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Osteoblastic cells colonization inside beta-TCP macroporous structures obtained by ice-templating
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Osteoblastic cells colonization inside beta-TCP macroporous structures obtained by ice-templating E. Meurice a , F. Bouchart a , J.C. Hornez a , A. Leriche a,∗ , D. Hautcoeur b , V. Lardot b , F. Cambier b , M.H. Fernandes c , F. Monteiro d a Laboratoire des Matériaux Céramiques et Procédés Associés (LMCPA), UVHC, Pôle Universitaire de Maubeuge, Boulevard Charles de Gaulle, F-59600 Maubeuge, France b Belgian Ceramic Research Centre (BCRC), Avenue Gouverneur Cornez, 4, B-7000 Mons, Belgium c Laboratory for Bone Metabolism and Regeneration, Faculdade de Medicina Dentária, Universidade do Porto, Rua Manuel Pereira da Silva, 4200-393 Porto, Portugal d INEB,Universidade do Porto, Rua do Campo Alegre, 823,4150-180 Porto, Portugal
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Article history: Received 17 August 2015 Received in revised form 12 October 2015 Accepted 21 October 2015 Available online xxx Keywords: Beta-tricalcium phosphate Ice-templating Scaffolds Orientated porosity Osteoblastic cell Colonisation
a b s t r a c t A casting method based on ceramic slurry ice-templating was used to create beta-tricalcium phosphate anisotropic oriented macroporous scaffolds. The freeze-casting method produces tubular interconnected pores with ellipsoidal shape. The tubular porosity structure is supposed to enhance cell invasion. Several samples with pore size ranging from 50 m to 350 m and porosity from 30% to 70% were synthesized using different processing parameters: dry matter and binder content of slurry and freezing rate. The osteoblastic cell colonisation into these samples was compared to that into classical scaffolds obtained from ceramic slurry impregnation of organic skeleton which allows the development of isotropic spherical porous structure with 250 to 600 m pore diameter size. The analysis by scanning electron microscopy of cell invaded samples shows that the size of the pores of the ice-templated scaffolds is sufficiently large to enable fast osteoblastic cell colonisation. © 2015 Published by Elsevier Ltd.
1. Introduction For several decades, hydroxyapatite and -tri-calcium phosphate have been used as substitutes for replacing bone defects in bone and joint surgery because their mineral composition is similar to bone and exhibits a very high bio-activity [1,2]. Compared to hydroxyapatite, -tri-calcium phosphate (-TCP) is more resorbable and can be rapidly replaced by new bone tissue [3,4]. To achieve a complete bone tissue reformation, bone cells, including osteoblast and endothelial cells, have to colonize the ceramic scaffolds which must present a porous structure favourable to cell invasion inside the total material volume and not only on the surface. To this purpose, several methods have been developed to mimic bone macroporous structure, including ceramic foams [5], solid reaction [6,7], ceramic slurry impregnation of polymer porosifier skeletons (sponge, PMMA bead scaffold, . . .) [8,9], ice-
∗ Corresponding author at: LMCPA-UVHC, Pôle Universitaire de Maubeuge, Boulevard Charles de Gaulle, Maubeuge F-59600, France. Fax: +33 327531667. E-mail address: [email protected] (A. Leriche).
templating [10] and additive manufacturing [11]. The last two methods produce oriented elongated and interconnected pores that could favour the cell colonisation and help endothelial cells to form the tubular structures required to capillary formation. This work is mainly devoted to test scaffolds obtained by the casting method based on ice-templating developed a few years ago, to create different morphological porous structures [10–12]. However, by this technique, the pore size is limited to a few tenth of a micron which could be deleterious to cell colonisation. Indeed, many investigations carried out on calcium phosphate macroporous materials presenting various porous architectures and different resorption rates and implantation sites and times suggested that cell colonization and bone ingrowth apparently occur if macropore size is greater than 100–150 m [13–17] with an interconnectivity between pores greater than 50 m [18]. In this work, the -TCP scaffolds obtained by ice-templating method was compared to commercially used scaffolds synthesized by slurry impregnation of organic bead skeleton in order to verify if the pore size of ice-templated scaffolds is large enough for cell colonisation.
http://dx.doi.org/10.1016/j.jeurceramsoc.2015.10.030 0955-2219/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: E. Meurice, et al., Osteoblastic cells colonization inside beta-TCP macroporous structures obtained by ice-templating, J Eur Ceram Soc (2015), http://dx.doi.org/10.1016/j.jeurceramsoc.2015.10.030
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Fig. 1. Schematic illustration of the freeze-casting equipment.
2. Materials and methods 2.1. ˇ-TCP powder synthesis The -TCP powder was prepared by aqueous precipitation technique using diammonium phosphate and calcium nitrate solutions following a method described elsewhere [9]. In this work, the specific surface area of calcined and ground -TCP was 6.2 m2 /g and the ultimate particle size ranged from 1 to 2 m with d50 = 1.5 m. 2.2. Processing of macroporous ceramic scaffolds 2.2.1. Scaffolds prepared by slurry impregnation of organic bead skeleton Polymethylmethacrylate (PMMA) beads (DiakonTM Ineos Acrylics, Holland) with different diameter sizes (200, 300, 400, 500, 600, 700 and 800 m) were chemically stuck together in order to manufacture an organic scaffold in the shape of a block. This chemical welding was ensured by aceton which causes a slow superficial dissolution of polymer and allows the formation of bridges at the contact points between beads. Voids between polymeric beads were then filled by an aqueous -TCP powder suspension. After drying of the samples in a plaster mould, a debinding treatment was carried out at low temperature (220 ◦ C during 30 h) to eliminate the porogen agent (PMMA). The residual organics were then eliminated by heating at 400 ◦ C during 5 h. After debinding treatment, samples were sintered at 1115 ◦ C during 3 h in order to consolidate the ceramic walls up to a relative density equal or higher than 99%. This value was measured by mercury porosimetry. The porosity of the as-obtained scaffold is closely controlled in terms of shape and size: the spherical pore size was determined by the PMMA bead diameter and the interconnection size between the pores by the neck size between PMMA beads. For this study, 5 samples were prepared with fixed grain size distribution: B250 formed from a mixture of 200 and 300 m diameter size beads, B350 from 300 and 400 m diameter size beads, B450 m from 400 and 500 m diameter size beads, B550 m from 500 and 600 m diameter size beads and B750 m from 700 and 800 m diameter sizes. 2.2.2. Scaffolds prepared by ice-templating The -TCP slurries were prepared by mixing distilled water and -TCP powder with a small quantity (2.25 wt.% based on powder content) of ammonium polymethacrylate (Dolapix CE64, from Zschimmer and Schwarz, Germany). Powder content ranged from 20 to 40 vol%. Slurries were ball milled for 24 h with zirconia balls to obtain better deagglomeration. After sieving to remove zirconia balls, the binder, polyethylene glycol (Mw = 1000 mol g−1 , from Merck, Germany) was added (1.5 < wt% <6). Slurries were casted into a cylindrical PTFE mould (60 mm outer diameter and 50 mm long) and placed in the freezing equipment shown in Fig.1. Such a mould allows obtaining 3 samples with a diameter of 8 mm (for
