Preparation and characterization of nano biphasic calcium phosphate/poly-L-lactide composite scaffold

2.1 Synthesis of nano-BCP particles with the sol-gel method

Trimethyl phosphite [(CH₃O)₃P, AR] and calcium nitrate tetrahydrate [Ca(NO₃)₂·4H₂O, AR] were firstly dissolved respectively in anhydrous ethanol to form a 2 mol/l solution. Deionized water was added to a trimethyl phosphite ethanol solution according to the volume ratio of 1:2.5 under constant stirring until uniformly mixed. After that, the above solution was mixed with calcium nitrate tetrahydrate ethanol solution with a starting Ca/P molar ratio of 1.60. The mixed solution was continuously stirred in a water bath at 50°C for 30 min and kept undisturbed at 50°C until a wet gel was formed. The wet gel was vacuum dried at 80°C for 48 h, and then the dried gel was calcined at 800°C for 3 h. (The influence of starting Ca/P molar ratio and heating temperature on the properties of as-prepared BCP will be reported elsewhere.) Finally, the products were ground into powders using an agate mortar. All the chemical reagents mentioned were manufactured from Kelong Chemical Reagent Company, Chengdu, China.

2.2 Preparation of nano-BCP/PLLA composite porous scaffold

BCP/PLLA composite was prepared by a solvent self-diffusion method. Briefly, PLLA (Shandong Institute of Medical Instruments, China; with coefficient of viscosity 2.67 dL/g) was dissolved in chloroform (CHCl₃) to form 6 wt% solution. Nano-BCP particles were ultrasonically homogenized with the above solution to form a suspension; the weight ratio of BCP to PLLA was 2:1. Then, the suspension was slowly poured into a mixed solution of acetone and anhydrous ethanol with continuous agitation. After that, the BCP/PLLA composite particles were deposited. The deposition was repeatedly washed with anhydrous ethanol and filtered. After vacuum drying at 65°C for 48 h, the product was placed into a glass desiccator until its weight became constant.

Composite scaffold was prepared by hot-pressing and particulate leaching process [12]. Sodium chloride (NaCl) with particle size ranging from 100 to 350 μm was used as porogen. As-prepared BCP/PLLA composite particles were homogeneously mixed with porogen NaCl. The mixture was then placed into a cylindrical mold (Φ=10 mm) and shaped by hot-pressing with 10 MPa pressure at 170°C for 10 min to form the samples. After cooling and porogen leaching in deionized distilled water for a week, porous samples were dried and stored for use.

Micro-BCP/PLLA composite prepared according to the same process described above was studied as the control sample. Micro-BCP particles (∼0.5 μm) were self-prepared via calcining calcium-deficient apatite synthesized using a chemical precipitation method reported elsewhere [15].

2.3 SBF soaking experiment

Composite cylindrical samples were soaked in SBF, as proposed by Kokubo et al. [13, 14], with the solution buffered at pH 7.40 with trimethanol animomethane-HCl at 37°C in a constant-temperature shaking system. The solution was replaced to be fresh every 48 h. At each retrieval time point, samples were removed, washed with deionized water, and vacuum dried at 80°C for 24 h. Finally, after being naturally cooled to room temperature, the samples were stored in a glass desiccator until constant weight was achieved. Weight changes in the samples retrieved at days 3, 5, 7, 10, 15, 20, 25, and 30 were recorded and mass loss rate was calculated by (M1-M2)/M1×100% (M1 represents the weight before immersion and M2 represents the weight after immersion). The results were expressed by the average mass loss rate of three samples at each time point (n=3). The cross-sectional microstructure of the composite before and after immersion in SBF and the corresponding Ca and P elemental analyses were carried out.

2.4 Characterization methods

The crystalline structure of as-prepared BCP powders was analyzed by X-ray diffraction (XRD; Cu-Ka radiation, DX-1000, Dandong Fangyuan Technology Co. Ltd., China). Compressive strength was characterized by an electromechanical system (AG-10TA, Shimadzu, Japan) (five samples of each material). Particle size of nano-BCP was observed with transmission electron microscope (TEM; Tecnai G2 F20, FEI Co., Hong Kong). The microstructure of the composite and the corresponding Ca and P elemental analyses were observed using scanning electron microscopy (SEM-EDX; JSM-5900LV, Japan Electric Corporation, Japan).

