Cellulose Acetate-Hydroxyapatite-Bioglass-Zirconia Nanocomposite Particles as Potential Biomaterial: Synthesis, Characterization, and Biological Properties for Bone Application

 

Nuha Al-Harbi,1, 2, Mahmoud Ali Hussein,3, 4 Yas Al-Hadeethi1* and Ahmad Umar5, 6*

 

1 Department of Physics, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia.

2 Department of Physics, Umm AL-Qura University, Makkah, Kingdom of Saudi Arabia.

3 Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Kingdom of Saudi Arabia.

4 Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt.

5 Department of Chemistry, Faculty of Science and Arts, Najran University, Najran-11001, Kingdom of Saudi Arabia.

6 Promising Centre for Sensors and Electronic Devices (PCSED), Najran University, Najran-11001, Kingdom of Saudi Arabia.

*Email: ahmadumar786@gmail.com (Prof. Ahmad Umar);

yalhadeethi@kau.edu.sa (Prof. Yas Al-Hadeethi)

 

Abstract

 

Biopolymer nanocomposites based cellulose acetate, hydroxyapatite, bioglass and zirconia have been synthesized using a solvent casting method for biological interest. Different concentrations of zirconia have been prepared and characterized. The results clarified that the increase in the concentration of ZrO2 improves the mechanical properties as the microhardness becomes 405.5 MPa with 11.76 wt% of ZrO2 instead of 69.4 MPa with no ZrO2 content. Additionally, introducing ZrO2 into the nanocomposite improve its wettability as contact angle is decreased from 65 for the pure sample to 38.4 for the composite with 1.3 wt% ZrO2. Moreover, the agar diffusion antimicrobial study showed that only sample with 3.22 wt% of ZrO2 nanocomposite has mild inhibitory responses against Pseudomonas aeruginosa, whereas the rest of the formula does not have any antibacterial activity. Furthermore, in-vitro cytotoxicity of the nanocomposite samples on the Vero cell line was also studied. These Vero normal cells were incubated with test materials for 72h at 37C/ 5% CO2, and cell viability was detected using the sulforhodamine B (SRB) assay. All Nanocomposites were mildly to non-cytotoxic to Vero cells with high concentration compared with inhibitory effect of doxorubicin, which was added with 10-fold lower than nanocomposites. With these findings, the proposed nanocomposite could be used in dental applications.

 

Table of Contents

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Keywords: Zirconia; Biopolymer nanocomposite; Mechanical properties; Antimicrobial Activity; Cytotoxicity.

 

 


1. Introduction

Bone problems due to trauma, illness, or surgery are a significant major issue with an increasing global population. In the coming years, regenerative medicine and the rising use of dental implants and replacements are projected to hit tremendous levels and could reach 20% of the elderly by 2050.[1] A variety of criteria must be fulfilled for the material utilized as a bone scaffold,[2] including biocompatibility,[3] biodegradation with vanishingly small toxicity,[4] sufficient porosity and mechanical characteristics, and the capacity to be incorporated into biological molecules or tissue regeneration.[57]

Bioactive glass (BG) is commonly considered an inorganic filler or additive in biopolymer composites.[8] Therefore, BGs have excellent surface area resulting in higher rate of dissolution and, as a result, faster apatite creation.[9] Subsequently, the mechanical characteristics of these composites for natural bones are shown to increase as well as provide biomimetic nano-structuring that enhances cell adhesion. The bioactive properties of glass are highly influenced by the composition and shape of glass as well as synthesis methods and the dissolutive rate of ionic dissolution.[10] Due to poor mechanical properties, as being very brittle, glass structure is not suitable for manufacturing porous scaffolds. Therefore, to enhance their mechanical properties, there is a need for adding BG with biopolymer and metal oxides.[11] Skallevold et al.,[10] reported the use of BG in different dental applications such as bone grafting, enamel re-mineralization, dentin hypersensitivity, air abrasion, restorative materials, pulp capping and root canal therapy, bone regeneration, in periodontics, and in implant dentistry.

Moreover, nanoparticles have been used in the creation of regenerative composite structures, such as the inclusion of materials containing nano metal oxides. In addition, dental adhesives were also integrated with nanoparticles.[12,13] The nanocomposites are made up of composites containing nanometric dimensional filler particles (less than 100 nm).[14] In addition, several experimental studies have shown that nanoparticles dispersion plays an important role in enhancing the physical particularly the mechanical properties of composites.

