Objectives: Critical-size defect models have been developed to assess the biologic potential, efficacy and safety of new regenerative approaches prior to their use in humans. The objective of this report is to describe the animal model used to study the regenerative potential of periodontal mesenchymal cells. Methods: 9 male beagle dogs aging 12-14 months were used for this investigation.After 3 weeks of quarantine, animals undergo defect creation surgery: mucoperiosteal flaps were raised and P1, P2 and M1 were extracted. Bilateral circumferential horizontal defects were surgically created around P3 and P4. Defects were standardized to have 6mm from the bone to the furcation fornix and the mesial and distal CEJ. Ligatures and a soft diet were used for 2 months to allow plague accumulation and defects chronification. 7 days prior to regenerative therapy oral hygiene was performed and systemic antibiotics were administered. After flap elevation, defect de-granulation and root notches creation for histological analysis, regeneration was performed with a xenogeneic bone substitute (Bioss-Collagen®) with or without canine periodontal mesenchymal cells. Post surgical regimen included broad-spectrum antibiotic and analgesics. Sutures were removed at 2 weeks and plaque control was maintained twice weekly. Results: At time of defect creation, the distance between cemento-enamel junction and bone crest (CEJ-AB) was 5.19±0.38 mm and the distance between furcation fornix and bone crest (F-AB) was 3.65±0.47 mm. Two months after ligature chronification, prior to performing the regenerative surgery the CEJ-AB was 4.64±0.46 and F-AB was 3.99±0,67, showing that periodontal defects remained with evident loss of attachment and furcation exposure. Four weeks after treatment healing occurred uneventfully. The histological analysis performed at 3 months showed that most of the furcation defects healed partially. Periodontal regeneration was mainly located at the most apical areas. Bioss-Collagen® underwent a considerable degree of resorption, residual graft particles were located mostly in the proximity of the basal bone. The formation of new acellular cementum was evident in all specimens at varying degrees. Conclusions: The chronified supra-alveolar periodontal defect in the beagle dog closely resembles challenging defects in humans and proved to be a valid model for the study of periodontal regeneration with cell therapy. Keywords: periodontal regeneration, regeneration materials, cell therapy