Production of porous Calcium Phosphate (CaP) ceramics with aligned pores using ceramic/camphene-based co-extrusion
© Choi et al. 2015
Received: 10 November 2014
Accepted: 9 June 2015
Published: 3 July 2015
Calcium phosphate (CaP) ceramics are one of the most valuable biomaterials for uses as the bone scaffold owing to their outstanding biocompatability, bioactivity, and biodegradation nature. In particular, these materials with an open porous structure can stimulate bone ingrowth into their 3-dimensionally interconnected pores. However, the creation of pores in bulk materials would inevitably cause a severe reduction in mechanical properties. Thus, it is a challenge to explore new ways of improving the mechanical properties of porous CaP scaffolds without scarifying their high porosity.
Porous CaP ceramic scaffolds with aligned pores were successfully produced using ceramic/camphene-based co-extrusion. This aligned porous structure allowed for the achievement of high compressive strength when tested parallel to the direction of aligned pores. In addition, the overall porosity and mechanical properties of the aligned porous CaP ceramic scaffolds could be tailored simply by adjusting the initial CaP content in the CaP/camphene slurry. The porous CaP scaffolds showed excellent in vitro biocompatibility, suggesting their potential as the bone scaffold.
Aligned porous CaP ceramic scaffolds with considerably enhanced mechanical properties and tailorable porosity would find very useful applications as the bone scaffold.
Porous bioceramics with an open porous structure have been widely examined as the scaffold for bone regeneration, since they can provide 3- dimensionally interconnected pores and biocompatible frameworks for cell attachment, proliferation and differentiation, as well as new bone formation in vivo [1, 2]. In particular, calcium phosphate (CaP) ceramics have gained much attention as a scaffold material on account of their similarity with natural bone in terms of chemical compositions and crystalline structure [3, 4]. These biomimetic physical and chemical characteristics allow porous CaP scaffolds to provide strong direct bond with the host bone in vivo as well as reasonable biodegradation nature when used as a bone scaffold .
Thus far, a variety of manufacturing techniques have been developed for the production of porous ceramic scaffolds , which include sponge replication , direct foaming techniques [8–12], vacuum-assisted foaming of a ceramic suspension (VFC) [13, 14], and freeze casting [15–17]. Fundamentally, the mechanical properties and biological function of porous ceramic scaffolds are strongly affected by their porous structure, such as porosity, pore geometry, pore size, and pore connectivity, as well as pore orientation [2, 18]. In general, high porosity is beneficial to bone ingrowth into 3-dimensionally interconnected pores but inevitably causes a severe reduction in mechanical strength [2, 19]. Thus, considerable effort has been made to improve the mechanical properties of porous ceramic scaffolds without sacrificing their high porosity. Unidirectional freeze casting is one of the most promising approaches for this goal, which can create aligned pores by inducing the preferential growth of ice dendrites along the direction of freezing [20–22]. The degree of pore alignment can be significantly enhanced by adopting polymeric additives [23–26], double-side cooling , and electric field [28, 29]. On the other hand, the use of camphene as a novel freezing vehicle allows for the production of porous ceramics with aligned pores even at room temperatures, which would provide more flexibility in manufacturing process [30–32]. Porous ceramic scaffolds with an aligned porous structure produced using these technique can have much higher compressive strengths than those with a random porous structure .
In this study, we produced porous CaP ceramics with aligned pores using ceramic/camphene-based co-extrusion, the basic concept of which was recently developed by our group [34, 35], and characterized their porous structure, mechanical properties, and in vitro biocompatibility for assessing their potential as a bone scaffold. The porous structure of porous CaP ceramic scaffolds (e.g. porosity, pore size, pore alignment, and pore connectivity) was characterized by field emission scanning electron microscopy (FE-SEM). The crystalline structure and phases were examined by X-ray diffraction (XRD). The compressive strength of the porous CaP scaffolds with aligned pores was measured to determine their structural integrity, while the in vitro biocompatibility of the scaffold was evaluated by in vitro cell tests using a pre-osteoblast cell line.
