Date of Award

9-27-2024

Department

Department of Orthodontics

First Advisor

Marie M. Tolarová

First Committee Member

Mirek Tolar

Abstract

Introduction: Orofacial clefts, including non-syndromic cleft lip and cleft palate (NCLP), are among the most common congenital anomalies affecting approximately 1 in 700 live births. The etiology of NCLP is multifactorial involving both genetic and environmental factors. MTHFR 677CT polymorphism belongs to a group of candidate genes. Deficiency of folate belongs to environmental factors contributing to etiology of NCLP. Folate plays a critical role in DNA synthesis and cellular methylations. It was feasible to investigate them together in human dental stem cells (hDSC). hDSC are derived from neural crest cells that are integral to craniofacial development. This study investigates the effects of folate on hDSC exposed to ischemia/reperfusion injury. It mimics a transient ischemic episode during embryonic development that was linked to NCLP development. Materials and Methods: hDSC were isolated from teeth extracted from patients in the Oral maxillofacial surgery clinic (IRB#2023-80). MTHFR677 genotypes (CC, CT, TT) were identified. Ischemia/reperfusion injury was induced using Billups-Rothenberg hypoxic chambers. Cytotoxic damage was assessed by measuring concentration of lactate dehydrogenase (LDH) released to medium using CyQuant LDH Cytotoxicity Assay (Life Technologies). Samples were collected at multiple time points: T0 (baseline, before hypoxia), T1 (immediately before hypoxia), T2 (24 hours post-hypoxia), and T3 (48 hours post-reperfusion). Total number of cells was assessed at baseline and after recovery from ischemia/reperfusion injury (CyQuant LDH assay after lysis of all cells). The experiments were done in triplicates in culture medium with added 2 μg/mL folic acid and in medium with no folic acid added. 3 Results: Cytotoxicity tests showed no significant differences between hDSC exposed to ischemia/reperfusion injury in medium with or without folic acid using hDSC with different MTHFR 677CT genotypes. Multiplication rates were found to be different in hDSC defined by MTHFR 677 CT genotypes. Multiplication rate equal to 1.2 was found in hDSC with MTHFR 677TT genotype, multiplication rate equal to 4.0 was found in hDSC with MTHFR 677CT genotype, and multiplication rate equal to16.8 was found in hDSC with MTHFR 677CC genotype. It seems that the lower activity of MTHFR conferred by T mutated allele was reflected in magnitude of hDSC multiplication rate. Conclusion: Cells with the MTHFR 677 CC genotype exhibited the highest proliferation rate, followed by CT, while TT genotypes showed the least multiplication rate. These findings suggest that hDSC multiplication rate depended on availability of active folate in the cells. Further research will be done to confirm these results on a larger sample. It will further explore the molecular mechanisms involved in prevention of NCLP by folic acid supplementation.

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