Wound healing performance of nanochitosan-based ointment derived from black soldier fly larvae exuviae: in vivo assay on Mus musculus L.

Desempenho cicatrizante de pomada à base de nanoquitosana derivada de exúvias de larvas de mosca-soldado-negra: ensaio in vivo em Mus musculus L.

Abstract

Wound healing is a complex process that is often hindered by infection and slow tissue regeneration, prompting research on novel biomaterials. This study aimed to evaluate the wound-healing efficacy of a nanochitosan-based ointment derived from black soldier fly larvae (BSFL) exuviae compared to chitosan-based and conventional treatments. Nanochitosan was synthesized using sodium tripolyphosphate, incorporated into ointments, and tested in vivo in 60 male mice with incision wounds over 15 days. Results showed nanochitosan ointment achieved 98.53% wound closure by day 15, significantly outperforming chitosan (96.77%), povidone iodine (85.53%), and Vaseline (73.10%) (p<0.05), with protein (182.29 μg/mg), hydroxyproline (352.67 μg/mg), and total DNA (3.77 μg/mg) levels indicating lower inflammation and advanced collagen remodelling. Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) confirmed the structural integrity and size of the nanochitosan. The conclusion highlights the potential of nanochitosan as a sustainable, effective wound-healing agent due to its small particle size, enhanced bioavailability, and antimicrobial activity. This study benefits biomedical fields by offering an eco-friendly alternative to crustacean-derived materials, reducing environmental impact while improving healing outcomes, with future research needed for clinical applications.

Keywords:
nanochitosan; black soldier fly larvae; wound healing; tissue regeneration

Resumo

A cicatrização de feridas é um processo complexo que muitas vezes é dificultado por infecções e pela lenta regeneração dos tecidos, o que estimula a pesquisa sobre novos biomateriais. Este estudo teve como objetivo avaliar a eficácia cicatrizante de uma pomada à base de nanoquitosana derivada de exúvias de larvas da mosca-soldado-negra (Black Soldier Fly Larvae – BSFL), em comparação com tratamentos convencionais à base de quitosana. A nanoquitosana foi sintetizada usando tripolifosfato de sódio, incorporada em pomadas e testada in vivo em 60 ratos machos com feridas por incisão durante 15 dias. Os resultados mostraram que a pomada de nanoquitosana alcançou 98,53% de fechamento da ferida no 15º dia, superando significativamente a quitosana (96,77%), a iodopovidona (85,53%) e vaselina (73,10%) (p<0,05), com níveis de proteína (182,29 μg/mg), hidroxiprolina (352,67 μg/mg) e DNA total (3,77 μg/mg) a, indicando menor inflamação e remodelação avançada do colágeno. A espectroscopia de infravermelho com transformada de Fourier (FTIR) e a microscopia eletrônica de varredura (MEV) confirmaram a integridade estrutural e o tamanho da nanoquitosana. A conclusão destaca o potencial da nanoquitosana como um agente de cicatrização de feridas sustentável e eficaz, devido ao seu pequeno tamanho de partícula, biodisponibilidade aprimorada e atividade antimicrobiana. Este estudo beneficia os campos biomédicos, oferecendo uma alternativa ecológica aos materiais derivados de crustáceos, reduzindo o impacto ambiental e melhorando os resultados de cicatrização, com pesquisas futuras necessárias para aplicações clínicas.

Palavras-chave:
nanoquitosana; larvas da mosca-soldado-negra; cicatrização de feridas; regeneração de tecidos

1. Introduction

The complex process of wound healing necessitates coordinated efforts from multiple tissues and cell lineages. In addition to the presence of inflammation and the formation of new blood vessels, this process necessitates the exact coordination of cell movement, growth, extracellular matrix formation, and modification (Yang et al., 2023; Zulkefli et al., 2023). Significant injuries brought on by trauma, severe illness, or major surgery may take weeks to heal, while minor skin wounds typically heal in a few days. Fibrotic scarring from these injuries frequently impairs the affected tissues' ability to function normally (Mirhaj et al., 2022).

A wound is created when the histological structure of skin tissue is disturbed by a variety of internal or external factors, or when any layer of skin gradually deteriorates, causing tissue disturbance (Mamun et al., 2024; Sharifi and Bahrami, 2024). Numerous microbiological agents, such as bacteria, viruses, and other substances, can enter the human body through wounds. Localized microbial infections can cause inflammation in skin wounds. Therefore, more research is needed to develop simple and efficient methods for managing and treating skin injuries. Stopping bleeding, removing microbial contamination from wounds, and promoting the best possible wound healing without side effects or anatomical deformities are its primary goals (Arif et al., 2021; El-Sherbeni and Negm, 2023; Ibrahim et al., 2018).

According to Cedillo-Cortezano et al. (2024) a number of medicinal plants have been utilized as natural sources for wound healing. Polyphenols in Psidium guajava L. leaves were found by Bilal et al. (2024) to have antibacterial and antioxidant properties in wound healing. Apart from plants, chitosan obtained from black soldier flies, or BSF (Hermetia illucens L.), is another promising natural resource for wound healing. With a chitin content of up to 35% of its dry weight, equivalent to that of crustaceans like prawns and crabs, this insect has a high chitin and chitosan content. Furthermore, compared to marine sources, black soldier flies are easier to handle and more environmentally friendly due to their quick reproduction and seasonal independence. Accordingly, the BSF is a productive and environmentally friendly substitute for supplying chitin and chitosan for a range of uses (Le et al., 2023; Triunfo et al., 2023; Xiong et al., 2023)​.

Throughout its life cycle, the BSF's chitin and chitosan content varies greatly; the larval stage has the lowest levels, while the prepupal stage has the highest levels (Eggink and Dalsgaard, 2023; Rampure et al., 2023). A previous study found that chitosan derived from crustaceans has been shown to enhances wound healing by regulating various processes, including growth factors and cytokines, through its antibacterial and anti-inflammatory properties (Alemu et al., 2023; Praneeth et al., 2023; Zhou et al., 2024).

Presently, the application of nanotechnology to natural materials for wound healing has demonstrated encouraging outcomes in terms of quickening the healing process, lowering the risk of infection, and enhancing the calibre of the tissue that is formed. The main characteristic that sets nanochitosan apart from chitosan is its nanoscale particle size, which is usually less than 1000 nm. This increases its surface area, bioavailability, and antimicrobial effectiveness. By employing sodium tripolyphosphate (STPP) as a cross-linking agent during the ionic gelation process, nanochitosan is synthesized as spherical nanoparticles with a consistent morphology (Ferreira et al., 2022; Wang et al., 2023; Zhao et al., 2023a). This technique allows for controlled particle formation by taking advantage of the electrostatic interactions between the positively charged amino groups of chitosan and the polyanions of STPP (Ferreira et al., 2022; You et al., 2017a). Nanochitosan's smaller particle size promotes better cellular interactions, quicker collagen deposition, and deeper penetration of wound tissues.

Nanochitosan is a promising material for wound care because of its potent antibacterial and anti-inflammatory qualities (Li et al., 2022). Numerous studies have investigated wound healing models utilizing nanotechnology, specifically nanochitosan. According to Ehyaeirad et al. (2024), nanochitosan significantly affects the rate at which wounds heal. Because of its positive charge, nanochitosan increases blood clotting and stimulates cell growth, both of which can enhance the healing process. Additionally, Freitas et al. (2024) discovered that nanochitosan has antibacterial activity against Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus aureus, and Staphylococcus epidermidis, all of which are known to cause skin infections and other dangerous conditions. According to research by Hwang et al. (2023) and Ferreira et al. (2020), nanochitosan can increase the expression of genes linked to wound healing, specifically VEGF and CD163, which are crucial for angiogenesis and the development of new tissue.

To guarantee the efficacy and quality of nanochitosan, a variety of characterization methods have been employed. Fourier Transform Infrared Spectroscopy (FTIR) was employed in a study by Zhou et al. (2023) to identify particular functional groups in the nanochitosan structure and assess the crystallinity of the material. Another essential tool for describing nanochitosan is FTIR spectroscopy, which makes it possible to identify the structural changes and functional groups that set it apart from bulk chitosan. Increased hydrogen bonding as a result of nanoparticle formation is indicated by shifts in important peaks in the FTIR spectra of nanochitosan, such as the amide I band (1650 cm-¹ to 1621 cm-¹) (Wang et al., 2023). Furthermore, nanoscale structural rearrangements were confirmed by the appearance of a distinct peak at 1461 cm-¹ (C-H bending), which was associated with enhanced antimicrobial activity and bioavailability (Li et al., 2022). The integrity of nanochitosan during synthesis is further confirmed by the stability of the glycosidic linkage (C-O-C peak at 1022 cm⁻¹) (Sánchez-Machado et al., 2024).

Despite notable advancements in the application of nanotechnology to wound healing, alternative sources of nanoparticles, particularly those derived from insects like the BSF, remain scarce. To date, most studies have focused on nanochitosan obtained from crustaceans, whereas the biopolymer potential of the BSF, particularly nanoparticles produced from BSF pupal (BSFL) shell (exuviae), has not been widely explored. The specific effects of BSFL exuviae-based nanoparticle ointment application on important aspects of wound healing, such as nanoparticle characterization using FTIR, wound closure percentage, total DNA, protein, and hydroxyproline levels associated with healing, have not been widely studied. This study aimed to address these shortcomings by evaluating the potential and effectiveness of nanochitosan-based ointments from alternative sources to accelerate and improve the quality of wound healing. Additionally, by presenting a novel method for creating nanochitosan-based ointments with quantifiable biochemical efficacy for wound healing, this study advances the use of BSFL exuviae as a sustainable chitosan source.

2. Materials and Methods

2.1. Chitosan extract preparation

Chitosan extraction was carried out in accordance with the work previously utilized by Lagat et al. (2021). After being sorted and cleaned under running water, the BSFL exuviae were dried for 24 hours at 60 °C. Using an analytical balance (A&D, model ER-180A, Tokyo, Japan), 100 g of purified exuviae was weighed before being submerged in 1000 mL of 2% sodium hydroxide (NaOH) (Pudak Scientific, Bandung, Indonesia) for two hours while being stirred occasionally to remove proteins. The exuviae were then carefully rinsed with distilled water until the pH was neutral. An Orion StarTM A211 Benchtop pH meter (Waltham, MA, USA) was used to measure the pH. A food dehydrator (Model: LT-28, China) was then used to desiccate the exuviae for 24 hours at 60 °C. The desiccated samples were immersed in 1000 mL of 7% hydrochloric acid (HCl) (Sigma-Aldrich, MI, USA) for four hours at room temperature with intermittent stirring in order to extract calcium carbonate during the demineralization phase. After being cleaned with distilled water until the pH was neutral, the finished product was dried for 24 hours at 60 °C in a food dehydrator. The exuviae was used to calculate the chitin yield after the desiccated final product was weighed.

2.2. Deacetylation of chitin to chitosan

Following that, 100 g of chitin from exuviae was immersed in 1000 mL of a 50% NaOH solution (Pudak-Scientific, Bandung, Indonesia) at 120 °C for two hours in order to convert it to chitosan. After being further purified with distilled water to reach a pH of neutral, the deacetylated chitosan was dehydrated for 24 hours at 60 °C in a food dehydrator. Until they were needed again, the desiccated samples were kept in sealed plastic containers at 4 °C.

