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Healing of critical sized bone defects represents a challenging issue in clinical and research fields. Current therapeutic techniques, such as bone grafts or bone grafts substitutes, still have limitations and drawbacks. Therefore, stem cell-based therapy provides a prospective approach to enhance bone regeneration. The present study aimed to assess the regenerative capacity of Gingival mesenchymal stem cells (GMSCs) as well as Bone marrow mesenchymal stem cells (BMSCs) loaded on NanoBone scaffold, in comparison to the unloaded one, in surgically created bone defects in rabbits' tibiae. To achieve this aim, critical sized bone defects, of 6-mm diameter each, were unilaterally created in tibiae of adult New Zeeland male white rabbits (n = 27). The rabbits were then divided randomly into three groups (9 each) and received the following: Group I: Unloaded NanoBone Scaffold, Group II: GMSCs Loaded on NanoBone Scaffold, and Group III: BMSCs Loaded on NanoBone Scaffold. Three rabbits from each group were then sacrificed at each time point (2, 4 and 6 weeks postoperatively), tibiae were dissected out to evaluate bone healing in the created bony defects; both histologically and histomorphometrically. The findings of this study indicate that both GMSCs and BMSCs exhibited fibroblast morphology and expressed phenotypic MSCs markers. Histologically, local application of GMSCs and BMSCs loaded on NanoBone scaffold showed enhanced the pattern of bone regeneration as compared to the unloaded scaffold. Histomorphometrically, there was astatistically insignificant difference in the new bone area % between the bony defects treated with GMSCs and BMSCs. Thus, GMSCs can be considered as a comparable alternative source to BMSCs in bone regeneration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)