## 1 Introduction  
Tissue engineering (TE) is advancing fast in dentistry aiming to guide cell destiny into differentiating into osteo/ odontoblastic cells. Successful tissue regeneration requires the presence of three main components: stem cells, growth factors, and a scaffold [1]. Preparing scaffolds seeded with different types of cells and growth factors is the most popular technique for TE [2]. Chitosan (CS) is a natural, nontoxic cationic biopolymer of glucosamine and \(N\)-acetylglucosamine obtained after partial alkaline deacetylation of chitin, the main component of crustacean exoskeletons [3]. Structurally, it is similar to glycosaminoglycans (GAGs), which are important structural elements of the extracellular matrix of many tissues [4]. Hence, it has generated great interest in multiple medical fields, particularly for TE and wound healing. CS is biocompatible and biodegradable into nontoxic components, and has hemostatic potential and anti-inflammatory and antimicrobial properties; it promotes cell adhesion, proliferation, and differentiation [3]. The adequate pore sizes of CS, and the ability to modify it and combine it with other polymers, have facilitated the development of a wide range of scaffolds for the regeneration of many tissues, including bone, neural and vascular tissues, cartilage, and skin [4]. Recently, a porous CS scaffold was produced and proven to be capable of increasing the regenerative potential of dental pulp cells and odontoblastic differentiation [5]. Furthermore, resolution of periapical lesions, increase in root thickness and length, and high vascularized connective tissues in the root canal space was shown histologically in regenerative endodontic procedures using chitosan scaffolds [6].  
Simvastatin (SIM), a 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor, is a widely used statin and a well-established cholesterol-lowering drug [7]. Pioneering work demonstrated that statins, particularly SIM and lovastatin, increase the expression of bone morphogenic protein 2 (BMP-2) in osteoblasts and encourage bone formation [8]. Subsequent studies supported these findings, showing that statins continuously stimulate the expression of bone anabolic factors (e.g., vascular endothelial growth factor and BMP-2) and promote osteoblast differentiation and mineralization [8; 9; 10]. Additionally, statins suppress osteoclast differentiation which reflects positively on bone pathologies, such as osteopororotic fracture [11; 12]. From an endodontic perspective, dental pulp stem cells treated with SIM showed increased odontogenic differentiation and accelerated formation of mineralized tissue, and are considered to be potential supplemental pulp-capping agents [13; 14]. To our knowledge, no study has investigated the effect of CS scaffold, alone or with SIM impregnation, on human bone-marrow mesenchymal stem cells (hBMMSCs) and on osteoblastic differentiation specifically nor compared it to the most commonly used scaffold in endodontic wound healing, CollaCote (CL). In addition, no study has defined a known reliable SIM effective dose for cell proliferation and osteoblastic differentiation. Thus, the aim of this study was to develop CS scaffolds, with and without SIM, evaluate their effect and the potential synergistic outcome of the two materials on hBMMSCs and compare it to CollaCote. Two null hypotheses were tested:  
1. There is no difference between the combined use of CS scaffold and SIM and their individual use in osteoblastic differentiation of hBMMSCs.
2. These scaffolds show no difference in osteoblastic differentiation when compared to CollaCote.