Development of Polymeric Nanocomposite (Xyloglucan-co-Methacrylic Acid/Hydroxyapatite/SiO2) Scaffold for Bone Tissue Engineering Applications—In-Vitro Antibacterial, Cytotoxicity and Cell Culture Evaluation

Advancement and innovation in bone regeneration, specifically polymeric composite scaffolds, are of high significance for the treatment of bone defects. Xyloglucan (XG) is a polysaccharide biopolymer having a wide variety of regenerative tissue therapeutic applications due to its biocompatibility, in-vitro degradation and cytocompatibility. Current research is focused on the fabrication of polymeric bioactive scaffolds by freeze drying method for nanocomposite materials. The nanocomposite materials have been synthesized from free radical polymerization using n-SiO2 and n-HAp XG and Methacrylic acid (MAAc). Functional group analysis, crystallinity and surface morphology were investigated by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD) and scanning electron microscopy (SEM) techniques, respectively. These bioactive polymeric scaffolds presented interconnected and well-organized porous morphology, controlled precisely by substantial ratios of n-SiO2. The swelling analysis was also performed in different media at varying temperatures (27, 37 and 47 °C) and the mechanical behavior of the dried scaffolds is also investigated. Antibacterial activities of these scaffolds were conducted against pathogenic gram-positive and gram-negative bacteria. Besides, the biological behavior of these scaffolds was evaluated by the Neutral Red dye assay against the MC3T3-E1 cell line. The scaffolds showed interesting properties for bone tissue engineering, including porosity with substantial mechanical strength, biodegradability, biocompatibility and cytocompatibility behavior. The reported polymeric bioactive scaffolds can be aspirant biomaterials for bone tissue engineering to regenerate defecated bone.

Nisbet et al. has used XG and functionalized it by positive charge molecules of poly(D-lysine), which showed an appropriate atmosphere for cell culture, migration and proliferation. Moreover, the thermo-responsive hydrogels have shown returns of spinal cord injuries treatments [18]. Shaw et al. prepared carboxymethyl tamarind gum-based films for skin tissue engineering by the phase-separation method. They found that enhanced proliferation of human keratinocytes than the control group. It was found that drug-loaded films showed good antimicrobial behavior against Escherichia coli and results showed that films are suitable as matrices for skin tissue engineering [19]. Yoganand and coworkers have synthesized glass-ceramics from natural bovine HAp/SiO 2 -CaO-MgO glass composites by the brisk process. They have performed biological studies on cultures of human fibroblast cells and found promoted adherence and growth [20]. K. Mediaswanti et al. have deposited SiO 2 /HAp onto a titanium and tantalum surface by electron beam evaporation and magnetron sputtering. They found that HAp/SiO 2 coated surface was less desirable results of bacteria adherence than non-coated surface [21].
Hence, considering the physicochemical and biomechanical behavior of biomaterials, the objectives of this article are to prepare, characterize and biological analysis of polymeric bioactive scaffolds (PBSs). Nanoparticles of SiO 2 (n-SiO 2 , auxiliary component) and nanoparticles of HAp (n-HAp, supplementary constituent) lodged into a grafted biopolymeric network during free-radical polymerization of XG with methacrylic acid (MAAc). According to our best knowledge, the methodology of polymeric bioactive scaffolds has never reported up till now. n-SiO 2 doped polymeric bioactive scaffolds have a porous, large surface and biocompatible properties for osteogenesis. The structure and morphology of polymeric bioactive scaffolds were analyzed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET). The mechanical behavior and swelling properties were analyzed using the universal testing machine (UTM) in water and phosphate buffer saline (PBS) solution, respectively. MC3T3-E1 cell line was employed to study the biological behavior of polymeric bioactive scaffolds in vitro. The results of our study revealed, bioactive scaffolds as potential biomaterials to regenerate and repair defective bone in tissue engineering. Figure 1 illustrates the schematic diagram of the reported studies.
Polymers 2019, 11, x FOR PEER REVIEW 3 of 17 thermo-responsive hydrogels have shown returns of spinal cord injuries treatments [18]. Shaw et al. prepared carboxymethyl tamarind gum-based films for skin tissue engineering by the phaseseparation method. They found that enhanced proliferation of human keratinocytes than the control group. It was found that drug-loaded films showed good antimicrobial behavior against Escherichia coli and results showed that films are suitable as matrices for skin tissue engineering [19]. Yoganand and coworkers have synthesized glass-ceramics from natural bovine HAp/SiO2-CaO-MgO glass composites by the brisk process. They have performed biological studies on cultures of human fibroblast cells and found promoted adherence and growth [20]. K. Mediaswanti et al. have deposited SiO2/HAp onto a titanium and tantalum surface by electron beam evaporation and magnetron sputtering. They found that HAp/SiO2 coated surface was less desirable results of bacteria adherence than non-coated surface [21]. Hence, considering the physicochemical and biomechanical behavior of biomaterials, the objectives of this article are to prepare, characterize and biological analysis of polymeric bioactive scaffolds (PBSs). Nanoparticles of SiO2 (n-SiO2, auxiliary component) and nanoparticles of HAp (n-HAp, supplementary constituent) lodged into a grafted biopolymeric network during free-radical polymerization of XG with methacrylic acid (MAAc). According to our best knowledge, the methodology of polymeric bioactive scaffolds has never reported up till now. n-SiO2 doped polymeric bioactive scaffolds have a porous, large surface and biocompatible properties for osteogenesis. The structure and morphology of polymeric bioactive scaffolds were analyzed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction analysis (XRD), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET). The mechanical behavior and swelling properties were analyzed using the universal testing machine (UTM) in water and phosphate buffer saline (PBS) solution, respectively. MC3T3-E1 cell line was employed to study the biological behavior of polymeric bioactive scaffolds in vitro. The results of our study revealed, bioactive scaffolds as potential biomaterials to regenerate and repair defective bone in tissue engineering. Figure 1 illustrates the schematic diagram of the reported studies.

