Polyphenols-loaded electrospun nanofibers in bone tissue engineering and regeneration

Bone is a complex structure with unique cellular and molecular process in its formation. Bone tissue regeneration is a well-organized and routine process at the cellular and molecular level in humans through the activation of biochemical pathways and protein expression. Though many forms of biomaterials have been applied for bone tissue regeneration, electrospun nanofibrous scaffolds have attracted more attention among researchers with their physicochemical properties such as tensile strength, porosity, and biocompatibility. When drugs, antibiotics, or functional nanoparticles are taken as additives to the nanofiber, its efficacy towards the application gets increased. Polyphenol is a versatile green/phytochemical small molecule playing a vital role in several biomedical applications, including bone tissue regeneration. When polyphenols are incorporated as additives to the nanofibrous scaffold, their combined properties enhance cell attachment, proliferation, and differentiation in bone tissue defect. The present review describes bone biology encompassing the composition and function of bone tissue cells and exemplifies the series of biological processes associated with bone tissue regeneration. We have highlighted the molecular mechanism of bioactive polyphenols involved in bone tissue regeneration and specified the advantage of electrospun nanofiber as a wound healing scaffold. As the polyphenols contribute to wound healing with their antioxidant and antimicrobial properties, we have compiled a list of polyphenols studied, thus far, for bone tissue regeneration along with their in vitro and in vivo experimental biological results and salient observations. Finally, we have elaborated on the importance of polyphenol-loaded electrospun nanofiber in bone tissue regeneration and discussed the possible challenges and future directions in this field.


Background
Plant polyphenols are excellent sources of natural antioxidants and antimicrobials, acting as potential drugs in modern biomedicine [1,2]. Tissue regeneration and remodeling is one of the tedious and complex processes in bone tissue regeneration. Polyphenols have been promising bioactive micronutrients to safeguard and maintain bone health [3][4][5]. Plenty of research works have been reported to study the intriguing effects of polyphenols, likely antimicrobial, antioxidant and anti-inflammatory activity playing a vital role in bone tissue engineering [6][7][8]. The polyphenols' action to maintain the balance is attributed to their hydroxyl substituents' hydrogen bond donating ability [9]. Maintaining redox equilibrium is a critical factor in tissue engineering during the angiogenesis process, an essential step to promote long-term survival and engraftment of bone. Polyphenols can be classified into four major groups, flavonoids, lignans, stilbenes, and phenolic acids depending on the number of reactive phenolic units [10]. The flavones and catechins are the most potent flavonoids to protect the body from the reactive oxygen species (ROS) [11]. The mechanisms of polyphenols' antioxidant action include (1) scavenging ROS, (2) up-regulation or protection of antioxidant defenses, and (3) suppression of ROS formation either by inhibition of enzymes or by chelating trace elements involved in the free radical generation [12]. Several polyphenolic compounds such as curcumin, quercetin, catechin, icariin, EGCG, and resveratrol have been studied to apply bone tissue engineering. They initiate upregulation of several biochemical pathways by scavenging free radicals and mediate the expression of inflammatory cytokines involved in bone tissue remodeling, as shown in Fig. 1 [14,15]. Characteristic inhibition of nuclear factor kappa-Β (NF-κB), cyclooxygenase-2 (COX-2), protein-lysine 6-oxidase (LOX), and inducible nitric oxide synthase (iNOS) and activation of activating protein-1 (AP-1), mitogen-activated protein kinase (MAPK), protein kinase C (PKC), nuclear factorerythroid 2-related factor 2 (Nrf2), and phase II antioxidant detoxifying enzymes are affiliated to the antiinflammatory activities of the polyphenols [16]. Fig. 1 Demonstration of molecular signaling pathways of polyphenols involved in bone tissue regeneration. ROS-reactive oxygen species; p53tumor suppressor; Gpx-1-glutathione peroxidase 1; SOD-superoxide dismutase; RANKL-receptor activator of nuclear factor kappa-Β ligand; NF-κBnuclear factor kappa-light-chain-enhancer of activated B cells; NFATc1-nuclear factor of activated T cells 1; c-Fos-proto-oncogene; MAPKsmitogen-activated protein kinases; MMPs-matrix metalloproteinases; ECM-extracellular matrix [13] Polyphenols demarcate the inflammatory responses, control the osteoclast's activation process, and activate the osteoblast's production through various signaling proteins such as RANKL, osteoprotegerin (OPG), etc. [17].
