2.1 FT-IR analysis
FT-IR analysis was carried out to verify the conversion between n-HA, CS and CSH in the reaction process, as shown in Fig. 1(a). For n-HA, the characteristic peaks of PO43− are at 473cm− 1, 567 cm− 1, 602 cm− 1, 962 cm− 1, 1042 cm− 1 and 1095 cm− 1, which were owing to the P-O stretching vibration of PO43− [39–41]. In addition, the peaks at 631 cm− 1 and 3571 cm− 1 were ascribed to the OH− of hydroxyapatite [42]. The spectrum of CS showed the characteristic peaks at 3444 cm− 1 (overlap of O-H and N-H stretch), 1654 cm− 1 (NH-CO stretch) and 1589 cm− 1 (N-H bend), respectively [43]. The absorption peaks at 894 cm− 1, 1083 cm− 1 and 1153 cm− 1 originated from the glycosyl of chitosan. Figure 1a was the infrared spectra of CS/n-HA composite microspheres (c). The IR spectra of CSH/CS/n-HA was shown in Fig. 1b. The characteristic absorption peaks of SO42− were observed at 602 and 1143 cm− 1. The peak of 3404 cm− 1 was caused by the symmetric and antisymmetric stretching of H2O.
2.2 XRD analysis
Figure 2a shows the XRD pattern of the n-HA material. As shown in the figure, diffraction peaks appeared at positions of 10.8°, 25.9°, 31.8°, 32.2°, 32.9°, 39.8°, 46.7°, 49.4°, 53.1°, and 64.1°, which were consistent with characteristic diffraction peaks of hydroxyapatite standards proving the successful preparation of hydroxyapatite. Figure 2b revealed that the XRD pattern of pure CS powder showed characteristic diffraction peaks at only 10.7° and 20° at 2Θ [44, 45]. From Fig. 2b, the characteristic diffraction peaks of CS and n-HA appeared in the spectrum of CS/n-HA composite microspheres, and the intensity of the characteristic diffraction peak of CS uplift was weakened which were ascribed to the Schiff base reaction reducing the crystallinity of CS during cross-linking [46]. Comparing the diffraction peaks of n-HA (a), the peak shape of the characteristic diffraction peak of n-HA was still obvious in the XRD pattern of CS/n-HA composite microspheres (eg 2Θ = 25.9°, 31.8°, 32.2°, 32.9°, 39.8°), but significantly weakened, indicating that the crystallinity of n-HA was reduced after the two materials combined. Figure 2c is an XRD pattern of a CSH/CS/n-HA composite. The overall intensity of the diffraction peaks of the CSH/CS/n-HA composites was weakened.
2.3 Morphology analysis
SEM image of n-HA microsphere prepared by ultrasound-assisted chemical precipitation method showed that the synthesized n-HA exhibited a rod-like morphology with a uniform particle size distribution (Fig. 3a). As can be seen from Figs. 4a, b, more holes were observed on the surface of the CSH/CS/n-HA composite scaffold, but the distribution was uneven and irregular. According to the results of electron micrograph, Fig. 4c showed that calcium sulfate hemihydrate was a material of sheet structure. At the same time, the two materials were successfully combined according to the TFSEM image of the composite scaffold indicating that more CS/n-HA composite microspheres were embedded in the flake calcium sulfate hemihydrate and embedded in the scaffold (Figs. 4d, e, f). Therefore, an intuitive morphological characterization can verify the successful preparation of porous CSH/CS/n-HA composite scaffolds.
2.4 Mechanical strength study
The mechanical strength results indicated that the addition of an appropriate amount of CS/n-HA composite microspheres could effectively increase the maximum compressive strength of the porous composite stent. Briefly, the five sets of samples were subjected to a compression test using a universal testing machine, and the results were shown in Table 1. Compared with CSH scaffolds (CS/n-HA content of 0%), the maximum compressive strength of the scaffolds increased with an increase of CS/n-HA content (2%, 4% and 6%). When the CS/n-HA content was 6%, the compressive strength of the composite scaffold reaches the highest, which is 17.46 ± 1.29 MPa. When the CS/n-HA content was increased to 8%, the compressive strength of the stent decreased, even lower than the pure CSH scaffold.
