyzer (Oxford 2.1. Ceramic foam processing Instruments, UK). The scaffolds were embedded in Ceramic scaffolds were prepared by the polymer a resin, ground and polished by the standard ceramo- replica technique. This method was chosen for the graphic methods. To quantify the pore sizes and their manufacturing of the bioceramic scaffolds because it distribution, image analysis of SEM micrographs was accurately mimics a trabecular bone macrostructure. done using the ImageJ software (National Institutes of Polyurethane foam (PU) with initial pore sizes of 45, 60, Health, US). 75 and 90 PPI (Bulpren S 28133, S 28089, S 31062, The total porosity was calculated from the geo- S 31048, Eurofoam, Czech Republic) were cut into metric volume, mass and theoretical density according cylinders of ø 7.5 × 10 mm (for a compressive test) to EN 623–2:1993: and ø 5 × 2 mm (for biological testing). Subsequently, ρ ρ t b they were immersed into ceramic slurries containing P ¼ � 100 (1) HA with 0, 5, 10, 15 and 20 wt. % silica. Two types of 358 L. NOVOTNA ET AL. Table 1. Nominal ion concentrations (in mM) of MEM in comparison with SBF solution and human blood plasma [34,35]. + + 2+ 2+ − − 2− 2− Na K Mg Ca Cl HCO HPO SO pH 3 4 4 DMEM 155.3 5.3 0.8 1.8 119.3 44 0.9 0.8 7.4 MEM 144.4 5.3 0.8 1.8 126.2 26.2 1.0 0.8 7.4 SBF 142.0 5.0 1.5 2.5 147.8 4.2 1.0 0.5 7.4 Blood plasma 142.0 5.0 1.5 1.5 103.0 27.0 1.0 0.5 7.2–7.4 where ρ is the theoretical density and ρ is the bulk Healthcare, USA). It can be a better choice in terms of t b density. The bulk density is defined as: simulating the in vivo environment [35] because it contains, except the ionic composition like SBF (see ρ ¼ (2) Table 1), other components occurring in in vivo sys- tems (such as glucose, amino acids and vitamins). The where m is the mass of the dry test piece and V is the b b principle of bone-like apatite formation on scaffold total geometrical volume (the sum of the volumes of surfaces is analogous to that in SBF solution and can the solid material, the open and the closed pores). 35–37 be found elsewhere [ ]. Scaffolds were incubated in Additionally, the apparent density as the ratio between the medium for 3 days at 37°C under a humidified weight and geometrical volume for each analyzed atmosphere of 95% air and 5% CO . After the removal scaffold prior to testing was individually calculated to from the medium and rinsing with deionized water, allow a better understanding of the mechanical prop- the scaffolds were dried at 25°C. The presence of the erties observed. apatite layer on the surface was examined using SEM. 2.3. Mechanical testing – compressive strength of 2.5. Assaying biocompatibility in vitro: metabolic scaffolds activity of cells The compressive strength of prepared scaffolds was The viability and proliferation of cells on TCP and determined using an Instron 8862 electromechanically TCP/SiO scaffolds were assessed in vitro by MTT driven testing system (Instron, USA) of nominal capa- assay. Cytotoxicity tests for scaffolds were performed city 100 kN and equipped with a 5 kN load cell and according to the ISO 10993–5:2009(E) Biological eva- precise clip-gauge for the deformation measurement. luation of medical devices – Tests for in vitro cytotoxi- Cylindrical scaffolds of nominal dimensions after sin- city guidelines. Two standardized cell lines were used tering ø 6 mm × 8 mm were inserted between com- to determine the cytotoxicity of the materials: L929 pressive platens with 1 mm thick leather spacers used cells (NCTC clone 929: CCL 1, LOT: 70026472, for a proper load transfer from the steel platen to the American Type Culture Collection [ATCC], Manassas, scaffold. A cross-head speed of 0.5 mm/min was used VA, USA), and more sensitive ARPE-19 cells (ARPE-19: for the loading. The compressive strength was calcu- CRL-2302, LOT: 70013110, American Type Culture lated from the force corresponding with the first peak Collection [ATCC], Manassas, VA, USA). on the loading curve (force vs displacement) followed by a significant drop in applied force and scaffold 2.6. Assaying biocompatibility in vitro: dimensions. This approach leads to an estimate of the morphology of cells growing on