biology tests) and one sample of 27 mm (for other analyses) before sintering. A slow cooling rate (1 or 5 K/min) was used with this device. A classic device was also used to obtain samples at rapid cooling rate (estimated to about 20 K/min), using nitrogen liquid as cooling agent [12]. The frozen samples were put into a freeze dryer (HETO CD8, from Heto Lab Equipment) for 24 h to remove ice at a pressure of 10 Pa. All green bodies were sintered with a constant heating rate of 5 K/min and kept during 2 h at 1100 ◦ C, followed by cooling rate of 5 K/min. Porosity and bulk density were measured by hydrostatic weighing (Archimedes’ method). Pore sizes were measured using image analysis from SEM pictures. For each composition, 10 micrographs were used and more than 100 pores were measured. The analysis was performed at 2 mm from the sample upper side. 2.3. Cellular colonization tests All tests were carried out with MG63 human osteoblastic cells. The MG63 cells were grown until 80% of confluence in a alpha-MEM culture medium containing 10% foetal bovine serum, 100 g/mL penicillin, 2.5 g/mL fungizone, and 50 g/mL ascorbic acid, at 37 ◦ C, in a humidified atmosphere of 5% CO2 in air. For a subculture, the cell monolayer was washed twice with a phosphate-buffered saline solution (PBS) and incubated with a trypsin-EDTA solution (0.05% trypsin, 0.25% EDTA) for 5 min at 37 ◦ C to ensure cell detachment. The -TCP samples 6 mm of diameter, 2 mm of high were placed in 48-cell culture plates and seeded with 105 /cm2 of cells. Cells colonization of the -TCP samples was followed by MTT coloration and scanning electron microscopy (SEM) after 1, 4 and 7 days. The MTT tests were performed by adding MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) at 0.5 mg/mL to the cultured samples, during 3 h at 37 ◦ C. Viable cells reduced the MTT to a blue formazan product. Colonization of the -TCP samples was observed with a stereomicroscope and digital camera Lan Optics. For SEM observation, the colonized -TCP samples were washed once in PBS and once in sodium cacodylate 0.14 M solution. They were then dehydrated by a ethanol gradient from 70% to 100%, followed by a gradient of EtOH/hexamethylsylanate from 50% to 100%. Samples were coated by sputtering with an Au/Pd thin film, using a SPI Module Sputter Coater equipment. The SEM/EDS examination was performed using a High resolution (Schottky) Environmental Scanning Electron Microscope with X-Ray Microanalysis and Electron Backscattered Diffraction analysis, (Quanta 400 FEG ESEM / EDAX Genesis X4 M). 3. Results and discussion 3.1. Characterisation of macroporous scaffolds Macroporous structures developed using the two methods are very different as it can be seen in Fig. 2. Scaffolds prepared from impregnation of organic bead skeleton by ceramic slurry present spherical pores homogeneously distributed in space with several interconnecting spherical holes, whereas the ice-templated scaffolds present z-axis oriented elongated (Fig. 2B) and ellipsoidal shaped pores with randomly orientation in x–y plan (Fig. 2C). Five samples presenting a total porosity volume of about 65% with different pore sizes from 250 m to 600 m were prepared by slurry impregnation of PMMA bead skeleton. An example of diamond saw cut scaffolds obtained from a mixture of 500 and 600 m PMMA beads is presented in Fig. 3. It can be seen that
Please cite this article in press as: E. Meurice, et al., Osteoblastic cells colonization inside beta-TCP macroporous structures obtained by ice-templating, J Eur Ceram Soc (2015), http://dx.doi.org/10.1016/j.jeurceramsoc.2015.10.030
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Fig. 2. SEM micrography of surface of ceramic slurry infiltration of organic beads skeleton (A) and of vertical (B) and cross (C) section of -TCP ice-templated scaffold.
Fig. 3. Micrograph of fractured scaffold prepared from slurry impregnation of a 500–600 m size PMMA bead skeleton.
the initial diameter of the pore is reduced after sintering from 500 to 600 m down to around 400 m with spherical interconnection ranging between 45 and 65 m. All scaffolds present spherical pores homogeneously distributed in space with several interconnecting spherical 50 m holes. As described before, two types of ice-templated samples were obtained, one with a large diameter and 3 others one with a small diameter. Fig. 4 shows SEM micrographs of each type of sample. It has to be noted that the structure was kept identical by changing the diameter. Cross section perpendicular to the freezing direction allows observing pores with an elliptical shape (Fig. 4). It is well known that this shape is characteristic of freeze casting by ice templating [10]. In the other direction, parallel to the freezing direction, a lamellar morphology with elongated pores was observed (Fig. 5A). As described in the literature [10], a denser part was observed at the bottom of each sample. This heterogeneous part was removed. A greater magnification on the lamellar allows the observation of the wall porosity (Fig. 5B). As noted by several authors, ceramic walls present a residual porosity [19,20] which could positively influence the biological response [21]. Table 1 summarizes the results obtained for the two sample diameters. These data correspond to a freezing rate of 1 K/min, a powder content of 30% vol. and a binder content of 3 wt%. In both cases, the results are very close. The large diameter size sample presents a mean pore size of 42 m and 240 m for the small (a) and large (b) diameter of the elliptic pore and the small diameter size sample presents a mean pore size of 43 m and 260 m for the