Commercial CaP powder (NT-BCP, OssGen Co., Korea) with a mean particle size of 0.5 μm was used as the starting material, while camphene (C10H16, Alfa Aesar/Avocado Organics, Ward Hill, MA, USA) were used as the freezing vehicle. CaP powder was comprised of hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) with a weight ratio of 60:40 (manufacturer’s specification).
Preparation of CaP/camphene slurries and extrusion process
Post treatment and sintering
To increase the pore size of porous CaP ceramics, the extruded CaP/camphene green samples were treated in an oven at 33 °C for 3 h in an oven for inducing overgrowth of the camphene dendrites . After which, the green bodies were freeze-dried to remove the camphene dendrites, followed by sintering at 1250 °C for 3 h to densify the CaP frameworks.
Characterization of porous structure and crystalline phases
The overall porosity of aligned porous CaP ceramics produced with various CaP contents (15 vol %, 20 vol %, and 25 vol%) was calculated from their dimensions and weight. The pore structure of the porous scaffolds was characterized by field emission scanning electron microscopy (FE-SEM; JSM-6701 F; JEOL Techniques, Tokyo, Japan). The pores sizes of the porous scaffolds before and after treatment at 33 °C for 3 h were also measured from their FE-SEM images. The crystalline structures and phases of the samples were characterized by X-ray diffraction (XRD, M18XHF-SRA, MacScience Co., Yokohama, Japan).
Measurement of compressive strength
Compressive strength tests were carried out to evaluate the mechanical properties of aligned porous CaP ceramics produced with various CaP contents (15 vol%, 20 vol%, and 25 vol%). The samples before and after treatment at 33 °C for 3 h were tested. Samples (~2.9 mm in diameter and ~ 6 mm in height) were uniaxially compressed at a constant crosshead speed of 1 mm/min using a screw driven load frame (OTU-05D; Oriental TM Corp., Korea). The mean value and standard deviation were obtained from five samples.
Assessment of in vitro biocompatibility
The in vitro biocompatibility of aligned porous CaP ceramics produced with various CaP contents (15 vol%, 20 vol%, and 25 vol%) was evaluated using a pre-osteoblast cell line (MC3T3-E1; ATCC, CRL-2593, Rockville, MD, USA). The MC3T3-E1 cells were plated at a density of 5 × 104 cells/mL and cultured in a humidified incubator in an atmosphere containing 5 % CO2 at 37 °C. Minimum essential medium (α-MEM: Welgene Co., Ltd., Seoul, Korea) supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin-streptomycin, 10 mM β-glycerophosphate (Sigma) and 10 μg mL−1 ascorbic acid was used as the culturing medium. The morphologies of the attached cells on the porous CaP ceramics after 24 h of culturing were examined by FE-SEM. In addition, cell viability after 3 days of culturing was examined using a MTS (methoxyphenyl tetrazolium salt) assay with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, WI, USA) for mitochondrial reduction .
All quantitative data were expressed in terms of mean ± standard deviation (SD) values. One-way ANOVA followed by Bonferroni’s post hoc comparison tests were performed in all statistical analyses and p < 0.05 was considered significant.
Results and discussion
Change in pore geometry by extrusion
Characterization of aligned porous structure
Effect of post treatment on pore size
Compressive strengths of aligned porous CaP scaffolds
Overall porosity of aligned porous CaP scaffolds produced with various CaP contents (15 vol%, 20 vol%, and 25 vol%)
Initial Cap Content [vol%]
Overall Porosity [vol%]
71 ± 4.6
63 ± 3.8
55 ± 6.6
In vitro biocompatibility
Porous CaP ceramic scaffolds with an aligned porous structure were sucesfully produced by ceramic/camphene-based co-extrusion. Highly aligned pores could be created by removing extensively elongated camphene dendrites formed via extrusion process. In addition, the pore size could be significantly increased through simple heat-treatment at 33 °C, which is close to the melting point of the CaP/camphene slurry. Interestingly, this heat-treatment led to a considerable improvement in compressive strength. The aligned porous CaP scaffolds showed excellent in vitro biocompatibility. All of these findings suggest that porous CaP scaffolds with a unique aligned porous structure, coupled with tailorable porosity, high mechanical properties, and excellent biocompatibility, would find very useful applications as a bone scaffold.
