Purpose this study was conducted between 2011 and 2017 at the faculty of Veterinary Medicine Cairo University and aimed to histologically and radiologically compare the efficiency of freshly isolated bone marrow mesenchymal stem cells (F-BMMSC) versus culture expanded bone marrow mesenchymal stem cells (E-BMMSC) when added to the mixture of autologous platelet rich fibrin (PRF), collagen with nano-hydroxyapatite in healing of critical bony mandibular defects in mandibular site in dogs. Material and methods 12 healthy adult Mongrel dogs were used in this study and bilateral rectangular full thickness mandibular inferior border critical bone defects (15X10 mm) just before the angle area were resected. Group A (The left side) contained: E-BMMSCs The expanded BMSC seeded on collagen sponge with Nano-Hydroxyapatite and PRF membrane. Group B (The right side) contained: F-BMMSCs The Gradient immediately separated BMSC seeded on collagen sponge with Nano-Hydroxyapatite and PRF membrane. Radiographic follow up was done immediate, 4, 8, 12 weeks Scarification was done 4, 8, 12 weeks postoperative. local bone mineral densities (BMD) were measured on a DXA system. Histological evaluation was done using H&E. Results the results showed that group B right defects had better healing results than group A right defects all over the follow-up period, but the differences were not statically significant. Conclusion the study findings indicate that The F-BMMSCs that seeded in nano-hydroxyapatite collagen type I n-HA-COL scaffolds combined with platelet rich fibrin PRF successfully repaired mandibular critical size defects, the same as the E-BMMSCs seeded in the same scaffold. .