Table 1
Comparison of compressive strength of porous composite scaffolds
Group | Number | CS/n-HA content (%) | Maximum compressive strength (MPa) |
1 | 3 | 0 | 11.02 ± 0.52 |
2 | 3 | 2 | 12.16 ± 1.03 |
3 | 3 | 4 | 13.32 ± 0.58 |
4 | 3 | 6 | 17.46 ± 1.29 |
5 | 3 | 8 | 10.92 ± 0.67 |
2.5 In-vitro degradation
Biodegradation is a vital factor to assess whether the scaffold can be implanted in human body which also should possess a controllable speed, biocompatible and non-toxic property. In this experiment, five groups of CSH/CS/n-HA composite scaffolds (cylinder, Φ10 mm×10 mm) with CS/n-HA content of 0%, 2%, 4%, 6% and 8% were immersed in SBF solution to initially evaluate its degradation performance. The weight was taken every 24 h and the fresh SBF solution was replaced. The daily weight variable rate of the stent samples with increasing soaking time is shown in Fig. 5a. During the experiment, the five groups of stents were immersed until the 15th day, and the remaining weight had dropped to 1–4% of the initial weight until the simulated body fluid immersion experiment was stopped. As shown in Fig. 5a, the trend line of the daily weight loss rate of the CSH stent decreases day by day, with no obvious fluctuations. In general, there was no significant difference in the trend between the other four groups of stent samples, and the fluctuations in the first 5 days were larger. After the sixth day, the daily weight loss rate tended to change smoothly and decreased as time flows. It was speculated that this was a tightly structured scaffold formed by the exquisite CSH powder attributed to the addition of CS/n-HA composite microspheres so that the gap of the CSH/CS/n-HA composite scaffold was increased, which was more conducive to the entry of the SBF solution into the scaffold promoting the degradation of the stent.
2.6 Concentration change of Ca in soaking liquid
The variation of Ca2+ concentration with the immersion time was observed as shown in Fig. 5c. The EDTA complexometric titration method was used to determine the concentration of calcium ions in SBF soaking solution of the five groups of samples, and the fresh SBF solution was set as the control (x = 0, y = 0.0026). During the immersion experiment, Ca2+ was released from the 5 sample holders, and the Ca2+ concentration was always higher than that of the SBF stock solution. However, the Ca2+ concentration of the soaking solution decreased day by day compared with the sample before immersion. The data shows that there is no significant difference in the trend line of Ca2+ concentration with time compared with the other four sets of composite scaffolds with different CS/n-HA content. Therefore, it can be considered that the Ca2+ released in the solution is mainly derived from the material Calcium sulfate hemihydrate and calcium sulfate dihydrate.
2.7 Concentration change of phosphorus in soaking liquid
The reagent blank was set as the control, and the PO43− solution with different concentration gradients was taken at 319 nm, and the absorbance values were determined by ultraviolet-visible spectrophotometer. The standard curve is fitted using the software Origin. The results are shown in Fig. 6. Within the measured concentration range, the fitting yields a linear equation y = 0.13036x with a correlation coefficient r of 0.99957.
The PO43− concentration change of the daily SBF soaking solution can be determined by the phosphorus-molybdenum yellow ultraviolet-visible spectrophotometry and determined by the above linear equation. As shown in Fig. 5d, it is a trend diagram of phosphorus content in SBF soaking solution with time. The SBF stock solution was set as the control (x = 0, y = 5.5036). In the process of simulating body fluid soaking, the concentration of PO43− was always lower than that of the SBF stock solution before the stent sample was completely degraded, and the steady decrease trend was observed. The scaffold sample was degraded to the 15th day, and the concentration of PO43− was increased to almost the same level as that in the SBF stock solution.
