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.
KeywordsCalcium phosphate Porous materials Pores Scaffold Bone regeneration
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).
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