scaffolds initial compressive strength of prepared scaffolds, which is important from the application point of Scaffolds containing 0 and 10 wt % of silica were view. Note that the determined strength here can be sterilized by UV-irradiation for 20 minutes in the flow slightly lower than the “effective” compressive box. Scaffolds were wet in DMEM-Glutamax (Life strength as determined from the plateau in the loading Technologies, Czech Republic) medium for 1 h and curve. A minimum of four scaffolds of the same pore centrifuged for 10 min to eliminate air bubbles from size, porosity and composition were measured. the material. Adipose-derived stromal cells (ADSCs) were isolated from adipose tissue by centrifugation and collagenase 2.4. Evaluation of bioactivity of scaffolds extraction, as described elsewhere [38]. Briefly, adipose The bioactivity potential, i.e. the bone-bonding ability, tissue was digested with 0.1% collagenase type IV for was studied by means of apatite formation on the 30 min at 37°C. After enzyme activity neutralization by scaffold surfaces. Instead of the typically used simu- DMEM-F12 (Life Technologies, Czech Republic) with lated body fluid (SBF) prepared following the Kokubo 10% fetal bovine serum (FBS), cells were separated by recipe [34], the epitaxial growth of apatite was studied centrifugation. The pellet was resuspended in cultiva- using Dulbecco’s Modified Eagle Medium – DMEM (GE tion medium (10% FBS, 0.5% penicillin/streptomycin JOURNAL OF ASIAN CERAMIC SOCIETIES 359 (GE Healthcare Life Sciences, USA) in DMEM Glutamax) of adsorbed water. At temperatures between 200°C and propagated on a culture dish coated with 0.01% and 550°C, the two-stage thermal decomposition gelatin. Subsequently, the cells were trypsinized and process of the PU foam template was observed. seeded on materials at a concentration of 50.000/ This decomposition behavior, typical of PUs, was 39–43 100 μL for analysis of cell viability and 1 million cells/ described in various studies [ ] as primarily 100 μL for evaluation of cell morphology. Scaffolds a polymer splitting process that begins at about were analyzed after 24 h of cultivation. 200°C. At this temperature, hard segments (related Due to the similar composition of scaffolds, the cell to urethane links) start to decompose, while viability was assessed on scaffolds with 75 PPI porosity the second step of degradation (350–550°C) is by fluorescent live/dead assay. The fluorescent stock solu- caused by oxidation of soft segments (related to tion was prepared by diluting 0.03% w/v of acridine the ether group) [44]. The exothermic peak on the orange and 0.1% w/v of ethidium bromide (both Sigma- DTA curve (see Figure 1) in the same temperature Aldrich, USA) into 2% ethanol in distilled water, with a final range confirmed that the degradation process of dilution of 1/1000 in 0.1 M phosphate buffer. The fluores - the PU occurred by an oxidation mechanism. cent solution was added to the specimens for 5 min 25°C Experimental data show that the weight loss of and live/dead cells were visualized using an epifluores - the specimen continued even above the tempera- cence microscope Cell^R (Olympus C&S Ltd., Japan). ture of 550°C, at which the PU was supposed to Cells cultivated for 24 h on tested specimens were have already burnt out. This was likely caused by fixed with 4% paraformaldehyde dissolved in 0.1 M the thermal transformation of the HA powder. The phosphate buffer (PBS) and permeabilized by 0.1% endothermic drop on the DTA curve around 800°C Triton TX-100 (Merck, Germany). Actin cytoskeleton was related to the thermal transformation of HA to was stained with 60 nM Phalloidin Rhodamine (R415, hydroxyoxyapatite (HOA) [45]. Lifetech, Czech Republic) dissolved in 0.1 M PBS, and The total weight loss of 15% below 1000°C corre- cell nuclei were stained with 1 μg/mL 4′,6-diamidino- sponded to the initial amount of PU in the composite, 2-phenylindole, (Sigma-Aldrich, USA). The specimens adsorbed water and weight loss of commercial HA were observed by the epifluorescence microscope caused by the reaction of secondary phase – monetite Cell^R (Olympus C&S Ltd., Japan). (see Chapter 3.2). 