# In Vitro Studies and Evaluation of Antibacterial Properties of Biodegradable Bone Joints Based on PLA/PCL/HA

authors:

Department of Biomedical Engineering , Science and Research Branch, Islamic Azad University , Tehran, Iran
Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran, Iran

how to cite: Dehghani Firoozabadi F, Ramazani Saadatabadi A, Asefnejad A. In Vitro Studies and Evaluation of Antibacterial Properties of Biodegradable Bone Joints Based on PLA/PCL/HA. J Clin Res Paramed Sci. 2022;11(1):e124080. doi: 10.5812/jcrps-124080.

### Abstract

#### Background:

Due to the history of using permanent implants and the ability of adaptations of polymers to physiological environments such as the body environment, the need to design a polymer implant with a new formulation for orthopedic applications was felt.

#### Methods:

Polymer joints in this study were made by solvent casting method. The mechanical properties of the samples were investigated by bending tests, before and after immersion in simulated body fluid (SBF). Morphology of nanocomposites, bioactivity of samples and initiation of degradation process were performed by field emission scanning electron microscope (FESEM). Toxicity test was performed to evaluate the toxicity of nanocomposites. The antibacterial properties of the samples were investigated by examining the zone of inhibition and measuring the photometric concentration. Biodegradability test was performed to prove the biodegradability of polymer joints.

#### Results:

It was found that the mechanical properties of nanostructures increased with the addition of nanoparticles. Also, the presence of oxide and graphene nanoparticles affected the antibacterial properties of the composite nanostructure. Immersion in SBF solution showed that the nanostructures were biodegradable and bioactive. The results of this study indicate that the optimal nanocomposite PLA-PCL-HA-1% ZNO-1% GR has a Young’s modulus close to spongy bone and reduces the stress shielding phenomenon. The flexural Yang modulus of the PLA-PCL-HA nanocomposite was 2139.037 ± 381.312 MPa. The presence of zinc oxide and graphene nanoparticles increased the Young’s modulus to 4363.636 ± 127.498 MPa. The optimal sample has the necessary lethality against two strains of gram-positive and gram-negative bacteria and due to its bioactivity is a suitable option for use in spongy bone tissue. In this study, the viability of fibroblast cells in the vicinity of the polymer matrix versus the optimal matrix increased from 22.14 ± 0.623 to 82.96 ± 1.101% after 72 hours.

#### Conclusions:

Improving cell viability indicates a reduction in the optimal matrix toxicity compared to the polymer matrix.

### 2. Objectives

The aim of this study was to obtain a biodegradable nanostructure with antibacterial properties that can have the required mechanical properties of hard tissue and is also biodegradable. To achieve this goal, 9 composite nanostructures were fabricated. According to this goal in this study, the antibacterial behavior of PLA/PCL/HA nanocomposites containing zinc oxide and graphene was investigated and also the physical and biological properties of the samples were investigated and tested. Finally, the optimal sample was selected according to the results. This study will help to achieve absorbable orthopedic joints.

### 3. Methods

Polylactic acid with a molecular weight of 182,000 g/mol and polycaprolactone with a molecular weight of 80,000 g/mol were prepared by Sigma Aldrich. Graphene nanosheets with 2 - 8 nm thickness and purity of more than 95% were prepared from Sigma Aldrich. Zinc oxide nanoparticles with a purity of more than 99% and a particle size of 35 - 45 nm were prepared from Sigma Aldrich. Hydroxyapatite nanoparticles and FBS solution were prepared from Pardis Pajouhan Fanavaran Yazd Company. Oleic acid and chloroform were obtained from Merck. Escherichia coli and S. aureus were used to perform photometric concentration measurement and zone of inhibition test, and fibroblast cells were used to evaluate the toxicity of nanostructures, all of which were obtained from the domestic market.

