Evaluating the Oral Wound Healing Properties and Antimicrobial Efficacy of Sulfonated Shrimp Chitosan

Abstract

Objective

This study evaluated the oral wound healing efficacy and fibroblast proliferation induced by sulfonated Vannamei shrimp chitosan, compared with commercial chitosan. Additionally, its antimicrobial activity against oral microorganism Streptococcus mutans, Staphylococcus aureus, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis was assessed.

Materials and Methods

In the wound healing study, three groups of rats were treated with Aquades (negative control), commercial chitosan (positive control), and sulfonated shrimp chitosan, with healing time and fibroblast proliferation measured. The antimicrobial study tested different concentrations (1, 3, 5, and 7%) using a disk diffusion method.

Results

Sulfonated shrimp chitosan showed the fastest wound healing (5 ± 0.47 days) compared with commercial chitosan (6 ± 0.0 days) and Aquades (6.6 ± 0.6 days) (p = 0.036), and significantly higher fibroblast counts (20.32 ± 0.688, p < 0.001). Antimicrobial activity was moderate, with the highest inhibition at 3 or 5%. The ANOVA (analysis of variance) test was used for hypothesis testing with the significance level at p <0.05, followed by a Kruskal–Wallis test.

Discussion

Sulfonated chitosan demonstrated superior wound healing properties, consistent with studies showing enhanced tissue regeneration by sulfonated chitosan derivatives. Antimicrobial effects were moderate, with peak activity at intermediate concentrations and a decrease at 7%, possibly due to a saturation effect.

Conclusion

These findings suggest sulfonated shrimp chitosan could be a promising agent in wound healing and infection control.

 

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Keywords

sulfonated chitosan - antimicrobial activity - wound healing

Introduction

The human body, when faced with an injury, will undergo a series of sequential phases—hemostasis, inflammation, proliferation, and remodeling—as a healing and recovery mechanism.[1] In the oral cavity, wounds are exposed to a constant flow of saliva, which is warm, nutrient-rich, and has a near-neutral pH, making it an ideal place for millions of microorganisms. In the inflammatory phase, there are complications due to the invasion of microorganisms into the oral cavity which can delay recovery. The inflammatory phase is responded to by the accumulation of macrophages, protective cells that promote the healing process.[2] [3] [4] The administration of wound healing agents has the potential to accelerate the hemostasis phase, prevent the penetration of microorganisms, support and accelerate the wound healing process. Wound healing agents are widely available on the market, but have disadvantages such as the potential to cause allergies, form granulomas, interfere with bone healing, and cause foreign-body reactions.[5] [6]

Chitosan made from shrimp and lobster shells yields the most common form of chitosan in nature, α-chitosan. Chitosan extracted from mollusks yields ɣ-chitosan, while chitosan extracted from squid yields β-chitosan.[7] [8] In many studies, chitosan has been investigated as a topical hemostatic agent due to its biodegradable, nontoxic, antioxidant, and antibacterial properties that can be absorbed by the body. Previous studies have shown that chitosan is nontoxic when taken orally. Currently, most chitosan is made from seafood waste, such as shrimp and crab shells.[9] Chitosan can be developed in the form of gels, sponges, tablets, and films. In dentistry, wounds are often accompanied by mucosal bleeding, therefore chitosan is an appropriate material to reduce inflammation and accelerate cell growth in wound healing especially in the proliferation phase. The biocompatible, anti-inflammatory, and antibacterial properties of chitosan are the main reasons for using this drug, which is easily available at affordable prices. The involvement of chitosan in promoting the healing process by stimulating the immune system to stimulate the cytokine Interleukin-1, which stimulates the proliferation of damaged tissue fibroblast cells through macrophages. Fibroblast cells stimulate the production of collagen, hyaluronic acid, elastin, fibronectin, and proteoglycans stimulated by the cytokine tumor growth factor (TGF-β), regenerating tissues, and contributing to the formation of new blood vessels. The production of fibroblast cells and new blood vessels slowly decreases as the wound healing process enters the regeneration phase.[10] [11]

