Chitosan–gelatin enhanced antibacterial and biological properties of PLA and PGA braided threads for juvenile pseudomyopia treatment

Abstract

Polylactic acid (PLA) and polyglycolide acid (PGA) have been the most widely used biomedical materials in the acupoint catgut-embedding therapy (ACET) fields. However, a lack of antibacterial and biological properties is a definite obstacle that hinders its further application in juvenile pseudomyopia treatment. Here, four different kinds of PLA and PGA threads were fabricated, and were then modified using chitosan–gelatin coating method. Afterwards, PLA and PGA braided threads were fully characterized with respect to structure, mechanical and biological properties. The results showed that the surface roughness of PLA and PGA had been greatly improved; FT-IR analysis proved the existence of chitosan and gelatin molecules, the modified PLA (M-PLA-a = 87.1° ± 1.5°, M-PLA-b = 83.5° ± 1.4°) had smaller contact angle values than that of non-modified PLA (PLA-a = 108.6° ± 2.6°, PLA-b = 116.2° ± 3.1°), and that of the modified PGA (M-PGA-a = 79.3° ± 3.2°, M-PGA-b = 73.8° ± 1.8°) was smaller compared with that of the non-modified PGA (PGA-a = 98.2° ± 1.7°, PGA-b = 83.5° ± 1.4°). Based on the mechanical testing results, the tensile property and flexibility of samples increased slightly, and their swelling behavior also increased correspondingly. All the prepared samples exhibited non-toxicity with cell viability of more than 75%, and samples M-PGA-b presented the largest cell attachment ratios. In sum, this work exhibited great potential in the ACET application for juvenile pseudomyopia treatment.

Keywords

Polylactic acid (PLA)polyglycolide acid (PGA)acupoint catgut-embedding therapy (ACET)chitosan–gelatin juvenile pseudomyopia

Introduction

In terms of adolescent diseases, juvenile pseudomyopia has become one of the most serious diseases that threaten teenagers.¹,² According to the World Health Organization, there are about 2.6 billion people worldwide suffering from myopia in 2019, of which 12% is juvenile pseudomyopia; especially in children and adolescents, the rate of myopia is maintaining a constant upward trend.³,⁴ Regarding the common therapeutic methods for this disease, surgical treatment presents a serious risk for the youths, and traditional medical treatment may have some side effects.⁵ Thankfully, acupoint catgut-embedding therapy (ACET), a novel approach through embedded biodegradable materials into some part, or acupoints, of the human body, has attracted increasing attention in the treatment of juvenile pseudomyopia due to its numerous advantages, including safety, stable and lasting effects, etc.^6–8

As the functional part, acupoint catgut-embedding material (ACEM) plays a great role in the pseudomyopia treatment process.⁹ In brief, ACEM can be divided into the natural and synthetic materials according to their essential attribute.¹⁰ Catgut is a typical ACEM material with many advantages, including good biodegradability and biocompatibility, but it is likely to cause some infections due to its protein property.¹¹ Lately, with the rapid development of polymer materials technology, synthetic materials such as polylactic acid (PLA),¹² polyglycolide acid (PGA)¹³ and polycaprolactone (PCL),¹⁴ etc., have become the preferred ACEM in the biomaterials market; they offer many good characteristics like non-toxicity, reliable and easy-forming ability, but they lack some functional features such as antibacterial and biological properties, which limits their further development in the treatment process of pseudomyopia.^15–17 Thus, it is essential for researchers to combine the advantages of natural and synthetic biomedical materials, and to explore the possible paths and methods to resolve the conflicts between ACEM.