Materials
XG was purchased from DSP Gokyo Food and Chemical Co. Ltd., Tokyo, Japan. MAAc and N,N'-methylene-bis-acrylamide (N, N-MBA) as a crosslinker, n-HAp (<100 nm particle size) and n-SiO 2 (10-20 nm particle size) in powder forms were purchased from Sigma-Aldrich, Selangor, Malaysia. Analytical graded Na 2 HPO 4 , NaCl, KCl, K 2 HPO 4 and HCl were obtained from Merck Darmstadt, Germany for the preparation of buffer saline solution. All chemicals were used without any purification.

Polymeric Bioactive Scaffolds Synthesis
Synthesis of nanocomposite to fabricate polymeric bioactive scaffolds, 2 g XG in deionized-water and shifted in round bottom two-neck flask. The reaction media temperature was held at 60 • C with constant stirring under N 2 atmosphere and 0,05 g potassium persulfate was used as an initiator. After 20 min, 0.40 mL MAAc and N, N-MBA (0.05% of MAAc) were poured into the reaction media. After 45 min, different quantities of n-SiO 2 (0.25, 0.50, 0.75 and 1.0 g) and coded as PBS1, PBS2, PBS3 and PBS4, respectively. Then, n-Hap (2 g) powders were added gradually with continuous stirring for 3 h. Hence, MAAc was grafted into XG using a free-radical polymerization method. As a result, n-SiO 2 and n-HAp were incorporated into the XG-graft-MAAc polymeric network. On completion, the reaction was halted and the gas flow was removed to cool the reaction media. The residual suspension was separated by vacuum filtration from the reactive mixture. The residues were washed with excessive deionized water, then dried overnight in the oven at 55 • C to obtain the nanocomposite powder. Then the composite powder was prepared with different compositions of n-SiO 2 , as summarized in Table 1. The polymeric nanocomposite powder (0.5 g) was homogeneously mixed in deionized water to form a uniform paste, filled in cylindrical molds (1.5 mm × 6 mm) and kept at −80 • C for 24 h. The cylindrical polymeric bioactive scaffolds were obtained through the freeze-drying method with no significant volume reduction or deformation. The porous morphology of the polymeric bioactive scaffolds was observed through Brunauer-Emmett-Teller (Micromeritics Gemini II 2370, Micromeritics, Norcross, GA, USA).