Suherman et al reported that chitosan from Vaname shrimp skin at concentrations of 1, 3, 5, and 7% was able to inhibit the growth of Staphylococcus epidermidis, Pseudomonas aeruginosa, Propionibacterium acnes, and Escherichia coli bacteria where the most effective concentration was at 7% chitosan concentration with an average inhibition zone diameter of approximately 15.21 mm.[12] Varun et al showed that chitosan from shrimp inhibits Enterococcus faecalis, E. coli, Staphylococcus aureus, Enterobacter aerogenes.[13] The aim of this study was to evaluate the oral wound healing efficacy and fibroblast proliferation stimulated by sulfonated Vaname shrimp chitosan in comparison to commercial chitosan, while also assessing its antimicrobial activity against key oral and wound pathogens (Streptococcus mutans, S. aureus, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis).

 

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Materials and Methods

Preparation of Chitosan

Vaname shrimp shells were dried using an oven at a temperature of 70°C, then mashed using a pestle and a large stone mortar and sieved using a 200 mesh sieve. The dried material was mixed with 0.1M NaOH solution with a ratio of 1:10 to obtain a solid solution ratio in the deproteinization process. To get the results of chitin, the chitin must go through mixing with a temperature of 70°C, then stirred for 1 hour and stirred for 1 hour, then stirred for 1 hour and filtered using gauze as many as four layers with the help of a vacuum pump on a hot plate. To neutralize the pH of chitin, distilled water is used so that Litmus paper as an indicator shows a neutral pH. The next process is the dehydration stage, at this stage the chitin product is mixed twice with methanol and once with acetone, then dried in the oven at 60°C for 1 hour in a glass tray. The dried chitin was then cooled in a desiccator and weighed with analytical scales to be stored in polyethylene bottles. Preparation of chitosan, by mixing the dried chitin with 45% NaOH slowly and gradually in a three-neck boiling flask to get a ratio of 1:20 which will be heated at 140°C for 1 hour. After obtaining the chitosan, it was dried in the oven for 24 hours at a temperature of 80°C. The sulfonation process was carried out using chlorosulfonic acid as a reagent in dichloromethane solvent for 6 hours at 65°C to get chitosan sulfonate.

 

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Fourier-Transform Infrared Spectroscopy

Fourier-transform infrared (FTIR) spectroscopy was used to measure the degree of deacetylation of chitosan. This spectrum was taken by scanning in the wavenumber region of 4,000–500 cm⁻¹. The baseline method can be used to analyze the FTIR results. The FTIR results show that there is a breakdown of chitin groups into chitosan for each treatment, even though not complete.[14]

 

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Antibacterial Test

Antibacterial tests against S. mutans (ATCC 31987), S. aureus (ATCC 25923), P. gingivalis (ATCC 33277), and A. actinomycetemcomitans (ATCC 29522) were performed.

The antibacterial activity test of sulfonated chitosan obtained from shrimp shell sources against S. mutans, S. aureus, P. gingivalis, and A. actinomycetemcomitans was performed using the well diffusion method on Mueller-Hinton Agar (MHA) media. The test was conducted with seven treatment groups, each treated four times. Wells were made on MHA media that had been inoculated with S. mutans, S. aureus, P. gingivalis, and A. actinomycetemcomitans with a perforator of 6 holes with a diameter of 6 mm and a depth of 4 mm. Vaname shrimp skin sulfonated chitosan with concentrations of 1, 3, 5, and 7%, as well as negative control distilled water and positive control were each added (0.5 µL) in the wells that had been made. Petri dishes filled with media, bacteria, and extracts were incubated at 37°C in an incubator for 1 × 24 hours. The zone of inhibition formed was measured using a caliper.

