[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
Jean Aimé Mono a,b,**, Sandrine Emvoutou Ndongo a,b, Odette Thérèse Adegono Assiene b,c,d, Armel Mewoli c,e, Rachelle Appolince Nguefack Assona a,b, Richard Hervé Bitete c, Giresse Ulrich Defo Tatchum c,d, Claude Takoumbe c,d,*

Keywords:
Natural fiber
Carica papaya
Agricultural waste
Chemical extraction
Water retting

Carica papaya pseudostems are widely available as biomass waste in Cameroon. These agricultural wastes can be effectively used as natural fibers in the manufacture of biocomposites. In this study, Carica papaya fibers were extracted from papaya pseudostems by retting with water and an alkaline sodium hydroxide (NaOH) solution at different concentrations (2.5 %, 5 %, and 7.5 %). An experimental campaign is being conducted on the physical, chemical, thermal, mechanical and morphological characteristics of Carica papaya fibers. Fourier transform infrared spectroscopy (FTIR) of Carica papaya fibers extracted by water retting and those extracted with NaOH indicates that the cellulose, hemicellulose, and lignin functional groups are present in the fibers and are dissolved considerably as the percentage of NaOH increases. Scanning electron microscopy (SEM) in the longitudinal plane gives a visual representation of the rough and irregular surfaces without the presence of impurities on the chemically extracted fibers compared to that extracted with water. In contrast to the decrease in diameter, the measured density of Carica papaya pseudostem fibers increased with NaOH concentration (0.633 – 1.522 g cm–3), all of which remained light fibers. Water absorption decreased from 159.36 % to 141.28 % with increasing NaOH, and relative humidity dropped to 6.41 %. The thermal stability of Carica papaya fibers extracted at concentrations of 2.5 % NaOH (215 °C), 5 % NaOH (200 °C), and 7.5 % NaOH (175 °C) showed a clear decrease compared to those extracted by water retting (220 °C). Fibers extracted at 2.5 % NaOH achieved a tensile strength of 287.55 ± 56 MPa, a tensile modulus of 8.271 ± 1.62, and 3.505 ± 1.01 % elongation. The tensile properties of these fibers showed great variability, and an influence of diameter was observed, indicating the need to study the influence of technique and NaOH mass concentration. The results show that Carica papaya fiber extracted at a concentration of 2.5 % will be the most suitable for biocomposite applications.

journal homepage: www.cell.com/heliyon

Research article

2405-8440/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Today, natural plant fibers are increasingly studied throughout the world to reduce domestic consumption and the carbon impact produced by synthetic materials [1,2]. They are widely used in many fields, including textiles, automobiles, construction, insulation, and furniture [1,3-5]. Cameroon’s biomass is rich in a variety of natural resources, some of which are unexploited and others little exploited [1,6]. It is worth continuing to study these available natural resources, most of which are wild or invasive plants that are particularly rich in fibers with interesting properties [6-8].

Papaya (Carica papaya) mainly belongs to the Caricaceae family, which comprises around 31 species [9]. It is a fast-growing herbaceous plant that can reach 10 m in height. The Carica papaya plant is mainly cultivated for its fruits, for their high nutritional and medicinal value [9]. Cameroon ranks 64th in the world in papaya production, with an annual output of 44 tons, which generates a large mass of Carica papaya (CP) trunk at the end of production. Once this has reached the end of its life cycle, it is cut down and disposed of in the wild, increasing the volume of agricultural waste in the environment.

With the current evolution of our society towards a more sustainable and eco-responsible economy, which favors the development of locally available renewable resources, Pawpaw pseudo-trunks, which are agricultural by-products rich in natural plant fibers, can be valorized and used on an industrial scale, both nationally and internationally. At the same time, these fibers could also diversify the world’s production of natural fibers, which is dominated by three fibers (flax, hemp, and coir) that alone account for over 80 % of production, excluding cotton, used in textiles and many other applications [10].

