Synthesis of Silver-Coated Bioactive Nanocomposite Scaffolds Based on Grafted Beta-Glucan/Hydroxyapatite via Freeze-Drying Method: Anti-Microbial and Biocompatibility Evaluation for Bone Tissue Engineering

Advancement and development in bone tissue engineering, particularly that of composite scaffolds, are of great importance for bone tissue engineering. We have synthesized polymeric matrix using biopolymer (β-glucan), acrylic acid, and nano-hydroxyapatite through free radical polymerization method. Bioactive nanocomposite scaffolds (BNSs) were fabricated using the freeze-drying method and Ag was coated by the dip-coating method. The scaffolds have been characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction analysis (XRD) to investigate their functional groups, surface morphology, and phase analysis, respectively. The pore size and porosity of all BNS samples were found to be dependent on silver concentration. Mechanical testing of all BNS samples have substantial compressive strength in dry form that is closer to cancellous bone. The samples of BNS showed substantial antibacterial effect against DH5 alpha E. coli. The biological studies conducted using the MC3T3-E1 cell line via neutral red dye assay on the scaffolds have found to be biocompatible and non-cytotoxic. These bioactive scaffolds can bring numerous applications for bone tissue repairs and regenerations.


Introduction
Bone tissue engineering is an advanced approach to treat bone defects caused by disease, aging, and accident by using fabricated scaffolds. It has brought numerous new biomaterials and methods developments for treating difficult segmental and contained skeletal defects. This concept has applied testing machine (UTM). The mouse pre-osteoblast cell line (MC3T3-E1) was used to investigate the biocompatibility of the scaffolds

Materials
The ®-Glucan (BG) was purchased from DSP Gokyo Food and Chemical Co. Ltd., Osaka Japan. Acrylic acid (AAc) and N,N'-methylene-bis-acrylamide (N, N-MBA), n-HAp powder (<100 nm particle size), and AgNO 3 were purchased from Sigma-Aldrich, Selangor, Malaysia. The phosphate buffer saline (PBS) solution was prepared using sodium hydrogen phosphate, potassium chloride, sodium chloride, di-potassium hydrogen phosphate, and hydrochloric acid obtained from Merck, Darmstadt, Germany and used without any purification.

Bioactive Nanocomposite Scaffold Fabrication
The biopolymeric nanocomposites were prepared by dissolving 2 g β-glucan (BG) in deionized-water and transferred into round bottom two-neck flask heated at 65 • C with constant stirring under an inert nitrogen atmosphere with addition of 0.05 g potassium persulfate as initiator. After 30 min, 0.40 mL of AAc as monomer and N, N-MBA crosslinker (0.05 % of AAc) were added into reaction media. After 45 min, (2.5 g) n-HAp powder was added gradually with continuous stirring for 3 h. Hence, grafting of acrylic acid into BG was done using free-radical polymerization. HAp nanoparticles have been trapped into the polymeric network of BG-graft-AAc on completion of the reaction. N 2 gas was removed from the reaction media, followed by cooling at ambient. The reaction suspension was filtered under vacuum to remove unreacted reactants and washed with deionized water later it dried at 55 • C overnight in the oven. The synthesis of bioactive nanocomposite powder and their name identification are presented in Table 1. The bioactive nanocomposite powder (5 g) was homogenized into deionized-water (10 mL) to form slurry. Then slurry was filled into cylindrical molds (2 cm × 6 cm) and kept at −40 • C for 24 h. The porous bioactive nanocomposite scaffolds were obtained by and freeze drying method. The porosity and pore size of the bioactive nanocomposite scaffolds were analyzed using Brunauer-Emmett-Teller (Micromeritics Gemini II 2370) given in Table 1.

Deposition of Silver Particles on Bioactive Nanocomposite Scaffolds
The bioactive scaffolds were washed with deionized water after treating with 10 wt. % NaOH aq . The solution was kept at ambient for 5 min. Initially, different concentrations of silver nitrate (AgNO 3 ) solution was prepared from 0.15 to 0.60 M. Dropwise aqua ammonia (25 wt. %) was added into AgNO 3 solutions. It was stirred to obtain a clear [Ag(NH 3 ) 2 ] + solution. Then, scaffolds were dipped into the [Ag(NH 3 ) 2 ] + solution for 20 s and oven-dried for 5 min at 100 • C. This process was repeated for 50 cycles to deposit Ag onto the scaffolds. Finally, Ag-coated bioactive nanocomposite scaffolds (BNS) were washed and dried as shown in the Figure 1.

