Flavonoid loaded mineral clay-based 3D scaffold for bone tissue regeneration

Flavonoid loaded mineral clay-based 3D scaffold for bone tissue regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Flavonoid loaded mineral clay-based 3D scaffold for bone tissue regeneration Logeshwaran A, Renold Elsen, Ashutosh D Bagde, Sunita nayak This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8679759/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract For bone tissue regeneration, 3D printing ceramic materials is beneficial, however material synthesis and commercially accessible composites raise demand and decrease cost-effectiveness. Implementing more composite materials to the defective site may triggers oxidative stress to the surrounding cells. This study focuses on the development of 3D printed BEN-HAP scaffold infilled with flavonoids conjugated silk fibroin for bone tissue replacement for controlling oxidative stress during tissue regeneration. The silk fibroin infilled regions in the scaffold showed a sponge like fibre matrix that facilitates cell adhesion and proliferation which were observed under FESEM. Also, flake-shaped appetite formation was observed higher in SF-infilled scaffolds, confirming the calcium and phosphate mineralization layer. The scaffolds showed improved surface properties that facilitated slow water permeation and absorption compared with BEN-HAP scaffold. The biocompatibility results showed no toxic effect on hWJ-MSCs and RBCs additionally, the quercetin and hesperetin loaded SF-BEN-HAP scaffold showed more than 60% closure promoting a high cell migration rate at 10 µM concentration in scratch test assay. And a maximum reduction in ROS activity in hWJ-MSCs was observed when treated with 20 µM flavonoids. Bentonite Silk fibroin 3D Bone scaffold Antioxidants Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Scaffolds have been utilized to develop various tissues and organs, including skin, trachea, bone, pharynx, and myocardium, for translational and preclinical models [ 1 ]. The difficulty in bone tissue engineering is creating a structure based on biomaterials that can replicate the various physiochemical characteristics of bone, including its shape, architecture, mechanical qualities, and physiology [ 2 ]. Complex tissue 3D reconstruction is a good fit for additive manufacturing techniques because of its layer-by-layer assembly approach. Additionally, the functionality of the 3D construct is improved by incorporating biological materials (cells, proteins, enzymes, and growth factors)[ 3 ]. The lack of bioactive molecules in 3D fabricated scaffolds could delay the regeneration process and may have possibilities for graft rejection. Increasing bioactivity of the ceramic composite 3D scaffold for bone tissue regeneration can be a concern that can be overcome by loading growth factors, polymers, and proteins. The ceramic-based 3D printed scaffolds can undergo different thermal treatments/sintering processes to attain mechanical properties such as compression strength, hardness, and density [ 4 ]. Common bioceramic 3D scaffolds such as hydroxyapatite and calcium phosphate are porous and hydrophilic and can support angiogenesis, biosorption, and bone regeneration [ 5 ][ 6 ]. Considering the contribution of scaffolding on cell adhesion, proliferation, and differentiation, scaffold rejection might also occur due to a lack of cell-scaffold interaction (bio-interaction), poor microenvironment, and bio interface [ 7 ]. Thus, incorporating polymers or protein compounds into the porous scaffold is essential to enhance the bioavailability. The incorporation of bioactive molecules significantly impacts the scaffold characteristics and cell behavior [ 8 ]. According to the tissue engineering perspective, bioactive molecules such as peptides, flavonoids, and growth factors are known to improve the scaffold characteristics, promote cellular activity, and enhance tissue regeneration [ 2 ]. Collagen, gelatin, chitosan, elastin, hyaluronic acid (HA), and silk fibroin are well-known biodegradable natural polymers frequently utilized for scaffold fabrication. Polymer infiltration in 3D printed porous scaffolds is a technique for improving the bioactivity of the scaffold, where they facilitate cell-scaffold interaction and enable the delivery of biomolecules such as peptides, growth factors, and flavonoids for different applications [ 9 ][ 10 ][ 11 ][ 12 ]. Despite having a wide range of polymers, silk fibroin is a naturally available fibrous protein with unique properties such as inducing mineralization, increasing biocompatibility, and delivery of therapeutic compounds [ 13 ][ 14 ]. Also, the presence of several peptides and amino acids enables interaction with other biomolecules creating bonding for drug delivery application [ 15 ]. Bentonite (BEN) is a clay mineral composite material with diverse mineral availability, biocompatibility, and structural stability [ 16 ]. Based on its properties, it can be combined with an osteoconductive bioceramic material, hydroxyapatite (HAP), to enhance the bone regeneration process [ 17 ][ 18 ]. The composite slurry also indicated advantages in the extrusion process and further fabrication of load-bearing biocompatible scaffolds, having a compression strength of 52 MPa with suitable physical characteristics ideal for bone tissue graft [ 19 ]. The implantation of bone grafts including the scaffolds has been associated with oxidative stress due to the mineral ions leaching at the site. Oxidative stress is an imbalance in ROS in mitochondria and their neutralization by protective mechanisms [ 20 ]. At minimal levels, ROS function as signaling molecules that are crucial for regulating cellular functions such as proliferation and differentiation [ 20 ] as well as modulating signaling functions in transduction, inflammation, and receptor activation [ 21 ]. However, increased levels of ROS at the damaged area and the scaffold implanted site may cause oxidative stress. Imbalances in this protective system can cause damage to DNA, proteins, and lipids [ 22 ] [ 23 ]. Thus, it is crucial to give bone graft materials antioxidative qualities to facilitate the early regeneration of bone [ 24 ]. Polyphenolic chemicals or biomolecules like flavonoids are known for their various health impacts including anti-inflammatory and antioxidant capabilities [ 25 ] [ 26 ]. Quercetin and hesperetin are cost-effective flavonoids that are natural antioxidants used to reduce oxidative stress. In this work, the 3D printed BEN-HAP composite scaffold was infilled with SF to improvise the properties such as mineralization, cell-scaffold interaction, hydrophilicity, and hemocompatibility for bone tissue engineering (BTE) application. The BEN acts as an inorganic mineral matrix-like bone (consisting of mineral ions such as iron, magnesium, sodium, and phosphorous), and SF acts as an organic matter like collagen, which makes them an ideal bone-mimicking organic-inorganic matrix for tissue development. 2. Materials and methods 2.1 Materials Quercetin (QTN) and Hesperetin (HTN) were purchased from Sigma Aldrich (India), and silk cocoons were procured from Karnataka, India. Lithium bromide and sodium carbonate were purchased from Himedia (India) for the preparation of the silk fibroin solution. For cell culture studies, Dulbecco’s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), Penicillin/streptomycin antibiotic solution, 4% paraformaldehyde solution, dimethyl sulfoxide (DMSO), and MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) were purchased from Hi-Media (India). The calcein-AM and propidium iodide (PI) were purchased from Sigma Aldrich (India). ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), DPPH (1,1-Diphenyl-2-picrylhydrazyl) and DCFDA (2',7'-dichlorofluorescin diacetate) were purchased from Sigma Aldrich (India). 2.2 Silk Fibroin Preparation and Infilling in Scaffold For SF preparation, the cocoons were cut and boiled with 0.02 M Sodium carbonate, the silk fibers were then dried at 37 ºC. The silk fibers were dissolved in 9.2 M of lithium bromide solution at 60ºC for 4 h in a hot air oven [ 27 ]. The supernatant was collected and dialyzed for 48 h, and the SF solution was stored at 4ºC. A known volume of SF was dried and weighed to determine the final concentration of prepared SF [ 28 ]. The 3D printing of the BEN-HAP scaffold was done as previously described in paper [ 19 ]. In brief, a 1:1 ratio of BEN and HAP was prepared into a slurry using 2% Carboxymethyl cellulose and extruded in a 0.8 mm nozzle at a speed of 2 mm/s with pressure 0.25 MPa. The infill ratio and scaffold dimensions were 60% and 10*10*6 mm, respectively. The printed samples were further sintered at 1000˚C for 2 hr. The scaffold is then infiltrated with silk fibroin solution using a syringe, followed by a lyophilization process to remove moisture. 2.3 Preparation of Flavonoid-Silk Fibroin Conjugate The conjugation of flavonoids (quercetin and hesperetin) with silk fibroin was done by a conventional mechanical stirring method. The stock concentration of quercetin and hesperetin (1 mg/ml) was prepared by dissolving the flavonoids separately in DMSO: PBS (pH 7.4) solution in a 1:4 ratio (ml) as per the manufacturer’s instruction. Quercetin and hesperetin concentrations of 1, 5, 10, and 20 µM were prepared and mixed separately with silk fibroin. The solution was blended at low rpm in a mechanical stirrer for 30 min at room temperature. The prepared conjugate solution was stored at 4 ºC [ 29 ][ 30 ][ 31 ]. And further infiltrated in BEN-HAP scaffold using a syringe followed by a lyophilization process to remove moisture. 2.4 FTIR Analysis The functional groups of the synthesized SF, and conjugates SF-QTN and SF-HTN were confirmed by Fourier Transform Infrared (FTIR) spectrum analysis by Cary 630 spectrometer (US) in the range of 400 to 4000 cm -1 . 2.5 FESEM Analysis The SF-infiltrated 3D printed BEN-HAP scaffolds (SF-BEN-HAP) were gold sputter coated and observed under FESEM (Field emission scanning electron microscopy) for morphological analysis. 2.6 Contact Angle Measurement The wettability of the BEN-HAP and SF-BEN-HAP scaffolds was investigated as per ASTM D7334–08 [ 32 ] Standards. A static angle with a contact angle meter (HO-IAD-CAM-01A) was used. The experiment was performed by the Sessile drop method, where approximately 5 µl of distilled water was added from the micro syringe onto the sample and the behavior was examined. 2.7 In vitro Mineralization Mineralization/Apatite formation of the BEN-HAP and SF-BEN-HAP scaffold was performed by incubating the scaffolds in Simulated body fluid (SBF) of pH 7.4 at 37°C for 14 days, maintaining in same volume (3 ml) at submerged conditions throughout the study [ 33 ]. The scaffolds were then removed, dried at room temperature, and observed in FESEM after gold sputter coating. Changes in mineral composition among the scaffold groups were evaluated in EDAX. 2.8 Antioxidant Assay of Flavonoids: ABTS and DPPH Radical Scavenging The stock concentration of quercetin and hesperetin (1 mg/ml) was prepared by dissolving the flavonoids separately in DMSO: PBS (pH 7.4) solution in a 1:4 ratio (ml) as per the manufacturer’s instruction. Different concentrations of flavonoids (1, 5, 10, 20, 40, 80, 160, and 320 µM) were made from the stock solution to measure the antioxidant activity. For the ABTS radical scavenging assay, the reagent was prepared by dissolving ABTS in potassium persulfate (2.45 mM) with the final concentration of 7 mM ABTS solution. The mixture was kept in dark conditions for 12–16 h. The reagent was diluted with water and checked for absorbance of 0.71 at a wavelength of 734 nm. The final concentration of ABTS was stored at 4ºC. 50 µl of different concentrations of quercetin and hesperetin were added to 150 µl of ABTS and incubated in the dark for 30 min. The reduction of the absorbance was measured in a microplate reader (Tecan, Switzerland) at a wavelength of 734 nm [ 34 ] [ 35 ]. For DPPH assay, 0.5 mM DPPH solution was prepared in methanol. 50 µl of different concentrations of quercetin and hesperetin were added to 150 µl of DPPH and incubated in the dark for 30 min. The reduction of the absorbance was measured in a microplate reader (Tecan, Switzerland) at a wavelength of 517 nm [ 36 ] [ 37 ]. Ascorbic acid was taken as standard for both ABTS and DPPH assay for evaluating the scavenging activity, percentage of activity was measured by the following Eq. ( 1 ), Where, A B is the absorbance of the blank and A F is the absorbance of the flavonoid sample. 2.9 Fabrication of Flavonoids-Loaded SF-BEN-HAP Scaffold Quercetin and hesperetin concentrations of 1, 5, 10, and 20 µM were prepared and mixed separately with silk fibroin. The solution mixture was conjugated by blending at low rpm in a mechanical stirrer for 30 min at room temperature [ 29 ][ 30 ][ 31 ]. The prepared conjugate solution was infilled and lyophilized in 3D-printed BEN-HAP scaffolds (S-10). 2.10 Biocompatibility For accessing the cytocompatibility, human Wharton’s jelly mesenchymal stem cells (hWJ- MSCs) (NCCS, India) were cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. After 65% confluency, the scaffolds were placed on the monolayer of the cell and incubated for 72 h with 5% CO 2 supply in an incubator at 37 ºC as per ASTM F813 [ 38 ]. The MTT assay was performed as per the manufacturer’s instructions (EZCount MTT Cell Assay Kit, HiMedia, India) and measured the absorbance at wavelength 570 nm using an ELISA plate reader (Robonik, India). For live and dead assay, 20 µl of 10 µM calcein acetoxymethyl (AM) and propidium iodide (PI) in PBS (7.4 pH) was added to the cells cultured after 24 h and 72 h in the presence of scaffolds. The cells were then observed under Fluorescence microscopy (EVOS M5000). The scratch test was performed to assess the effect of in vitro cell migration in the presence of scaffold leach-out (72 h scaffold leach-out). The hWJ-MSCs cells were cultured in 24-well plate with Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. After 90% confluency, a perpendicular scratch was made using a sterile pipet tip in the middle of each well. Fresh media, along with 200 µl scaffold leach-out, was added to each well. The cell migration was observed at 0 h and 24 h under optical microscopy [ 39 ] [ 40 ]. 