| The role of silicon in the formation of new bone was established in the early 1970s. Silicon was found to be involved in the early stages of bone calcification. Silica-based materials, such as dicalcium silicate (Ca2SiO4, C2S), release silicon ions, which have important roles in skeletal development and repair. C2S possesses excellent bioactivity when used as a coating material for titanium alloy substrates. The in vitro and in vivo bioactivities and biocompatibilities of such implants increase when they are coated with a-tricalcium phosphate (aTCP) doped with C2S. In addition, aTCP doped with C2S promotes the differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts and supports human adipose stem cell adhesion and spreading. A-TCP cements modified with β-C2S exhibit the best properties in vitro and in vivo. Furthermore, electrospun poly (L-lactic acid) (PLLA) scaffolds containing C2S were shown to markedly promote the proliferation and osteogenic differentiation of MC3T3-E1 cells. Tissue engineering scaffolds containing C2S significantly enhanced the proliferation of MG-63 cells by stimulating the transcription of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7). This research shows that C2S has the potential to be used as a bone substitute. In dentistry, C2S cement has adequate biological properties and can be used as a root-end filling material and a pulp capping material, as it exhibits good bioactivity and biocompatibility in vitro studies. In addition, C2S cement exhibits high apatite-forming activity and low degradation in acidic environments when used as a root-end filling material Regarding its cytotoxicity, C2S cement is significantly superior to the traditional root-end filler, mineral trioxide aggregate (MTA). C2S cement is also a model system for drug release. These findings support the broad use of C2S in future orthopedic and dentistry applications.Numerous coating materials and bone substitute biomaterials in orthopedic and dental surgery possess potential proinflammatory effects. For example, hydroxyapatite (Ca10 (PO4)6(OH)2, HA) is considered relatively safe due to its bioactivity and biocompatibility. However, particle release from prostheses can cause aseptic inflammation depending on its amount and frequency. The interactions between HA particles and human monocytes lead to the production of inflammatory mediators, such as tumor necrosis factor alpha (TNF-a) and metalloproteinases. These inflammatory mediators trigger an immune response. The released particles also trigger the release of immune mononuclear cells, leading to phagocytosis and the activation of associated immune signaling pathways. Bone substitute materials, including biphasic calcium phosphate, titanium alloys, polymethylmethacrylate (PMMA) and aluminum, have been shown to induce different proinflammatory mediators. These proinflammatory effects have been studied using mixed cell popμ Lations, such as peripheral blood mononuclear cells, which include both lymphocytes and monocytes. However, the cytotoxicity of C2S particles in cell culture remains unclear because most previous studies have only focused on the safety of C2S coatings, biomaterials doped with C2S and C2S cements. Direct contact between cells and particles may cause a different effect. Furthermore, the potential proinflammatory effects of C2S particles have never been examined in immune cells. In the present study, we investigated the safety of C2S particles when directly cultured with immune and osteoblast-like cells. Second, we evaluated whether the particles induced the release of proinflammatory mediators and whether these effects were different from those caused by HA particles. Finally, we compared the abilities of C2S and HA to promote osteogenic effects. There are three parts in our work, which are briefly described as follows.Part I Cell proliferation and apoptosis profiles of C2S and HAThe cell viabilities in all experiments were greater than 95% (before and after powder exposure). Cell proliferation was evaluated using CCK-8 assays. The Relative growth rate (RGR) of PBMC culture with C2S after 24 h is 95.38±3.79% at SAR 10:1, 93.88±4.67% at SAR 5:1%,98.05±1.39% at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of PBMC culture with HA after 24 h is 90.00±7.61% at SAR 10:1,94.93±2.08% at SAR 