Recent studies [11–13] have demonstrated the effectiveness of Carica papaya fibers in composite reinforcement. However, it has been previously shown that fiber from the pseudo-trunk of the Indian Carica papaya has a cellulose content of 38 %, 11.8 % hemicellulose, a crystallinity index of 56.34 %, and a density of 943 kg m⁻³ [9]. With a thermal stabilization temperature of between 120 °C and 220 °C and a fiber constituent degradation temperature of 322 °C. Its microstructure does not have a good surface finish. According to the authors, it will be important to improve the surface finish by fiber treatments for use in composites [9]. Similar observations have been reported by Refs. [14,15]. The results obtained on Brazilian papaya trunk fibers give a density of 1.12 g cm⁻³, a cellulose content of 62 %, a lignin content of 15 %, a thermal stability temperature of 254 °C, and a degradation temperature of 346 °C, with a less rigorous surface finish than that obtained on Indian papaya fibers [16]. In view of the above, it is clear that the properties of natural fibers, such as morphology, biochemical composition, and internal structure of the natural fiber, such as microfibril angle and crystallinity index (CrI), vary with the maturity of climatic conditions, geographical location, and extraction conditions, as well as the fiber preparation stages, in particular the extraction and drying processes [17–19]. This justifies further studies on fibers derived from Carica papaya pseudo-trunks, and in particular extraction techniques, to further enrich the data available in the literature for use in composites or as textile fibers.

Lignocellulose fibers are a natural composite in which cellulose fibrils are embedded in a lignin matrix. In addition to cellulose, lignin, hemicellulose, pectin, and wax are other important components of lignocellulose fibers [20]. Traditionally, lignocellulosic and bast fibers were extracted by the dew retting process. Dew retting is also an alternative and sustainable method of extracting lignocellulosic fibers. However, due to the longer lead times and water pollution problems associated with water retting [1,21] researchers have developed other fiber extraction methods, including decortication, post-decortication cleaning, enzymatic retting, and chemical retting [11]. Chemical extraction using alkaline NaOH treatment is a widely used technique [9,22]. It can be effective when the elements (cellulose, hemicellulose, lignin, and pectin) contained in the fiber dissolve without risk of fiber damage [20,23]. Hemicellulose is highly hydrophilic and dissolves easily under alkaline conditions. Lignin, being entirely amorphous and hydrophobic, is soluble under warm alkaline conditions [18,22]. Several factors influence the extraction process (extraction time and NaOH concentration percentage) of the fibers. Some authors [19,22,24,25] point out that beyond 5 % NaOH during the extraction process, the mechanical characteristics in traction (tenacity) decrease by degrading the crystalline structure of the fiber [1,19].

In this study, we investigate the extraction of fibers from its pseudo-trunk of Cameroonian origin. Physical tests (density, yield, and absorption), chemical and microstructural tests (FTIR, ATG, and SEM), and mechanical tensile tests will be carried out. Two extraction techniques will be used in this study (water and chemical techniques).

Carica papaya pseudostem stems were collected at the ENSET site of the University of Douala (Wouri Department, Littoral Region, Cameroon). Sections 50 ± 5 mm long were cut using a hand saw and then washed with water. Sodium hydroxide (NaOH) concentrated to 99 % and acetic acid were supplied by Laboratoire PYCNOLAB (Douala, Cameroon). Fibers are extracted by water retting (Water. Ext), water supplied by CAMWATER, and chemically with NaOH at concentrations of 2.5 % NaOH, 5 % NaOH, and 7.5 % NaOH chosen according to the work of Soppie et al. [23] and Obame et al. [19].