3. Results and discussion 3.2. Phase composition 3.1. Thermal analysis X-ray diffraction (XRD) patterns of sintered scaf- folds are shown in Figure 2. The commercial cera- TGA curves of the PU foam template coated with mic powder was composed of HA and monetite. HA powder reinforced with 10 wt % SiO (the The quantitative analysis showed about 13 wt % of overall weight of the system with respect to PU is monetite as can be seen in Table 2. In the first therefore as follows: PU (10 wt %), HA (81 wt %) instance, the HA was thermally decomposed to and SiO (9 wt %)) are given in Figure 1. The HOA with the following decomposition to the TCP minimum weight loss (~1%) at temperatures from phase according to the following equations [45]. 40 to 200°C was caused mainly by the evaporation Figure 1. Thermogravimetric analysis of commercial reticulated polyurethane foam coated with HA powder reinforced with 10 wt % SiO . 2 360 L. NOVOTNA ET AL. Figure 2. X-ray diffraction patterns of initial HA powder and TCP/SiO composite scaffolds containing 0–20 wt % SiO , sintered at 2 2 1200°C for 3 h. 1000°C. This phenomenon can be attributed to the Table 2. Quantitative analysis results for initial ceramic powder presence of impurities such as the monetite phase. and tricalcium phosphates reinforced with silica. Further increase in temperature led to the transforma- 0 wt % 5 wt % 10 wt 15 wt 20 wt tion of the β-TCP into the α–phase. Newly formed TCPs Material (wt %) powder SiO SiO % SiO % SiO % SiO 2 2 2 2 2 hydroxyapatite 87 are believed to be more soluble in the body fluid than monetite 13 stoichiometric HA [49]. α-TCP 42 52 55 55 54 The phase composition of scaffolds containing silica β-TCP 58 44 37 33 29 cristobalite 4 8 12 17 was much more complex. Besides the α- and β- TCP, a new crystalline phase was formed after scaffold sin- tering. With increasing concentration of silica, the intensity of new strong diffraction at about 21.7° and 2Ca ðPO Þ OH $ Ca ðPO Þ O ðOHÞ þ xH O; 0 5 4 10 4 x 2 3 6 2ð1 xÞ two weak diffractions at about 28.2° and 35.8° � x � 1 increased and they were identified as cristobalite (P4 (3) 2 2 space group). The weight percentage of cristoba- lite within crystalline phases was roughly equivalent to Ca ðPO Þ O ðOHÞ $ 3Ca ðPO Þ þ CaO 10 4 x 3 4 6 2ð1 xÞ 2 the amount of colloidal silica in the initial slurry as þð1 xÞH O (4) documented by XRD quantitative analysis in Table 2. The results of the XRD analysis also showed that the The monetite phase was most likely decomposed amount of the α and β TCP phases were almost equal during the sintering process to calcium pyrophosphate in the absence of silica. However, the ratio between α- according to decomposition reaction [46]: TCP and β-TCP significantly increased with the increas- 2CaHPO $ Ca P O þ H O (5) 4 2 2 7 2 ing amount of silica. The addition of silica significantly reduced the amount of β-TCP phase (from 58 to 29 wt Then the resulting calcium oxide and calcium pyropho- %, see Table 2) whereas the α-TCP phase has been sphate reacted to the TCP phase: fixed at around 55 wt %. This behavior can be attrib- Ca P O þ CaO $ Ca ðPO Þ (6) 2 2 7 3 4 uted to Si doping into α-TCP structure and formation of the most stable phase. Some studies [17,27,50] con- Therefore, the original ceramic powder (without silica) firmed that the addition of silica shifts the temperature was fully decomposed into α- and β-TCP in the scaffold of HA→ α-TCP transformation to lower values. The after sintering at 1200°C (see Figure 2). In the literature, stable α-TCP phase could be formed even during sin- there is a vast discrepancy in the temperatures at tering above 700°C [17,27,28,50,51]. It was further which the decomposition of HA starts. According to reported that HA sintered in the presence of silica many authors [47,48], HA should remain stable up to at transformed to silica-substituted tricalcium phosphate least 1300°C. In our experiments (data are not shown (Si-α-TCP) with formula Ca (P Si O ) [17,28] here), a commercial HA started to decompose at about 3 1-x x 4-x/2 2 according to the following equation [50] 800°C; almost half of the powder was transformed at JOURNAL OF ASIAN CERAMIC SOCIETIES 361 Table 3. Crystallographic