Materials
The -Glucan (BG) was purchased from DSP Gokyo Food and Chemical Co. Ltd., Osaka Japan. Acrylic acid (AAc) and N, N'-methylene-bis-acrylamide (N, N-MBA), n-HAp powder (<100 nm particle size), and AgNO3 were purchased from Sigma-Aldrich, Selangor, Malaysia. The phosphate buffer saline (PBS) solution was prepared using sodium hydrogen phosphate, potassium chloride, sodium chloride, di-potassium hydrogen phosphate, and hydrochloric acid obtained from Merck, Darmstadt, Germany and used without any purification.

Bioactive Nanocomposite Scaffold Fabrication
The biopolymeric nanocomposites were prepared by dissolving 2 g β-glucan (BG) in deionizedwater and transferred into round bottom two-neck flask heated at 65 °C with constant stirring under an inert nitrogen atmosphere with addition of 0.05 g potassium persulfate as initiator. After 30 min, 0.40 mL of AAc as monomer and N, N-MBA crosslinker (0.05 % of AAc) were added into reaction media. After 45 min, (2.5 g) n-HAp powder was added gradually with continuous stirring for 3 h. Hence, grafting of acrylic acid into BG was done using free-radical polymerization. HAp nanoparticles have been trapped into the polymeric network of BG-graft-AAc on completion of the reaction. N2 gas was removed from the reaction media, followed by cooling at ambient. The reaction suspension was filtered under vacuum to remove unreacted reactants and washed with deionized water later it dried at 55 °C overnight in the oven. The synthesis of bioactive nanocomposite powder and their name identification are presented in Table 1. The bioactive nanocomposite powder (5 g) was homogenized into deionized-water (10 mL) to form slurry. Then slurry was filled into cylindrical molds (2 cm × 6 cm) and kept at −40 °C for 24 h. The porous bioactive nanocomposite scaffolds were

Fourier Transform Infrared Spectroscopy
Chemical composition of BNS was assessed using FTIR (Shimadzu FTIR-8100A, Tokyo, Japan) with a resolution of 4 cm −1 and a frequency region from 400 to 4000 cm −1 , and 16 scans were accumulated per sample. The samples were reduced to powder and analyzed as potassium bromide (KBr) pellets.

X-ray Diffraction
Phase analysis of the BNS was studied using Advance X-ray diffractometer (Bruker AXS D8, Kontich, Belgium) spectrometry. The XRD analysis was conducted through a diffractometer at a voltage of 40 kV and a current of 40 mA with Cu Kα radiation (1.540 Å) at 2θ range of 10-80 • to determine the structure and peak data.

Scanning Electron Microscope/ Energy Dispersive Spectroscopy
Morphology of the BNS was observed by SEM (JEOL-JSM-6480, Peabody, Massachusetts, USA) coupled with EDS for elemental quantitative analysis. The samples were gold-sputtered.

Mechanical Testing
Compression strength of the BNS was analyzed in a dry state by UTM (Testometrics, Rochdale, UK) with a compression loading rate of 5 mm/min. All cylindrical BNS (4 mm height and 10 mm diameter) were analyzed until failure. The elastic modulus was determined using the slope of the stress-strain curve. Five replicates per sample were tested.

Swelling Analysis
Scaffold swelling analysis was analyzed using water and phosphate buffer saline (PBS) solution by maintaining 7.4 pH at a temperature of 37 • C. The well dried scaffolds were soaked in solutions by keeping the conditions as mentioned above. The scaffolds were withdrawn from the solutions at different time periods and additional solvent concentrations were eliminated to have exact weight. The percentage of scaffold swelling has been estimated by Equation (1).
where, Ws = weight of swelling scaffolds and W D = weight of dried scaffolds at various time intervals.
3.6. In Vitro Studies

Anti-Microbial Activities
An in vitro antimicrobial activity assay was conducted by agar disc-diffusion assay using gram-negative model bacterium Escherichia coli DH5 alpha. These bacterial strains were incubated at 37 • C to analyze antimicrobial activities of materials. Bacterial culture was spread uniformly using sterile glass rod over solidified agar [35]. Then 90 mL of each scaffold extract was placed over the bacterial Petri-plate. The Petri-plate was kept into an oven incubated for 24 h at 37 • C.

