4 Results and Discussion
4.3 Polylactide (PLA) and PLA-SiO 2 Supraparticles for Bone Grafting
4.3.7 Degradation Behavior and Hydroxyapatite Formation
In this chapter, the degradation behavior, and the ability of the printed specimens to form hydrox-yapatite are investigated. These two factors significantly influence the performance and mechan-ical properties of the bone replacement material in the body.11,76–78 The degradation behavior of the fabricated PLA and 90:10 PLA-HAP multilayer specimens via the PBF-LB/P process was in-vestigated via an accelerated degradation study. Moreover, the printed specimens were placed in simulated body fluid (SBF) to see whether hydroxyapatite would be formed on the square speci-mens.
Degradation Behavior of Multilayered PLA and PLA-HAP Specimens
The degradation behavior of PLA and 90:10 PLA-HAP multilayered specimens was investigated in PBS buffer under accelerated conditions at 70 °C for 70 days (Figure 37). It should be noted that the results under these conditions do not represent the degradation rates in the human body, however, they give a first indication of the degradation behavior.233,327 For both specimens, it was observed that the pH value of the PBS buffer decreased over 70 days (Figure 37 e). The highest decrease was observed for the PLA specimen where the pH-value decreased from pH = 7.2 to pH = 6.3 and the 90:10 PLA-HAP specimen showed a decrease to pH = 6.8 after 70 days incubation at 70 °C (Figure 37 e). This decrease in the pH value of the PBS buffer is caused by the hydrolytic degradation of the polylactide.116,234 For this degradation to occur water needs to react with the ester bonds in the biopolymer and lactic acid is released into the surrounding media, thus increas-ing its proton concentration.11,116 The smaller decrease in the pH-value for the 90:10 PLA-HAP specimens can be attributed to the smaller mass fraction of PLA present in the specimen.
After the incubation in the PBS buffer, the PLA specimens had a mean water uptake of wu = 32.5 ± 3.1 %, which was slightly lower than the water uptake for the 90:10 PLA-HAP speci-mens with wu = 39.9 ± 0.2 % (Figure 37 f). This could be explained due to the higher porosity of the 90:10 PLA-HAP specimen given by the lower sintering degree of the surface in the PBF-LB/P process (Figure 37 h). After a drying step to remove the residual water after the incubation, the PLA specimens had lost ml = 9.7 ± 0.2 % of their initial mass and the 90:10 PLA-HAP specimens ml = 5.2 ± 0.1% (Figure 37 f). Furthermore, the SEM images of the surface for the PLA specimen showed many cracks after the incubation due to the hydrolytic degradation of the PLA (Figure 37 b).100,328 The surface of the 90:10 PLA-HAP specimen showed the presence of needles in the upper micron range (Figure 37 d), which could be attributed towards HAP primary particles that had grown in size.
Figure 37: Degradation behavior of square multilayered PLA and 90:10 PLA-HAP specimens produced in the PBF-LB/P process in PBS buffer under accelerated conditions of 70 °C for 70 days. Macroscopic images and SEM images of the surface of square multilayer PLA (a,b) and 90:10 PLA-HAP (c,d) specimens before (a,c) and after (b,d) the degradation. e) Change of the pH value over 70 days during the degradation of PLA and 90:10 PLA-HAP specimens in PBS buffer indicating the formation of lactic acid. f) Results for water uptake and mass loss of the PLA and PLA HAP specimens after the degradation time.
Testing of Bioactivity in Simulated Body Fluid
In the following step the bioactivity of the binary Ca-SiO2 primary particles (Chapter 4.3.2), the square 90:10 PLA-HAP, and PLA specimens was investigated (Figure 38). For this reason, the respective samples were incubated in simulated body fluid (SBF) at 37 °C for three weeks. After this time, the samples were washed in bidistilled water and subsequently analyzed.
The Ca-SiO2 primary particles (Figure 38 a) had before the incubation in SBF a mean particle size of x50,3 = 0.4 µm with a span of 1.2 (Figure 26 a). The EDX analysis of the sample confirmed the presence of calcium (Ca), silicon (Si), and oxygen (Figure 38 b,i). After the three weeks incubation of the Ca-SiO2 primary particles in the SBF a cauliflower-like rough structure and no individual Ca-SiO2 primary particles were observed (Figure 38 e). In the EDX analysis, the elements phos-phor (P) and calcium (Ca) were detected strongly alongside with oxygen (O) and silicon (Si) (Fig-ure 38 f,i). These findings indicate that a calcium phosphate layer was formed on top of the Ca-SiO2 primary particles and they can be regarded as bioactive.228,232
After the three weeks of incubation within the simulated body fluid, the 90:10 PLA-HAP square specimen also showed the formation of a calcium phosphate layer (Figure 38 g,j). The square specimen had before incubation a smooth surface topography with occasional pores (Figure 38 c). The EDX analysis revealed a strong carbon (C) and oxygen (O) peak for the polylactide (PLA) and the presence of the nanoscale hydroxyapatite (HAP) was indicated through two small peaks for phosphor (P) and calcium (Ca) (Figure 38 j, orange).
After the incubation, the surface of the 90:10 PLA-HAP specimen was fully covered with cauli-flower-like structures (Figure 38 g). Additionally, it was observed that the caulicauli-flower-like struc-tures were grown from needle-shaped precipitates (Figure 38 g). The EDX analysis showed after incubation of three weeks two distinct peaks for phosphor (P) and calcium (Ca), which indicate the formation of a calcium phosphate layer (Figure 38 j, blue).228,329
To this end, it can be said that the 90:10 PLA-HAP square specimens are bioactive and show the formation of a calcium phosphate layer, which is critical for the successful integration of an im-plant in the bone healing process.102,103 For the square 100:0 PLA-HAP specimens no change in the surface structure before and after incubation in the simulated body fluid was observed (Figure 38 d,h).
In summary, it was shown that the printed PLA and 90:10 PLA-HAP specimens degraded by ml = 5.2 - 9.7% of their initial mass over 70 days in PBS buffer by hydrolytic cleavage of the poly-mer matrix (Figure 37).101,116 This degradation of the PLA matrix should allow the immigration of bone cells and enable new bone formation.10,11,76 Furthermore, the binary Ca-SiO2 primary parti-cles and the printed PLA-HAP specimens were shown to be bioactive and allow calcium phosphate layer formation (Figure 38). This should improve the incorporation of the bone substitute into the existing bone and enhance the mechanical properties.76,78,82
Figure 38: Bioactivity study in simulated body fluid (SBF) for 3 weeks at 37°C for binary calcium silica (Ca-SiO2) primary particles, 90:10 PLA-HAP, and 100:0 PLA-HAP square specimens. SEM images for binary calcium silica primary particles (a,e) and square 90:10 PLA-HAP (c,g) and 100:0 PLA-HAP (d,h) specimens before (a,c,d) and after (e,g,h) incubation for 3 weeks in SBF (inserts show magnifications of the surface). EDX mapping (b,f) and spectra for binary calcium-silica primary particles (i) and 90:10 PLA-HAP square specimen (j) before and after incubation in SBF.