parameters of pure and Si- Table 4. Pore sizes of the fabricated scaffolds. substituted calcium phosphate phases. Pore size (µm) Space Scaffold 45 PPI 60 PPI 75 PPI 90 PPI Phase Lattice parameters group Ref. 0 wt % SiO 710 ± 65 550 ± 70 540 ± 60 330 ± 30 measured α–TCP a = 12.878 Å; b = 27.291 Å; P2 /a – 5 wt % SiO 715 ± 70 450 ± 30 420 ± 40 300 ± 30 c = 15.257 Å; β = 126.32° 10 wt % SiO 720 ± 70 470 ± 30 440 ± 30 310 ± 30 measured Si–α– a = 12.856 Å; b = 27.337 Å; P2 /a – 15 wt % SiO 725 ± 80 480 ± 20 450 ± 30 315 ± 30 TCP (10 wt %) c = 15.218 Å; β = 126.35° 20 wt % SiO 770 ± 50 525 ± 30 490 ± 30 350 ± 30 measured β–TCP a = b = 10.4263 Å; c = 37.423 Å R3c – measured Si–β– a = b = 10.4381 Å; c = 37.4933 Å R3c – TCP (10 wt %) α–TCP a = 12.887 Å; b = 27.280 Å; P2 /a [52] viscosity of the slurry, by repeating the coating process c = 15.219 Å; β = 126.20° and, finally, by the efficiency of removing the extra Si–α–TCP (0.87 wt a = 12.875 Å; b = 27.372 Å; P2 /a [28] slurry by compressed air. If the total porosity was % Si) c = 15.225 Å; β = 126.30° Si–TCP a = 12.863 Å; b = 27.357 Å; P2 /a [50] lower than 50%, the macropores were almost closed c = 15.232 Å; β = 126.38° and the remaining pores were too small for efficient β–TCP a = b = 10.4352(2) Å; R3c [53] c = 37.4029(5) Å cell ingrowth. According to the Jodati review [56] an optimal porosity for osteogenesis appeared to be approximately 60%. In our case, the porosity seemed 6Ca ðPO Þ OHþ 2SiO ! 10Ca P Si O 5 4 2 3 1 x x 4 x=2 to be ideal in the range of 65–80% as regards the þ 3H O (7) scaffold morphology (interconnected macropores) and strength. If the porosity exceeded 90%, the PU Being of the same space group, the Si-substituted α- template was almost perfectly replicated, but such TCP can be distinguished from its stoichiometric coun- scaffolds were fragile with no sufficient manipulation terpart by different lattice parameters [17,28]. strength due to very thin struts. Measured and theoretical (α-TCP [52], Si-α-TCP An overview of the macropore sizes of scaffolds [28,50], β-TCP [53]) lattice parameters are compared reinforced with 10 wt % SiO prepared from PU foam in Table 3 (data shown for scaffold containing 10 wt % 2 templates with initial pore sizes of 45 PPI, 60 PPI, 75 SiO ). The obtained data of the lattice parameters PPI, and 90 PPI and having 75% porosity is shown in (showing an increase of the b-axis length and β- Table 4 and Figure 3. The pore size of sintered scaffolds angle) [27,28,54,55] indicate that our α-TCP was pre- was dependent on the initial pore size of the PU foam sumably substituted. Diffraction peak shifts of Si sub- template, the thickness of the struts and shrinkage stituted TCP compared to undoped TCP confirm the during their sintering. The most convenient pore size lattice parameter changes, as also evident from for the applications in tissue engineering, i.e. from 150 Figure 2. to 500 µm [9,57], was observed for scaffolds prepared The shift of diffraction patterns to lower angles and from the PU templates having a porosity of 60 to 90 change of the lattice parameter in c-axis (see Table 3) PPI, where the measured pore size was in the range indicates that β-TCP might also have been silica- from 300 to 550 µm. Such range is ideal for cell migra- substituted. This partial substitution may have tion as it was also reported in the work of Karageorgiou occurred by diffusion of silicate ions into the already et al. [7]. transformed β-TCP phase. Nevertheless, further mea- In terms of the microstructure of struts, the differ - surements are needed to confirm this assumption. ences between individual compositions were more significant (see Figure 4). Pure TCP scaffolds exhibited 3.3. Characterization of structure and high microporosity in the struts (based on their low morphology of scaffolds density <65 vol %) and overall higher geometrical dimensions (lower shrinkage) indicating insufficient Ceramic scaffolds were prepared in a wide range of particle packing during processing and sintering. The porosities and pore sizes. Porosity (40–98%) was easily scaffolds containing silica had noticeably lower tunable by the initial PPI of the PU template, by the Figure 