Since the 1970s, bone replacement research has focused on calcium phosphates as degradable inorganic materials for clinical applications [1–3]. One of the most comprehensively investigated degradable calcium phosphates is β-tricalcium phosphate (β-TCP) which is proven to be a biocompatible, biodegradable and osteoconductive material [4–6]. Besides the choice of the bone replacement material, specific parameters such as, the percentage of porosity , the pore size  and the interconnectivity  are crucial for a complete in-vivo degradation of the bone replacement material. Most studies suggest a pore diameter of 100–1220 µm to ensure adequate degradation, remodelling and vascularisation [8, 10–13]. Furthermore, the volume percentage of the total porosity should be higher than 60% . Besides the requirement for a specific macroporosity of the ceramic scaffold, a certain percentage of microporosity is important for osteogenesis and the anchoring of the implant to surrounding bone tissue . Therefore, several techniques were developed to integrate the desired porous structure into calcium phosphate scaffolds [15–26]. One of these techniques is to coat polymeric sponges with a ceramic slurry. The resulting ceramic-coated polymeric template is subsequently dried and pyrolyzed through careful heating and sintering. This technique, however, shows the disadvantage of cracks caused by thermal expansion of the polymeric template and the shrinkage of the ceramic coating during the sintering process . Another technique for integrating porosity is to fill the whole polymeric foam with a ceramic slurry. The resulting part is sintered whereby the pyrolized foam generates the desired porosity . Using these prosification techniques the amount of porosity and the pore size are restricted by to the polymeric foam and cannot be adjusted to a specific value. Moreover, the pore size and the volume percentage of the porosity cannot be modified independently from one another. Another technique to produce porous structures is to use foaming agents in a cement paste during its setting time . However, the pore size and the percentage of porosity can hardly be controlled during this process. Besides the application of artificial porosification materials, techniques have been developed using natural structures (e.g., cuttlefish) to create porous ceramic structures . The porous structure is created by the natural material and the porosity, and the volume percentage of porosity can neither be varied nor fixed at specific value.
To address this problem, this paper introduces ammonium hydrogen carbonate (NH4HCO3) as an alternative porosification agent in varying particle size fractions and varying percentages in combination with β-tricalcium phosphate (β-TCP) material to fabricate porous β-TCP scaffolds. Until now, NH4HCO3 has only been used to porosify metal parts . One important advantage of using NH4HCO3 as a porosification agent is its low decomposition temperature (60°C). Due to this low decomposition temperature, no cracks would be expected as the result of thermal expansion of the porosifying agent and the β-TCP. Furthermore, the solid NH4HCO3 can be sieved to a specific particle size range, which should lead to a specific pore size range in the fabricated β-TCP scaffolds. In addition the volume percentage of porosity ought to be easily adjustable by varying the ratios by weight of NH4HCO3 and β-TCP.
Materials and sample characterization
The particle size distribution of the β-TCP slurry prior the spray drying showed a mean particle size after the milling process of 4.39 µm; whereas 10 vol.% of the particles where smaller than 1.45 µm and 10 vol.% of the particles were larger than 11.46 µm. Furthermore, there were no particles larger than 20 µm detectable. The shrinkage of the different samples was calculated from the diameter size of the samples before and after sintering (Table 1). Table 1 shows the mean shrinkage value and standard deviation of 10 samples, respectively. The volume percentage of porosity of the samples was calculated by the diameter, height and weight of the samples after the sintering process (Table 2). Table 2 shows the mean volume percentage of porosity and standard deviation of 10 samples, respectively.
Microstructure and phase analysis
The microstructure of the sintered β-TCP samples without the porosification agent NH4HCO3 depicted only a microporosity of 2–10 µm (Figure 1A, B). Besides the micropores of 2–10 µm the sintered samples prepared with the porosification agent showed macropores caused by the decomposed NH4HCO3. After sintering, the smallest particle size fraction of NH4HCO3 (45–63 µm) left behind the smallest macropores of 50 µm (Figure 2A, B). The particle size fraction of NH4HCO3 (200–224 µm) resulted in medium-sized macropores of 200 µm (Figure 3A, B) and the particle size fraction of NH4HCO3 (224–500 µm) showed the largest macropore size up to 500 µm (Figure 4A, B). The sintered samples prepared with a small range (45–63 µm as well as 200–224 µm) of the particle size fraction of NH4HCO3 left behind pores of the same range. Moreover, the larger particle size fraction with a wider range (224–500 µm) yielded also pore sizes of a this same range. Furthermore, the analysis of the microstructure showed a homogeneous distribution of the pores in the samples regardless of the particle size fraction utilized. Neither the volume percentage of NH4HCO3 nor the particle size range of the NH4HCO3 chosen affected these results. The powder X-ray diffraction pattern of a fabricated sample was compared to the powder diffraction file of pure β-TCP. Using the information of the Powder Diffraction File PDF #9-169 , no redundant peaks were found. Hence, β-TCP was the only crystalline phase present in the scaffolds.
