The Research Of The Bioactive Glass Microspheres With Different Particle Sizes And Their Interaction With MC3T3-E1 Cells In Vitro

Mono-disperse spherical mesoporous nano- and micro- bioactive glass particles(NMBGs) have potential application in gene and drug delivery, bone tissue engineering and wound healing However, their size-dependent interaction with osteoblasts has never been studied.NMBGs were successfully synthesized through the modified sol-gel method using DDA as a hydrolysis catalyst. By changing the concentration of DDA, NMBGs with different diameters(61 nm, 174 nm, 327 nm, 484 nm, 647 nm, 743 nm, 990 nm and 1085 nm) were obtained. NMBGs were largely mono-dispersed, spherical with hierarchical pores structure with a relatively narrow particle size distribution.Ca atoms located close to the surface of NMBGs, which formed a Ca ionic shell-model. Ionic dissolution products from inorganic materials are informative and critical for understanding the behavior of these materials in vitro and in vivo. Degradation experiments in SBF showed that Ca ion concentration reached a peak in 1 day; Si ion reached a stable stage after 3 d of releasing. On the particle surface, a large number of needle-like crystalization of hydroxyapatite particles was formed and the internal structure of the particles remained intact after 3 d. We found that cell cytotoxicity and proliferation showed a general tendency of size-, concentration- and time-dependency in all NMBGs groups. The larger particle size and higher concentration of NMBGs leaded to greater toxicity. Surprisingly, we also found that the cell viability will be recovered over time when cultured with lower concentration and smaller particles. It demonstrated that the effect of NMBG particles on cell viability wasn’t permanent and reversible in some cases. The cells apoptotic was only induced by NMBGs at the concentration of 150 μg/m L. The amount of apoptotic cells increased along with the increase of particles size. Meanwhile, the morphological change of MC3T3-E1 cells was also size-, concentration- and time-dependent in all groups. The larger particles(Groups-743 nm, 990 nm, 1085 nm) tended to result in more cell apoptosis manifested by cell volume reduction, nuclear condensation, cytoplasmic vacuoles, cell shrinkage, and formation of apoptotic bodies or cellular debris. However, the morphological changes of MC3T3-E1 cells cultured with the smaller particles(Group-61 nm, Group-174 nm and Group-327 nm) could be recovered over time. Microstructure changes of MC3T3-E1 cells were also size-dependent. With the size increase, the transport vesicle increased, the microvillus of the surface decreased, and the ridges of mitochondria damaged.NMBGs adhereing to the cell membrane above the periphery of the cells can initiate endocytosis. Once internalized, the sizes of NMBGs played a significant role in determining their intracellular localization. When the NMBGs were smaller than 174 nm, they were transported via the lysosomal pathway and phagocytized in lysosomes, resulting in little cytotoxicity. In contrary, larger NMBGs(over 327 nm) escaped from the lysosomes after endocytosis, and were re-localized inside the intra-cytoplasmic vacuoles or random presence in the cytoplasm of the cells. This escape phenomenon from lysosomes handling and induced cell apoptosis and the cytotoxicity.The NMBGs were uptake by MC3T3-E1 cells which can host the particles for 7, 10, 14, 35 and 49 days. The results suggested that the NMBGs gradually degraded over time, which can take 49 days or longer. Calcium ions were the first to release from NMBGs, and could be completely released within 10 days.Phosphate was dissociated from the silicate network and released into the cytoplasm during a course of 7 days. The decrease of the Ca, Si and P on the particle surface resulted in a reduced silicate network connectivity. In this process, NMBGs maintained approximately a spherical or ellipsoidal morphology, and the surface became rougher. At last, the particle collapsed and disappeared in situ, and only trace debris was found in the cytoplasm. The localization of NMBGs in the cells dictates the degree of the degradation; and the NMBGs degrade faster in the endosome than in the cytoplasm.Cell adhesion experiments showed that the cells were completely spreadout around the NMBGs surface. The degree of cells spreading on NMBGs(except Group-1085 nm group) was greater than in Blank group. The cells attached to the Group-1085 nm and Group-77 S were triggered to rupture and die. It indicated that the large particles are not conducive to cell adhesion. Cell adhesion behaved different from above description following endocytosis of NMBGs. Some particles could be internalized in 30 min. Cell adhesion after uptaking NMBGs was size-dependent at 150 μg/mL and the number of cells adhesion declines with increase of particle size, especially for Group-990 nm and Group-1085 nm in which both had statistically significant cell adhesion(p <0.05) compared with Blank.MC3T3-E1 cells with internalized NMBGs exhibited disrupted and poorly organized F-actin cytoskeleton. The results showed a size- and dose-dependent pattern that the larger particle sizes and higher concentrations resulted in greater damage or poorer formation of the microfilaments. The degree of damaged cell microfilaments was in correlated with cell migration. Cell migration experiment with 10% FBS was mainly affected by particle size. The result showed that the migration of cells was size-dependent and the migration speed decreased with increase of the particle size. Cell-cell adhesion of NMBGs groups is more compact than Group-77 S in which the cell-cell adhesion failed, and the particle deposition in the scratch area hindered the leading cell migration.Our work shows NMBGs with a smaller size(61-327 nm) at lower doses(below 100 μg/m L) were minimally cytotoxic and may have biomedical applications such as in bone tissue engineering scaffolds and gene or drug delivery.