3. Macrostructure of TCP/SiO composite scaffold (10 wt % SiO ) having 75 vol % porosity sintered at 1200°C/3 h; pore sizes 2 2 of the PU foam template from left to right: 45 PPI, 60 PPI, 75 PPI, and 90 PPI. 362 L. NOVOTNA ET AL. Figure 4. SEM cross-section micrographs of struts in scaffolds (top row – overall overview, bottom row – detailed images at higher magnification). Individual phases are marked by arrows (white areas – TCP, gray areas – cristobalite, black areas – micropores). microporosity in the struts. This can be attributed to colloidal silica particles, which filled the spaces between HA particles and transformed into the cristo- balite during processing and sintering. Its amount (the darker area) in Figure 4 grew proportionally with increasing initial silica content. For the lowest silica content, it was located in small areas near the grain boundaries. With increasing silica concentration, it formed a continuous network around the TCP grains. The grains were smaller in the presence of silica; prob- ably due to the grain boundary pinning effect when a low concentration of silica was present or suppressed diffusion at higher concentrations of silica. The micro- pores were open and interconnected in all tested scaf- Figure 5. Dependence of compressive strength on total por- folds. The size of the micropores in the struts varied osity for TCP and TCP/SiO composite scaffolds. between 0.5 and 20 µm. Microporosity can negatively influence mechanical properties, but it is essential for protein adhesion, cell migration, and osseointegration occurs in a relatively mechanically weak matrix. Ansari [58,59]. et al. [61] reported a more than threefold increase in tensile strength for 20 vol % of cristobalite in hydroxyl- terminated polydimethylsiloxane. In the work of Li 3.4. Mechanical properties et al. [62], the cristobalite enhanced the compressive The influence of the concentration of silica, i.e. pre- strength of geothermal geopolymer. However, the sence of the cristobalite in the microstructure, on the detail information about the influence of cristobalite compressive strength, was evaluated for scaffolds of on the mechanical properties of ceramic materials is various PU template pore sizes (45, 60, 75 and 90 PPI). still poorly discussed in the literature. Generally, in the The results are summarized in Figure 5. The compar- case of ceramics, especially hydroxyapatite or trical- ison of dependence of compressive strength of pure cium phosphate, a certain amount of glassy phase TCP and TCP/SiO composites on their total porosity is may be advantageous, since the glass is expected to presented. Not surprisingly, the compressive strength have a positive effect on the sintering behavior, densi- increased exponentially with decreasing porosity by fication and mechanical properties of the composite two orders of magnitude from 0.3 MPa to almost 30 with respect to the original bioceramics [63]. Our MPa. The shift in the composite strength (gray sym- results support this statement. bols) to higher values compared with the pure TCP Due to the elimination of the influence of the total foams (white symbols) for the same densities is distin- porosity (as a parameter having the most significant guishable, reaching more than threefold enhance- effect) on the compressive strength, a new set of scaf- ment; however, the scatter of compressive strength folds having the same porosity were prepared. values is significant. Such difference can be attributed A detailed view of the influence of the microstructure to the presence of cristobalite in the microstructure composition and the template pore size on the com- (see Figures 2 and 4). It was reported that the cristo- pressive strength of the optimized scaffolds at balite present in the tough matrix rather deteriorate a nominal porosity of approximately 75% is given in mechanical properties [60]. The opposite phenomenon Figure 6. The trend in the obtained data is reaching the JOURNAL OF ASIAN CERAMIC SOCIETIES 363 Figure 6. Compressive strength at 75% porosity for TCP and TCP/SiO composite scaffolds prepared from initial PU foam templates with pore sizes: 45 PPI, 60 PPI, 75 PPI, and 90 PPI. maximum (in