Sample Preparation for Cell Culture
BNS3 extract was selected for cell culture observation. The bottom of each well of 24-well plate was finely coated with scaffold and UV-light sterilized for 1 h and used to study morphological changes of the cells. Different concentrations of BNS3 extract from 0.125 to 2.00 mg/mL were prepared to evaluate cell viability and uncoated wells were used as control.

Cell Morphological Analysis
The MC3T3-E1 mouse pre-osteoblast cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured on coated wells of 24 well plates at the density of approximately 5000 cells per cm 2 in α-MEM supplemented with 10% FBS (Fetal Bovine Serum Gibco™ 12662011, Gibco laboratories, Gaithersburg, MD, USA, 100 U/mL Penicillin and 0.1 mg/mL Streptomycin solution (Gibco™ 15140122, ATCC, Manassas, VA, USA). The cells were incubated for 72 h in 5% CO 2 with 90% humidity at 37 • C. The cell morphology was analyzed using a Nikon ECLIPSE TS100 (ATCC, Manassas) inverted fluorescence microscope with live cells stained using 10 µg/mL of Fluorescein diacetate solution in complete growth medium to minimize the background scaffold coating and highlight only living viable cells under 488 nm excitation wavelength.

Cytotoxicity Using the Neutral Red Assay
The pre-osteoblast cell viability assay was performed by seeding cells in a 12-well plate with approximately 5,000 cells per cm 2 for 24h. Different concentrations of BNS3 extract (from 0.125 to 2.00 mg mL −1 ), dimethyl sulfoxide (DMSO) (1%) and non-treated cells were taken as negative and positive control, respectively. The neutral red assay of cells was performed after 24, 48, and 72 h, reported by Repetto [36]. The treated and control cells were incubated in 40 µg/mL neutral red in complete growth medium for 2 h and washed with phosphate buffer saline (BS). Picked up dye was released in a de-staining solution consisting of 1% glacial acetic acid, 49% distilled water, and 50% ethanol for 5 min at room temperature. The optical density was measured at 540 nm using a spectrophotometer and cell viability (%) by Equation (2).
whereas OD S represents Optical Density in treated samples, and OD C designates Optical Density in positive control.

SEM Sample Preparation
The images of the cultured cell on BNS3 were taken using SEM (JSM 6940A, Jeol, Tokyo, Japan) with a different period; 24 h, 48 h, 72 h, and 7 days. The cultured cells were washed with PBS and fixed by absolute ethanol for 7 min and kept at 4 • C. Before SEM analysis, the well dried samples were gold-sputtered and analyzed with 1 kV voltage, 7 × 10 −2 bar pressure and 20 mA deposition current for 2.0 min.

Statistical Analysis
The quantitative data were obtained in triplicate form and expressed as the mean ± standard deviation (SD). The statistical analysis conducted using statistical analysis system software (IBM SPSS Statistics 21, SPSS Inc., New York, NY, USA) and value of p < 0.05 and n = 3 taken as statistically significant.

Results and Discussion
The freeze-dried porous BNS samples have been prepared using n-HAp in the grafted natural polymer. The acrylic acid was grafted into β-glucan through the free-radical polymerization process and, n-HAp has been trapped into the polymeric matrix of grafted BG during the reaction. Figure 2 shows the spectral peaks at 1093 cm −1 are described triply degenerated P-O stretching [37]. Whereas, peaks at 603 and 569 cm −1 describes the bending mode of O-P-O. The absorption band in the region from 560 to 600 and from 1000 to 1100, cm −1 were attributed to the presence of calcium phosphate moiety of HAp [37,38]. Hence, presence of n-HAp has been confirmed by PO 4 −3 at 630 cm −1 into BNS [39][40][41]. The peak at 1220 cm −1 , 906 cm −1 , and 1033 cm −1 attributed to C-O cyclic, pyranose, and functional group of acrylic. These vibrations might due to the formation of covalent bond between BG and AAc [42]. The band at 1740 cm −1 corresponds to the stretching vibration of AAc carbonyl group. The peaks/bands between 1430 and 1450 cm −1 are the result of C-O stretching and C-O-H bending vibrations. Consequently, the presence of all these peaks/bands and the demise of BG's O-H bending vibration is confirmation that AAc grafted the BG polysaccharide on the O-H site. These peaks/bands confirm the grafting of AAc on the backbone of the polysaccharide [43]. The adsorption peak at 947 cm −1 is due to C-O stretching [44]. Stretching vibration at 412 cm −1 is the characteristic of silver and hydroxyl (OH − ) has a steric effect on coordination between oxygen (O) and Ag-particles, the electronegativity of oxygen is higher due to its donating ability [45]. The absorption bands from 3600 to 3100 cm −1 ( Figure 2) attribute to the hydroxyl stretching vibrations. The absorption at 2950-2850 cm −1 expresses the aliphatic C-H stretching vibration.