An ideal scaffold for the guided bone regeneration should be biocompatible, space-making, and permeable to fluids but acting as barriers for cells, slowly resorbable, bone-promoting, coupled with exceptional biological properties including antimicrobial ability, and commercially inexpensive [18][19][20][21]. The scaffold with these properties can be achieved in the electrospun nanofibrous membrane of the biomaterials [22][23][24][25]. During the last two decades, many researchers have shown increasing interest in the fabrication of nanofibers for bone tissue engineering applications. They develop nanofibers through multiple techniques such as electrospinning (conventional or coaxial) [26], self-assembly [27], vapor phase polymerization [28], and phase separation [29].
Among these methods, the electrospinning method is a versatile technique, which has been widely utilized to fabricate nano-fibrous scaffolds with nanosized pores and fiber diameter. The electrospun nanofibers thus prepared closely imitate extracellular matrix (ECM) with suitable mechanical property, porosity, and surface-areato-volume ratio, which supports enhanced cell adhesion, spreading, growth, and proliferation [30]. The functional electrospun scaffold can be produced by incorporating desired biomolecules and nanoparticles into the polymeric solution (Fig. 2). The porosity and fiber diameter of electrospun fibers can be tuned by altering the parameters such as voltage, needle to collector distance, injection rate, roller speed, etc. [32]. Core-shell nanofibers are generated using a specialized coaxial electrospinning method, which uses two aligned needles that can concurrently spin two different polymer solutions [33].
The advantage of electrospun nanofibers as a drug carrier is that a greater number of drugs can be  [31]. b Though polyphenols alone can help bone tissue regeneration, electrospun nanofiber containing polyphenols shows enhanced wound healing due to the sustained release of bioactive molecules from the scaffold encapsulated into the scaffold compared to other forms of nanocarriers such as micelles, nanoparticles, hydrogels, etc. [29]. Further, the nanofibers can demonstrate a sustained drug release preserving the bioavailability of active drugs like polyphenols [4,34], antibiotics [35], oligopeptides [36], medicative ingredients [37], and growth factors [38]. Various drug loading strategies lead to different kinds of interaction between drugs and nanofibers, observing different drug-releasing kinetics [39]. A curcumin-loaded PCL/gum tragacanth electrospun nanofiber was demonstrated to improve the bioavailability of curcumin, heal the wound faster, and enhance fibroblast proliferation and collagen deposition [40]. A core-shell electrospun nanofiber of PVA and PLGA loaded with naringin and metronidazole improved nanofibers' antibacterial action, cell mobility, proliferation, and mineralization in dental application [41]. A biodegradable electrospun scaffold incorporated with transforming growth factor β-3 improved stiffness of the nanofiber and modulated chondrogenesis, and increased collagen I protein expression [42].
Bone is a prominent exoskeletal framework safeguarding the vital organs inside the body. The complex cellular architecture of the bone comprised 35% of organic and 65% of inorganic materials and can be classified into micro and nanocomposite tissues [43][44][45][46][47]. A series of biological processes, bone resorption, and bone formation make up the skeletal system, in which four major cells are involved in maintaining multiple extracellular and intracellular signaling networks (Fig. 3). Among the cells, osteoclasts and osteoblasts are responsible for bone resorption and the formation of bone matrix, respectively. These cell structures can withstand the physical pressure and maintain phosphocalcic homeostasis [49,50]. Another cell type formed from the maturate phase of osteoblast is the osteocyte, which acts as a sensor for the endocrine responses [51]. The bone resorption and formation could be integrated using the bone cell lining, called osteogenic cells.
