The HA discs were prepared according to the previous study^{[ 13]}. HA powder (Engineering Research Center in Biomaterials, Sichuan University) was put into a model, and certain kind of pore-forming particles were spread on the surface. Then the powder was compacted into HA disc-shaped pellets by single-axis pressing at 100 MPa followed by sintering process to remove the pore-forming particles. Finally, HA discs with different surface topographies were obtained as (1) HA-S: smooth surface, no pore-forming particles used, polished after the sintering process; (2) HA-R50: regular and hemisphere-like micropore, by using polymethyl methacrylate (PMMA) particles with 50 μm diameter as porogen; (3) HA-R200: regular and hemisphere-like macropore structure, by using polystyrene resin (PS) particles with 200 μm diameter as porogen; (4) HA-R500: regular and hemisphere-like macropore, by using polystyrene resin (PS) with 500 μm diameter as porogen; (5) HA-MIR: irregular micropore, by treating HA-S with 1 mol/L hydrochloric acid for 10 min; (6) HA-IR; irregular macropore, by adding NH₄HCO₃ with irregular shape as porogen before pressing.

For different tests, different sizes of discs were prepared: for basic cells test, φ12 mm×2 mm; for genetic test: φ 20 mm×2 mm. After sintering or post treating, the phase composition was assessed by X-ray diffraction (XRD, Dandong Fangyuan DX-1000). The surface topography was characterized by scanning electron microscope (SEM, JSM-5900LM, JEOL, Japan) and Smileview software was used to analyze the surface pore size of the materials.

Human osteosarcoma cells (MG-63) were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The MG-63 cells were seeded at a concentration of 2×10⁴ cells/well to the surface of HA discs in 24-well culture plates (Falcon, USA) and cells cultured on blank wells were used as a control group.

Cell morphology on different material surface was observed by SEM at day 3. MTT assay was used to measure cell viability after being cultured on the discs for 2, 4 and 6 d. The absorbance of the resulting solution was measured at 570 nm using a multi-detection microplate reader (Bio-Rad 550).

The activity of intracellular alkaline phosphatase (ALP) was measured with a SensoLyte pNPP Alkaline Phosphatase Assay Kit (AnaSpec, Inc.). Aliquots of 500 µL of cell lysate were obtained according to the procedure described and mixed with an equal amount of pNPP working solution in 96-well microplate. Mixtures were then incubated for 30 min at 37℃. The reaction was stopped by addition of 50 µL stop solution to each well and the resulting optical densities were measured at 405 nm with a microplate photometer (Thermo Multiskan MK3, USA). Measurements were compared to an alkaline phosphatase standard and normalized using the total protein amounts which were measured with a BCA protein assay kit (Pierce, USA).

The expressions of osteogenesis-related genes of Cbfa1 and Collagen-I were investigated by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). After 3 d, total RNA was extracted from samples using Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. And then it is converted into complementary DNA (cDNA) using ReverTra Ace® qPCR RT Kit (TakaRa, Japan). The sequences of primers for core-binding factor α1 (Cbfa1), collagen Ⅰ (Col-Ⅰ) and GAPDH genes were given in Table 1. A quantitative real-time PCR reaction was performed using CFX96TM real-time PCR detection system (Bio-Rad, CFX960) with SsoFastTM EvaGreen® Supermix (Bio-Rad). GAPDH was used as the housekeeping gene to normalize results.

Table 1

Table 1

Table 1 Primers sequences for target genes Symbol The left primers The right primers Product size Col-Ⅰ 5´-CAAGAACCCCAAGGACAAGAG-3´ 5´-CTTGCAGTGGTAGGTGATGTTC-3´ 169 bp Runx2 (Cbfa1) 5´-CACTGGCGCTGCAACAAGA-3´ 5´-CATTCCGGAGCTCAGCAGAATAA-3´ 127 bp GADPH 5´-GCACCGTCAAGGCTGAGAAC-3´ 5´-TGGTGAAGACGCCAGTGGA-3´ 138 bp

Table 1 Primers sequences for target genes

Figure 1 showed the XRD patterns of the prepared HA samples. The six samples exhibited the same typical phase composition of hydroxyapatite, which indicated that the porogens were successfully removed by calcination and the processing procedures did not change the phase composition of samples.