Furthermore, Cellulose acetate (CA) and hydroxyapatite (HAP) composite has been hypothesized to be a promising bioactive covering substance used throughout the long-term biomedical implants.[15] CA is a renewable material that, as a biodegradable, non-corrosive, non-toxic, and biocompatible material, is gradually drawing researchers' attention due to its potential benefits. It is usually used as a dispersing agent and to distribute nanoparticles evenly in a solution.[16] HAP is similar in composition as the mineral component of bones and hard tissues in mammals. HAP is a filler with molecular formula of Ca10(PO4)6(OH)2. It is a white powder with a needle-shape and an average size ranging from 80 to 100 nm and width = 15 25 nm.[17]

BGs are given considerable importance because of their ability for biomedical applications such as enhancing the bond strength in bone formation, speeding healing time, etc.[18] These factors have made them a promising choice to be used as a coating on suitable substrates such as ZrO2-based for biomedical applications. In other words, to achieve a desired implant dental treatment, Zhang and Le developed new ZrO2/BG composites with excellent mechanical and bioactivity features.[18] In addition, Odermatt et al. reported that the nanosized BG particles improve the alkalizing ability of the composite while having little adverse effect on their specific properties.[19]

Hence, because of its non-toxic properties, chemical stability in the human body, and good mechanical properties, ZrO2 is commonly used in orthopedics. ZrO2 is a metal oxide with a white color, excellent strength, and resistance to corrosion that is an example of high ceramic material and particularly biocompatible implant materials.[20] Also, high thermal stability, low thermal conductivity, high fracture durability, and relatively high ion conductivity are indeed benefits of ZrO2.[21] Therefore, ZrO2 is the broad material in the field of dentistry. Currently, the high strength of ZrO2 means that it is difficult to process and shape.[22] Nano- ZrO2 reinforcement of the repair material can dramatically boost the transverse strength of several broken denture polymers as reported by Gad et al.[23] In addition, Bacakova et al. reported that ZrO2 nanoparticles have non-toxicity properties.[24] Kumar et al. observed that the mechanical performance and thermal properties of the scaffolds are improved by adding ZrO2 nanoparticles into the BG matrix, and the scaffolds have acceptable antibacterial capabilities against certain bacterial strains.[25] They suggested that it's a great tissue engineering replacement. Additionally, An et al. reported that the ZrO2-HAP composite scaffold has good mechanical features and cellular/tissue compatibility, making it a suitable candidate for large-scale bone restoration as well as regeneration.[26] Moreover, when ZrO2-HAP-BG was utilized, Bian et al.[27] found that the scaffold obtained outstanding mechanical characteristics, bioactivity, and strong cytocompatibility. The composite scaffold represents the potential to be used as a bone implant in the near term.

The objective of this study has been oriented mostly on the impact of ZrO2 nanoparticles in various proportions on the physical properties of dental composites. It is a spherical-like shape with an average size of 25 nm. Five composite samples comprising 0, 1.3, 3.2, 6.3, and 11.76 wt% of different ZrO2 nanoparticles were combined with a matrix (where the CA

wt% is two times of the BG-HAP wt%) and characterized using different methods. Then, the contact angle, the mechanical and bioactivity properties of the composite with different concentrations of ZrO2 are explored.

 

2. Materials and Methods

2.1. Materials

All the chemicals are of analytical grade purity. We purchased CA powder from PDH Company, United Kingdom (UK) and BG nanoparticles (BGNPs), and HAP nanoparticles (HAPNPs) from NanoTech Company Limited Egypt. ZrO2 nanoparticles (ZrO2NPs) were purchased from PDH Company, United Kingdom (UK). They have a spherical geometry and a size of 25 nm. BGs are amorphous non-crystalline solids made mostly of silica-based compounds with modest additions. Moreover, BG has a composition of 45SiO224.5Na2O24.5CaO6P2O5 (wt%).[28] BGNPs (its average size is 5.03 nm) are white powder with a spherical-like shape.

 

2.2. Preparation and fabrication of CA-HAP-BG-ZrO2 nanocomposites

Initially, the process involving the determination and evaluation of the weight fractions needed for a suitable composite is as the following: CA was dissolved in 10 mL of acetone and continuously stirred for 10 min at a temperature of 50 C. A sample of 100 wt% of CA is considered as a pure sample. Then, the nanofillers were poured into the CA that had dissolved. Moreover, using a tabletop ultrasonic cleaner, the mixture was sonicated to ensure the proper uniform mixing and dissolution of the filler in the polymer matrix (CA). The well-mixed composite is then casted into a circular glass dish and was allowed to set overnight at room temperature, as seen in Fig. S1. The samples with their different weight fractions, compositions and densities are shown in Table 1.


Table 1. The different weight fractions, compositions, and densities of CA-HAP-BG-ZrO2 NPs.