Fourier Transform Infrared Spectroscopy
Functional groups of the polymeric bioactive scaffolds were investigated through IR Prestige-21, Shimadzu (Kyoto, Japan) with a frequency range of 4000−400 cm −1 and resolution of 4.0 cm −1 with 200 scans average per spectrum.

Scanning Electron Microscope/Energy Dispersive Spectroscopy
Morphologies of the polymeric bioactive scaffolds have been analyzed by scanning electron microscope (SEM, JEOL-JSM-6480, Akishima, Japan) coupled with energy dispersive spectroscopy (EDS) for elemental quantitative analysis. Cross-sectional pieces of the scaffolds were sliced and placed on a stub and located into the vacuum chamber to collect images at different magnifications.

Mechanical Testing
A universal testing machine (UTM), (Testometrics, UK) was employed for mechanical testing with a loading rate of 5 mm/min. Ultimate compression strength (UCS) of the samples was determined using triplicate.

Swelling Test
Dried specimens of scaffolds were weighted (W D ) at pH 7.4 and 27, 37 and 47 • C, swelling behavior of dried specimens of scaffolds was conducted in deionized H 2 O and PBS solution. PBS solution was prepared in the laboratory by standard method. The pH of the solution was maintained at 7.4 by adding hydrochloric acid (0.1 mol L −1 ) dropwise and raised the volume of PBS solution to 1 L by adding deionized water. Then, scaffolds specimens were soaked and liquid-immersed into pores until equilibrium reached. The swelled specimens of scaffolds took out from liquid and removed the excess surface water to determine their weights (W S ). The swelling (%) of the scaffolds was evaluated using Equation (1).
Whereas, W S = scaffold weight, W D = Weight of dry scaffold

In Vitro Studies
In vitro studies were conducted from the extracts of polymeric bioactive scaffolds to investigate antibacterial activities and biological activities against various bacterium (E. coli, S. aureus and P. Aeruginosa) and mouse pre-osteoblast (MC3T3-E1) cell lines.

Antibacterial Activities
The antibacterial activities of all samples were studied by the disc diffusion method against gram-negative and gram-positive model bacterial strains (E. coli, S. aureus and P. Aeruginosa). These bacterial strains were incubated at 37 • C to study antibacterial activities of all polymeric bioactive scaffolds. Hot molten (15 mL) of agar was poured into three sterile Petri-plates and left them for solidification. After that, these bacterial cultures were spread uniformly using sterile cotton swab over solidified agar [22]. 85 µL of each scaffold extract was put over bacterial cultured plates and incubated into the oven for 24 h at 37 • C.

Extract of Scaffold Preparation
All polymeric bioactive scaffolds exhibited characteristics like porosity, swelling and mechanical properties. Five different concentrations (0.125-2.00 mg mL −1 ) were prepared from all samples of polymeric bioactive scaffolds in liquid to evaluate cell viability. The bottoms of 24 well plates were fine coated for all samples in triplicate and sterilized under UV-light for 1 h.

Cell Culture and Morphological Analysis
Mouse pre-osteoblast (MC3T3-E1) cell line obtained from ATCC (American Type Culture Collection) was kept in α-MEM (without ascorbic acid Gibco™ A10490-9, New York, NY, USA) with 10% FBS (Fetal Bovine Serum Gibco™ 12662011, New York, NY, USA) and 100 U/mL Penicillin/Streptomycin solution (Gibco™ 15140122, New York, NY, USA). To conduct microscopy-based morphological analysis, all wells of 24 well plates were seeded with approximately 5000 cells per cm 2 for five different concentrations using the same media in triplicate and incubated at 37 • C for 72 h in 5% CO 2 with 90% humidity. The Nikon Eclipse TS100 inverted microscope (Nikon Instruments Inc, New York, NY, USA) was employed to observe cell morphology and cell viability assay for 72 h changes.

Cell Cultural and Morphological Studies
Cell culture images of the polymeric bioactive scaffold were captured using SEM (JSM 6940A, Jeol, Tokyo, Japan). At room temperature, the attached cells were washed with PBS solution using absolute ethanol for 7 min. Then, samples were gold-sputtered and analyzed under 1 kV voltage, 7 × 10 −2 bar pressure and 20 mA deposition current for 2.0 min.

