Biological effects of cementum and bone extracts on human periodontal fibroblasts

Background: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activit y of many types of mesenchymal cells. This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosp hatase (ALPase) activity and in vitro mineralization of cultured human peri odontal fibroblasts (hPF). Methods: Alveolar bone and cementum obtained from clinically healthy subjec ts were extracted by a solution containing 0.5 M acetic acid and enzyme inh ibitors. Osteoblastic phenotypes of hPF were assayed by ALPase activity, ge ne expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 mug/ml ascorbic acid, 10 mM s odium beta -glycerophosphate, and 10(-7) M dexamethasone. The effects of ce mentum and bone extracts on the expression of osteoblastic phenotypes in hP F were also determined. Results: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts. The hPF cells were found to exhibit a moderate ALPase activi ty when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same ex perimental conditions. Gene expression for ALPase, osteocalcin bone sialopr otein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells. The confluent hPF and ROS 17/2.8 cells showed eviden ce of calcium deposition in the extracellular milieu at 30 and 15 to 30 day s' cultures, respectively, under a mineralization medium. The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells. The addition of low c oncentrations (5 mug/ml) of either cementum or bone extract produced an inc rease in the size and number of mineralization spots, as well. as greater A LPase activity in both hPF and ROS 17/2.8 cultures during the observation p eriods. Conclusions: These results suggest that hPF possess certain mineralizing ph enotypes, and that acetic acid extracts of bone and cementum contain compon ents capable of stimulating osteogenic differentiation of hPF.