Fig. 1

	Figure Option ViewDownloadNew Window
	Fig. 1 XRD patterns of the prepared HA

SEM images of surface structure of HA discs were shown in Fig. 2(A-F), and the measured average pore size was listed in Table 2. The SEM images showed that a layer of regular and closely connected hemispherical shallow pores with different pore size was covered on the surface of sample HA-R50, HA-R200 and HA-R500. While on the surface of HA-IR, the shallow pores were extremely irregular and disorderly distributed with an average pore size of 473.7 μm which was similar to HA-R500. HA-MIR showed a honeycomb multi-level irregular pore structure with the average pore size of about 30 μm while the HA-S surface was dense and smooth.

Table 2

Table 2

Table 2 Pore size on the surface of HA materials(mean±SD) Sample HA-R50 HA-R200 HA-R500 HA-IR HA-MIR Mean pore size/μm 52.96±8.67 203.6±36.5 507.7±30.2 473.7±133.7 27.6±5.91

Table 2 Pore size on the surface of HA materials(mean±SD)

The result indicated that HA discs with different surface topography were successfully constructed by using single-axis pressing methods with the assistance of proper porogen. The size and morphology could be designed evenly and precisely. Obviously, by controlling the size and morphology of porogen, the pores with corresponding size and morphology were fabricated precisely as shown in Fig. 2 (HA-R50, HA-R200, HA-R500 and HA-IR). The advantage of this method is that surface topography of the ceramics can be easily designed at the scale from 50 μm to 500 μm. Since it is difficult to get proper pores under 50 μm, acid erosion is employed to obtain a micropore structure in the range of 20-30 μm as shown in sample HA-MIR.

Fig. 2

	Figure Option ViewDownloadNew Window
	Fig. 2 SEM images of HA surface topography without cells (A-F) and with MG-63 cells on HA surface (a1-f1,a2-f2). Arrows: Cells.(A, a1) HA-R50, (B, b1), HA-R200, (C, c1) HA-R500, (D, d1) HA-IR, (E, e1) HA-MIR and (F, f1) HA-S(a2), (b2), (c2), (d2), (e2) and (f2) are the higher magnification images of (a1), (b1), (c1), (d1), (e1) and (f1), respectively

Figure 3 showed the cell viability of MG-63 cells cultured on different HA discs after 2, 4 and 6 d. Obvious cell proliferation was observed on all of the six discs, indicating that all the materials were biocompatible and could support cell proliferation which was consistent with the previous studies^{[ 14]}. However, the data also showed that the proliferation behavior varied with the surface structure of the ceramics.

Fig. 3

	Figure Option ViewDownloadNew Window
	Fig. 3 Cell proliferation on different surface structure of HA evaluated by MTT

HA-S shows lower viability compared to other materials (#: p < 0.05) at day 2 and HA-MIR shows lower than others at day 6(*: p < 0.05)

MG-63 cells on HA-S showed statistically lower viability comparing to other materials at the early days. But it increased dramatically with prolonging the culture time which was similar to the control group, indicating that a smooth surface was more conducive to cell proliferation. This may be due to a fact that smooth surface have less stimulation on cell skeleton, and cells can attach, spread easily, thus the proliferation process was not significantly affected.

Compared with other groups, MG-63 cells on HA-R50 and HA-MIR showed different proliferation trend: The cells proliferated rapidly at day 2 and day 4 while gradually slowed down at day 6. This may be related to the surface pore structure and pore size. For both the groups, the pore size was less than 100 μm which was similar to the size of MG-63 cell. This micropore would promote cell proliferation at the early days and achieved a maximum value at day 4, while on the sixth day when the cells showed contact inhibition, cell proliferation slowed down. The lowest cell viability of HA-MIR group at day 6 may be related to its irregularly honeycombed porous structure.

Cell morphology was examined by SEM after 3 d cell seeding to reveal the cell adhesion on the surfaces of the materials. As shown in Fig. 2 (a1-f1, a2-f2), it was evident that cells spread well on all materials.

Corresponding to the different cell proliferation trend on HA-R50 and HA-MIR, the cell adhesion on these two groups were also different with other groups. For both materials, cells closely attached on the edge of the pores and extended to the center of the pore, trying to cover the micropores. While for other groups, cells closely attached to the pore wall and growth in monolayer. Single cells exhibited a polygonal or star-shaped morphology with many extended pseudopodia when firmly attached to the material substrate. Moreover, cells on materials have been connected into a sheet covering the surface of the material especially on the HA-R200 and HA-S.It is difficult to observe a single cell morphology.

The results clearly proved that cell morphology varied with the surface structure of material. Cells on materials with smaller surface pores showed obviously different morphology with that of bigger surface pores. As MG-63 cell belongs to the family of cells that need an appropriate substrate to attach to so that they can get fully stretched^{[ 15]}. Therefore, for group HA-200, HA-500 and HA-IR, when the pore size is much larger than MG-63 cells, it can provide a proper base for cell attachment where cells can spread well on the pore wall. While for HA-R50 and HA-MIR, the wall surface of pore were much smaller than cells and the cells were likely to attach on the edge of the pore and cover the micropores.