Bone tissue regeneration is the critical process to maintain the bone mass by repairing and regeneration. For years, 25% of trabecular bone and 3% of cortical bone have been removed and replaced through the bone regeneration process in human beings [52]. Inflammation, renewal, and bone remodeling are the three interconnecting phases involved in the bone tissue regeneration process. The inflammatory phase begins within 24 h of bone fracture or damage and continues up to a week. The blood flows into the damaged site leading to coagulation and inflammation as the immediate response after the fracture [53]. At this juncture, a series of complex signals such as proinflammatory signals and growth factors are released in a spatially controlled way [54]. Several inflammatory mediators, including interleukin-1 (IL-1, IL-6, IL-11, and IL-18) and tumor necrosis factor-α, are significantly elevated, leading to angiogenesis through inflammatory cells [55]. The platelets are activated by the damaged blood vessels to  [48] release transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor [56]. Bone morphogenetic proteins are expressed by the osteoprogenitor cells at the fracture site. Inflammatory mediators along with these factors facilitate the proliferation and differentiation of mesenchymal stem cells. Stem cells differentiate into osteoblasts at the periphery of the fracture site during the renewal phase. Intramembranous ossification takes place to develop bone formation after 7 to 10 days of the bone defect. Chondrogenesis occurs at the bulk of the injured tissue, which is mechanically less stable. Endochondral bone and cartilaginous callus formation are initiated through several molecular signaling pathways, and subsequently, the calcified cartilage is replaced with woven bone [57]. In the remodeling phase, the osteoblasts with the restorative ability and the osteoclasts with resorptive ability substitute the already formed bone. Firming up of the fractured callus with a faster healing rate is observed, controlled by the proinflammatory signals and growth hormones. Within some weeks of fracture, the mechanical strength and structure are reinstated, while the molecular and cellular signaling proceeding could take up many years to restore. In a human hip fracture, the bone metabolism controlling hormone's level remains spiked up for over a year [58]. The present review elaborates on the role of polyphenol in bone tissue engineering and their sustained activity of in-loaded electrospun nanofibers. Further, possible challenges and future directions have been discussed in this field.

Contribution of polyphenols in bone tissue regeneration
Several in vitro and in vivo studies have been reported by many researchers to study the potential role of polyphenols in bone-related cells and bone defect models of experimental animals, respectively [59][60][61][62][63][64][65][66]. We have compiled, in this section, the source of availability, experimental parameters, and salient outcomes of polyphenols in bone tissue regeneration (Table 1).
Curcumin, extracted from the rhizome of Curcuma longa, has become a subject matter as a potential therapeutic agent in the orthopedic field [67]. Curcumin supplementation has been proven to be efficient in preventing and managing osteopenia and has been reported to have beneficial effects on fat metabolism and bone health [68]. The potential mechanisms of curcumin include inhibition of nuclear factor NF-κB, RANKL, inflammatory cytokine synthesis, and the generation of reactive oxygen species and nitric oxide [69,70]. Ahmed et al. studied the effect of curcumin (CR group) on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and mouse embryonic fibroblasts (MEFs) compared with all-trans-retinoic acid (ATRA group) and osteogenic medium only as control (OM group). Curcumin stimulated osteogenic differentiation at the cellular and molecular level and increased the expression of osteogenic differentiation markers such as Runx2, osterix, and BMP2. The positive effect of curcumin showed a strong ALP staining intensity, higher mineralization, and upregulation of osteo specific bone markers, confirming an improved osteogenic differentiation of BMSCs compared with ATRA and control (Fig. 4). Moreover, it enhanced the osteogenic differentiation in MEFs reprogrammed with the osteogenic factor hLMP 3, participating in the regulation of bone remodeling [59]. Safali et al. examined the effects of curcumin on bone healing using a total rat femur fracture injury model. Unexpectedly, they found that curcumin had no effect on fracture healing based on biomechanical, radiological, and histological evaluations on 14 and 28 days of investigation. However, they suggested that curcumin's impact may be more noticeable in long-term follow-up investigations because of its potential positive effects, such as activation of cell migration and autophagy during the remodeling phase [60].
