Markers of bone and cementum formation accumulate in tissues regenerated in periodontal defects treated with expanded polytetrafluoroethylene membranes

Abstract

Guided tissue regeneration (GTR) is a concept that evolved from the development of membrane-barrier techniques, which allow the repopulation of periodontal wounds by specific cells, resulting in a new attachment apparatus. To help understand the biological mechanisms involved in membrane barrier-led periodontal healing, the present study investigated the macromolecules phenotypic of bone and cementum formation in tissues grown under the GTR barrier by immunolocalization. Periodontal regeneration was initiated by placing barriers on experimentally induced periodontal defects in a Rhesus monkey model. Samples were harvested 6 wk after healing and sections of soft tissues grown under GTR barriers (membrane tissue) were stained with antibodies to bone morphogenetic proteins-2 and 4 (BMP-2, BMP-4), bone morphogenetic protein-7 (OP-1), cementum attachment protein (CAP), osteonectin (OTN) and bone sialoprotein (BSP). Tissues grown in the absence of any barrier device served as a control (control tissue). Membrane periodontal tissues from beneath the ePTFE membrane were comprised of spindle-shaped fibroblast-like cells encased in a dense fibrillar extracellular matrix (ECM). Round-shaped cells aggregated to form nodules. Newly formed hard tissue was conspicuous. A similar, but very disorganized, fiber network was observed in control tissues, but neither nodule formation nor hard tissue was observed. Osteonectin staining was observed in the ECM of membrane tissues and particularly in the area of the connective tissue adjacent to newly formed hard tissue. The dense network of connective tissue fibers was also stained. In control tissues, cells and fiber network had a significantly weaker signal for osteonectin. An intense reaction was observed in membrane tissues stained for BSP, particularly the connective tissue adjacent to the newly formed hard tissue, while the control tissues did not stain for BSP. Cementum attachment protein (CAP) was observed in the connective tissue adjacent to the newly formed hard tissue of the membrane tissues whereas control tissues exhibited no CAP staining. In membrane tissues, BMP-2 and 4 distribution was found to concentrate in nodule areas, in the newly formed hard tissue and in the fiber network, while very faint staining was observed in control sections. The distribution of OP-1 in membrane and control tissues was found to mimic the BMP-2 pattern, but staining was more distributed in hard tissue matrix. When the profile of BMP-2, BMP-4, OP-1, OTN, CAP and BSP staining was analyzed on membrane tissue sections, striking similarities were noted in the connective tissue adjacent to the newly formed hard tissue and in nodular areas. In addition, the localization of BMP-2 and BMP-4 mRNA was investigated in both tissues by in situ hybridization. An intense expression of BMP-2 and 4 transcripts was observed in membrane tissues while control tissues never yielded any positive hybridization signal. The correlation between these histochemical findings strongly suggests that the forming soft tissues under ePTFE membranes contain cells and ECM macromolecules normally associated with bone and cementum.