Figure 4 displayed the ALP activity of MG-63 affected by the surface topography. Generally, the ALP activity of each group increased with the culture time. HA-MIR group had the highest ALP activity, followed by the HA-S, HA-R50 and HA-R200 group, while HA-R500 and HA-IR group owned the lowest value. It can be inferred that surface structure of HA affected the intracellular alkaline phosphatase activity significantly.

Fig. 4

	Figure Option ViewDownloadNew Window
	Fig. 4 Alkaline phosphatase (ALP) activity of MG-63 on HA materials after 3, 6 and 9 d

HA-MIR has the highest ALP activity (*, p < 0.05); HA-IR has the lowest ALP activity at day 9(#, p < 0.05)

ALP expression served as an important index of early osteoblast differentiation^{[ 16]}. The highest ALP expression of cells on HA-MIR indicated that the small and shallow micropore structures might have the ability to facilitate the early differentiation process of MG-63. The higher ALP activity on HA-S, HA-R50 and HA-R200 groups also indicated a higher degree of differentiation than cells on HA-R500 and HA-IR groups.

HA-R500 and HA-IR groups showed high cell viability while the expression of alkaline phosphatase was very low. This might be due to a greater surface area of the two materials would provide a larger number of cell adhesion points and allow the continuous proliferation of cells while cell differentiation was inhibited. In comparison, HA-R200 had a moderate pore structure, not only accommodated the appropriate number of cells, but also can promote cell differentiation, and therefore increased alkaline phosphatase activity.

Among all the groups, HA-MIR and HA-R50 groups have the highest alkaline phosphatase activity, indicating that pore size (less than 100 μm) at cell-scale would promote osteogenic differentiation of MG-63 cells. For HA-S and the control group, a high ALP expression was also found which means a spontaneous osteogenic differentiation of MG-63 cells, indicating that the smooth surface have no significant effect on osteogenic differentiation process. However, it still required further confirmation on the cellular and molecular level by using RT-PCR means.

The expression of Cbfa1 and Col-Ⅰgenes of MG-63 cells seeded on the HA was shown in Fig. 5. The surface structure of HA had great influence on the expression of Cbfa1. There was a low expression of Cbfa1 genes in HA-R500 and HA-IR compared with HA-S which had a smooth surface. Much higher Cbfa1 gene expression was found in HA-R50 and HA-R200, and HA-MIR was the highest. The result was consistent with the ALP activity. In contrast, the expression of Col-Ⅰshowed almost similar level on all the materials, indicating that the surface structure of HA had little influence on the expression of Col-Ⅰ gene. This might be related to the different signal pathway that modulated the expression of Col-Ⅰand Cbfa1 genes.

Fig. 5

	Figure Option ViewDownloadNew Window
	Fig. 5 Cbfa1 and Col-Ⅰ gene expressions of the MG-63 cells cultured on HA for 3 d GAPDH is used as the house-keeping gene to normalize results

Cbfa1 is an osteoblast-specific transcription factor and a key regulator of osteoblast differentiation and function. The expression of Cbfa1 indicates the early osteoblast differentiation of the cells^{[ 17]}. The significantly higher expression of Cbfa1 in HA-MIR, HA-R50 and HA-R200, which was consistent with that of ALP activity, indicating that these surface topographies were more likely to up-regulate Cbfa1 expression thus supporting adherent cells differentiation along osteogenic lineage.

According to the ALP activity and Cbfa1 gene expression, it can be concluded that the osteoblast differentiation of MG-63 was highly affected by the surface topography of materials. HA-MIR showed a highest differentiation, followed by HA-R50, HA-200 , HA-S, HA-IR and HA-R500. This trend was contrary to that of cell proliferation. The low Cbfa1 expression of HA-R500 and HA-IR might be due to the large pore size with little stimulation on cell differentiation, meanwhile, the large pores could promote cell division, growth and prolong the proliferative phase of MG-63. Finally the osteogenic differentiation of cells was inhibited.

However, other three groups with small pore size (<200 μm) had positive effect on cell differentiation. The expression of Cbfa1 gene increased with the decrease of pore size. MG-63 cells could be more easily identified by small pore and promote the osteogenic differentiation. On the other hand, the irregular alveolate surface of HA-MIR improved the expression of Cbfa1.