**1. Introduction**

A variety of techniques have been tried for the regeneration of bone defects, including bone grafting, distraction osteogenesis, and guided bone regeneration (GBR) [1–3]. Among them, GBR has been used as an efficient method for both the reconstruction of the structure of, and reestablishment of the function in, bone defects and damaged tissues. The GBR approach principally uses a membrane acting as a barrier membrane in between soft tissue and bone tissue defect. GBR membranes prevent the migration of faster-growing fibrous tissue into the defect and secure osteoconduction (bone growth) to promote bone regeneration [3]. Thus, GBR membranes need to have biocompatibility to support bone tissue reconstruction and mechanical stability to maintain a protective space during bone restoration process.

Nanofibrous membranes have structural similarity to the extracellular matrix (ECM) and high specific surface area [4]. GBR membranes composed of ultrafine nanofibers are more suitable for cell attachment and proliferation than conventional membranes with large scale structures. Various

strategies—such as self-assembly, wet-spinning, and electrospinning—have been used to fabricate GBR membranes [4–10]. Of these strategies, electrospinning has attracted a great deal of attention because this method is very simple and versatile, and can produce various polymer nanofibers. Electrospun polymer nanofibers have unique advantages in the biomedical fields such as drug delivery and tissue engineering, as they can form porous structures that simulate the features of the ECM [2,4,11,12].

Poly(vinylidene fluoride) (PVDF) is an attractive semicrystalline polymers for use in biomedical applications because of its efficient piezoelectricity and biocompatibility [13,14]. The strong piezoelectric effect observed in PVDF is directly linked to the crystalline phase arrangement. PVDF exits in one of five polymorphic crystalline phases (α, β, γ, δ, and ε) dependent on its distinct chain conformation [15]. The nonpolar α-phase does not exhibit an efficient conformation for piezoelectric application, but it does present good mechanical properties and thermal stability. On the other hand, β- and γ-phases exhibit electroactive and polar properties. Among them, the β-phase has a higher density net dipole moment and exhibits more promising piezoelectric properties. Therefore, increasing the piezoelectric β-phase content in PVDF is very important for its biomedical application because piezoelectric materials can provide electrical stimulation to cells to promote tissue formation and can use as a scaffold for tissue engineering [14,16]. This phenomenon is very similar that electrically charged ECM stimulates the cell growth during tissue regeneration [14]. In particular, bone tissue and its constituent collagen fibers possess intrinsic piezoelectricity to play a significant role in regeneration of the damaged bone, and thus a variety of piezoelectric materials have been applied to promote bone regeneration both in vitro and in vivo [16,17].

Polyhedral oligomeric silsesquioxane (POSS) molecules have a unique nanostructure comprised of an inorganic silsesquioxane cage core and an organic functional group shell [18]. A variety of POSS molecules contain reactive functionalities, making them suitable precursors for grafting. Therefore, they have been used in various biomedical applications including tissue engineering due to their distinctive structure and excellent biocompatibility [19,20]. Epigallocatechin gallate (EGCG) is a polyphenolic flavonoid derived from a variety of plants and, in particular, is a major bioactive component in green tea, which has been shown to possess a variety of pharmacological functions; for instance, it exhibits antioxidant, anticancer, and anti-inflammatory properties [21–23]. EGCG inhibits lipopolysaccharide (LPS)-induced osteoclastic bone resorption and attenuates inflammatory bone loss in bone metabolism [24,25]. Moreover, EGCG increases osteoblast proliferation and alkaline phosphatase (ALP) activity, which leads to increased osteoblastic bone formation [26].

Piezoelectric materials can change their surface charge without external energy power source [14]. Several research papers have reported an observed enhancement of the piezoelectric β-phase content in PVDF fabricated by electrospinning with the incorporation of fillers, such as metal nanoparticles, ceramics, and inorganic salts [27–30]. In addition, EGCG-incorporated membranes have been fabricated and have been observed to promote osteoblastic proliferation and prevent inflammatory responses [26]. Therefore, in this study, we fabricated PVDF/POSS–EGCG conjugate composite nanofibers, which showed improved piezoelectric, antioxidant, and anti-inflammatory properties, to support bone tissue regeneration using an electrospinning method. We systematically examined the effect of the POSS–EGCG conjugate content on the structure and physicochemical properties of the composite nanofibers obtained. Furthermore, the in vitro bioactivity of the composite nanofibers was investigated through the cell proliferation, ALP activity, and bone mineralization assays.

#### **2. Materials and Methods**

#### *2.1. Materials*

PVDF (*Mw* = 275000 g/mol), EGCG, lipopolysaccharide (LPS), horseradish peroxidase (HRP), hydrogen peroxide (H2O2, 30 wt% in H2O), dimethyl sulfoxide (DMSO), *N,N*-dimethylformamide (DMF), tetrahydrofuran (THF), and isopropyl alcohol (IPA) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Xanthine oxidase (XO from buttermilk, EC 1.1.3.22), xanthine, and nitro

blue tetrazolium (NBT) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Aminopropylisobutyl polyhedral oligomeric silsesquioxane (API-POSS) was purchased from Hybrid Plastics (Hattiesburg, MS, USA). Osteoblast-like cell line MC3T3-E1 derived from mouse calvaria was acquired from the American Type Culture Collection (Manassas, VA, UAS) and a murine macrophage cell line RAW 264.7 was purchased from the Korean Cell Line Bank (Seoul, South Korea). All materials were used as received without further purification.