XRD
The XRD pattern is shown in Figure 3 for organic and inorganic phase analysis of the BNS. The low intensity of organic substances (BG and AAc) has exhibited at a characteristic peak near 2θ of 26° with reduced crystallinity [46]. This reduced crystallinity is due to the H-bond formation and chemical reaction of BG and AAc that results in reduced crystallinity of BG in BNS (observed in FTIR Spectra). The presence of the n-HAp into the polymeric matrix of grafted BG leads towards noncrystalline behavior of the polymeric region [47,48]. The observed sharp peaks of HAp are at an angle of 2ѳ of 25.3°, 31.4°, 46.6 o , 49.6°, 51.3°, and 61.3° (ICPDS-04-0783) with their corresponding diffraction planes of (213), (112), (222), (213), (140), and (304) as shown in Figure 3. The peak at 39.69° indicates the existence of Ag-coated with corresponding (102) diffraction plane which matched with standard card (ICPDS-04-932). Furthermore, the calculated cell parameters BNS are (a = b = 9.4000) and (c = 6.9300), which is perfectly match with standard data (PDF-4-932). The detailed calculated parameters of prepared samples shown in the Table 2 below.

XRD
The XRD pattern is shown in Figure 3 for organic and inorganic phase analysis of the BNS. The low intensity of organic substances (BG and AAc) has exhibited at a characteristic peak near 2θ of 26 • with reduced crystallinity [46]. This reduced crystallinity is due to the H-bond formation and chemical reaction of BG and AAc that results in reduced crystallinity of BG in BNS (observed in FTIR Spectra). The presence of the n-HAp into the polymeric matrix of grafted BG leads towards non-crystalline behavior of the polymeric region [47,48]. The observed sharp peaks of HAp are at an angle of 2

XRD
The XRD pattern is shown in Figure 3 for organic and inorganic phase analysis of the BNS. The low intensity of organic substances (BG and AAc) has exhibited at a characteristic peak near 2θ of 26° with reduced crystallinity [46]. This reduced crystallinity is due to the H-bond formation and chemical reaction of BG and AAc that results in reduced crystallinity of BG in BNS (observed in FTIR Spectra). The presence of the n-HAp into the polymeric matrix of grafted BG leads towards noncrystalline behavior of the polymeric region [47,48]. The observed sharp peaks of HAp are at an angle of 2ѳ of 25.3°, 31.4°, 46.6 o , 49.6°, 51.3°, and 61.3° (ICPDS-04-0783) with their corresponding diffraction planes of (213), (112), (222), (213), (140), and (304) as shown in Figure 3. The peak at 39.69° indicates the existence of Ag-coated with corresponding (102) diffraction plane which matched with standard card (ICPDS-04-932). Furthermore, the calculated cell parameters BNS are (a = b = 9.4000) and (c = 6.9300), which is perfectly match with standard data (PDF-4-932). The detailed calculated parameters of prepared samples shown in the Table 2 below.