During the simulated experiment, the Ca2+ concentration released in the soaking solution decreased day by day, the PO43− concentration also decreased steadily, with white sediment at the bottom of beakers, as shown in Fig. 7a. It is used and verified by means of infrared and X-ray diffraction analysis, and the results are shown in Figs. 7b, c. The FT-IR spectrum showed characteristic absorption peaks of phosphate (PO43−) at positions 565 cm− 1, 603 cm− 1 and 1043 cm− 1. Figure 7c showed the XRD pattern of the uncalcined deposits. The crystallinity is low, and the diffraction peaks of hydroxyapatite are still observed at 25.9°, 31.8°, 49.4° and 53.1°. It is speculated that during the soaking process, hydroxyapatite was formed due to the dynamic changes of Ca2+ and PO43−.
2.8 In-vitro hemolysis study
The hemolysis rates of the different concentrations of n-HA, CS/n-HA and CSH/CS/n-HA suspensions were less than 5% in accordance with the Standard Practice of American Society for Testing and Materials Designation (ASTM:F 756-00), when the hemolysis rate is below 2% and 2% between 5%, those materials were viewed as non-hemolytic and slightly hemolytic, respectively. The results of in-vitro hemolysis experiments are shown in Fig. 8. Overall, the hemolysis rate of the three materials increased slightly with the increase of concentration of the suspension, and the maximum concentration of material hemolysis rate is less than 5%, indicating that no hemolysis occurs in the three concentrations of the material in the experimental concentration range, which meets the hemolysis experiment requirements and national standards of medical materials.
2.9 Discussion
FTIR spectra of CS/n-HA composite microspheres (Fig. 1a), which compared with the spectra of n-HA and pure CS powder. Some characteristic peaks of n-HA and CS overlap on spectra of CS/n-HA. The peak at 1045 cm− 1 was the overlap of the C-O-C of chitosan and the PO43− of n-HA (stretching vibration). And the overlap of the O-H between n-HA and CS induced the blue shift. The peak at 1639 cm− 1 was attributed to the C = N formed by schiff reaction of amino group and glutaraldehyde. The above IR spectrum analysis results show that the CS/n-HA composite microspheres contain both CS and n-HA, and no other impurity peaks appear. The infrared spectral characteristic absorption peaks of CSH/CS/n-HA composites fully illustrated the successful preparation of composites.
XRD pattern further confirmed the existence of CSH/CS/n-HA composites. The characteristic peak shape of n-HA was sharper, indicating that the crystallinity of n-HA synthesized by ultrasonic-assisted chemical precipitation was high. The above XRD pattern analysis showed that the CS/n-HA composite microspheres were further confirmed to contain both CS and n-HA components, and no other impurity peaks appeared. The phase results of CS/n-HA showed that the diffraction peak intensity of the XRD pattern was weak. Therefore, the overall intensity of the diffraction peak of the CSH/CS/n-HA composite is weakened attributed to the addition of the CS/n-HA composite.
From mechanical strength results, it is know that when the content of CS/n-HA increases to 8%,, the adhesion between the materials decreased, and the stent was more easily loosened, resulting in a decrease in the compressive strength of the sample. The in-vitro degradation illustrated that the cumulative trend of the weight loss rate of the five groups of samples showed no significant difference with time, increasing day by day, and almost completely degraded by soaking until the 15th day. Therefore, the results revealed that the CSH/CS/n-HA composite scaffolds with different CS/n-HA content are biodegradable, and the addition of CS/n-HA does not affect the degradation rate.
The results of in vitro hemolysis experiments illustrated that the suspensions of n-HA, CS/n-HA and CSH/CS/n-HA were in the range of 0.025 ~ 0.8 mg/mL, and the hemolysis rate was less than 5%. There is no hemolysis, and both meet the requirements of hemolysis experiments and national standards for medical materials. Through the above experimental research, it can be preliminarily believed that these three self-made materials will not cause acute hemolysis and have good biosafety.