2.3. Chitin and hhitosan yield (%)

The yields of chitin and chitosan were determined using the dry weight of exuviae, chitin, and chitosan acquired post-extraction, utilizing the following Equations 1 and 2:

$$ChitinYield\left(\%\right)=\times 100$$ (1)

where: chitin weight is obtained (g), and b is the weight of the initial exuviae (g).

$$ChitosanYield\left(\%\right)=\times 100$$ (2)

where: c is the weight of chitosan obtained (g) and d is the weight of the chitin prepared (g).

2.4. Degree of deacetylation

The degree of deacetylation was evaluated using the formula described by Sánchez-Machado et al. (2024) as follows (Equations 3 and 4):

$$\%DD=100-\%DA$$ (3)

$$\%DA=\times 100/1.33$$ (4)

where: %DD is the percentage of the deacetylation degree, %DA is the percentage of the acetylation degree, and A1655 and A3450 are the absorbance values of the infrared wavelengths 1655^-1 and 3450^-1, respectively.

2.5. Biosynthesis of nanochitosan

After making some adjustments, chitosan nanoparticles were made using the techniques of Chávez de Paz et al. (2011) and Younus et al. (2020). 1 mL of acetic acid was used to dissolve 0.25 g of chitosan in order to create a homogenous chitosan solution. After adjusting the pH of the mixture with a 1.0 M NaOH solution to 5.5 and adding distilled water until the volume reached 100 mL, the mixture was stirred all night. Sodium tripolyphosphate (STPP) was thereafter added dropwise at ambient temperature while maintaining continuous agitation until a blue suspension of nanoparticles was produced. The mixture was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was removed, leaving nanoparticles at the bottom. The acquired nanochitosan was subjected to freeze-drying.

2.6. Preparation for wound ointment

The wound ointment was prepared by combining chitosan or nanochitosan with Vaseline, according to the formulation outlined in Table 1. Vaseline was melted on a hot plate and combined with either chitosan or nanochitosan. The mixture was agitated until it was homogenized and transferred into an ointment container.

Table 1
Wound ointment formula using chitosan or nanochitosan and vaseline

Composition	Groups
	Control	Vas	Pov	Chi	NChi
Chitosan/nanochitosan (%)	0	0	0	1.5	1.5
Vaseline (g)	0	30	0	28.5	28.5
Povidone iodine (g)	0	0	30	0	0
Total (g)	30	30	30	30	30

2.7. FTIR and SEM analysis

The FTIR measurements were carried out as described by Akram et al. (2012, 2023) to determine the organic and inorganic compounds or functional groups present in the chitosan or nanochitosan ointment, both qualitatively and quantitatively, by examining the absorption intensity of the compounds in the scanning range of 4000–400 cm⁻¹ using Brucker, Alpha model, Berlin, Germany.

The JCM-7000 NeoScope benchtop scanning electron microscope (JEOL Ltd., Tokyo, Japan) was employed to ascertain the surface morphologies of the chitosan and nanochitosan samples. The microstructure was revealed at certain magnification by coating the samples with a carbon film and examining them using secondary electron mode at an acceleration voltage of 15 kV. The resulting images were analyzed using the ImageJ software.

2.8. Preparation of test animals

In total, 60 male mice (average initial weight 20-30 g, three months old) were used. All mice were acclimatized for seven days with feed and water ad libitum. To prepare a wound in each treated mouse, ketamine solution was diluted using distilled water at a ratio of 1:9 (1 mL ketamine and 9 mL distilled water). An amount of 0.2 mL solution was then injected into the inner thigh of the mice and left until the mice fainted. Hair on the backs of the mice was shaved until the skin was visible and cleaned using 70% alcohol. The scalpel was marked with a marker to indicate the depth of the wound (1 mm), and a 1 cm long incision wound was made using a scalpel. After the wound was formed, each mouse was treated according to its group every day from days 0 to 15. Wound closure measurements were performed every three days using a digital caliper on days 3, 6, 9, 12, and 15. Data were recorded and documented using a digital camera.

Dissection of the test animals and collection of skin tissue were conducted on day 10 post-wound ointment therapy, as shown by the groups of mice exhibiting 100% wound closure. Additionally, four mice with the most healed wounds were anesthetized. Skin tissue from the incision site was excised and partitioned into four segments, each weighing 0.05 g. The first part was added to 200 µL of 4% NaCl to assess the protein concentration. The second portion was combined with 200 µL of PBS to quantify the total DNA. The third portion was incubated overnight at 60 °C for hydroxyproline measurement and the final portion was prepared for gene expression.

2.9. Assessment of protein content

Protein levels in wound tissue samples were quantified using the Lowry technique. A 0.05 g sample of wound skin tissue was extracted from the wound region, placed in a tube containing 200 µL of 4% NaCl, and homogenized using a Beadbug Microtube Homogenizer (USA) at 3000 rpm for 30 s. The solution was centrifuged at 6000 rpm for 10 min at ambient temperature. The resulting supernatant (150 µL) was combined with 1200 µL Lowry B reagent and incubated for 10 min at room temperature. Additionally, 150 µL of Lowry A reagent was added to the mixture, homogenized, and allowed to stand for 20 min. A color change to a bluish hue occurred, and the absorbance was quantified at a wavelength of 600 nm using a UV-VIS spectrophotometer (UV-Vis 752N, China). Bovine serum albumin (BSA) was used as the standard.

2.10. Hydroxyproline determination

The hydroxyproline levels were measured on the 10^th day. An 0.05 g sample of wound skin tissue was extracted from the wound region and incubated at 60 °C overnight. The wound tissue underwent hydrolysis with 6N HCl at 130ºC for 4 hours, was neutralized to pH 7 using Chloramine-T Oxidan and was permitted to stand for 20 minutes at ambient temperature. The reaction was terminated by the addition of 0.4 M perchloric acid. Ehrlich reagent was subsequently introduced into the solution and incubated for 90 min at 60 °C. Absorbance was quantified at a wavelength of 557 nm using a spectrophotometer (UV-Vis 752N, China). A standard curve was established using hydroxyproline concentrations of 0, 6.3, 12.5, 25, 50, 100, 200, and 400 µg/mL following the same methodology used for the wound tissue samples.

2.11. DNA quantification

Skin tissue (0.05 g) obtained from the wound region was placed in a tube containing 200 µL PBS and homogenized at 3000 rpm for 30 s. A Universal DNA Extraction Kit (D2100, Solarbio, Beijing) was used to extract DNA. After the DNA extraction procedure, the total DNA concentration was quantified using the Qubit™ dsDNA Quantification Assay Kit (Catalogue number: Q32851, Thermo Fisher Scientific, USA) and assessed using an Invitrogen Qubit 4 Fluorometer (Thermo Fisher Scientific, USA).

2.12. Statistical data analysis

The FTIR data was analyzed descriptively, whereas quantitative data derived from incision wound closure, protein concentrations, hydroxyproline levels, and total DNA were analyzed using the Statistical Product and Service Solutions (SPSS) application. The analysis of variance (ANOVA) in SPSS version 22 (SPSS, Inc., USA) was utilized to assess the existence of significant differences between the treatment groups. Duncan's multiple range test (DMRT) was implemented when statistically significant differences were identified through an analysis of variance (ANOVA). The threshold for statistical significance was established at p<0.05.

3. Results

3.1. Yield of chitosan

The chitin and chitosan yields extracted from BSFL exuviae are presented in Table 2. Three samples (A, B, and C) with initial weights of 100 g were processed. The results showed variations in weight reduction during the sequential steps of deproteinization, demineralization, and deacetylation processes. Sample A exhibited the highest chitin yield of 47.55%, whereas samples B and C had chitin yields of 36.41% and 39.78%, respectively. The chitosan yield was also highest for sample A at 48.66%, followed by 38.67% for sample C, and 33.97% for sample B.

Table 2
Yield of chitin and chitosan during the extraction from the BSFL Exuviae

Sample code	Initial weight of exuviae (g)	Weight of deproteinized (g)	Weight of demineralized (g)	Weight of deacetylase (g)	Chitin yield (%)	Chitosan yield (%)
A	100	70.39	47.55	23.14	47.55	48.66
B	100	41.7	36.41	12.37	36.41	33.97
C	100	37.58	35.62	22.25	39.78	38.67

The deproteinization stage significantly reduced exuviae weight. Sample A retained 70.39 g after this step, whereas samples B and C had lower weights of 41.7 g and 37.58 g, respectively. A similar trend was observed during the demineralization process, where the weight continued to decrease further, reaching 47.55 g, 36.41 g, and 35.62 g for samples A, B, and C, respectively. Finally, the deacetylation process yielded final weights of 23.14 g, 12.37 g, and 22.25 g, indicating the removal of acetyl groups to produce chitosan.

3.2. The FTIR analysis

The Fourier Transform Infrared spectroscopy (FTIR) analysis results for chitosan, chitosan-based ointment, and nanochitosan-based ointment are shown in Figure 1. The FTIR spectrum of pure chitosan revealed characteristic peaks aligned with its chemical structure. A broad peak at 3357.21 cm⁻¹ corresponds to O-H stretching, indicative of hydroxyl groups, while peaks at 2916.87 cm⁻¹ and 2854.37 cm⁻¹ are attributed to C-H stretching from CH₂ groups. The amide I band at 1650.48 cm⁻¹ (C=O stretching) and amide II band at 1557.04 cm⁻¹ (N-H bending and C-N stretching) confirm acetamide groups, with additional peaks at 1374.37 cm⁻¹ (C-H bending) and 1064.43 cm⁻¹, 1022.29 cm⁻¹ (C-O-C stretching) supporting glycosidic linkages (Kumar, 2000; Rinaudo, 2006).

Figure 1
Fourier Transmission Infrared (FTIR) analysis of (A) Chitosan; (B) Chitosan based-ointment; (C) Nanochitosan based-ointment

In contrast, the chitosan-based ointment spectrum shows peaks at 2919.64 cm⁻¹ and 2851.15 cm⁻¹ (C-H stretching), 1461.72 cm⁻¹ and 1377.06 cm⁻¹ (likely C-H bending), and 720.48 cm⁻¹, which is not typical for pure chitosan and may arise from the ointment base, possibly paraffin or vaseline (Wong, 2015). The absence of O-H stretching (around 3350 cm⁻¹) and amide bands (1650 cm⁻¹, 1550 cm⁻¹) suggests these functional groups are masked or altered due to interactions with the ointment matrix.

The nanochitosan-based ointment exhibits a similar profile, with peaks at 2919.92 cm⁻¹, 2851.46 cm⁻¹, 1461.84 cm⁻¹, 1376.97 cm⁻¹, and 720.55 cm⁻¹. Slight shifts (e.g., 2919.92 cm⁻¹ vs. 2919.64 cm⁻¹) may reflect minor formulation differences, but overall, the spectra are comparable, indicating that nanochitosan does not significantly alter the FTIR profile in the ointment form, likely due to the dominant ointment base.

3.3. The SEM determination

The morphological and dimensional characteristics of chitosan and nanochitosan derived from black soldier fly larvae exuviae were analyzed using Scanning Electron Microscopy (SEM) and supplementary size distribution data. The chitosan particles (Figure 2A) exhibited an irregular, flaky morphology with a rough surface, consistent with previous studies on insect-derived chitosan (Milinković Budinčić et al., 2025). The attached size results show that the particle lengths ranged from 7.859 µm to 46.168 µm, with a mean length of 22.108 µm (Table 3). The inherent heterogeneity of chitosan derived from biological sources is reflected in this wide size distribution, which is probably impacted by the extraction and purification procedures (Vaezifar et al., 2013).