small and large pore diameters. The thickness of the lamellar walls and the porosity of the sample were kept at of about 40 m and 54%, respectively. It can be concluded that the sample size change does not affect the porosity characteristics. Characteristics of the seven samples prepared by the ice-templating method using different process parameters are presented in Table 2. Each parameter influences the size of the pores, which allow us to control the structure. For instance, a higher freezing rate results in obtaining a finer structure without changing the porosity. The pore sizes are ranging from 40 to 6 m and 200 to 13 m for the small and large diameters, respectively and from 34 to 7 m for the lamellar thickness. This can be clearly observed in Fig. 6A and B presenting the same magnification of micrographs following x–y plane of samples synthesized at 5 and 20 K/min freezing rate, respectively. The decrease of pore size versus freezing rate increase confirms the previous results obtained for freeze casted hydroxyapatite by Deville et al. [10] which showed a decrease from 600 m down to 150 m with cooling rate increase from 1 up to 10 K/min. For the suspension parameters, two processing conditions were modified: slurry dry matter content and binder content. The dry matter content strongly influences the porosity level which decreases from 67 to 36 % when the dry matter content increases from 20 up to 40 %. Simultaneously, the pore size decreases from 329 to 149 m for b axis values (comparison between IT6, IT5 and IT7) and no relevant influence was noted on the wall thickness. The binder content increase from 1.5 to 6 wt% (comparison between IT4, IT1 and IT5) does not influence the global porosity volume which remains about 50 vol.% but simply allows a better sample handling. The ice-templating method allows obtaining a larger range of porosity level than the slurry impregnation of PMMA bead scaffold with a minimum value of 36% for IT7 sample and maximum value of 67% for IT6 sample, the porosity value being always around 65% for the PMMA bead method. However, the highest porous scaffold IT6 has been disintegrated very rapidly in physiological medium during the cell impregnation tests. The value of the small diameter of the pores in the structures obtained by ice-templating (35–50 m) corresponds to the size of the interconnection between the spherical pore of scaffolds prepared from the impregnation of PMMA beads which could lead to a good cell invasion. 3.2. Cell colonization inside the ceramic scaffolds To perform colonisation experiments, MG63 osteoblasts from human cell line were used. All scaffolds obtained by the two previously described methods were as supports for osteoblast growth during 1, 4 and 7 days and cell invasions were checked by optical microscopy and by SEM. The micrographs of cut samples after colonization tests are presented in Fig. 7 for the ice-templated scaffolds and Fig. 8 for slurry impregnated PMMA bead skeleton scaffolds. It is observed by optical microscopy that MG63 osteoblasts characterized by having 50 m length and a 10 m width (black points
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Fig. 4. Micrographs of different size ice-templated samples prepared with the same processing parameters (A): large size sample (27 mm Ø) et (B) small size sample (8 mm Ø).
Fig. 5. (A) SEM image showing the lamellar morphology parallel to the freezing direction; (B) SEM image showing the porosity of ceramic lamellars.
Table 1 Porosity characteristics of ice-templated samples of different diameters. Sample Large 27 mm Ø Small 8 mm Ø
Pore size (m) a
b
42 ± 14 43 ± 14
240 ± 150 260 ± 100
Lamellar thickness (m)
Total porosity (%)
40 ± 18 38 ± 16
54 ± 1 55 ± 1
Table 2 Porosity characteristics of ice-templated samples synthesized from different process parameters. Sample
Conditions
IT 1 IT 2 IT 3 IT 4 IT 5 IT 6 IT 7
30%/3%/1 K/min 30%/3%/5 K/min 30%/3%/20 K/min 30%/1.5%/1 K/min 30%/6%/1 K/min 20%/3%/1 K/min 40%/3%/1 K/min
Pore size (m) a
b
41 ± 12 37 ± 14 6±2 54 ± 16 53 ± 17 67 ± 27 44 ± 11
209 ± 80 198 ± 110 13 ± 5 366 ± 200 277 ± 160 329 ± 140 149 ± 60
Lamellar thickness (m)
Porosity (%)
34 ± 14 38 ± 13 7±3 52 ± 15 37 ± 13 39 ± 18 43 ± 29
51 ± 1 52 ± 1 53 ± 1 51 ± 1 50 ± 1 67 ± 1 36 ± 1
Fig. 6. Micrographs of the ice-templated scaffolds prepared at different process parameters: (A) IT2: 30% Dry matter, 3% binder, 1 K/min freeze rate (B) IT3: 30% Dry matter, 3% binder, 20 K/min freeze rate showing the pore size decrease with freeze rate increase.
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Fig. 7. Micrographs of cut ice-templated samples by optical microscopy and SEM.
on micrographs) are able to colonise the surface of all samples independently of the preparation method of scaffolds. The tests show that the porosity size larger than 100 m of ice-templated samples is able to let the cells invade into the pores. Indeed, it is clear that the cells are not able to enter into the scaffold IT3, presenting smaller pores, even after 7 days. This is confirmed by the SEM image of the IT3 sample surface presented in Figs. 7 and 9E showing that cells have colonized the surface of the scaffold and appeared to be larger than the pore diameter and unable to enter into the material. This is in agreement with a pore size limit for cell colonisation higher than 100 m announced in the literature [13–17]. In the case of scaffolds prepared by slurry impregnated PMMA beads
method, all of them are colonized by the cells, as shown in Fig. 8. A different invasive behavior between the two different types of scaffolds can be outlined: in the tubular porous structure, the cells penetrate individually into the scaffolds whereas in the spherical porous structure, the cells are grouped and very few isolated cells are observed. It is difficult to quantify the cell invasion depth but a rough estimation shows the presence of cells only inside the first rank of pores for the spherical macroporosity, which corresponds approximately to 150 m in the B250 sample after 7 days, whereas the cells penetrate down to 250 m for columnar porosity in the IT1 ice-templated sample after the same duration. Scanning Electron Microscopy (SEM) performed on these samples confirms the
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Fig. 8. Micrographs of cut slurry impregnated PMMA beads samples by optical microscopy and SEM.
Fig. 9. SEM of ice-templated sample cultured with MG63 cells for 4 days. (A) Surface of IT1 sample with cells on surfaced (arrows) and inside the pores (circle); (B) high magnification image of cut IT1 with cells inside the pores; (C) homogeneous cell spreading on IT2 sample surface; (D) high magnification image of IT2 sample surface displaying the close interaction between cells and material surface; (E) surface of IT3 sample showing that individual cells were larger that the pores.