SEM-EDX
The SEM analysis has been performed to investigate the pore size and surface morphology of BNS, as depicted in Figure 4. All of the BNS samples show porous and rough surface morphology. The irregular, non-smooth, and rough surface morphology help cell adhesion, while the porosity with appropriate pore size encourages cell proliferation and adhesion [49,50]. Additionally, the SEM profile has presented a different pore size to promote cell adhesion, proliferation, diffraction, and extracellular matrix secretion. The BNS almost have an almost uniform pore size from 83 ± 2 m to 175 ± 2 m, whereas, BNS3 had 108 ± 1 m with an average pore size suitable for osteointegration. However, 50-200 m is an appropriate pore size for osteoblast cell attachment, proliferation [51,52]. Hence, BNS2 with proper pore size has encouraged cell proliferation and cell culture (evident from Figure 9). The conducted elemental analysis of the bioactive scaffold confirmed the presence of HAp by available carbon (31.26%), oxygen (24.56%), calcium (14.18%), phosphorus (12.64%), and silver (8.99%) into BNS3 as shown Figure 4 The overall SEM-EDX results like: morphology, pore size,

XRD
The XRD pattern is shown in Figure 3 for organic and inorganic phase analysis of the BNS. Th low intensity of organic substances (BG and AAc) has exhibited at a characteristic peak near 2θ of 2 with reduced crystallinity [46]. This reduced crystallinity is due to the H-bond formation an chemical reaction of BG and AAc that results in reduced crystallinity of BG in BNS (observed in FTI Spectra). The presence of the n-HAp into the polymeric matrix of grafted BG leads towards no crystalline behavior of the polymeric region [47,48].

SEM-EDX
The SEM analysis has been performed to investigate the pore size and surface morphology of BNS, as depicted in Figure 4. All of the BNS samples show porous and rough surface morphology. The irregular, non-smooth, and rough surface morphology help cell adhesion, while the porosity with appropriate pore size encourages cell proliferation and adhesion [49,50]. Additionally, the SEM profile has presented a different pore size to promote cell adhesion, proliferation, diffraction, and extracellular matrix secretion. The BNS almost have an almost uniform pore size from 83 ± 2 µm to 175 ± 2 µm, whereas, BNS3 had 108 ± 1 µm with an average pore size suitable for osteointegration. However, 50-200 m is an appropriate pore size for osteoblast cell attachment, proliferation [51,52]. Hence, BNS2 with proper pore size has encouraged cell proliferation and cell culture (evident from Figure 9). The conducted elemental analysis of the bioactive scaffold confirmed the presence of HAp by available carbon (31.26%), oxygen (24.56%), calcium (14.18%), phosphorus (12.64%), and silver (8.99%) into BNS3 as shown Figure 4 The overall SEM-EDX results like: morphology, pore size, porosity, and available elements show that these scaffolds are helpful to regenerate and have vital role for bone tissue engineering [16].
Materials 2020, 13, x FOR PEER REVIEW 9 of 18 porosity, and available elements show that these scaffolds are helpful to regenerate and have vital role for bone tissue engineering [16].

Mechanical Testing
Mechanical strength of all BNS samples have been studied in dry and wet form as shown in Figure 5. Deionized water (H2O) was used as media used for wet analysis. The coating concentrations of AgNO3 have changed the mechanical feature of the BNS. It was found that increasing concentrations leads to enhance compressive strength. It was also found that mechanical strength of these scaffold samples and exhibited lower mechanical strength in wet environment due to release of Ag-particle into aqueous media [53,54]. Ag-coating has a vital role in improving the mechanical strength of BNS samples with suitable porosity for tissue engineering [55]. The polymeric matrix has a different chemical structure and impact over the grain boundary. Hence, Ag-coating has the capability of inducing functional properties like porosity, pore size, grain boundary, which also affects the mechanical strength [56].

Mechanical Testing
Mechanical strength of all BNS samples have been studied in dry and wet form as shown in Figure 5. Deionized water (H 2 O) was used as media used for wet analysis. The coating concentrations of AgNO 3 have changed the mechanical feature of the BNS. It was found that increasing concentrations leads to enhance compressive strength. It was also found that mechanical strength of these scaffold samples and exhibited lower mechanical strength in wet environment due to release of Ag-particle into aqueous media [53,54]. Ag-coating has a vital role in improving the mechanical strength of BNS samples with suitable porosity for tissue engineering [55]. The polymeric matrix has a different chemical structure and impact over the grain boundary. Hence, Ag-coating has the capability of inducing functional properties like porosity, pore size, grain boundary, which also affects the mechanical strength [56].