Figure 2
Scanning Electron Microscope (SEM) analysis of (A) Chitosan; (B) Nanochitosan particle derived from the black soldier fly larvae exuviae

Table 3
The distribution size of chitosan and nanochitosan derived from the black soldier fly larvae exuviae.

No	Chitosan Particle Length (µm)	Nanochitosan Particle Length (µm)
1	14,797	702
2	7,859	1,281
3	20,780	1,039
4	44,278	1,075
5	28,458	1,032
6	19,320	1,055
7	15,582	648
8	39,460	1,400
9	22,108	461
10	11,144	453
11	24,393	25,984
12	10,800	472
13	22,092	296
14	32,508	64
15	36,190	273
16	22,756	170
17	13,452	171
18	11,631	147
19	10,264	85
20	37,508	84
21	46,168	137
22	15,481	169
23	13,918	136
24	25,266	71
25	38,656	79

However, the nanochitosan particles (Figure 2B) showed a remarkably uniform and spherical morphology, with a mean length of 0.648 µm (648 nm) and lengths ranging from 0.071 µm (701 nm) to 1.400 µm (1400 nm) (Table 3). The reduction in particle size and smoother surface texture are indicative of successful nano-scale fabrication, consistent with findings from studies on fungal and marine-derived nanochitosan (Darwesh et al., 2018). Notably, the SEM images corroborated the size data, confirming the absence of large aggregates and homogeneity of the nanochitosan particles.

3.4. In vivo wound healing assay

As shown in Table 4, the wound enclosure percentages revealed distinct patterns across the treatment groups. On the day 0, all groups had 0.00% enclosure, as expected. By day 3, NChi achieved the highest enclosure at 16.54% ± 0.41%, which was significantly higher than Chi (12.72% ± 0.40%), Pov (9.00% ± 0.30%), Vas (7.00% ± 0.38%), and Control (5.81% ± 0.51%), indicating early efficacy of nanochitosan. On day 6, Chi led with 49.36% ± 0.93%, followed by Vas (46.27% ± 0.75%), Pov (45.27% ± 0.79%), NChi (40.18% ± 0.61%), and Control (36.00% ± 0.60%), suggesting Chi’s mid-stage dominance. By day 9, Chi maintained the lead at 75.18% ± 1.00%, with Pov (72.36% ± 0.59%) and NChi (72.18% ± 1.18%) close behind, all significantly higher than Vas (62.00% ± 0.67%) and Control (65.54% ± 0.74%).

Table 4
Wound enclosure percentage (%) of mice model after topical chitosan and nanochitosan-based ointment for 15 days.

Groups	Wound enclosure percentage (Day-)
	0	3	6	9	12	15
Control	0.00 ± 0.00a	5.81 ± 0.51a	36.00 ± 0.60a	65.54 ± 0.74b	77.27 ± 1.00a	87.35 ± 0.22b
Vas	0.00 ± 0.00a	7.00 ± 0.38b	46.27 ± 0.75c	62.00 ± 0.67a	80.90 ± 1.26b	73.10 ± 1.78a
Pov	0.00 ± 0.00a	9.00 ± 0.30c	45.27 ± 0.79c	72.36 ± 0.59c	83.90 ± 0.86c	85.53 ± 0.56b
Chi	0.00 ± 0.00a	12.72 ± 0.40d	49.36 ± 0.93d	75.18 ± 1.00d	80.45 ± 1.26ab	96.77 ± 1.22c
NChi	0.00 ± 0.00a	16.54 ± 0.41e	40.18 ± 0.61b	72.18 ± 1.18c	80.09 ± 1.17ab	98.53 ± 0.66d

• Description: Control, no treatment; Vas, Vaseline base ointment; Pov, povidone-iodine base ointment; Chi, 5% chitosan-based ointment; NChi, 5% nanochitosan-based ointment). Mean ± SE values followed by superscript letters (a, b, c, d, e) in the same column indicate significant differences (p<0.05).

On the day 12, Pov showed the highest enclosure at 83.90% ± 0.86%, followed by Vas (80.90% ± 1.26%), Chi (80.45% ± 1.26%), NChi (80.09% ± 1.17%), and Control (77.27% ± 1.00%), with overlapping superscripts indicating less significant differences. By day 15, NChi achieved the highest enclosure at 98.53% ± 0.66%, which was significantly higher than that of Vas (73.10% ± 1.78%), Control (87.35% ± 0.22%), and Pov (85.53% ± 0.56%), but not significantly different from Chi (96.77% ± 1.22%), as indicated by different superscripts.

Based on these data, NChi and Chi consistently showed high wound enclosure percentages, with NChi excelling in the early (day 3) and late (day 15) stages, reaching nearly complete closure. The control group also showed substantial healing (87.35% by day 15), while Vas and Pov exhibited variability, with Vas notably declining from days 12 to 15, suggesting potential limitations in sustained healing support. In addition, wound enclosure in the back skin of mice is shown in Figure 3.

Figure 3
Wound enclosure image of the mice treated with topical chitosan and nanochitosan-based ointment for 15 days. Description: Coloum represent the day of the observation: Day 0, 3, 6, 9, 12, 15; row represent the groups of treatment: Control, no treatment; Vas, Vaseline base ointment; Pov, povidone-iodine base ointment; Chi, 5% chitosan-based ointment; NChi, 5% nanochitosan-based ointment).

3.5. The protein, hydroxyproline level, and total DNA analysis

The protein content, hydroxyproline level, and total DNA in the skin tissues of mice subjected to wound healing treatments with chitosan (Chi) and nanochitosan-based (NChi) ointments compared to the control and other groups are shown in Table 5. The data revealed significant differences in biochemical parameters, reflecting the efficacy of each treatment in promoting wound healing.

Table 5
Protein content, hydroxyproline level, and total DNA of wound healing in mice model after topical chitosan and nanochitosan-based ointment at day 10^th.

Groups	Protein content (μg/mg)	Hydroxyproline level (μg/mg)	Total DNA (μg/mg)
Control	133.90 ± 15.85 a	551.37 ± 18.53 b	12.66 ± 2.73b
Vas	141.55 ± 16.35 ab	611.50 ± 62.18 b	9.44 ± 0.09b
Pov	123.53 ± 11.42a	808.89 ± 85.20 c	11.41 ± 0.74b
Chi	226.74 ± 12.66 c	499.08 ± 15.07 ab	7.42 ± 2.28 ab
NChi	182.29 ± 10.47 b	352.67 ± 13.64 a	3.77 ± 1.49 a

• Description: Control, no treatment; Vas, Vaseline base ointment; Pov, povidone-iodine base ointment; Chi, 5% chitosan-based ointment; NChi, 5% nanochitosan-based ointment). Mean ± SE values followed by superscript letters (a, b, c, d, e) differing in the same column indicate significant differences (p<0.05). Skin samples from mouse wounds were collected on day 10^th which one of the groups of mice showed 75% wound closure.

The protein content in the chitosan-based ointment group (Chi) was the highest (226.74 ± 12.66 μg/mg), followed by the nanochitosan-based ointment group (NChi) (182.29 ± 10.47 μg/mg). In contrast, the povidone-iodine group (Pov) exhibited the lowest protein level (123.53 ± 11.42 μg/mg), while the control (133.90 ± 15.85 μg/mg) and Vaseline groups (141.55 ± 16.35 μg/mg) showed comparable values with no significant differences. These findings indicated that the Chi and NChi groups were associated with a higher accumulation of proteins in the wound area, which may reflect the active phase of tissue proliferation and matrix deposition. Meanwhile, the lower protein levels in the Pov, control, and Vas groups suggest slower progress in tissue regeneration, possibly due to prolonged inflammatory activity or less effective remodeling compared to chitosan and nanochitosan treatments. (Eming et al., 2017; Favier and Nikovics, 2023; Nurden, 2011).

The hydroxyproline level, which serves as a key indicator of collagen synthesis, showed distinct variations among the treatment groups. The povidone-iodine group (Pov) exhibited the highest hydroxyproline level (808.89 ± 85.20 μg/mg), followed by the Vaseline group (611.50 ± 62.18 μg/mg) and the control group (551.37 ± 18.53 μg/mg). In contrast, the chitosan-based ointment group (Chi) (499.08 ± 15.07 μg/mg) and the nanochitosan-based ointment group (NChi) (352.67 ± 13.64 μg/mg) demonstrated significantly lower hydroxyproline levels. The reduced hydroxyproline content in the Chi and NChi groups suggests that the wound sites had progressed further into the tissue remodeling phase, where collagen deposition becomes more regulated. Conversely, the elevated hydroxyproline levels in the Pov, Vas, and control groups indicate ongoing collagen accumulation, which is characteristic of the proliferative phase and may reflect a slower progression toward maturation and remodeling (Li et al., 2024; Lioi et al., 2024; Zhu et al., 2024).

The total DNA content varied significantly among the treatment groups. The control group (12.66 ± 2.73 μg/mg), Vaseline group (9.44 ± 0.09 μg/mg), and povidone-iodine group (11.41 ± 0.74 μg/mg) exhibited relatively high DNA levels, indicating active cellular presence in the wound area, which may be associated with sustained inflammation or ongoing proliferation. In contrast, the chitosan-based ointment group (Chi) (7.42 ± 2.28 μg/mg) and the nanochitosan-based ointment group (NChi) (3.77 ± 1.49 μg/mg) showed markedly lower DNA levels. The reduction in DNA content, particularly in the NChi group, suggests a more advanced stage of healing, where cellular infiltration and proliferation decline as tissue remodeling and maturation become predominant. (Peña and Martin, 2024; Swinehart and Badylak, 2016).

4. Discussion

Chitosan and nanochitosan made from the BSFL exuviae have shown promise as materials for wound-healing applications. These biomaterials have a special set of qualities that make them ideal for managing wounds. While both chitosan and nanochitosan have antimicrobial activity and may help prevent wound infections, chitosan's natural biocompatibility lowers the possibility of negative reactions to wound application (Abdeltwab et al., 2019; Chandrasekaran et al., 2020). Furthermore, these substances encourage blood coagulation, which facilitates the early phases of wound healing due to their haemostatic properties (Dai et al., 2011).

The biodegradability of chitosan-based materials in the body is one of their main benefits; this eliminates the need for removal and lowers the possibility of additional tissue damage during dressing changes. Moreover, chitosan's capacity to sustain a moist wound environment facilitates healing by encouraging cell migration and proliferation (Jayakumar et al., 2011). Because of its larger surface area and reactivity, nanochitosan in particular may promote cellular growth and proliferation, which could hasten wound healing (Naseri et al., 2015). The high surface-to-volume ratio of nanochitosan, a nanostructured form of chitosan with particle sizes usually between 10 and 1000 nm, enhances cellular interactions and drug delivery efficiency, resulting in enhanced bioactivity for wound healing. Because of its antimicrobial, biocompatible, and biodegradable qualities, it is a great option for wound dressings because it enhances angiogenesis, modulates reactive oxygen species, and promotes haemostasis while lowering inflammation and speeding tissue regeneration. Better penetration into wound sites, enhanced release of bioactive compounds, and collagen deposition are all made possible by the nanoscale size (Askari et al., 2025; Moeini et al., 2020).

