Abstract
Aims: This study aimed to expand previous projects to assess the impact of freezing media incorporating high molecular weight hyaluronic acid (HA) (1.5 MDa) at 0.1% concentration and dimethyl sulfoxide (DMSO) as a cryoprotectant (CPA) at 3% concentration on Dental Pulp Stem Cells (DPSCs).
Conclusion: In this research, we focused on monitoring the effect of freezing media containing hyaluronic acid (HA) of high molecular weight (1.5 MDa) at a concentration of 0.1% and dimethyl sulfoxide (DMSO) as a cryoprotectant (CPA) at a concentration of 3% on stem cells isolated from dental pulp (DPSCs). The goal was to reduce the concentration of DMSO in the used cryopreservation media. The impact on the number, viability, phenotype, and differentiation ability into osteogenic and chondrogenic cell lines was observed in comparison to two control groups - DPSCs standardly cryopreserved using 10% DMSO (control group 1), and DPSCs cryopreserved using 3% DMSO without the influence of hyaluronic acid (control group 2). The combination of low molecular and high molecular weight cryoprotectants appears to be a way to protect cells both intra- and extracellularly during temperature drops. For DPSC cryopreserved using 3% DMSO and 0.1% HA, we observed the highest average viability over 89%, for control group 1 over 87%, and control group 2 over 85%. No change in shape was observed in any group. The cells maintained a fibroblast-like shape throughout the cultivation period after thawing. For DPSCs cryopreserved using only 3% DMSO without HA, it took twice as long to reach 70-90% confluence after thawing, and even then, the average number of recovered cells was lower compared to control group 1 or group 3. From the data obtained, it can be inferred that 3% DMSO as a cryoprotectant without combining hyaluronic acid is insufficient to protect cells during temperature drops well below the freezing point. The yield of viable cells after thawing is low, and it takes longer for DPSCs to restore their 'functional' health. During phenotype analysis, we observed a decrease in the expression of CD31, CD73, and CD117 in all groups, and a more significant decrease in CD146 expression in groups 2 and 3 compared to the analysis before cryopreservation. Conversely, we observed an increased expression of the CD49f marker in group 3 after cryopreservation. There were no significant differences except for the mentioned increased expression of the CD49f marker in subgroup 3. CD49f belongs to the group of integrins, membrane receptors that bind to the extracellular matrix or other cells. They enable cell adhesion to substrates and also cell migration, indicating their role in tissue development and healing. CD49f is expressed in many types of stem cells and can be considered a 'stemness' marker, as its increasing expression also increases the ability for self-renewal, proliferation, and differentiation capacity of SCDP. DPSCs from all groups retained the ability to differentiate into cells similar to osteoblasts and chondroblasts.