Storage conditions of avulsed teeth affect the phenotype of cultured human periodontal ligament cells.

After severe injury to the periodontal ligament (PL), the phenotypes of cells recolonizing root surfaces influence the extent and type of repair processes. In teeth that are replanted following avulsion injury, recolonization of the PL space by osteogenic cells instead of by PL fibroblasts may favor bone formation (i.e. ankylosis) instead of PL regeneration. We consider here that recolonization processes depend in part on the storage conditions of the teeth following avulsion. We used an in vitro cell culture model to assess the effect of storage conditions on immunohistochemical staining of several marker proteins that are expressed by osteogenic cells (osteopontin and alkaline phosphatase) and fibroblasts (alpha-smooth muscle actin, type III and XII collagens). Prior to cell culture, extracted human premolar teeth were stored in air ("dry") or in alpha-MEM ("wet") for either 30 or 120 min as surrogate conditions for the variations of extra-alveolar tooth storage that may occur following avulsion. Collagenase/trypsin-digested suspensions of PL cells were prepared from the tissue adherent to the extracted root surface. Passage #2 or #3 cultures were immunostained and examined by fluorescence microscopy. For type XII collagen, cells from wet samples displayed perinuclear staining while cells from 30-min dry samples showed only isolated foci. The staining for 120-min dry samples was weak and non-specific. alpha-Smooth muscle actin was not incorporated into stress fibers in wet samples, whereas dry samples demonstrated prominent stress fibers stained for alpha-smooth muscle actin. Detached cytoplasmic fragments resembling cell processes that stained for alpha-smooth muscle actin were abundant in dry samples, indicating the presence of highly contractile cells. The staining for osteopontin was mainly perinuclear but was more intense in dry samples. The focal adhesion pattern of osteopontin staining in 120-min dry samples resembled that of migrating osteogenic cells. The pattern of staining did not vary for type III collagen or alkaline phosphatase, although staining for alkaline phosphatase was more intense in samples stored under dry conditions. We conclude that prolonged extra-alveolar dry storage favors increased in vitro growth of contractile cells expressing osteogenic cell markers while storage in cell culture medium favors growth of cells with the classical phenotype of PL fibroblasts.