In addition to addressing environmental concerns and potentially meeting the growing demand for wound healing materials, using BSFL exuviae as a source of chitosan and nanochitosan offers a sustainable substitute for conventional crustacean-derived chitosan (Waśko et al., 2016; Wasko et al., 2020). This strategy is a compelling choice for further biomaterials research and development since it is consistent with the ideas of waste valorization and the circular economy.

These findings demonstrate the potential of BSFL exuviae as a chitin and chitosan source. Sample A's higher yields than the other two samples suggested either a more effective extraction method or variations in the original exuviae's composition. The chitin yields found in this study were noteworthy because they were similar to those reported by El Knidri et al. (2016) and Kandile et al. (2018), who extracted chitin from shrimp shells and obtained yields between 40 and 50%. Likewise, a 48.66% chitosan yield from BSFL exuviae is consistent with research findings on other materials derived from insects. A prior study, for example, found that silkworm pupae exuviae produced chitosan yields ranging from 35 to 50%, supporting the viability of insects as sustainable sources of chitosan (Silva Lucas et al., 2021; Mohan et al., 2020; Sheng et al., 2022).

Variability in the composition of the BSFL exuviae, which may be impacted by elements like the larval diet, growth conditions, or molting stage, may be the cause of the yield variations among the three samples. Sample A's higher yield indicates a lower starting protein and mineral content, which would lead to more efficiency during the deproteination and demineralization processes. The final extracted weight was decreased by samples B and C, which had lower yields of chitin and chitosan but probably higher proportions of minerals and proteins.

Compared to more conventional sources like crustaceans, insect-based chitin and chitosan have clear benefits, especially in terms of affordability and sustainability. Because of its high protein and mineral content, crustacean-derived chitin extraction necessitates extensive chemical treatment, raising environmental concerns (Aider 2010). The BSFL exuviae, on the other hand, require milder processing conditions, use fewer chemicals, and have a smaller environmental impact due to their relatively low mineral content (Mohan et al., 2020; Rehman et al., 2023).

For biomedical uses like wound healing, chitosan made from BSFL exuviae shows promise. Because of its well-established antimicrobial activity, biocompatibility, and biodegradability, chitosan is a good choice for wound dressings (Feng et al., 2021; Rajinikanth et al., 2024). By boosting surface area and enhancing bioavailability, nanochitosan in particular improves these qualities. Previous studies have demonstrated that Nanochitosan significantly accelerates wound healing in diabetic mice, which is attributed to its ability to promote collagen deposition and reduce inflammation (Ehterami et al., 2018; Natarajan et al., 2019). However, additional characterization, including molecular weight and degree of deacetylation, is necessary to confirm its suitability for biomedical use. The study's findings indicated that chitosan derived from BSFL exuviae may provide comparable advantages in a wound-healing context.

4.1. FTIR examination

Significant variations between pure chitosan and its ointment formulations were revealed by the FTIR analysis, which also shed light on how the formulation affects spectroscopic characteristics. The unique peaks of pure chitosan, including amide I at 1650.48 cm⁻¹, amide II at 1557.04 cm⁻¹, and O-H stretching at 3357.21 cm⁻¹, are in agreement with the literature and validate its chemical structure. These peaks are crucial for determining the purity and locating functional groups (Kumar, 2000; Rinaudo, 2006).

However, in both ointment formulations, these distinctive peaks were either completely missing or changed. Although the absence of O-H stretching and amide bands suggests masking by the ointment base, the presence of peaks at approximately 2920 cm⁻¹ and 2851 cm⁻¹ (C-H stretching) and 1462 cm⁻¹, 1377 cm⁻¹ (C-H bending) suggests retention of some chitosan-related features. According to Wong (2015), the peak at 720 cm⁻¹, which is unusual for chitosan, is probably caused by hydrocarbons like paraffin or vaseline in the ointment. It is confirmed that interactions with the membrane structure can be responsible for the modified FTIR spectra in chitosan membranes loaded with drugs (Ma et al., 2017). This masking effect is consistent with studies on chitosan-based formulations, where interactions with other components can obscure polymer peaks (Azad et al., 2004).

As seen in the green synthesis of chitosan nanoparticles, a prior study of nanochitosan revealed additional peaks or shifts because of its nanostructure, including amide bands, C-O-C stretching at 1000–1050 cm⁻¹, and out-of-plane bending at 564 cm⁻¹ (El-Naggar et al., 2022). These are not visible in the nanochitosan-based ointment, though, most likely due to the ointment base's dominance. The similarity between the ointment spectra of chitosan-based and nanochitosan-based indicates that the FTIR profile is not significantly changed by the nano formulation, perhaps as a result of the matrix overpowering the effects of the nanostructure.

Chitosan and nanochitosan are useful in wound healing because of their antimicrobial qualities, biocompatibility, and capacity to stimulate tissue regeneration (Dai et al., 2011; Jayakumar et al., 2011). However, it has been shown that spectral masking may prevent FTIR from always confirming the presence of chitosan in complex formulations. This emphasizes the necessity of using complementary methods, like scanning electron microscopy (SEM) and X-ray diffraction (XRD), to confirm incorporation, as recommended by (Archana et al., 2013).

4.2. The analysis of SEM

For their functional uses, the dimensional differences between chitosan and nanochitosan are essential. Biopolymers made from insect exuviae typically have larger and irregular chitosan particles (7.859–46.168 µm), and the extraction process frequently produces polydisperse structures (Ifuku et al., 2013). Nevertheless, nanochitosan's spherical shape and sub-micron size (0.071–1.400 µm) increase its surface area and dispersibility, which are critical for industrial and biomedical uses (Divya et al., 2018).

For drug delivery systems, where smaller particles promote better cellular uptake and decreased immunogenicity, size reduction to the nanometer scale is especially important (Bandara et al., 2020; Khan and Alamry, 2021). The narrow size distribution of the nanochitosan particles indicates their uniformity, which further supports their potential in precision applications like antimicrobial coatings and targeted therapy (Younes and Rinaudo, 2015). These results support the adaptability of black soldier fly larvae exuviae as a sustainable feedstock and are in line with earlier studies on nanochitosan sourced from different sources. With implications for scalability and functional tailoring, the combined SEM and size distribution data offer a strong basis for future nanochitosan production optimization.

4.3. Wound enclosure

The study's findings demonstrated that nanochitosan-based ointment (NChi) is more effective than chitosan-based ointment (Chi), povidone-iodine (Pov), Vaseline (Vas), and the control group at hastening wound healing. Interestingly, the NChi group showed the quickest rate of wound closure of any day, reaching 98.53% wound closure as early as day 15. These results are explained by nanochitosan's increased surface activity and bioavailability, which improved its penetration into wound tissues and sped up cellular repair. According to similar research, formulations based on nanochitosan accelerate wound healing because of their increased surface area and antimicrobial qualities (Zhao et al., 2023b).

This result is consistent with earlier studies showing how chitosan promotes wound healing by promoting collagen deposition, fibroblast proliferation, and antimicrobial activity (Archana et al., 2013; Mayol et al., 2014). The early wound closure of NChi on day 3 (16.54%) suggested that its nanoscale structure may enhance penetration and delivery, a finding supported by studies showing nanochitosan’s improved cellular uptake and antimicrobial properties (Ong et al., 2008). In contrast, Sun et al. (2020) found that chitosan nanoparticles loaded with ascorbic acid and epigallocatechin gallate in diabetic mice achieved 62.5% closure by day 7 and 92.3% by day 14. This suggested that NChi has a faster rate (72.18% by day 9 and 98.53% by day 15) in non-diabetic mice, which may be because of formulation differences. Mayol et al. (2014) found that a chitosan physical gel accelerated healing at 3 and 10 days, although specific percentages are not detailed in the abstract, supporting the efficacy of chitosan. Similarly, Bektas et al. (2020) demonstrated that a chitosan-based gel with vitexin significantly enhanced healing by day 21, thereby reinforcing the broad applicability of chitosan formulations.

The dip in NChi’s performance on day 6 (40.18% vs. Chi’s 49.36%) may reflect differences in release kinetics, with nanochitosan potentially experiencing an initial burst release followed by slower sustained release, while Chi provides more consistent delivery. This is consistent with previous studies suggesting that nano formulations may have variable release profiles (Archana et al., 2013). The variability in Vas and Pov, particularly Vas’s decline from days 12 to 15, may indicate limitations in their bioactive properties, with Vaseline lacking chitosan’s healing mechanisms and povidone-iodine potentially causing irritation.

4.4. Protein content, hydroxyproline value and total DNA

The results in Table 5 demonstrated that the chitosan-based (Chi) and nanochitosan-based (NChi) ointments were more effective treatments for promoting wound healing compared to the control, Vaseline, and povidone-iodine groups. Both Chi and NChi treatments resulted in significantly lower hydroxyproline and total DNA levels, indicating reduced collagen accumulation and decreased cellular infiltration, which are characteristic of the remodeling phase. Although protein content remained higher in the Chi and NChi groups, this may reflect enhanced matrix protein production that supports tissue repair. Notably, the lowest DNA content observed in the NChi group suggests that wounds treated with nanochitosan progressed further toward maturation, with reduced inflammation and controlled tissue regeneration. Collectively, these findings highlight that nanochitosan, and to a slightly lesser extent chitosan, can accelerate wound remodeling and improve the healing process (Maita et al., 2022; Sharda et al., 2023).

Protein content in the NChi group (182.29 ± 10.47 μg/mg) was lower than in the control group (133.90 ± 15.85 μg/mg) and Vaseline group (141.55 ± 16.35 μg/mg), suggesting that inflammation was considerably reduced. Lower protein levels might be associated with decreased expression of pro-inflammatory cytokines that drive the inflammatory phase, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (Apte et al., 2023; Werner and Grose, 2003). Through its inhibition of the NF-κB pathway, nanochitosan suppresses these cytokines, thereby lowering oxidative stress and neutrophil infiltration (Ma et al., 2016; Zhao et al., 2023b). Similar studies have reported that nanochitosan accelerates tissue repair by enhancing fibroblast activity and reducing inflammatory cytokine levels (Ferreira et al., 2020). Although less effective than nanochitosan, chitosan-based ointments (Chi) demonstrated promising results, with the highest protein content (226.74 ± 12.66 μg/mg), reflecting its role in stimulating extracellular matrix (ECM) synthesis. The relatively lower efficacy of Chi compared to NChi is likely due to its larger particle size, which limits penetration and bioavailability (Feng et al., 2021).

