Human Calvarial Osteoblasts (HCO)

E. faecalis Infection Triggered Apoptosis in Primary Osteoblasts

Chronic apical periodontitis (PAP) results in bone destruction because of an imbalance in bone turnover and development, typically with accelerated osteoblast apoptosis. One of the most significant microbes linked to PAP is Enterococcus faecalis, which causes osteoblast death. It is not clear whether it is involved in primary human osteoblast apoptosis, although the BCL-2 family is known to modulate apoptotic processes.

In this work, Li's team characterized the apoptotic activity of E. faecalis OG1RF in primary human calvarial osteoblasts in an attempt to discover targets for PAP. For measuring apoptosis in osteoblasts infected with E. faecalis OG1RF, they stained them with Annexin V-FITC/PI by flow cytometry (Fig. 1a). Apoptotic cells increased rapidly at 6 hours for a MOI of 500 and 1,000, and increased MOI-dependently at 12 hours (Fig. 1b). Early apoptosis reached a maximum at 6 hours with an MOI of 500, while late apoptosis emerged at MOIs of 500 and 1,000 at 6 and 12 hours respectively. JC-1 staining for mitochondrial membrane potential (ΔΨm) measurements indicated a significant decrease in (ΔΨm) over increasing MOIs, demonstrating intrinsic apoptosis activation (Fig. 2). For 12 hours, Caspase-3, 8/9 activity was high at MOI 1000 indicating that both intrinsic and extrinsic apoptotic pathways were involved (Fig. 3).

Fig. 1. Analysis of E. faecalis-induced apoptotic osteoblasts in early and late phases using Annexin V-FITC/PI staining (Li Y, Sun S, et al., 2022).

Fig. 2. Analysis of the ΔΨm in primary osteoblasts infected with E. faecalis OG1RF using JC-1 staining (Li Y, Sun S, et al., 2022).

Fig. 3. Intrinsic and extrinsic apoptotic pathways were involved in E. faecalis-infected osteoblasts (Li Y, Sun S, et al., 2022).

Effects of Equibiaxial Mechanical Stretch on Cell Viability in Human Calvarial Osteoblasts

Craniofacial sutures are essential for bone formation, and they can also carry mechanical stretch, which regulates bone development by acting on the sutures before they close. Orthodontists use mechanical stretch to repair craniofacial sutures. These remodeling activities involve osteoblast responses to mechanical stretching, such as differentiation, apoptosis and ECM production.

Zhang's team explored the effect of mechanical stretch on cell viability in human calvarial osteoblasts. Morphological alterations occurred in human calvarial osteoblasts (HCObs) after 12 hours of equibiaxial stretch. Light microscopy showed that the stretched cells expanded and shifted away from the force direction, but not the randomly placed control cells (Fig. 4). HCObs (area, perimeter, width, length) were bigger in the stretch-treated patients than in controls. At 12 and 24 hours, caspase 3/7 was significantly lower in stretch-treated HCObs than in controls (P < 0.05) (Fig. 5A). 24 hours stretching also reduced caspase 3/7 activity. The MTT results showed that 24 hours of stretching led to higher HCObs proliferation compared with control (Fig. 5B), more prolific at 24 hours than at 6 and 12 hours. CellTiter-Glo tests showed a significant increase in cell viability between stretch-treated HCObs and controls 6 and 24 hours later, which suggests that 2% lengthening at 0.2 Hz increases HCObs viability (Fig. 5C). 

Fig. 4. Human calvarial osteoplasts (HCObs) exhibited altered morphology after an intermittent deformation of 2% for 5 s (0.2 Hz) every 60 s for 12 h (Zhang J, Xu S, et al., 2019).

Fig. 5. (A) Effect of mechanical stretch on the activities of caspase 3/7. (B) Effect of mechanical stretch on cell proliferation. (C) Effect of mechanical stretch on cell viability (Zhang J, Xu S, et al., 2019).