#### 3.1. Fabrication of Polymer Scaffolds by Solution Method

To prepare nanocomposite samples, 40 g of polylactic acid and 10 g of polycaprolactone were first vacuumed at 80°C for 24 hours to dehumidify the granules. In this study, chloroform was selected as the solvent and suspending agent for nanoparticles. Nine samples were prepared for the preparation of pure and reinforced nanocomposites. To prepare a pure sample, 5 g of polylactic acid with 1.2 g of polycaprolactone was poured into 100 mL of chloroform. The mixture was stirred at 60°C for 6 hours to dissolve completely. The sample was then exposed to ultrasonic waves at a frequency of 45 kHz, at room temperature and with a power of 60 w. After 30 minutes, the sample was poured into a petri dish and placed under the hood to completely remove the solvent. To add 1 wt% of hydroxyapatite nanoparticles to the polymer matrix, first 0.062 g of hydroxyapatite nanoparticles in 10 ml of chloroform were placed on a stirrer for 10 minutes. Then oleic acid and chloroform with a ratio of 0.6% v/v was placed on the stirrer for 5 minutes at 70°C. The mixture was then added to a container containing polymers and solvents prepared in accordance with the sample. The sample was exposed to ultrasound for 30 minutes under similar conditions. The nanocomposite was then transferred to a petri dish and placed under the hood to remove the solvent.

#### 3.1.1. Modification of Composite Scaffold with Zinc Oxide Nanoparticles

To make samples containing zinc oxide nanoparticles, 3 samples containing 0.1, 0.5 and 1 wt% of nanoparticles were added to the composite matrix. To make nanocomposites containing zinc oxide, the base matrix was prepared according to the steps mentioned above. After fabrication of the nanocomposite, 0.1% zinc oxide nanoparticles were poured into 10 cc of chloroform and irradiated with ultrasound for 15 minutes under the same conditions as other samples. After homogeneous dispersion of the nanoparticles in the solvent, the mixture was added to a polymer mixture containing hydroxyapatite. The sample was irradiated with ultrasound for 15 minutes in an ultrasonic bath. The sample was then transferred to a petri dish and placed under the hood for 48 hours to completely remove the solvent. The same steps were performed to make other weight percentages.

#### 3.1.2. Modification of Composite Scaffolds with Graphene Nanosheets

The same process was repeated to make composite nanostructures containing graphene nanosheets. The weight percentages of graphene nanosheets added to the polymer matrix are 0.1, 0.5 and 1 wt%.

### 4. Results

#### 4.1. Characterization of Composite Nanostructures

Field emission scanning electron microscopy (FESEM) method was used to study the morphology and distribution of nanoparticles in nanocomposites. In this study, Tescan VEGA II field emission scanning electron microscope was used. To confirm the presence of nanoparticles, X-ray diffraction spectroscopy (EDS) test was prepared from cross-section of PLA-PCL-HA-1% ZNO-1 Gr% nanocomposite. Also, to investigate the distribution of nanoparticles in the polymeric matrix, elemental analysis maps of the cross-section of the mentioned nanocomposite were prepared. As shown in Figure 1A - D, the surface of the nanocomposites is completely porous. The size of the pores on the surface was measured in the range of 1.5 to 3.5 μm. The porosities are uniformly observed on the surface of the nanocomposite and the size of the porosities is close to each other. A number of porosities are interconnected. The surface is free of cracks and fractures. By adding zinc oxide nanoparticles in the polymer matrix, bumps and white spots are observed on the surface, which indicates the presence of nanoparticles. By adding graphene nanosheets, the surface becomes more integrated and the porosity of the surface is reduced. Figure 2A-E and H shows cross-sectional images of composite nanostructures. As can be seen in the images, the nanoparticles of hydroxyapatite, zinc oxide and graphene are marked with yellow arrows in the image. Graphene nanosheets in the polymer matrix are seen as clear sheets in the polymer matrix. As you can see in the pictures, the graphene plates are fully open and have a good distribution. To investigate the presence of nanoparticles, X-ray energy diffraction (EDS) spectroscopy was performed at the same time as FESEM test. Its diagram is shown in Figure 3. As shown in Figure 3, the distribution of calcium, phosphorus and zinc oxide nanoparticles is shown in the elemental analysis map. Scattering of nanoparticles is well observed on the surface of composite nanostructures (14).

#### 4.2.1. Bending Test

Equations 1, have been used to calculate the flexural properties under three-point loading and the modulus of elasticity.

