Abstract
Periodontal diseases are a group of infectious inflammatory conditions that result in bone destruction. Three members of the tumour necrosis factor (TNF) family have been demonstrated to play important roles in bone healing: RANK (receptor activator of NF-kB), RANKL (receptor activator of NF-kB ligand) and its inhibitor Osteoprotegerin (OPG). The expression of these cytokines in the periodontium during wound healing is poorly understood. Furthermore, it has been suggested that this expression may be modified by chronic periodontitis, altering the normal healing of bone.
Aim: To characterise the presence of RANKL, RANK and OPG proteins in a sheep furcation, and to relate this to the temporal expression pattern in surgically created periodontal defects in a sheep model.
Methodology: The sheep periodontal furcation model was used. Two control animals were sacrificed without any surgical intervention at baseline. In the test group, ten sheep had Class II furcation defects surgically created on the buccal surface of the mandibular second premolars and permitted to heal naturally. Two animals per time interval were sacrificed at six hours, one, two, four and six weeks post-surgery. Specimens were resected en bloc, formalin-fixed, demineralised, paraffin embedded and followed by serial sectioning. Immunohistochemical (IHC) staining protocols for RANKL, RANK, OPG were developed and optimised. Protocols to identify osteoclasts using Tartrate Resistant Acid Phosphatase (TRAP) staining were refined. To permit spatial identification of protein expression, IHC-labelled sections were matched with Haematoxylin & Eosin-stained step-serial sections.
Results: After six weeks, the test furcations filled with new bone that was less dense than the unwounded controls. Osteoclasts were prominent at six hours, whilst osteoblasts began to populate the wounds from two weeks post-op. The expression of RANKL, RANK and OPG in the control and test sites differed. RANK staining was most intense at six hours, corresponding to osteoclast activation and bone resorption. RANKL expression increased from four weeks and peaked after six weeks, as osteoblasts numbers increased. This suggests RANKL may be important in bone remodeling during initial healing of an osseous wound. OPG, a negative regulator of the RANKL/RANK pathway, was highest in the unwounded controls.
Conclusions: The temporal expression pattern of RANKL, RANK and OPG proteins during healing of a surgical periodontal wound has been characterised. Future investigations will examine the expression of these cytokines in chronic periodontal lesions.