In recent years, natural or some highly water-soluble polymers such as chitosan,¹⁸ gelatin,¹⁹ hyaluronic acid,²⁰ etc., have been widely used for coating materials. The main advantages of these biomass materials are cheap, rich resources and easy availability.²¹ Due to the recognition of the issues of natural and synthetic ACEM materials, a series of strategies had been proposed to enhance their surface functional properties.^22–24 Hongsriphan and Sanga²⁵ applied chitosan solutions with different concentrations (0.25, 0.50, 1.00, and 2.00% w/v) to deposit an antibacterial layer on the surface of PLA; the results showed that the chitosan coating had enhanced the water vapor transmission rate, mechanical properties, and antibacterial activity against Staphylococcus aureus and Escherichia coli. Stoleru et al.²⁶ adopted cold radiofrequency plasma in a nitrogen atmosphere to first activate surface active sites of PLA, and then to graft chitosan molecular via carbodiimide chemistry, achieving stable antibacterial activity, surface performance and wet hydrophilicity properties. Zhang et al.²⁷ developed a chitosan-based tubular scaffold by chitosan/gelatin (4:1, w/w) complex solution coating, which showed a wall of 1.0 mm in thickness with a sandwich structure and a porosity of 81.2%, and vascular smooth muscle cells were observed to spread well on the scaffold. Hseh et al.²⁸ selected PGA to modify chitosan matrices, and fabricated three different types of both dense and porous composite matrices; these results indicated that the surface hydrophilicity, water absorption rate and swelling ratio were greatly improved after modification. However, the surface modification process and its further mechanism in PLA and PGA have not been clarified clearly, and most of the studied objects are patches, stents or films; the braided threads used for ACET are seldom mentioned.

In this work, four kinds of PLA and PGA braided threads with different ratios were fabricated, and then subjected to surface coating with chitosan–gelatin antibacterial agents. Their comprehensive properties, including surface, mechanical and biological properties were fully studied, and the feasibility of PLA and PGA applied for ACET were estimated according to these experiment results.

Materials and methods

Materials

PLA multifilament (Linear density = 2.74 tex, number of fiber bundles = 6), PGA multifilament (Linear density = 3.18 tex, number of fiber bundles = 6), chitosan powder with a degree of 85% deacetylation was provided by Alfa Aesar Co., Ltd (Haverhill, United states), gelatin powder and acetic acid 99.5% (V/V) were provided by Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). All chemicals are analytically pure and used without further purification.

Fabrication of PLA and PGA braided threads

Figure 1 shows the fabrication process of PLA and PGA braided threads. Four kinds of braided threads were prepared using a 2D spindle braiding machine (Figure 1(a)), and six shell strands made up the threads of PLA and PGA. Moreover, the carriers were taken along the PLA or PGA multifilaments to form braided threads with the motion of twirling around. In this work, four kinds of threads with different diameters were fabricated, which were named as PLA-a, PLA-b, PGA-a and PGA-b (Figure 1(b)).

Figure 1.

Schematic diagram illustrating the fabrication process of PLA and PGA braided threads: (a) 2 D Spindle braiding machine, (b) braiding areas of multifilament.

Surface modification of PLA and PGA braided threads

Figure 2 shows the surface modification of PLA and PGA braided threads. Chitosan powder was dissolved in acetic acid solution to form an antibacterial coating agent with a concentration of 2%, and gelatin power was dissolved in deionized water, and mixed with chitosan agents with a ratio of 1:1 to prepare coating agents. Gelatin solution was prepared using gelatin power at the temperature of 60°C, and chitosan–gelatin modified agents were prepared at 80°C. Meanwhile, alcohol (7%(v/v)) was used to remove dust and oil stains from the surface of PLA and PGA braided threads. Afterward, PLA and PGA threads were immersed into the mixed chitosan/gelatin coating solutions for 30 min, and dried in the drying oven to remove the water molecules on the surface and interior of samples. In the present work, four kinds of antibacterial coating samples were prepared and coded as M-PLA-a, M-PLA-b, M-PGA-a and M-PGA-b.

Figure 2.

Schematic diagram of surface modification of PLA and PGA braided threads.

Characterizations

Structural properties

Surface morphology

A scanning electron microscope (SEM) Hitachi TM3000 at 15kV was adopted to evaluate the surface morphology of samples, and the non-modifed and modified samples were evaluated by comparing the sample images.

FT-IR analysis

The chemical structures of the samples were tested by FT-IR spectrometer (Spectrum two; PerkinElmer, UK). Samples (2 g) were cut into pieces and ground into powder, and then mixed with potassium bromide powder; FT-IR spectrum was recorded with a resolution of 4 cm⁻¹ at room temperature over 16 cumulative scans in a wave number range of 4000 cm⁻¹ to 450 cm⁻¹.

