Improvement of ECM-based bioroot regeneration via N-acetylcysteine-induced antioxidative effects

Abstract

Background: The low survival rate or dysfunction of extracellular matrix (ECM)-based engineered organs caused by the adverse effects of unfavourable local microenvironments on seed cell viability and stemness, especially the effects of excessive reactive oxygen species (ROS), prompted us to examine the importance of controlling oxidative damage for tissue transplantation and regeneration. We sought to improve the tolerance of seed cells to the transplant microenvironment via antioxidant pathways, thus promoting transplant efficiency and achieving better tissue regeneration.

Methods: We improved the antioxidative properties of ECM-based bioroots with higher glutathione contents in dental follicle stem cells (DFCs) by pretreating cells or loading scaffolds with the antioxidant NAC. Additionally, we developed an in situ rat alveolar fossa implantation model to evaluate the long-term therapeutic effects of NAC in bioroot transplantation.

Results: The results showed that NAC decreased H₂O₂-induced cellular damage and maintained the differentiation potential of DFCs. The transplantation experiments further verified that NAC protected the biological properties of DFCs by repressing replacement resorption or ankylosis, thus facilitating bioroot regeneration.

Conclusions: The following findings suggest that NAC could significantly protect stem cell viability and stemness during oxidative stress and exert better and prolonged effects in bioroot intragrafts.

Keywords: Bioroot regeneration; Dental follicle stem cell; N-Acetylcysteine; Oxidative stress; Treated dentin matrix.

Fig. 1

Characterization of DFCs, establishment of H₂O₂-induced oxidative stress model, and determination of particular NAC treatment dose. a Characterization of DFCs. Primary DFCs were adherent to plastic and showed colony-forming abilities. TEM evaluations showed homogeneous electron-dense granules without membranous structures (indicated by yellow arrow). After being cultured in osteogenesis and adipogenesis-inducing media for 14 days and in neuronal-inducing medium for 2 h, the cultured DFCs showed mineralized nodules, lipid clusters, and positive βIII-tubulin expression, respectively. b Effect of different concentrations of H₂O₂ on the viability of hDFCs. c Effect of different concentrations of NAC on the viability of hDFCs. d Effect of NAC on the viability of hDFCs under oxidative stress conditions induced by 150 μm H₂O₂. e Effect of NAC on the proliferation of hDFCs for 1 week. f Assessment of ROS levels using fluorescence microscopy and statistical analysis of DCFH fluorescence from three experiments. Asterisks indicate statistically significant differences compared with the control group. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001

Fig. 2

The effect of NAC and the H₂O₂-induced intracellular oxidative stress status on TDM/DFCs system. a Protein release of the TDM scaffold. b Effect of different concentrations of hTDM protein on the viability of hDFCs. c GSH level. d The level of ROS in hDFCs was measured by DCFH-DA with a fluorescence microscope. e statistical analysis of (d)

Fig. 3

The effect of NAC on the proliferation, migration and osteogenic differentiation of hDFCs under H₂O₂-induced oxidative stress. a Effect of combination drug treatment on the viability of hDFCs. b After 24 h of culture in Transwell plates, crystal violet staining was performed. c Pretreatment with NAC led to increased hDFC migration to the opposite side of the membrane compared with that observed in the H₂O₂-treated groups. d The effect of NAC on the osteogenic differentiation of hDFCs stimulated with H₂O₂, as determined by Alizarin Red staining. e RT-PCR analysis of Collagen I, ALP, RunX2, and Periostin mRNA expression. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001

Fig. 4

Allogeneic transplantation of bioroot composites in a Sprague-Dawley rat bone defect model for 8 weeks. A (a) GFP-labelled rDFCs. A (b) Construction of cell sheets. A (c) Histological section of the TDM scaffold combined with intrinsic fibre three-dimensional dental pulp extracellular matrix, as determined by haematoxylin and eosin (HE) staining. Images of PI staining to detect the survival of grafted cells on B day 1 postimplantation and C day 3 postimplantation. TDM, treated dentin matrix; DPEM, dental pulp extracellular matrix

Fig. 5

Allogeneic transplantation of bioroot composites in the alveolar fossa of Sprague-Dawley rats for 8 weeks. Images of TRAP, HE, and Masson staining to detect the efficiency of transplantation at a 1 week postimplantation and b 2 months postimplantation. TDM, treated dentin matrix; AB, alveolar bone; CE, cementum; DP, dental pulp; margin of the TDM scaffold (white dashed line); neovascularization (yellow arrows)