 

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Preparation of Chitosan Gel

Chitosan was mixed with Na-CMC, then added propylene glycol, glycerin, nipagin, and triethanolamine (TEA) and stirred until homogeneous. Chitosan was dissolved with distilled water at 50°C in another mortar, Na-CMC mucilage was added, and stirred for 15 minutes until a gel mass was formed. After that, the gel was stored in a vial bottle.[5]

 

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Wound Making in Wistar White Rats

The subjects were 15 Wistar white rats (Rattus norvegicus) aged 12 to 14 weeks and weighing 200 g obtained from the Animal House of the Faculty of Veterinary Medicine, Universitas Syiah Kuala. The Wistar rats were divided into three groups with details of the treatment group, positive control group, and negative control. All rats were given food and water and placed in insulated polypropylene cages with 12 hours of light and 12 hours of darkness. The rats were given xylazine (1–2 mg) and ketamine (10 mg/kg/body weight) intramuscularly to induce relaxation and sedation effects. A 5 mm incision was made in the mandibular gingival mucosa of the rats, with the tissue depth reaching the alveolar bone.[5] [15]

 

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Procedure of Chitosan Application on Wound Incision

Immediately after the incision was made, chitosan gel was applied to the mandibular gingival mucosa directly topically along the wound, and then for the penetration of chitosan gel in the gingival tissue of the rats, the lower lip was retracted for 1 minute. The chitosan gel was applied twice daily in the morning and evening with an interval of 8 hours. The administration of chitosan gel was performed every day for 14 consecutive days regularly. The positive control group of rats was given commercial chitin chitosan, and the negative control group was given distilled water.

 

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Histological Wound Healing Observation

Histological preparation was performed after collection of gingivae of euthanized rats. Observation of fibroblast cell density was performed with a light microscope at 400 times magnification. Observations were made based on the calculation of each field of view until five fields of view were counted with Top View software.[15]

 

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Ethical Issues

Ethical eligibility was approved by the Ethics Commission of the Faculty of Dentistry, Syiah Kuala University (ethics number 543/KE/FKG/2024).

 

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Data Analysis

The research data were analyzed using SPSS and hypothesis testing was performed with the ANOVA (analysis of variance) test with significance level at p <0.05, followed by the Kruskal– Wallis test.

 

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Results

FTIR Analysis

Antimicrobial Activity

The antimicrobial activities of S. mutans, S. aureus, A. actinomycetemcomitans, and P. gingivalis are provided in this article.

 

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Discussion

The results of FTIR analysis in [Fig. 1](A,B) show that the degree of deacetylation obtained in the sulfonated chitosan used for research is 47.62%. The absorption at wavenumbers 3,000 to 3,600 cm⁻¹ shows –OH and N–H stretching vibrations of primary and secondary amine functional groups and hydrogen bonds in the chitosan structure. The absorption at wavenumbers 2,800 to 3,000 cm⁻¹ with peaks at wavenumbers 2,833, 2,873, and 2,966 cm⁻¹ showed C–H stretching vibrations of asymmetric CH₂, C–H of pyranose rings, and CH₃ (NHCOCH₃).[14] [16] The presence of amide I, amide II, and amide III is indicated by absorption with peaks at wavenumbers 1,658, 1,550, and 1,259 cm⁻¹ derived from C = O stretching vibrations, N–H bending vibrations, and C–N stretching vibrations from NHCOCH₃ functional groups. Bending vibrations of C–H are shown at wavenumbers 1,421, 1,371, and 1,320 cm⁻¹ from the functional groups CH₂ (e.g., CH₂OH), CH₃ (e.g., NHCOCH₃), and pyranose ring. Symmetric and asymmetric C–O–C stretching vibrations are shown at wavenumber peaks of 1,153 and 1,072 cm⁻¹ (glycosidic bond). C–O stretching vibrations derived from primary and secondary −OH functional groups were shown at wavenumbers 1,008 and 950 cm⁻¹. The presence of pyranose ring is evidenced by the peak at wavenumber 894 cm⁻¹.[14] [16]

Fig. 1 FTIR spectrum of (A) chitosan from Vaname shrimp, and (B) sulfonated chitosan. FTIR, Fourier-transform infrared.

