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Hyaluronic Acid Induction Promotes the Differentiation of HumanNeural Crest-like Cells into Periodontal Ligament Stem-like Cells

Dr. Jun Tomoshikiyo et al. of Graduate School of Dentistry, Hokkaido University presented

Hyaluronic Acid Induction Promotes the Differentiation of HumanNeural Crest-like Cells into Periodontal Ligament Stem-like Cells

Our NANON was used in the paper “Hyaluronic Acid Induction Promotes the Differentiation of HumanNeural Crest-like Cells into Periodontal Ligament Stem-like Cells! The following is a summary of the paper.

When the PDL is damaged beyond repair, periodontitis can progress and teeth can be lost. Periodontal tissue contains periodontal tissue stem cells (PDLSCs) that have the ability to regenerate periodontal tissue, but the population of PDLSCs in periodontal tissue is thought to be very small, limiting the transplantation of autologous PDLSCs.

Neural crest (NC) cells can differentiate into a variety of cells and tissues, and some NC cells can remain in cranial NCs to give rise to PDLSCs. This suggests that differentiation of NC cells into PDLSCs in periodontal tissues may promote periodontal tissue regeneration.

However, the difficulty in obtaining large numbers of NC cells is the main factor limiting the application of NC cells in regeneration research.

Therefore, we focused on SK-N-SH cells, a neuroblastoma cell line, as a method to culture periodontal tissue stem cells for periodontal tissue regeneration, and developed a new scaffold for cell culture.

I want to take good care of my teeth because I don’t want them to disappear, but it is also difficult to take care of them.
If there is a way to regenerate them, it would be very helpful!


First, PDL tissue was detached from the root surfaces of human teeth and cultured to obtain human PDLCs (HPDLCs). This HPDLC was further cultured, processed and detached to obtain plates coated with HPDLC-derived ECM (HPDLCs-ECM: HPDLCs-ECM).

SK-N-SH cells were seeded onto these plates and cultured to obtain SK-N-SH cell-derived PDLSC cells (SK-PDLSCs). qPCR analysis showed that the expression of PDL-related genes was predominantly higher in SK-PDLCs compared to SK-N-SH cells. PDL-related genes were found to be predominantly higher in SK-PDLCs compared to SK-N-SH cells.

We also analyzed the expression levels of osteoblast- and adipocyte-related genes to determine the multipotent differentiation potential of SK-PDLSCs. The expression levels of both osteoblast- and adipocyte-related genes were predominantly higher in SK-PDLSCs. These results prove that SK-N-SH cells are a useful model for inducing differentiation of PDLSCs from NCs.

Previous studies have shown that differentiation of iPS cell-derived NCs (iNCs) into iPDLSCs resulted in higher expression of hyaluronic acid (HA)-related genes. In the current study, we also analyzed the expression levels of HA-related genes in iNCs and iPDLSCs, and found that the expression of HA-related genes was the same as in previous studies. Based on this finding, we observed the expression of HA-related genes in SK-N-SH cells and SK-PDLSCs, and found that HA-related genes were more highly expressed in SK-PDLSCs than in SK-N-SH cells.

CD44, a surface receptor for HA, induces the initiation of intracellular signaling pathways when activated. This CD44 antigen is expressed in periodontal tissues, and HA-CD44 interactions have been associated with PDL cell proliferation and calcification.

We used CD44-expressing SK-N-SH cells to investigate the involvement of HA signaling in differentiation into SK-PDLSCs; when CD44+ SK-N-SH cells were cultured on wells coated with HPDLSCs-ECM, PDL-related gene expression of PDL-related genes was significantly higher. In addition, experiments using siRNA confirmed that inhibition of CD44 reduced differentiation ability.

Based on these results, CD44 was determined to be an important regulator of SK-N-SH cell to SK-PDLSC differentiation.

From previous studies, it has been reported that HA molecules affect the differentiation of stem and progenitor cells of various species. Based on this finding, we investigated the effect of HA on SK-PDLSCs differentiation from SK-N-SH cells using high molecular weight hyaluronan (HMWHA) and low molecular weight hyaluronan (LMWHA) in the presence of HPDLCs-ECM and LMWHA we found that SK-PDLSCs cultured in the presence of HPDLCs-ECM and LMWHA showed predominantly higher expression levels of CD44 and PDL genes upon initial stimulation with LMWHA. Next, nanofiber membranes were prepared from PCL solution mixed with LMWHA using electrospinning technology.

Made with our NANON Electrospinning nanofiber membranes!

     

After confirming that the concentration of HA released from the nanofiber membrane was sufficient, cell culture was performed by placing the nanofiber membrane on cells in wells seeded with SK-N-SH cells, and the expression of CD44 and PDL-related genes was examined from the cultured cells. The results showed that SK-N-SH cells cultured in the presence of HPDLCs-ECM and LMWHA mixed nanofiber membranes had higher expression of CD44 and PDL-related genes. This may be due to the release of HA in the membrane and increased expression of PDL-related genes upon continuous stimulation.

Nanofibers are effective for drug delivery systems because of their high specific surface area and good cell adhesion.

In summary, we found that SK-N-SH cells have utility as a cell culture model for periodontal tissue regeneration. In addition, the use of biodegradable scaffolds that release LMWHA is expected to activate and differentiate NCs in the PDL, increase the number of PDLSCs, and regenerate destroyed periodontal tissue.
They plan to continue their research by conducting experiments to see if it can be adapted in vivo.

Nanofibers are often used in cell culture scaffolds, but the idea of using nanofibers mixed with particles to stimulate cell expression by adding stimulation through a drug delivery system in addition to culture was interesting and very informative! I am looking forward to seeing the future development.


M.Anas Alhasan , Atsushi Tomokiyo,et al. (2023)MDPI. Hyaluronic Acid Induction Promotes the Differentiation of HumanNeural Crest-like Cells into Periodontal Ligament Stem-like Cells (https://www.mdpi.com/2073-4409/12/23/2743)