2.11 Antioxidant Property of Scaffold: ROS Estimation using DCFDA Assay For evaluating the antioxidant properties, hWJ-MSCs (NCCS, India) were seeded in 24-well plate and cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. The hWJ-MSCs cells were then treated with 100 µM of H 2 O 2 for 6 h to induce oxidative stress [ 41 ]. The quercetin and hesperetin-conjugated SF-BEN-HAP scaffolds (1, 5, 10, and 20 µM) were added to the cells and incubated for 24 h with 5% CO 2 supply in an incubator at 37 ºC as per ASTM F813 [ 38 ]. A stock solution of DCFDA (20 mM) was prepared in methanol, and the solution was diluted in deionized water (Milli-Q water) to prepare a 20 µM working concentration. The media was removed from wells, and the ROS was estimated by staining the cells with 100 µl of 20 µM DCFDA (2',7'-dichlorofluorescin diacetate) stain and incubated for 30 min at 37°C in the dark. The cells were observed under confocal microscopy (Olympus, Fluoview Fv3000, Japan). The fluorescence intensity was calculated by using a plate reader under an excitation wavelength of 492 nm and an emission wavelength of 520 nm [ 42 ]. 2.12 Statistical Analysis All experimental data was the average from five tested samples which were statistically analyzed and expressed in mean±standard deviation. The significant difference between the tested samples was analyzed by using a t-test and one-way ANOVA using GraphPad Prism software (version 8.0.1). P values, P > 0.05, P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001 are considered as non-significant (ns), significant (*), very significant (**), and highly significant (***) respectively. 3. Results and discussion 3.1 FTIR Characterization of SF and Flavonoid-SF Conjugate FTIR is used for the conformation of SF, exhibiting a particular absorption band of vibrational regions along with distinct peaks that correspond to amide groups as shown in Figure. 1. The synthesized SF was characterized in FTIR analysis and conformed with the presence of β–sheet (motif of the regular protein secondary structure) related with three amide groups where the amide I at 1654 cm − 1 attributed to C = O stretching, amide II at 1554 cm − 1 corresponds to N-H bending and amide III at 1257 cm − 1 attributed to C-N stretching [ 27 ] [ 43 ]. Also, it was observed that an amide group overlapped with O-H hydroxyl stretching at 3282 cm − 1 [ 44 ]. Hydroxyl and carbonyl group stretching vibration peaks were observed at 3294.20 and 1671.24 cm − 1 , and cyclobenzene stretching vibration peaks were observed at 1614.62, 1513.53, and 1457.72 cm − 1 attributed to quercetin (QTN) as shown in Figure. 2. The C-O bands at 1025, 1260, and 1310 cm − 1 , carbonyl group (C = O) at 1634.43 cm − 1 , and -OH stretching at 3497.17 cm − 1 confirms hesperetin (HTN) [ 28 ] [ 45 ]. The SF conjugated with quercetin and hesperetin shows OH stretching and vibration at 3280.12 cm − 1 and the carboxyl groups at 951.40 and 1011.97 cm − 1 bond formation with SF as shown in Figure. 2. 3.2 SF Infilling and Morphological Analysis of 3D Scaffold The FE-SEM showed a rough morphology surface of both BEN-HAP and SF-BEN-HAP scaffold groups. The SF-infilled scaffold group shows fibrous/sponge regions inside and slightly at the top layer of the scaffold, as shown in Figure. 3. The rough surface morphology can facilitate cell adhesion and proliferation [ 46 ], and the fibrous mesh region of SF could improve cell attachment and biocompatibility, by entrapping more cells and facilitating proliferation at the site [ 14 ] [ 47 ]. The scaffolds were also shown to exhibit micro and nanoporous structures. 3.3 Contact Angle Measurement Silk fibroin is a biopolymer with hydrophilic (–OH and –COOH) and hydrophobic groups having repeating sequences of residues (glycine, alanine, and serine) [ 48 ]. Despite having properties such as porous and permeability, the ceramic scaffolds after being infilled with SF show slightly compromised hydrophilicity compared to BEN-HAP. However, both scaffold groups are hydrophilic in nature, still the water permeation of the BEN-HAP scaffold group was very high which may be due to high surface energy that affects the surface tension of liquid. The SF-BEN-HAP scaffolds showed gradual water permeation where the surface energy could be less compared to BEN-HAP scaffolds, as shown in Figure. 4a. This hydrophilicity of SF-BEN-HAP can be tuned with change in concentration of silk fibroin [ 49 ]. The contact angle of the scaffold groups with the respective time point is mentioned in Figure. 4b. 3.4 In Vitro Mineralization After 14 days of incubation in SBF solution, the scaffolds showed hydroxyapatite crystal growth in both groups. HAP is composed of calcium and phosphate, with a Ca/P ratio near 1.67 molar ratio in bone, and are the most essential ions for mineralization [ 50 ]. This calcium (Ca) and phosphate (P) mineralization was observed like flake-shaped crystals on the surface [ 51 ], which are higher in SF-infilled scaffold as shown in Figure. 5. This shows the potential of silk fibroin to modulate the growth of hydroxyapatite crystals through the Ca 2+ interaction and surface interactions [ 52 ][ 53 ]. Further, the change in Ca:P ratios was analyzed in EDAX and found to be 9.89:6.44 wt % in BEN-HAP and 13.21:9.11 wt % in SF-BEN-HAP scaffold. Flavonoids are widely known for their radical-scavenging ability and are highly used in tissue repair and wound-healing applications. DPPH and ABTS are free radical compounds that are widely used to test the free radical scavenging ability [ 54 ]. Also, the ABTS assay is more sensitive due to fast reaction kinetics compared to DPPH [ 55 ]. The antioxidant property of quercetin and hesperetin neutralizes the ABTS and DPPH by the transfer of an electron or hydrogen atom [ 56 ]. The unpaired electrons are delocalized by the phenol compounds that can react with them within the aromatic ring and can form stable intermediates by undergoing resonance stabilization within the molecule [ 57 ]. The reduction capacity in ABTS was determined by colour changes from blue-green to colourless at 734 nm and DPPH observed colour changes from deep violet to yellow by reading at 517 nm. The radical scavenging activity of quercetin and hesperetin was detected and compared with the standard antioxidant, Ascorbic acid. In the ABTS assay, the quercetin and hesperetin have scavenging activity of 5 % and 1 % at 10µM. At igher concentrations, 320 µM has scavenging activity of 81 % and 86 % whic are cloer to the ascorbic acid (87 %). In the DPPHassay, quercetin and ascorbic have 5 % scavenging acivity at 10 µM. Quercetin and hesperetin were observed to have 24 % and 12 % scavnging acivity at a concentration of 320 µM as shown in Figure. 6. The electron transfer is generally instantaneous and hydrogen transfer is comparatively slower. In the ABTS assay, this initial electron transfer takes place significantly more quickly [ 56 ]. Since the DPPH radical site has steric hindrance, it may cause difficulty in accessing phenols. This could be the case where the lower concentration of both hesperetin and quercetin has lesser activity along with the ascorbic acid (control) compared to ABTS. Also, the lower concentrations of these compounds may cause very minimum electron transfer which cannot have enough scavenging activity. However, the efficacy of antioxidants to scavenge radicals is largely dependent on their steric accessibility to the radical site in DPPH [ 58 ]. After investigation, it was observed that both compounds had a strong antioxidant capacity that varied with different concentrations (dose-dependent) compared to control. 3.6 Biocompatibility of Flavonoids conjugated SF-BEN-HAP scaffolds The MTT experiment conducted on hWJ-MSC cells treated with different concentrations of quercetin and hesperetin reveals no harmful effects of the flavonoids present in the SF-BEN-HAP scaffolds as shown in Figure. 7a. SF-BEN-HAP scaffold without flavonoids was taken as control. In addition, the flavonoid-loaded scaffolds showed improved cell growth and viability than the BEN-HAP and SF-BEN-HAP scaffolds. The MSC cell viability and proliferation rate are increased when quercetin and hesperetin are present at lower concentrations [ 29 ]. The higher concentration (20 µM) reviled cell damage that might be due to the solvent residues. The live and dead staining of hWJ-MSCs cells after incubation with quercetin and hesperetin-loaded SF-BEN-HAP scaffolds shows no notable toxicity and cell damage, in lower concentrations as shown in Figure. 7b. The cell proliferation of hWJ-MSCs was evaluated by scratch test assay. It was observed that the flavonoids-loaded SF-BEN-HAP scaffolds show improved cell migration which can be due to an effective biomolecule (flavonoids and silk fibroin) in the leaching solution. Among different concentrations, 10 µM of quercetin and hesperetin was observed to have more than 60% closure and promoted a high cell migration rate as shown in Figure. 8. The percentage of gap closure by cell migration was measured using ImageJ software. 3.7 Antioxidant Properties of Flavonoids-SF-BEN-HAP: DCFDA Oxidative stress occurs due to a high percentage of the reactive oxygen species (ROS) and their ratio may rise under physiological conditions like injuries. Mesenchymal stem cells (MSCs) are multipotent stromal cells that reside in adipose tissue and bone marrow and are highly known to contribute to tissue regeneration applications [ 59 ]. Based on their ability to differentiate into osteoprogenitor cells and bone cells [ 60 ], MSCs are used in this study to evaluate the antioxidant properties of the flavonoid-loaded SF-BEN-HAP scaffolds. The H 2 O 2 -induced intracellular ROS production in hWJ-MSCs was measured by the DCFH-DA fluorescent probe and observed under confocal microscopy (Olympus, Fluoview Fv3000, Japan). The intracellular ROS level is determined based on the fluorescence intensity. Compared with the control group, the H 2 O 2 treated cells were observed to have increases intracellular ROS levels and observed having high fluorescence intensity as shown in Figure. 9. The SF-BEN-HAP scaffold loaded with different concentrations of quercetin and hesperetin indicated that the level of ROS gradually decreases when the concentration increases (1, 5, 10, and 20 µM). Especially, the SF-BEN-HAP scaffolds with 20 µM concentration of quercetin and hesperetin exhibited the strongest antioxidant activity and effectively suppressed the H 2 O 2 -induced intracellular ROS production in hWJ-MSCs. DCF-DA does not emit fluorescence yet is permeable within the cells. In the presence of ROS, it oxidizes and generates fluorescein, which emits light, implying that the more ROS in the cells, the higher the emission. Following acetate group breakage and oxidation by intracellular esterases, the non-fluorescent H2DCFDA converts into the highly fluorescent 2,7-dichlorofluorescein (DCF) [ 42 ]. Thus, the fluorescent intensity of the cells is proportional to the intracellular ROS levels. Specifically, an increase in the fluorescence intensity of DCF indicates the high ROS generation in the cells. When compared to the control (H 2 O 2 -treated cells), 1 µM and 5 µM concentrations showed minimal activity in inhibiting ROS production. However, 10 µM and 20 µM concentrations of both quercetin and hesperetin showed much greater antioxidant activity as shown in Figure. 10. Following to that, comparatively, even at low concentrations, quercetin inhibits ROS production more effectively than hesperetin-loaded scaffolds. The 20 µM dose of quercetin and hesperetin resulted in maximal ROS suppression, but significantly reduced cell migration. Based on the cell viability, anti-oxidative potential, and cell migration, 10 µM of quercetin-loaded SF-BEN-HAP can be an effective scaffold for bone tissue regeneration with antioxidative ability to reduce the oxidative stress at the implant site. Conclusion Bone consists of organic and inorganic components which provide mechanical strength and a biologically niche environment for cell growth. In the 3D fabricated scaffold, BEN-HAP acts as an inorganic mineral matrix-like bone (consists of mineral ions such as calcium, phosphate, iron, magnesium, and sodium), and SF acts as an organic matter like collagen which makes them an ideal bone mimicking organic-inorganic matrix for bone tissue repair. The infilling of silk fibroin in BEN-HAP scaffold, results with a fibrous mesh-like structure. The presence of silk fibroin in the scaffold influences the hydrophilicity and in vitro mineralization, improves cell attachment and proliferation. Recent literature on tissue engineering addresses the problem of oxidative stress which is linked with the potential cause of implant loosening and hinders new bone formation. To prevent the scaffold from loosening and reduce the oxidative stress on the nearby cells, the fabricated scaffold can be conjugated with antioxidants. Quercetin and hesperetin are cost-effective flavonoids that are natural antioxidants used to reduce oxidative stress based on concentration (dose-dependent). The radical scavenging activity of quercetin and hesperetin was investigated and observed to have a strong antioxidant capacity that varied with dose (1, 5, 10, 20, 40, 80, 160, and 320 µM). The scaffolds consisting of 10 µM of flavonoids were observed to have more than 60% closure and promoted high cell migration compared to scaffolds with 20 µM flavonoids. We anticipated the potential of flavonoid-loaded scaffolds to reduce the oxidative stress in the hWJ-MSCs cells which are artificially induced for intracellular ROS by H 2 O 2 treatment. As a result, the dose-dependent reduction in ROS was observed through DCFH-DA stain and visualized under confocal microscopy. Among the different concentrations used, 10 and 20 µM scaffolds exhibited strong antioxidative response in hWJ-MSCs cells. Since a significant reduction in cell viability and cell migration was observed in 20 µM scaffold groups, flavonoids with 10 µM scaffold groups can be considered more suitable for bone tissue repair. Declarations Declaration of interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This work was funded by DST-NewGen IEDC, National Science & Technology Entrepreneurship Development Board (NSTEDB), Department of Science and Technology, Govt. of India, New Delhi through Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. The authors are grateful to DST-SERB (Grant no: SRG/2019/002038) for financial support for the development of ceramic 3D printer. Author contributions: CRediT Methodology, Writing - original draft preparation: Logeshwaran A 1 ; Formal analysis and investigation: Renold Elsen 2 ; Funding acquisition, Resources: Ashutosh D. Bagde 3a,b ; Conceptualization, Supervision, Writing - review and editing: Sunita Nayak *1 References Y. Seol, H. Kang, S.J. Lee, A. Atala, J.J. Yoo, Bioprinting technology and its applications, pp. 1–7, 2014, 10.1093/ejcts/ezu148 A. Szwed-Georgiou et al., Bioactive Materials for Bone Regeneration: Biomolecules and Delivery Systems. ACS Biomater. Sci. Eng. 9 (9), 5222–5254 (Sep. 2023). 