The deacetylation process determines the percentage of chitosan formed. The higher the percentage of deacetylation degree, the better the chitosan produced. The degree of deacetylation value ranging from 40 to 100% is said to be chitosan. A low degree of deacetylation indicates that the disconnection of acetyl groups on chitin does not occur completely. Chitosan is said to be completely deacetylated if the deacetylation degree value is >90%. The degree of deacetylation is influenced by various factors, such as the concentration of base used, temperature, reaction time, the ratio between chitin and alkaline solution, and particle size. Process time that is too short can cause the deacetylation process to not take place completely, where the hydroxyl addition from NaOH does not have enough time to eliminate the acetyl group. The longer the process time, the longer the reaction that takes place, so that more NaOH molecules are added to the chitin molecule, causing more acetyl groups to be released, thereby the degree of deacetylation increases.[17]

The level of deacetylation reveals the percentage of glucosamine units present in the chitosan molecule. Chitosan has inherent antibacterial properties, preventing bacterial growth. The binding of chitosan chains to negatively charged bacterial cell walls causes cell rupture and membrane permeability alterations. The stage that followed involves binding to DNA, which limits replication and causes cell death. Chemical alterations to chitosan, such as sulfonation, can drastically modify its antibacterial properties. Chitosan interacts differently with gram-positive and gram-negative bacteria due to their unique cell-wall compositions. According to certain studies, chitosan has a stronger bactericidal impact on gram-negative bacteria than gram-positive bacteria due to the increased attraction of amino groups for anionic radicals in the cell wall.[18] Chitosan with a deacetylation degree of 47.62% showed the strongest antibacterial activity against the gram-negative bacteria A. actinomycetemcomitans in this investigation. Compared with gram-positive bacteria, gram-negative bacteria have more hydrophilic and negatively charged cell walls. Chitosan thus showed enhanced interaction with gram-negative bacteria, boosting its antibacterial effectiveness against them.[19]

The antibacterial mechanism of chitosan may include interaction of cationic chitosan with anionic cell surfaces, increased membrane permeability, and leakage of cellular material from cells. Chitosan can also interfere with mRNA synthesis and the adherence of protein synthesis. Chitosan nanoparticles inhibit bacterial adherence and prevent biofilm development according to the study by Valian et al.[20] Chitosan works by a more intricate mechanism than straightforward electrostatic interactions. They enter the cell wall and affect the synthesis of proteins or DNA/RNA. Chitosan (≤50 kDa) can impede DNA transcription by penetrating the cell wall.[21] Numerous microorganisms have been shown to be inhibited by chitooligomers. The antimicrobial action of chitosan oligomers may result from attaching to the bacteria's surface and interfering with their metabolism, or from binding to the DNA after entering the cell and preventing the transcription of DNA and RNA.[13]

In this investigation on gram-positive bacteria, sulfonated chitosan of concentration 3% inhibited S. Aureus more strongly than S. mutans. The study found that sulfonated chitosan 5% was more effective against gram-negative bacteria A. actinomycetemcomitans than P. gingivalis ([Table 1]). The current 1 to 7% concentration gradient exhibits maximum activity at 3 to 5% but decreased efficacy at 7% ([Fig. 2]); according to earlier studies, chitosan's antibacterial efficacy against gram-positive S. aureus rose as the molecular weight increased. Furthermore, chitosan's antibacterial efficacy for gram-negative E. coli rose as molecular weight decreased.[22] A higher inhibition zone may occur at a concentration of 3 to 5% because the solution's viscosity is still low, allowing it to diffuse better in the agar medium than at a concentration of 7%, which has a high viscosity, making it too thick and difficult to diffuse well in the agar medium.[23]

Fig. 2 Antimicrobial effect from four bacterial species.