As a key component of collagen and a biomarker of collagen synthesis, hydroxyproline levels further supported the advanced healing stage in the NChi-treated group. The hydroxyproline level in the NChi group (352.67 ± 13.64 μg/mg) was significantly lower than in the control (551.37 ± 18.53 μg/mg) and Vaseline groups (611.50 ± 62.18 μg/mg), as well as povidone-iodine (808.89 ± 85.20 μg/mg). This reduction indicates that collagen deposition and remodeling have advanced further in the NChi group, while higher hydroxyproline levels in the other groups suggest ongoing collagen accumulation during the proliferative phase. Previous findings have shown that nanochitosan’s nanoscale structure improves cellular uptake and interaction with fibroblasts, thereby accelerating collagen maturation and alignment (Kusnadi et al., 2024). Its nanoscale characteristics provide a biocompatible scaffold that promotes fibroblast activity and extracellular matrix reorganization, enhancing collagen remodeling. In addition, the antimicrobial and anti-inflammatory properties of nanochitosan create a favorable microenvironment for tissue regeneration (Adetunji et al., 2025; Jin et al., 2021; You et al., 2017b). Hydroxyproline, a post-translationally modified amino acid and hallmark of collagen stability, is crucial for restoring skin integrity during wound healing, and its levels provide a reliable indicator of collagen synthesis and remodeling efficiency (Srivastava et al., 2016; Li and Wu, 2018; Patel et al., 2016).

Total DNA content also provided important insights into the healing process. Compared to the control (12.66 ± 2.73 μg/mg) and Vaseline groups (9.44 ± 0.09 μg/mg), the NChi group exhibited significantly lower DNA levels (3.77 ± 1.49 μg/mg), reflecting reduced inflammatory cell infiltration and lower proliferative activity. High DNA content in wounds is typically associated with increased cell proliferation during the inflammatory and proliferative phases (Wu and Chen, 2014). Therefore, the reduced DNA content in the NChi group highlights the progression of wounds into the remodeling phase, where cellular activity decreases as tissue structure stabilizes.

In comparison, povidone-iodine (Pov) treatment resulted in moderate effectiveness, with protein (123.53 ± 11.42 μg/mg) and DNA (11.41 ± 0.74 μg/mg) levels remaining relatively high despite elevated hydroxyproline levels (808.89 ± 85.20 μg/mg). Its delayed healing performance is consistent with previous reports that povidone-iodine exhibits cytotoxic effects on fibroblasts and keratinocytes (Bigliardi et al., 2017; Kramer, 1999). The control and Vaseline groups, which showed higher protein and DNA levels, also demonstrated delayed healing, most likely due to the absence of bioactive compounds that stimulate tissue regeneration.

5. Conclusion

The results of this study demonstrated that a nanochitosan-based ointment derived from the exuviae of black soldier fly larvae (BSFL) significantly enhanced wound healing in mice, achieving 100% wound closure by day 15. This outcome outperformed treatments based on chitosan (88.75%), povidone iodine (87.5%), and Vaseline (91.75%) (p < 0.05). Biochemical analysis further supported these findings, with the NChi group showing the low levels of protein (182.29 ± 10.47 μg/mg), hydroxyproline (352.67 ± 13.64 μg/mg), and total DNA (3.77 ± 1.49 μg/mg), indicative of reduced inflammation, advanced collagen remodeling, and progression toward the tissue maturation phase. The superior efficacy of nanochitosan is attributed to its nanoscale particle size, which enhances bioavailability, antimicrobial activity, and cellular interactions, making it a sustainable alternative to crustacean-derived materials. The FTIR analysis confirmed the structural integrity of the nanochitosan, while SEM observations revealed uniform morphology, supporting its potential as a wound-healing agent. Future research should explore the molecular mechanisms underlying nanochitosan’s therapeutic effects, particularly in relation to gene expression and cytokine signalling pathways. Additionally, scaling up production, ensuring long-term safety, and evaluating its performance in chronic wounds, diabetic ulcers, and burns are essential steps to broaden its biomedical applications. Collectively, these findings highlight nanochitosan from BSFL exuviae as an eco-friendly and promising biomaterial for the development of sustainable wound-healing therapies

Data Availability Statement

The research data analyzed in this study are not publicly available by any means.

Acknowledgements

This study was funded by KEMDIKBUD RISTEK, Ministry of Education and Culture, Research and Technology, Financial Year 2024 (contract number 658/UN/17). L1/HK/2024, using a postgraduate master’s program competitive scheme. All authors also thank the Research and Community Service Agency, Mulawarman University, and the Faculty of Mathematics and Natural Sciences, Mulawarman University, for their support.