The β-TCP scaffolds prepared without NH4HCO3 showed the highest strengths, whereas the specimens fabricated with 40% β-TCP and 60% NH4HCO3 (224–500 µm) showed the lowest ones (Table 3). The stress-strain curves for the tested specimens prepared without NH4HCO3 showed a linear increase and a total fracture at highest load, while the samples prepared with NH4HCO3 showed an irregular curve (Figure 5).
The particle size of the β-TCP slurry prior the spray drying process fulfilled the requirement of a particle size smaller than 20 µm. Consequently, the β-TCP slurry did not clog the nozzle of the spray dryer, due to large particles. The shrinkage of the sintered β-TCP scaffolds prepared with the NH4HCO3 was higher than that of the sintered β-TCP samples without NH4HCO3. This higher shrinkage is attributed to the macropores resulting from the decomposed NH4HCO3. The shrinkage of the mixture of 40 wt.% β-TCP and 60 wt.% NH4HCO3 tended to be greatest with the largest particle size fraction of NH4HCO3 (224–500 µm) and smallest at the smallest particle size fraction of NH4HCO3 (45–63 µm). However, this tendency was not detectable for the mixture of 50 wt.% β-TCP and 50 wt.% NH4HCO3. The measured shrinkage values for all the samples agree well with those values for processing porous ceramics published by others .
The microstructure of the sintered β-TCP samples prepared without NH4HCO3 showed only micropores (2–10 µm). In agreement with other fabrication techniques, these pores were caused by the sintering process . In addition, the sintered β-TCP samples prepared with NH4HCO3 showed macropores with a diameter in the same particle size range of the NH4HCO3 utilized. No agglomeration of NH4HCO3 appeared during the mixing or pressing of the NH4HCO3- β-TCP. Thus, the resulting pore size of the sintered β-TCP scaffolds correspond to the specific particle size range of the porosification agent. Since the particles of the NH4HCO3 were homogenously distributed in the pressed samples before the sintering process, there was a homogeneous distribution of resulting pores in the β-TCP scaffolds. This homogeneity was found for all particle sizes and for all volume percentages of NH4HCO3 tested. In addition, the volume percentage of porosity can be controlled by the ration by weight of NH4HCO3to β-TCP. More NH4HCO3 resulted in a higher volume percentage of porosity and this volume percentage of porosity was independent of the particle size range of the NH4HCO3 porosification agent.
Unlike other porosification agents which decompose at higher temperatures (e.g., about 600°C for carbon based porosification agents), the NH4HCO3 porosification agent immediately begins to decompose at a relatively low temperature of 60°C. The low decomposition temperature is advantageous, because it prevents cracking of the resulting ceramic that typically occurs at much higher temperatures at the sintering process. The first sintering step at 90°C for 1 h is important to decompose the NH4HCO3 material. If the NH4HCO3 has not fully dissipated until its melting point is reached of 106°C, the melt will deform the pressed samples before the beginning of the sintering process. The low decomposition temperature of the NH4HCO3 and the specific sintering curve implies no visible cracks of the fabricated β-TCP scaffolds. Therefore, NH4HCO3 is a viable alternative to naphthalene as a porosification agent for calcium phosphates . In contrast to NH4HCO3, naphthalene is disadvantageous: its decomposition temperature of 80°C is 20°C higher than that of NH4HCO3. Moreover, it is a toxic and carcinogenic .
Using the presented manufacturing process, it is also possible to mix several particle size fractions of NH4HCO3 to create various pore size ranges in the β-TCP scaffolds. This fact is relevant for designing tailored degradable bone replacement materials with improved osseointegration properties and optimized degradations kinetics in a subsequent in-vivo application [8, 31].
The compression test of the sintered β-TCP samples clearly showed that samples fabricated with a specific particle size range of the NH4HCO3 resulted in higher strengths for the mixtures of 50 wt.% of NH4HCO3 than for the mixtures of 60 wt.% of NH4HCO3. The lesser percentage of porosity of the 50 wt.% mixture thus resulted in a higher mechanical strength. On the other hand, the results of the compression test showed that the pore size of the sintered β-TCP samples affects also the strength of the β-TCP scaffolds. For both mixtures (50 wt.% of NH4HCO3 and 60 wt.% of NH4HCO3), the samples prepared with the particle size fraction of 45–63 µm NH4HCO3 showed the lowest strength. The highest compression strength for the β-TCP scaffolds prepared within the 50 wt.% of NH4HCO3, 50 wt.% of β-TCP mixture was reached using the particle size fraction of 200–224 µm NH4HCO3 and, for the 60 wt.% of NH4HCO3, 40 wt.% of β-TCP, with the particle size fraction of 224–500 µm NH4HCO3. Thus, the compression strength was not directly related to the pore size. The compression strength of the β-TCP scaffolds was within order of magnitude as that of cancellous bone . Due to the high volume percentage of porosity of the β-TCP scaffolds, cracks occur along the whole length of the specimens and resulted in an irregular stress-strain curve.
