Enamel renal syndrome (ERS) is a rare recessive disorder caused by loss-of-function mutations in FAM20A (family with sequence similarity 20 member A, OMIM #611062). Enamel renal syndrome is characterized by amelogenesis imperfecta, delayed or failed tooth eruption, intrapulpal calcifications, gingival overgrowth and nephrocalcinosis. Although gingival overgrowth has consistently been associated with heterotopic calcifications the pathogenesis, structure and interactions of the mineral deposits with the surrounding connective tissue are largely unknown. We here report a novel FAM20A mutation in exon 1 (c.358C > T) introducing a premature stop codon (p.Gln120*) and resulting in a complete loss of FAM20A. In addition to the typical oral findings and nephrocalcinosis, ectopic calcified nodules were also seen in the cervical and thoracic vertebrae regions. Histopathologic analysis of the gingiva showed an enlarged papillary layer associated with aberrant angiogenesis and a lamina propria displaying significant changes in its extracellular matrix composition, including disruption of the collagen I fiber network. Ectopic calcifications were found throughout the connective gingival tissue. Immunomorphological and ultrastructural analyses indicated that the calcification process was associated with epithelial degeneration and transformation of the gingival fibroblasts to chondro/osteoblastic-like cells. Mutant gingival fibroblasts cultures were prone to calcify and abnormally expressed osteoblastic markers such as RUNX2 or PERIOSTIN. Our findings expand the previously reported phenotypes and highlight some aspects of ERS pathogenesis.

Cell culture is an important tool in medical, odontological and biological research laboratories, supporting cell therapies and tissue bioengineering strategies. It is used as a means for in vitro testing of the biocompatibility of resin polymers used in dentistry. The majority of cells are cultured with Dulbecco's modified Eagle's medium (DMEM) or RPMI supplemented with fetal bovine serum. Several cell types are being studied including gingival fibroblasts. Gingival fibroblasts are the main cells of gingival connective tissue. These cells play an active and important role in almost all coating fabric processes, and its involvement in various pathophysiological conditions, including, healing, repair, aging, psoriasis, cancer among others, is only beginning to be understood. DMEM is the most widely used fibroblastic culture medium. This model describes a method for obtaining and cultivating human gingival fibroblasts, by explants derived from surgical discards. Fibroblasts were isolated mechanically and cultured in RPMI 1640 culture medium supplemented with fetal bovine serum 10%, Penicillin (10000 U/ml)/Streptomycin (10 mg/ml) 1% and L-Glutamine (200 mM) 1%. The culture medium is replaced every two days. Cells forming a fairly dense network were observed after a period of 4 days of culture. Human gingival fibroblasts can be cultured by direct explant technique with RPMI 1640 culture medium supplemented with fetal bovine serum and antibiotics.