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results obtained by optical microscopy: cells seem healthy and are able to colonize surface (Fig. 9A, arrows) and pores. Comparison of different sample cross sections after 4 and 7 culture days reveals that cell invasion inside the ceramic structure is stopped for all samples because of cell proliferation on the surface which obstruct the pores. To compare the influence of the ceramic substrate architecture for longer incubation times, it would be necessary to perform tests with continuous medium flow. The SEM picture of IT2 sample after 4 days, presented in Fig. 9C shows a homogeneous cell spreading on the scaffold surface and an invasion inside the scaffold with sticking on the microporous ceramic walls. The cells are able to develop cytoplasmic filaments between the ceramic walls in ice-templated samples (circle in Fig. 9A). On the contrary, such a behavior is not observed in slurry impregnated PMMA beads scaffolds. To explain why the cytoplasmic extension phenomenon was observed only in ice-templated porous structures, it is suggested that the samples prepared by slurry impregnation of impregnated PMMA bead skeletons present a too large distance between the pore walls to permit the cytoplasmic extension. It has also to be noted that the elliptic shape of the pore could have a positive effect on the cell affinity as already observed by Lasgorceix [22]. This means that ice-templated pores could be more easily filled by a real osteoblastic tissue than the other porous structures, and then colonization of materials and mechanical properties after cell colonization could be better for the ice-templated materials. The tubular porous scaffolds obtained by ice-templating would also be favourable to invasion of other cells like endothelial cells which have difficulties to completely form the characteristic tubular tissue in ceramic bone substitutes and enhance tissue formation. According to the pore characteristics obtained by the two scaffolds preparation methods, it could be envisaged in the future to combine them to simultaneously mimic the compact and spongious parts of the bone with the possibility to create Havers and Wolkmann channels versus longitudinal or lateral directions. 4. Conclusion In this work, two types of -TCP macroporous scaffolds structures were processed. The method of ceramic slurry impregnation into polymeric beads allows the formation of 65% porosity scaffolds constituted by interconnected spherical pores with a close control of pore and interconnection diameters, whereas the ice-templating method leads to larger porosity distribution, from 36 to 67 volume percent, constituted by porous continuous channels, the thickness of which can be controlled from process parameters adjustment. The evaluation of osteoblast cell invasion ability into these different scaffolds has been carried out by static tests which have shown that the two porous structure types allow a good cell invasion until 4 days of incubation provided that the pore size is higher than 100 m. After this time, the first porosity layers are completely filled by the cells and the cell progression is stopped. Cell invasion tests for longer durations would need experimentation under continuous flow to mimic the in-vivo conditions. Nevertheless, these first tests have demonstrated the cell affinity with the -TCP substrates, particularly in the case of ice-template porous structures, through the observation of cytoplasmic extension phenomenon.
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Acknowledgements Dominique Hautcoeur thanks the Walloon Region (BE) for a grant (First Doca nr ECV320600FD007F/1017208-Ecopor). Edwige Meurice thanks the COST Action MP1301 “NEWGEN” for a STSM performed in Porto University. References [1] K.J.L. Burg, S. Porter, J.F. Kellam, Biomaterial developments for bone tissue engineering, Biomaterials 21 (2000) 2347–2359. [2] L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (1998) 1705–1728. [3] J. Lu, M. Descamps, J. Dejou, G. Koubi, P. Hardouin, J. Lemaître, The biodegradation mechanism of calcium phosphate biomaterials in bone, J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 408–412. [4] J. Lu, A. Gallur, B. Flautre, K. Anselme, M. Descamps, B. Thierry, Comparative study of tissue reaction to calcium phosphate ceramics among cancellous, cortical and medullar bone sites in rabbits, J. Biomed. Mater. Res. 42 (1998) 357–367. [5] B.V. Rejda, J.G. Peelen, K. de Groot, Tri-calcium phosphate as a bone substitute, J. Bioeng. 1 (1977) 93–97. [6] S. Pollick, E.C. Shors, R.E. Holmes, R.A. Kraut, Bone formation and implant degradation of coralline porous ceramics placed in bone and ectopic sites, J. Oral. Maxillofac. Surg. 53 (1995) 915–922. [7] D.M. Roy, S.K. Linnehon, Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange, Nature 247 (1974) 220–222. [8] D.M. Liu, Fabrication of hydroxyapatite with controlled porosity, J. Mater. Sci. Mater. Med. 8 (1997) 227–232. [9] M. Descamps, T. Duhoo, F. Monchau, J. Luc, P. Hardouin, J.C. Hornez, A. Leriche, Manufacture of macroporous -tricalcium phosphate bioceramics, J. Eur. Ceram. Soc. 28 (2008) 149–157. [10] S. Deville, E. Saiz, A.P. Tomsia, Freeze casting of hydroxyapatite scaffolds for bone tissue engineering, Biomaterials 27 (2006) 5480–5489. [11] J.M. Tabaos, R.D. Maddox, P.H. Krebsbach, S.J. Hollister, Indirect solid free form fabrication of local and global porous biomimetic and composite 3D polymer-ceramic scaffolds, Biomaterials 24 (2003) 181–194. [12] T. Fukasawa, Z.-Y. Deng, M. Ando, Pore structure of porous ceramics synthesized from water-based slurry by freeze–dry process, J. Mater. Sci. 36 (2001) 2523–2527. [13] S.F. Hulbert, J.J. Klawitter, R. Leonard, W.W. Kriegel, H. Palmours, Ceramics in Severe Environments, Plenum Press, New York, 1971, pp. 417. [14] J.J. Klawitter, S.F. Hulbert, Application of porous ceramics for the attachment of load boring orthopaedic applications, Biomed. Mater. Symp. 2 (1971) 161–167. [15] O.P. Frayssinet, J.L. Trouillet, N. Rouquet, E. Azimus, Autefage, Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition, Biomaterials 14 (1993) 423–429. [16] B. Flautre, M. Descamps, C. Delecourt, M. Blary, P. Hardouin, Porous ceramic for bone replacement: role of the pores and interconnections-experimental study in the rabbit, J. Mater. Sci. Mater. Med. 12 (2001) 679–682. [17] P.S. Eggli, W. Mueller, R.K. Schenk, Porous hydroxyapatite and tricalcium phosphate cylinders with two different macropore size ranges implanted in the cancellous bone of rabbits, Clin. Orthop. 232 (1998) 127–138. [18] J. Lu, B. Flautre, K. Anselme, A. Gallur, M. Descamps, B. Thierry, Role of the porous interconnections in porous bioceramics on bone recolonization in vitro and in vivo, J. Mater. Sci. Mater. Med. 10 (1999) 111–120. [19] P.M. Hunger, A.E. Donius, U.G.K. Wegst, Structure–property-processing correlations in freeze-cast composite scaffolds, Acta Biomater. 9 (2013) 6338–6348. [20] E. Landi, D. Sciti, C. Melandri, V. Medri, Ice templating of ZrB2 porous architectures, J. Eur. Ceram. Soc. 33 (2013) 1599–1607. [21] H. Yuan, K. Kurashina, J.D. de Brujin, Y. Li, K. de Groot, X. Zhang, A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics, Biomaterials 20 (1999) 1799–1806. [22] M. Lasgorceix, Mise en forme par microstéréolithographie et frittage de céramiques macro-micro-poreuses en hydroxyapatite silicatée et évaluation biologique, PhD thesis le 17 juillet 2014 Université de Limoges.
