Phenotypic comparison of periodontal ligament cells in vivo and in vitro

Abstract

The mammalian periodontal ligament contains heterogeneous populations of connective tissue cells, the precise function of which is poorly understood. Despite close proximity to bone and the application of high amplitude physical forces, cells in the periodontal ligament (PL) are capable of expressing regulatory factors that maintain PL width during adult life. The study of PL homeostasis and PL cell differentiation requires culture and phenotypic methods for precise characterization of PL cell populations, in particular those cells with an inherently osteogenic program. Currently it is unknown if cells cultured from the PL are phenotypically similar to the parental cells that are present in the tissues. We have compared the phenotype of cells in vivo with cells derived from the PL and expanded in vitro to assess the general validity of in vitro models for the study of phenotypic regulation in vivo. Rat PL cells were isolated by either scraping the root of the extracted first mandibular molars (Group A), or by scraping the alveolar socket following extraction of first mandibular molars (Group B), or by obtaining a mixture of cells after disaggregating a block of tissue consisting of first mandibular molar, PL and the surrounding alveolar bone (Group C). Cultured cells at confluence were fixed and immunostained for alpha-smooth muscle actin (alpha-SMA), osteopontin (OPN), alkaline phosphatase (AP), or bone sialoprotein (BSP). For in vivo assessments, frontal sections of rat first mandibular molar were immunostained for alpha-SMA, OPN, AP and BSP. We examined osteogenic differentiation of cultured PL cell cultures by bone nodule-forming assays. In vivo and at all examined sites, > 68% of PL cells were immunostained for AP; approximately 50% and approximately 51% for OPN and alpha-SMA (p = 0.3), respectively, while only approximately 8% were positively stained for BSP (p < 0.01). Analysis of cultured PL cells in Groups A, B and C showed 54%, 53%, and 56% positive staining for alpha-SMA respectively; 51%, 56%, 54% for OPN; 66%, 70%, 69% for AP and 2.2%, 1.4% and 2.8% for BSP. The mean percentage of PL cells in situ stained for the different markers was similar to that of cultured PL cells (Group A approximately Group B approximately Group C in situ for p > 0.2) except for BSP which was 3 to 4 fold higher in vitro (p < 0.01). PL cell cultures treated with dexamethasone showed mineralized tissue formation for all groups (A, B, C), but no mineralized tissue formation was detected in the absence of dexamethasone. As PL cells express quantitatively similar phenotypes in vitro and in vivo, we conclude that the in vitro models used here for assessment of PL cell differentiation appear to be appropriate and are independent of the cell sampling method. Further, dexamethasone-dependent progenitors are present both on the root and bone-related sides of the PL.

Highly osteogenic PDL stem cell clones specifically express elevated levels of ICAM1, ITGB1 and TERT

“…A total number of 12 clones, namely PC1-PC12, were cloned from primary PDL-derived cells, which are believed to comprise progenitor cells of PDL-producing fibroblasts, of cementoblats (cells that form cementum, a mineralized matrix layer covering the tooth root) and of bone forming osteoblasts (Ivanovski et al 2006, Kramer et al 2004, Lekic et al 2001, McCulloch and Bordin 1991, Melcher 1976, Seo et al 2004. Although all 12 clones had an ability to form CFU-F, only PDL clones PC2, PC3, PC4, PC8 and PC12 underwent adipogenic and osteoblastic differentiation, suggesting stem cell-like characteristics of these clones.…”

Estrogen Deficiency Leads to Impaired Osteogenic Differentiation of Periodontal Ligament Stem Cells in Rats

“…It has been demonstrated that human PDL contains a heterogeneous population of cells (Lekic et al 2001;Murakami et al 2003) capable of differentiating into cementoblasts or osteoblasts in vitro. Recently, Seo et al (2004) proved that human periodontal ligament cells contained stem cells (periodontal ligament stem cells, PDLSCs) and the application of these stem cells is a promising therapeutic approach for reconstruction of tissues destroyed by periodontal diseases.…”

Multipotent Dental Stem Cells: An Alternative Adult Derived Stem Cell Source for Regenerative Medicine

“…It is responsible for anchoring and supporting the tooth within the tooth socket. The periodontal ligament is composed of a heterogeneous population of cells containing fibroblasts, osteoblasts and cementoblasts (Bartold et al, 2000;Gay et al, 2007;Lekic et al, 2001;Seo et al, 2004;Shimono et al, 2003). Early studies have suggested that the PDL tissue had regenerative or repair abilities when an injury was incurred by the periodontal tissue (Reviewed in Bartold et al (2005) and Shimono et al (2003)).…”