The musculoskeletal system is essential for its structural, protective, and support roles in the body as well as being a mineral source that facilitates movement [
1]. Injuries to the musculoskeletal system are common, debilitating, and expensive to treat. Skeletal muscle injuries resulting in tissue loss are distinctively challenging in terms of surgical repair. Although the skeletal muscle is potentially regenerative, skeletal myofibers do not completely grow to fill the injured area, in case of losing a significant amount of tissue. If the defect does not exceed a certain volume, the healthy bone has the potency to regenerate [
2]. However, in cases with extensive defects, bone graft biomaterials can be used for recovery of the defects and to facilitate bone formation in the defective regions [
3]. Although, these traditional treatments have some limitations, such as disease transfer, histo-incompatibilities, limited autograft tissue supply, and insufficient mechanical support of implants or synthetic grafts. It has been shown that bone tissue engineering, as a new therapeutic strategy, can be used for bone regeneration [
4‐
6]. Due to the drawbacks of the traditional therapeutic approaches, tissue engineering is being applied to look for new strategies to design an artificial biomaterial scaffold containing regenerating competent cells. Bone tissue engineering complex inclusive osteoconductive scaffolds, cells and osteogenic growth factors [
7]. Among these three components, scaffolds play significant roles since they maintain the transplanted cells and lead their functions effectively [
8‐
11]. For the artificial bone transplant, materials or the device must be non-toxic in interaction with body function [
12,
13]. Hydroxyapatite (HA) (Ca10(PO
4)6(OH)2) is known as the most biocompatible replacement biomaterial among the developed artificial bones, and it is a constituent of 70% of human [
14,
15]. HA, as an alkaline calcium phosphate, is extremely bioactive and biocompatible due to its similarities to the bone tissue and mineral components of the tooth in the human body [
16‐
20]. Furthermore, HA is the most widely used material for coating the hard tissue and metal implant due to being non-toxic and its ability to promote osteoinductivity [
21,
22]. Poly(lactic-co-glycolic acid) (PLGA), as a copolymer of PGA and PLA, has significant properties, such as being mechanically strong and biodegradable as well as being Food and Drug Administration (FDA) approved. One of the most important advantages of PLGA is that its biodegradation can be controlled by altering the ratio of PLA and PGA; therefore, it has been widely used in the medical field [
23]. Various fabrication methods have been evolved for constructing the scaffolds. Among these conventional methods, salt leaching, solvent casting, fiber bonding, phase separation processes, and membrane lamination approaches are currently used to fabricate scaffolds with irregular pore sizes and porosity [
4]. Conventional methods, such as emulsion freeze-drying technique, were used for fabrication of highly porous PLGA scaffolds with an interconnected porous structure which is highly potential for bone tissue engineering [
1]. Scaffolds with porosity greater than 90% and a pore size ranging from 20 to 200 μm can be fabricated using this method [
3]. The pore size can be controlled by the freezing rate and pH; a faster freezing rate results in smaller pores [
6]. Electrospinning is one of the widely used techniques for the preparation of nanofibrous materials with an ultrafine diameter (the diameter of the fibrous can range from few nanometers to several hundred nanometers or even micrometers), wide surface area per unit mass, and small interfibrous pore size [
24]. Electrospinning has unique advantages over some other techniques that are used to fabricate scaffolds; for instance, the porous structures created using this method can potentially mimic the natural ECM of the biological tissues [
25]. Electrospun nanofiber of biocompatible polymers is particularly used in drug delivery bioengineering, adhesion of biomacromolecules or cells, wound dressing, etc. [
26]. It has recently been shown by several that mesenchymal stem cells (MSCs), embryonic stem cells (ES), and hematopoietic stem cells (HSC) can differentiate into osteoblast cells [
27,
28]. Human endometrial stem cells (EnSCs) are an alternative for osteogenic differentiation due to their dynamic nature [
29,
30]. The endometrial stem cells have shown to have a great multipotency potential. The human endometrium includes a few mesenchymal stem cells (MSCs) that can provide an easily accessible source of MSC. This study investigated the effect of the scaffold architecture on the adhesion, proliferation, and osteogenic differentiation of hEnSC-derived osteoblast cells cultured on PLGA/HA scaffolds which were fabricated using either freeze-drying or electrospinning techniques.