Expression of Bone Morphogenetic Protein Inhibitors by Rat Gingival Fibroblasts

Objectives: Observation suggests the extent of periodontal coronal bone regeneration is limited by encroachment of the gingival lamina propria. We propose bone formation may be suppressed by constitutive expression of specific Bone Morphogenetic Protein (BMP) inhibitors in these tissues.

Methods: The constitutive expression of 9 BMP inhibitors by rat gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) was determined by Taqman real-time RT-PCR. Effects of BMP-stimulation on inhibitor expression was tested by stimulation of cultures with 100ng/ml rhBMP-2 at 2, 6 and 24hrs. Reactions were normalized to GAPDH and EifA2 expression. To test total BMP-inhibitory activity of rat GF, conditioned medium (CM) was collected from GF cultures over 48 hours and compared with CM from rat PDLF. Untreated serum-free media used as controls. ROS17/2.8 (ROS) cells were stimulated with rhBMP-2 at doses up to 10ng/ml, together with the BMP inhibitor Noggin (up to 100ng/ml), CM or controls. BMP-induced osteoblastic commitment was assessed after 72 hours by measuring total alkaline phosphatase activity spectrophotometrically.

Results: 7 out of 9 BMP inhibitors tested were constitutively expressed by gingival fibroblasts. The inhibitors Gremlin-1(2.52x c.f. controls), DAN(0.64x) and Gremlin-2(0.23x) had particularly high levels of basal expression. BMP-stimulation had little effect on inhibitor expression. 10ng/ml BMP-2 increased ALP expression in ROS cells increased by 76% (Controls 2.37±0.25 OD units, BMP-2 4.16±0.37 OD units). The effect of BMP was totally inhibited by 100ng/ml noggin. CM from gingival cells also blocked BMP activity equivalent to 50ng/ml Noggin. In contrast CM from PDL cells stimulated ALP activity.

Conclusion: A range of BMP inhibitors are constitutively expressed in gingival connective tissues, and factors secreted by gingivae inhibit BMP-induced bone formation. This inhibitory activity may limit the coronal bone regenerative potential during oral and periodontal surgery.

Supported by UK MRC G0601408 and ITI Research Foundation.