the range of 1–3 MPa) for scaffolds hav- One of the requirements imposed on the materials ing initially 10 wt % content of silica and a typical used in tissue engineering are properties similar to those macropore size of 440 µm. A similar limit was reported of replaced tissues. The reported compressive strength of by Oktar and Göller [64] for a glass-reinforced HA. cancellous bone lays between 1.5 and 38 MPa [13], typi- A closer look at each material composition suggests cally 2–20 MPa [12]. Therefore, the strength-porosity rela- that smaller initial pores resulted in higher compres- tionship of TCP/SiO scaffolds indicates that the optimal sive strength. However, this behavior takes place up to strength for the bone tissue replacement can be reached the initial 10 wt % content of silica. The opposite trend using an optimized preparation method with 10 wt % of was observed for scaffolds containing 15 and 20 wt % silica, as was demonstrated here. of silica. From the mechanical strength point of view, the porosity was significantly reduced by the SiO addition to the concentration of 10 wt % silica offering 3.5. Biological properties – bioactivity the best ratio between reduced overall porosity in the assessment in Dulbecco’s Modified Eagle Medium struts and only isolated cristobalite structure, as can be The bioactivity of prepared scaffolds was evaluated seen in Figure 4. This explains the maximum strength using the immersion test in a DMEM. Figure 7 presents achieved. Since the higher concentration of the initial an overview of the surface morphology of the scaffolds silica (15 and 20 wt %) in the scaffold led to the before and after soaking in the medium. After 3 days, formation of the cristobalite interconnected network almost the entire surface of scaffolds was covered with around the TCP grains, the scaffolds became more a newly formed apatite layer. This layer, nucleated brittle. Figure 7. Surfaces of TCP and TCP/SiO composite scaffolds before and after 3-day incubation in MEM. 2 364 L. NOVOTNA ET AL. under in-vivo-simulated conditions, indicates good results further revealed that neither composition nor bioactivity, i.e. bone-bonding ability, of all prepared pore size had significant effect on the cell viability. The scaffolds. viability of all tested samples was above the 70% via- 65–67 According to the literature [17, ], silica incorpo- bility threshold and within a 15% standard deviation rated into the ceramic structure containing calcium range from the negative control. That means that no phosphates can enhance the biomimetic precipitation tested scaffold has proven any cytotoxic potential. on the surface of specimens immersed in the simulated body fluid. This can happen by two mechanisms. First, silicon can promote biomimetic precipitation on Si-α- 3.7. Biological properties – morphology of cells TCP by the higher solubility of the material due to growing on scaffolds defects in the lattice [17,68,69]. Second, the higher Human ADSCs were seeded into calcium phosphate biological activity can be influenced by the surface scaffolds to further test capability of material to sup- charge, which is here negative due to the substitution port cell growth. These cells are well suited for analyz- 4- 3- of SiO for PO ions, and can facilitate surface adhe- 4 4 ing clinically relevant cell–material interaction because sion leading to the rapid biomimetic precipitation of their human origin, non-cancerous nature, and mul- [17,70]. The bioactive behavior of some types of sili- tilineage differentiation potential. At 24 hours after con-based ceramics was described in numerous stu- seeding, majority of cells on all specimens exhibited dies [17,25,71,72]. green fluorescence signal, indicating their viability, with only few cells (less than 1%) being dying/dead, as demonstrated by red fluorescence signal. Such pro- 3.6. Biological properties – metabolic activity of portions are typical for in vitro cultured cells, so that cells ADSCs obviously did not undergo extensive material- The potential cytotoxicity of TCP and TCP/SiO scaf- induced cell death. Such finding was typical for all the folds of different pore sizes were assessed in vitro by