Surface hydrophilicity

Surface hydrophilicity of samples was measured by an OCA15EC water contact angle measuring instrument (Defei Instrument Co. Limited, Shanghai, China). Onto the surface of the sample was dropped small amount of deionized water, and three separated points were measured by microscope and the average contact angle of samples was calculated.

Mechanical properties

Tensile property

The tensile properties of samples were evaluated by an electronic single-yarn tensile tester (YG061f, Shandong Laizhou electron instrument Co., Ltd, Shanghai, China) according to the standard GB/T14344.²⁹ Breaking strength and breaking elongation were adopted to evaluate the tensile properties of samples. The final result was expressed with an average value and a standard deviation of 10 replicates.

Flexibility

Based on the studied literature,³⁰ bending stiffness was used to evaluate the flexibility of samples, and it was measured according to the cantilever beam principle. The calculation formula (equation (1)) is as follows:

B = \frac{(F \times L^{2})}{f}

(1)

In which: B (cN·cm²) refers to the bending stiffness; L (cm) refers to the length of monofilament hanging in the air; F (cN) refers to the loading force on the hanging part of monofilaments; f (cm) refers to the deflection of monofilaments loading end.

Swelling behavior

Swelling ratio was used to evaluate the swelling behavior of samples. Samples were immersed in phosphate buffered saline (PBS) solutions, and then incubated at 37°C in a shaker bath at 100 RPM for a certain time. The swelling ratio was calculated by equation (2) as follows:

Swelling ratio (%) = \frac{D - d}{d} \times 100 %

(2)

where: D (mm) refers to the diameter of monofilament after incubation time; d (mm) refers to the diameter of monofilament before incubation time.

Biological properties

Antibacterial activity

The antibacterial activity of modified PLA and PGA threads was measured by the agar culture method, which was based on the standard ISO 20645:2004. Escherichia coli and Staphylococcus aureus were selected as they are commonly seen in infection issues. A sterilized agar plate was used for Escherichia coli and Staphylococcus aureus with a concentration of 1 × 10⁶ CFU/ml. The tested threads were placed against the agar surface to ensure close contact, and the plates were incubated at 37°C for 24 h. Finally, the zones of inhibition of samples were achieved, and calculated by equation (3) as follows:

Z = \frac{D - d}{2}

(3)

In which: Z (mm) is the inhibition zone, D (mm) is the total diameter of the specimen and inhibition zone, and d (mm) is the total diameter of the specimen.

Cell viability

A cell-counting kit-8 (CCK-8) cell proliferation method was adopted to estimate the cell viability of threads. Samples were immersed in an alcohol solution (99.5% (v/v)) for 3 h to remove the extra impurities, and then washed with PBS solution. The leach liquor was gained after culturing samples in the Dulbecco’s modified Eagle’s medium (DMEM) for 48 h, and second-generation rat fibroblasts with a concentration of 2 × 10⁴ ml were seeded into a 96-well plate. Afterwards, the leach liquor was dropped into the well plate, and the cell viability was tested by the CCK-8 assay; the optical density (OD) value was obtained using a microplate reader at a wavelength of 450 nm. Finally, the relative growth rate (RGR) was calculated by equation (4) as follows:

RGR (%) = \frac{{O D}_{1}}{{O D}_{2}} \times 100 %

(4)

In which: OD₁ refers to the OD values of the sample; OD₂ refers to the OD values of the control.

Cell attachment and its morphology

Cell attachment ratio and its morphology were used to evaluate the cell attachment property of samples. A detailed description can be found in our previous work,³¹ and cell attachment and morphology were observed with a phase contrast microscope (Nikon). The percentage of C2C12 coverage on control and modified samples were calculated and analyzed using image 6.0 software. This experiment was repeated three times and each specimen was measured at five different positions.

Statistical analysis

Statistical analysis was performed using Origin (Origin lab, USA). Results were averaged and expressed as means and standard deviation. Analysis of variance (ANOVA) was used for determining the statistical difference between samples. The data in the discussion of results are marked by “for p < 0.05.”