Vester et al. examined the dose-and time-dependent effect of green tea extracts (GTE) in human osteoblasts, isolated from femoral heads of patients undergoing total hip replacement. They performed RT-PCR to access the combined effects of GTE (0.01, 0.1, and 1 μg/ml) and H 2 O 2 (50 μM) on the osteogenic genes and found significant expression of bone-related genes such as osteocalcin and collagen1α1 during osteoblast differentiation. They reported that GTE, at all the concentrations studied, enhanced the mineralized matrix development despite H 2 O 2 treatment. Further, GTE significantly reduced oxidative stress improving cell viability, suggesting that dietary supplementation of GTE could reduce inflammatory reactions in bone-related diseases such as osteoporosis. Osteoporosis is characterized by structural deterioration of bone tissue and low bone mass causing bone fragility [61]. Shen et al. investigated whether green tea polyphenol (GTP) has the potential to restore bone microstructure in both estrogen adequate (sham group) and estrogen-deficient (OVX group) middle-aged female rats. According to HPLC-ECD and HPLC-UV analyses, GTP (1000 mg) contained a mixture of epigallocatechin gallate (480 mg), epicatechin gallate (160 mg), epicatechin (60 mg), epigallocatechin (103 mg), and catechin (30 mg). The analyses of dual-energy X-ray absorptiometry, micro-computed tomography, and histomorphometry revealed that GTP supplementation increased trabecular thickness, volume, and number and periosteal bone formation rate of tibia shaft, cortical thickness, and femur area. Meanwhile, GTP decreased bone erosion of proximal tibia, trabecular separation, and endocortical bone erosion of the tibia shaft. OVX rat groups demonstrated a dose-dependent increase with GTP in the parameters such as mineralized bone surface and bone formation rate. In contrast, SH rat groups with GTP did not show any significant difference [62].
A new ceramic granulated biomaterial (Synergoss Red, SR) was functionalized with red grape pomace extract containing polyphenolic mixture to study its regeneration effect on periodontal tissues [63]. The primary polyphenols present in pomace extract, including quercetin [71], kaempferol [72], and catechins [73], were shown to direct osteogenic differentiation in different mesenchymal stem cell types. The bone filler, SR, was synthesized from the mixture of 47 wt% of hydroxyapatite (HA) and tricalcium phosphate (β-TCP) powders with the binding agent, 3 wt% of poly(vinyl alcohol). A 0.2 g of Synergoss Red was capable of adsorbing around 0.951 mg of polyphenols. Several studies have confirmed that polyphenols exhibit antioxidant properties naturally and involve many bone regeneration mechanisms [74,75]. In the present study, the compound showed free radical inhibition by 72.8%, characterized by a DPPH assay. Polyphenols in Synergoss Red significantly reduced the level of iNOS expression (p < 0.0001) compared to control (bone filler alone). The antiinflammatory and antioxidant properties of polyphenols in pomace extract exerted a protective role in bone loss by reducing osteoclastogenesis and enhancing osteoblastogenesis.
Naringin, a dihydrotestosterone flavonoid compound, has been reported to improve bone density, inhibit bone loss, and augment biomechanical anti compression performance [76]. Fan et al. studied the osteogenic differentiation ability of naringin in BMSCs collected from the lateral tibial tubercle of the white rabbit. The treatment of BMSCs with 0.1, 1, and 10 μM naringin for 48 h significantly increased the mRNA expression levels of OC, ALP, and Col I, compared to control (without additive). The western blot and RT-PCR analyses showed a decreased PPARγ protein expression and an increased miR-20a marker expression in BMSCs when the cells were treated with 1 μM of naringin for 21 days. The results suggested that naringin, as a potential drug, may promote BMSCs' differentiation into the osteoblasts during osteoporosis treatment [64]. Apigenin (4′,5,7-trihydroxyflavone), a member of the flavone family of flavonoid compounds, was reported to possess remarkable anti-carcinogenic, antioxidant, and estrogenic properties [77]. Zhang et al. studied the transducing ability of apigenin in hMSCs into osteoblasts and reported that apigenin significantly increased activity of ALP and the mineralized nodule formation in a dose-dependent manner [65]. The cells treated with 5 μM of apigenin significantly increased Runx2 and OSX protein expression through JNK and p38 MAPK pathways, which play a pivotal role in regulating the osteogenic differentiation of MSCs [78].