Cell Culture Viability
Mouse pre-osteoblast (MC3T3-E1) cell line obtained from ATCC was kept in α-MEM (without ascorbic acid Gibco™ A10490-9, New York, NY, USA) with 10% FBS (Fetal Bovine Serum Gibco™ 12662011, New York, NY, USA) and 100 U/mL Penicillin/Streptomycin solution (Gibco™ 15140122, New York, NY, USA). Microscopic based cell morphology was observed. Approximately 5,000 cells per cm 2 were seeded into wells of 12 well plates with five different concentrations of PBS3 and incubated at 37 • C for 72 h in 5% CO 2 with 95% humidity. Gelatin coating is frequently used to enhance surface cell adherence. Herein, gelatin with a concentration of 0.1% was used as a positive control. The cells were seeded with five different concentrations (0.125-2.00 mg mL −1 ) with 1% dimethyl sulfoxide (DMSO) as a negative control and non-treated cells as a positive control. Cells were incubated for 24, 48, 72 h and the Neutral Red uptake assay was performed reported by Repetto et al. [23]. Every day, three wells for each concentration and three for negative control were taken out. The treated and control cells were incubated in neutral red medium (40 µgmL −1 Neutral Red medium) for 2 h. Then, the incubated cells were washed with an excessive amount of PBS solution to remove the Neutral Red medium and all samples were then transferred to a de-staining solution (1% glacial acetic acid 49% distilled water and 50% ethanol) for 5 min at room temperature. The spectrophotometer was used to measure optical density at 540 nm and cell viability (%) was determined by Equation (2).
where OD S and OD C for sample concentration and OD C are OD for positive control having untreated cells.

Statistical Analysis
Experimental data was conducted in triplicate form and presented with mean standard errors (S.E). The statistical analysis was calculated using statistical analysis system software (IBM, SPSS Statistics 21). The means and standard errors of means (mean ± S.E) were calculated for every analysis and S.E values have displayed as Y-error bars in Figures. The error bars displayed standard deviations (p < 0.05 (5 %); size of the sample n = 3).