Results and discussion

Structure properties of the prepared samples

Surface morphology

Figure 3 shows the SEM images of the longitudinal views of PLA and PGA braided threads. The non-modified samples (PLA-a, PLA-b, PGA-a, PGA-b) had relatively smooth surface morphologies, while the modified samples (M-PLA-a, M-PLA-b, M-PGA-a, M-PGA-b) presented more complex surfaces, including coating layer and some fragments. Moreover, the modified samples showed larger diameters than the non-modified ones, which was caused by the chitosan–gelatin coating treatment, and the non-modified samples with different multifilament achieved a variety of diameter changes after the modification process.

Figure 3.

SEM images of the longitudinal views of non-modified and modified PLA and PGA braided threads.

FT-IR analysis of the prepared samples

Figure 4 shows the FT-IR analysis of non-modified and modified PLA and PGA braided threads. PLA braided threads were observed to have four absorption bands at 1089.58 cm⁻¹, 1766.48 cm⁻¹, 2915.84 cm⁻¹ and 3461.59 cm⁻¹. The sharp band at 1089.58 cm⁻¹ was attributed to the bending vibration of (C-O), and the absorption bands at 1766.48 cm⁻¹, 2915.84 cm⁻¹ and 3461.59 cm⁻¹ confirmed the presence of (C = O), (C-H) and (O-H). Hence, PLA braided threads presented the typical structure of aliphatic polyester. Moreover, the modified PLA braided threads had changing curves similar to the PLA ones, while some new peaks including 1186.01 cm⁻¹, 1463.71 cm⁻¹ and 1641.12 cm⁻¹ appeared, attributed to the presence of (C-O-C), (N-H) amide II bending groups, and (C = O) amide I in the chitosan and gelatin structures. In addition, the modified PGA showed sharp peaks at 3046.97 cm⁻¹, 1702.84 cm⁻¹, 1403.92 cm⁻¹, 1159.01 cm⁻¹ and 1062.68 cm⁻¹. Among these, the sharp band at 1702.84 cm⁻¹ was attributed to the bending vibration of (C = O), and the absorption bands at 1403.92 cm⁻¹, 1159.01 cm⁻¹ confirmed the presence of (-CH2) and (-CH2-COO-). These findings revealed that chitosan–gelatin coating agents had successfully covered the surface of PLA and PGA, and no interactions existed between the fibers and coating layers.

Figure 4.

FT-IR analysis of non-modified and modified PLA and PGA braided threads.

Surface hydrophilicity

Figure 5 shows the contact angle values of PLA and PGA braided threads. The modified threads had smaller contact angle values than the controls, indicating that the surface hydrophilicity of samples had improved after the coating treatment. In consideration of PLA threads, samples PLA-a (108.6° ± 2.6°) and PLA-b (116.2° ± 3.1°) showed significant difference from samples M-PLA-a (87.1° ± 1.5°, p < 0.05) and M-PLA-b (83.5° ± 1.4°, p < 0.05), respectively. Meanwhile, similar was observed among the PGA threads: sample PGA-a (98.2° ± 1.7°) presented the significant larger contact angle value compared with sample M-PGA-a (79.3° ± 3.2°, p < 0.05), while the contact angle of PGA-b (91.4° ± 2.5°) was larger than that of sample M-PGA-b (73.8° ± 1.8°, p < 0.05). In conclusion, these findings illustrated that the contact angle values of PLA and PGA became smaller after modification, and the PGA threads showed better hydrophilicity than the PLA ones.

Figure 5.

Contact angle values of non-modified and modified PLA and PGA braided threads.

Mechanical properties of the prepared samples

Tensile performance

Figure 6 shows the tensile properties of non-modified and modified PLA and PGA threads. Both the breaking strength and breaking elongation values of PLA and PGA were observed to increase after coating treatment, while the changes in tensile properties were different. Taking PLA as example, the non-modified PLA threads (PLA-a and PLA-b) presented the breaking strength values at 42.73 ± 3.61 cN/dtex, 58.34 ± 1.58 cN/dtex, (p < 0.05), respectively. The modified PLA threads (M-PLA-a and M-PLA-b) were 53.17 ± 2.34 cN/dtex, 69.15 ± 3.42 cN/dtex, (p < 0.05), respectively. These findings revealed that the breaking strength of samples could be enhanced by the chitosan–gelatin coating treatment, which was caused by the chitosan–gelatin layer and some fragments covering their surfaces. This coating treatment could also improve the molecular arrangement of threads, further causing the increase of breaking strength values.