materials examined here (TCP and TCP/SiO with 10 wt MTT assay. MTT test is routinely used for measurement % of silica) (see Figure 9). of viability and proliferation of standardized cell lines Besides viability, morphological features of in vitro by colorimetric assessment of the metabolic ADSCs have been also studied to evaluate behavior activity of the cells. Figure 8 shows the MTT assay of cells growing on calcium phosphate scaffolds. results for scaffolds of three different pore sizes (60, As determined by visualizing nuclei and cytoskele- 75 and 90 PPI) containing 0 and 10 wt % of silica. As it is tal elements, the cells adhered and became evenly evident from the scaffold interactions with cells of distributed on internal surfaces of the scaffolds, both lines L929 and ARPE-1 (see Figure 8), all scaffolds with fully respecting details of scaffold morpholo- indicated similar or higher cell viability compared to gies. The cells grew in monolayer similarly to stan- cells growing under standard 2D conditions. The dard 2D culture in Petri dish, with producing Figure 8. Relative viability of tested cell lines ((a) L929 cell line; (b) ARPE-19 cell line) when compared to the non-treated negative control cells. Positive controls were treated with medium with added DMSO (percentage values in brackets refer to percentage of DMSO in medium). Viability of all tested samples was above the 70% viability threshold and within 15% standard deviation range from the negative control. No tested sample have proven any cytotoxic potential. JOURNAL OF ASIAN CERAMIC SOCIETIES 365 Figure 9. Cell viability assessment after 24 hours of cultivation. Cells seeded on scaffolds exhibited green fluorescence, indicating viable cells (merged images of red and green fluorescence) for both materials: (a) TCP, and (b) TCP/SiO composite scaffold (10 wt % SiO ). protrusions (filopodia) in some parts of scaffolds. pores (porosity 90 PPI, both TCP and TCP/SiO , These protrusions, indicating active interaction green arrows in Figure 10. Also importantly, the with materials, were mainly detected on scaffolds cells were void of blebbing of their cytoplasmic with larger pores (45 and 75 PPI), as marked in the membranes, and they had regularly shaped figures by white arrows (see Figures 10 and 11). It nuclei and well-developed network of actin, is of note that filopodia were more frequently seen underlying their vital contacts with the support- in TCP/SiO materials; further analyses of cell beha- ing scaffold. vior (e.g. proliferation and formation of focal adhe- From the above described findings made using sion) should be performed for more detailed ADSCs, we conclude that our newly developed materi- characterization. als have no adverse effects on normal human cells, and Another significant phenomenon was penetra- instead they behave highly supportive so that they tion of cells through the pores and forming cell may represent a proper technological step toward sheets, typically seen in the scaffolds with small clinical application. Figure 10. Morphological features of the cells grown in TCP scaffolds with initial porosity of (a) 45 PPI, (b) 75 PPI, (c) 90 PPI, and TCP/SiO (10 wt % SiO ) composite scaffolds with initial porosity of (d) 45 PPI, (e) 75 PPI, and (f) 90 PPI. Red color stains the actin 2 2 and the blue visualizes the chromatin. White arrows indicate the formation of filopodia, green arrows indicate cells forming sheets over the scaffold pores. 366 L. NOVOTNA ET AL. Figure 11. Detail representation of cellular adhesion and spreading of adipose-derived stromal cells on TCP scaffolds with initial porosity of (a) 45 PPI, (b) 75 PPI, (c) 90 PPI, and TCP/SiO (10 wt % SiO ) composite scaffolds with initial porosity of (d) 45 PPI, (e) 75 2 2 PPI, and (f) 90 PPI. White arrows indicate the formation of filopodia. 