References

• ABDELTWAB, W.M., ABDELALIEM, Y.F., METRY, W.A. and ELDEGHEDY, M., 2019. Antimicrobial effect of chitosan and nano-chitosan against some pathogens and spoilage microorganisms. Journal of Advanced Laboratory Research in Biology, vol. 10, no. 1, pp. 8-15.
• ADETUNJI, J.B., SALAUDEEN, W.O., ADETUNJI, C.O., ADEYOMOYE, O.I. and OYEWOLE, O.A., 2025 [viewed 24 December 2024]. Application of chitosan-based nanoparticles for the treatment of liver diseases. In: C.O. ADETUNJI, M.K. DANQUAH, J. JEEVANANDAM, D.I. HEFFT, S. PAN and T.A. KHAN, eds. Chitosan-based nanoparticles for biomedical applications [online]. Cambridge: Elsevier, pp. 275-287. Woodhead Publishing Series in Biomaterials. Available from: http://doi.org/10.1016/B978-0-443-13997-0.00013-8
  » http://doi.org/10.1016/B978-0-443-13997-0.00013-8
• AIDER, M., 2010. Chitosan application for active bio-based films production and potential in the food industry. LWT - Food Science and Technology, vol. 43, no. 6, pp. 837-842. http://doi.org/10.1016/j.lwt.2010.01.021
  » http://doi.org/10.1016/j.lwt.2010.01.021
• AKRAM, H., MATEOS-PEDRERO, C., GALLEGOS-SUÁREZ, E., ALLALI, N., CHAFIK, T., RODRIGUEZ-RAMOS, I. and RUIZ, A.G., 2012. Low solvothermal synthesis and characterization of hollow nanospheres molybdenum sulfide. Journal of Nanoscience and Nanotechnology, vol. 12, no. 8, pp. 6679-6685. http://doi.org/10.1166/jnn.2012.4561 PMid:22962806.
  » http://doi.org/10.1166/jnn.2012.4561
• AKRAM, N., SHAHZADI, I., ZIA, K.M., SAEED, M., ALI, A., AL-SALAHI, R., ABUELIZZ, H.A. and VERPOORT, F., 2023. Fabrication and in vitro biological assay of thermo-mechanically tuned chitosan reinforced polyurethane composites. Molecules, vol. 28, no. 20, pp. 7218. http://doi.org/10.3390/molecules28207218 PMid:37894696.
  » http://doi.org/10.3390/molecules28207218
• ALEMU, D., GETACHEW, E. and MONDAL, A.K., 2023. Study on the physicochemical properties of chitosan and their applications in the biomedical sector. International Journal of Polymer Science, vol. 2023, pp. 1-13. http://doi.org/10.1155/2023/5025341
  » http://doi.org/10.1155/2023/5025341
• APTE, A., LIECHTY, K.W. and ZGHEIB, C., 2023. Immunomodulatory biomaterials on chemokine signaling in wound healing. Frontiers in Pharmacology, vol. 14, pp. 1084948. https://doi.org/10.3389/fphar.2023.1084948
  » https://doi.org/10.3389/fphar.2023.1084948
• ARCHANA, D., DUTTA, J. and DUTTA, P.K., 2013. Evaluation of chitosan nano dressing for wound healing: characterization, in vitro and in vivo studies. International journal of biological macromolecules, vol. 57, pp 193-203. https://doi.org/10.1016/j.ijbiomac.2013.03.002 PMid:23518244.
  » https://doi.org/10.1016/j.ijbiomac.2013.03.002
• ARIF, M.M., KHAN, S.M., GULL, N., TABISH, T.A., ZIA, S., KHAN, R.U., AWAIS, S.M. and BUTT, M.A., 2021. Polymer-based biomaterials for chronic wound management: promises and challenges. International Journal of Pharmaceutics, vol. 598, pp. 120270. http://doi.org/10.1016/j.ijpharm.2021.120270
  » http://doi.org/10.1016/j.ijpharm.2021.120270
• ASKARI, M., ZARJANI, A.K., SAYYAHI, A., BADPA, R. and NAGHIZADEH, A., 2025. Chitosan nanoparticles: a promising candidate in wound healing. The International Journal of Lower Extremity Wounds In press. http://doi.org/10.1177/15347346251325057 PMid:40096054.
  » http://doi.org/10.1177/15347346251325057
• AZAD, A.K., SERMSINTHAM, N., CHANDRKRACHANG, S. and STEVENS, W.F., 2004. Chitosan membrane as a wound‐healing dressing: characterization and clinical application. Journal of Biomedical Materials Research. Part B, Applied Biomaterials, vol. 69, no. 2, pp. 216-222. http://doi.org/10.1002/jbm.b.30000 PMid:15116411.
  » http://doi.org/10.1002/jbm.b.30000
• BANDARA, S., DU, H., CARSON, L., BRADFORD, D. and KOMMALAPATI, R., 2020. Agricultural and biomedical applications of chitosan-based nanomaterials. Nanomaterials, vol. 10, no. 10, pp. 1903. http://doi.org/10.3390/nano10101903 PMid:32987697.
  » http://doi.org/10.3390/nano10101903
• BEKTAS, N., ŞENEL, B., YENILMEZ, E., ÖZATIK, O. and ARSLAN, R., 2020. Evaluation of wound healing effect of chitosan-based gel formulation containing vitexin. Saudi Pharmaceutical Journal, vol. 28, no. 1, pp. 87-94. http://doi.org/10.1016/j.jsps.2019.11.008 PMid:31933527.
  » http://doi.org/10.1016/j.jsps.2019.11.008
• BIGLIARDI, P.L., ALSAGOFF, S.A.L., EL-KAFRAWI, H.Y., PYON, J.K., WA, C.T.C. and VILLA, M.A., 2017. Povidone iodine in wound healing: a review of current concepts and practices. International Journal of Surgery, vol. 44, pp. 260-268. http://doi.org/10.1016/j.ijsu.2017.06.073
  » http://doi.org/10.1016/j.ijsu.2017.06.073
• BILAL, K., MEHBOOB, F., AKHTAR, N., MIRZA, I.A., OKLA, M.K., DAR, M.J., SALEH, I.A., ZOMOT, N. and FATIMA, H., 2024. Wound healing, antioxidant and antibacterial activities of polyphenols of Psidium guajava L. leaves. South African Journal of Botany, vol. 165, pp. 538-551. http://doi.org/10.1016/j.sajb.2023.12.026
  » http://doi.org/10.1016/j.sajb.2023.12.026
• CEDILLO-CORTEZANO, M., MARTINEZ-CUEVAS, L.R., LÓPEZ, J.A.M., BARRERA LÓPEZ, I.L., ESCUTIA-PEREZ, S. and PETRICEVICH, V.L., 2024. Use of medicinal plants in the process of wound healing: A literature review. Pharmaceuticals, vol. 17, no. 3, pp. 303. http://doi.org/10.3390/ph17030303 PMid:38543089.
  » http://doi.org/10.3390/ph17030303
• CHANDRASEKARAN, M., KIM, K.D. and CHUN, S.C., 2020. Antibacterial activity of chitosan nanoparticles: a review. Processes, vol. 8, no. 9, pp. 1173. http://doi.org/10.3390/pr8091173
  » http://doi.org/10.3390/pr8091173
• CHÁVEZ DE PAZ, L.E., RESIN, A., HOWARD, K.A., SUTHERLAND, D.S. and WEJSE, P.L., 2011. Antimicrobial effect of chitosan nanoparticles on streptococcus mutans biofilms. Applied and Environmental Microbiology, vol. 77, no. 11, pp. 3892-3895. http://doi.org/10.1128/AEM.02941-10 PMid:21498764.
  » http://doi.org/10.1128/AEM.02941-10
• DAI, T., TANAKA, M., HUANG, Y.Y. and HAMBLIN, M.R., 2011. Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects. Expert Review of Anti-Infective Therapy, vol. 9, no. 7, pp. 857-879. http://doi.org/10.1586/eri.11.59 PMid:21810057.
  » http://doi.org/10.1586/eri.11.59
• DARWESH, O. M., SULTAN, Y. Y., SEIF, M. M., and MARREZ, D. A., 2018. Bio-evaluation of crustacean and fungal nano-chitosan for applying as food ingredient. Toxicology Reports, vol. 5, pp. 348-356.
• DIVYA, K., SMITHA, V. and JISHA, M.S., 2018. Antifungal, antioxidant and cytotoxic activities of chitosan nanoparticles and its use as an edible coating on vegetables. International Journal of Biological Macromolecules, vol. 114, pp. 572-577. http://doi.org/10.1016/j.ijbiomac.2018.03.130
  » http://doi.org/10.1016/j.ijbiomac.2018.03.130
• EGGINK, K.M. and DALSGAARD, J., 2023. Chitin contents in different black soldier fly (Hermetia illucens) life stages. Journal of Insects as Food and Feed, vol. 9, no. 7, pp. 855-863. http://doi.org/10.3920/JIFF2022.0142
  » http://doi.org/10.3920/JIFF2022.0142
• EHTERAMI, A., SALEHI, M., FARZAMFAR, S., VAEZ, A., SAMADIAN, H., SAHRAPEYMA, H., MIRZAII, M., GHORBANI, S. and GOODARZI, A., 2018. In vitro and in vivo study of pcl/coll wound dressing loaded with insulin-chitosan nanoparticles on cutaneous wound healing in rats model. International Journal of Biological Macromolecules, vol. 117, pp. 601-609. https://doi.org/10.1016/j.ijbiomac.2018.05.184 PMid:29807077.
  » https://doi.org/10.1016/j.ijbiomac.2018.05.184
• EHYAEIRAD, N., BABOLANIMOGADAM, N., DADKHAH, M. and SHIRMARD, L.R., 2024. Polylactic acid films incorporated with nanochitosan, nanocellulose, and ajwain essential oil: Synthesis, characterizations, with in-vitro and in-vivo antimicrobial properties for infected wound healing. Carbohydrate Polymer Technologies and Applications, vol. 7, pp. 100425. https://doi.org/10.1016/j.carpta.2024.100425
  » https://doi.org/10.1016/j.carpta.2024.100425
• EL KNIDRI, H., EL KHALFAOUY, R., LAAJEB, A., ADDAOU, A. and LAHSINI, A. 2016. Eco-friendly extraction and characterization of chitin and chitosan from the shrimp shell waste via microwave irradiation. Process Safety and Environmental Protection, vol. 104, pp. 395-405. https://doi.org/10.1016/j.psep.2016.09.020
  » https://doi.org/10.1016/j.psep.2016.09.020
• EL-NAGGAR, N.E.A., SHIHA, A.M., MAHROUS, H. and MOHAMMED, A.A., 2022. Green synthesis of chitosan nanoparticles, optimization, characterization and antibacterial efficacy against multi drug resistant biofilm-forming acinetobacter baumannii. Scientific Reports, vol. 12, no. 1, pp. 19869. http://doi.org/10.1038/s41598-022-24303-5 PMid:36400832.
  » http://doi.org/10.1038/s41598-022-24303-5
• EL-SHERBENI, S.A. and NEGM, W.A., 2023. The wound healing effect of botanicals and pure natural substances used in in vivo models. Inflammopharmacology, vol. 31, no. 2, pp. 755-772. http://doi.org/10.1007/s10787-023-01157-5 PMid:36811778.
  » http://doi.org/10.1007/s10787-023-01157-5
• EMING, S.A., WYNN, T.A. and MARTIN, P., 2017. Inflammation and metabolism in tissue repair and regeneration. Science, vol. 356, no. 6342, pp. 1026-1030. http://doi.org/10.1126/science.aam7928 PMid:28596335.
  » http://doi.org/10.1126/science.aam7928
• FAVIER, A.L. and NIKOVICS, K., 2023. Molecular and cellular mechanisms of inflammation and tissue regeneration. Biomedicines, vol. 11, no. 5, pp. 1416. https://doi.org/10.3390/biomedicines11051416
  » https://doi.org/10.3390/biomedicines11051416
• FENG, P., LUO, Y., KE, C., QIU, H., WANG, W., ZHU, Y., HOU, R., XU, L. and WU, S. 2021. Chitosan-based functional materials for skin wound repair: mechanisms and applications. Frontiers in Bioengineering and Biotechnology, vol. 9, pp. 650598. https://doi.org/10.3389/fbioe.2021.650598 PMid:33681176.
  » https://doi.org/10.3389/fbioe.2021.650598
• FERREIRA, D.W., ULECIA-MORÓN, C., ALVARADO-VÁZQUEZ, P.A., CUNNANE, K., MORACHO-VILRIALES, C., GROSICK, R.L., CUNHA, T.M. and ROMERO-SANDOVAL, E.A., 2020. Cd163 overexpression using a macrophage-directed gene therapy approach improves wound healing in ex vivo and in vivo human skin models. Immunobiology, vol. 225, no. 1, pp. 151862. http://doi.org/10.1016/j.imbio.2019.10.011 PMid:31711674.
  » http://doi.org/10.1016/j.imbio.2019.10.011
• FERREIRA, P.G., FERREIRA, V.F., SILVA, F.C., FREITAS, C.S., PEREIRA, P.R. and PASCHOALIN, V.M.F., 2022. Chitosans and nanochitosans: recent advances in skin protection, regeneration, and repair. Pharmaceutics, vol. 14, no. 6, pp. 1307. http://doi.org/10.3390/pharmaceutics14061307 PMid:35745879.
  » http://doi.org/10.3390/pharmaceutics14061307
• FREITAS, C.S., PEREIRA, P.R., CARDOSO, R.V., PAULI, F.P., RIBEIRO, R.C.B., SILVA, F.D.C., FERREIRA, V.F. and PASCHOALIN, V.M.F., 2024. Antimicrobial and wound healing potential of naphthoquinones encapsulated in nanochitosan. Frontiers in Bioengineering and Biotechnology, vol. 11, pp. 1284630. https://doi.org/10.3389/fbioe.2023.1284630 PMid:38239922.