10.1021/ACSBIOMATERIALS.3C00609 . /ASSET/IMAGES/LARGE/AB3C00609_0003.JPEG M.M. Germaini, S. Belhabib, S. Guessasma, R. Deterre, P. Corre, P. Weiss, Additive manufacturing of biomaterials for bone tissue engineering – A critical review of the state of the art and new concepts, Prog Mater Sci , vol. 130, p. 100963, Oct. 2022, 10.1016/J.PMATSCI.2022.100963 G. Lee, M. Carrillo, J. McKittrick, D.G. Martin, E.A. Olevsky, Fabrication of ceramic bone scaffolds by solvent jetting 3D printing and sintering: Towards load-bearing applications. Addit. Manuf. 33 , 101107 (May 2020). 10.1016/J.ADDMA.2020.101107 H. Budharaju et al., Ceramic materials for 3D printing of biomimetic bone scaffolds – Current state-of-the-art & future perspectives. Mater. Des. 231 , 112064 (Jul. 2023). 10.1016/J.MATDES.2023.112064 H.L. Jaber, T.A. Kovács, Preparation and synthesis of hydroxyapatite bio-ceramic from bovine bone by thermal heat treatment. Epitoanyag - J. Silicate Based Compos. Mater. 71 (3), 98–101 (2019). 10.14382/epitoanyag-jsbcm.2019.18 K. Dave, V.G. Gomes, Interactions at scaffold interfaces: Effect of surface chemistry, structural attributes and bioaffinity. Mater. Sci. Engineering: C 105 , 110078 (Dec. 2019). 10.1016/J.MSEC.2019.110078 M. Tallawi et al., Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review. J. R Soc. Interface. 12 , 20150254 (Jul. 2015). 10.1098/RSIF.2015.0254 S. Du et al., Sep., Bioactive polymer composite scaffolds fabricated from 3D printed negative molds enable bone formation and vascularization, Acta Biomater , vol. 186, pp. 260–274, 2024, 10.1016/J.ACTBIO.2024.07.038 L. Hodásová et al., Polymer infiltrated ceramic networks with biocompatible adhesive and 3D-printed highly porous scaffolds. Addit. Manuf. 39 , 101850 (Mar. 2021). 10.1016/J.ADDMA.2021.101850 F.J. Martínez-Vázquez, F.H. Perera, P. Miranda, A. Pajares, F. Guiberteau, Improving the compressive strength of bioceramic robocast scaffolds by polymer infiltration, Acta Biomater , vol. 6, no. 11, pp. 4361–4368, Nov. 2010, 10.1016/J.ACTBIO.2010.05.024 X. Shi et al., 3D printed intelligent scaffold prevents recurrence and distal metastasis of breast cancer. Theranostics. 10 , 10652 (2020). 10.7150/THNO.47933 J.E. Song, N. Tripathy, D.H. Lee, J.H. Park, G. Khang, Quercetin Inlaid Silk Fibroin/Hydroxyapatite Scaffold Promotes Enhanced Osteogenesis. ACS Appl. Mater. Interfaces. 10 , 32955–32964 (Oct. 2018). 10.1021/ACSAMI.8B08119/ASSET . /IMAGES/MEDIUM/AM-2018-081198_0006.GIF E. Wenk, H.P. Merkle, L. Meinel, Silk fibroin as a vehicle for drug delivery applications. J. Controlled Release. 150 (2), 128–141 (Mar. 2011) S.A.L. Matthew, F.P. Seib, Silk Bioconjugates: From Chemistry and Concept to Application. ACS Biomater. Sci. Eng. 10 (1), 12–28 (Jan. 2024). 10.1021/ACSBIOMATERIALS.2C01116 . /ASSET/IMAGES/LARGE/AB2C01116_0006.JPEG M.A. Sakr et al., Development of bentonite-gelatin nanocomposite hybrid hydrogels for tissue engineering. Appl. Clay Sci. 199 , 105860 (Dec. 2020). 10.1016/J.CLAY.2020.105860 C. Shuai, W. Yang, P. Feng, S. Peng, H. Pan, Accelerated degradation of HAP/PLLA bone scaffold by PGA blending facilitates bioactivity and osteoconductivity. Bioact Mater. 6 (2), 490–502 (2021). 10.1016/j.bioactmat.2020.09.001 B. Banimohamad-Shotorbani, A. Rahmani, Del A. Bakhshayesh, S. Mehdipour, Jarolmasjed, H. Shafaei, The efficiency of PCL/HAp electrospun nanofibers in bone regeneration: a review. https://doi.org/10.1080/03091902.2021 . 1893396 , vol. 45, no. 7, pp. 511–531, 2021, 10.1080/03091902.2021.1893396 A. Logeshwaran, R. Elsen, S. Nayak, Mechanical and biological characteristics of 3D fabricated clay mineral and bioceramic composite scaffold for bone tissue applications, J Mech Behav Biomed Mater , vol. 138, no. December 2022, p. 105633, 2023. 10.1016/j.jmbbm.2022.105633 T. Zhou, Y. Yan, C. Zhao, Y. Xu, Q. Wang, N. Xu, Resveratrol improves osteogenic differentiation of senescent bone mesenchymal stem cells through inhibiting endogenous reactive oxygen species production via AMPK activation. Redox Rep. 24 (1), 62–69 (2019). 10.1080/13510002.2019.1658376 S. Abdulrahman Hussain et al., Efficacy and safety of co-administration of resveratrol with meloxicam in patients with knee osteoarthritis: A pilot interventional study. Clin. Interv Aging. 13 , 1621–1630 (2018). 10.2147/CIA.S172758 R. Hameister, C. Kaur, S.T. Dheen, C.H. Lohmann, G. Singh, Reactive oxygen/nitrogen species (ROS/RNS) and oxidative stress in arthroplasty. J. Biomed. Mater. Res. B Appl. Biomater. 108 (5), 2073–2087 (Jul. 2020). 10.1002/JBM.B.34546 A.E. Bădilă, D.M. Rădulescu, A. Ilie, A.G. Niculescu, A.M. Grumezescu, A.R. Rădulescu, Bone Regeneration and Oxidative Stress: An Updated Overview. Antioxid. (Basel). 11 (2) (Feb. 2022). 10.3390/ANTIOX11020318 H. Hu et al., Regulation of hypoxic stress and oxidative stress in bone grafting: Current trends and future perspectives. J. Mater. Sci. Technol. 157 , 144–153 (2023). 10.1016/j.jmst.2023.01.055 M.F. Manchope et al., Naringenin mitigates titanium dioxide (TiO 2)-induced chronic arthritis in mice: role of oxidative stress, cytokines, and NFκB. Inflamm. Res. 67 , 11–12 (Dec. 2018). 10.1007/S00011-018-1195-Y/METRICS T. yang Wang, Q. Li, K. shun Bi, Bioactive flavonoids in medicinal plants: Structure, activity and biological fate. Asian J. Pharm. Sci. 13 (1), 12–23 (Jan. 2018). 10.1016/J.AJPS.2017.08.004 S. Chakravarty, N. Bhardwaj, B.B. Mandal, N. Sen Sarma, Silk fibroin-carbon nanoparticle composite scaffolds: A cost effective supramolecular ‘turn off’ chemiresistor for nitroaromatic explosive vapours. J. Mater. Chem. C Mater. 4 (38), 8920–8929 (2016). 10.1039/C6TC03337G I. D’Onofrio et al., Inhalable drug-loaded silk fibroin carriers for pulmonary drug delivery. RSC Adv. 14 (37), 27288–27297 (Aug. 2024). 10.1039/D4RA03324H D. Xue et al., The role of hesperetin on osteogenesis of human mesenchymal stem cells and its function in bone regeneration. Oncotarget. 8 , 21031 (Mar. 2017). 10.18632/ONCOTARGET.15473 P.A. Miguez et al., Mar., Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo, Int J Mol Sci , vol. 22, no. 6, pp. 1–13, 2021, 10.3390/IJMS22063223 K.H. Huang, C.Y. Chen, C.Y. Chang, Y.W. Chen, C.P. Lin, The synergistic effects of quercetin-containing 3D-printed mesoporous calcium silicate/calcium sulfate/poly-ε-caprolactone scaffolds for the promotion of osteogenesis in mesenchymal stem cells, Journal of the Formosan Medical Association , no. xxxx, 2021, 10.1016/j.jfma.2021.01.024 (ASTM), ASTM D7334-08 Standard Practice for Surface Wettability of Coatings, Substrates and Pigments by Advancing Contact Angle Measurement, ASTM International , vol. 08, no. Reapproved, pp. 1–3, 2008, 10.1520/D7334-08R22.Copyright M.N. Gómez-Cerezo, D. Lozano, D. Arcos, M. Vallet-Regí, C. Vaquette, The effect of biomimetic mineralization of 3D-printed mesoporous bioglass scaffolds on physical properties and in vitro osteogenicity, Materials Science and Engineering: C , vol. 109, p. 110572, Apr. 2020, 10.1016/J.MSEC.2019.110572 X. Zhang et al., Three-Dimensional Printed Cell Culture Model Based on Spherical Colloidal Lignin Particles and Cellulose Nanofibril-Alginate Hydrogel. Biomacromolecules. 21 (5), 1875–1885 (2020). 10.1021/acs.biomac.9b01745 G.E. Fawal, H. Hong, X. Mo, H. Wang, Fabrication of scaffold based on gelatin and polycaprolactone (PCL) for wound dressing application. J. Drug Deliv Sci. Technol. 63 , 102501 (2021). 10.1016/j.jddst.2021.102501 K.M. Sadek, W. Mamdouh, S.I. Habib, M.E. Deftar, A.N.A. Habib, In Vitro Biological Evaluation of a Fabricated Polycaprolactone/Pomegranate Electrospun Scaffold for Bone Regeneration, 2021. 10.1021/acsomega.1c04608 J.Y. Lee et al., Design of a 3D BMP-2-delivering tannylated PCL scaffold and its anti-oxidant, anti-inflammatory, and osteogenic effects in vitro, 2018. 10.3390/ijms19113602 F. Astm, 813-01: Standard Practice for Direct Contact Cell Culture Evaluation of Materials for, 24 , pp. 1652–1653, 2001, 10.1520/F0813-20.1 Z. Xu, Y. Sun, H. Dai, Y. Ma, H. Bing, Engineered 3D-Printed Polyvinyl Alcohol Scaffolds Incorporating β-Tricalcium Phosphate and Icariin Induce Bone Regeneration in Rat Skull Defect Model. Molecules. 27 (14) (Jul. 2022). 10.3390/MOLECULES27144535 W. Liu et al., Electrospun porous poly (3-hydroxybutyrate-co-4-hydroxybutyrate)/lecithin scaffold for bone tissue engineering. RSC Adv. 12 (19), 11913–11922 (2022) K. Pal et al., Folic acid conjugated curcumin loaded biopolymeric gum acacia microsphere for triple negative breast cancer therapy in invitro and invivo model, Materials Science and Engineering C , vol. 95, no. November, pp. 204–216, 2019, 10.1016/j.msec.2018.10.071 P. Kumar, I. Rupmanjari, K. Ajay, R.P. Mariadhas, V. Arasu, Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells. Mol. Biol. Rep. 48 (8), 5857–5872 (2021). 10.1007/s11033-021-06580-x H. Hong et al., Cytocompatibility of modified silk fibroin with glycidyl methacrylate for tissue engineering and biomedical applications. Biomolecules. 11 (1), 1–18 (Jan. 2021). 10.3390/BIOM11010035 S. Das, D. Pati, N. Tiwari, A. Nisal, S. Sen Gupta, Synthesis of silk fibroin-glycopolypeptide conjugates and their recognition with lectin. Biomacromolecules. 13 (11), 3695–3702 (Nov. 2012). 10.1021/BM301170U/SUPPL_FILE/BM301170U_SI_001.PDF E.T. Baran, K. Tuzlakoǧlu, J.F. Mano, R.L. Reis, Enzymatic degradation behavior and cytocompatibility of silk fibroin–starch–chitosan conjugate membranes. Mater. Sci. Engineering: C 32 (6), 1314–1322 (Aug. 2012). 10.1016/J.MSEC.2012.02.015 R. Ferracini, I. Martínez Herreros, A. Russo, T. Casalini, F. Rossi, G. Perale, Scaffolds as Structural Tools for Bone-Targeted Drug Delivery, Pharmaceutics , vol. 10, no. 3, Sep. 2018, 10.3390/PHARMACEUTICS10030122 R. Deshpande, S. Shukla, A. Kale, N. Deshmukh, A. Nisal, P. Venugopalan, Studies, ACS Biomater. Sci. Eng. 8 (3), 1226–1238 (Mar. 2022). 10.1021/ACSBIOMATERIALS.1C01103 . /SUPPL_FILE/AB1C01103_SI_001.PDF K.A. Burke, D.C. Roberts, D.L. Kaplan, Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins. Biomacromolecules. 17 (1), 237 (Jan. 2016). 10.1021/ACS.BIOMAC.5B01330 S. Kaushik, P.D. Thungon, P. Goswami, Silk Fibroin: An Emerging Biocompatible Material for Application of Enzymes and Whole Cells in Bioelectronics and Bioanalytical Sciences, ACS Biomater Sci Eng , vol. 6, no. 8, pp. 4337–4355, Aug. 2020, 10.1021/ACSBIOMATERIALS.9B01971/ASSET /IMAGES/MEDIUM/AB9B01971_0009.GIF X. Wu, K. Walsh, B.L. Hoff, G. Camci-Unal, Mineralization of Biomaterials for Bone Tissue Engineering, Bioengineering 2020, Vol. 7, Page 132, vol. 7, no. 4, p. 132, Oct. 2020. 10.3390/BIOENGINEERING7040132 K. Rodríguez, J. Sundberg, P. Gatenholm, S. Renneckar, Electrospun nanofibrous cellulose scaffolds with controlled microarchitecture. Carbohydr. Polym. 100 , 143–149 (2014). 10.1016/J.CARBPOL.2012.12.037 Y. Jin, B. Kundu, Y. Cai, S.C. Kundu, J. Yao, Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering. Colloids Surf. B Biointerfaces. 134 , 339–345 (Oct. 2015). 10.1016/J.COLSURFB.2015.07.015 Z.J. Chen et al., Mineralized self-assembled silk fibroin/cellulose interpenetrating network aerogel for bone tissue engineering. Biomaterials Adv. 134 , 112549 (Mar. 2022). 10.1016/J.MSEC.2021.112549 M. Olszowy, A.L. Dawidowicz, Is it possible to use the DPPH and ABTS methods for reliable estimation of antioxidant power of colored compounds? Chem. Pap. 72 (2), 393–400 (Feb. 2018). 10.1007/S11696-017-0288-3/TABLES/3 K.J. Lee, Y.C. Oh, W.K. Cho, J.Y. Ma, Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay, Evid Based Complement Alternat Med , vol. 2015, p. 165457, 2015. 10.1155/2015/165457 J. Rumpf, R. Burger, M. Schulze, Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins. Int. J. Biol. Macromol. 233 , 123470 (Apr. 2023). 10.1016/J.IJBIOMAC.2023.123470 C.M.C. Andrés, J.M. Pérez de la Lastra, C.A. Juan, F.J. Plou, E. Pérez-Lebeña, Polyphenols as Antioxidant/Pro-Oxidant Compounds and Donors of Reducing Species: Relationship with Human Antioxidant Metabolism. Processes 2023. 11 (9), 2771 (Sep. 2023). 