5:1,91.73±3.74% at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of PBMC culture with C2S after 48 h is 88.48±11.79% at SAR 10:1,88.17±9.57% at SAR 5:1,92.81±18.52% at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of PBMC cμ Lture with HA after 48 h is 91.94±24.37% at SAR 10:1,95.95±6.50% at SAR 5:1,95.36±23.85% at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of THP-1 culture with C2S after 24 h is 89.10±13.02% at SAR 10:1,84.44±12.86% at SAR 5:1,86.85±20.41% at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of THP-1 culture with HA after 24 h is 99.23±27.40% at SAR 10:1, 97.14±7.88% at SAR 5:1,91.44±28.56%at SAR 1:1. The Classification of cytotoxicity is level 1. The Relative growth rate (RGR) of THP-1 culture with C2S after 48 h is 92.03±3.68% at SAR 10:1,98.49±7.00% at SAR 5:1, The Classification of cytotoxicity is level 1.109.52±3.06% at SAR 1:1. The Classification of cytotoxicity is 0. The Relative growth rate (RGR) of THP-1 culture with HA after 48 h is 105.24±2.39% at SAR 10:1,110.01±6.95% at SAR 5:1,103.46±2.45% at SAR 1:1. The Classification of cytotoxicity is 0. The Relative growth rate (RGR) of MG-63 culture with C2S after 24 h is 125.57±8.90%, at SAR 10:1,119.59±11.32% at SAR 5:1,108.36±3.06% at SAR 1:1. The Classification of cytotoxicity is 0. The Relative growth rate (RGR) of MG-63 culture with HA after 24 h is 120.09±9.15% at SAR 10:1,110.06±5.43% at SAR 5:1,119.73±15.20% at SAR 1:1. The Classification of cytotoxicity is level O.The Relative growth rate (RGR) of MG-63 culture with C2S after 48 h is 87.05±1.35%, at SAR 10:1,95.88±3.16% at SAR 5:1,93.86±3.30% at SAR 1:1. The Classification of cytotoxicity is 1. The Relative growth rate (RGR) of MG-63 culture with HA after 48 h is 84.50±4.00% at SAR 10:1,88.60±6.14% at SAR 5:1,95.59±4.06% at SAR 1:1. The Classification of cytotoxicity is level 1.No apparent cytotoxicity was detected after primary human monocytes, THP-1 cells and MG-63 cells were exposed to C2S and HA for 24 and 48 h. More than 85% of the cells were viable in all experimental groups. Flow cytometry was performed using a FITC Annexin V Apoptosis Detection Kit to confirm the CCK-8 viability result. The result showed no obvious apoptosis in primary human monocytes treated with different SARs. Approximately 4% of THP-1 cells in the C2S group and 2% of cells in the HA group underwent apoptosis at an SAR of 10:1. No obvious apoptosis was observed at SARs of 5:1 and 1:1 in the C2S or HA group. Approximately 3% of MG-63 cells in the C2S and HA groups underwent apoptosis at an SAR of 10:1. The rates of apoptosis in the C2S and HA groups did not differ from that of the control group at SARs of 5:1 and 1:1. For the remaining experiments, an SAR of 1:1 was chosen based on a previous study and on the actual amounts released from implants.Part Ⅱ expression and production of proinflammatory mediators in THP-1 cellsA key issue related to biomaterials is the degradation of the extracellular matrix by proteases secreted by inflammatory cells in response to these materials. To examine the effects of C2S and HA particles on MMP expression in THP-1 cells, the steady-state mRNA levels of MMP-2 and MMP-9 were determined using qRT-PCR analyses. Cells were harvested after cultivation for 6 or 24 h. LPS- and zymosan-treated cells were used as positive control groups. Constitutive low levels of MMP-2 and MMP-9 mRNA were observed in the C2S and HA groups after 6 h (P>0.05). At this time point, high levels of MMP-2 and MMP-9 mRNA were observed in the positive controls and LPS-stimulated cells in the presence of particles (P<0.05). After 24 h, MMP-2 and MMP-9 mRNA expression significantly increased in the C2S and HA groups (P<0.05), and the C2S particles had a significantly weaker effect on MMP-9 mRNA expression compared with HA particles (P<0.05). No significant difference was observed in the MMP-2 mRNA expression between C2S and HA groups (P>0.05). The positive control group and the LPS-stimulated cells expressed higher levels of MMP-2 and MMP-9 after 24 h of exposure to particles versus 6 h of exposure to particles. C2S and HA had no modifying effect on MMP-2 and MMP-9 mRNA expression in LPS-stimulated cells.Next, we estimated the levels of the MMP inhibitors TIMP-2 and TIMP-1. No significant decreases in TIMPs were detected after 6 h of cultivation