Figure 6.

Tensile properties of non-modified and modified PLA and PGA braided threads.

Regarding the breaking elongation of PLA and PGA, the modified PLA threads (M-PLA-a =38.75% ± 4.76%, M-PLA-b = 43.16% ± 3.72%) exhibited much larger breaking elongation values than the non-modified PLA threads (PLA-a = 27.82% ± 2.31%, PLA-b = 37.29% ± 1.28%, p <0.05). M-PGA-a (58.12% ± 4.26%) had larger breaking elongation values compared with PGA-a (42.17% ± 2.18%, p < 0.05). These findings illustrated that the chitosan–gelatin coating was beneficial for the breaking elongation of PLA and PGA, and PLA showed larger breaking elongation values than PGA.

Swelling behavior

Figure 7 shows the swelling behavior of non-modified and modified PLA and PGA threads. It is obvious that PGA presented a larger swelling ratio value compared with PLA. For example, sample PGA-b (11.49% ± 2.15%) showed a larger swelling ratio than sample PLA-b (3.65% ± 0.27%, P < 0.05), and the swelling ratio value of sample M-PGA-b (28.19% ± 1.54%) was larger than that of sample M-PLA-b (8.53% ± 1.54%, P < 0.05). Moreover, sample M-PLA-b exhibited larger swelling ratio values compared with sample PLA-b, revealing that the coating modification was beneficial for the swelling behavior of PLA. A similar effect was observed for PGA; for instance, sample M-PGA-a (21.36% ± 0.61%) presented much difference from that of sample PGA-a (10.25% ± 1.87%, p < 0.05). This phenomenon could be explained by the arrangement of molecular chains mechanism for PLA and PGA; this modification was beneficial for the inner distance among the molecular chains of samples, further enhancing the swelling ability of PLA and PGA. In sum, the coating treatment could enhance the swelling behavior of both PLA and PGA, and the modified PGA exhibited a larger swelling ratio compared with that of modified PLA.

Figure 7.

Swelling behavior of non-modified and modified PLA and PGA braided threads.

Flexibility property

Figure 8 shows the flexibility properties of non-modified and modified PLA and PGA braided threads. In this research, bending stiffness was adopted to evaluate the flexibility of PLA and PGA. PGA exhibited larger bending stiffness values compared with PLA. In particular, M-PGA-b (28.19 ± 1.54 cN·mm²) exhibited larger bending stiffness than M-PLA-b (8.53 ± 1.16 cN•mm², p < 0.05), and M-PGA-a (21.36 ± 0.61 cN·mm²) presented a much larger bending stiffness value than M-PLA-a (7.52 ± 1.26 cN·mm², p < 0.05). In addition, M-PGA-b was observed to have a much larger bending stiffness than PGA-b (12.73 ± 2.18 cN·mm², p < 0.05), while that of M-PGA-a was larger compared with PGA-a (10.25 ± 1.87 cN•mm², p < 0.05). These results illustrate that the chitosan–gelatin coating treatment could improve the flexibility of PLA and PGA, and PGA offered greater stiffness and less flexibility compared with PLA. The molecular arrangement of PLA and PGA become much more ordered after modification, and this coating modification method could enhance the rigidity of both the PLA and PGA.

Figure 8.

Flexibility properties of non-modified and modified PLA and PGA braided threads.

Biological properties

Antibacterial activity

Figure 9 showed the zones of inhibition values of non-modified and modified PLA and PGA braided threads. The non-modified samples (PLA-a, PLA-b, PGA-a, PGA-b) were observed to present no antibacterial activity, while the coated samples (M-PLA-a, M-PLA-b, M-PGA-a, M-PGA-b) showed effective antimicrobial activity against Escherichia coli and Staphylococcus aureus. These findings revealed that the chitosan–gelatin coating treatment could improve the antibacterial activity of PLA and PGA, and the modified PLA showed much larger zones of inhibition compared with the non-modified PLA. Meanwhile, the modified PGA presented larger zones of inhibition compared with non-modified PGA, indicating that the PGA achieved better antibacterial capacity after surface modification. The surface of PGA was covered by more chitosan coating compared with that of PLA, enhancing the antibacterial activity of the threads.

Figure 9.

