In vitro fatique

The load-bearing implant materials must be evaluated not only by their strength as described previously but also by their resistance to fatigue in solution because of the cyclic nature of in vivo loading. However, to the knowledge of the authors, relatively little information is available in the published literature concerning fatigue behavior in a physiological solution for bio-inspired ceramic/polymer composites. The results of fatigue experiments are presented as S-N diagrams (the so-called fatigue life diagram), where S is the maximum stress in a cyclic loading and N is the number of cycles until fracture. Fig. 7 is a plot of the maximum stress applied in the compressive cyclic fatigue against the number of cycles to failure at 37ºC in SBF. The graph showed that the stability of the composites was apparently affected by the cyclic loading with a remarkable decrease in the strength as the number of cycles increased. For example, the control fatigued in SBF for 2 ×10³cycles had a significant degradation down to 35% of the original strength. When a loading stress of 37 MPa was applied, the specimen containing 10 wt% gelatin (CSG10) lasted approximately 40 min in the in vitro fatigue test until failure occurred. As for the 0, 5, and 15 wt% gelatin-containing specimens, 40-min fatigue-induced failure in SBF solution required only 27, 32, and 26 MPa, respectively.

When subjected to fatigue testing in SBF under a dynamic condition, the detrimental effect on the specimens is seen, consistent with a previous study [23]. In addition to the crack growth that occurs under stress, the decrease in strength of the bio-inspired materials is also caused by environmental factors, such as the penetration of water/ions [23, 24]. Water/ions can easily infiltrate the inner portion of the specimens through structural imperfections, particularly under applied stress, resulting in weakened bond strength due to particle dissolution. The addition of gelatin can also increase the ratio of water absorption of the composite, which reduced the retention of the mechanical properties of the CaSi-gelatin composite fatigued under moisture conditions. The conclusion may be drawn that the present specimens have a limited resistance to fatigue failure. However, it provides that the fatigue properties of composites should be a noticeable factor.

5.8. In vitro bioactivity and degradation

Broad face and cross-sectional SEM micrographs of the bone grafts after soaking in a SBF solution for 1 day are shown in Fig. 8. The surface morphology of CSG0 immersed for 1

day was similar to those of the other three specimens (Fig. 8 a-d). It is clear that precipitation took place on all specimen surfaces which were covered with clusters of precipitated spherulites. From the cross-sectional SEM micrograms (Fig. 8 e-h), it can be seen that such a precipitated layer with a distinct contrast was observed in all specimens, confirming the broad face examination. The precipitated apatite layer was smoother and denser than the original specimen structure. The average thickness of precipitated apatite layers was approximately 220, 200, 160, 100 nm, respectively, for CSG0, CSG5, CSG10, and CSG15. To further confirm that the observed layer was indeed ascribed to apatite precipitated from the SBF solution, SEM/EDS analyses were performed on the 1-day-immersed specimens, in addition to the XRD analysis. Ca/P ratios of the SBF-immersed specimens were 4.0, 4.5, 5.8, and 10.6 for CSG0, CSG5, CSG10, and CSG15, respectively, which significantly (p < 0.05) increased with increasing gelatin content. The reason for the much lower apatite precipitation rate on CSG15 was possibly due to the presence of gelatin in its as-prepared material. Nevertheless, all the bonegraftsshowed astrong tendency for“attracting”apatiteprecipitateonto theirsurfaces, as evidenced by the Ca/P molar ratio. The much higher Ca/P ratio (compared to the 1.67 stoichiometric Ca/P ratio of apatite) on 1-day-immersed surfaces was not surprising due to the fact that a large quantity of calcium originating from the underlying specimens was detected.

The lower Ca/P ratio on the surface of the CSG0 control without gelatin was possibly due to the faster apatite precipitation rate, consistent with the thickness of the precipitated layer. The in vitro bioactivity of the SiO₂–CaO-based materials indicates that the presence of PO43

ions in the composition is not an essential requirement for the development of an apatite layer, which consumes the calcium and phosphate ions. This is because PO43

ions originate from the in vitro assay solution. An increase in the pH of SBF at different time intervals was attributed to the release of Ca(OH)₂, which is conducive for the formation of apatite precipitation. The results of the higher pH value in the CSG0 control-immersed SBF paralleled the apatite precipitation rate.

It was of interest to immerse specimens in an SBF solution for extended time (up to 180 days) to investigate the variations in the activity and degradation of the composites. After soaking for 180 days, the surface morphology of the specimens was significantly altered in the presence of the etching-induced micropores on the apatite layer (Fig. 9). It appears that during the immersion test dissolution of the surface had taken place. To further understand the etching effects, porosity measurements were conducted using a liquid displacement technique.