2.5 Cytocompatibility of nano-BCP/PLLA composite scaffold

Cytocompatibility was evaluated by MG-63 cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were derived from human osteosarcoma and expressed the characteristic features of osteoblasts. MG-63 cells were routinely grown in Ham’s F12 medium (Gibco, UK), which was supplemented with 15 vol% calf serum, 10³ U/ml penicillin, and 103 U/ml streptomycin in a 37°C incubator with 5% CO₂ and 95% relative humidity.

Nano-BCP/PLLA scaffold samples were sterilized with 70% ethanol solution, soaked with phosphate buffered saline (PBS), and washed every 1 h for 3 times. Then, the sterilized samples were transferred into a 24-well polystyrene plate and incubated with culture medium for 24 h. Then, MG-63 cells were seeded onto the samples at a density of 1×10⁵ cells/well. After that, the culture plates were cultured in a humidified incubator containing 5% CO₂ at 37°C. The medium was replaced every 48 h.

After 24, 72, and 120 h of incubation, the samples were taken out, washed with PBS twice, transferred to a new 24-well plate, and allowed to react with 20 μl 0.5% MTT dye solution. After another 4-h culture, MTT solution and the medium were removed, and 200 μl of dimethyl sulfoxide was added into each well. The plates were slightly shaken for 10 min to ensure crystal dissolution. After that, absorbance was measured at the wavelength of 490 nm on an enzyme analyzer. Results were expressed in percentages compared with the control group. Cells incubated in the medium without composite were used as control group. Each result consisted of three repeated measurements (n=3). The microscale BCP/PLLA composite was tested with the same method as the comparative study. The morphologies of cells after culture were observed by a phase contrast optical microscopy (TE2000-U, Nikon Co., Japan).

3.1 Nano-BCP particles

The XRD pattern of as-prepared powders is shown in Figure 1. It can be seen from the pattern that the component of the sample was BCP phase consisting of β-TCP (Whitlockite, JCPD 70-2065) and HA (JCPD 72-1243). The Ca/P molar ratio, which can be calculated from the XRD pattern by JADE software (Materials Data, Inc., USA), was 1.625.

Figure 1

XRD pattern of sol-gel-derived BCP powder.

The SEM image and EDX spectrum of as-prepared BCP powders are shown in Figure 2A. Particle size shown in the SEM image was below 100 nm, and the particles formed agglomerate because of the high surface energy of nanoparticles. The Ca/P molar ratio of the sample based on EDX was 1.629, which was almost in accordance with the calculated value. TEM micrographs are shown in Figure 2B. The diameter of the powder distributed between 50 and 90 nm, which agreed with the SEM result.

Figure 2

(A) SEM image (scale bar, 100 nm) and corresponding EDX spectrum of as-prepared BCP powder. (B) TEM micrograph of as-prepared BCP powder.

3.2 Nano-BCP/PLLA composite scaffold

The SEM image of the cross-section of nano-BCP/PLLA composite scaffold is shown in Figure 3A. It reveals a suitable porous structure of typical bone scaffold materials. The pores possessed a size ranging from 300 to 500 μm; Figure 3B and C shows the pore wall structure of nano-BCP/PLLA composite and micro-BCP/PLLA composite (the control group), respectively.

Figure 3

SEM image of (A) the cross-section of nano-BCP/PLLA composite scaffold showing the porous structure of the materials (porosity is about 50%), (B) the pore wall of nano-BCP/PLLA composite scaffold, and (C) the pore wall of micro-BCP/PLLA composite scaffold.