Results and Discussion
Polymeric bioactive scaffolds were produced using the freeze-drying method. The doped n-HAp and n-SiO 2 were accompanied through a free-radical polymerization process in a grafted polymer matrix of XG and MAAc. Figure 2 indicates the relationships between the materials and possible chemical reactions.  Figure 2. shows the proposed chemical interactions among XG, MAAc, N, N-MBA, n-HAp and n-SiO2. Stretching vibrations at 1220 cm −1 ,1033 cm −1 and a weak peak at 906 cm −1 are due to CO cyclic, acyclic and pyranose, respectively and it might be due to formation of the covalent bond between XG and MAAc as shown in Figure 2 [24]. FTIR spectra represent specific functional groups in polymeric bioactive scaffolds. Bands from 3600 to 3100 cm −1 (Figure 3) are due to hydroxyl stretching vibrations. The band absorption at 29502850 cm −1 shows the aliphatic CH stretching vibration. The peak features at 1093 cm −1 are due to triply degenerated PO stretching components, while peaks at 603 and 569 cm −1 are attributed to OPO bending mode. Hence, FTIR spectra present the characteristic bands in regions 560-600 cm −1 and 1000-1100 cm −1 are assigned to calcium phosphate moiety of n-HAp [25]. Furthermore, the absorption band at 630 cm −1 confirms the presence of n-HAp in all the fabricated polymeric bioactive scaffolds [26,27]. The 947 cm −1 band defines the formation of SiO hydrogen-bonding with XG hydroxyl groups. Rising peak intensity from 947 to 921 cm −1 was observed as n-SiO2 increased. The H-bond formation between the oxygen atom of n-SiO2 and hydrogen atoms of the hydroxyl group of XG is presented in Figure 3. The characteristic peak is shown by n-SiO2 from 600 to 800 cm −1 presenting the stretching vibration of SiOSi for polymeric bioactive scaffolds [28]. Analysis of the FT-IR spectral profile confirms the successful synthesis of nanocomposite for the development of bioactive scaffolds.  Figure 2 shows the proposed chemical interactions among XG, MAAc, N, N-MBA, n-HAp and n-SiO 2 . Stretching vibrations at 1220 cm −1 ,1033 cm −1 and a weak peak at 906 cm −1 are due to C-O cyclic, acyclic and pyranose, respectively and it might be due to formation of the covalent bond between XG and MAAc as shown in Figure 2 [24]. FTIR spectra represent specific functional groups in polymeric bioactive scaffolds. Bands from 3600 to 3100 cm −1 (Figure 3) are due to hydroxyl stretching vibrations. The band absorption at 2950-2850 cm −1 shows the aliphatic C-H stretching vibration. The peak features at 1093 cm −1 are due to triply degenerated P-O stretching components, while peaks at 603 and 569 cm −1 are attributed to O-P-O bending mode. Hence, FTIR spectra present the characteristic bands in regions 560-600 cm −1 and 1000-1100 cm −1 are assigned to calcium phosphate moiety of n-HAp [25]. Furthermore, the absorption band at 630 cm −1 confirms the presence of n-HAp in all the fabricated polymeric bioactive scaffolds [26,27]. The 947 cm −1 band defines the formation of Si-O hydrogen-bonding with XG hydroxyl groups. Rising peak intensity from 947 to 921 cm −1 was observed as n-SiO 2 increased. The H-bond formation between the oxygen atom of n-SiO 2 and hydrogen atoms of the hydroxyl group of XG is presented in Figure 3. The characteristic peak is shown by n-SiO 2 from 600 to 800 cm −1 presenting the stretching vibration of Si-O-Si for polymeric bioactive scaffolds [28]. Analysis of the FT-IR spectral profile confirms the successful synthesis of nanocomposite for the development of bioactive scaffolds.  Figure 4 illustrates the compression strength of bioactive scaffolds samples in our study. It is highly challenging but important to maintain the mechanical strength of our scaffolds during cell growth and proliferation. It not only physically safeguards the cells but also provides compatible mechanical strength to mimic in vivo environments that have stronger influences over cell proliferation and differentiation. The ceramic phase (n-HAp and n-SiO2) has an extraordinary role to increase biomechanical features of several polymeric materials [29,30]. The incorporation of n-SiO2 increased the compression strength of the polymeric bioactive scaffolds (UCS of PBS1 = 2.4 MPa, PBS2 = 3.2 MPa, PBS3 = 5.7 MPa, PBS4 = 6.9 MPa). The sample PBS3 has better efficacy to bear the load with suitable porosity levels that eventually increased the mechanical properties of the fabricated polymeric bioactive scaffold. All polymers have different chemical structures that affect the grain boundary of the matrix. The properties of ceramic material (n-HAp and n-SiO2) is a function of pore size, porosity and grain boundary that is decreased with decreasing in grain size.   Figure 4 illustrates the compression strength of bioactive scaffolds samples in our study. It is highly challenging but important to maintain the mechanical strength of our scaffolds during cell growth and proliferation. It not only physically safeguards the cells but also provides compatible mechanical strength to mimic in vivo environments that have stronger influences over cell proliferation and differentiation. The ceramic phase (n-HAp and n-SiO 2 ) has an extraordinary role to increase biomechanical features of several polymeric materials [29,30]. The incorporation of n-SiO 2 increased the compression strength of the polymeric bioactive scaffolds (UCS of PBS1 = 2.4 MPa, PBS2 = 3.2 MPa, PBS3 = 5.7 MPa, PBS4 = 6.9 MPa). The sample PBS3 has better efficacy to bear the load with suitable porosity levels that eventually increased the mechanical properties of the fabricated polymeric bioactive scaffold. All polymers have different chemical structures that affect the grain boundary of the matrix. The properties of ceramic material (n-HAp and n-SiO 2 ) is a function of pore size, porosity and grain boundary that is decreased with decreasing in grain size.   Figure 4 illustrates the compression strength of bioactive scaffolds samples in our study. It is highly challenging but important to maintain the mechanical strength of our scaffolds during cell growth and proliferation. It not only physically safeguards the cells but also provides compatible mechanical strength to mimic in vivo environments that have stronger influences over cell proliferation and differentiation. The ceramic phase (n-HAp and n-SiO2) has an extraordinary role to increase biomechanical features of several polymeric materials [29,30]. The incorporation of n-SiO2 increased the compression strength of the polymeric bioactive scaffolds (UCS of PBS1 = 2.4 MPa, PBS2 = 3.2 MPa, PBS3 = 5.7 MPa, PBS4 = 6.9 MPa). The sample PBS3 has better efficacy to bear the load with suitable porosity levels that eventually increased the mechanical properties of the fabricated polymeric bioactive scaffold. All polymers have different chemical structures that affect the grain boundary of the matrix. The properties of ceramic material (n-HAp and n-SiO2) is a function of pore size, porosity and grain boundary that is decreased with decreasing in grain size.   Figure 5 discusses the swelling analysis of polymeric bioactive scaffolds in deionized-water and phosphorous buffer saline solution. Identical amounts of scaffolds were immersed in deionized water and PBS solution. The polymer bioactive scaffolds are found to have diverse swelling patterns in different media at different temperatures, because of different n-SiO 2 concentrations. Osmotic pressure may increase or decrease because of the hydrogen bonding (H-bonding) among the scaffold and media at varying temperatures. The swelling rate decreased and osmotic forces are balanced at equilibrium after increasing the time. The extraordinary difference in water absorption is not observed with time until it has reached a plateau [31]. Initially, more water molecules interacted with porous scaffolds and decreased when the equilibrium point is near. Sample (PBS1) with the maximum amount of n-SiO 2 has maximum swelling in water media at 47 • C. Whereas, sample (PBS4) with a little amount of silica at 27 • C has the least swelling. It is obvious ( Figure 5) that swelling has an inverse relationship to the ceramic quantity within the polymeric network. Due to the relatively hydrophobic nature of ceramic materials in XG-graft-MAAc and ceramic materials, the different swelling behaviors of these bioactive scaffolds can react as a crosslinker. As a consequence, by increasing ceramic quantities into a polymeric network, which generates additional crosslink points in polymer networking and decreases elasticity which decreases swelling behavior.

