Temperature effect on the porosity of hydroxyapatite scaffolds and its use in tissue engineering

The search for a suitable bone replacement is of great importance due to the difficulty to use autologous transplants. Hence, the objective of this work is to compare the temperature effect on the porosity and average pore diameter of hydroxyapatite porous scaffolds fabricated by the salt leaching method.  Hydroxyapatite porous scaffolds fabricated by the salt leaching technique were sintered from ~150 to 1000 °C. Synthesized hydroxyapatite was assessed by X-ray diffraction (XRD). Zeta potential at different temperatures was evaluated. Specimens were characterized using scanning electron microscopy (SEM) and Raman analysis. The results showed that significant porosity (57%) and pore size (49 µm) occurred with a thermal treatment above ~ 850 °C for scaffolds that were pre-sintered at 1050 °C.


Introduction
In tisssue enginneering applications, autologous bone grafting procedure is the current treatment for bone injuries, but this procedure has several limitations, which include additional surgical procedures, chronic pain after surgery, donor site morbidity and lack of tissue availability [1]. To overcome the problems associated with autograft use, alloplastic materiales have been developed. Alloplastics are materials with unlimited availability, no risk of disease transmission and osteoconductive properties [2]. The most common alloplastic material used in biomedical applications is hydroxiapatite [3].
The use of hydroxyapatite as porous scaffolds or as a bioactive coating material in medical devices is justified because ceramics resist oxidation and corrosion in the physiological environment and possess great resistance to friction and wear; however, hydroxyapatite by itself has poor biomechanical properties, its ability to withstand flexion and compression stresses is very low, causing it to fracture easily. Given these drawbacks, in recent decades several organic compounds of the extracellular matrix such as fibronectin, vitrionectin, osteopontin, growth factors and type I collagen, among others, have been added to hydroxyapatite coatings in order to improve osteoconduction, cell adhesion and the mechanical properties of the coating [4][5][6][7][8][9]. A relatively new promising material that can improve the mechanical properties of the coating is graphene [10], since it has great flexibility and mechanical rigidity, in addition to its properties as an electrical conductor that could help coating methods based on voltage, such as electrodeposition and electrophoresis [11,12].
It has been reported that the most suitable pore size for optimal vascularization is 100-500 µm, which also provides an area of adhesion to osteogenic cells [13,14], and that percentage porosity values above 10% in ceramic materials indicate pore interconnectivity, the more percentage of porosity, the more probability of pore interconnection. The porosity values present in trabecular bone vary between 30 and 90% with interconnected porosity between 50 and 90%. The more porosity and interconnectivity, the more ease of cell proliferation and migration, aswell as greater nutrient transport [15].
The objective of this work is to compare the temperature effect on the porosity and average pore diameter of HAp porous scaffolds manufactured by the salt leaching method, were this technique is easy to perform, affordable and promote the pososity of a compressed inorganics powders as hydroxyapatite.

Hydroxyapatite (HAp) synthesis
Hydroxyapatite powder (HAp) was synthesized using the methodology reported by Guillen-Romero, L, et al. [16], were an H3PO4 solution was blended with Ca (OH)2 w/v in a relation 1:1.67 with a constant stirring during 7 days. After that, the solution was washed 3 times by centrifugation at 3000 rpm for 5 min. The pellet was resuspended and filtered off through Buchner funnel and washed again with ethanol. The filtered sample was left to dry at 80 ᵒC for 5 days in an oven. Finally, half a gram of the resulting HAp was sintered at four different temperatures (~150 ° C, ~450 ° C, ~850 ° C and ~1000 ° C) for 2 h to observe the influence of the temperature on the fabrication of the sintered hydroxyapatite (sHA) scaffolds [16,17].

HAp and sHA porous scaffold preparation
Potassium chloride (KCl) was added to HAp and sHA in a KCl/HAp-sHA (1/1.85 w/w). The mixture was homogenized by grinding them together in a porcelain mortar. Then, the samples were compacted using a hydraulic press into cylindrical scaffolds with a force of 5000 lbs for 2 min. HAp and sHA scaffolds were sintered in different temperatures ranging from ~150 to 1000 °C for 2 h in an oven. Finally, HAp and pHA scaffolds were placed in a drip leaching system. The volume of liquid solvent used in the leaching process was 10 mL of distilled water for all samples. All sHA scaffolds disintegrated in contact with water during the drip leaching process. However, HAp scaffolds maintained their consistency. For this reason, the dripleaching process was only applied to the HAp scaffolds [16,18].

X-ray diffraction (XRD)
The equipment used for this analysis was the Bruker D8 Advance diffractometer, with the powder methodology. A metallic holder was used, and was set with few samples, enough to cover the surface of the holder (1 cm lenght x 3 mm width). After that, the holder was located inside the equipment with the following conditions: 15 and 30 rpm, lamp of copper (Cu) at 30 kV [16].