Sample #

Compositions

Density (kg/m3)

Pure

100 wt% CA

1280

CHBZ1

65.8 wt% CA, 16.45 wt% HAPNPs

16.45 wt % BGNPs, 1.3 wt% ZrO2 NPs

1950

CHBZ2

64.5 wt % CA, 16.13 wt% HAPNPs

16.13 wt% BGNPs, 3.22 wt% ZrO2 NPs

2215

CHBZ3

62.5 wt% CA, 15.62 wt% HAPNPs

15.62 wt% BGNPs, 6.25 wt% ZrO2 NPs

2321

CHBZ4

58.82 wt% CA, 14.7 wt% HAPNPs

14.7 wt% BGNPs, 11.76 wt% ZrO2 NPs

2436


2.3 Characterization Techniques

The synthesized nanocomposites, previously described, were subjected to structure characterization using different techniques. The physical and chemical properties were evaluated using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and thermal analysis. A short summary about the analysis methods will be described as follows: The phase composition of the ZrO2/CA/BG-HAPNPs composite was studied by X-ray diffraction analysis (XRD, Bruker D8 advance diffractometer, Germany). The x-ray tube parameters used in this analysis were 40 kV applied voltage and 15 mA beam current with diffraction patterns obtained at 25 C and over a 20 to 80 angular range. A scanning electron microscope (SEM, Carl-Zeiss Sigma 500 VP, Germany) is used to investigate the morphology of the nanocomposite surface. In this study, the images were acquired at different magnifications at 20-25 keV with the nanocomposite surface coated with a gold/palladium thin alloy film to prevent surface charging and activate the emission of secondary electrons.

Furthermore, the ZrO2/CA/BG-HAP composite elemental evaluation was carried out using an Energy-Dispersive X-ray (EDX) detector (EDS, EDAX Inc., Mahwah, NJ, USA) linked to the SEM machine. Then, the screened data was post-analyzed using APEXTM EDS software. The surface elemental composition was investigated using X-ray photoelectron spectroscopy (XPS, Perkin Elmer PHI 5600, USA). To classify and quantify the existing elements on the surface of the specimen, large energy spectra of the prepared composites were analyzed over a range of energies of 01350 eV. Fourier transform infrared spectrometer (FTIR, Bruker Tensor 27, Germany) was used to identify the functional groups of the synthesized nanocomposites. Its spectral range was 400 to 4000 cm-1, with resolution of more than 1 cm-1. The thermal analysis of ZrO2 nanocomposites samples is measured using Shimadzu (TGA-50) Thermogravimetric analyzer. The particle size of synthesized composites dispersion in acetone was recorded by dynamic light scattering (DLS) system using Microtrac Nanotrac Wave. The technique is also referred to as photon correlation spectroscopy (PCS) in which particle size is determined by measuring the random changes in the intensity of light scattered from a suspension or solution. The sample is illuminated by a laser beam followed by detection of the resultant fluctuations of the scattered light at a known scattering angle θ by a fast photon detector.

 

2.3.1 Mechanical Properties by Ultrasonic Testing

The mechanical properties were examined using the ultrasonic system (UT) to calculate wave velocities using the pulse-echo technique. These velocities were measured using an ultrasonic flaw detector (SIUI ultrasonic flaw detector) connected to an oscilloscope. In addition, the uncertainty was estimated to be about 9 m/s and 6 m/s for longitudinal and transverse velocities, respectively. For longitudinal waves, oil was used as a coupling medium, while varnish was used for shear waves. Moreover, the mechanical properties were calculated using the equations in[29,30] with the help of the estimation of shear velocity, longitudinal velocity, and density (Table 1).

 

2.3.2 Contact Angle Measurements

Contact angle measurements are carried out using the SEO Phoenix-10 contact angle analyzer for different composite samples. Low-bond axisymmetric drop shape analysis (LB-ADSA) plug-in tool for Java image processing; ImageJ software is used for contact angle estimation. In LB-ADSA, the shape matching adjustment of the droplet image on the sample by shifting their corresponding sliders would expect its shape to be closely matched to the drop image.

 

2.3.3 Biological Test

2.3.3.1 Antimicrobial Activity Assays

We used a diffusion-growth kinetic technique in agar to assess the antibacterial activity of the synthesized nanocomposites. We concluded this experiment according to Jaramillo et al. procedure.[31] A total of five bacterium strains included P. Aeruginosa, E. Coli (ATCC 25922), S. Typhi, S. aureus (ATCC 25923), and B. Subtilis (ATCC), tested and obtained from Microbial Biotechnology and Molecular Biology lab (Egyptian Atomic Energy Authority, Egypt).