Antibacterial activity of non-modified and modified PLA and PGA braided threads.

Cell viability

Figure 10 shows the cell viability of non-modified and modified PLA and PGA braided threads. The cell cytotoxicity of prepared samples was determined by their leach liquors after incubation with C2C12 rat fibroblasts for 48 h. Both the PLA and PGA presented non-toxicity, with more than 75% of cells being viable. The modified samples showed much larger cell viability values compared with the non-modified ones. Sample M-PLA-a (90.4 ± 2.5%) had larger cell viability than sample PLA-a (80.3 ± 1.3%), and M-PGA-a (91.5 ± 2.4%) presented larger cell viability compared with sample PGA-a (80.6 ± 1.8%). These findings were consistent with the theory that chitosan could promote cell proliferation and biocompatibility of PLA and PGA, and the modified PGA with better cell viability showed greater potential in the application of ACET.^32–34

Figure 10.

Cell viability of non-modified and modified PLA and PGA braided threads.

Cell attachment

Figure 11 shows the cell attachment of non-modified and modified PLA and PGA braided threads. In this work, the cell attachments of the modified and non-modified samples were evaluated by fluorescent staining, and expressed as fluorescence area proportion values of the whole area. In detail, sample M-PLA-b (62.4% ± 1.5%) exhibited significant larger cell attachment than sample PLA-b (40.3% ± 3.6%, p < 0.05), while the cell attachment of sample M-PLA-a (54.7% ± 4.2%) was larger than that of sample PLA-a (35.2% ± 1.4%, p < 0.05). Moreover, the results in PGA threads were found to be similar to those in PLA threads; sample M-PGA-b showed the largest cell attachment of 74.5% ± 4.6%, which was much larger than that of sample PGA-b (56.8% ± 2.9%, p < 0.05), and the cell attachment of sample M-PGA-a (68.3% ± 3.7%) was much larger compared with that of sample PGA-a (41.5% ± 2.4%, p < 0.05). These results indicate that the chitosan–gelatin coating modification had effectively improved the surface roughness and hydrophilicity of PLA and PGA, further enhancing the cell attachment of samples.

Figure 11.

Cell attachment of non-modified and modified PLA and PGA braided threads.

Figure 12 shows the cell morphology of non-modified and modified PLA and PGA braided threads. The non-modified samples were observed to attach relatively small number of cells, while sample PLA showed little difference to sample PGA cultured for 48 h. In consideration of modified threads, cells attached to the surface of samples were increased, and cell growth around the modified PGA was slightly larger compared with that of PLA. These findings revealed that the chitosan–gelatin coating treatment had successfully enhanced the cytocompatibility of samples, and PGA offered better cell attachment ability compared with that of PLA.

Figure 12.

Cell morphology of non-modified and modified PLA and PGA braided threads.

Conclusion

Different types of PLA and PGA embedding materials were prepared using a chitosan–gelatin coating method, and their characteristics such as structural properties, mechanical properties and biological properties were tested to verify their feasibility. The surfaces of both PLA and PGA were observed to be covered by chitosan–gelatin coating layers and some fragments. The modified samples exhibited larger diameters compared with non-modified ones, while the non-modified samples gained varied of diameter changes after the chitosan–gelatin modification process. The mechanical performance of samples was greatly enhanced after modification, and PLA presented larger breaking elongation values compared with PGA. The biological properties revealed that all the samples were non-toxic, and the modified PGA with a greater amount of chitosan–gelatin coating had better antibacterial activity, including antibacterial efficacy against Escherichia coli and Staphylococcus aureus, than that of PLA. Sample M-PGA-b, with the largest cell attachment value at 74.5% ± 4.6%, had greatest alternative potential for application on ACEM.

In sum, this work may promote the development of PLA and PGA with good antibacterial and biological properties, satisfying the clinical application for treatment of juvenile pseudomyopia.

Footnotes

Acknowledgements

This work is Sponsored by Shanghai Sailing Program (20YF1401000), the Fundamental Research Funds for the Central Universities, the Initial Research Funds for Young Teachers of Donghua University, Engineering Research Center of Technical Textiles, Ministry of Education, Donghua University and the 111 Project (BP0719035).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Shaoju Fu

Peihua Zhang

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