SEM-EDX
The surface morphology of the bioactive polymeric scaffolds was examined using SEM as shown in Figure 6. The various magnifications exhibit rough and porous topography of polymeric bioactive scaffolds (evident from the XRD results), which remarkably encourage cell adhesion and proliferation on the scaffold [32,33]. At higher magnification, however, the SEM images show a specific porous Figure 5. The swelling analysis was carried out at different temperatures (27,37 and 47 • C) and found that not only is the quantity of silica responsive to swelling changes but the increasing temperature is also responsible for increased swelling. (A) In deionized-water (B) In phosphorous buffer saline solution.

SEM-EDX
The surface morphology of the bioactive polymeric scaffolds was examined using SEM as shown in Figure 6. The various magnifications exhibit rough and porous topography of polymeric bioactive scaffolds (evident from the XRD results), which remarkably encourage cell adhesion and proliferation on the scaffold [32,33]. At higher magnification, however, the SEM images show a specific porous morphology which also supports cell infiltration, adhesion and extracellular matrix secretion. The bioactive polymeric scaffolds show different pore sizes distributed uniformly through the samples. The average pore size of PBS3 is 80-140 µm, which is ideal for osteointegration and 50-200 µm is optimal for cell attachment, proliferation and osteoblast cells. The PBS3 sample has a uniform distribution of pore size in the structure and is ideal for significant cell proliferation demonstrating excellent cell growth capability. [34]. During the SEM analysis, n-SiO 2 was found to have imparted its role in porosity, as the increasing quantity of n-SiO 2 caused increased pores and porosity. The PBS3 sample was chosen because of the uniform pore size used for chemical composition analysis with EDX [33]. EDX study reveals the different amounts of elements present in sample PBS3. Results show the presence in the porous bioactive scaffold of carbon (C), oxygen (O), silica (Si), calcium (Ca) and phosphorus (P) elements ( Figure 6), which help grafted-XG in manufactured bioactive polymer scaffolds.

In-vitro Study
The polymeric bioactive scaffolds samples were selected for in-vitro analysis due to their optimum characteristics. 4.5.1. Antibacterial Activities

In-Vitro Study
The polymeric bioactive scaffolds samples were selected for in-vitro analysis due to their optimum characteristics.

