Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains

Abstract

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

Membrane proteome characterization of periodontal ligament cell sets from deciduous and permanent teeth

“…Overall, clear differences have been found in the production of matrix metalloproteinases and their extracellular inhibitors, transcriptomic profile, and the receptor activator nuclear kappa B ligand (RANKL)/osteoprotegerin (OPG) ratios in the PDL tissues harvested from deciduous and permanent teeth. In addition, for the first time, our group showed potential constitutive dissimilarities between the PDL of deciduous and permanent teeth that may account for their distinct physiological roles . Comparative histology is available in a previous paper for gross observation …”

“…In addition, for the first time, our group showed potential constitutive dissimilarities between the PDL of deciduous and permanent teeth that may account for their distinct physiological roles . Comparative histology is available in a previous paper for gross observation …”

“…Primary PDL cells were obtained from completely erupted/root‐formed and not‐resorbed permanent (n = 3, two males and one female, aged 19 to 23 years) and deciduous (n = 3, two males and one female, aged 8 to 10 years) molars extracted from clinically healthy subjects and characterized . Additionally, tissues were obtained for RNA and protein isolation (n = 8, one male and three females, aged 18 to 35 years; and two males and two females, aged 5 to 8 years).…”

“…Collected teeth were placed in biopsy media (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 250 mg/mL gentamicin sulfate, 5 mg/mL amphotericin B, and 1% penicillin/streptomycin . Cell isolation and culture were performed as previously described elsewhere . Cells at two to four passages were used in our experiments, which were performed in triplicates simultaneously for both groups with cells within the same passage (see supplementary Appendix in online Journal of Periodontology ).…”

“…Supplementary Appendix Table describes primers’ details. PICALM, LRRC15, and extended‐synaptotagmin 2 (ESYT2) protein levels in tissues and cells were assessed by Western blotting . Membranes were incubated with anti‐PICALM (1:1000 ab172962), anti‐LRRC15 (1:500, ab150376), and anti‐ESYT2 (1:250, ab88900) antibodies (see supplementary Appendix).…”