4. Conclusions osseointegration. The compressive strength increased exponentially with decreasing porosity by two orders In this work, scaffolds based on calcium phosphates of magnitude from 0.3 MPa to almost 30 MPa. The were prepared by the polymer replica technique. The presence of cristobalite in the composite scaffold pure and silica-rich (5–20 wt %) scaffolds with a 3D structure led to a twofold increase in the compressive interconnected porosity (40–98%) were fabricated by strength (1–3 MPa) compared to pure calcium phos- templating the polyurethane foam with initial pore phate scaffolds at a nominal porosity of 75%, which is sizes of 45, 60, 75 and 90 PPI. The XRD analysis showed in the range of the cancellous bone. The presence of the total decomposition of the HA into α-TCP and β- cristobalite in the structure after sintering did not TCP after sintering. Also, the strong diffraction of cris- negatively affect the biological properties of scaffolds. tobalite was measured with an increasing concentra- In vitro tests demonstrated that all the scaffolds pre- tion of silica in original scaffolds. It was proved that the pared were bioactive, as evidenced by the formation of silica significantly contributed to the phase transforma- an apatite layer on the surface after 72 h immersion in tion of HA to α-TCP. Moreover, the obtained data of the the medium. The scaffolds containing 0 and 10 wt % lattice parameters indicate that α-TCP had presumably silica were not cytotoxic as demonstrated by MTT been substituted by Si ions. Macropore dimensions of assay. The content of 10 wt % silica may even be sintered scaffolds (between 300 and 770 µm) were beneficial to cells as it was indicated by cell viability dependent on the pore size of replicated templates. assay using adipose-derived stromal cells. Such bene- The measured average pore size of 300–550 µm in ficial effect was maximally pronounced in materials scaffolds prepared from the PU templates having a 60 with the smallest pores (90 PPI). Overall morphology to 90 PPI porosity was considered ideal for cells pene- of adipose-derived stromal cells growing on materials tration based on available literature data. In terms of confirmed their proper supportive action. the struts microstructure, the scaffolds containing cris- Collectively, concerning phase composition, micro- tobalite had a noticeably higher density of struts with structure, compressive strength and biological proper- micropores in the range of 0.5 to 20 µm, which is essential for protein adhesion, cell migration and ties, the promising candidates for potential application JOURNAL OF ASIAN CERAMIC SOCIETIES 367 in bone tissue engineering are TCP/SiO scaffolds hav- [10] Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials. 2000;21(24):2529–2543. ing 60–80 vol % porosity. [11] Carter DR, Hayes WC. Bone compressive strength: the influence of density and strain rate. Science. 1976;194 (1):174–1176. Acknowledgments [12] Olszta MJ, Cheng X, Jee SS, et al. Bone structure and formation: a new perspective. Mater Sci Eng R Rep. This work was supported by the Ministry of Education, Youth 2007;58(3–5):77–116. and Sports under Grant COST LD14072; Czech Health [13] Alessandro R, Brucato V, Rimondini L, et al. I materiali Research Council under Grant NU20-08-00402; Ministry of biocompatibili per la medicina [Biomaterials for med- Education, Youth and Sports under Grant CZ.02.1.01/0.0/0.0/ icine]. Palermo: Atti del Convegno Nazionale della 16_026/0008451; Czech Science Foundation under Grant Società Italiana Biomateriali; 2014. Italian. p 15–18. GA18-05510S; Ministry of Education, Youth and Sports under [14] Lei C, Cao Y, Hosseinpour S, et al. Hierarchical Grant CZ.02.1.01/0.0/0.0/15_003/0000492 and Ministry of dual-porous hydroxyapatite doped dendritic meso- Education, Youth and Sports under Grant LM2018110. porous silica nanoparticles based scaffolds promote osteogenesis in vitro and in vivo. Nano Res. 2020;14 Disclosure statement (3):770–777. [15] Nikom J, Charoonpatrapong-Panyayong K, No potential conflict of interest was reported by the author(s). Kedjarune-Leggat U, et al. 3D interconnected por- ous HA scaffolds with SiO2 additions: effect of SiO2 content and macropore size on the viability of Funding human osteoblast cells. J Biomed Mater Res A. 2013 Aug;101(8):2295–2305. This work was supported by the Agentura Pro Zdravotnický [16] Lodoso-Torrecilla I, Klein Gunnewiek R, Grosfeld EC, Výzkum České Republiky [NU20-08-00402]; Ministerstvo et al. 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