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.
There are wide variations of gene expression and strikingly different responses to extracellular signals among different fibroblast populations. This has prompted a large number of in vitro studies which suggest that fibroblasts are not homogeneous but instead comprise multiple subpopulations with extensive site-to-site and intra-site variations. Conceivably, either fibroblasts are not all created equal, or, alternatively, discrete subpopulations may emerge in development, inflammatory lesions, or wound healing. While the heterogeneous nature of cultured fibroblasts has been known for some time, are these variations relevant to our understanding of the biology of oral tissues, their involvement in disease, and their response to therapy? Since fibroblasts are the predominant cell type in soft connective tissue matrices, the regulation of their proliferative, synthetic, and degradative behavior is likely to be important in tissue physiology and pathology. In this review, we use the current literature to assess whether fibroblast subpopulations really make a difference in the health and disease of periodontal tissues. We address the following questions: (1) Is fibroblast heterogeneity a real in vivo phenomenon? (2) How can we advance our knowledge of phenotypic variations and the regulation of fibroblast differentiation? (3) Could a knowledge of fibroblast heterogeneity have an impact on the development of new approaches to pathogenesis and the treatment of periodontal tissues?
Cells in mechanically challenged environments must cope with high amplitude forces to maintain cell viability and tissue homeostasis. Currently, force-induced cell death and the identity of mechanoprotective factors are not defined. We examined death in cultured periodontal fibroblasts, connective tissue cells that are exposed to heavy applied forces in vivo. Static tensile forces (0.48 piconewtons/m 2 cell area) were applied through magnetite beads coated with collagen or bovine serum albumin. There was a time-dependent increase of the percentage of propidium iodide-permeable cells in force-loaded cultures incubated with collagen but not bovine serum albumin beads, indicating a role for integrins. Cells exhibited reduced mitochondrial membrane potential, increased caspase-3 activation, nuclear condensation, terminal deoxynucleotidyl transferase nick end labeling staining, and detachment from the culture dish. The caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde reduced detachment 3-fold. There was a rapid (<10-s) decrease in plasma membrane potential after force application, which, in filamin A-deficient melanoma cells, contributed to irreversible cell depolarization. In fibroblast cultures, cells with increased permeability to propidium iodide exhibited ϳ2-fold less filamin A content than impermeable cells. Fibroblasts transfected with antisense filamin A constructs or with filamin A constructs without an actin-binding domain exhibited 2-3-fold increased proportions of dead cells relative to controls. We conclude that high amplitude forces delivered through integrins can promote apoptosis in a proportion of cells and that filamin A confers mechanoprotection by preventing membrane depolarization.
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