10.3390/PR11092771 M.S. Shojaee, M. Moeenfard, R. Farhoosh, Kinetics and stoichiometry of gallic acid and methyl gallate in scavenging DPPH radical as affected by the reaction solvent, Scientific Reports 2022 12:1 , vol. 12, no. 1, pp. 1–9, May 2022, 10.1038/s41598-022-12803-3 A. Deb, How Stem Cells Turn into Bone and Fat, N Engl J Med , vol. 380, no. 23, p. 2268, Jun. 2019, 10.1056/NEJMCIBR1905165 M. Ponzetti, N. Rucci, Osteoblast Differentiation and Signaling: Established Concepts and Emerging Topics. Int. J. Mol. Sci. 22 , 6651 (Jul. 2021). 10.3390/IJMS22136651 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 26 Apr, 2026 Reviews received at journal 25 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviews received at journal 09 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 15 Mar, 2026 Reviewers invited by journal 15 Mar, 2026 Editor assigned by journal 29 Jan, 2026 Submission checks completed at journal 29 Jan, 2026 First submitted to journal 23 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8679759","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":607081678,"identity":"7e8021a1-2d03-47a4-87eb-32b1b32b5d08","order_by":0,"name":"Logeshwaran A","email":"","orcid":"","institution":"Vellore Institute of Technology University","correspondingAuthor":false,"prefix":"","firstName":"Logeshwaran","middleName":"","lastName":"A","suffix":""},{"id":607081679,"identity":"7096a9e1-a900-4ea1-8b56-bc1d4137738d","order_by":1,"name":"Renold Elsen","email":"","orcid":"","institution":"Vellore Institute of Technology University","correspondingAuthor":false,"prefix":"","firstName":"Renold","middleName":"","lastName":"Elsen","suffix":""},{"id":607081680,"identity":"a5e90e0d-6d1c-4fa4-a5ce-5a12509535f3","order_by":2,"name":"Ashutosh D Bagde","email":"","orcid":"","institution":"Datta Meghe Institute of Higher educational and research","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"D","lastName":"Bagde","suffix":""},{"id":607081681,"identity":"c9140434-1fa9-4de0-9484-4a32811a33ee","order_by":3,"name":"Sunita nayak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie2PsYrCQBCGJxxsFbBNlfgIE6zlXiUi5Jp4eN2BFlZJZ517izzCyEBsImlXtLjKa65YOLhKjtsQU9isKQX3awaG/2P+AbBY7hRqh3A+Aa82BqVNiCcExH7KJSGEB90ZE0E+27BaHgPclvHCnZ9hkJHDc4OC8jUiKk9hUcXlwdXFvCoCzk2KlyCRYKegl3SfN79IAHaNxRrlj5+L+it9a5TglgJSK5uUJ4WMS1BawVsKVt9IuzVPP+Rp6ikcuWE1WZmLZclIvf/yeF3HoYrOvu9vmX+MxTqG1E4ddlZ9BH2uZ85isVgekH84olcskGvKsgAAAABJRU5ErkJggg==","orcid":"","institution":"Vellore Institute of Technology University","correspondingAuthor":true,"prefix":"","firstName":"Sunita","middleName":"","lastName":"nayak","suffix":""}],"badges":[],"createdAt":"2026-01-23 13:39:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8679759/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8679759/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104827213,"identity":"fef704b6-aaa3-4636-b260-c4eb43367473","added_by":"auto","created_at":"2026-03-17 15:41:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":296699,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of synthesis of Silk fibroin (a) and FTIR characterization (b)\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/fafca05c4a6390f004bfbdd2.png"},{"id":104827157,"identity":"c2d94a39-338f-40f5-a367-98f30ded3f42","added_by":"auto","created_at":"2026-03-17 15:41:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":307813,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR analysis of quercetin (QTN), hesperetin (HTN), and silk fibroin-conjugated quercetin (SF-QTN) and hesperetin (SF-HTN)\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/9b8fce0837b6d3638d38ca12.png"},{"id":104827163,"identity":"fea0423b-148f-4f1e-a51d-1841af8d9ab7","added_by":"auto","created_at":"2026-03-17 15:41:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":960186,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of BEN-HAP and SF infilled scaffold\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/00b43d7bff6eade79a1e444a.png"},{"id":104827207,"identity":"ae19caee-c0a3-4ad5-8c37-5adb5035e256","added_by":"auto","created_at":"2026-03-17 15:41:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":265918,"visible":true,"origin":"","legend":"\u003cp\u003eWettability (a) and contact angle measurement (b) of BEN-HAP and SF-BEN-HAP scaffold\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/4f5adde6e788e3ce6c3a42be.png"},{"id":104827158,"identity":"01338d81-9284-4ea2-b6e0-e1d4f63b8b51","added_by":"auto","created_at":"2026-03-17 15:41:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":888234,"visible":true,"origin":"","legend":"\u003cp\u003eMorphology of BEN-HAP and SF-BEN-HAP scaffold under FE-SEM after mineralization and EDAX analysis (Yellow arrows represents the flake-shaped apatite formation)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/83a07772c1ac6e23ebb765b9.png"},{"id":104827297,"identity":"c62a89c6-ae2d-4125-aae7-37530804c897","added_by":"auto","created_at":"2026-03-17 15:42:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77202,"visible":true,"origin":"","legend":"\u003cp\u003eDPPH and ABTS radical scavenging assay of quercetin and hesperetin (P ≤ 0.0001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/5a1de56c40eee969d45f8bb4.png"},{"id":105562585,"identity":"34aea61b-9e48-4c7a-8e39-25446db5e714","added_by":"auto","created_at":"2026-03-27 12:43:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":847377,"visible":true,"origin":"","legend":"\u003cp\u003eMTT assay (P ≤ 0.05) (a) and Live and dead assay of hWJ-MSCs in quercetin and hesperetin-loaded SF-BEN-HAP scaffolds.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/a3d5a1d5115bcae90d3175f5.png"},{"id":104827159,"identity":"e9e2dce8-4ba9-4082-9e26-fd0a87a1b0e3","added_by":"auto","created_at":"2026-03-17 15:41:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":928455,"visible":true,"origin":"","legend":"\u003cp\u003eImage of scratch test assay using hWJ-MSCs (a), The percentage of healing area after 24 h incubation (b) (P ≤ 0.0001)\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/3d17f12c791afd8047bcadff.png"},{"id":104827299,"identity":"076824de-4f52-419c-87d8-e50d29c2970c","added_by":"auto","created_at":"2026-03-17 15:42:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":810302,"visible":true,"origin":"","legend":"\u003cp\u003eDCFH-DA staining of hWJ-MSCs after incubation with flavonoid-loaded SF-BEN-HAP scaffold\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/c00c3a9937a450e05eee98ac.png"},{"id":104827250,"identity":"67a5cb8c-2868-4187-b050-f504587e5d75","added_by":"auto","created_at":"2026-03-17 15:41:57","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":139384,"visible":true,"origin":"","legend":"\u003cp\u003eROS fluorescence intensity of hWJ-MSCs after incubation with quercetin-loaded SF-BEN-HAP scaffold (P ≤ 0.05)\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/dd0f985ca832a5b0a47909c9.png"},{"id":105568733,"identity":"60d6678e-8bb7-4240-afd3-9019dfd94aec","added_by":"auto","created_at":"2026-03-27 13:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6215370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8679759/v1/46eeed46-fea3-459a-8fd4-dbd19e4b7d0c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Flavonoid loaded mineral clay-based 3D scaffold for bone tissue regeneration","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eScaffolds have been utilized to develop various tissues and organs, including skin, trachea, bone, pharynx, and myocardium, for translational and preclinical models [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The difficulty in bone tissue engineering is creating a structure based on biomaterials that can replicate the various physiochemical characteristics of bone, including its shape, architecture, mechanical qualities, and physiology [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Complex tissue 3D reconstruction is a good fit for additive manufacturing techniques because of its layer-by-layer assembly approach. Additionally, the functionality of the 3D construct is improved by incorporating biological materials (cells, proteins, enzymes, and growth factors)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The lack of bioactive molecules in 3D fabricated scaffolds could delay the regeneration process and may have possibilities for graft rejection. Increasing bioactivity of the ceramic composite 3D scaffold for bone tissue regeneration can be a concern that can be overcome by loading growth factors, polymers, and proteins. The ceramic-based 3D printed scaffolds can undergo different thermal treatments/sintering processes to attain mechanical properties such as compression strength, hardness, and density [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Common bioceramic 3D scaffolds such as hydroxyapatite and calcium phosphate are porous and hydrophilic and can support angiogenesis, biosorption, and bone regeneration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Considering the contribution of scaffolding on cell adhesion, proliferation, and differentiation, scaffold rejection might also occur due to a lack of cell-scaffold interaction (bio-interaction), poor microenvironment, and bio interface [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, incorporating polymers or protein compounds into the porous scaffold is essential to enhance the bioavailability. The incorporation of bioactive molecules significantly impacts the scaffold characteristics and cell behavior [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. According to the tissue engineering perspective, bioactive molecules such as peptides, flavonoids, and growth factors are known to improve the scaffold characteristics, promote cellular activity, and enhance tissue regeneration [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Collagen, gelatin, chitosan, elastin, hyaluronic acid (HA), and silk fibroin are well-known biodegradable natural polymers frequently utilized for scaffold fabrication.\u003c/p\u003e \u003cp\u003ePolymer infiltration in 3D printed porous scaffolds is a technique for improving the bioactivity of the scaffold, where they facilitate cell-scaffold interaction and enable the delivery of biomolecules such as peptides, growth factors, and flavonoids for different applications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Despite having a wide range of polymers, silk fibroin is a naturally available fibrous protein with unique properties such as inducing mineralization, increasing biocompatibility, and delivery of therapeutic compounds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Also, the presence of several peptides and amino acids enables interaction with other biomolecules creating bonding for drug delivery application [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBentonite (BEN) is a clay mineral composite material with diverse mineral availability, biocompatibility, and structural stability [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Based on its properties, it can be combined with an osteoconductive bioceramic material, hydroxyapatite (HAP), to enhance the bone regeneration process [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e][\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The composite slurry also indicated advantages in the extrusion process and further fabrication of load-bearing biocompatible scaffolds, having a compression strength of 52 MPa with suitable physical characteristics ideal for bone tissue graft [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The implantation of bone grafts including the scaffolds has been associated with oxidative stress due to the mineral ions leaching at the site. Oxidative stress is an imbalance in ROS in mitochondria and their neutralization by protective mechanisms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. At minimal levels, ROS function as signaling molecules that are crucial for regulating cellular functions such as proliferation and differentiation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] as well as modulating signaling functions in transduction, inflammation, and receptor activation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, increased levels of ROS at the damaged area and the scaffold implanted site may cause oxidative stress. Imbalances in this protective system can cause damage to DNA, proteins, and lipids [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Thus, it is crucial to give bone graft materials antioxidative qualities to facilitate the early regeneration of bone [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Polyphenolic chemicals or biomolecules like flavonoids are known for their various health impacts including anti-inflammatory and antioxidant capabilities [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Quercetin and hesperetin are cost-effective flavonoids that are natural antioxidants used to reduce oxidative stress.\u003c/p\u003e \u003cp\u003eIn this work, the 3D printed BEN-HAP composite scaffold was infilled with SF to improvise the properties such as mineralization, cell-scaffold interaction, hydrophilicity, and hemocompatibility for bone tissue engineering (BTE) application. The BEN acts as an inorganic mineral matrix-like bone (consisting of mineral ions such as iron, magnesium, sodium, and phosphorous), and SF acts as an organic matter like collagen, which makes them an ideal bone-mimicking organic-inorganic matrix for tissue development.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eQuercetin (QTN) and Hesperetin (HTN) were purchased from Sigma Aldrich (India), and silk cocoons were procured from Karnataka, India. Lithium bromide and sodium carbonate were purchased from Himedia (India) for the preparation of the silk fibroin solution. For cell culture studies, Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), Penicillin/streptomycin antibiotic solution, 4% paraformaldehyde solution, dimethyl sulfoxide (DMSO), and MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) were purchased from Hi-Media (India). The calcein-AM and propidium iodide (PI) were purchased from Sigma Aldrich (India). ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), DPPH (1,1-Diphenyl-2-picrylhydrazyl) and DCFDA (2',7'-dichlorofluorescin diacetate) were purchased from Sigma Aldrich (India).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Silk Fibroin Preparation and Infilling in Scaffold\u003c/h2\u003e \u003cp\u003eFor SF preparation, the cocoons were cut and boiled with 0.02 M Sodium carbonate, the silk fibers were then dried at 37 \u0026ordm;C. The silk fibers were dissolved in 9.2 M of lithium bromide solution at 60\u0026ordm;C for 4 h in a hot air oven [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The supernatant was collected and dialyzed for 48 h, and the SF solution was stored at 4\u0026ordm;C. A known volume of SF was dried and weighed to determine the final concentration of prepared SF [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The 3D printing of the BEN-HAP scaffold was done as previously described in paper [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In brief, a 1:1 ratio of BEN and HAP was prepared into a slurry using 2% Carboxymethyl cellulose and extruded in a 0.8 mm nozzle at a speed of 2 mm/s with pressure 0.25 MPa. The infill ratio and scaffold dimensions were 60% and 10*10*6 mm, respectively. The printed samples were further sintered at 1000˚C for 2 hr. The scaffold is then infiltrated with silk fibroin solution using a syringe, followed by a lyophilization process to remove moisture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of Flavonoid-Silk Fibroin Conjugate\u003c/h2\u003e \u003cp\u003eThe conjugation of flavonoids (quercetin and hesperetin) with silk fibroin was done by a conventional mechanical stirring method. The stock concentration of quercetin and hesperetin (1 mg/ml) was prepared by dissolving the flavonoids separately in DMSO: PBS (pH 7.4) solution in a 1:4 ratio (ml) as per the manufacturer\u0026rsquo;s instruction. Quercetin and hesperetin concentrations of 1, 5, 10, and 20 \u0026micro;M were prepared and mixed separately with silk fibroin. The solution was blended at low rpm in a mechanical stirrer for 30 min at room temperature. The prepared conjugate solution was stored at 4 \u0026ordm;C [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. And further infiltrated in BEN-HAP scaffold using a syringe followed by a lyophilization process to remove moisture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 FTIR Analysis\u003c/h2\u003e \u003cp\u003eThe functional groups of the synthesized SF, and conjugates SF-QTN and SF-HTN were confirmed by Fourier Transform Infrared (FTIR) spectrum analysis by Cary 630 spectrometer (US) in the range of 400 to 4000 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 FESEM Analysis\u003c/h2\u003e \u003cp\u003eThe SF-infiltrated 3D printed BEN-HAP scaffolds (SF-BEN-HAP) were gold sputter coated and observed under FESEM (Field emission scanning electron microscopy) for morphological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Contact Angle Measurement\u003c/h2\u003e \u003cp\u003eThe wettability of the BEN-HAP and SF-BEN-HAP scaffolds was investigated as per ASTM D7334\u0026ndash;08 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Standards. A static angle with a contact angle meter (HO-IAD-CAM-01A) was used. The experiment was performed by the Sessile drop method, where approximately 5 \u0026micro;l of distilled water was added from the micro syringe onto the sample and the behavior was examined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 \u003cem\u003eIn vitro\u003c/em\u003e Mineralization\u003c/h2\u003e \u003cp\u003eMineralization/Apatite formation of the BEN-HAP and SF-BEN-HAP scaffold was performed by incubating the scaffolds in Simulated body fluid (SBF) of pH 7.4 at 37\u0026deg;C for 14 days, maintaining in same volume (3 ml) at submerged conditions throughout the study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The scaffolds were then removed, dried at room temperature, and observed in FESEM after gold sputter coating. Changes in mineral composition among the scaffold groups were evaluated in EDAX.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Antioxidant Assay of Flavonoids: ABTS and DPPH Radical Scavenging\u003c/h2\u003e \u003cp\u003eThe stock concentration of quercetin and hesperetin (1 mg/ml) was prepared by dissolving the flavonoids separately in DMSO: PBS (pH 7.4) solution in a 1:4 ratio (ml) as per the manufacturer\u0026rsquo;s instruction. Different concentrations of flavonoids (1, 5, 10, 20, 40, 80, 160, and 320 \u0026micro;M) were made from the stock solution to measure the antioxidant activity. For the ABTS radical scavenging assay, the reagent was prepared by dissolving ABTS in potassium persulfate (2.45 mM) with the final concentration of 7 mM ABTS solution. The mixture was kept in dark conditions for 12\u0026ndash;16 h. The reagent was diluted with water and checked for absorbance of 0.71 at a wavelength of 734 nm. The final concentration of ABTS was stored at 4\u0026ordm;C. 50 \u0026micro;l of different concentrations of quercetin and hesperetin were added to 150 \u0026micro;l of ABTS and incubated in the dark for 30 min. The reduction of the absorbance was measured in a microplate reader (Tecan, Switzerland) at a wavelength of 734 nm [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For DPPH assay, 0.5 mM DPPH solution was prepared in methanol. 50 \u0026micro;l of different concentrations of quercetin and hesperetin were added to 150 \u0026micro;l of DPPH and incubated in the dark for 30 min. The reduction of the absorbance was measured in a microplate reader (Tecan, Switzerland) at a wavelength of 517 nm [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Ascorbic acid was taken as standard for both ABTS and DPPH assay for evaluating the scavenging activity, percentage of activity was measured by the following Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e),\u003c/p\u003e \n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"609\" height=\"55\"\u003e\u003c/p\u003e\n\u003cp\u003eWhere, A\u003csub\u003eB\u003c/sub\u003e is the absorbance of the blank and A\u003csub\u003eF\u003c/sub\u003e is the absorbance of the flavonoid sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Fabrication of Flavonoids-Loaded SF-BEN-HAP Scaffold\u003c/h2\u003e \u003cp\u003eQuercetin and hesperetin concentrations of 1, 5, 10, and 20 \u0026micro;M were prepared and mixed separately with silk fibroin. The solution mixture was conjugated by blending at low rpm in a mechanical stirrer for 30 min at room temperature [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The prepared conjugate solution was infilled and lyophilized in 3D-printed BEN-HAP scaffolds (S-10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Biocompatibility\u003c/h2\u003e \u003cp\u003eFor accessing the cytocompatibility, human Wharton\u0026rsquo;s jelly mesenchymal stem cells (hWJ-\u003cem\u003eMSCs)\u003c/em\u003e (NCCS, India) were cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. After 65% confluency, the scaffolds were placed on the monolayer of the cell and incubated for 72 h with 5% CO\u003csub\u003e2\u003c/sub\u003e supply in an incubator at 37 \u0026ordm;C as per ASTM F813 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The MTT assay was performed as per the manufacturer\u0026rsquo;s instructions (EZCount MTT Cell Assay Kit, HiMedia, India) and measured the absorbance at wavelength 570 nm using an ELISA plate reader (Robonik, India). For live and dead assay, 20 \u0026micro;l of 10 \u0026micro;M calcein acetoxymethyl (AM) and propidium iodide (PI) in PBS (7.4 pH) was added to the cells cultured after 24 h and 72 h in the presence of scaffolds. The cells were then observed under Fluorescence microscopy (EVOS M5000). The scratch test was performed to assess the effect of \u003cem\u003ein vitro\u003c/em\u003e cell migration in the presence of scaffold leach-out (72 h scaffold leach-out). The hWJ-MSCs cells were cultured in 24-well plate with Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. After 90% confluency, a perpendicular scratch was made using a sterile pipet tip in the middle of each well. Fresh media, along with 200 \u0026micro;l scaffold leach-out, was added to each well. The cell migration was observed at 0 h and 24 h under optical microscopy [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Antioxidant Property of Scaffold: ROS Estimation using DCFDA Assay\u003c/h2\u003e \u003cp\u003eFor evaluating the antioxidant properties, hWJ-MSCs (NCCS, India) were seeded in 24-well plate and cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% FBS and 1% penicillin/streptomycin solution. The hWJ-MSCs cells were then treated with 100 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 6 h to induce oxidative stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The quercetin and hesperetin-conjugated SF-BEN-HAP scaffolds (1, 5, 10, and 20 \u0026micro;M) were added to the cells and incubated for 24 h with 5% CO\u003csub\u003e2\u003c/sub\u003e supply in an incubator at 37 \u0026ordm;C as per ASTM F813 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. A stock solution of DCFDA (20 mM) was prepared in methanol, and the solution was diluted in deionized water (Milli-Q water) to prepare a 20 \u0026micro;M working concentration. The media was removed from wells, and the ROS was estimated by staining the cells with 100 \u0026micro;l of 20 \u0026micro;M DCFDA (2',7'-dichlorofluorescin diacetate) stain and incubated for 30 min at 37\u0026deg;C in the dark. The cells were observed under confocal microscopy (Olympus, Fluoview Fv3000, Japan). The fluorescence intensity was calculated by using a plate reader under an excitation wavelength of 492 nm and an emission wavelength of 520 nm [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data was the average from five tested samples which were statistically analyzed and expressed in mean\u0026plusmn;standard deviation. The significant difference between the tested samples was analyzed by using a t-test and one-way ANOVA using GraphPad Prism software (version 8.0.1). P values, P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, P\u0026thinsp;\u0026le;\u0026thinsp;0.05, P\u0026thinsp;\u0026le;\u0026thinsp;0.01, and P\u0026thinsp;\u0026le;\u0026thinsp;0.001 are considered as non-significant (ns), significant (*), very significant (**), and highly significant (***) respectively.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 FTIR Characterization of SF and Flavonoid-SF Conjugate\u003c/h2\u003e \u003cp\u003eFTIR is used for the conformation of SF, exhibiting a particular absorption band of vibrational regions along with distinct peaks that correspond to amide groups as shown in Figure. 1. The synthesized SF was characterized in FTIR analysis and conformed with the presence of β\u0026ndash;sheet (motif of the regular protein secondary structure) related with three amide groups where the amide I at 1654 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributed to C\u0026thinsp;=\u0026thinsp;O stretching, amide II at 1554 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponds to N-H bending and amide III at 1257 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributed to C-N stretching [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Also, it was observed that an amide group overlapped with O-H hydroxyl stretching at 3282 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHydroxyl and carbonyl group stretching vibration peaks were observed at 3294.20 and 1671.24 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and cyclobenzene stretching vibration peaks were observed at 1614.62, 1513.53, and 1457.72 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e attributed to quercetin (QTN) as shown in Figure. 2. The C-O bands at 1025, 1260, and 1310 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, carbonyl group (C\u0026thinsp;=\u0026thinsp;O) at 1634.43 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and -OH stretching at 3497.17 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e confirms hesperetin (HTN) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The SF conjugated with quercetin and hesperetin shows OH stretching and vibration at 3280.12 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the carboxyl groups at 951.40 and 1011.97 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bond formation with SF as shown in Figure. 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 SF Infilling and Morphological Analysis of 3D Scaffold\u003c/h2\u003e \u003cp\u003eThe FE-SEM showed a rough morphology surface of both BEN-HAP and SF-BEN-HAP scaffold groups. The SF-infilled scaffold group shows fibrous/sponge regions inside and slightly at the top layer of the scaffold, as shown in Figure. 3. The rough surface morphology can facilitate cell adhesion and proliferation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and the fibrous mesh region of SF could improve cell attachment and biocompatibility, by entrapping more cells and facilitating proliferation at the site [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The scaffolds were also shown to exhibit micro and nanoporous structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Contact Angle Measurement\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSilk fibroin is a biopolymer with hydrophilic (\u0026ndash;OH and \u0026ndash;COOH) and hydrophobic groups having repeating sequences of residues (glycine, alanine, and serine) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Despite having properties such as porous and permeability, the ceramic scaffolds after being infilled with SF show slightly compromised hydrophilicity compared to BEN-HAP. However, both scaffold groups are hydrophilic in nature, still the water permeation of the BEN-HAP scaffold group was very high which may be due to high surface energy that affects the surface tension of liquid. The SF-BEN-HAP scaffolds showed gradual water permeation where the surface energy could be less compared to BEN-HAP scaffolds, as shown in Figure. 