Storage conditions of avulsed teeth affect the phenotype of cultured humanperiodontal ligament cells

After severe injury to the periodontal ligament (PL), the phenotypes of cel ls recolonizing root surfaces influence the extent and type of repair proce sses. 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 he re that recolonization processes depend in part on the storage conditions o f the teeth following avulsion. We used an in vitro cell culture model to a ssess the effect of storage conditions on immunohistochemical staining of s everal 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 tee th 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 stor age that may occur following avulsion. Collagenase/trypsin-digested suspens ions of PL cells were prepared from the tissue adherent to the extracted ro ot surface. Passage #2 or #3 cultures were immunostained and examined by fl uorescence microscopy. For type XII collagen, cells from wet samples displa yed perinuclear staining while cells from 30-min dry samples showed only is olated 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 f or alpha-smooth muscle actin. Detached cytoplasmic fragments resembling cel l 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 f or alkaline phosphatase was more intense in samples stored under dry condit ions. We conclude that prolonged extra-alveolar dry storage favors increase d in vitro growth of contractile cells expressing osteogenic cell markers w hile storage in cell culture medium favors growth of cells with the classic al phenotype of PL fibroblasts.