The research group of Zhao et al. evaluated the osteogenic effect of icariin through in vitro and in vivo biological characterizations [66]. Icariin, a flavonoid glycoside isolated from the herb of Epimedium pubescens, has been reported to have potential therapeutic effects on a rat model of osteoporosis induced by ovariectomy [79,80]. Preosteoblast MC3T3-E1 and fibroblast NIH3T3 cells were used for the in vitro osteogenesis analysis in work. MC3T3-E1 cells treated with 10 − 5 M of icariin exhibited a significant increase in ALP activity, Runx2, bone sialoprotein (BSP), and osteocalcin (OCN) expression at day 3. In contrast, icariin-treated fibroblast cell line NIH3T3 had not shown remarkable ALP and protein expression. Two types of animal

Advantages of polyphenol-loaded electrospun nanofibers
Biocompatible and naturally available biopolymers and synthetic polymers have been widely used to prepare electrospun nanofibrous mats for tissue engineering applications [81][82][83]. The current situation demands the fabrication of highly bioactive scaffolds with superior biocompatibility, mechanical properties, and remodeling potential to repair the damaged tissues. The same can be achieved by either surface functionalization or Fig. 4 In vitro mineralization assay of bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation following treatment with curcumin (CR) and all-trans-retinoic acid (ATRA) groups. BMSCs in the osteogenic medium were indicated by the OM group. Alkaline phosphatase (ALP), Alizarin red (ALZ), and von Kossa (VK) staining results were obtained after 1, 3, and 4 weeks of post-induction, respectively. Quantification of staining intensity was performed with ImageJ software. The level of calcium deposition reflects the extent of mineralization, which was higher in the CR group than in the OM and ATRA groups. ATRA group did not show any symptoms of mineralization. The data were represented as the mean ± standard deviation (n = 3). The statistical significance was defined using a one-way analysis of variance, followed by Tukey's post hoc test. *p < 0.05, **p < 0.01 and, ***p < 0.001 [59] incorporation of bioactive materials in the nanofiber membrane. The nanofiber scaffold's primary goal is to provide an appropriate microenvironment for bone tissue to restore and facilitate the bone tissue regeneration process [84]. Ideally, the fabrication of polyphenolloaded electrospun nanofibers scaffolds has some advantages in bone tissue regeneration applications. They exert anti-inflammatory and antioxidant activity, improve bioavailability, and release the polyphenols at a sustained level in the cell differentiation site. They provide an active shield against infection, minimize toxicity to other tissues, and enhance the bone remodeling process via calcium deposition and activation of several bone-specific proteins [85]. The incorporated biomolecules into the scaffold can interact with the biomaterial's surface through various physical and chemical forces, including hydrogen bonding, hydrophobic interaction, and Van der Waals force [86]. It was reported that the ion complexation property of bioactive molecules could cause protein deactivation and denaturation [87,88].
The surface-functionalization of nanofibers with bioactive molecules via non-covalent immobilization techniques protects the nature of the bioactive molecules and the structure of biomaterials [89,90]. Further, the bioactive molecules improve hydrophilicity and surface charge of the nanofiber's surface, establishing a favorable milieu, enhancing the protein adsorption on its surface [91,92]. Henceforth, the fabrication of electrospun nanofibers using biocompatible polymer or specific polyphenols expands their mechanical, biological, and functional properties, leading to cell attachment, cell migration, and cell proliferation [93]. A list of polyphenols incorporated electrospun nanofibers and their application in bone tissue regeneration has been provided in Table 2. Jain et al. prepared curcumin-loaded PCL electrospun nanofibers (CU1 and CU5) to investigate the influence of curcumin drug release from the scaffold on osteogenesis and compare the results with PCL scaffold without drugs (CU0). It was found that both fiber mats released around 18% of the drugs on day 3. However, CU1 and CU5 showed different drug releases of 42 and 50%, respectively, on day 6 of the investigation. The in vitro results using MC3T3-E1 mouse pre-osteoblasts demonstrated that ALP activity of the scaffolds was found in the order of CU1 > CU0 > CU5. The optimized concentration of curcumin and sustained drug release from CU1 helped increase osteogenic expression compared to CU5, which had a high drug loading content [94]. Sedghi et al. developed bioactive molecule-loaded coaxial electrospun nanofibrous scaffolds with antiinfective properties to prove effective bone tissue regeneration. The bioactive complex was composed of zinccurcumin (Zn-CUR) and graphene oxide. The developed core-shell nanofiber membrane comprised a blend of polyvinyl alcohol and carboxymethyl chitosan (PVA/ CMCh) in the shell and 4armPCL/Zn-CUR in the core part. Cellular morphology and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide) assay showed that Zn-CUR-containing scaffolds substantially supported cellular adhesion, spreading, and proliferation compared to drug-free scaffolds. Moreover, the Zn-CUR complex in the scaffolds increased ALP activity and matrix mineralization and reduced postoperative infection with an excellent antibacterial activity as the metalorganic complex improves the bioavailability of curcumin. Further, complex localization into the core part of the core-shell nanofiber leads to its controlled release, enhancing its therapeutic efficiency [95].