For the antibacterial diffusion growth kinetics on agar test,[32] strains struck separately on Nutrient Agar (NA) medium, followed by incubation overnight at 37 C. We choose a well-isolating colony within each strain using an inoculating loop, then introduced to one of 50 ml falcon tubes which contain 10 ml of Nutrient agar broth. This broth then incubated at room temperature overnight till its turbidity obtained by a turbidimeter reached 0.5 on McFarland scales. The cells were suspended to 1:10, yielding 107 CFU/mL as the final concentration of inoculum. Every inoculum of the five strains was sown equally on nutrient agar (NA) dishes. After that, every nanocomposite thin sheet (3 mm3 mm), as well as a CA pure sample, has been attached to the top of TSA plates to confirm good adhesion. The control was placed in the center of the plate, and each one of nanocomposites CHBZ1-4 was placed surrounding the control. Finally, the plates were incubated for 24 hours at room temperature.

 

2.3.3.2 In-Vitro Cell Viability

Four samples of nanocomposites CHBZ1-4 with different concentrations have been tested individually in synchronization with the impact of the CA effect alone. In-vitro cytotoxicity against normal Vero cells (Green monkey kidney cell line) was used.[33] Green monkey kidney has obtained from (Nawah Scientific Inc., Mukatam, Cairo, Egypt). SRB (Sulforhodamine B) assay was used to determine the Vero cells viability. The aliquots of 100 μL Vero-cell suspension containing approximately 5 103 cells were inserted in 96-well plates, and then incubated for one day in DMEM medium. In a humidified, 5% (v/v) CO2 environment at room temperature, these cells were inserted into 100 units/mL penicillin and 10% heat-inactivated fetal bovine serum. We added 100 μL of media containing 100 ug/ml of nanocomposites CHBZ1-4 to the cells and the cells were exposed for 72 hours to the drug. We treated the samples by replacing the medium with 150 L of 10% TCA (trichloroacetic acid) before incubation for 1 hour at 4 C, then TCA sol emptied, and the cells were cleaned for five times with ddH2O. A 70 μL SRB sol (0.4 % w/v) was applied to the cells and allowed to brood for 10 minutes inside the dark conditions at 37 C. Dishes were rinsed three times with 1 % of acetic acid then dried overnight. We applied 150 L of TRIS (10 mM) to solubilize the protein bound SRB stain and used a Microplate Reader (model ELx800, Biotek, USA) to detect the absorption at 540 nm. We carried out this experiment in parallel with testing the viability of normal cells against 10 ug/ml Doxorubicin (DOX) as a positive control.

 

3. Results and Discussion

3.1 X-ray Diffraction (XRD) Analysis

XRD measurements of the synthesized nanocomposites are shown in Figs. 1 (a-e). The diffractogram of the CA indicates amorphous behavior patterns as shown Figs. 1 (a-e).[34,35] Figs. 1 (a-e) shows the XRD patterns of the synthesized nanocomposites with different contents of ZrO2 ranging from 1.3 to 11.76 wt%, respectively. As a result, it is clear that all of the samples' diffraction characteristic peaks for ZrO2 crystals were in strong alignment with the International Center for Diffraction Data (ICDD) (PDF: 49-1642). ZrO2 has diffraction peak values at 30, 34, 50, and 59.

Also, Figs. 1 (a-e) shows that the main composition in the composites is HAP. It is because there exist the semi crystalline peaks at 25.9, 31.8, 32.0, 33.0, and 40.0, which constitute the structure of HAP crystals, also, this figure shows that most diffraction peaks of BGs appeared as stated as.[36,37] It can be seen that the major composition of the composites is still HAP. Generally, the introduction of ZrO2 into the composites can enhance the decomposition of HAP into tricalcium phosphate (TCP) and CaO because CaO can be incorporated into the ZrO2 lattice in agreement with.[38] The obtained data from XRD measurements agreed with the obtained data from microhardness measurements. The addition of ZrO2 nanoparticles into the composites is good for the enhancement of the mechanical performance of the nanocomposites as stated as.[39]

Fig. 1 XRD patterns of the synthesized nanocomposites with different weight percentages of ZrO2: (a) Pure, (b) CHBZ1, (c) CHBZ2, (d) CHBZ3, and (e) CHBZ4 samples.