Table 1 Inhibitory value of sulfonated chitosan in four bacterial species

Species	Concentration	R1	R2	R3	R4	Sum	Mean ± SD
S. mutans	Negative control	0	0	0	0	0	0
	Amoxicillin	47.9	43.3	44	15.5	151.05	37.7 ± 12.7
	1%	10.65	0	4.35	4.85	19.85	4.9 ± 3.7
	3%	5.85	8.75	5.2	9.25	29.05	7.2 ± 1.7
	5%	9.05	7.5	0	10	26.55	6.6 ± 3.9
	7%	0	0	4.35	0	4.35	1.08 ± 1.8
S. aureus	Negative control	0	0	0	0	0	0
	Ciprofloxacin	23.35	23.6	24.4	22.85	94.2	23.55 ± 0.6
	1%	9.35	6.75	8.6	10.05	34.75	8.7 ± 1.4
	3%	14.15	8.4	9.15	8.7	40.4	10.1 ± 2.7
	5%	7.9	10.9	10.25	10.25	39.3	9.8 ± 1.3
	7%	10.05	8.95	10.05	8.55	37.6	9.4 ± 0.7
A. actinomycetemcomitans	Negative control	0	0	0	0	0	0
	Tetracycline	29.9	26.05	25.25	26.75	107.95	26.98 ± 2.04
	1%	8.35	9	16.5	0	33.9	8.47 ± 7.38
	3%	6.35	9.9	8.33	10.9	35.48	8.87 ± 1.99
	5%	7.2	0	17.5	17.8	42.5	10.6 ± 8.63
	7%	4.9	10.9	12.4	0	28.2	7.05 ± 5.71
P. gingivalis	Negative control	0	0	0	0	0	0
	Metronidazole	25.6	24.2	26.25	19.95	96	24 ± 4.90
	1%	0	10.7	5.9	0	16.6	4.15 ± 8.96
	3%	6.4	7.25	4.55	9.3	27.5	6.87 ± 3.41
	5%	7.95	9.5	7.75	3.15	28.35	7.08 ± 4.74
	7%	7.45	8.45	0	8.45	24.35	6.08 ± 7.07

Abbreviations: R, replication; SD, standard deviation.

The inhibitory effect against S. mutans was reported to be that chitosan interfered with S. mutans adhesion and primary biofilm formation for up to 1 week with little or no decrease in efficiency. In addition, chitosan caused a significant decrease in the survival of mature biofilms.[24] Chitosan works against S. aureus by altering its cellular ultrastructure, which results in thicker chromatin, collapsing cell walls, and a rise in intracellular structures including vacuoles and cell detritus.[25] Chitosan's capacity to inhibit A. actinomycetemcomitans reduces the quantity of colonies.[26] The inhibitory mechanism of chitosan against P. gingivalis bacteria involves electrostatic interactions between the polycationic structure and anionic-dominant components on the surface of P. gingivalis, such as gram-negative lipopolysaccharide and cell-surface proteins play a major role in antibacterial activity.[27] Chitosan is directly related to the absorption of polysaccharides in bacteria and causes changes in the cell-wall structure and increases the permeability of the cell membrane, causing cell death.[28]

The results of this study demonstrate that sulfonated Vaname shrimp chitosan significantly accelerates wound healing and promotes fibroblast proliferation in comparison to both a negative control (distilled water) and a positive control (commercial chitosan; [Fig. 3A], [B]). The faster wound healing observed in the sulfonated shrimp chitosan group is consistent with the known bioactive properties of modified chitosan derivatives, which have been shown to enhance wound closure. The wound healing time for each group is presented in [Fig. 3A]. The sulfonated shrimp chitosan group exhibited the fastest wound healing compared with both the negative control (distilled water) and the positive control (commercial chitosan). A Kruskal–Wallis test confirmed significant differences between the groups (H = 6.641, df = 2, p = 0.036), indicating that the wound healing time varied significantly across treatments. Fibroblasts play a critical role in the wound healing process by synthesizing extracellular matrix (ECM) proteins and collagen, which are essential for tissue repair. Our study showed that the number of fibroblasts in the proliferation phase of wound healing was significantly higher in the sulfonated shrimp chitosan group compared with both control groups (p < 0.001). [Fig. 3B] present the fibroblast count for each group. An ANOVA test revealed a significant difference between the groups (F = 162.069, p < 0.001), with a large effect size (η ² = 0.964), suggesting a substantial impact of the treatment on fibroblast proliferation. This suggests that sulfonated shrimp chitosan is more effective than both the positive and negative control groups in promoting faster wound healing and increasing fibroblast proliferation during the healing process ([Table 2]).