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Osteoblastic cells colonization inside beta-TCP macroporous structures obtained by ice-templating E. Meurice a , F. Bouchart a , J.C. Hornez a , A. Leriche a,∗ , D. Hautcoeur b , V. Lardot b , F. Cambier b , M.H. Fernandes c , F. Monteiro d a Laboratoire des Matériaux Céramiques et Procédés Associés (LMCPA), UVHC, Pôle Universitaire de Maubeuge, Boulevard Charles de Gaulle, F-59600 Maubeuge, France b Belgian Ceramic Research Centre (BCRC), Avenue Gouverneur Cornez, 4, B-7000 Mons, Belgium c Laboratory for Bone Metabolism and Regeneration, Faculdade de Medicina Dentária, Universidade do Porto, Rua Manuel Pereira da Silva, 4200-393 Porto, Portugal d INEB,Universidade do Porto, Rua do Campo Alegre, 823,4150-180 Porto, Portugal
a r t i c l e
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Article history: Received 17 August 2015 Received in revised form 12 October 2015 Accepted 21 October 2015 Available online xxx Keywords: Beta-tricalcium phosphate Ice-templating Scaffolds Orientated porosity Osteoblastic cell Colonisation
a b s t r a c t A casting method based on ceramic slurry ice-templating was used to create beta-tricalcium phosphate anisotropic oriented macroporous scaffolds. The freeze-casting method produces tubular interconnected pores with ellipsoidal shape. The tubular porosity structure is supposed to enhance cell invasion. Several samples with pore size ranging from 50 m to 350 m and porosity from 30% to 70% were synthesized using different processing parameters: dry matter and binder content of slurry and freezing rate. The osteoblastic cell colonisation into these samples was compared to that into classical scaffolds obtained from ceramic slurry impregnation of organic skeleton which allows the development of isotropic spherical porous structure with 250 to 600 m pore diameter size. The analysis by scanning electron microscopy of cell invaded samples shows that the size of the pores of the ice-templated scaffolds is sufficiently large to enable fast osteoblastic cell colonisation. © 2015 Published by Elsevier Ltd.
1. Introduction For several decades, hydroxyapatite and -tri-calcium phosphate have been used as substitutes for replacing bone defects in bone and joint surgery because their mineral composition is similar to bone and exhibits a very high bio-activity [1,2]. Compared to hydroxyapatite, -tri-calcium phosphate (-TCP) is more resorbable and can be rapidly replaced by new bone tissue [3,4]. To achieve a complete bone tissue reformation, bone cells, including osteoblast and endothelial cells, have to colonize the ceramic scaffolds which must present a porous structure favourable to cell invasion inside the total material volume and not only on the surface. To this purpose, several methods have been developed to mimic bone macroporous structure, including ceramic foams [5], solid reaction [6,7], ceramic slurry impregnation of polymer porosifier skeletons (sponge, PMMA bead scaffold, . . .) [8,9], ice-
∗ Corresponding author at: LMCPA-UVHC, Pôle Universitaire de Maubeuge, Boulevard Charles de Gaulle, Maubeuge F-59600, France. Fax: +33 327531667. E-mail address: [email protected] (A. Leriche).
templating [10] and additive manufacturing [11]. The last two methods produce oriented elongated and interconnected pores that could favour the cell colonisation and help endothelial cells to form the tubular structures required to capillary formation. This work is mainly devoted to test scaffolds obtained by the casting method based on ice-templating developed a few years ago, to create different morphological porous structures [10–12]. However, by this technique, the pore size is limited to a few tenth of a micron which could be deleterious to cell colonisation. Indeed, many investigations carried out on calcium phosphate macroporous materials presenting various porous architectures and different resorption rates and implantation sites and times suggested that cell colonization and bone ingrowth apparently occur if macropore size is greater than 100–150 m [13–17] with an interconnectivity between pores greater than 50 m [18]. In this work, the -TCP scaffolds obtained by ice-templating method was compared to commercially used scaffolds synthesized by slurry impregnation of organic bead skeleton in order to verify if the pore size of ice-templated scaffolds is large enough for cell colonisation.
http://dx.doi.org/10.1016/j.jeurceramsoc.2015.10.030 0955-2219/© 2015 Published by Elsevier Ltd.
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Fig. 1. Schematic illustration of the freeze-casting equipment.
2. Materials and methods 2.1. ˇ-TCP powder synthesis The -TCP powder was prepared by aqueous precipitation technique using diammonium phosphate and calcium nitrate solutions following a method described elsewhere [9]. In this work, the specific surface area of calcined and ground -TCP was 6.2 m2 /g and the ultimate particle size ranged from 1 to 2 m with d50 = 1.5 m. 2.2. Processing of macroporous ceramic scaffolds 2.2.1. Scaffolds prepared by slurry impregnation of organic bead skeleton Polymethylmethacrylate (PMMA) beads (DiakonTM Ineos Acrylics, Holland) with different diameter sizes (200, 300, 400, 500, 600, 700 and 800 m) were chemically stuck together in order to manufacture an organic scaffold in the shape of a block. This chemical welding was ensured by aceton which causes a slow superficial dissolution of polymer and allows the formation of bridges at the contact points between beads. Voids between polymeric beads were then filled by an aqueous -TCP powder suspension. After drying of the samples in a plaster mould, a debinding treatment was carried out at low temperature (220 ◦ C during 30 h) to eliminate the porogen agent (PMMA). The residual organics were then eliminated by heating at 400 ◦ C during 5 h. After debinding treatment, samples were sintered at 1115 ◦ C during 3 h in order to consolidate the ceramic walls up to a relative density equal or higher than 99%. This value was measured by mercury porosimetry. The porosity of the as-obtained scaffold is closely controlled in terms of shape and size: the spherical pore size was determined by the PMMA bead diameter and the interconnection size between the pores by the neck size between PMMA beads. For this study, 5 samples were prepared with fixed grain size distribution: B250 formed from a mixture of 200 and 300 m diameter size beads, B350 from 300 and 400 m diameter size beads, B450 m from 400 and 500 m diameter size beads, B550 m from 500 and 600 m diameter size beads and B750 m from 700 and 800 m diameter sizes. 2.2.2. Scaffolds prepared by ice-templating The -TCP slurries were prepared by mixing distilled water and -TCP powder with a small quantity (2.25 wt.% based on powder content) of ammonium polymethacrylate (Dolapix CE64, from Zschimmer and Schwarz, Germany). Powder content ranged from 20 to 40 vol%. Slurries were ball milled for 24 h with zirconia balls to obtain better deagglomeration. After sieving to remove zirconia balls, the binder, polyethylene glycol (Mw = 1000 mol g−1 , from Merck, Germany) was added (1.5 < wt% <6). Slurries were casted into a cylindrical PTFE mould (60 mm outer diameter and 50 mm long) and placed in the freezing equipment shown in Fig.1. Such a mould allows obtaining 3 samples with a diameter of 8 mm (for