Swelling Analysis
The swelling behavior of scaffold has a vital role in biological activities like in vitro studies. The body fluid contains cell nutrients and metabolites that absorbs during swelling of scaffold. More is the swelling and more transfer of body fluids. The porosity, pore size, and surface area of scaffolds increases due to swelling that helps cell migration, adhesion, and proliferation [57,58]. Moreover, controlled swelling of scaffolds under physiological conditions strengthen the scaffolds structure with controlled degradation that plays a key role for bone regeneration [59].
The swelling effects of scaffolds were analyzed under various physiological conditions, such as temperatures, pH levels, and time intervals. Figure 6A,B shows that these scaffolds exhibited different rate of swelling in aqueous media and PBS solution at a temperature of 37 °C and pH of 7.4 and continued to swell until equilibrium. At these conditions, all scaffolds were showing different increasing rate of swelling. The highest and lowest swelling rate were observed for PNS1 and PNS4, respectively. The different swelling behavior of these scaffolds was due to coating of various silver concentrations that cause increase or decrease in ionic swelling pressure in both media [60]. Swelling rate of any material mainly depends upon the osmotic pressure. The increasing concentration of silver solution also responsible in decreasing rate of swelling [61]. The swelling of these scaffolds are different in aqueous media and PBS solution due to different hydrogen bonding, hydrophilic and hydrophobic interactions, and other forces of attraction that cause different osmotic pressure [62]. So, there is an inverse relation between silver concentration and rate of swelling.

Swelling Analysis
The swelling behavior of scaffold has a vital role in biological activities like in vitro studies. The body fluid contains cell nutrients and metabolites that absorbs during swelling of scaffold. More is the swelling and more transfer of body fluids. The porosity, pore size, and surface area of scaffolds increases due to swelling that helps cell migration, adhesion, and proliferation [57,58]. Moreover, controlled swelling of scaffolds under physiological conditions strengthen the scaffolds structure with controlled degradation that plays a key role for bone regeneration [59].
The swelling effects of scaffolds were analyzed under various physiological conditions, such as temperatures, pH levels, and time intervals. Figure 6A,B shows that these scaffolds exhibited different rate of swelling in aqueous media and PBS solution at a temperature of 37 • C and pH of 7.4 and continued to swell until equilibrium. At these conditions, all scaffolds were showing different increasing rate of swelling. The highest and lowest swelling rate were observed for PNS1 and PNS4, respectively. The different swelling behavior of these scaffolds was due to coating of various silver concentrations that cause increase or decrease in ionic swelling pressure in both media [60]. Swelling rate of any material mainly depends upon the osmotic pressure. The increasing concentration of silver solution also responsible in decreasing rate of swelling [61]. The swelling of these scaffolds are different in aqueous media and PBS solution due to different hydrogen bonding, hydrophilic and hydrophobic interactions, and other forces of attraction that cause different osmotic pressure [62]. So, there is an inverse relation between silver concentration and rate of swelling.

Anti-Microbial Activity
Antimicrobial activities of BNS samples (A) and individual materials (B) were studied using agar well-diffusion assay against E. coli DH5 alpha. It was found that silver has an important role in antimicrobial activities as shown in Figure 7A,B [63]. The anti-microbial activity of silver nanoparticles attributes was due to adsorption into the cell wall and interaction with cellular protein.
Denaturation of proteins was a result of sliver-nanoparticle permeability and accretion of an external fluid, owing to better antimicrobial activities of Ag-coated scaffolds [64]. The charged component of the bacterial surface membrane, i.e., phospholipids and lipopolysaccharides interacted with several available functional groups of BNS silver nanoparticles [65,66]. The active sites of hydrogels and nano-Ag endorsed their changes into the bacteria membrane, hindering the microbe's activity. The bacterial growth was also delayed due to the bonding between BG and bacterial DNA, thus leading to inhibition of bacterial transcription and translation. The molecular alteration in DNA due to Agcoated bioactive scaffolds resulted in little or no growth of bacteria. Hence, the sample BNS4 showed maximum antimicrobial activity that is due to higher concentration of Ag-coating over the scaffold.