Equation 1.$σf=3FL2bd2$
Equation 2.$εf=6bdl2$
Equation 3.$E=L34bd3$

Where F represents the force, L is the length of the specimen between the two supports, b is the width of the specimen, is rectangular, and d is the thickness of the specimen. Where σf, εf and E are the flexural stress and strain and the Young’s modulus, respectively. The calculated values before immersion in SBF solution are given in Table 1. Due to the similarity of the ions in SBF solution compared to human blood plasma and the creation of similar conditions when the implant was exposed to body fluids, the behavior of the composites in the bending test after immersion in SBF solution was performed and the calculated values after immersion in SBF solution are given in Table 2. The strain stress curves before and after immersion in the simulated body fluid are shown in Figures 4 and 5 (14, 15).

Table 1. Results of Bending Test of Nanocomposites Before Immersion in SBF Solution
SampleYoung’s Flexural (Mpa) ModulusProbability
PLA-PCL-HA2352.94 ± 138.2610.017
PLA-PCL-HA-0.1%ZNO3336.898 ± 276.3490.015
PLA-PCL-HA-0.5%ZNO312.833 ± 3337.6790.013
PLA-PCL-HA-1%ZNO3508.021 ± 378.1260.008
PLA-PCL-HA-0.1%ZNO-0.1%Gr4705.882 ± 426.7690.005 >
PLA-PCL-HA-0.5ZNO-0.5%Gr5219.251 ± 467.9240.005 >
PLA-PCL-HA-1%ZNO-1%Gr5475.935 ± 520.4890.005 >
Table 2. Calculated Values of Bending Test of Composite Nanostructures After 15 Days of Immersion in SBF Solution
SampleYoung’s Flexural (Mpa) ModulusProbability
PLA-PCL-HA2139.037 ± 381.3120.021
PLA-PCL-HA-0.1%ZNO2652.406 ± 294.3470.019
PLA-PCL-HA-0.5%ZNO2737.967 ± 129.3680.016
PLA-PCL-HA-1%ZNO2994.652 ± 403.3850.012
PLA-PCL-HA-0.1%ZNO-0.1%Gr3850.267 ± 167.4110.007
PLA-PCL-HA-0.5%ZNO-0.5%Gr4064.171 ± 498.472< 0.005
PLA-PCL-HA-1%ZNO-1%Gr4363.636 ± 127.498< 0.005

Stress-strain curve of the bending test of composite nanostructures before and after immersion in SBF solution are shown in Figures 2 and 3, respectively.

#### 4.3. Investigation of Bioactivity of Composite Nanostructures

In order to evaluate the bioactivity of the polymer matrix and composite nanostructures, the growth rate of apatite on the surface of the samples was investigated. If apatite grows on the surface of the samples, it can be said that the polymer matrix and the made nanocomposites are bioactive and can be placed in the host body and can stimulate bone formation. Also, by observing cracks on the surface of the polymer matrix and composite nanostructures, the beginning of the degradation process of the samples was determined. To investigate this issue, FESEM images of the samples were examined after 1 month of immersion in (SBF). The results are shown in Figure 6 (15).

#### 4.5. Toxicity Test

To evaluate the biocompatibility and cytotoxicity of the polymer matrix and the optimal nanostructure, the viability of L929 fibroblast cells in the presence of the polymer matrix and the optimal composite nanostructure was investigated. The results are shown as the percentage of L929 fibroblast cell survival after 1 day and 3 days in Table 3 and inverted light microscope images after 24 hours in Figure 8. The percentage of cell viability was calculated from Equation 4.

Table 3. Percentage of L929 Cell Viability in the Presence of optimal polymer matrix and Composite Nanostructure After 24 and 72 Hours
SampleSurvival Rate After 24 HoursSurvival Rate After 72 HoursProbability
PLA-PCL25.82 ± 0.78222.14 ± 0.623< 0.005
PLA-PCL-HA-1% ZNO-1%Gr84.96 ± 1.00682.96 ± 1.101< 0.005
Equation 4.

Examination of cell survival results shows that the cell survival rate in the vicinity of the optimal composite matrix after 24 hours was 84.96 ± 1.006% and with the passage of time up to 72 hours this rate decreased to 82.96 ± 1.101%. The survival rate of cells against polymer matrix was25.82 ± 0.782% after 24 hours and 22.14 ± 0.623% after 72 hours. Cell viability in the presence of optimal composite shows a significant increase due to surface modification and improvement of surface properties and the positive effect of the presence of nanoparticles in the polymer matrix (17-20).