 

3.2 Scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) analysis

The observed images of the synthesized nanocomposites are shown in Figs. 2a and 2b. The obtained images of EDX mapping are presented in Figs. 2c and 2d. As shown from Fig. 2(a), the synthesized nanocomposites with 3.22 wt% of ZrO2 appeared as circular spots distributed uniformly over entire surface, while the synthesized nanocomposites with 11.76 wt% of ZrO2 show variation in morphological features and appeared as irregular forms and agglomeration with inter-granular micropores and small amounts of porosity; the ZrO2 grains were found with lighter color ratios as shown in Fig. 2(b).[40-42] The SEM-EDX analysis was performed to determine the surface elemental composition of the synthesized composites. The elemental mapping for Zr, O, C, P, Ca, Na, and Si elements for the synthesized composites with 3.22 and 11.76 wt% of ZrO2 is shown in Figs. S2 and S3. From Figs. S2 and S3, it was observed that all the samples contain Zr, O, C, P, Ca, Na, and Si in certain contents as stated above.[43]


Fig. 2 SEM images of the synthesized composites with (a) CHBZ2, (b) CHBZ4, (c) analysis EDX for CHBZ2 and (d) EDX for CHBZ4 samples.


3.3 Dynamic Light Scattering

Dynamic light scattering is popularly used for the determination of the size of particles in suspension, emulsions, colloids, polymer etc. The advantage of this is it allows the particle sizing down to 1 nm diameter. The acetone suspended nanocomposite samples were diluted and analyzed by the DLS particle size analyzer. The size distribution of the synthesized composites is in the nano range as presented in Fig. S4. The data clearly reveals that the sizes of the synthesized nanocomposite are in the nanoscale. The average particle sizes are 387, 550, 509, 474, and 984 nm for pure, CHBZ1, CHBZ2, CHBZ3, and CHBZ4 samples, respectively.

 

3.4 X-ray Photoelectron Spectroscopy

The chemical composition of the surface of the synthesized composites and the states of the material coating were determined by using X-ray photoelectron spectroscopy (XPS), and the obtained data is presented in Figs. 3 (a-d).

The survey wide energy spectra of applied composites were taken over an energy range of 01350 eV in order to identify and quantify the present elements on the composite surface. A typical XPS spectrum of CHBZ1 composite is shown in Fig. 3 (a). The plot exhibits seven signals, in this low-resolution spectrum, at 103.59, 190.66, 287.12, 348.25, 133.03, 533.67, and 1072.27 eV. These signals could be attributed to electrons ejected from Si2p, Zr3d, C1s, Ca2p, P2p, O1s, and Na1s orbitals.[44]

While a typical XPS spectrum of CHBZ2 composite is shown in Fig. 3 (b), the plot also exhibits seven signals, in this low-resolution spectrum, at 103.83, 191.11, 287.02, 348.12, 133.96, 533.24, and 1072.27 eV with low shift, respectively in agreement with.[44] Also, typical XPS spectra of CHBZ3 and CHBZ4 composites are shown in Figs. 3c and 3d. The percentage of the different elements present in all nanocomposites was determined and listed in Table 2.



Table 2. Atomic percentage ratio of the elements on the surfaces of CHBZ1, CHBZ2, CHBZ3, and CHBZ4 nanocomposites measured by XPS.

Concentration

O%

C%

Na%

Ca%

Si%

P%

Zr%

CHBZ1

36.94

54.74

3.12

2.26

1.88

1.05

0.1

CHBZ2

38.11

52.79

3.1

2.73

1.51

1.5

0.26

CHBZ3

39.83

47.94

3.35

3.89

2.84

1.8

0.33

CHBZ4

38.33

52.54

2.97

2.33

1.6

1.55

0.68


Fig. 3 XPS spectra for (a) CHBZ1, (b) CHBZ2, (c) CHBZ3, and (d) CHBZ4 nanocomposite samples.


The high-resolution signals, corresponding to these elements, are shown in Fig. 3, the C1s and O1s that represent the main constituents of CA. It is worth noting that C1s signal while the main peak was detected at 287.12 eV, which could be related to C=O species.[45] It is clarified that the peaks at 103.59 and 1072.27 eV were attributed to Si and Na atoms in the structure of SiO and NaO from SiO2 and Na2O, respectively of the bioactive glass. In addition, the peak at 348.25 eV was attributed to the Ca atom in the structure of CaO of HAP in the composite. Moreover, the peak at 533.67 eV was assigned to the O atom in the structure of O-CO and that is related to metal oxides of Na2O and ZrO2, respectively. Thus, it emphasizes the presence of these metals as oxides and other explanations for hydroxyl adsorption present on the composite.[46] Furthermore, the peak at 190.66 eV corresponded to ZrO2.[47] Therefore, the P2p core level peak located at 133 eV is attributed to PO bonds in PO43− that is present in HAP in agreement with.[43]

Fig. 4 FTIR spectrum of the synthesized nanocomposites.

