**1. Introduction**

Bone tissue regeneration represents one of the major challenges of biomedicine. As in other areas of biomedicine, efforts are being conducted on replacing conventional approaches with more biomimetic ones. In this scope, tissue-specific active scaffolds are being developed, combining stem or precursor cells and physic/chemical cues, that synergistically stimulate the repairing process, eventually being replaced by the patient's own tissue [1].

Bone can be differentiated, according to the macrostructure, in trabecular (porous) and cortical (compact). At the cellular and molecular levels, bone is composed of cells (osteoblasts, osteoclasts, osteocytes, and bone lining cells) merged in a non-oriented collagen type I matrix, mineralized by hydroxyapatite (HA) that is responsible for toughening the bone [2]. When an injury takes place, a defective microenvironment compromises the normal resorption and regrowth of bone tissue, and consequently its regeneration.

Investigations are being developed based on different strategic cues, such as electromechanical [3,4], chemical [5,6], and morphological [7,8], in an attempt to recreate tissue-specific microenvironments and thus trigger their natural recovery. Morphological cues have been demonstrated to effectively influence cellular proliferation and differentiation, the cell–scaffold interaction triggering a series of physical-chemical reactions. Cells sense the site they are attached to and mechanically transduce that information (hardness, curvature, and shape) into morphological changes [9]. When favorable topographical signals are presented

at the surface of the scaffold, they can trigger the initiation of mechanosensitive cell cascades and thus a cell's differentiation signaling pathways [10–12]. However, the effective mechanism by which morphological cues regulate cell fate, in terms of orientation, morphology, proliferation, and differentiation, is still barely understood. Given the topographic complexity of its natural microenvironment, bone cells are adaptable to different scaffolds' architectures, although it is known that their phenotype is not favored in aligned morphologies, unlike for instance in the case of myoblasts or neurons. Different structures have been developed to grow bone tissue, but only a few trials with micropatterned scaffolds have been reported so far. Micropatterned scaffolds based on polycaprolactone (PCL)/polylactic-co-glycolic acid (PLGA) have been applied for periodontal tissue regeneration [13], demonstrating that micropatterning can effectively enhance tissue responses. HA ceramics with surface micropatterning have been demonstrated to promote the osteogenic differentiation [14]. In addition to the influence of the morphology, studies have demonstrated that piezoelectric biomaterials, capable of providing mechano-electrical stimuli, can enhance bone cell differentiation and regeneration [15,16], as those electro active stimuli effectively mimic the natural cell's microenvironment.

As previously shown [7], pre-osteoblast cells maintain their phenotype when adhered to a scaffold with isotropic hexagonal surface topography, unlike what happens in the linear topography. The influence of both morphologies, hexagonal and linear, on pre-osteoblasts proliferation and differentiation is here studied in an attempt to determine whether it is possible to physically induce the differentiation of bone precursor cells, avoiding the use of biochemical differentiation factors. In addition, it is known that bone tissue presents inherent piezoelectricity, and therefore, morphological features were patterned on a piezoelectric polymer, using the non-biodegradable polymer of poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE) once it presented the highest piezoelectric coefficient among all the polymers [17]. This was done to allow the development of electroactive platforms for bone tissue engineering that combines the morphology and the possibility of further mechano-electrical stimuli to the cells.

## **2. Results and Discussion**
