Cryopreservation of human teeth using vitrification method with cryoprotectant cocktails and N-acetylcysteine for banking and clinical applications.

Preserving freshly-extracted healthy human teeth offers an optional resource for potential tooth transplantation and cell therapy. This study aimed to assess the impact of vitrification, utilizing a blend of cryoprotectant agents and N-acetylcysteine (NAC), on the cryopreservation of periodontal ligament tissues, and investigate the underlying mechanisms of NAC on the tooth cryopreservation. Periodontal ligament cells were isolated from freshly-extracted healthy human permanent teeth, and cell sheets of PDLCs were fabricated. The samples including cell sheets, freshly-extracted human and rat teeth were cryopreserved with or without NAC for three months. The viability, ROS level, gene expressions and microstructure of PDLCs within cell sheets were assessed. The expression of SOD-2, Caspase3, LC3A/B and Catalase were evaluated through western blotting. Histological assessments of cryopreserved cell sheets and teeth were conducted. PDLCs were isolated from cryopreserved teeth, and their immunophenotype and differentiation ability were evaluated. The data was analyzed using one-way analysis of variance. The vitrification method showed good performance in preserving the viability and differentiation potential of PDLCs. Cryopreservation supplemented with NAC improved the survival rate of PDLCs, enhanced osteogenic differentiation ability, upregulated the expression of SOD-2 and Catalase, and inhibited cell apoptosis. Additionally, mRNA sequencing analysis revealed a significant activation of the PI3K-AKT pathway following cryopreservation via vitrification. Adding a PI3K-AKT activator improved the survival rates of PDLCs post-cryopreservation. The vitrification strategy combining various CPAs and NAC proved to be feasible for tooth cryopreservation. Targeting the PI3K-AKT pathway may improve the efficacy of tooth cryopreservation.

The role of EMILIN-1 in the osteo/odontogenic differentiation of dental pulp stem cells.

BACKGROUND: Human dental pulp stem cells (hDPSCs) may be the best choice for self-repair and regeneration of teeth and maxillofacial bone tissue due to their homogeneous tissue origin, high proliferation and differentiation rates, and no obvious ethical restrictions. Recently, several studies have shown that extracellular matrix (ECM) proteins can effectively regulate the proliferation and differentiation fate of mesenchymal stem cells (MSCs). However, the role of elastin microfibril interface-located protein-1 (EMILIN-1), a new ECM glycoprotein, in osteo/odontogenic differentiation of hDPSCs has not been reported. The aim of this study was to explore the effect of EMILIN-1 during osteo/odontogenic differentiation of hDPSCs. METHODS: hDPSCs were cultured in osteo/odontogenic induction medium. qPCR and Western blot analysis were performed to detect osteo/odonto-specific genes/proteins expression as well as the expression of EMILIN-1. After knockdown of Emilin-1 in hDPSCs with small interfering RNA and exogenous addition of recombinant human EMILIN-1 protein (rhEMILIN-1), Cell Counting Kit-8 assay, alkaline phosphatase staining, alizarin red S staining, qPCR and Western blot were performed to examine the effect of EMILIN-1 on proliferation and osteo/odontogenic differentiation of hDPSCs. RESULTS: During the osteo/odontogenic induction of hDPSCs, the expression of osteo/odonto-specific genes/proteins increased, as did EMILIN-1 protein levels. More notably, knockdown of Emilin-1 decreased hDPSCs proliferation and osteo/odontogenic differentiation, whereas exogenous addition of rhEMILIN-1 increased them. CONCLUSIONS: These findings suggested that EMILIN-1 is essential for the osteo/odontogenic differentiation of hDPSCs, which may provide new insights for teeth and bone tissue regeneration.

Advanced antibacterial activity of biocompatible tantalum nanofilm via enhanced local innate immunity.

Tantalum (Ta) has been shown to enhance osseointegration in clinical practice, yet little is known about whether Ta nanofilms can be used as antimicrobial coatings in vivo. A highly biocompatible Ta nanofilm was developed using magnetron sputtering technology to further study the mechanism of its antibacterial effects in vivo and elucidate its potential for clinical translation. The Ta nanofilms exhibited effective antimicrobial activity against soft tissue infections but did not show an intrinsic antimicrobial effect in vitro. This inconsistency between the in vivo and in vitro antimicrobial effects was further investigated using ex vivo models. The Ta nanofilms could enhance the phagocytosis of bacteria by polymorphonuclear neutrophils (PMNs, neutrophils), reduce the lysis of neutrophils and enhance the proinflammatory cytokine release of macrophages. This accumulative enhancement of the local host defenses contributed to the favorable antibacterial effect in vivo. The alleviated osteolysis observed in the presence of the Ta nanofilms in the osteomyelitis model further proved the practicality of this antibacterial strategy in the orthopedic field. In summary, Ta nanofilms show excellent biocompatibility and in vivo antimicrobial activity mediated by the enhancement of local innate immunity and are promising for clinical application. STATEMENT OF SIGNIFICANCE: In this study, Ta nanofilms were deposited on titanium substrate by magnetron sputtering. Ta nanofilms exhibited excellent in vivo and in vitro biocompatibility. In vivo antimicrobial effects of Ta nanofilms were revealed by soft tissue infection and osteomyelitis models, while no direct antibacterial activity was observed in vitro. Comprehensive ex vivo models revealed that Ta nanofilms could enhance the phagocytosis of bacteria by neutrophils, reduce the lysis of neutrophils and promote the release of proinflammatory cytokines from macrophages. This immunomodulatory effect helps host to eliminate bacteria. In contrast to traditional antimicrobial nanocoatings which apply toxic materials to kill bacteria, this work proposes a safe, practical and effective Ta nanofilm immunomodulatory antimicrobial strategy with clinical translational prospect.