Periodontal disease affects about 80% of dogs, highlighting the importance of addressing periodontitis in veterinary dental care. The periodontal ligament (PDL) is a key structure holding the potential to regenerate the entire periodontal complex. This work presents an in vitro model of canine PDL-derived cell cultures that mimic the PDL’s regenerative capacity for both mineralised and soft tissues. Explant outgrowth-derived PDL cells were cultured under standard conditions in osteoinductive medium and with hydroxyapatite nanoparticles (Hap NPs). Cell behaviour was assessed for viability/proliferation, morphology, growth patterns, and the expression of osteogenic and periodontal markers. Osteogenic conditions, either achieved with osteoinducers or an osteoconductive biomaterial, strongly promoted PDL-derived cells’ commitment towards the osteogenic phenotype and significantly increased the expression of periodontal markers. These findings suggest that cultured PDL cells replicate the biological function of the PDL, supporting the regeneration of both soft and hard periodontal tissues under normal and demanding healing conditions. This in vitro model will offer a platform for testing new regenerative treatments and materials, ultimately contributing to canine dental care and better outcomes.