Raman spectroscopy
In the case of Raman spectroscopy, it was analyzed using an FRA 106/S FT-Raman, Bruker with an Nd: Y AG laser source operating at 1200 nm with a 180° back scattering geometry, spectral width 1 cm −1 , and power of the laser beam 250 mW reaching the sample [19].

Scanning electron microscopy (SEM)
Specimens were characterized by scanning electron microscopy (SEM). SEM images were obtained using secondary electron detector (SED). Percentage of porosity and average pore diameter were measured using the software Fiji ImageJ using images with a magnification of 1000 x.

Z potential studies
A Z potential analysis was performed using the HORIBA SZ-100 zetameter by taking 1 mg of the hydroxyapatite, 1 mmol of KCl and dissolving them in 100 mL of 70% ethanol using the BRANSONIC 2510R-MT sonifier. To determine the particle size, the same equipment was used with a solution of 10 mg in 100 mL of ethanol. Both Z potential and particle size analysis of HAp treated at different temperatures were also determined. For that, 1 gr of HAp, 0.001 mol of KCl and 100 mL of 70 % ethanol were used in order to make the Z potential analysis and 10 mL of HAp with 100 mL of ethanol were used in the particle size analysis. Misonix Branson 2510R-MT ultrasonic cleaner was used to mix the samples [16].

Raman spectroscopy
The Raman spectrum showed in figure 1, that the obtained signals are according with literature [16][17][18], demonstrating the HAp presence in the samples, validating its synthesis.
In the Raman spectra all signals were assigned to the internal vibrational modes of the PO 4group.
The intense signal at 960 cm −1 matches with the symmetric stretching mode ν1 of the tetrahedron

Scanning electron microscope (SEM)
The microstructure of HAp and sHA porous scaffolds was examined using scanning electron microscopy "figure 3 A-C" and "figure 3 E-G" respectively. The heat treatment applied to HAp and sHA scaffolds were carried out using temperatures from ~150 to 1000 °C. Porosity and pore size were estimated using the ImageJ software "table 1". ISSN: 2594-1925  Based on the results obtained by scanning electron microscopy, it is concluded that all specimens that were evaluated presented pore interconnectivity based in the fact that percentage porosity in both HAp and sHA scaffolds were above 10 %. According to some studies, porosity values above 10 % indicate interconnectivity between pores in ceramic materials [22] and a higher porosity translates into higher cellular proliferation and nutrient transport.
In addition, microporosity and macroporosity was perceived in all samples. Pores between 9 and 308 µm were found. Sequeda reported that the most suitable pore size range for ease of cell
Thermal treatment at higher temperature (~1000°C) of HAp scaffolds resulted in a considerable increase of average pore diameter compared with the rest. It can be observed, that the particle size of HAp decreases when the temperature increment. The particle size results are shown in "table 2".  [24]. With the thermal treatments of the sHA it was achieved a low particle size diameter (~8-12 µm), which are interesting sizes for tissue engineering [24]. At ~1000° C where obtained the lowest particle size (~8 µm). In the case of the particle size obtained (~30 µm) from the HAp, its higher size can be attributed to the existence of coordinated H20 molecules that remain when were allowed to dry at a temperature of ~80 °C. The particle size is suitable to use in coatings [16], because at a size of 10 µm allows cell adhesion, however, is not as feasible to be used in other medical applications, because the size of the particle would complicate of the construction of seeding channels in the bone.

Z potential analysis
On the other hand, low zeta potentials values promote the differentiation of osteogenic cells at the surface's material and negatively charged surfaces have excellent biocompatibility [25]. Table 3 show the lowest zeta potential results for HAp at higher temperature. At the opposing, the effect of heat temperature on HAp and sHA at ~150 °C produced less percentage of porosity and higher zeta potential compared to ~850 and ~1000 °C treatments. As a result, sHA scaffolds treated at ~1000 °C can have potential properties for cell adhesion and proliferation [26][27][28]. According to a study, the Z potential value for hydroxyapatite at this pH should be in the range of 15 mV as seen in the sintered sample at ~850 °C, however given the favorable results of the sample at ~1000 °C [16].

Conclusions
The porosity and average pore diameter of HA porous scaffolds fabricated by the salt leaching method can be controlled by thermal treatments. In this work, hydroxyapatite was successfully synthesized using the wet precipitation method. The higher porosity ratio was obtained at ~850 °C treatment, but lower Z potential of HAp was at ~1000 °C indicating that any of these two sHA sample can be adequate cell adhesion and proliferation properties. Further studies have to be done to find a balance with adequate % porosity and lower z potential.
Its important to denote, that all the obtained HAp and sHA scaffolds presented a superior porosity than 30 %, been suitable for trabecular bone replacement applications. Still, future tests are necessary to complement this study in order to propose these HAp and sHA scaffolds for tissue engineering applications.