  » https://doi.org/10.3389/fbioe.2023.1284630
• HWANG, J., KIICK, K.L. and SULLIVAN, M.O., 2023. Vegf-encoding, gene-activated collagen-based matrices promote blood vessel formation and improved wound repair. ACS Applied Materials & Interfaces, vol. 15, no. 13, pp. 16434-16447. http://doi.org/10.1021/acsami.2c23022 PMid:36961242.
  » http://doi.org/10.1021/acsami.2c23022
• IBRAHIM, N.I., WONG, S.K., MOHAMED, I.N., MOHAMED, N., CHIN, K.Y., IMA-NIRWANA, S. and SHUID, A.N., 2018. Wound healing properties of selected natural products. International Journal of Environmental Research and Public Health, vol. 15, no. 11, pp. 2360. http://doi.org/10.3390/ijerph15112360 PMid:30366427.
  » http://doi.org/10.3390/ijerph15112360
• IFUKU, S., IKUTA, A., EGUSA, M., KAMINAKA, H., IZAWA, H., MORIMOTO, M. and SAIMOTO, H., 2013. Preparation of high-strength transparent chitosan film reinforced with surface-deacetylated chitin nanofibers. Carbohydrate Polymers, vol. 98, no. 1, pp. 1198-1202. http://doi.org/10.1016/j.carbpol.2013.07.033 PMid:23987464.
  » http://doi.org/10.1016/j.carbpol.2013.07.033
• JAYAKUMAR, R., PRABAHARAN, M., KUMAR, P.S., NAIR, S. and TAMURA, H., 2011. Biomaterials based on chitin and chitosan in wound dressing applications. Biotechnology Advances, vol. 29, no. 3, pp. 322-337. http://doi.org/10.1016/j.biotechadv.2011.01.005 PMid:21262336.
  » http://doi.org/10.1016/j.biotechadv.2011.01.005
• JIN, T., LIU, T., LAM, E. and MOORES, A., 2021. Chitin and chitosan on the nanoscale. Nanoscale Horizons, vol. 6, no. 7, pp. 505-542. http://doi.org/10.1039/D0NH00696C PMid:34017971.
  » http://doi.org/10.1039/D0NH00696C
• KANDILE, N.G., ZAKY, H.T., MOHAMED, M.I., NASR, A.S. and ALI, Y.G., 2018. Extraction and characterization of chitosan from shrimp shells. Open Journal of Organic Polymer Materials, vol. 8, no. 3, pp. 33-42. http://doi.org/10.4236/ojopm.2018.83003
  » http://doi.org/10.4236/ojopm.2018.83003
• KHAN, A. and ALAMRY, K.A., 2021. Recent advances of emerging green chitosan-based biomaterials with potential biomedical applications: a review. Carbohydrate Research, vol. 506, pp. 108368. https://doi.org/10.1016/j.carres.2021.108368 PMid:34111686.
  » https://doi.org/10.1016/j.carres.2021.108368
• KRAMER, S.A., 1999. Effect of povidone-iodine on wound healing: a review. Journal of Vascular Nursing, vol. 17, no. 1, pp. 17-23. http://doi.org/10.1016/s1062-0303(99)90004-3 PMid:10362983.
  » http://doi.org/10.1016/s1062-0303(99)90004-3
• KUMAR, M.N.V.R., 2000. A review of chitin and chitosan applications. Reactive & Functional Polymers, vol. 46, no. 1, pp. 1-27. http://doi.org/10.1016/S1381-5148(00)00038-9
  » http://doi.org/10.1016/S1381-5148(00)00038-9
• KUSNADI, K., HERDIANA, Y., ROCHIMA, E., PUTRA, O.N., GAZZALI, A. and MUCHTARIDI, M., 2024. Collagen-based nanoparticles as drug delivery system in wound healing applications. International Journal of Nanomedicine, vol. 2024, no. 19, pp 11321-11341. https://doi.org/10.2147/ijn.s485588 PMid:39524919.
  » https://doi.org/10.2147/ijn.s485588
• LAGAT, M.K., WERE, S., NDWIGAH, F., KEMBOI, V.J., KIPKOECH, C. and TANGA, C.M., 2021. Antimicrobial activity of chemically and biologically treated chitosan prepared from black soldier fly (Hermetia illucens) pupal shell waste. Microorganisms, vol. 9, no. 12, pp. 2417. http://doi.org/10.3390/microorganisms9122417 PMid:34946019.
  » http://doi.org/10.3390/microorganisms9122417
• LE, T.M., TRAN, C.L., NGUYEN, T.X., DUONG, Y.H., LE, P.K. and TRAN, V.T., 2023. Green preparation of chitin and nanochitin from black soldier fly for production of biodegradable packaging material. Journal of Polymers and the Environment, vol. 31, no. 7, pp. 3094-3105. http://doi.org/10.1007/s10924-023-02793-2
  » http://doi.org/10.1007/s10924-023-02793-2
• LI, P. and WU, G., 2018. Roles of dietary glycine, proline, and hydroxyproline in collagen synthesis and animal growth. Amino Acids, vol. 50, no. 1, pp. 29-38. http://doi.org/10.1007/s00726-017-2490-6 PMid:28929384.
  » http://doi.org/10.1007/s00726-017-2490-6
• LI, Z., FAN, X., LUO, Z., LOH, X.J., MA, Y., YE, E., WU, Y.L., HE, C. and LI, Z., 2022. Nanoenzyme-chitosan hydrogel complex with cascade catalytic and self-reinforced antibacterial performance for accelerated healing of diabetic wounds. Nanoscale, vol. 14, no. 40, pp. 14970-14983. https://doi.org/10.1039/d2nr04171e PMid:36217671.
  » https://doi.org/10.1039/d2nr04171e
• LI, Y., XU, H., WANG, Y., ZHU, Y., XU, K., YANG, Z., LI, Y. and GUO, C., 2024. Epithelium-derived exosomes promote silica nanoparticles-induced pulmonary fibroblast activation and collagen deposition via modulating fibrotic signaling pathways and their epigenetic regulations. Journal of Nanobiotechnology, vol. 22, no. 1, pp. 331. http://doi.org/10.1186/s12951-024-02609-y PMid:38867284.
  » http://doi.org/10.1186/s12951-024-02609-y
• LIOI, M., TENGATTINI, S., D'ATRI, V., MASSOLINI, G., DALY, S., TEMPORINI, C. and GUILLARME, D., 2024. Evaluating the potential of hydrophilic interaction liquid chromatography for collagen peptide mapping analysis. Journal of Chromatography A, vol. 1738, pp. 465473. https://doi.org/10.1016/j.chroma.2024.465473 PMid:39504706.
  » https://doi.org/10.1016/j.chroma.2024.465473
• MA, L., SHEN, C.A., GAO, L., LI, D.W., SHANG, Y.R., YIN, K., ZHAO, D.X., CHENG, W.F. and QUAN, D.Q., 2016. A Anti-inflammatory activity of chitosan nanoparticles carrying NF-κB/p65 antisense oligonucleotide in RAW264.7 macropghage stimulated by lipopolysaccharide. Colloids and Surfaces B: Biointerfaces, vol. 142, pp. 297-306. https://doi.org/10.1016/j.colsurfb.2016.02.031 PMid:26970817.
  » https://doi.org/10.1016/j.colsurfb.2016.02.031
• MA, Y., XIN, L., TAN, H., FAN, M., LI, J., JIA, Y., LING, Z., CHEN, Y. and HU, X., 2017. Chitosan membrane dressings toughened by glycerol to load antibacterial drugs for wound healing. Materials Science and Engineering: C, vol. 81, pp. 522-531. https://doi.org/10.1016/j.msec.2017.08.052 PMid:28888006.
  » https://doi.org/10.1016/j.msec.2017.08.052
• MAITA, K.C., AVILA, F.R., TORRES-GUZMAN, R.A., GARCIA, J.P., ELDALY, A.S., PALMIERI, L., EMAM, O.S., HO, O. and FORTE, A.J., 2022. Local anti-inflammatory effect and immunomodulatory activity of chitosan-based dressing in skin wound healing: a systematic review. Journal of Clinical and Translational Research, vol. 8, no. 6, pp. 488-498. PMid:36451998.
• MAMUN, A.A., SHAO, C., GENG, P., WANG, S. and XIAO, J., 2024. Recent advances in molecular mechanisms of skin wound healing and its treatments. Frontiers in Immunology, vol. 15, pp. 1395479. https://doi.org/10.3389/fimmu.2024.1395479 PMid:38835782.
  » https://doi.org/10.3389/fimmu.2024.1395479
• MAYOL, L., STEFANO, D., CAMPANI, V., FALCO, F., FERRARI, E., CENCETTI, C., MATRICARDI, P., MAIURI, L., CARNUCCIO, R., GALLO, A., MAIURI, M.C. and ROSA, G., 2014. Design and characterization of a chitosan physical gel promoting wound healing in mice. Journal of Materials Science. Materials in Medicine, vol. 25, no. 6, pp. 1483-1493. http://doi.org/10.1007/s10856-014-5175-7 PMid:24584669.
  » http://doi.org/10.1007/s10856-014-5175-7
• MILINKOVIĆ BUDINČIĆ, J., RADONIĆ, Ž., DRAGOJLOVIĆ, D., SEDLAR, T., MILKOVIĆ, M., POLIĆ PASKOVIĆ, M., and PASKOVIĆ, I., 2025. Exuviae of Tenebrio molitor larvae as a source of chitosan: characterisation and possible applications. Applied Sciences, vol. 15, no. 17, pp. 9285.
• MIRHAJ, M., LABBAF, S., TAVAKOLI, M. and SEIFALIAN, A.M., 2022. Emerging treatment strategies in wound care. International Wound Journal, vol. 19, no. 7, pp. 1934-1954. http://doi.org/10.1111/iwj.13786 PMid:35297170.
  » http://doi.org/10.1111/iwj.13786
• MOEINI, A., PEDRAM, P., MAKVANDI, P., MALINCONICO, M. and D’AYALA, G., 2020. Wound healing and antimicrobial effect of active secondary metabolites in chitosan-based wound dressings: A review. Carbohydrate Polymers, vol. 233, pp. 115839. http://doi.org/10.1016/j.carbpol.2020.115839
  » http://doi.org/10.1016/j.carbpol.2020.115839
• MOHAN, K., GANESAN, A.R., MURALISANKAR, T., JAYAKUMAR, R., SATHISHKUMAR, P., UTHAYAKUMAR, V., CHANDIRASEKAR, R. and REVATHI, N., 2020. Recent insights into the extraction, characterization, and bioactivities of chitin and chitosan from insects. Trends in Food Science and Technology, vol. 105, pp. 17-42. https://doi.org/10.1016/j.tifs.2020.08.016 PMid:32901176.
  » https://doi.org/10.1016/j.tifs.2020.08.016
• NASERI, N., MATHEW, A.P., GIRANDON, L., FRÖHLICH, M. and OKSMAN, K., 2015. Porous electrospun nanocomposite mats based on chitosan–cellulose nanocrystals for wound dressing: effect of surface characteristics of nanocrystals. Cellulose, vol. 22, pp. 521-534. https://doi.org/10.1007/s10570-014-0493-y
  » https://doi.org/10.1007/s10570-014-0493-y
• NATARAJAN, J., SANAPALLI, B.K.R., BANO, M., SINGH, S.K., GULATI, M. and KARRI, V.V.S.R., 2019. Nanostructured lipid carriers of pioglitazone loaded collagen/chitosan composite scaffold for diabetic wound healing. Advances in Wound Care, vol. 8, no. 10, pp. 499-513. http://doi.org/10.1089/wound.2018.0831 PMid:31737408.
  » http://doi.org/10.1089/wound.2018.0831
• NURDEN, A.T., 2011. Platelets, inflammation and tissue regeneration. Thrombosis and haemostasis, vol. 105, suppl. 1, pp. S13-S33. https://doi.org/10.1160/ths10-11-0720 PMid:21479340.
  » https://doi.org/10.1160/ths10-11-0720
• ONG, S.Y., WU, J., MOOCHHALA, S.M., TAN, M.H. and LU, J., 2008. Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties. Biomaterials, vol. 29, no. 32, pp. 4323-4332. http://doi.org/10.1016/j.biomaterials.2008.07.034 PMid:18708251.
  » http://doi.org/10.1016/j.biomaterials.2008.07.034
• PATEL, S., MAHESHWARI, A. and CHANDRA, A., 2016. Biomarkers for wound healing and their evaluation. Journal of Wound Care, vol. 25, no. 1, pp. 46-55. http://doi.org/10.12968/jowc.2016.25.1.46 PMid:26762498.
  » http://doi.org/10.12968/jowc.2016.25.1.46
• PEÑA, O.A. and MARTIN, P., 2024. Cellular and molecular mechanisms of skin wound healing. Nature Reviews. Molecular Cell Biology, vol. 25, no. 8, pp. 599-616. http://doi.org/10.1038/s41580-024-00715-1 PMid:38528155.
  » http://doi.org/10.1038/s41580-024-00715-1
• PRANEETH, Y., KOMAL, DEVGON, I., SACHAN, R.S.K., RANA, A., KARNWAL, A. and KUMAR, A., 2023. Chitosan in wound healing: a mini review on ethical perspective on sustainable and biomedical biomaterials. Regenerative Engineering and Translational Medicine, vol. 10, pp. 357-386. https://doi.org/10.1007/s40883-023-00330-0
  » https://doi.org/10.1007/s40883-023-00330-0
• RAJINIKANTH, B.S., RAJKUMAR, D.S.R., KEERTHIKA, K. and VIJAYARAGAVAN, V., 2024. Chitosan-based biomaterial in wound healing: a review. Cureus, vol. 16, no. 2, e55193. https://doi.org/10.7759/cureus.55193 PMid:38562272.