Before soaking in SBF, the porosities were 16%, 12%, 10%, and 11% for the specimens containing 0, 5, 10, and 15 wt % gelatin, respectively. On day 180, the porosities became 17%, 22%, 23%, and 28%, respectively. Significant differences (p < 0.05) between the porosities before and after soaking were found in the gelatin-containing composites.

Four soaking regimes up to 180 days were selected for testing compressive strength of the specimens, as shown in Fig. 10. The results revealed that all the four different types of bone grafts gradually lost their strengths with the increase in soaking time. After immersion for 180 days, the strength values of immersed specimens were in the range of 77−39 MPa, lower than respective strength values on day 0. It is worth noting that the gelatin-containing composites had a significantly lower strength (p < 0.05) compared to corresponding as-prepared composites, but not for the CSG control. Additionally, CSG0, CSG5, and CSG10 had a similar strength at day 180. It is surprising that the CSG0 control has insignificantly bond degradability (p > 0.05) when immersed in SBF solution up to 180 days. The statistical analysis using Scheffé multiple comparison test showed that the strength of CSG15 significantly declined from 98 MPa, the as-prepared strength, down to 39 MPa with a

reduction of approximately 60% after immersion for 180 days (p < 0.05). CSG5 and CSG10 composites lost 30 and 47% in compressive, respectively, after 180-day immersion. This deterioration in strength seems unavoidable for gelatin-containing composites immersed in SBF.

The effect of the soaking time on the changes in the weight of the bone graft specimens are presented in Fig.11. After soaking for 15 days, gelatin-containing composites showed a small amount of weight loss (~2%), whereas the CSG0 control gained weight. All the specimens exhibited an increased weight loss with an increase in the soaking time, reaching a weight loss of up to about 1–5% after 90 days depending on the type of specimen. At the end of the soaking experiment (180 days), weight loss of approximately 6%, 8%, 10%, and 18%

were observed for CSG0, CSG5, CSG10, and CSG15, respectively, which indicated a significant difference (p < 0.05). The degradation of the current bone graft systems was a slow process with the exception of CSG15, and the degree of the degradation was time dependent.

When soaked in SBF, the three gelatin-containing bone graft systems were associated with a relatively small degree of weight loss of about 2% after a 30-day soaking time; the CSG0 control even exhibited a weight increase. The few changes in sample weight may be explained by the formation of apatite, which was consistent with the morphology results. The four specimens continued to dissolve without stopping accompanied by continued weight loss after 90 days. This may be due to the release of soluble fractions (mainly gelatin). Because gelatin is biocompatible and has been classified as a bioresorbable material, its presence or dissolution is not expected to cause biological problems. Immersed CSG15 reached its maximal weight loss of 18% on day 180. It is important to note that 10 wt % gelatin (CSG10) led to a weight loss of 10% even after soaking in an SBF solution for 180 days. Moreover, the trend in weight loss of gelatin-containing composites was similar to the changes in the compressive strength. High physiochemical activity and low degradability were the characteristics of the present SiO₂– CaO-based material, which are of utmost importance and a necessity for successful results of bone graft substitutes for vertebroplasty applications [25]. The designed composites were expected to have an optimal mechanical performance, a controllable degradation rate, and eminent bioactivity, which will be of great importance for bone remodeling and growth.

However, the degradation rate and mechanical strength may be improved to support large defect sites for long-lasting and permanent implantation applications.

While soaking in the SBF solution, all bone grafts caused the pH of the solution to increase during the first 15 days, as shown in Fig. 12. By day 30, the pH of the solution approached a steady state value ranging from 8.4 to 9.0 depending on the type of bone grafts.

A greater amount of gelatin in the composite produced a lower pH value of the SBF solution.

On day 180, the pH of the solution containing CSG0 (pH 9.1) was significantly (p < 0.05) higher than the pH of the solution containing the gelatin-containing composites (pH 8.48.7).

5.9. Cytotoxicity

A common objective in orthopedic and dental fields is the design of biomaterials that supports cell and tissue growth, improving fracture healing and bone defect filling. Cell viability and function on a bone graft are closely related to the physical, chemical and biological characteristics of the materials used. This study supports the hypothesis that a natural polymer such as gelatin successfully improves the mechanical properties of calcium silicate ceramics. More specifically, this investigation will show that the newly developed composites enhance the cell functions.

The results of the alamarBlue assay for cytotoxicity are shown in Fig.13. The cells on the

positive control (RM-A) showed a high degree of cytotoxicity with an increase in the culture time while OD values decreased. In contrast, the negative control (RM-C) exhibited an increased OD level of mitochondrial activity with an increasing culture time. When the cells were seeded on different bone grafts, no depression of cellular activity occurred. More importantly, the absorbance values of all the tested bone grafts were significantly (p < 0.05) higher than those obtained from the negative control after 24 and 48 h of culture.