Dhand et al. fabricated catecholamine contained collagen nanofiber with excellent mechanical property without interfering with hydrophilicity of the nanofiber surface [102]. The in vitro cell viability and calcium deposition analysis confirmed that the highly biocompatible catecholamine contained composite nanofiber enhanced calcium mineralization (Fig. 5). Lee et al. prepared catechin coated functional PCL nanofibrous scaffolds with antioxidative property and calcium-binding ability to achieve an enhanced osteogenic differentiation of human adipose-derived stem cells (hADSCs). The scaffold was reported to significantly promote in vivo bone formation in a critical-sized calvarial bone defect. The scaffolds were divided into five different groups, no treatment, PCL scaffold (PCL), catechin coated PCL scaffold (PCL-Cat), PCL with hADSCs (PCL-hADSC), and catechin-coated PCL scaffold with hADSCs (PCL-Cat-hADSC). The results of micro-CT images and histological examination demonstrated that PCL-Cat-hADSC showed an improved tissue regenerative efficacy by the influence of catechin (Fig. 6) [96].
Jeong et al. developed polyhedral oligomeric silsesquioxane-epigallocatechin gallate (POSS-EGCG) loaded poly (vinylidene fluoride) electrospun nanofiber to investigate bone tissue regeneration [97]. Epigallocatechin gallate (EGCG), a polyphenolic flavonoid derived from a variety of plants, has been reported to impede lipopolysaccharide (LPS)-stimulated osteoclastic bone resorption and reduce inflammatory bone loss in bone metabolism [103,104]. It was found that the concentration of 6 wt% POSS-EGCG conjugates (PE06) loaded PVDF nanofibrous scaffold showed betterment in physiochemical and mechanical properties than 0, 2, and 4 wt% conjugate loaded scaffolds (PVDF, PE02, and PE04, respectively). The in vitro osteogenic and osteoblast differentiation results confirmed that PE06 exhibited a higher ALP activity and bone mineralization than other scaffolds. Our recent work demonstrated a promising PCL-gelatin-(Zn + Q(PHt)) nanofibrous scaffold to    [101] increase osteoblastogenesis, leading to bone formation. The metal-organic complex (Zn + Q(PHt)) was synthesized by refluxing a methanolic mixture of phenanthroline (PHt), quercetin, and Zn 2+ ions. The fibers diameter distribution of PCL-gelatin-(Zn + Q(PHt)) scaffold was 250-500 nm with 72% of bioactive complex entrapped into nanofiber matrix. The in vitro and in vivo biological studies indicated that the scaffold exhibited a better osteogenic differentiation with a large amount of Runx2 and type 1 collagen mRNAs expression than the PCLgelatin nanofiber alone [98]. Resveratrol (RSV), a natural polyphenolic compound, is present in numerous plant products, including red wine. Its biological effects are antioxidant, anticancer, anti-inflammatory, cardiovascular protection, antiaging, and bone-protective property [105,106]. It stimulates osteoblast differentiation in a dose-dependent manner activating MAPK signaling pathway and further inhibits bone resorption by constraining RANKL-induced osteoclast differentiation [107]. RSV has been used for the alveolar socket reduction and remodeling of the dental implant after removing the tooth [108,109]. However, the oral bioavailability of RSV is limited due to its low water solubility, poor pharmacokinetics, and instant metabolism. Hence, it is required to formulate a sound delivery system to deliver RSV at the target site [110]. Riccitiello et al. prepared RSV-loaded defect-free PCL (PCL-RSV) and poly(lactic) acid electrospun nanofibers (PLA-RSV) for the treatment of alveolar bone defect. Both PCL-RSV and PLA-RSV promoted human dental  pulp stem cells (DPSCs) differentiation into osteoblastlike phenotype, triggering the expression of early (Runx2 and OSX) and late (OCN, ONN, OPN, and BSP) osteoblast differentiation markers. Though PLA-RSV contained a lower number of drugs than PCL-RSV, only the former scaffold could induce osteoblast differentiation and inhibit osteoclast differentiation, suggesting its use in preserving the post-extraction alveolar ridge volume during bone resorption and new bone formation [99]. Gong et al. fabricated an icariin-loaded core-shell electrospun membrane to imitate artificial periosteum for bone tissue regeneration. They prepared different core-shell type electrospun nanofibrous scaffolds from PCL and gelatin polymeric components and labeled them as PG (without