Extraction took place in two phases, the first of which involved immersing (Fig. 1a) the papaya sections in a tank containing water at room temperature. After fourteen (14) days [9], decomposition of the sections due to microbial degradation [15] was observed (Fig. 1b). The resulting mattress was rinsed with ambient water (Fig. 1c). The second phase involved immersing the resulting mat

(Fig. 1c), after cutting it finely to facilitate extraction, in ambient water (Fig. 1d) for a further two weeks (Fig. 1d). After two weeks, the fibers could be extracted before being rinsed and sun-dried for 24 h to remove moisture [21].

## 2.3. Chemical extraction in sodium hydroxide (NaOH)

Sections of papaya pseudostem were cut into small pieces to facilitate extraction (Fig. 2a). As shown in Fig. 2b, the cut pseudostems were immersed in 2.5%, 5%, and 7.5% NaOH solutions by weight and boiled in a 1:20 (w/v) solid/liquid bath for 2 h (Fig. 2b). The fibers were successively washed several times with demineralized water before being washed in a solution containing 1% acetic acid to neutralize the effects of NaOH, followed by a thorough rinse with running water to remove any additional alkaline chemicals adhering to the surface until a pH of 7 was obtained [19,23]. The fibers extracted by the different techniques were dried at 28 °C in the sun for 72 h to reduce the water content. These were fibers extracted by water retting and fibers extracted chemically at concentrations of 2.5%, 5%, and 7.5% NaOH by weight, respectively, as shown in Fig. 3a for water extraction, Fig. 3b–c, and Fig. 3d for 2.5% NaOH, 5% NaOH, and 7.5% NaOH, respectively. The water-extracted fibers are whitish in color, unlike the chemically extracted fibers, which are yellowish. It is also observed that this coloration increases with the percentage of NaOH.

To determine the extraction yield of the fibers, the masses before extraction (Me) and the dried masses of the fibers after drying for 72 h (Ms) were recorded. These masses were measured using a digital balance. Fig. 3a shows fibers extracted with water; Fig. 3b with 2.5% NaOH; Fig. 3c with 5% NaOH; and Fig. 3d with 7.5% NaOH. The extraction yield (Rdt) was calculated using equation (1) [7].

$$
\text{Rdt} (\%) = \frac{M_s}{M_e} \times 100
$$

## 2.4. Physical characterization of Carica papaya fibers

### 2.4.1. Determination of fiber diameter

Apparent diameter was measured according to the method reported in the literature [21] at three points spaced 5 mm apart on a 40 mm-long fiber using a micrometer. Twenty-five (25) samples were tested for each extraction technique, making a total of one hundred (100) samples.

### 2.4.2. Determination of water absorption degree

Five constant mass packages per extraction process are tested, for a total of 20 samples. Samples are dried at 105 °C for 3 h to render them anhydrous. The anhydrous mass (Mi) of the samples was measured on an electronic balance before being immersed in distilled water (30 °C) for 24 h [8]. Once removed from the water, a dry cotton cloth is used to remove surface water before being reweighed and the final mass (Mf) recorded. The method used was gravimetric [26–28]. The absorption degree (W) is calculated by relation 2 [8].

$$
W (\%) = \left(\frac{M_f}{M_i} - 1\right) \times 100
$$

With W (%): water absorption degree; Mf: mass of water-saturated fibers (g); Mi: mass of fibers in anhydrous state (g).

### 2.4.3. Determination of relative humidity recovery degree

The relative humidity recovery degree was determined using a gravimetric technique [6]. Five fiber bundles of constant mass per extraction process were used, for a total of twenty bundles, and dried at 105 °C in an oven to make them anhydrous. Once anhydrous, the dry mass is recorded (Mi), then the bundles were conditioned in an adiabatic chamber saturated with sodium chloride at a relative humidity of 75% [29]. The fiber bundles remained in the chamber for 48 h. After this time, the samples were removed from the chamber and weighed to record the wet mass (Mf), and the relative humidity, a, was calculated using relation 2.

|                                  |                              |                     |                                  |
| -------------------------------- | ---------------------------- | ------------------- | -------------------------------- |
| (a) Submerged papaya trunk       | (b) Decomposing trunks       | (c) Fiber mat       | (d) Mat submerged in water       |

Fig. 1. Stagnant water extraction; (a): Submerged papaya trunk; (b): Decomposing trunks; (c): Fiber mat; (d): Mat submerged in water.