 

3.5 FTIR Analysis

FTIR is considered the most powerful tool to identify the chemical bonds (functional groups) and is used to detect possible changes in the functional groups in structure. The wavelength of absorbed light is characteristic of the chemical bond as can be seen in the spectra throughout the range of 4004000 cm−1.The FTIR spectra of the synthesized composites are presented in Fig. 4.

The spectra show broad peaks around the wavenumber 3424-3450 cm−1 for each spectrum due to the presence of hydroxyl (OH) groups on the surface of the synthesized composites.[48] In addition, the peaks detected at ~ 2930 cm-1 may be due to CH stretching vibrations of CA in agreement with.[49,50] Moreover, the bands revealed in 1747 and 1639 cm−1 could be attributed to the stretching vibrations of carbonyl (C=O) and C=C groups, respectively.[51,52]

Furthermore, the absorption peaks at 1420 and 1380 cm-1 correspond to CH2 bending vibration of CA.[53] Also, the absorbance bands around 9501250 cm−1 were determined to be the presence of all 6 types of composites which signify the CH stretching and CO groups.[53-55] It is clear that these peaks were observed in all spectra with small shifts. The strong interaction between the PO group and calcium on an appetite surface demonstrates this shift, indicating that these obtained materials are indeed composites.

In addition, an absorption bands in the range 600 to 400 cm−1 may be assigned to the metal-oxygen (MO) as CaO, ZrO and PO stretching mode as shown in CHBZ1, CHBZ2, CHBZ3, and CHBZ4 samples. [5658]

 

3.6 Mechanical Properties by Ultrasonic Testing

The objective of this section is to evaluate the effects of zirconia nanoparticle addition at low concentrations (up to 12 wt%) to biopolymer on the mechanical properties, such as micro-hardness and Youngs modulus with the help of ultrasonic technique.

Fig. 5(a) shows the effects of the presence of ZrO2 nanoparticles onto the micro-hardness of the synthesized composites. As shown in this figure, the micro-hardness of the synthesized composites increases with the increase of ZrO2 content and have been improved greatly after introducing ZrO2 nanoparticles into biopolymer nanocomposites as compared with those of unmodified composites.

It can be observed from this figure; the microhardness is increased from 245.8 MPa at 1.3 wt% to 405.5 MPa at 11.76 wt% of ZrO2 as compared with the unmodified ones that show low micro-hardness (69.4 MPa).[29] This is due to the fact that Zirconia, has a high flexural strength and micro-hardness, as well as the decrease of porosity. Furthermore, zirconia shows excellent biocompatibility compared to other ceramic materials such as alumina and found.[59,60] Also, the density of the synthesized composites increased from 1280 to 2436 kg/m3 with increasing ZrO2 content as shown in Table 1; consequently, the micro-hardness of biopolymer composites was increased.[39] The addition of zirconia in biopolymer between 1.3 wt% and 11.76 wt% zirconia would provide the optimum mechanical properties suitable for denture base applications similar.[29]

Fig. 5(b) shows Youngs modulus as a function of ZrO2 weight fraction (wt%). It can be detected from this figure that Youngs modulus increases from 5181.6 to 6467.3 MPa with increasing ZrO2 weight fraction from 1.3 to 11.76 wt%, respectively. In other words, Youngs modulus increases with the increase in the ZrO2 weight fraction (wt%).

 

3.7 Contact Angle Measurements

It is of substantial significance to identify and describe the wettability of solid surfaces for the material characterization. Wettability of solid-fluid surface mechanism is also established by calculating the contact angle shaped between a water droplet and a solid surface.[61] Low wettability and unsatisfied adhesion characteristics also exist on the polymer surface. Therefore, the polymer surface has to be adjusted and controlled. Different fillers and composites with different


Fig. 5 (a) Micro-Hardness of composites with different weight percentages of ZrO2 and (b) Youngs modulus as a function of ZrO2 weight fraction (wt%).

 


weight fraction contents are used in this analysis. Wettability is obtained by calculating the liquid drop interface angle with the biopolymer substrate. Low contact angle (<< 90) refers to high wettability, whereas low wettability is detected for higher contact angle (>> 90).[62]

The contact angle is carried out using a contact angle analyzer system (model SEO Phoenix-10). It consists of Phoenix 300 instrument and ImageJ software [63] where the measurements were carried out at room temperature (20-25˚C).

A HHD camera is used to record a drop image within 10 sec of water deposition. In this study, a low-bond axisymmetric drop shape analysis (LBADSA) plug-in,[64] based on the Young-Laplace equation, is used to measure the contact angle here. This plug-in in ImageJ software calculates the contact angle of a drop image on a flat surface using the ellipse approximation.