This work was supported by the Korea Healthcare Technology R&D Project (contract grant number: HI11C0388) funded by Ministry of Health & Welfare (Republic of Korea).
- Jones JR, Hench LL. Regeneration of trabecular bone using porous ceramics. Curr Opin Solid St M. 2003;7:301–7.View ArticleGoogle Scholar
- Hing KA. Bioceramic bone graft substitutes: influence of porosity and chemistry. Int J Appl Ceram Tech. 2005;2:184–99.View ArticleGoogle Scholar
- Dorozhkin SV. Calcium orthophosphates as bioceramics: state of the art. J Funct Biomater. 2010;1:22–107.View ArticleGoogle Scholar
- Dorozhkin SV. Bioceramics of calcium orthophosphates. Biomaterials. 2010;31:1465–85.View ArticleGoogle Scholar
- Dorozhkin SV. Biphasic, triphasic and multiphasic calcium orthophosphates. Acta Biomater. 2012;8:963–77.View ArticleGoogle Scholar
- Studart AR, Gonzenbach UT, Tervoort E, Gauckler LJ. Processing routes to macroporous ceramics: a review. J Am Ceram Soc. 2006;89:1771–89.View ArticleGoogle Scholar
- Jo IH, Shin KH, Soon YM, Koh YH, Lee JH, Kim HE. Highly porous hydroxyapatite scaffolds with elongated pores using stretched polymeric sponges as novel template. Mater Lett. 2009;63:1702–4.View ArticleGoogle Scholar
- He X, Zhang YZ, Mansell JP, Su B. Zirconia toughened alumina ceramic foams for potential bone graft applications: fabrication, bioactivation, and cellular responses. J Mater Sci Mater Med. 2008;19:2743–9.View ArticleGoogle Scholar
- Akartuna I, Studart AR, Tervoort E, Gauckler LJ. Macroporous ceramics from particle-stabilized emulsions. Adv Mater. 2008;20:4714–8.View ArticleGoogle Scholar
- Barg S, Soltmann C, Andrade M, Koch D, Grathwohl G. Cellular ceramics by direct foaming of emulsified ceramic powder suspensions. J Am Ceram Soc. 2008;91:2823–9.View ArticleGoogle Scholar
- Barg S, Moraes EG, Koch D, Grathwohl G. New cellular ceramics from high alkane phase emulsified suspensions (HAPES). J Eur Ceram Soc. 2009;29:2439–46.View ArticleGoogle Scholar
- Montufar EB, Traykova T, Gil C, Harr I, Almirall A, Aguirre A, et al. Foamed surfactant solution as a template for self-setting injectable hydroxyapatite scaffolds for bone regeneration. Acta Biomater. 2010;6:876–85.View ArticleGoogle Scholar
- Ahn MK, Shin KH, Moon YW, Koh YH, Choi WY, Kim HE. Highly porous biphasic calcium phosphate (BCP) ceramics with large interconnected pores by freezing vigorously foamed BCP suspensions under reduced pressure. J Am Ceram Soc. 2011;94:4154–6.View ArticleGoogle Scholar
- Ahn MK, Moon YW, Koh YH, Kim HE. Use of glycerol as a cryoprotectant in vacuum-assisted foaming of ceramic suspension (VFC) technique for improving compressive strength of porous biphasic calcium phosphate (BCP) ceramics. J Am Ceram Soc. 2012;95:3360–2.View ArticleGoogle Scholar
- Deville S, Saiz E, Nalla RK, Tomsia AP. Freezing as a path to build complex composites. Science. 2006;311:515–8.View ArticleGoogle Scholar
- Tang YF, Miao Q, Qiu S, Zhao K, Hu L. Novel freeze-casting fabrication of aligned lamellar porous alumina with a centrosymmetric structure. J Eur Ceram Soc. 2014;34:4077–82.View ArticleGoogle Scholar
- Ghazanfari SMH, Zamanian A. Effect of nanosilica addition on the physicomechanical properties, pore morphology, and phase transformation of freeze cast hydroxyapatite scaffolds. J Mater Sci. 2014;49:5429–504.View ArticleGoogle Scholar
- Loh QL, Choong C. Three-dimensional scaffolds for tissue engineering applications: role of porosity and pore size. Tissue Eng Part B Rev. 2013;19:485–502.View ArticleGoogle Scholar