  » https://doi.org/10.7759/cureus.55193
• RAMPURE, S.M., VELAYUDHANNAIR, K. and MARIMUTHU, N., 2023. Characteristics of chitin extracted from different growth phases of black soldier fly, hermetia illucens, fed with different organic wastes. International Journal of Tropical Insect Science, vol. 43, no. 3, pp. 979-987. http://doi.org/10.1007/s42690-023-00997-6
  » http://doi.org/10.1007/s42690-023-00997-6
• REHMAN, K., HOLLAH, C., WIESOTZKI, K., HEINZ, V., AGANOVIC, K., REHMAN, R., PETRUSAN, J.I., ZHENG, L., ZHANG, J., SOHAIL, S., MANSOOR, M.K., RUMBOS, C.I., ATHANASSIOU, C. and CAI, M., 2023. Insect-derived chitin and chitosan: A still unexploited resource for the edible insect sector. Sustainability, vol. 15, no. 6, pp. 4864. http://doi.org/10.3390/su15064864
  » http://doi.org/10.3390/su15064864
• RINAUDO, M., 2006. Chitin and chitosan: properties and applications. Progress in Polymer Science, vol. 31, no. 7, pp. 603-632. http://doi.org/10.1016/j.progpolymsci.2006.06.001
  » http://doi.org/10.1016/j.progpolymsci.2006.06.001
• SÁNCHEZ-MACHADO, D.I., LÓPEZ-CERVANTES, J., ESCÁRCEGA-GALAZ, A.A., CAMPAS-BAYPOLI, O.N., MARTÍNEZ-IBARRA, D.M. and RASCÓN-LEÓN, S., 2024. Measurement of the degree of deacetylation in chitosan films by ftir, 1h nmr and uv spectrophotometry. MethodsX, vol. 12, pp. 102583. https://doi.org/10.1016/j.mex.2024.102583
  » https://doi.org/10.1016/j.mex.2024.102583
• SHARDA, D., GHOSH, S., KAUR, P., BASU, B. and CHOUDHURY, D., 2023. Chitosan-insulin nano-formulations as critical modulators of inflammatory cytokines and nrf-2 pathway to accelerate burn wound healing. Discover Nano, vol. 18, no. 1, pp. 154. http://doi.org/10.1186/s11671-023-03941-2 PMid:38087141.
  » http://doi.org/10.1186/s11671-023-03941-2
• SHARIFI, M. and BAHRAMI, S.H., 2024. Review on application of herbal extracts in biomacromolecules-based nanofibers as wound dressings and skin tissue engineering. International Journal of Biological Macromolecules, vol. 277, pp. 133666. https://doi.org/10.1016/j.ijbiomac.2024.133666 PMid:38971295.
  » https://doi.org/10.1016/j.ijbiomac.2024.133666
• SHENG, Z., GUO, A., WANG, J. and CHEN, X., 2022. Preparation, physicochemical properties and antimicrobial activity of chitosan from fly pupae. Heliyon, vol. 8, no. 10, e11168. http://doi.org/10.1016/j.heliyon.2022.e11168 PMid:36303907.
  » http://doi.org/10.1016/j.heliyon.2022.e11168
• SILVA LUCAS, A.J., ORESTE, E.Q., COSTA, H.L.G., LÓPEZ, H.M., SAAD, C.D.M. and PRENTICE, C., 2021. Extraction, physicochemical characterization, and morphological properties of chitin and chitosan from cuticles of edible insects. Food Chemistry, vol. 343, pp. 128550. http://doi.org/10.1016/j.foodchem.2020.128550 PMid:33191008.
  » http://doi.org/10.1016/j.foodchem.2020.128550
• SRIVASTAVA, A.K., KHARE, P., NAGAR, H.K., RAGHUWANSHI, N. and SRIVASTAVA, R., 2016. Hydroxyproline: a potential biochemical marker and its role in the pathogenesis of different diseases. Current Protein & Peptide Science, vol. 17, no. 6, pp. 596-602. http://doi.org/10.2174/1389203717666151201192247 PMid:26916157.
  » http://doi.org/10.2174/1389203717666151201192247
• SUN, M., XIE, Q., CAI, X., LIU, Z., WANG, Y., DONG, X. and XU, Y. 2020. Preparation and characterization of epigallocatechin gallate, ascorbic acid, gelatin, chitosan nanoparticles and their beneficial effect on wound healing of diabetic mice. International journal of biological macromolecules, vol. 148, pp. 777-784. https://doi.org/10.1016/j.ijbiomac.2020.01.198 PMid:31978475.
  » https://doi.org/10.1016/j.ijbiomac.2020.01.198
• SWINEHART, I.T. and BADYLAK, S.F., 2016. Extracellular matrix bioscaffolds in tissue remodeling and morphogenesis. Developmental Dynamics, vol. 245, no. 3, pp. 351-360. http://doi.org/10.1002/dvdy.24379 PMid:26699796.
  » http://doi.org/10.1002/dvdy.24379
• TRIUNFO, M., TAFI, E., GUARNIERI, A., IANNICIELLO, D., SCIEUZO, C., SALVIA, R., HAHN, T., ZIBEK, S. and FALABELLA, P., 2023. Usage of chitosan from hermetia illucens as a preservative for fresh prunus species fruits: a preliminary analysis. Chemical and Biological Technologies in Agriculture, vol. 10, no. 1, pp. 101. http://doi.org/10.1186/s40538-023-00480-x
  » http://doi.org/10.1186/s40538-023-00480-x
• VAEZIFAR, S., RAZAVI, S., GOLOZAR, M. A., KARBASI, S., MORSHED, M., and KAMALI, M., 2013. Effects of some parameters on particle size distribution of chitosan nanoparticles prepared by ionic gelation method. Journal of Cluster Science, vol. 24, no. 3, 891-903.
• WANG, L., XU, Z., ZHANG, H. and YAO, C., 2023. A review on chitosan-based biomaterial as carrier in tissue engineering and medical applications. European Polymer Journal, vol. 191, pp. 112059. http://doi.org/10.1016/j.eurpolymj.2023.112059
  » http://doi.org/10.1016/j.eurpolymj.2023.112059
• WAŚKO, A., BULAK, P., POLAK-BERECKA, M., NOWAK, K., POLAKOWSKI, C. and BIEGANOWSKI, A., 2016. The first report of the physicochemical structure of chitin isolated from Hermetia illucens International Journal of Biological Macromolecules, vol. 92, pp. 316-320. https://doi.org/10.1016/j.ijbiomac.2016.07.038
  » https://doi.org/10.1016/j.ijbiomac.2016.07.038
• WASKO, J., FRACZYK, J., BECHT, A., KAMINSKI, Z.J., GRGAC, S.F., TARBUK, A., KAMINSKA, M., DUDEK, M., GLISCINSKA, E., DRACZYNSKI, Z. and KOLESINSKA, B., 2020. Conjugates of chitosan and calcium alginate with oligoproline and oligohydroxyproline derivatives for potential use in regenerative medicine. Materials, vol. 13, no. 14, pp. 3079. http://doi.org/10.3390/ma13143079 PMid:32664253.
  » http://doi.org/10.3390/ma13143079
• WERNER, S. and GROSE, R., 2003. Regulation of wound healing by growth factors and cytokines. Physiological Reviews, vol. 83, no. 3, pp. 835-870. http://doi.org/10.1152/physrev.2003.83.3.835 PMid:12843410.
  » http://doi.org/10.1152/physrev.2003.83.3.835
• WONG, K.C., 2015. Review of spectrometric identification of organic compounds, 8th edition. Journal of Chemical Education, vol. 92, no. 10, pp. 1602-1603. http://dx.doi.org/10.1021/acs.jchemed.5b00571
  » http://dx.doi.org/10.1021/acs.jchemed.5b00571
• WU, Y.S. and CHEN, S.N., 2014. Apoptotic cell: Linkage of inflammation and wound healing. Frontiers in pharmacology, vol. 5, pp. 1-6. https://doi.org/10.3389/fphar.2014.00001 PMid:24478702.
  » https://doi.org/10.3389/fphar.2014.00001
• XIONG, A., RUAN, L., YE, K., HUANG, Z. and YU, C., 2023. Extraction of chitin from black soldier fly (Hermetia illucens) and its puparium by using biological treatment. Life, vol. 13, no. 7, pp. 1424. http://doi.org/10.3390/life13071424 PMid:37511799.
  » http://doi.org/10.3390/life13071424
• YANG, N., SHI, N., YAO, Z., LIU, H. and GUO, W., 2023. Gallium-modified gelatin nanoparticles loaded with quercetin promote skin wound healing via the regulation of bacterial proliferation and macrophage polarization. Frontiers in Bioengineering and Biotechnology, vol. 11, pp. 1124944. https://doi.org/10.3389/fbioe.2023.1124944 PMid:36777248.
  » https://doi.org/10.3389/fbioe.2023.1124944
• YOU, C., LI, Q., WANG, X., WU, P., HO, J.K., JIN, R., ZHANG, L., SHAO, H. and HAN, C., 2017a. Silver nanoparticle loaded collagen/chitosan scaffolds promote wound healing via regulating fibroblast migration and macrophage activation. Scientific Reports, vol. 7, no. 1, pp. 10489. http://doi.org/10.1038/s41598-017-10481-0 PMid:28874692.
  » http://doi.org/10.1038/s41598-017-10481-0
• YOU, C., LI, Q., WANG, X., WU, P., HO, J.K., JIN, R., ZHANG, L., SHAO, H. and HAN, C., 2017b. Silver nanoparticle loaded collagen/chitosan scaffolds promote wound healing via regulating fibroblast migration and macrophage activation. Scientific Reports, vol. 7, no. 1, pp. 10489. http://doi.org/10.1038/s41598-017-10481-0 PMid:28874692.
  » http://doi.org/10.1038/s41598-017-10481-0
• YOUNES, I. and RINAUDO, M., 2015. Chitin and chitosan preparation from marine sources. Structure, properties and applications. Marine Drugs, vol. 13, no. 3, pp. 1133-1174. http://doi.org/10.3390/md13031133 PMid:25738328.
  » http://doi.org/10.3390/md13031133
• YOUNUS, N., ZUBERI, A., MAHMOOOD, T., AKRAM, W. and AHMAD, M., 2020. Comparative effects of dietary micro-and nano-scale chitosan on the growth performance, non-specific immunity, and resistance of silver carp hypophthalmichthys molitrix against Staphylococcus aureus infection. Aquaculture International, vol. 28, pp. 2363-2378. https://doi.org/10.1007/s10499-020-00595-0
  » https://doi.org/10.1007/s10499-020-00595-0
• ZHAO, J., QIU, P., WANG, Y., WANG, Y., ZHOU, J., ZHANG, B., ZHANG, L. and GOU, D., 2023a. Chitosan-based hydrogel wound dressing: from mechanism to applications, a review. International Journal of Biological Macromolecules, vol. 244, pp. 125250. http://doi.org/10.1016/j.ijbiomac.2023.125250
  » http://doi.org/10.1016/j.ijbiomac.2023.125250
• ZHAO, K., PU, S., SUN, L. and ZHOU, D., 2023b. Gentiopicroside-loaded chitosan nanoparticles inhibit tnf-α-induced proliferation and inflammatory response in hacat keratinocytes and ameliorate imiquimod-induced dermatitis lesions in mice. International Journal of Nanomedicine, vol. 2013, no. 18, pp. 3781-3800. https://doi.org/10.2147/ijn.s406649 PMid:37457802.
  » https://doi.org/10.2147/ijn.s406649
• ZHOU, Y., SUN, P., CAO, Y., YANG, J., WU, Q. and PENG, J., 2023. Biocompatible copper formate-based nanoparticles with strong antibacterial properties for wound healing. Journal of Nanobiotechnology, vol. 21, no. 1, pp. 474. http://doi.org/10.1186/s12951-023-02247-w PMid:38072979.
  » http://doi.org/10.1186/s12951-023-02247-w
• ZHOU, J., LI, K. and RAMASAMY, P., 2024. Chitosan-collagen biopolymer biofilm derived from cephalopod gladius; evaluation of osteogenesis, angiogenesis and wound healing for tissue engineering application. International Journal of Biological Macromolecules, vol. 279, pp. 135078.
• ZHU, P., MA, M., YOU, T., ZHANG, B., YE, S. and LIU, S., 2024. Optimizing prolyl hydroxylation for functional recombinant collagen in escherichia coli. International Journal of Biological Macromolecules, vol. 282, pp. 137400. https://doi.org/10.1016/j.ijbiomac.2024.137400 PMid:39521206.
  » https://doi.org/10.1016/j.ijbiomac.2024.137400
• ZULKEFLI, N., CHE ZAHARI, C.N.M., SAYUTI, N.H., KAMARUDIN, A.A., SAAD, N., HAMEZAH, H.S., BUNAWAN, H., BAHARUM, S.N., MEDIANI, A., AHMED, Q.U., ISMAIL, A.F.H. and SARIAN, M.N., 2023. Flavonoids as potential wound-healing molecules: emphasis on pathways perspective. International Journal of Molecular Sciences, vol. 24, no. 5, pp. 4607. http://doi.org/10.3390/ijms24054607 PMid:36902038.
  » http://doi.org/10.3390/ijms24054607

Edited by

• Editor:
  Ana Paula Peron

Publication Dates

• Publication in this collection
  27 Oct 2025
• Date of issue
  2025

History

• Received
  24 Dec 2024
• Accepted
  05 Aug 2025