4a. This hydrophilicity of SF-BEN-HAP can be tuned with change in concentration of silk fibroin [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The contact angle of the scaffold groups with the respective time point is mentioned in Figure. 4b.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cem\u003eIn Vitro\u003c/em\u003e Mineralization\u003c/h2\u003e \u003cp\u003eAfter 14 days of incubation in SBF solution, the scaffolds showed hydroxyapatite crystal growth in both groups. HAP is composed of calcium and phosphate, with a Ca/P ratio near 1.67 molar ratio in bone, and are the most essential ions for mineralization [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. This calcium (Ca) and phosphate (P) mineralization was observed like flake-shaped crystals on the surface [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], which are higher in SF-infilled scaffold as shown in Figure. 5. This shows the potential of silk fibroin to modulate the growth of hydroxyapatite crystals through the Ca\u003csup\u003e2+\u003c/sup\u003e interaction and surface interactions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e][\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Further, the change in Ca:P ratios was analyzed in EDAX and found to be 9.89:6.44 wt % in BEN-HAP and 13.21:9.11 wt % in SF-BEN-HAP scaffold.\u003c/p\u003e \u003cp\u003eFlavonoids are widely known for their radical-scavenging ability and are highly used in tissue repair and wound-healing applications. DPPH and ABTS are free radical compounds that are widely used to test the free radical scavenging ability [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Also, the ABTS assay is more sensitive due to fast reaction kinetics compared to DPPH [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The antioxidant property of quercetin and hesperetin neutralizes the ABTS and DPPH by the transfer of an electron or hydrogen atom [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The unpaired electrons are delocalized by the phenol compounds that can react with them within the aromatic ring and can form stable intermediates by undergoing resonance stabilization within the molecule [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The reduction capacity in ABTS was determined by colour changes from blue-green to colourless at 734 nm and DPPH observed colour changes from deep violet to yellow by reading at 517 nm. The radical scavenging activity of quercetin and hesperetin was detected and compared with the standard antioxidant, Ascorbic acid. In the ABTS assay, the quercetin and hesperetin have scavenging activity of 5 % and 1 % at 10\u0026micro;M. At igher concentrations, 320 \u0026micro;M has scavenging activity of 81 % and 86 % whic are cloer to the ascorbic acid (87 %). In the DPPHassay, quercetin and ascorbic have 5 % scavenging acivity at 10 \u0026micro;M. Quercetin and hesperetin were observed to have 24 % and 12 % scavnging acivity at a concentration of 320 \u0026micro;M as shown in Figure. 6. The electron transfer is generally instantaneous and hydrogen transfer is comparatively slower. In the ABTS assay, this initial electron transfer takes place significantly more quickly [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Since the DPPH radical site has steric hindrance, it may cause difficulty in accessing phenols. This could be the case where the lower concentration of both hesperetin and quercetin has lesser activity along with the ascorbic acid (control) compared to ABTS. Also, the lower concentrations of these compounds may cause very minimum electron transfer which cannot have enough scavenging activity. However, the efficacy of antioxidants to scavenge radicals is largely dependent on their steric accessibility to the radical site in DPPH [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. After investigation, it was observed that both compounds had a strong antioxidant capacity that varied with different concentrations (dose-dependent) compared to control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Biocompatibility of Flavonoids conjugated SF-BEN-HAP scaffolds\u003c/h2\u003e \u003cp\u003eThe MTT experiment conducted on hWJ-MSC cells treated with different concentrations of quercetin and hesperetin reveals no harmful effects of the flavonoids present in the SF-BEN-HAP scaffolds as shown in Figure. 7a. SF-BEN-HAP scaffold without flavonoids was taken as control.\u003c/p\u003e\u003cp\u003eIn addition, the flavonoid-loaded scaffolds showed improved cell growth and viability than the BEN-HAP and SF-BEN-HAP scaffolds. The MSC cell viability and proliferation rate are increased when quercetin and hesperetin are present at lower concentrations [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The higher concentration (20 \u0026micro;M) reviled cell damage that might be due to the solvent residues.\u003c/p\u003e \u003cp\u003eThe live and dead staining of hWJ-MSCs cells after incubation with quercetin and hesperetin-loaded SF-BEN-HAP scaffolds shows no notable toxicity and cell damage, in lower concentrations as shown in Figure. 7b. The cell proliferation of hWJ-MSCs was evaluated by scratch test assay. It was observed that the flavonoids-loaded SF-BEN-HAP scaffolds show improved cell migration which can be due to an effective biomolecule (flavonoids and silk fibroin) in the leaching solution. Among different concentrations, 10 \u0026micro;M of quercetin and hesperetin was observed to have more than 60% closure and promoted a high cell migration rate as shown in Figure. 8. The percentage of gap closure by cell migration was measured using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Antioxidant Properties of Flavonoids-SF-BEN-HAP: DCFDA\u003c/h2\u003e \u003cp\u003eOxidative stress occurs due to a high percentage of the reactive oxygen species (ROS) and their ratio may rise under physiological conditions like injuries. Mesenchymal stem cells (MSCs) are multipotent stromal cells that reside in adipose tissue and bone marrow and are highly known to contribute to tissue regeneration applications [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Based on their ability to differentiate into osteoprogenitor cells and bone cells [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], MSCs are used in this study to evaluate the antioxidant properties of the flavonoid-loaded SF-BEN-HAP scaffolds. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced intracellular ROS production in hWJ-MSCs was measured by the DCFH-DA fluorescent probe and observed under confocal microscopy (Olympus, Fluoview Fv3000, Japan). The intracellular ROS level is determined based on the fluorescence intensity. Compared with the control group, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treated cells were observed to have increases intracellular ROS levels and observed having high fluorescence intensity as shown in Figure. 9. The SF-BEN-HAP scaffold loaded with different concentrations of quercetin and hesperetin indicated that the level of ROS gradually decreases when the concentration increases (1, 5, 10, and 20 \u0026micro;M). Especially, the SF-BEN-HAP scaffolds with 20 \u0026micro;M concentration of quercetin and hesperetin exhibited the strongest antioxidant activity and effectively suppressed the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced intracellular ROS production in hWJ-MSCs.\u003c/p\u003e \u003cp\u003eDCF-DA does not emit fluorescence yet is permeable within the cells. In the presence of ROS, it oxidizes and generates fluorescein, which emits light, implying that the more ROS in the cells, the higher the emission. Following acetate group breakage and oxidation by intracellular esterases, the non-fluorescent H2DCFDA converts into the highly fluorescent 2,7-dichlorofluorescein (DCF) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, the fluorescent intensity of the cells is proportional to the intracellular ROS levels. Specifically, an increase in the fluorescence intensity of DCF indicates the high ROS generation in the cells. When compared to the control (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e -treated cells), 1 \u0026micro;M and 5 \u0026micro;M concentrations showed minimal activity in inhibiting ROS production. However, 10 \u0026micro;M and 20 \u0026micro;M concentrations of both quercetin and hesperetin showed much greater antioxidant activity as shown in Figure. 10. Following to that, comparatively, even at low concentrations, quercetin inhibits ROS production more effectively than hesperetin-loaded scaffolds. The 20 \u0026micro;M dose of quercetin and hesperetin resulted in maximal ROS suppression, but significantly reduced cell migration. Based on the cell viability, anti-oxidative potential, and cell migration, 10 \u0026micro;M of quercetin-loaded SF-BEN-HAP can be an effective scaffold for bone tissue regeneration with antioxidative ability to reduce the oxidative stress at the implant site.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBone consists of organic and inorganic components which provide mechanical strength and a biologically niche environment for cell growth. In the 3D fabricated scaffold, BEN-HAP acts as an inorganic mineral matrix-like bone (consists of mineral ions such as calcium, phosphate, iron, magnesium, and sodium), and SF acts as an organic matter like collagen which makes them an ideal bone mimicking organic-inorganic matrix for bone tissue repair. The infilling of silk fibroin in BEN-HAP scaffold, results with a fibrous mesh-like structure. The presence of silk fibroin in the scaffold influences the hydrophilicity and \u003cem\u003ein vitro\u003c/em\u003e mineralization, improves cell attachment and proliferation. Recent literature on tissue engineering addresses the problem of oxidative stress which is linked with the potential cause of implant loosening and hinders new bone formation. To prevent the scaffold from loosening and reduce the oxidative stress on the nearby cells, the fabricated scaffold can be conjugated with antioxidants. Quercetin and hesperetin are cost-effective flavonoids that are natural antioxidants used to reduce oxidative stress based on concentration (dose-dependent). The radical scavenging activity of quercetin and hesperetin was investigated and observed to have a strong antioxidant capacity that varied with dose (1, 5, 10, 20, 40, 80, 160, and 320 \u0026micro;M). The scaffolds consisting of 10 \u0026micro;M of flavonoids were observed to have more than 60% closure and promoted high cell migration compared to scaffolds with 20 \u0026micro;M flavonoids. We anticipated the potential of flavonoid-loaded scaffolds to reduce the oxidative stress in the hWJ-MSCs cells which are artificially induced for intracellular ROS by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment. As a result, the dose-dependent reduction in ROS was observed through DCFH-DA stain and visualized under confocal microscopy. Among the different concentrations used, 10 and 20 \u0026micro;M scaffolds exhibited strong antioxidative response in hWJ-MSCs cells. Since a significant reduction in cell viability and cell migration was observed in 20 \u0026micro;M scaffold groups, flavonoids with 10 \u0026micro;M scaffold groups can be considered more suitable for bone tissue repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by\u0026nbsp;DST-NewGen\u0026nbsp;IEDC, National Science \u0026amp;\u0026nbsp;Technology Entrepreneurship Development Board (NSTEDB), Department of Science and Technology, Govt. of India, New Delhi through Datta Meghe Institute of Higher Education and Research, Wardha, Maharashtra, India. The authors are grateful to DST-SERB (Grant no: SRG/2019/002038) for financial support for the development of ceramic 3D printer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: CRediT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMethodology, Writing - original draft preparation:\u0026nbsp;Logeshwaran A\u003csup\u003e1\u003c/sup\u003e; Formal analysis and investigation:\u0026nbsp;Renold Elsen\u003csup\u003e2\u003c/sup\u003e; Funding acquisition, Resources:\u0026nbsp;Ashutosh D. Bagde\u003csup\u003e\u0026nbsp;3a,b\u003c/sup\u003e; Conceptualization, Supervision, Writing - review and editing:\u0026nbsp;Sunita Nayak\u003csup\u003e*1\u003c/sup\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eY. Seol, H. Kang, S.J. Lee, A. Atala, J.J. Yoo, Bioprinting technology and its applications, pp. 1\u0026ndash;7, 2014, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/ejcts/ezu148\u003c/span\u003e\u003cspan address=\"10.1093/ejcts/ezu148\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Szwed-Georgiou et al., Bioactive Materials for Bone Regeneration: Biomolecules and Delivery Systems. ACS Biomater. Sci. Eng. \u003cb\u003e9\u003c/b\u003e(9), 5222\u0026ndash;5254 (Sep. 2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACSBIOMATERIALS.3C00609\u003c/span\u003e\u003cspan address=\"10.1021/ACSBIOMATERIALS.3C00609\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/ASSET/IMAGES/LARGE/AB3C00609_0003.JPEG\u003c/span\u003e\u003cspan address=\"http:///ASSET/IMAGES/LARGE/AB3C00609_0003.JPEG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.M. Germaini, S. Belhabib, S. Guessasma, R. Deterre, P. Corre, P. Weiss, Additive manufacturing of biomaterials for bone tissue engineering \u0026ndash; A critical review of the state of the art and new concepts, \u003cem\u003eProg Mater Sci\u003c/em\u003e, vol. 130, p. 100963, Oct. 2022, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.PMATSCI.2022.100963\u003c/span\u003e\u003cspan