RELATIONSHIP OF CLONOGENIC CAPACITY TO PLATING EFFICIENCY AND VITAL DYE STAINING OF HUMAN PERIODONTAL-LIGAMENT CELLS - IMPLICATIONS FOR TOOTH REPLANTATION

The survival rate of avulsed permanent teeth following replantation is affected primarily by the duration of the extra-alveolar period and t he nature of the storage conditions. These factors are believed to str ongly affect the viability of periodontal ligament (PL) cells but in v itro assays of cell viability based on vital dye assays are only weakl y correlated with the tooth survival rate after replantation. The aim of the present study was to examine the relative dependence of cell me mbrane integrity, attachment and clonogenic capacity of human PL cells on the temperature and duration of the extra-alveolar period and the type of storage medium. Twenty-four premolar teeth were extracted for orthodontic reasons from 9 patients 11-18 years of age. Teeth were mai ntained at 4 degrees C or 23 degrees C for 15, 30, 60 or 120 min in ei ther milk or dry conditions. Cell membrane integrity was determined by BCECF/AM dye inclusion. Plating efficiency was determined by measurem ent of cell attachment at 3 and 6 h. The clonogenic capacity of progen itor cells was estimated by limiting dilution and colony counts, For a ll assays teeth stored in milk at 4 degrees C showed the highest perce ntages of BCECF positive, attached cells with clonogenic capacity. Inc reased storage time (15-120 min) was associated with a 50% relative re duction of BCECF staining and a 5-fold relative reduction of cell atta chment regardless of storage conditions. However, the clonogenic capac ity of progenitor cells decreased 25-fold over the same duration of st orage. These data demonstrate that in vitro assays of clonogenic capac ity are much more sensitive to extra-oral storage rime and storage con ditions than dye inclusion or cell attachment. We suggest that in comp arison with in vitro measures of cell membrane integrity, the clonogen ic capacity of PL cells is more closely linked to tooth survival rate, probably reflecting the capacity of PL progenitor cells to recolonize the root surface after replantation.