biology tests) and one sample of 27 mm (for other analyses) before sintering. A slow cooling rate (1 or 5 K/min) was used with this device. A classic device was also used to obtain samples at rapid cooling rate (estimated to about 20 K/min), using nitrogen liquid as cooling agent [12]. The frozen samples were put into a freeze dryer (HETO CD8, from Heto Lab Equipment) for 24 h to remove ice at a pressure of 10 Pa. All green bodies were sintered with a constant heating rate of 5 K/min and kept during 2 h at 1100 ◦ C, followed by cooling rate of 5 K/min. Porosity and bulk density were measured by hydrostatic weighing (Archimedes’ method). Pore sizes were measured using image analysis from SEM pictures. For each composition, 10 micrographs were used and more than 100 pores were measured. The analysis was performed at 2 mm from the sample upper side. 2.3. Cellular colonization tests All tests were carried out with MG63 human osteoblastic cells. The MG63 cells were grown until 80% of confluence in a alpha-MEM culture medium containing 10% foetal bovine serum, 100 g/mL penicillin, 2.5 g/mL fungizone, and 50 g/mL ascorbic acid, at 37 ◦ C, in a humidified atmosphere of 5% CO2 in air. For a subculture, the cell monolayer was washed twice with a phosphate-buffered saline solution (PBS) and incubated with a trypsin-EDTA solution (0.05% trypsin, 0.25% EDTA) for 5 min at 37 ◦ C to ensure cell detachment. The -TCP samples 6 mm of diameter, 2 mm of high were placed in 48-cell culture plates and seeded with 105 /cm2 of cells. Cells colonization of the -TCP samples was followed by MTT coloration and scanning electron microscopy (SEM) after 1, 4 and 7 days. The MTT tests were performed by adding MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) at 0.5 mg/mL to the cultured samples, during 3 h at 37 ◦ C. Viable cells reduced the MTT to a blue formazan product. Colonization of the -TCP samples was observed with a stereomicroscope and digital camera Lan Optics. For SEM observation, the colonized -TCP samples were washed once in PBS and once in sodium cacodylate 0.14 M solution. They were then dehydrated by a ethanol gradient from 70% to 100%, followed by a gradient of EtOH/hexamethylsylanate from 50% to 100%. Samples were coated by sputtering with an Au/Pd thin film, using a SPI Module Sputter Coater equipment. The SEM/EDS examination was performed using a High resolution (Schottky) Environmental Scanning Electron Microscope with X-Ray Microanalysis and Electron Backscattered Diffraction analysis, (Quanta 400 FEG ESEM / EDAX Genesis X4 M). 3. Results and discussion 3.1. Characterisation of macroporous scaffolds Macroporous structures developed using the two methods are very different as it can be seen in Fig. 2. Scaffolds prepared from impregnation of organic bead skeleton by ceramic slurry present spherical pores homogeneously distributed in space with several interconnecting spherical holes, whereas the ice-templated scaffolds present z-axis oriented elongated (Fig. 2B) and ellipsoidal shaped pores with randomly orientation in x–y plan (Fig. 2C). Five samples presenting a total porosity volume of about 65% with different pore sizes from 250 m to 600 m were prepared by slurry impregnation of PMMA bead skeleton. An example of diamond saw cut scaffolds obtained from a mixture of 500 and 600 m PMMA beads is presented in Fig. 3. It can be seen that
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Fig. 2. SEM micrography of surface of ceramic slurry infiltration of organic beads skeleton (A) and of vertical (B) and cross (C) section of -TCP ice-templated scaffold.
Fig. 3. Micrograph of fractured scaffold prepared from slurry impregnation of a 500–600 m size PMMA bead skeleton.
the initial diameter of the pore is reduced after sintering from 500 to 600 m down to around 400 m with spherical interconnection ranging between 45 and 65 m. All scaffolds present spherical pores homogeneously distributed in space with several interconnecting spherical 50 m holes. As described before, two types of ice-templated samples were obtained, one with a large diameter and 3 others one with a small diameter. Fig. 4 shows SEM micrographs of each type of sample. It has to be noted that the structure was kept identical by changing the diameter. Cross section perpendicular to the freezing direction allows observing pores with an elliptical shape (Fig. 4). It is well known that this shape is characteristic of freeze casting by ice templating [10]. In the other direction, parallel to the freezing direction, a lamellar morphology with elongated pores was observed (Fig. 5A). As described in the literature [10], a denser part was observed at the bottom of each sample. This heterogeneous part was removed. A greater magnification on the lamellar allows the observation of the wall porosity (Fig. 5B). As noted by several authors, ceramic walls present a residual porosity [19,20] which could positively influence the biological response [21]. Table 1 summarizes the results obtained for the two sample diameters. These data correspond to a freezing rate of 1 K/min, a powder content of 30% vol. and a binder content of 3 wt%. In both cases, the results are very close. The large diameter size sample presents a mean pore size of 42 m and 240 m for the small (a) and large (b) diameter of the elliptic pore and the small diameter size sample presents a mean pore size of 43 m and 260 m for the
small and large pore diameters. The thickness of the lamellar walls and the porosity of the sample were kept at of about 40 m and 54%, respectively. It can be concluded that the sample size change does not affect the porosity characteristics. Characteristics of the seven samples prepared by the ice-templating method using different process parameters are presented in Table 2. Each parameter influences the size of the pores, which allow us to control the structure. For instance, a higher freezing rate results in obtaining a finer structure without changing the porosity. The pore sizes are ranging from 40 to 6 m and 200 to 13 m for the small and large diameters, respectively and from 34 to 7 m for the lamellar thickness. This can be clearly observed in Fig. 6A and B presenting the same magnification of micrographs following x–y plane of samples synthesized at 5 and 20 K/min freezing rate, respectively. The decrease of pore size versus freezing rate increase confirms the previous results obtained for freeze casted hydroxyapatite by Deville et al. [10] which showed a decrease from 600 m down to 150 m with cooling rate increase from 1 up to 10 K/min. For the suspension parameters, two processing conditions were modified: slurry dry matter content and binder content. The dry matter content strongly influences the porosity level which decreases from 67 to 36 % when the dry matter content increases from 20 up to 40 %. Simultaneously, the pore size decreases from 329 to 149 m for b axis values (comparison between IT6, IT5 and IT7) and no relevant influence was noted on the wall thickness. The binder content increase from 1.5 to 6 wt% (comparison between IT4, IT1 and IT5) does not influence the global porosity volume which remains about 50 vol.% but simply allows a better sample handling. The ice-templating method allows obtaining a larger range of porosity level than the slurry impregnation of PMMA bead scaffold with a minimum value of 36% for IT7 sample and maximum value of 67% for IT6 sample, the porosity value being always around 65% for the PMMA bead method. However, the highest porous scaffold IT6 has been disintegrated very rapidly in physiological medium during the cell impregnation tests. The value of the small diameter of the pores in the structures obtained by ice-templating (35–50 m) corresponds to the size of the interconnection between the spherical pore of scaffolds prepared from the impregnation of PMMA beads which could lead to a good cell invasion. 3.2. Cell colonization inside the ceramic scaffolds To perform colonisation experiments, MG63 osteoblasts from human cell line were used. All scaffolds obtained by the two previously described methods were as supports for osteoblast growth during 1, 4 and 7 days and cell invasions were checked by optical microscopy and by SEM. The micrographs of cut samples after colonization tests are presented in Fig. 7 for the ice-templated scaffolds and Fig. 8 for slurry impregnated PMMA bead skeleton scaffolds. It is observed by optical microscopy that MG63 osteoblasts characterized by having 50 m length and a 10 m width (black points
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Fig. 4. Micrographs of different size ice-templated samples prepared with the same processing parameters (A): large size sample (27 mm Ø) et (B) small size sample (8 mm Ø).
