DOI: 10.14457/tu.the.2022.1468
title: Effects of biosynthesis oroxylum indicum/silver nanoparticles on biological behaviors of the encapsulated stem cells

description: Background: Biosynthesized silver nanoparticles (AgNPs) with Oroxylum indicum (Pheka) as reducing agent (Pheka/AgNPs) were introduced as alternative biomaterial to combat tissue destruction caused by bacterial toxins and inflammatory cytokines and promote bone regeneration in peri-implantitis defects. The goal was to improve treatment outcomes for peri-implantitis and promote alveolar bone regeneration. Objective: To investigate and compare biologic effects of the extracts from the stem bark of the Oroxylum indicum (Pheka) and biosynthesis silver nanoparticles (Pheka/AgNPs) on human periodontal ligament stem cells (hPDLSCs) that were under oxidative stress and inflammatory stimulus, including growth and osteogenic differentiation of hPDLSCs on two-dimensional cell culture and the hydrogel encapsulated cells. Materials and methods: Results were compared with effects of quercetin (QT) as a reference flavonoid. Cytotoxicity, antioxidative effects, and anti-inflammatory effects and effects on growth and osteogenic differentiations of Pheka and Pheka/AgNPs on hPDLSCs were investigated in two- and three-dimensional cell cultures. The present research was divided into two parts: I) two-dimensional (2D) cell culture and II) three-dimensional (3D) cell culture or cell encapsulation. According to different cell culture medium supplements, four study groups were created: A) growth medium alone (negative control); B) growth medium with Oroxylum indicum crude extract (Pheka); C) biosynthesized Pheka/AgNPs; and D) Quercetin (positive(2)control). Approved by the Ethical Review Sub-Committee Board for Human Research Involving Sciences, Thammasat University, No.3 and patient informed consent, third molars and premolars were collected from 10 healthy patients aged 18 to 25 undergoing surgical removal or tooth extraction at the Dental Clinic, Thammasat University Hospital. In Part I, hPDLSCs were exposed to culture media supplemented with Pheka or Pheka/AgNPs according to study groups, and cell viability assay was performed to determine working concentrations for each supplement. Human PDLSCs were stimulated with oxidative stress and inflammatory stimulus caused by hydrogen peroxide (H2O2) and purified Porphyromonas gingivalis lipopolysaccharide (LPS), respectively. In addition, Pheka/AgNP endocytosis was examined by transmission electron microscopy (TEM). In Part II, hPDLSCs were encapsulated in an in-house thermosensitive 2% (w/v) nano-hydroxyapatite – 2% (w/v) calcium carbonate microcapsules - 10% beta-glycerophosphate- 4:1 (w/w) chitosan/collagen hydrogel (nHA-CaCO3-Chitosan/collagen hydrogel) to recapitulate natural restricted environment of human mesenchymal stem cells (hMSCs) during stem cell transplantation. Then hydrogel-cell suspensions were seeded on a 24-well cell culture plate and developed in group A, B, C and D culture mediums for 21 days. After that, live/dead cell staining and cell viability assays, TEM and enzyme-linked immunosorbent assays (ELISA) of secreted inflammatory cytokines, interleukin-1 beta (IL-1β), prostaglandin E2 (PGE2) and transforming growth factor-beta 1 (TGF-β1) in culture mediums and characterizations of osteogenic differentiation of hPDLSCs were performed (p&lt;0.05, N=3-5, Mean±SD). Results: 1) Working concentrations of Pheka, Pheka/AgNPs were 5 parts per million (ppm) and 20 ppm and for QT, 2.5 ppm, and 5 ppm; 2) biosynthesized Pheka/AgNPs were non-cytotoxic and internalized into cell cytoplasm and endosomes. Human PDLSCs with AgNPs endocytosis exhibited strong cell viability, intact cell and nuclear membranes, and exosomes for AgNPs exportation from cells as exhibited by TEM; 3) Pheka and Pheka/AgNPs could decrease adverse effects of oxidative stress and inflammatory stimulus on cell viability of hPDLSCs similarly to a reference flavonoid, QT; 4) Pheka and Pheka/AgNPs could promote growth and osteogenic differentiation of hPDLSCs on cell culture plates and hydrogel encapsulated cells; and 5)(3)Pheka/AgNPs enhanced biological functions of Pheka on hPDLSCs. Conclusions: Biosynthesized Pheka/AgNPs were non-cytotoxic to hPDLSCs, and biosynthesized silver nanoparticles could enhance bioactivity of the flavonoid Pheka. It was hypothesized that hydrogel impregnated with Pheka/AgNPs could decrease degrees of inflammatory response and tissue damage caused by bacterial toxins and inflammatory cytokines in periodontal defects as well as promote osteogenic differentiation of host and transplanted cells in skeletal defects. Effects on bone regeneration of biosynthesized Pheka/AgNPs in peri-implantitis in an animal model should be further investigated.