Fig. 6 shows contact angle (a) images of different weight fraction of ZrO2 in biopolymer nanocomposite. It is observed that zirconium oxide addition enhances the wettability. In other words, contact angle is decreased from 65 for the pure sample to 38.4 for the composite with 1.3 wt% ZrO2. This leads to improvement in the hydrophilicity of the biopolymer matrix under the addition of ZrO2 NPs. However, as ZrO2 is applied to the biopolymer matrix, an extra hydroxyl group on ZrO2 and HAPs can begin to bind with water and increase hydrophilicity.[36]

Furthermore, as shown in Fig. 7, the fraction of ZrO2 in the nanocomposite polymer has insignificant effect on the contact angle improvement.

 

3.8 Thermal Analysis

Measurements of thermogravimetric analysis (TGA) were carried out using a Shimadzu TGA/DTA-50 system from Kyoto, Japan. The phase changes and weight losses of the samples were determined by heating at a range of 10C.min-1 in presence of nitrogen gas to avoid thermal oxidation of the powder sample. The thermal measurements were done up to a temperature of 700 oC, using α-Al2O3 as a reference.

The thermal decomposition and thermal stability of polymers are most effectively assessed by TGA. TGA of the synthesized nanocomposites and was performed at temperature ranging from 25 up to 700 oC. The obtained thermograms are presented in Figs. S5(a-e). In this figure, TGA of the pure, CHBZ1, CHBZ2, CHBZ3, and CHBZ4 nanocomposites showed three weight losses on the TGA profile. The first weight loss was observed with rising temperature up to about 220 oC. The four composites exhibiting a loss in weight amounted to 6.3, 7.2, 5.5, and 8.5 %, respectively for CHBZ1, CHBZ2, CHBZ3, and CHBZ4 nanocomposites. Also, the first weight loss of the pure CA sample was observed with rising temperature up to ~290 oC and amounted to 4.8 %. This loss could be attributed to the evaporation of loosely bound moisture on the surface and the intermolecular hydrogen bonded chemisorbed water.[65] The second weight loss is a sharp decrease in weight in a temperature range of 220350 oC.


Text, surface chart

Description automatically generated

Fig. 6 Images of drop on biopolymer nanocomposite under different weight fraction of ZrO2.


Fig. 7 Contact angle as a function of wt% weight fraction of ZrO2 on the biopolymer nanocomposite.



This weight loss amounted to ~ 81.7, 48.6, 47.5, 45, and 53 %, respectively for pure, CHBZ1, CHBZ2, CHBZ3, and CHBZ4 nanocomposites and could be thermal depolymerization of hemicellulose acetate and the breakdown of glycosidic linkages of CA.[66] The third weight loss in the temperature range of 350-425 oC was amounted to ~ 13.5, 28.1, 26.5, 18.5, and 32 %, respectively for pure, CHBZ1, CHBZ2, CHBZ3, and CHBZ4 nanocomposites and could be attributed to the removal of the residual oxygenated groups, such as hydroxyl, carbonyl, and M-O groups on the surface of the synthesized nanocomposites.[65] It is clear that synthesized nanocomposites are stable at a temperature less than 225 C.

Also, it is clear that synthesized CHBZ3 nanocomposite is more thermally stable compared to the other three composites. In the Differential thermal analysis (DTA or DrTGA) curve, an endothermic peak appeared at 280 - 320 C, presumably caused by the decomposition of the carbonate groups and shows degradation of the nanocomposites.[36] Also, an endothermic peak appeared at 386 - 410 C, which shows the crystallization would start in composite samples or may be because of the presence of various oxygen functional groups from its structure with different thermal stabilities and their cleavage occurring at different temperatures.[67]

The TGA results of the synthesized nanocomposites have been tabulated in Table 3, giving clear information on the degradation pathway of these materials. From Table 3, the T10 and T50 symbols refer to the corresponding temperature where 10% (as the measures of the onset) and 50% (as half degradation temperature) mark the weight losses, respectively. Noticeably, the values of T10 and T50 show diverse thermal performances according to the loading amounts of ZrO2.



Table 3. TGA results of synthesized nanocomposites with different ratios of ZrO2.

Concentration

Temperature (C) for different percentage decompositions

 

T10

T50

PDTmax

(C)a

CDTfinal

(C)b

CHBZ1

305

344

345

475

CHBZ2

244

360

318

420

CHBZ3

235

356

280

442

CHBZ4

249

396

292

425

a Values are from the DTG curve.

b Values are from the TGA curve.


