Abstract
ObjectivesThe inhibitory action of the superficial gingival connective tissues may limit the regenerative potential of alveolar bone in periodontal therapy or dental implant applications. The aims of this study were to investigate the hypothesis that gingival fibroblasts (GF) can inhibit bone morphogenetic protein (BMP)‐induced osteoblastic differentiation, to determine their expression of BMP inhibitors, and finally to determine whether reduction of these inhibitors can relieve suppression of osteoblastic differentiation.MethodsGingival fibroblasts were co‐cultured either directly or indirectly with calvarial osteoblasts to assess alkaline phosphatase inhibitory activity, a marker of osteoblastic differentiation. To test total BMP‐inhibitory activity of rat GF, conditioned media (GFCM) were collected from cultures. ROS 17/2.8 osteoblastic cells were stimulated with BMP2, together with GFCM. Inhibitor expression was tested using RT‐qPCR, Western blotting and in situ hybridization. Removal of inhibitors was carried out using immunoprecipitation beads.ResultsCo‐culture experiments showed GF‐secreted factors that inhibit BMP‐stimulated ALP activity. 10 ng/ml BMP2 increased alkaline phosphatase expression in ROS cells by 41%. GFCM blocked BMP activity which was equivalent to the activity of 100 ng/ml Noggin, a well‐described BMP inhibitor. Cultured gingival fibroblasts constitutively expressed BMP antagonist genes from the same subfamily, Grem1, Grem2 and Nbl1 and the Wnt inhibitor Sfrp1. Gremlin1 (6.7 × reference gene expression) had highest levels of basal expression. ISH analysis showed Gremlin1 expression was restricted to the inner half of the gingival lamina propria and the PDL. Removal of Gremlin1 protein from GFCM eliminated the inhibitory effect of GFCM on ALP activity in ROS cells. Subsequent addition of recombinant Gremlin1 restored the inhibitory activity.ConclusionsFactors secreted by gingival fibroblasts inhibit BMP‐induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues. These inhibitors, particularly Gremlin1, may limit coronal alveolar bone regenerative potential during oral and periodontal surgery.
Highlights
New bone formation in periodontal regenerative treatments is generally restricted to infrabony sites
Factors secreted by gingival fibroblasts inhibit Bone morphogenetic proteins (BMPs)-induced bone formation and a range of BMP inhibitors are constitutively expressed in gingival connective tissues
In ROS cells, the inhibitory activity of GFCM was completely reversed by addition of 25 ng/ml BMP2. We investigated whether this effect was similar to that caused by known BMP antagonists. 100 ng/ml Gremlin[1] and 100 ng/ ml Noggin inhibited osteoblastic differentiation to a similar extent as that caused by GFCM (Figure 3B). 100 ng/ml noggin completely inhibited the stimulatory effect of 10 ng/ml BMP2 in control cultures (Figure 3C), whereas addition of 10 ng/ml BMP2 completely overcame inhibition of GFCM (Figure 3D)
Summary
New bone formation in periodontal regenerative treatments is generally restricted to infrabony sites. With a horizontal pattern of bone loss, regeneration is typically limited by encroachment of the overlying gingival connective tissues. Undesirable invasion by soft connective tissue negatively influences both the extent and nature of the bone formed;[1] whilst elsewhere in the body clinical, developmental and experimental observations suggest that bone formation can be inhibited by non-ossifying connective tissues.[2] The biological basis for this effect may be due to competition for the available space adjacent to bone; an alternative hypothesis is that fibroblasts constitutively exert inhibitory activity that limits bone growth. BMP activity is tightly regulated at both the extra and intracellular levels, including by secretion of BMP antagonists such as Gremlin[1] and Noggin.[8] it is suggested that osteogenic inhibitory influences from connective tissue may involve these inhibitors
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