in either the C2S or HA group. The positive control group and the LPS-stimulated cells expressed lower levels of TIMP-2 and TIMP-1 mRNA in the presence of particles (P<0.05). After 24 h, the expression of TIMP-2 and TIMP-1 mRNA clearly decreased in the positive control group and in LPS-stimulated cells in the presence of particles (P<0.05). TIMP-1 mRNA expression significantly decreased in the C2S and HA groups (P<0.05). Although the TIMP-2 mRNA expression decreased in the C2S and HA groups, no significant differences were observed when these groups were compared with the control group.Subsequently, TNF-α mRNA expression was detected. Zymosan and LPS induced high levels of TNF-α expression after 6 and 24 h. No differences were noted in the C2S and HA groups after 6 h (P>0.05). After 24 h, C2S and HA promoted TNF-α mRNA expression significantly (P<0.05). C2S particles had a weaker effect on TNF-α mRNA expression compared with HA particles (P<0.05). Based on our qRT-PCR results, ELISAs were used to detect the effects of C2S particles on the production of MMPs and their inhibitors (TIMPs) after 24 h. The MMP-2 and MMP-9 concentrations were increased in the C2S and HA groups compared with the control group (P<0.05). The same result were observed when mRNA expression was analyzed. No significant differences in the MMP-2 concentration were observed between the C2S and HA groups (control:1.55±1.16 ng/mL, C2S:15.84±3.5 ng/mL, HA:13.71±2.76 ng/mL). Nevertheless, the HA particles induced a 2-fold increase in the MMP-9 concentration compared with the C2S particles (control:2.66±0.47 ng/mL, C2S:15.16±1.81 ng/mL, HA:36.02±1.69 ng/mL, P<0.05). The C2S and HA particles did not alter the MMP-2 and MMP-9 concentrations in LPS-stimulate cells. Next, we examined TIMP-2 and TIMP-1 levels. The TIMP-2 concentration was not greatly altered in the presence of C2S or HA particles. TIMP-1 is an important MMP-9-specific inhibitor, and a clear decrease in TIMP-1 secretion was observed in the C2S and HA groups in vitro (P<0.05). HA particles triggered a stronger reduction in TIMP-1 expression (P<0.05, control: 4.13±0.50 ng/mL, C2S:1.71±0.39 ng/mL, HA:0.96±0.20 ng/mL). A balanced MMP: TIMP ratio plays a significant role in bone remodeling; therefore, the MMP:TIMP ratios were evaluated. The MMP-2:TIMP-2 and MMP-9:TIMP-1 ratios were increased in the C2S and HA groups. HA induced a larger imbalance in the MMP-9: TIMP-1 ratio compared with C2S.Finally, we measured the TNF-a concentration in all groups. The HA particles induced a 2-fold increase in TNF-α production compared with the C2S particles (P<0.05, control:100.07±35.51 pg/mL, C2S:228.44±39.68 pg/mL, HA: 419.80±89.94 pg/mL). LPS and zymosan were stronger inducers of TNF-α. The C2S and HA particles did not alter the effects of LPS on the monocytes.In this study, we demonstrated that C2S particles are not cytotoxic to primary human monocytes, THP-1 cells or osteoblast-like cells. These particles increased the production of TNF-α, MMP-2 and MMP-9 and decreased the production of their inhibitors, TIMP-2 and TIMP-1. Furthermore, compared with HA particles, C2S particles have less influence on MMP-9, TIMP-2 and TNF-α, indicating that C2S may be a much safer biomaterial. Hence, these findings provide evidence supporting the adoption and widespread use of C2S as coatings for prostheses or dental implants and as bone substitute materials.Part Ⅲ cytotoxicity of C2S coating and 45S5 coatingAn atmosphere plasma spray (APS) system was applied to fabricate coating on Ti-6A1-4V substrate with dimensions of 10 mm×10 mm×1mm. Characterizations of dicalcium silicate and 45 S5 coating was analysied.To evaluate cytotoxicity induced by dicalcium silicate disc and 45S5 disc, mouse macrophage cell line RAW 264.7 were used. The viability of cells was evaluated by trypan blue exclusion test. The viability was>85% in all of the experiments (before and after exposure to coating discs). The cell proliferation effects were evaluated by CCK-8 assays kit. No obvious cytotoxicity was detected after RAW 264.7 macrophages cultured with dicalcium silicate and 45S5 coating discs after 24 h and 48 h.In this study, we demonstrated that dicalcium silicate and 45S5 coating disc have no cytotoxicity when contact with macrophages. |
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