As shown in Figure 3B and C, BCP particles were distributed homogeneously in the PLLA matrix to form BCP/PLLA composite. Compared with the pore wall structure of micro-BCP/PLLA composite (Figure 3C), much more tiny crystallites were exposed in the surface of the pore wall in nano-BCP/PLLA scaffold. A larger area of the bioactive phase exposed in the pore surface was supposed to facilitate ion exchange and protein absorption and thus promote biomineralization and cell proliferation in implantation environment.

Table 1 shows the compressive strength of BCP/PLLA composite scaffold. The compressive strength of BCP/PLLA composite scaffold was related to such factors as porosity, the molecular weight of PLLA, and processing condition. Porosity in the present work was controlled mainly by the amount of porogen added. All tested composites were prepared with BCP particles of the same Ca/P mole ratio and PLLA of the same molecular weight under the same processing conditions.

Generally, in order to meet the requirement of the scaffold for bone tissue engineering, the porosity should be at least 45% [16, 17]. It can be seen from Table 1 that the compressive strength of all the tested samples met the needs of the compressive strength for bone tissue engineering scaffolds (5–8 MPa [16, 18]). The strength of scaffolds would be further increased with new bone ingrowth after implantation. The compressive strength of nano-BCP/PLLA scaffolds reached 10.25 and 12.73 MPa at porosities of 60% and 50%, respectively. As for micro-BCP/PLLA samples, the compressive strengths were 7.34 and 8.46 MPa at porosities of 60% and 50%, respectively. Obviously, both nano-BCP groups demonstrated at least 40% compressive strength improvement.

The enhanced mechanical properties can be attributed to the larger interfacial area between the nano-BCP particles and the PLLA matrix and the more uniform distribution of the nano-BCP particles in the PLLA matrix. Nano-BCP particles have strong interface with and are stronger than the PLLA matrix. Microvoids will unlikely initiate from the nano-particles or the interface between nanoparticles and the PLLA matrix. Compared with micro-BCP/PLLA composite, internal cavitation formation or interface debonding will hardly happen for nano-BCP/PLLA composite, and the connection between the surface of nanoparticles and the polymer chain is more intimate. Upon loading, stress concentrates around the nanoparticles. The stress fields overlap each other and highly constrain the development of the local crack dilatation in the matrix. Hence, by adding nano-BCP fillers instead of the micrometer counterpart into the PLLA matrix, the mechanical strength can be enhanced.

3.3 SBF soaking experiment

It was reported by Kokubo and Takadama [13] that the examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material. Thus, this approach was adopted in the present study.

Figure 4 shows that mass loss increased when the immersion time was prolonged. The weight change of the composite in SBF was due to the degradation of the composite materials and the biomineralized apatite formation process. PLLA degrades fast in an aqueous environment, and the incorporation of inorganic particles could regulate the degradation rate of the composite. As shown in Figure 5, the degradation speed increased fast within the initial days of degradation, reaching peak values of 4.3% for nano-BCP/PLLA on the fifth day and 4.5% for micro-BCP/PLLA on the seventh day. Before the peak value, the mass loss rate was predominantly due to the degradation of PLLA and the dissolution of BCP. After that, the mass loss rate decreased because more new apatite particles deposited. Obviously, the turning point of the mass loss rate for nano-BCP/PLLA was much earlier. This indicates that more biomineralized apatite particles would deposit on the pore-wall surface for nano-BCP/PLLA scaffold, which has more bioactive BCP particles exposing on the surface and has finer exposed BCP particles (as shown in Figure 3). After 20 days, the mass loss tendency of nano-BCP/PLLA composite almost remained stable, which was similar to that of micro-BCP/PLLA composite.

Figure 4

Mass loss rate of nano-BCP/PLLA composite and micro-BCP/PLLA composite during immersion.

Figure 5

SEM micrographs of nano-BCP/PLLA composite scaffold after immersion in SBF: (A) 3 days, (B) 5 days, (C) 10 days, (D) 20 days, and (E) 30 days.