Ammonium hydrogen carbonate (NH4HCO3) is an excellent porosification agent to construct β-tricalcium phosphate scaffolds of defined pore architecture. The volume percentage of porosity can be controlled by using different weight ratios of NH4HCO3 and β-TCP. In addition, the pore size can be optimally controlled by using a specific particle size range of NH4HCO3 before the sintering process. This porosification agent allows the independent setting of a specific pore size range as well as a specific percentage of porosity. Consequently, the proposed porosification technique is very promising for the fabrication of tailored biodegradable β-TCP scaffolds which will improve both osseointegration and degradation kinetics in in-vivo applications.
Materials and methods
Synthesis of β-TCP/NH4HCO3 composite material
Tricalcium phosphate powder (art-no. 1.02143.9026, VWR, Darmstadt, Germany) was calcined at 1000°C for 1 h to synthesize pure β-TCP. After the calcination process a slurry was prepared. The β-TCP powder was milled in a ball mill (Table 4) using water, ZrO2 milling balls (1 mm diameter/art no. 19TS05/001, Krahn Chemie, Hamburg, Germany) and a dispersant (Dolapix CE64, Zschimmer & Schwarz, Lahnstein, Germany). The particle size distribution of the prepared slurry was measured using a particle size analyzer (Mastersizer 2000, Malvern, Worcestershire, UK). The milling process was required to achieve a slurry with particle sizes smaller than 20 µm to avoid blocking of the nozzle in the subsequent spray drying process. The spray drying process was performed in a conventional spray dryer (Mobile minor 2000, Niro, Soeborg, Denmark) using the displayed parameters (Table 5). The porosification agent NH4HCO3 (art-no. 21218.298, VWR, Darmstadt, Germany) was sieved to different particle size distributions using sieves (VWR, Darmstadt, Germany) with a varying mesh size of 45, 63, 200, 224 and 500 µm, respectively A stack of two sieves with a mesh size of 45 µm and a 63 µm each was used to obtain a small particle size distribution of NH4HCO3 ranging from 45 µm to 63 µm. A stack of two sieves with a mesh size of 200 µm and 224 µm each was applied to achieve a medium particle size distribution of NH4HCO3 ranging from 200 µm to 224 µm. Furthermore, a stack of two sieves with a mesh size of 224 µm and 500 µm each was used to achieve a large particle size distribution of NH4HCO3 from ranging 224 µm to 500 µm.
The sieved quantity of each NH4HCO3 fraction was mixed with the β-TCP granules of the spray drying process by either using 50 wt.% of NH4HCO3 and 50 wt.% of β-TCP or 60 wt.% of NH4HCO3 and 40 wt.% of β-TCP. Each of these six mixtures was filled in a PE-bottle and the bottle was rotated on a rolling platform for 30 min.
The various mixtures of NH4HCO3 and β-TCP were pressed by a uniaxial pressing machine (Z030, Zwick, Ulm, Germany) (Table 6). After the cylindrical specimens were removed from the mould the green parts were measured in diameter using a micrometer dial indicator. The subsequent thermal treatment (Figure 6) decomposed the NH4HCO3 porosification agent and subsequently sintered the β-TCP. The sintered samples were measured again in diameter to calculate the shrinkage. The weight and the height of every cylinder were also measured to calculate the volume percentage of porosity of the sintered samples based on the bulk density of β-TCP [3.14 g/cm³; 33].
Microstructure and phase analysis
The microstructure of the manufactured samples, in particular, the porous structure and the pore distribution, was analyzed using scanning electron microscopy (FEI ESEM XL30 FEG, Philips, Eindhoven, Netherlands). The samples were cut perpendicular to the cylinder axis. The pore sizes resulting of the different particle size distributions of the NH4HCO3 porosification agent were compared with each other, and with samples manufactured without the porosifying agent. The volume percentage of porosity was measured by gravimetry . The phase analysis of the manufactured samples was performed using powder X-ray diffraction (X’Pert System, Philips, Almelo, Netherlands). Monochromized Cu-Kα1 radiation (λX-ray=1.5418 Å) was used for the measurement in the range from 2θ=10° up to 2θ=70°. The obtained X-ray powder diffraction pattern was compared to the data content of the powder diffraction file database  and was also compared to the diffraction pattern of pure β-TCP.
The cylindrical samples were mechanically tested, whereby the strength of the specimens was analyzed in a compression test. Seventy samples grouped in seven batches were prepared to analyze the strength of each sample depending on the pore size and the volume percentage of porosity (Table 7). The samples prepared without NH4HCO3 were tested with the same device (Z030, Zwick, Ulm, Germany) which was used for pressing the cylinders. The strength of the cylinders fabricated with NH4HCO3 was analyzed using a universal testing machine (Z2,5, Zwick, Ulm, Germany) that can precisely measure also low strengths. Both testing devices were set to a cross-head speed of 1 mm/min and the abort criteria for the compression test was a change in the length of the specimen of 1 mm.
The authors appreciate the funding by the BMBF (German Federal Ministry of Education and Research) (03G0820A) for this study.
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About the article
Published Online: 2013-05-25
Published in Print: 2013-09-01