Characterization of Fibronectin Adhesion Isolated Dental Pulp Progenitor Cell Populations

" Background: The DPSC constitutes a significantly small population, comprising approximately 1% of the total cells in the pulp tissue. Therefore, it is imperative to successfully culture and populate DPSCs in vitro before utilizing them for therapeutic purposes. Understanding DPSC heterogeneity is crucial for innovative regenerative therapies, prompting the investigation of mixed populations using fibronectin as a biological marker. It is reported that cellular phenotype and biological function may undergo alterations as cells replicate in a cultured environment or the substrate to which the cells adhere. Objectives: In this study, we compared and analyzed the impact of various isolation methods on the expansion, diversity, and variability in the expression of mesenchymal cell surface markers of DPSCs as well as the cellular expression during osteodifferentiation. The study aims to examine the relationship between fibronectin adherent, fibronectin non-adherent, and explant DPSC populations in terms of mesenchymal cell surface markers and mineralizing lineage differentiation potential. Identifying markers that consistently and specifically represent distinct subtypes of DPSCs within mixed populations will streamline direct purification and will help us to give more insight into DPSC heterogeneity. Methods: Two distinct sets of dental pulp stem cells (DPSC) were derived from separate donors using enzymatic digestion or explant outgrowth methods. The experiments were replicated for each set. DPSC underwent a selection process with fibronectin coating on plates, resulting in two variables: fibronectin-adherent cells and non-adherent cells. DPSCs went through passages, population doublings were calculated, and the proliferation rate was evaluated. Flow cytometric analysis was conducted to assess mesenchymal cell surface marker expression for each group. After treatment with osteogenic differentiation media, DPSCs' total RNA was extracted. A cDNA library was generated using RT-PCR, and qPCR assessed gene expression related to the mineralization lineage pathway. Results: The DPSCs isolated through fibronectin adhesion had greater cell growth than those without selection (P-value <0.05). Flow cytometry analysis of DPSCs from various donors, including HADAN II and HADAN III, demonstrated comparable percentages of quadruplicate positive cells cell surface markers and negative markers in both fibronectin-adherent and nonfibronectin cells. HADAN II exhibited reduced osteopontin (OPN) expression levels and elevated osteocalcin (OCN) expression levels (P-value <0.05). Conversely, both the HADAN III fibronectin-adherent isolate and non-adherent isolates displayed increased OPN expression, with no significant difference in BGLAP mRNA expression levels. OPN is recognized as an early marker of mineralization pathways, while OCN is acknowledged as a later marker in the osteogenic process. The data suggests that DPSCs in HADAN III may be less differentiated than those in HADAN II after a 7-day of incubation in the osteogenic medium. While both types of isolates demonstrated osteodifferentiation capacity, the fibronectin-adherent isolate exhibited a higher level of osteogenic differentiation markers compared to non-adherent dental pulp progenitor cells. Conclusion: While all isolates of dental pulp progenitor cells displayed similar proportions of mesenchymal stem cell markers, those selected through fibronectin exhibited an accelerated division rate and demonstrated potentially enhanced osteogenic potential across two donors. Our findings support fibronectin as a valuable selection tool capable of promoting MSC expansion and increased differentiation potential without compromising their stem cell characteristic.Nevertheless, it is essential to recognize the substantial individual variations in mesenchymal progenitor proportions, phenotype, and behavior. In addition, the inherent heterogeneity of human dental pulp stem cells (hDPSCs) presents a barrier to discerning the quality of stem cells solely through the examination of a single cellular marker. This challenge complicates the effective characterization and study of DPSCs.