Figure 5 shows the SEM micrographs of nano-BCP/PLLA composite scaffold after immersion in SBF within 30 days. After immersion in SBF, white flocculent particles appeared and aggregated on the surface of the porous scaffold. The increasing trend of the aggregation from 3 days to 10 days was remarkable. On the fifth day, there were plenty of apatite crystals that deposited and almost completely covered the surface of the materials (Figure 5C). After that, the precipitated bio- apatite groups accumulated, became larger in size, and formed a kind of typical “cauliflower” morphology (Figure 5D and E). The apatite morphology on the 20th day and that in the 30th day had no marked difference. Accordingly, the mass loss rate tendency was kept stable in these periods, as shown in Figure 4.

The corresponding EDX analysis of Ca and P elements on the surface precipitate layer after immersion is shown in Figure 6. In the initial period, the Ca/P ratio was not stable and the surface apatite granules were deposited, accompanying the material degradation and ion exchange with SBF. Ca element accumulated rapidly, and the Ca/P ratio increased from 1.475 on the 5th day to 1.834 on the 15th day. The Ca/P ratio started to decrease after 15 days and remained almost stable after the 20th day. As described above, the mass loss tendency remained stable and the morphology of mineralized apatite became steady 20 days after immersion. The final Ca/P ratio on the 30th day was 1.627 (Figure 5), which indicates that the final formed apatite was calcium deficient (the ideal Ca/P mole ratio of HA is 1.667). The rapid formation of a surface nonstoichiometric apatite layer suggests good bioactivity of nano-BCP/PLLA composite scaffold.

Figure 6

Corresponding EDX analysis of nano-BCP/PLLA composite scaffold after immersion in SBF (n=3).

3.4 Cytocompatibility of the scaffold

The MTT results of MG-63 cell growth on nano-BCP/PLLA and microscale BCP/PLLA scaffolds are shown in Figure 7A. The increasing trend of the cell viability of both tested materials with time was remarkable, which shows that the tested materials exhibited an appropriate microenvironment benefit for the proliferation of the cells. It is also obvious that the cell viability of nano-BCP/PLLA scaffold was significantly higher than that of micro-BCP/PLLA scaffold (p<0.05). The cell viability of nano-BCP/PLLA at 120 h was 96.4%, which almost reached the cell proliferation of the control group (100%).

Figure 7

(A) Cell viability of MG-63 cells analyzed by MTT assay. Results are expressed in percentages compared with the control group. Values are mean±SD of three samples. (B) Morphologies of the MG-63 cells after culturing with nano-BCP/PLLA scaffold for 120 h (200×).

The morphology of the cells after culturing with nano-BCP/PLLA scaffold for 120 h is shown in Figure 7B. Shuttle or polygon-shaped cells were well grown, spread, and proliferated.

The morphology and microstructure of the scaffold have important influence on the cells’ responses to the scaffolds [19, 20]. As described before, much more inorganic particles (BCP) are exposed on the surface of nano-BCP/PLLA composite. The size of BCP in nano-BCP/PLLA composite was 50–90 nm, which was much smaller than that of micro-BCP/PLLA composite (0.3 μm). The crystallinity of the inorganic phase (HA and β-TCP) in nano-BCP/PLLA composite is lower. Thus, the adhesion of cells on nano-BCP surface is much better than that on micro-BCP surface [21]. In addition, there may be more surface defects (such as edge sites, particle boundaries, and delocalized regions) in the interface between nano-BCP particles and the PLLA matrix or on the surface of the nanoparticles [22, 23]. Therefore, nano-BCP crystals have stronger capability to absorb some special peptide sequence (e.g., arginine-glycine-aspartic [RGD] sequence) and promote interactions of select serum protein(s) with nanocrystals [24]. When more RGD is absorbed to the surface of nano-BCP/PLLA composite, the adhesion ability of MG-63 cells is increased [25].

Therefore, behaviors such as cell adhesion, protein adsorption, and cell proliferation of nano-BCP/PLLA composite could be enhanced. It could be expected that the addition of nano-BCP particles in the PLLA matrix would enhance the formation of new bone tissue after implantation by increasing osteoblast adhesion, protein adsorption, osteointegration, and the bio-apatite mineralization on its surface.