address=\"10.1016/J.PMATSCI.2022.100963\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Lee, M. Carrillo, J. McKittrick, D.G. Martin, E.A. Olevsky, Fabrication of ceramic bone scaffolds by solvent jetting 3D printing and sintering: Towards load-bearing applications. Addit. Manuf. \u003cb\u003e33\u003c/b\u003e, 101107 (May 2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ADDMA.2020.101107\u003c/span\u003e\u003cspan address=\"10.1016/J.ADDMA.2020.101107\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Budharaju et al., Ceramic materials for 3D printing of biomimetic bone scaffolds \u0026ndash; Current state-of-the-art \u0026amp; future perspectives. Mater. Des. \u003cb\u003e231\u003c/b\u003e, 112064 (Jul. 2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.MATDES.2023.112064\u003c/span\u003e\u003cspan address=\"10.1016/J.MATDES.2023.112064\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH.L. Jaber, T.A. Kov\u0026aacute;cs, Preparation and synthesis of hydroxyapatite bio-ceramic from bovine bone by thermal heat treatment. Epitoanyag - J. Silicate Based Compos. Mater. \u003cb\u003e71\u003c/b\u003e(3), 98\u0026ndash;101 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.14382/epitoanyag-jsbcm.2019.18\u003c/span\u003e\u003cspan address=\"10.14382/epitoanyag-jsbcm.2019.18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Dave, V.G. Gomes, Interactions at scaffold interfaces: Effect of surface chemistry, structural attributes and bioaffinity. Mater. Sci. Engineering: C \u003cb\u003e105\u003c/b\u003e, 110078 (Dec. 2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.MSEC.2019.110078\u003c/span\u003e\u003cspan address=\"10.1016/J.MSEC.2019.110078\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Tallawi et al., Strategies for the chemical and biological functionalization of scaffolds for cardiac tissue engineering: a review. J. R Soc. Interface. \u003cb\u003e12\u003c/b\u003e, 20150254 (Jul. 2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1098/RSIF.2015.0254\u003c/span\u003e\u003cspan address=\"10.1098/RSIF.2015.0254\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Du et al., Sep., Bioactive polymer composite scaffolds fabricated from 3D printed negative molds enable bone formation and vascularization, \u003cem\u003eActa Biomater\u003c/em\u003e, vol. 186, pp. 260\u0026ndash;274, 2024, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ACTBIO.2024.07.038\u003c/span\u003e\u003cspan address=\"10.1016/J.ACTBIO.2024.07.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL. Hod\u0026aacute;sov\u0026aacute; et al., Polymer infiltrated ceramic networks with biocompatible adhesive and 3D-printed highly porous scaffolds. Addit. Manuf. \u003cb\u003e39\u003c/b\u003e, 101850 (Mar. 2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ADDMA.2021.101850\u003c/span\u003e\u003cspan address=\"10.1016/J.ADDMA.2021.101850\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF.J. Mart\u0026iacute;nez-V\u0026aacute;zquez, F.H. Perera, P. Miranda, A. Pajares, F. Guiberteau, Improving the compressive strength of bioceramic robocast scaffolds by polymer infiltration, \u003cem\u003eActa Biomater\u003c/em\u003e, vol. 6, no. 11, pp. 4361\u0026ndash;4368, Nov. 2010, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.ACTBIO.2010.05.024\u003c/span\u003e\u003cspan address=\"10.1016/J.ACTBIO.2010.05.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Shi et al., 3D printed intelligent scaffold prevents recurrence and distal metastasis of breast cancer. Theranostics. \u003cb\u003e10\u003c/b\u003e, 10652 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7150/THNO.47933\u003c/span\u003e\u003cspan address=\"10.7150/THNO.47933\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.E. Song, N. Tripathy, D.H. Lee, J.H. Park, G. Khang, Quercetin Inlaid Silk Fibroin/Hydroxyapatite Scaffold Promotes Enhanced Osteogenesis. ACS Appl. Mater. Interfaces. \u003cb\u003e10\u003c/b\u003e, 32955\u0026ndash;32964 (Oct. 2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACSAMI.8B08119/ASSET\u003c/span\u003e\u003cspan address=\"10.1021/ACSAMI.8B08119/ASSET\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/IMAGES/MEDIUM/AM-2018-081198_0006.GIF\u003c/span\u003e\u003cspan address=\"http:///IMAGES/MEDIUM/AM-2018-081198_0006.GIF\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Wenk, H.P. Merkle, L. Meinel, Silk fibroin as a vehicle for drug delivery applications. J. Controlled Release. \u003cb\u003e150\u003c/b\u003e(2), 128\u0026ndash;141 (Mar. 2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.A.L. Matthew, F.P. Seib, Silk Bioconjugates: From Chemistry and Concept to Application. ACS Biomater. Sci. Eng. \u003cb\u003e10\u003c/b\u003e(1), 12\u0026ndash;28 (Jan. 2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACSBIOMATERIALS.2C01116\u003c/span\u003e\u003cspan address=\"10.1021/ACSBIOMATERIALS.2C01116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/ASSET/IMAGES/LARGE/AB2C01116_0006.JPEG\u003c/span\u003e\u003cspan address=\"http:///ASSET/IMAGES/LARGE/AB2C01116_0006.JPEG\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.A. Sakr et al., Development of bentonite-gelatin nanocomposite hybrid hydrogels for tissue engineering. Appl. Clay Sci. \u003cb\u003e199\u003c/b\u003e, 105860 (Dec. 2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.CLAY.2020.105860\u003c/span\u003e\u003cspan address=\"10.1016/J.CLAY.2020.105860\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC. Shuai, W. Yang, P. Feng, S. Peng, H. Pan, Accelerated degradation of HAP/PLLA bone scaffold by PGA blending facilitates bioactivity and osteoconductivity. Bioact Mater. \u003cb\u003e6\u003c/b\u003e(2), 490\u0026ndash;502 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bioactmat.2020.09.001\u003c/span\u003e\u003cspan address=\"10.1016/j.bioactmat.2020.09.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Banimohamad-Shotorbani, A. Rahmani, Del A. Bakhshayesh, S. Mehdipour, Jarolmasjed, H. Shafaei, The efficiency of PCL/HAp electrospun nanofibers in bone regeneration: a review. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03091902.2021\u003c/span\u003e\u003cspan address=\"https://doi.org/10.1080/03091902.2021\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003cem\u003e1893396\u003c/em\u003e, vol. 45, no. 7, pp. 511\u0026ndash;531, 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/03091902.2021.1893396\u003c/span\u003e\u003cspan address=\"10.1080/03091902.2021.1893396\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Logeshwaran, R. Elsen, S. Nayak, Mechanical and biological characteristics of 3D fabricated clay mineral and bioceramic composite scaffold for bone tissue applications, \u003cem\u003eJ Mech Behav Biomed Mater\u003c/em\u003e, vol. 138, no. December 2022, p. 105633, 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmbbm.2022.105633\u003c/span\u003e\u003cspan address=\"10.1016/j.jmbbm.2022.105633\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. Zhou, Y. Yan, C. Zhao, Y. Xu, Q. Wang, N. Xu, Resveratrol improves osteogenic differentiation of senescent bone mesenchymal stem cells through inhibiting endogenous reactive oxygen species production via AMPK activation. Redox Rep. \u003cb\u003e24\u003c/b\u003e(1), 62\u0026ndash;69 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/13510002.2019.1658376\u003c/span\u003e\u003cspan address=\"10.1080/13510002.2019.1658376\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Abdulrahman Hussain et al., Efficacy and safety of co-administration of resveratrol with meloxicam in patients with knee osteoarthritis: A pilot interventional study. Clin. Interv Aging. \u003cb\u003e13\u003c/b\u003e, 1621\u0026ndash;1630 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/CIA.S172758\u003c/span\u003e\u003cspan address=\"10.2147/CIA.S172758\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Hameister, C. Kaur, S.T. Dheen, C.H. Lohmann, G. Singh, Reactive oxygen/nitrogen species (ROS/RNS) and oxidative stress in arthroplasty. J. Biomed. Mater. Res. B Appl. Biomater. \u003cb\u003e108\u003c/b\u003e(5), 2073\u0026ndash;2087 (Jul. 2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/JBM.B.34546\u003c/span\u003e\u003cspan address=\"10.1002/JBM.B.34546\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA.E. Bădilă, D.M. Rădulescu, A. Ilie, A.G. Niculescu, A.M. Grumezescu, A.R. Rădulescu, Bone Regeneration and Oxidative Stress: An Updated Overview. Antioxid. (Basel). \u003cb\u003e11\u003c/b\u003e(2) (Feb. 2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ANTIOX11020318\u003c/span\u003e\u003cspan address=\"10.3390/ANTIOX11020318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Hu et al., Regulation of hypoxic stress and oxidative stress in bone grafting: Current trends and future perspectives. J. Mater. Sci. Technol. \u003cb\u003e157\u003c/b\u003e, 144\u0026ndash;153 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jmst.2023.01.055\u003c/span\u003e\u003cspan address=\"10.1016/j.jmst.2023.01.055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.F. Manchope et al., Naringenin mitigates titanium dioxide (TiO 2)-induced chronic arthritis in mice: role of oxidative stress, cytokines, and NFκB. Inflamm. Res. \u003cb\u003e67\u003c/b\u003e, 11\u0026ndash;12 (Dec. 2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/S00011-018-1195-Y/METRICS\u003c/span\u003e\u003cspan address=\"10.1007/S00011-018-1195-Y/METRICS\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. yang Wang, Q. Li, K. shun Bi, Bioactive flavonoids in medicinal plants: Structure, activity and biological fate. Asian J. Pharm. Sci. \u003cb\u003e13\u003c/b\u003e(1), 12\u0026ndash;23 (Jan. 2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.AJPS.2017.08.004\u003c/span\u003e\u003cspan address=\"10.1016/J.AJPS.2017.08.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Chakravarty, N. Bhardwaj, B.B. Mandal, N. Sen Sarma, Silk fibroin-carbon nanoparticle composite scaffolds: A cost effective supramolecular \u0026lsquo;turn off\u0026rsquo; chemiresistor for nitroaromatic explosive vapours. J. Mater. Chem. C Mater. \u003cb\u003e4\u003c/b\u003e(38), 8920\u0026ndash;8929 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/C6TC03337G\u003c/span\u003e\u003cspan address=\"10.1039/C6TC03337G\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI. D\u0026rsquo;Onofrio et al., Inhalable drug-loaded silk fibroin carriers for pulmonary drug delivery. RSC Adv. \u003cb\u003e14\u003c/b\u003e(37), 27288\u0026ndash;27297 (Aug. 2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/D4RA03324H\u003c/span\u003e\u003cspan address=\"10.1039/D4RA03324H\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Xue et al., The role of hesperetin on osteogenesis of human mesenchymal stem cells and its function in bone regeneration. Oncotarget. \u003cb\u003e8\u003c/b\u003e, 21031 (Mar. 2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18632/ONCOTARGET.15473\u003c/span\u003e\u003cspan address=\"10.18632/ONCOTARGET.15473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP.A. Miguez et al., Mar., Hesperidin Promotes Osteogenesis and Modulates Collagen Matrix Organization and Mineralization In Vitro and In Vivo, \u003cem\u003eInt J Mol Sci\u003c/em\u003e, vol. 22, no. 6, pp. 1\u0026ndash;13, 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/IJMS22063223\u003c/span\u003e\u003cspan address=\"10.3390/IJMS22063223\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.H. Huang, C.Y. Chen, C.Y. Chang, Y.W. Chen, C.P. Lin, The synergistic effects of quercetin-containing 3D-printed mesoporous calcium silicate/calcium sulfate/poly-ε-caprolactone scaffolds for the promotion of osteogenesis in mesenchymal stem cells, \u003cem\u003eJournal of the Formosan Medical Association\u003c/em\u003e, no. xxxx, 2021, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jfma.2021.01.024\u003c/span\u003e\u003cspan address=\"10.1016/j.jfma.2021.01.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e(ASTM), ASTM D7334-08 Standard Practice for Surface Wettability of Coatings, Substrates and Pigments by Advancing Contact Angle Measurement, \u003cem\u003eASTM International\u003c/em\u003e, vol. 08, no. Reapproved, pp. 1\u0026ndash;3, 2008, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1520/D7334-08R22.Copyright\u003c/span\u003e\u003cspan address=\"10.1520/D7334-08R22.Copyright\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.N. G\u0026oacute;mez-Cerezo, D. Lozano, D. Arcos, M. Vallet-Reg\u0026iacute;, C. Vaquette, The effect of biomimetic mineralization of 3D-printed mesoporous bioglass scaffolds on physical properties and in vitro osteogenicity, \u003cem\u003eMaterials Science and Engineering: C\u003c/em\u003e, vol. 109, p. 110572, Apr. 2020, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.MSEC.2019.110572\u003c/span\u003e\u003cspan address=\"10.1016/J.MSEC.2019.110572\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Zhang et al., Three-Dimensional Printed Cell Culture Model Based on Spherical Colloidal Lignin Particles and Cellulose Nanofibril-Alginate Hydrogel. Biomacromolecules. \u003cb\u003e21\u003c/b\u003e(5), 1875\u0026ndash;1885 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acs.biomac.9b01745\u003c/span\u003e\u003cspan address=\"10.1021/acs.biomac.9b01745\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG.E. Fawal, H. Hong, X. Mo, H. Wang, Fabrication of scaffold based on gelatin and polycaprolactone (PCL) for wound dressing application. J. Drug Deliv Sci. Technol. \u003cb\u003e63\u003c/b\u003e, 102501 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jddst.2021.102501\u003c/span\u003e\u003cspan address=\"10.1016/j.jddst.2021.102501\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.M. Sadek, W. Mamdouh, S.I. Habib, M.E. Deftar, A.N.A. Habib, In Vitro Biological Evaluation of a Fabricated Polycaprolactone/Pomegranate Electrospun Scaffold for Bone Regeneration, 2021. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/acsomega.1c04608\u003c/span\u003e\u003cspan address=\"10.1021/acsomega.1c04608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ.Y. Lee et al., Design of a 3D BMP-2-delivering tannylated PCL scaffold and its anti-oxidant, anti-inflammatory, and osteogenic effects in vitro, 2018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms19113602\u003c/span\u003e\u003cspan address=\"10.3390/ijms19113602\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF. Astm, 813-01: Standard Practice for Direct Contact Cell Culture Evaluation of Materials for, \u003cb\u003e24\u003c/b\u003e, pp. 1652\u0026ndash;1653, 2001, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1520/F0813-20.1\u003c/span\u003e\u003cspan address=\"10.1520/F0813-20.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ. Xu, Y. Sun, H. Dai, Y. Ma, H. Bing, Engineered 3D-Printed Polyvinyl Alcohol Scaffolds Incorporating β-Tricalcium Phosphate and Icariin Induce Bone Regeneration in Rat Skull Defect Model. Molecules. \u003cb\u003e27\u003c/b\u003e(14) (Jul. 2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/MOLECULES27144535\u003c/span\u003e\u003cspan address=\"10.3390/MOLECULES27144535\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW. Liu et al., Electrospun porous poly (3-hydroxybutyrate-co-4-hydroxybutyrate)/lecithin scaffold for bone tissue engineering. RSC Adv. \u003cb\u003e12\u003c/b\u003e(19), 11913\u0026ndash;11922 (2022)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Pal et al., Folic acid conjugated curcumin loaded biopolymeric gum acacia microsphere for triple negative breast cancer therapy in invitro and invivo model, \u003cem\u003eMaterials Science and Engineering C\u003c/em\u003e, vol. 95, no. November, pp. 204\u0026ndash;216, 2019, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.msec.2018.10.071\u003c/span\u003e\u003cspan address=\"10.1016/j.msec.2018.10.071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP. Kumar, I. Rupmanjari, K. Ajay, R.P. Mariadhas, V. Arasu, Insulin signaling pathway assessment by enhancing antioxidant activity due to morin using in vitro rat skeletal muscle L6 myotubes cells. Mol. Biol. Rep. \u003cb\u003e48\u003c/b\u003e(8), 5857\u0026ndash;5872 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11033-021-06580-x\u003c/span\u003e\u003cspan address=\"10.1007/s11033-021-06580-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH. Hong et al., Cytocompatibility of modified silk fibroin with glycidyl methacrylate for tissue engineering and biomedical applications. Biomolecules. \u003cb\u003e11\u003c/b\u003e(1), 1\u0026ndash;18 (Jan. 2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/BIOM11010035\u003c/span\u003e\u003cspan address=\"10.3390/BIOM11010035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Das, D. Pati, N. Tiwari, A. Nisal, S. Sen Gupta, Synthesis of silk fibroin-glycopolypeptide conjugates and their recognition with lectin. Biomacromolecules. \u003cb\u003e13\u003c/b\u003e(11), 3695\u0026ndash;3702 (Nov. 2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/BM301170U/SUPPL_FILE/BM301170U_SI_001.PDF\u003c/span\u003e\u003cspan address=\"10.1021/BM301170U/SUPPL_FILE/BM301170U_SI_001.PDF\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE.T. Baran, K. Tuzlakoǧlu, J.F. Mano, R.L. Reis, Enzymatic degradation behavior and cytocompatibility of silk fibroin\u0026ndash;starch\u0026ndash;chitosan conjugate membranes. Mater. Sci. Engineering: C \u003cb\u003e32\u003c/b\u003e(6), 1314\u0026ndash;1322 (Aug. 2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.MSEC.2012.02.015\u003c/span\u003e\u003cspan address=\"10.1016/J.MSEC.2012.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Ferracini, I. Mart\u0026iacute;nez Herreros, A. Russo, T. Casalini, F. Rossi, G. Perale, Scaffolds as Structural Tools for Bone-Targeted Drug Delivery, \u003cem\u003ePharmaceutics\u003c/em\u003e, vol. 10, no. 3, Sep. 2018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/PHARMACEUTICS10030122\u003c/span\u003e\u003cspan address=\"10.3390/PHARMACEUTICS10030122\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR. Deshpande, S. Shukla, A. Kale, N. Deshmukh, A. Nisal, P. Venugopalan, Studies, ACS Biomater. Sci. Eng. \u003cb\u003e8\u003c/b\u003e(3), 1226\u0026ndash;1238 (Mar. 2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACSBIOMATERIALS.1C01103\u003c/span\u003e\u003cspan address=\"10.1021/ACSBIOMATERIALS.1C01103\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/SUPPL_FILE/AB1C01103_SI_001.PDF\u003c/span\u003e\u003cspan address=\"http:///SUPPL_FILE/AB1C01103_SI_001.PDF\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.A. Burke, D.C. Roberts, D.L. Kaplan, Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins. Biomacromolecules. \u003cb\u003e17\u003c/b\u003e(1), 237 (Jan. 2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACS.BIOMAC.5B01330\u003c/span\u003e\u003cspan address=\"10.1021/ACS.BIOMAC.5B01330\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS. Kaushik, P.D. Thungon, P. Goswami, Silk Fibroin: An Emerging Biocompatible Material for Application of Enzymes and Whole Cells in Bioelectronics and Bioanalytical Sciences, \u003cem\u003eACS Biomater Sci Eng\u003c/em\u003e, vol. 6, no. 8, pp. 4337\u0026ndash;4355, Aug. 2020, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/ACSBIOMATERIALS.9B01971/ASSET\u003c/span\u003e\u003cspan address=\"10.1021/ACSBIOMATERIALS.9B01971/ASSET\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e/IMAGES/MEDIUM/AB9B01971_0009.GIF\u003c/span\u003e\u003cspan address=\"http:///IMAGES/MEDIUM/AB9B01971_0009.GIF\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eX. Wu, K. Walsh, B.L. Hoff, G. Camci-Unal, Mineralization of Biomaterials for Bone Tissue Engineering, \u003cem\u003eBioengineering\u003c/em\u003e 2020, Vol. 7, Page 132, vol. 7, no. 4, p. 132, Oct. 2020. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/BIOENGINEERING7040132\u003c/span\u003e\u003cspan address=\"10.3390/BIOENGINEERING7040132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Rodr\u0026iacute;guez, J. Sundberg, P. Gatenholm, S. Renneckar, Electrospun nanofibrous cellulose scaffolds with controlled microarchitecture. Carbohydr. Polym. \u003cb\u003e100\u003c/b\u003e, 143\u0026ndash;149 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.CARBPOL.2012.12.037\u003c/span\u003e\u003cspan address=\"10.1016/J.CARBPOL.2012.12.037\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eY. Jin, B. Kundu, Y. Cai, S.C. Kundu, J. Yao, Bio-inspired mineralization of hydroxyapatite in 3D silk fibroin hydrogel for bone tissue engineering. Colloids Surf. B Biointerfaces. \u003cb\u003e134\u003c/b\u003e, 339\u0026ndash;345 (Oct. 2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.COLSURFB.2015.07.015\u003c/span\u003e\u003cspan address=\"10.1016/J.COLSURFB.2015.07.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ.J. Chen et al., Mineralized self-assembled silk fibroin/cellulose interpenetrating network aerogel for bone tissue engineering. Biomaterials Adv. \u003cb\u003e134\u003c/b\u003e, 112549 (Mar. 2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.MSEC.2021.112549\u003c/span\u003e\u003cspan address=\"10.1016/J.MSEC.2021.112549\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Olszowy, A.L. Dawidowicz, Is it possible to use the DPPH and ABTS methods for reliable estimation of antioxidant power of colored compounds? Chem. Pap. \u003cb\u003e72\u003c/b\u003e(2), 393\u0026ndash;400 (Feb. 2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/S11696-017-0288-3/TABLES/3\u003c/span\u003e\u003cspan address=\"10.1007/S11696-017-0288-3/TABLES/3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.J. Lee, Y.C. Oh, W.K. Cho, J.Y. Ma, Antioxidant and Anti-Inflammatory Activity Determination of One Hundred Kinds of Pure Chemical Compounds Using Offline and Online Screening HPLC Assay, \u003cem\u003eEvid Based Complement Alternat Med\u003c/em\u003e, vol. 2015, p. 165457, 2015. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2015/165457\u003c/span\u003e\u003cspan address=\"10.1155/2015/165457\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Rumpf, R. Burger, M. Schulze, Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins. Int. J. Biol. Macromol. \u003cb\u003e233\u003c/b\u003e, 123470 (Apr. 2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/J.IJBIOMAC.2023.123470\u003c/span\u003e\u003cspan address=\"10.1016/J.IJBIOMAC.2023.123470\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eC.M.C. Andr\u0026eacute;s, J.M. P\u0026eacute;rez de la Lastra, C.A. Juan, F.J. Plou, E. P\u0026eacute;rez-Lebe\u0026ntilde;a, Polyphenols as Antioxidant/Pro-Oxidant Compounds and Donors of Reducing Species: Relationship with Human Antioxidant Metabolism. Processes 2023. \u003cb\u003e11\u003c/b\u003e(9), 2771 (Sep. 2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/PR11092771\u003c/span\u003e\u003cspan address=\"10.3390/PR11092771\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.S. Shojaee, M. Moeenfard, R. Farhoosh, Kinetics and stoichiometry of gallic acid and methyl gallate in scavenging DPPH radical as affected by the reaction solvent, \u003cem\u003eScientific Reports 2022 12:1\u003c/em\u003e, vol. 12, no. 1, pp. 1\u0026ndash;9, May 2022, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-022-12803-3\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-12803-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eA. Deb, How Stem Cells Turn into Bone and Fat, \u003cem\u003eN Engl J Med\u003c/em\u003e, vol. 380, no. 23, p. 2268, Jun. 2019, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMCIBR1905165\u003c/span\u003e\u003cspan address=\"10.1056/NEJMCIBR1905165\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM. Ponzetti, N. Rucci, Osteoblast Differentiation and Signaling: Established Concepts and Emerging Topics. Int. J. Mol. Sci. \u003cb\u003e22\u003c/b\u003e, 6651 (Jul. 2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/IJMS22136651\u003c/span\u003e\u003cspan address=\"10.3390/IJMS22136651\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-porous-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopo","sideBox":"Learn more about [Journal of Porous Materials](http://link.springer.com/journal/10934)","snPcode":"10934","submissionUrl":"https://submission.nature.com/new-submission/10934/3","title":"Journal of Porous Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bentonite, Silk fibroin, 3D Bone scaffold, Antioxidants","lastPublishedDoi":"10.21203/rs.3.rs-8679759/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8679759/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFor bone tissue regeneration, 3D printing ceramic materials is beneficial, however material synthesis and commercially accessible composites raise demand and decrease cost-effectiveness. Implementing more composite materials to the defective site may triggers oxidative stress to the surrounding cells. This study focuses on the development of 3D printed BEN-HAP scaffold infilled with flavonoids conjugated silk fibroin for bone tissue replacement for controlling oxidative stress during tissue regeneration. The silk fibroin infilled regions in the scaffold showed a sponge like fibre matrix that facilitates cell adhesion and proliferation which were observed under FESEM. Also, flake-shaped appetite formation was observed higher in SF-infilled scaffolds, confirming the calcium and phosphate mineralization layer. The scaffolds showed improved surface properties that facilitated slow water permeation and absorption compared with BEN-HAP scaffold. The biocompatibility results showed no toxic effect on hWJ-MSCs and RBCs additionally, the quercetin and hesperetin loaded SF-BEN-HAP scaffold showed more than 60% closure promoting a high cell migration rate at 10 \u0026micro;M concentration in scratch test assay. And a maximum reduction in ROS activity in hWJ-MSCs was observed when treated with 20 \u0026micro;M flavonoids.\u003c/p\u003e","manuscriptTitle":"Flavonoid loaded mineral clay-based 3D scaffold for bone tissue regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 15:40:26","doi":"10.21203/rs.3.rs-8679759/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-26T11:04:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T06:15:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"33674269350897059067821354533857689111","date":"2026-04-13T08:55:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T06:19:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T07:04:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296446831412233935507617440967965831713","date":"2026-03-18T16:52:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252251257827411442499577889442474203383","date":"2026-03-16T16:33:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111184001501140393683511113575551064970","date":"2026-03-16T03:37:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T03:15:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T12:08:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-29T12:08:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Porous Materials","date":"2026-01-23T13:16:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-porous-materials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopo","sideBox":"Learn more about [Journal of Porous Materials](http://link.springer.com/journal/10934)","snPcode":"10934","submissionUrl":"https://submission.nature.com/new-submission/10934/3","title":"Journal of Porous Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"88edc8bf-736a-4424-9077-311b4ea84e04","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T21:38:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 15:40:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8679759","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8679759","identity":"rs-8679759","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}