Specific cementum attachment protein enhances selectively the attachment and migration of periodontal cells to root surfaces.

A specific cementum attachment protein (CAP) was identified in human cementum and found to bind with high affinity to non-demineralized root surfaces, hydroxyapatite and fibronectin. Attempting to elucidate the biological function of this protein and its possible role in cementogenesis the capacity of CAP to promote selective cell migration towards and attachment of various periodontal derived cell populations to root surfaces in vitro was assessed. Human gingival fibroblasts (HGF), periodontal ligament cells (HPC), and alveolar bone cells (HABC) were labeled with [3H]Thymidine during their exponential growth phase. Root slices, 300 microns thick, were incubated with increasing concentrations of CAP. Untreated and fibronectin (FN) treated root slices served as negative and positive controls, respectively. Migration was assessed by placing root slices on confluent layers of labeled cells maintained in serum free medium and determining the number of cells migrated onto the root surface 3 days thereafter. Attachment was assessed by incubating root slices with labeled cell suspensions for 2 h and determining the number of attached cells. CAP promoted both cell migration and attachment dose dependently. HABC responded better than HPC and HGF to CAP treated root slices, and HPC response was higher than that of HGF. Cell attachment was dose dependently inhibited by synthetic RGD peptides. FN did not affect the migration of HGF, barely enhanced that of HABC, and was less potent than CAP at enhancing the migration of HPC. FN was more effective than CAP in promoting the attachment of HGF to root slices, but it was as potent as CAP in supporting the attachment of HPC and HABC.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of a cementum attachment protein to extracellular matrix components and to dental surfaces.

Cementum proteins (CP) have been shown to mediate cell attachment. Among these, a 55 kDa protein was isolated. The purpose of the present study was to assess the capacity of CP to bind to non-demineralized and demineralized root surfaces and to support cell attachment to dentin. CP were prepared by sequential extraction of bovine cementum with 25 mM EDTA, 0.5 M acetic acid followed by 4 M guanidine HCl. The latter was subjected to ion exchange chromatography on a DEAE-3SW column and eluted stepwise with a 0-0.5 M NaCl gradient. CP were labelled with 125I and the capacity of 125I-CP to bind to mineralized and partially demineralized dentin, synthetic hydroxyapatite, collagen, fibronectin and fibrillar collagen-fibronectin complex was assessed. It was found that CP bind specifically to mineralized dentin and synthetic hydroxyapatite but not to demineralized dentin. The specific binding was 60% of the total binding. SDS-PAGE analysis of the proteins bound to dentin indicated that the main bound protein had a molecular weight of 55 kDa. CP exhibited high affinity for fibronectin (kD = 1.56 x 10(-10) M) and fibronectin-collagen complex, but their binding to either molecular or fibrillar collagen was negligible. It is suggested that CP may play an important role in the attachment of cells of the periodontium to cementum extracellular matrix during homeostasis and regeneration.

Mechanism of endotoxin inhibition of human gingival fibroblast attachment to type I collagen.

Bacterial endotoxin inhibits the attachment of human gingival fibroblasts to collagen. The present study attempted to elucidate the possible mechanism of this inhibition. Two mechanisms were considered: direct toxicity to the cells and steric interference. Collagen substrates were prepared by rat type I collagen being air-dried in the wells of 24 multi-well plates. Experimental collagen substrates were treated with 50 micrograms of endotoxin/well, while untreated collagen substrates served as controls. Two mL of cell suspension (10(4) cells/mL) was added to each well, and these were incubated at 37 degrees C for two h. The average cell number/mm2 attached to experimental and control substrates was determined. Cell attachment to endotoxin-treated collagen was inhibited by 78%, compared with that to untreated collagen. The washing of the endotoxin-treated collagen for two h did not affect the inhibition of cell attachment, whereas after 24 h of washing, cell attachment was inhibited by 54%, compared with that to untreated collagen. Pre-incubation of the cells in endotoxin for two h did not affect their attachment to collagen. The addition of fetal calf serum (15%) to the experimental system completely reversed the inhibition of fibroblast attachment to endotoxin-treated collagen. These findings suggest that endotoxin interferes with fibroblast attachment to collagen through a steric phenomenon, possibly by blocking the binding sites on the collagen molecule recognized by the membrane receptor for collagen.