drug), PGM (MOX loaded), PGI (icariin loaded), and PGMI (dual drug-loaded). All the prepared scaffolds showed membrane degradation up to 60-80% for over 2 months. The sustained release of icariin from the nanofiber PGI helped augment osteogenic differentiation, especially ALP expression and Ca 2+ deposition. However, dual drug-loaded PGMI was effective in showing more expression of OCN and COL I. The histology, immunohistochemical and radiographic results demonstrated that the quality of bone formation and the quantity of bone mass was highly enhanced in the PGMI group than that of PG and untreated rabbit groups [100]. In another research work, the same researcher prepared various PCL/gelatin nanofibrous scaffolds with different concentrations of icariin viz. 0, 1.2, 2.4, 12, 24, and 120 mg/ml and labeled as PGI0, PGI0.005, PGI0.01, PGI0.05, PGI0.1, and PGI0.5, respectively. They found that PGI0.05 possessed exceptional overall performances related to bone regeneration by accelerating OCN, ALP, COL I, and calcium expression. Further, the degradation behavior and mechanical strength of PGI0.05 were also reported to fulfill the requirements of an artificial periosteum. Hence, the PGI0.05 scaffold was recommended as a potential artificial periosteum to repair large-sized bone defects [101]. The compilation of research materials demonstrates that polyphenols contribute to enhancing bone tissue regeneration as the drug molecules alone or the active components in scaffolds like electrospun nanofibers.

Conclusion and future perspectives
This review demonstrated the in vitro and in vivo bone tissue regenerating ability of polyphenols with or without the electrospun nanofibrous scaffold. The beneficial properties of polyphenols loaded nanofibrous scaffolds are antioxidant property, biocompatibility, porosity, flexibility, tensile strength, cell proliferation, and osteogenic differentiation. However, there are some issues to be addressed in this field. (1) Though many reports are available to study polyphenols loaded incorporated electrospun nanofiber for bone tissue regeneration, the nanofibers prepared in their studies are mostly accomplished using the conventional electrospinning method. Only a few reports are available to prepare core-shell nanofibers using coaxial methods, releasing polyphenols with a desired drug delivery profile. According to the literature reports, the core-shell structured nanofiber permits the encapsulation of sensitive bioactive molecules into the core portion for better loading and controlled long-term release compared to normal nanofiber [95]. Hence, the researchers should focus on loading polyphenols into differently structured nanofibers to increase their therapeutic efficacy.
(2) The researchers should conduct drug loading/release profiles using different polyphenols in the nanofibrous mats to provide comparative data. (3) The bone tissue regeneration phase, in which the polyphenols contribute more effectively, should be investigated through many systematic biological studies. (4) The selection of suitable polyphenols for scaffold fabrication is still an open question in most cases due to the non-specific regulations and theoretically indistinguishable structure-function performance. So, more works need to be carried out to identify which polyphenols category is most suitable for bone tissue engineering applications. (5) The nature and functionality of the polyphenols should be analyzed when they are released from the nanofibrous mats because the polyphenols may alter their molecular structures and dissolution depending on the external medium. We are suggesting the following comments for the future direction in this field. (1) Characterizing more in vitro and in vivo biological studies based on polyphenol-loaded electrospun nanofibrous mats prepared by the coaxial method. (2) Tracking the presence of polyphenols and determining their quantity in different bone tissue regeneration phases using appropriate sophisticated methods. (3) Analyzing the nature of polyphenols before and after loading into the scaffolds using spectral characterizations such as HPLC and NMR. (4) Performing more molecular level studies to gain insights into the cellular mechanism in which polyphenols are involved. We anticipate that researchers with interdisciplinary backgrounds will develop bone tissue regeneration by emphasizing polyphenols' importance and suggestions.