Fig. 5. (A) SEM image showing the lamellar morphology parallel to the freezing direction; (B) SEM image showing the porosity of ceramic lamellars.
Table 1 Porosity characteristics of ice-templated samples of different diameters. Sample Large 27 mm Ø Small 8 mm Ø
Pore size (m) a
b
42 ± 14 43 ± 14
240 ± 150 260 ± 100
Lamellar thickness (m)
Total porosity (%)
40 ± 18 38 ± 16
54 ± 1 55 ± 1
Table 2 Porosity characteristics of ice-templated samples synthesized from different process parameters. Sample
Conditions
IT 1 IT 2 IT 3 IT 4 IT 5 IT 6 IT 7
30%/3%/1 K/min 30%/3%/5 K/min 30%/3%/20 K/min 30%/1.5%/1 K/min 30%/6%/1 K/min 20%/3%/1 K/min 40%/3%/1 K/min
Pore size (m) a
b
41 ± 12 37 ± 14 6±2 54 ± 16 53 ± 17 67 ± 27 44 ± 11
209 ± 80 198 ± 110 13 ± 5 366 ± 200 277 ± 160 329 ± 140 149 ± 60
Lamellar thickness (m)
Porosity (%)
34 ± 14 38 ± 13 7±3 52 ± 15 37 ± 13 39 ± 18 43 ± 29
51 ± 1 52 ± 1 53 ± 1 51 ± 1 50 ± 1 67 ± 1 36 ± 1
Fig. 6. Micrographs of the ice-templated scaffolds prepared at different process parameters: (A) IT2: 30% Dry matter, 3% binder, 1 K/min freeze rate (B) IT3: 30% Dry matter, 3% binder, 20 K/min freeze rate showing the pore size decrease with freeze rate increase.
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Fig. 7. Micrographs of cut ice-templated samples by optical microscopy and SEM.
on micrographs) are able to colonise the surface of all samples independently of the preparation method of scaffolds. The tests show that the porosity size larger than 100 m of ice-templated samples is able to let the cells invade into the pores. Indeed, it is clear that the cells are not able to enter into the scaffold IT3, presenting smaller pores, even after 7 days. This is confirmed by the SEM image of the IT3 sample surface presented in Figs. 7 and 9E showing that cells have colonized the surface of the scaffold and appeared to be larger than the pore diameter and unable to enter into the material. This is in agreement with a pore size limit for cell colonisation higher than 100 m announced in the literature [13–17]. In the case of scaffolds prepared by slurry impregnated PMMA beads
method, all of them are colonized by the cells, as shown in Fig. 8. A different invasive behavior between the two different types of scaffolds can be outlined: in the tubular porous structure, the cells penetrate individually into the scaffolds whereas in the spherical porous structure, the cells are grouped and very few isolated cells are observed. It is difficult to quantify the cell invasion depth but a rough estimation shows the presence of cells only inside the first rank of pores for the spherical macroporosity, which corresponds approximately to 150 m in the B250 sample after 7 days, whereas the cells penetrate down to 250 m for columnar porosity in the IT1 ice-templated sample after the same duration. Scanning Electron Microscopy (SEM) performed on these samples confirms the
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Fig. 8. Micrographs of cut slurry impregnated PMMA beads samples by optical microscopy and SEM.
Fig. 9. SEM of ice-templated sample cultured with MG63 cells for 4 days. (A) Surface of IT1 sample with cells on surfaced (arrows) and inside the pores (circle); (B) high magnification image of cut IT1 with cells inside the pores; (C) homogeneous cell spreading on IT2 sample surface; (D) high magnification image of IT2 sample surface displaying the close interaction between cells and material surface; (E) surface of IT3 sample showing that individual cells were larger that the pores.