- Gibson LJ. Biomechanics of cellular solids. J Biomech. 2005;38:377–99.View ArticleGoogle Scholar
- Deville S, Saiz E, Tomsia AP. Freeze casting of hydroxyapatite scaffolds for bone tissue engineering. Biomaterials. 2006;27:5480–9.View ArticleGoogle Scholar
- Deville S, Saiz E, Tomsia AP. Ice-templated porous alumina structures. Acta Mater. 2007;55:1965–74.View ArticleGoogle Scholar
- Liu G, Zhang D, Meggs C, Button TW. Porous Al2O3-ZrO2 composites fabricated by an ice template method. Scripta Mater. 2010;62:466–8.View ArticleGoogle Scholar
- Fu Q, Rahaman MN, Dogan F, Bal BS. Freeze casting of porous hydroxyapatite scaffolds-I. processing and general microstructure. J Biomed Mater Res B. 2008;86B:125–35.View ArticleGoogle Scholar
- Pekor CM, Kisa P, Nettleship I. Effect of polyethylene glycol on the microstructure of freeze-cast alumina. J Am Ceram Soc. 2008;91:3185–90.View ArticleGoogle Scholar
- Munch E, Saiz E, Tomsia AP, Deville S. Architectural control of freeze-cast ceramics through additives and templating. J Am Ceram Soc. 2009;92:1534–9.View ArticleGoogle Scholar
- Zuo KH, Zeng YP, Jiang DL. Effect of cooling rate and polyvinyl alcohol on the morphology of porous hydroxyapatite ceramics. Mater Design. 2010;31:3090–4.View ArticleGoogle Scholar
- Waschkies T, Oberacker R, Hoffmann MJ. Control of lamellae spacing during freeze casting of ceramics using double-side cooling as a novel processing route source. J Am Ceram Soc. 2009;92:S79–84.View ArticleGoogle Scholar
- Zhang YM, Hu LY, Han JC. Preparation of a dense/porous bi-layered ceramic by applying an electric field during freeze casting. J Am Ceram Soc. 2009;92:1874–6.View ArticleGoogle Scholar
- Tang YF, Zhao K, Wei JQ, Qin YS. Fabrication of aligned lamellar porous alumina using directional solidification of aqueous slurries with an applied electrostatic field. J Eur Ceram Soc. 2010;30:1963–5.View ArticleGoogle Scholar
- Yoon BH, Choi WY, Kim HE, Kim JH, Koh YH. Aligned porous alumina ceramics with high compressive strengths for bone tissue engineering. Scripta Mater. 2008;58:537–40.View ArticleGoogle Scholar
- Soon YM, Shin KH, Koh YH, Lee JH, Kim HE. Compressive strength and processing of camphene-based freeze cast calcium phosphate scaffolds with aligned pores. Mater Lett. 2009;63:1548–50.View ArticleGoogle Scholar
- Soon YM, Shin KW, Koh YH, Choi WY, Kim HE. Assembling uinidirectionally frozen alumina/camphene bodies for aligned porous alumina ceramics with larger dimensions. J Eur Ceram Soc. 2011;31:415–9.View ArticleGoogle Scholar
- Halloran J. Materials science. Making better ceramic composites with ice. Science. 2006;311:479-80.View ArticleGoogle Scholar
- Moon YW, Shin KH, Koh YH, Choi WY, Kim HE. Production of highly aligned porous alumina ceramics by extruding frozen alumina/camphene body. J Eur Ceram Soc. 2011;31:1945–50.View ArticleGoogle Scholar
- Moon YW, Shin KH, Koh YH, Choi WY, Kim HE. Porous alumina ceramics with highly aligned pores by heat-treating extruded alumina/camphene body at temperature near its solidification point. J Eur Ceram Soc. 2012;32:1029–34.View ArticleGoogle Scholar
- Lei B, Shin KH, Noh DY, Jo IH, Koh YH, Choi YW, et al. Nanofibrous gelatin–silica hybrid scaffolds mimicking the native extracellular matrix (ECM) using thermally induced phase separation. J Mater Chem. 2012;22:14133–40.View ArticleGoogle Scholar
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