Antibacterial Activities
Antibacterial activities of scaffolds were performed via agar disc-diffusion assay against various sever pathogens (E. coli, S. aureus and P. aeruginosa) and zone of inhibition was measured as seen in Figure 7 [35]. Antibacterial activity of scaffolds is due to the penetration of silica and hydroxyapatite nano-particles into bacteria to interact with the cellular protein. The denatured proteins were caused by silica and hydroxyapatite nanoparticles and by the accretion of external fluids to give silica/hydroxyapatite scaffolds better antibacterial activities. The charged bacterial surface membrane (phospholipids and lipopolysaccharides) interacts with several functional groups of bioactive polymeric scaffolds [36,37]. These active sites of bioactive polymeric scaffolds allowed their changes into the bacterial membrane to obstruct bacterial activity. Bacterial growth is overdue due to the interaction between the polymeric portion of scaffold and bacterial DNA. Molecular alteration in DNA because of polymeric and SiO 2 /HAp nanoparticles of bioactive polymeric scaffolds that caused little to no bacterial growth. All samples of polymeric bioactive scaffolds, therefore, exhibited antibacterial activity due to components of polymeric bioactive scaffolds and also increasing quantities of silica nanoparticles [38]. fluids to give silica/hydroxyapatite scaffolds better antibacterial activities. The charged bacterial surface membrane (phospholipids and lipopolysaccharides) interacts with several functional groups of bioactive polymeric scaffolds [36,37]. These active sites of bioactive polymeric scaffolds allowed their changes into the bacterial membrane to obstruct bacterial activity. Bacterial growth is overdue due to the interaction between the polymeric portion of scaffold and bacterial DNA. Molecular alteration in DNA because of polymeric and SiO2/HAp nanoparticles of bioactive polymeric scaffolds that caused little to no bacterial growth. All samples of polymeric bioactive scaffolds, therefore, exhibited antibacterial activity due to components of polymeric bioactive scaffolds and also increasing quantities of silica nanoparticles [38].

Cytotoxicity
All scaffolds were incubated into culture medium at 37 • C for 24, 48 and 72 h. Culture media contained 90% DMEM glucose followed by 2 mmol L −1 glutamine, 10% FBS and 1% penicillin. Attached cells demonstrate ideal MC3T3-E1 cell line morphology for all samples of polymeric bioactive scaffolds, where cells are cylindrical with proper cell bodies. Cells attached to the well-bottom are comparatively less retained morphology as compared to control [33,39]. Figure 8, on the contrary, detects a particular pattern. PBS3 is found to have become more supportive of cells as it has added a large number of cells to the bottom of the well plate without any deformation of cell morphology compared to +ve-control. Whereas PBS1 has the least growth in cells compared to PBS3 because of an increasing amount of the n-SiO 2 . The increasing amount of silica makes scaffolds more biocompatible and increases the growth of MC3T3-E1 over time. In comparison, the negative control depicted smaller or no cells growth. It is therefore evident from the results that grafted XG-based biomaterials strengthened osteoblast cell attachment by increasing the n-SiO 2 content.

Neutral Red Assay
Cytotoxicity assay was carried out using the pre-osteoblast mouse (MC3T3-E1) cells by rising against all scaffolds. Cell viability was evaluated for each concentration of all samples and recorded at different time intervals using Neutral red uptake assay by incubation (24,48 and 72 h) [31,33]. As the amount of n-SiO 2 increases, polymeric bioactive scaffolds show greater cell viability and non-toxicity against pre-osteoblast mouse (MC3T3-E1) cells (Figure 9). PBS3 has no or less toxic cell viability that promotes pre-osteoblast (MC3T3-E1) cell differentiation to help the bone formation and plays an important role in tissue engineering for bone regeneration. at different time intervals using Neutral red uptake assay by incubation (24,48 and 72 h) [31,33]. As the amount of n-SiO2 increases, polymeric bioactive scaffolds show greater cell viability and nontoxicity against pre-osteoblast mouse (MC3T3-E1) cells (Figure 9). PBS3 has no or less toxic cell viability that promotes pre-osteoblast (MC3T3-E1) cell differentiation to help the bone formation and plays an important role in tissue engineering for bone regeneration. The PBS3 sample was selected for SEM analysis because of optimal swelling, mechanical, porosity, pore size, antibacterial and biocompatibility. Adherence of MC3T3-E1 pre-osteoblast cells to PBS3 was analyzed using SEM ( Figure 8) and presented via micrographs (A) PBS3, (B) Positive control (C) after 48 h, 72 h and 7 days of cultivation, respectively [31,33]. After specific incubation time (48 h, 72 h and 7 days), the pre-osteoblast MC3T3-E1 cells responded to PBS3 by exhibiting bioactive and unique cell attachment, which spread all over the surface ( Figure 10). Hence, the PBS3 was found to good in cell adherence and proliferation [33,39].