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results obtained by optical microscopy: cells seem healthy and are able to colonize surface (Fig. 9A, arrows) and pores. Comparison of different sample cross sections after 4 and 7 culture days reveals that cell invasion inside the ceramic structure is stopped for all samples because of cell proliferation on the surface which obstruct the pores. To compare the influence of the ceramic substrate architecture for longer incubation times, it would be necessary to perform tests with continuous medium flow. The SEM picture of IT2 sample after 4 days, presented in Fig. 9C shows a homogeneous cell spreading on the scaffold surface and an invasion inside the scaffold with sticking on the microporous ceramic walls. The cells are able to develop cytoplasmic filaments between the ceramic walls in ice-templated samples (circle in Fig. 9A). On the contrary, such a behavior is not observed in slurry impregnated PMMA beads scaffolds. To explain why the cytoplasmic extension phenomenon was observed only in ice-templated porous structures, it is suggested that the samples prepared by slurry impregnation of impregnated PMMA bead skeletons present a too large distance between the pore walls to permit the cytoplasmic extension. It has also to be noted that the elliptic shape of the pore could have a positive effect on the cell affinity as already observed by Lasgorceix [22]. This means that ice-templated pores could be more easily filled by a real osteoblastic tissue than the other porous structures, and then colonization of materials and mechanical properties after cell colonization could be better for the ice-templated materials. The tubular porous scaffolds obtained by ice-templating would also be favourable to invasion of other cells like endothelial cells which have difficulties to completely form the characteristic tubular tissue in ceramic bone substitutes and enhance tissue formation. According to the pore characteristics obtained by the two scaffolds preparation methods, it could be envisaged in the future to combine them to simultaneously mimic the compact and spongious parts of the bone with the possibility to create Havers and Wolkmann channels versus longitudinal or lateral directions. 4. Conclusion In this work, two types of -TCP macroporous scaffolds structures were processed. The method of ceramic slurry impregnation into polymeric beads allows the formation of 65% porosity scaffolds constituted by interconnected spherical pores with a close control of pore and interconnection diameters, whereas the ice-templating method leads to larger porosity distribution, from 36 to 67 volume percent, constituted by porous continuous channels, the thickness of which can be controlled from process parameters adjustment. The evaluation of osteoblast cell invasion ability into these different scaffolds has been carried out by static tests which have shown that the two porous structure types allow a good cell invasion until 4 days of incubation provided that the pore size is higher than 100 m. After this time, the first porosity layers are completely filled by the cells and the cell progression is stopped. Cell invasion tests for longer durations would need experimentation under continuous flow to mimic the in-vivo conditions. Nevertheless, these first tests have demonstrated the cell affinity with the -TCP substrates, particularly in the case of ice-template porous structures, through the observation of cytoplasmic extension phenomenon.
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Acknowledgements Dominique Hautcoeur thanks the Walloon Region (BE) for a grant (First Doca nr ECV320600FD007F/1017208-Ecopor). Edwige Meurice thanks the COST Action MP1301 “NEWGEN” for a STSM performed in Porto University. References [1] K.J.L. Burg, S. Porter, J.F. Kellam, Biomaterial developments for bone tissue engineering, Biomaterials 21 (2000) 2347–2359. [2] L. Hench, Bioceramics, J. Am. Ceram. Soc. 81 (1998) 1705–1728. [3] J. Lu, M. Descamps, J. Dejou, G. Koubi, P. Hardouin, J. Lemaître, The biodegradation mechanism of calcium phosphate biomaterials in bone, J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 408–412. [4] J. Lu, A. Gallur, B. Flautre, K. Anselme, M. Descamps, B. Thierry, Comparative study of tissue reaction to calcium phosphate ceramics among cancellous, cortical and medullar bone sites in rabbits, J. Biomed. Mater. Res. 42 (1998) 357–367. [5] B.V. Rejda, J.G. Peelen, K. de Groot, Tri-calcium phosphate as a bone substitute, J. Bioeng. 1 (1977) 93–97. [6] S. Pollick, E.C. Shors, R.E. Holmes, R.A. Kraut, Bone formation and implant degradation of coralline porous ceramics placed in bone and ectopic sites, J. Oral. Maxillofac. Surg. 53 (1995) 915–922. [7] D.M. Roy, S.K. Linnehon, Hydroxyapatite formed from coral skeletal carbonate by hydrothermal exchange, Nature 247 (1974) 220–222. [8] D.M. Liu, Fabrication of hydroxyapatite with controlled porosity, J. Mater. Sci. Mater. Med. 8 (1997) 227–232. [9] M. Descamps, T. Duhoo, F. Monchau, J. Luc, P. Hardouin, J.C. Hornez, A. Leriche, Manufacture of macroporous -tricalcium phosphate bioceramics, J. Eur. Ceram. Soc. 28 (2008) 149–157. [10] S. Deville, E. Saiz, A.P. Tomsia, Freeze casting of hydroxyapatite scaffolds for bone tissue engineering, Biomaterials 27 (2006) 5480–5489. [11] J.M. Tabaos, R.D. Maddox, P.H. Krebsbach, S.J. Hollister, Indirect solid free form fabrication of local and global porous biomimetic and composite 3D polymer-ceramic scaffolds, Biomaterials 24 (2003) 181–194. [12] T. Fukasawa, Z.-Y. Deng, M. Ando, Pore structure of porous ceramics synthesized from water-based slurry by freeze–dry process, J. Mater. Sci. 36 (2001) 2523–2527. [13] S.F. Hulbert, J.J. Klawitter, R. Leonard, W.W. Kriegel, H. Palmours, Ceramics in Severe Environments, Plenum Press, New York, 1971, pp. 417. [14] J.J. Klawitter, S.F. Hulbert, Application of porous ceramics for the attachment of load boring orthopaedic applications, Biomed. Mater. Symp. 2 (1971) 161–167. [15] O.P. Frayssinet, J.L. Trouillet, N. Rouquet, E. Azimus, Autefage, Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition, Biomaterials 14 (1993) 423–429. [16] B. Flautre, M. Descamps, C. Delecourt, M. Blary, P. Hardouin, Porous ceramic for bone replacement: role of the pores and interconnections-experimental study in the rabbit, J. Mater. Sci. Mater. Med. 12 (2001) 679–682. [17] P.S. Eggli, W. Mueller, R.K. Schenk, Porous hydroxyapatite and tricalcium phosphate cylinders with two different macropore size ranges implanted in the cancellous bone of rabbits, Clin. Orthop. 232 (1998) 127–138. [18] J. Lu, B. Flautre, K. Anselme, A. Gallur, M. Descamps, B. Thierry, Role of the porous interconnections in porous bioceramics on bone recolonization in vitro and in vivo, J. Mater. Sci. Mater. Med. 10 (1999) 111–120. [19] P.M. Hunger, A.E. Donius, U.G.K. Wegst, Structure–property-processing correlations in freeze-cast composite scaffolds, Acta Biomater. 9 (2013) 6338–6348. [20] E. Landi, D. Sciti, C. Melandri, V. Medri, Ice templating of ZrB2 porous architectures, J. Eur. Ceram. Soc. 33 (2013) 1599–1607. [21] H. Yuan, K. Kurashina, J.D. de Brujin, Y. Li, K. de Groot, X. Zhang, A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics, Biomaterials 20 (1999) 1799–1806. [22] M. Lasgorceix, Mise en forme par microstéréolithographie et frittage de céramiques macro-micro-poreuses en hydroxyapatite silicatée et évaluation biologique, PhD thesis le 17 juillet 2014 Université de Limoges.
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