Anti-Microbial Activity
Antimicrobial activities of BNS samples (A) and individual materials (B) were studied using agar well-diffusion assay against E. coli DH5 alpha. It was found that silver has an important role in antimicrobial activities as shown in Figure 7A,B [63]. The anti-microbial activity of silver nanoparticles attributes was due to adsorption into the cell wall and interaction with cellular protein. Denaturation of proteins was a result of sliver-nanoparticle permeability and accretion of an external fluid, owing to better antimicrobial activities of Ag-coated scaffolds [64]. The charged component of the bacterial surface membrane, i.e., phospholipids and lipopolysaccharides interacted with several available functional groups of BNS silver nanoparticles [65,66]. The active sites of hydrogels and nano-Ag endorsed their changes into the bacteria membrane, hindering the microbe's activity. The bacterial growth was also delayed due to the bonding between BG and bacterial DNA, thus leading to inhibition of bacterial transcription and translation. The molecular alteration in DNA due to Ag-coated bioactive scaffolds resulted in little or no growth of bacteria. Hence, the sample BNS4 showed maximum antimicrobial activity that is due to higher concentration of Ag-coating over the scaffold.

Anti-Microbial Activity
Antimicrobial activities of BNS samples (A) and individual materials (B) were studied using agar well-diffusion assay against E. coli DH5 alpha. It was found that silver has an important role in antimicrobial activities as shown in Figure 7A,B [63]. The anti-microbial activity of silver nanoparticles attributes was due to adsorption into the cell wall and interaction with cellular protein.
Denaturation of proteins was a result of sliver-nanoparticle permeability and accretion of an external fluid, owing to better antimicrobial activities of Ag-coated scaffolds [64]. The charged component of the bacterial surface membrane, i.e., phospholipids and lipopolysaccharides interacted with several available functional groups of BNS silver nanoparticles [65,66]. The active sites of hydrogels and nano-Ag endorsed their changes into the bacteria membrane, hindering the microbe's activity. The bacterial growth was also delayed due to the bonding between BG and bacterial DNA, thus leading to inhibition of bacterial transcription and translation. The molecular alteration in DNA due to Agcoated bioactive scaffolds resulted in little or no growth of bacteria. Hence, the sample BNS4 showed maximum antimicrobial activity that is due to higher concentration of Ag-coating over the scaffold.

Cytotoxicity
BNS3 has been selected due to its adequate porosity and compressive strength for biological activities. Figure 8 shows the cytotoxicity response of the bioactive nanocomposite scaffold against different concentrations (from 0.125 to 2.00 mg mL −1 ) of BNS3 extract against MC3T3-E1 cell line. Considering the control cell viability as 100% the cell viability of bioactive scaffolds was relatively calculated. The BNS3 has shown excellent cell viability with only 6-15% cell loss in the process.
Materials 2020, 13, x FOR PEER REVIEW 12 of 18 4.6.2. Cytotoxicity BNS3 has been selected due to its adequate porosity and compressive strength for biological activities. Figure 8 shows the cytotoxicity response of the bioactive nanocomposite scaffold against different concentrations (from 0.125 to 2.00 mg mL −1 ) of BNS3 extract against MC3T3-E1 cell line. Considering the control cell viability as 100% the cell viability of bioactive scaffolds was relatively calculated. The BNS3 has shown excellent cell viability with only 6-15% cell loss in the process.

Conclusions
We have fabricated Ag-coated BG/n-Hap bioactive nanocomposite scaffold via the freeze-dried method to mimic the functionalization of the extracellular matrix for bone tissue engineering. Agcoated scaffolds are mechanically robust and have sufficient compression strength near to the cancellous bone. Higher concentration of Ag will decrease the pore size but elevate the mechanical strength and anti-microbial activities of BNS samples. The biological studies have confirmed that BNS3 has potential for growth of the MC3T3-E1 cell line by maintaining their cell density without compromising the cell viability. Hence, the presented BNS samples have potential applications for bone tissue engineering.

Conclusions
We have fabricated Ag-coated BG/n-Hap bioactive nanocomposite scaffold via the freeze-dried method to mimic the functionalization of the extracellular matrix for bone tissue engineering. Ag-coated scaffolds are mechanically robust and have sufficient compression strength near to the cancellous bone. Higher concentration of Ag will decrease the pore size but elevate the mechanical strength and anti-microbial activities of BNS samples. The biological studies have confirmed that BNS3 has potential for growth of the MC3T3-E1 cell line by maintaining their cell density without compromising the cell viability. Hence, the presented BNS samples have potential applications for bone tissue engineering.