Table 2 Healing time and fibroblast count

Healing time	Mean	Standard deviation	p-Value	p-Value	Kruskal–Wallis H
			Normalitya	Homogeneityb
				0.200	0.036c
Commercial chitosan	6.00	0.00	0.119
Sulfonated chitosan	5.00	0.47	0.821
Negative control	6.60	0.60	0.552
Fibroblast	Mean	Standard deviation	p -Value	p -Value	ANOVA
			Normalitya	Homogeneityb
				0.072	<0.001c
Commercial chitosan	10.88	0.14	0.22
Sulfonated chitosan	20.32	0.68	0.30
Negative control	11.68	0.10	0.72

Abbreviation: ANOVA, analysis of variance.

^anormality distribution; ^bhomogeneity of variance; ^csignificance level.

Fig. 3 (A) Fibroblast count across treatment groups and (B) wound healing time across treatment groups.

Chitosan from Vaname shrimp was found to have antibacterial properties in this study, which prevented the inflammatory phase of the wound healing process from being postponed. This means that Protein S-100A8, a calprotectin secreted by neutrophils, becomes a pro-inflammatory mediator in both acute and chronic conditions. This protein is a chemotactic molecule that macrophages, active monocytes, and neutrophils express.[29] When released into cells, extracellular calgranulin acts as an injury-related molecule, activating macrophages. During the inflammatory response, platelets and macrophages create chemoattractants such platelet-derived growth factor, transforming growth factor-β, basic fibroblast growth factor, interleukin-1β, and tumor necrosis factor-α, which attract fibroblasts to the wound area. Fibroblasts travel to the wound, multiply, and create matrix metalloproteinase (MMPs), which destroy the temporary ECM. Fibroblast activity decreases and binds fibrin clots in the wound via multiple integrins in the focal adhesion. Fibroblasts also manufacture MMPs. To create the granulation tissue that will seal the wound, fibroblasts replace the temporary ECM with a new one made up of collagen, proteoglycans, hyaluronic acid, glycosaminoglycans, and fibronectin. Type I collagen, which has a significantly higher tensile strength but takes longer to deposit, replaces type III collagen, which is generated quickly. The original matrix serves as a barrier against pathogens and prevents the loss of serum and fluid. Proteases then break down the matrix and fibroblasts repair it.[30] In this investigation, sulfonated chitosan-treated wounds had more fibroblast cells ([Fig. 4]).

Fig. 4 Comparison of fibroblast cell count between sulfonated chitosan, commercial chitosan as positive control and negative control.

The limitations of the in vivo model in this work are the rat gingival wound model does not evaluate oral-specific variables like saliva rinsing or microbial interactions. The study is still in the experimental stage, with no preclinical safety assessments such as sensitization tests or toxicology investigations.

 

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Conclusion

Based on this study, it can be informed that sulfonated shrimp chitosan demonstrated superior wound healing capabilities, significantly reducing healing time and enhancing fibroblast proliferation compared with commercial chitosan. Additionally, it exhibited moderate antimicrobial activity, with the highest effectiveness observed at intermediate concentrations (3% or 5%). These findings highlight sulfonated shrimp chitosan's potential as a potent wound healing agent with moderate antibacterial properties, suggesting its application in wound care and infection prevention.

 

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Acknowledgment

This research was funded by Universitas Syiah Kuala, Kementerian Pendidikan, Kebudayaan, Riset dan Teknologi Indonesia (Grant Number: 303/UN11.2.1/PG.01.03/SPK/PTNBH/2024).

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Publikationsverlauf

Artikel online veröffentlicht:
08. September 2025