The test was carried out in the PYCNOLAB Laboratory, with samples made anhydrous in a ventilated oven in accordance with ISO 3344 [30]. The mass of the anhydrous sample was measured (m₀) on a digital scale. The pycnometer is then filled with demineralized and degassed water up to meniscus zero, and stabilized at reference temperature before weighing (m₁). The sample is introduced into the pycnometer, and the water level is adjusted using a propipette after the sample has been immersed for 24 h. The sample is stabilized at the reference temperature before being weighed (m₂). Equation (3) [2] is used to calculate the absolute density. Five samples were used per extraction process for a total of twenty samples.

\[
\rho_{abs} = \frac{m_0 * \rho_{eau}}{m_0 + m_1 - m_2}
\]
(3)

m₀: the mass of the sample in the anhydrous state; m₁: the mass of the Gay-Lussac pycnometer filled with water; m₂: the mass of the Gay-Lussac pycnometer containing the sample and water after 24 h immersion in the water bath.

The functional chemical groups were identified using a Shimadzu IRAffinity⁻¹ CE instrument equipped with an ATR module and controlled by Opus/Mentor software. Samples of a few milligrams of powder (0.3 μm) were scanned over a spectral region from 4000 to 400 cm⁻¹ with an accumulation of 24 scans and a resolution of 4 cm⁻¹. The FTIR spectra were recorded in absorbance mode.

Thermal degradation of Carica papaya pseudostem fibers was carried out in a TGA Q50-0836 thermal analyzer with an open platinum crucible. Approximately 20 mg of extracted fiber powder, ground to a size of 0.3 μm, was heated from room temperature to 650 °C at a heating rate of 10 °C·min⁻¹ under a nitrogen atmosphere (flow rate = 10 ml min⁻¹).

The longitudinal surface of the extracted fibers was observed using a Hitachi S-3500N scanning electron microscope (SEM). Prior to

observation, the fibers were sputtered with a thin layer of gold/palladium.

### 2.5.4. Mechanical characterization of Carica papaya fibers by tensile testing

Tensile tests were carried out in accordance with the protocol of standard NF T25-501-2 [1,12,21]. Fiber samples were produced with a length L₀ of twenty (20) mm and subjected to a load of 5 kN at a loading speed of 5 mm min⁻¹ at room temperature. Twenty-five (25) samples were tested on each extraction process for a total of one hundred samples for all processes. Stress at break (σr) was calculated from equation (4) and strain at break εr: by equation (5) [6]. Young's modulus is the linear part of the stress-strain curve.

\[
σ = \frac{F}{S}
\]
(4)

\[
ε = \frac{Δl}{l}
\]
(5)

With F: Force (N); S: Cross-sectional area (mm²); σ: Breaking stress (MPa); ε: Strain; Δl: Comparator displacement; L₀: Working length.

### 2.6. Statistical analysis

A multivariate study of variance (MANOVA) was carried out to determine the influence of the extraction method on the physical and mechanical responses of Carica papaya fibers. A confidence level of 95% was set to test the hypothesis that the extraction method did not influence the observed properties. IBM SPSS 27 software was used.

## 3. Results and discussions

### 3.1. Physical results of Carica papaya fibers

#### 3.1.1. Extraction yield

Fig. 3 shows that fiber yield (Fig. 4) and texture are influenced by the parameters used to obtain the fibers. Yield is lower for fibers extracted by retting with stagnant water (17.98%) and whitish in color (Fig. 3a); it changes with increasing NaOH (19.92%, 24.39%, and 28.63%), respectively, for fibers extracted with 2.5%, 5%, and 7.5% NaOH with a change in color (Fig. 3b, c, and 3d). The change in color is attributed to the effectiveness of NaOH in eliminating non-cellulosic elements and thus favoring fiber recovery [31]. Similar results were obtained for banana skirt, kenaf, and flax fibers [7,31,32]. The low amount of fiber obtained with water extraction may be due to the additional extraction process, the first phase of which initially produces a honeycomb. Unlike chemical extraction, which is carried out directly. This was reported by Hamidon et al. [33] on kenaf fibers.

