Activation and Biological Properties of Human β Defensin 4 in Stem Cells Derived From Human Exfoliated Deciduous Teeth

Abstract

Pulpitis in primary teeth, a condition caused by presence of bacteria, is highly prevalent worldwide. The use of biocompatibility materials with anti-inflammatory, anti-bacterial, and regenerative properties is critical for prognosis of this endodontic disease. This study aimed to identify expression of human β defensin 4 (HBD4) in stem cells derived from human exfoliated deciduous teeth (SHED) and characterize the effects of HBD4 on SHED. Quantitative polymerase chain reaction (qPCR) was used to detect HBD4 expression in SHED and the effect of HBD4 on inflammatory factors in lipopolysaccharide (LPS)-stimulated SHED. Affinity measurement was made by the Fortebio Octet System to explore the potential interaction between LPS and HBD4. Western blot analysis was used to explore the effect of HBD4 on mitogen-activated protein kinase (MAPK) pathway. Colony-forming unit methods and scanning electron microscopy were applied to study antimicrobial effect of HBD4 on Fusobacterium nucleatum and Porphyromonas gingivalis. Alkaline phosphatase staining, alizarin red staining, qPCR and western blot were taken to detect effects of HBD4 on osteoblast/odontoblast differentiation of SHED. RT2 Profiler PCR Array was used to explore the potential signaling pathways involved in the osteogenic/odontogenic differentiation. HBD4 was highly expressed in SHED stimulated by TNF-α and IL-1α. HBD4 could bind to LPS directly and down-regulate IL-1α, IL-1β, IL-6, TNF-α in LPS-stimulated SHED, thus the activation of MAPK pathway decreased. HBD4 was sensitive to P. gingivalis and enhanced osteoblast/odontoblast differentiation potential of SHED by modulating Notch pathway. HBD4 was highly expressed in SHED stimulated by proinflammatory cytokines, and possessed anti-inflammatory, anti-bacterial activity. HBD4 promoted osteogenic/odontogenic differentiation of SHED. HBD4 may thus represent a suitable agent for vital pulp therapy in future clinic application.

Potential Application of Human β-Defensin 4 in Dental Pulp Repair

“…HBD4 was significantly upregulated in DPSC following stimulation with inflammatory factors, as previously observed in SHED (Zhai et al, 2019), and was mainly expressed in the cytoplasm of DPSC. HBD4 possessed strong anti-inflammatory activity in vitro.…”

“…cell phenotype differentiation of DPSC by HBD4 was related to Notch signaling pathway. However, the anti-inflammatory mechanism of HBD4 in DPSC differed from that observed in SHED: in SHED, HBD4 mainly exerts its anti-inflammatory action via p42/44 MAPK (Zhai et al, 2019), whereas we found HBD4 appears to act on p38 MAPK and the NF-κB signaling pathway in DPSC. The specific mechanism of inhibition of NF-κB pathway by HBD4 is not clear, but the inhibition effect by other members of HBD family can provide us with clues: C-terminal peptides within HBD3 could penetrate the outer membrane of LPS-treated RAW 264.7 macrophages and downregulate NF-κBdependent inflammation by directly suppressing the degradation of phosphorylated-IκBα and by downregulating active NF-κB p65 (Lee et al, 2015).…”

“…The cells were cultured in four groups: osteogenic induction medium, osteogenic induction medium + 10 μg/mL HBD4, osteogenic induction medium + 1 μg/mL LPS, osteogenic induction medium + 1 μg/mL LPS + 10 μg/mL HBD4. The concentration of LPS used in this study was based on previous data in our laboratory ( Zhai et al, 2019 ). Alkaline phosphatase (ALP) activities of DPSC in the different groups were measured after 7 days incubation with the Alkaline Phosphatase Assay Kit (Beyotime) following the manufacturer’s protocol.…”

“…Moreover, we found HBD4 promoted osteogenic/odontogenic differentiation of SHED. Therefore, HBD4 may represent a suitable agent for VPT in primary teeth ( Zhai et al, 2019 ).…”

Fabrication and Characterization of Calcium-Phosphate Lipid System for Potential Dental Application

“…Irreversible pulpitis in an immature permanent tooth will interrupt root development, causing worse function, and shorter survival time of the tooth. Dental pulps with pulpitis suffer higher expressions of proinflammatory cytokines (IL-1α, IL-1β, IL-6, and TNF-α) and innate immune response (TLR2, TLR4) than pulps without pulpitis (Zhai et al, 2019). It is a great challenge to find a bioactive and anti-inflammatory material for dental pulp regeneration if failed to preserve vital pulp tissue.…”