Should cementoblasts express alkaline phosphatase activity? Preliminary study of rat cementoblasts in vitro

Background: A well-characterized cell culture model for cementoblasts is essential to understand the mechanisms of periodontal ligament (PDL) reattachment and regeneration. Whether cementoblasts express alkaline phosphatase (ALP) activity in vivo and in vitro remains to be determined. Methods: Using a 2-step method of enzyme digestion/ explant culture, osteoblasts, gingival/PDL fibroblasts, and cementoblasts were obtained from alveolar bone, gingiva, and the root surface of rat first molars and cultured. Initially, bone sialoprotein (BSP) was immunolocalized on tissue sections of periodontium and on cultured cells to distinguish mineral-forming cells from fibroblasts. Proteins were extracted from these cells to assess ALP activity by using an enzyme assay. RNA was extracted from the same cell source to detect ALP mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). Results: Cultured PDL/gingival fibroblasts were spindle shaped. Osteoblasts were irregularly shaped, and cell clusters/nodules were observed as they approached confluence. The cementoblasts manifested a polygonal shape and had two morphotypes: osteoblast-like and cuboidal or stratified. BSP was localized within the mineralized tissues and in osteoblasts and cementoblasts in culture and in tissue sections. The highest level of ALP activity was found in osteoblasts, a moderate level in PDL fibroblasts, and the lowest level in gingival fibroblasts. The cementoblasts lacked ALP activity, and this was reflected by a very weak signal (or no signal at all) for ALP mRNA in the cementoblasts. Conclusions: These studies indicate that cells consistent with a cementoblast-like phenotype may be successfully cultured, and that they lack ALP activity.