SEM Analysis of Cell Culture
The PBS3 sample was selected for SEM analysis because of optimal swelling, mechanical, porosity, pore size, antibacterial and biocompatibility. Adherence of MC3T3-E1 pre-osteoblast cells to PBS3 was analyzed using SEM ( Figure 8) and presented via micrographs (A) PBS3, (B) Positive control (C) after 48 h, 72 h and 7 days of cultivation, respectively [31,33]. After specific incubation time (48 h, 72 h and 7 days), the pre-osteoblast MC3T3-E1 cells responded to PBS3 by exhibiting bioactive and unique cell attachment, which spread all over the surface ( Figure 10). Hence, the PBS3 was found to good in cell adherence and proliferation [33,39].

Conclusions
Polysaccharides are prominent polymeric biomaterials for many biomedical applications. We have used XG to fabricate polymeric bioactive scaffolds for bone tissue engineering applications. The different amounts of n-SiO2 have different porosity, pore size, swelling and biomechanical behavior of these polymeric bioactive scaffolds for support and growth bones. The ultimate compression strength of PBS3 is ~5.7 MPa, which is closer to cancellous bone strength. PBS3 polymeric bioactive scaffold exhibited adequate porosity (86.5% ± 1%) with a pore size (105 ± 2 µm) that is essential for osteogenesis. PBS3 was found to be antibacterial against several infections causing pathogens. PBS3 is a more biologically active scaffold that encouraged osteoblast cell line growth with adequate cell viability and cell growth. The overall outcome has verified that polymeric bioactive scaffolds are potential biomaterial for bone tissue engineering.

Limitations
The increasing amount of ceramic contents (n-SiO2) changes behavior from polymeric to amorphous that also changed the physicochemical properties of the scaffolds. Further increasing amount of n-SiO2 causes a bigger pore size that fails mechanical properties that may not support these scaffolds mechanically. Since we are investigating other possible material to increase more mechanical properties by enhancing physicochemical and biological properties. Funding: This research has received financial support from Universiti Teknologi Malaysia research grants (15J82, 01M48 and 08G00). The authors also acknowledge the support from Innotech Horizon Sdn. Bhd., Malaysia for providing characterizing facilities.

Conflicts of Interest:
The authors have declared no conflict of interest.

Conclusions
Polysaccharides are prominent polymeric biomaterials for many biomedical applications. We have used XG to fabricate polymeric bioactive scaffolds for bone tissue engineering applications. The different amounts of n-SiO 2 have different porosity, pore size, swelling and biomechanical behavior of these polymeric bioactive scaffolds for support and growth bones. The ultimate compression strength of PBS3 is~5.7 MPa, which is closer to cancellous bone strength. PBS3 polymeric bioactive scaffold exhibited adequate porosity (86.5% ± 1%) with a pore size (105 ± 2 µm) that is essential for osteogenesis. PBS3 was found to be antibacterial against several infections causing pathogens. PBS3 is a more biologically active scaffold that encouraged osteoblast cell line growth with adequate cell viability and cell growth. The overall outcome has verified that polymeric bioactive scaffolds are potential biomaterial for bone tissue engineering.

Limitations
The increasing amount of ceramic contents (n-SiO 2 ) changes behavior from polymeric to amorphous that also changed the physicochemical properties of the scaffolds. Further increasing amount of n-SiO 2 causes a bigger pore size that fails mechanical properties that may not support these scaffolds mechanically. Since we are investigating other possible material to increase more mechanical properties by enhancing physicochemical and biological properties.