Great stability was detected for 6.25% loading of ZrO2 nanocomposite. The final composite degradation temperature (CDTfinal).[68] As presented in Table 3, is recognized from the TGA curve. The CDTfinal values take place from 405 C to 475 C. Also, PDTmax represents the maximum temperature accomplishment when the sample decomposition was recognized.[69] From these findings, it was confirmed that the thermal stability of synthesized nanocomposites was enhanced excellently by the incorporation of proper loading of ZrO2.

 

3.9 Effect of Nanocomposite on Vero Cells Viability

All nanocomposites CHBZ1-4 were tested separately in-vitro for their cytotoxic activity against Vero cells (Green monkey kidney), normal cell lines using SRB assay, which run on parallel with examining cells with 10 ug/ml DOX. Table 4 represents the percentage of cell viabilities after treatment with 100 ug/ml of nano formula, where the percentages ranging between 56.6079 % for CHBZ3 and 79.2287 % in case of CHBZ4. On the other hand, only 10 ug/ml (only one

cell line, for in-vitro evaluation. All data showed that mild to non-cytotoxic effects have been seen for the nano formulas tested compared to inhibitory effect of DOX to cells, although it was added with concentrations 10 folds lower than that of our target nanocomposite to tenth of the concentration of NCs) of DOX were enough to kill about 96.5% of cells in the same conditions.

Fig. 8 represents an image for each cell line after treatment with each formula separately, contrary to the image of cells treated with DOX. Growth inhibition has been seen obviously in case of DOX, while cell density after the same time and condition seems to be good. This study is designed to investigate the cytotoxicity of novel Nanocomposites CHBZ1-4 on normal cells. We used Vero cells, the available normal.


Table 4. Values of Vero cells viability % after treatment with 10 ug/ml of NCs formulas separately in comparison with treatment with 10 ug/ml DOX.

NCs Formula

CHBZ1

CHBZ2

CHBZ3

CHBZ4

DOX

Cell Viability %

78.334

74.0074

56.6079

79.2287

3.5673

 

Background pattern

Description automatically generated

Fig. 8 (a) Morphology of Vero cells of untreated cells (pure sample), (b) image of cells treated with only 10 ug/ml DOX, and Vero cells after treated with 1 mg of (c) CHBZ1, (d) CHBZ2, (e) CHBZ3, and (f) CHBZ4 samples.

 

CHBZ

Organism name

(P. Aeruginosa,)

(B. Subtilis)

(S. Aureus)

(E. Coli)

(S. Typhi)

Fig. 9 Results of antibacterial testing for nano composites CHBZ1-4 against five pathogenic bacterial strains (Pseudomonas. Aeruginosa, Bacillus. Subtilis, (Bacillus. Subtilis, Escherichia. Coli, and Salmonella. Typhi). Only Formula CHBZ2 Pic No.1. shows antibacterial activity against Pseudomonas. Aeruginosa, while other formulas do not exhibit any activity towards the rest of the strains.

 


3.8 Antibacterial Activity Results

In Fig. 9, the antibacterial examination for the target nano composites shows no activity against any of the five pathogenic bacterial strains; only mild inhibitory activity is shown against P. Aeruginosa by nano composite CHBZ2. Being noble and nearly not active against these already resistant pathogenic bacterial strains or mammalian normal cells never decreases its importance as potent and safe nanocomposite formulas as a dental filler, but it may be highly recommended for use due to its safe properties.

 

4. Conclusion

In this work, it was concluded that the nanosized composite based on cellulose acetate, hydroxyapatite, bioglass with different loading of ZrO2 were synthesized with solvent casting method. The target materials were also characterized by different techniques including: FTIR, SEM with EDX analyses, thermal analyses (TGA, DTA), and XRD. Also, the mechanical, the contact angle, and bioactivities measurements have been conducted for these synthesized nanocomposites. It could be observed that with the increase of the ZrO2 content, the microhardness is increased from 245.8 MPa at 1.3 wt% to 405.5 MPa at 11.76 wt% of ZrO2 as compared with those which are unmodified, show low microhardness (69.4 MPa), and lead to a high value of modulus. It can be revealed that because of the characterization of the prepared samples, all the contributed composite nanoparticles have appeared, and a semi crystalline pattern has been detected. Moreover, the results clarified that the increase in the ZrO2 content have led to an improvement in the two mechanical and thermal properties and a decrease in the contact angle. From our results, we believe that the proposed synthesized nanocomposite could serve as a potential candidate material for orthopedic and dental implants. Under the conditions of this in vitro study, using these novel Nanocomposite materials will be safe. Particularly, it did not show cytotoxic effects on normal mammalian cells, which is important for clinical applications of these materials.

 

 

Supporting information

Applicable.

 

Conflict of interest

There are no conflicts to declare.

 

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