#### 4.5. Zone of Inhibition Test

In order to investigate the antibacterial properties of the polymer matrix and the optimal composite nanostructure, the Zone of Inhibition test was performed. Gram-positive and gram-negative bacteria of S. aureus and E. coli were used for this purpose. As can be seen in the images, in the PLA-PCL polymer matrix sample, no growth inhibition zone is formed around the polymer matrix. Of course, no bacterial colonies are observed on the surface of the polymer matrix, which indicates the proper structure of the matrix. In the optimal sample, due to the presence of oxidized nanoparticles and graphene, the growth inhibition zone is clearly observed. The sharp edges of graphene nanosheets also cause the bacterial membrane to rupture, and resulting in bacterial death. The growth inhibition zone of E. coli is slightly lower than that of S. aureus, which is due to the structure of the membrane of the two layers of gram-negative bacteria against the monolayer wall of gram-positive bacteria. The images of the zone of inhibition of the optimal nanocomposite and polymer matrix are given in Figure 9. These results are consistent with the results of previous research. The antibacterial activity of nanocomposites not only involves the direct action of zinc oxide nanoparticles, but is also enhanced by the mechanism of release of zinc ions as well as the production of reactive oxygen species (ROS). In addition, graphene is the storage site for zinc ions, which are released from zinc oxide nanoparticles and, by contact with negative bacteria, increase the permeability of cells, which ultimately causes cell deformation and then leakage. Graphene can also improve the electron transfer rate due to its unique structure, which makes this nanocomposite have higher antibacterial activity (21, 22).

#### 4.6. Photometric Concentration Measurement Test

To measure the antibacterial percentage of the samples, photometric concentration was measured on two samples of polymer matrix and optimal sample. The mortality rate was calculated from Equation 5. The results are given in Table 4.

Equation 5.
Table 4. Results of Photometric Concentration Test of PLA-PCL, PLA-PCL-HA-1% ZNO-1% Gr Against Staphylococcus aureus and Escherichia coli
SampleAntibacterial Percentage of the Sample Against Escherichia coliAntibacterial Percentage of the Sample Against Staphylococcus aureusProbability
PLA-PCL25.16 ± 0.209%%27.14 ± 0.218< 0.005
PLA-PCL-HA-1% ZNO-1%Gr%76.87 ± 0.249%79.17 ± 0.320< 0.005

As shown in Table 4, the antibacterial content of the optimal sample against the polymeric matrix has almost tripled, that resulting in the release of zinc ions from the nanocomposite surface as well as the presence of graphene nanosheets. The result of this test is also consistent with the result of the zone of inhibition test (21, 22).

### 5. Discussion

In a study by Pietrzykowska et al., The flexural Young’s modulus of pure polylactic acid was 1603 ± 175 MPa (23). While with adding HA nanoparticle to polymeric matrix flexural Young’s modulus increased to 8104 ± 38 Mpa (23). In a study conducted by Sadudeethanakul et al., the flexural strength of polylactic acid-hydroxyapatite nanocomposite was investigated. It was found that the best result was obtained by adding 5% hydroxyapatite (24). In a study by Ko et al., the flexural modulus of polylactic acid nanocomposites containing hydroxyapatite was reduced compared to pure polylactic acid, because the interfacial adhesion was not sufficiently improved (25). In this study, the addition of nanoparticles also improved the flexural modulus. The addition of graphene and zinc oxide nanoparticles also improved the antibacterial properties of the optimal sample against the control sample. The results of this study are consistent with the results of research conducted by other researchers.

#### 5.1. Conclusions

The composite nanostructure is biodegradable, so there is no need for re-surgery to remove the implant from the body after repair. The flexural Yang modulus of the PLA-PCL-HA nanocomposite was 2139.037 ± 381.312 MPa. The presence of zinc oxide and graphene nanoparticles increased the Young’s modulus to 4363.636 ± 127.498 MPa. The Young’s modulus of the optimal sample close to the Young’s modulus of spongy bone. Due to the biodegradability of the implant and due to the temporary presence of the implant in the body tissue, it will cause a minimal immune response. Also, due to the antibacterial properties of the nanocomposite, the patient needs to take antibiotics is reduced. Therefore, this nanocomposite has sufficient potential for use in orthopedic surgeries in spongy bone.

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