#### 3.1.2. Fiber diameter

The curve in Fig. 5 shows the results for fiber diameter. A decrease of 302.02 ± 4 μm for fibers extracted by water retting and 196.33 ± 4 μm - 221.94 ± 6 μm - 219.31 ± 4 μm respectively for fibers extracted with 2.5%, 5%, and 7.5% NaOH. This indicates that the NaOH extraction process creates a narrowing of diameters due to the removal of non-cellulosic material. This was reported by Cesarino et al. [34] and corroborated by Betene et al. [30], Mono et al. [6]. This narrowing of fiber diameters is likely to make them denser, less hydrophilic due to their porous nature resulting from the dissolution of the hemicelluloses contained in the fiber, and is in agreement with the results of the degree of moisture absorption and recovery, less resistant, and less tenacious [6,19,30,34]. The

| Fig. 4. Extraction yield distribution curve. |            |           |         |           |
| -------------------------------------------- | ---------- | --------- | ------- | --------- |
|                                              | Water Ret  | 2.5% NaOH | 5% NaOH | 7.5% NaOH |
| Extraction yield (%)                         | 17.98      | 19.92     | 24.39   | 28.63     |

diameters are smaller than those of plantain pseudostem fibers (117–247 μm) [21] for water-extracted fibers and are of the same order for NaOH-extracted fibers. They are smaller than those of cylindrical loofah fibers from Cameroon (233–419 μm) [6]. Dispersions around diameters due to their natural character and the various defects obtained during their extraction process are generally observed in the literature on plant fibers [6,19,23]. These dispersions of fiber diameter values could lead to heterogeneous properties in the production of composites with polymers or in the textile yarns that can be manufactured.

The evolution of the distribution (Fig. 6) presents the results of the water absorption degree. Like other plant fibers, Carica papaya pseudostem fiber has a hydrophilic character, which can be explained by its porous structure [35]. This may explain why it absorbs 159.36 % water in dry mass gain for fibers extracted by water retting and [141.28–153.64 %] for chemically extracted fibers. An overall decrease in the water absorption degree was observed with increasing NaOH concentration. Fibers extracted by water retting are more hydrophilic than those extracted chemically, which can be justified by their higher hemicellulose content [36,37]. Similar observations have been made of pineapple, jute, hemp, sisal, and loofah fibers [6,30,36,38]. Capacity to absorb water most often leads to a reduction in the mechanical strength and rigidity of the fibers, and can lead to the appearance of cracks in the production of biocomposites [30,39,40]. The results obtained are of the same order of magnitude as those for palm nut mesocarp fiber (OPMF) [41], plantain pseudostem fiber [2,21], and Triumfetta pentendra fibers [42]. Increasing the percentage of NaOH considerably reduces the rate of water uptake. This shows that NaOH is effective in dissolving the amorphous hemicellulose contained in papaya fibers [19], and that the use of treated fibers could limit shrinkage, swelling, and cracking phenomena in the production of fiber biocomposites with possible applications in wetlands.

The moisture absorption degree (Fig. 7) follows the same profile as that of water absorption. It decreases with increasing NaOH percentage, in particular from 9.14 % (fibers extracted by water retting) and from 9.09 % to 6.41 % (fibers extracted with NaOH) in gain of their dry masses over a period of 48 h. This water content confirms the hydrophilic behavior of these fibers and is in agreement with the results obtained on water absorption, confirming the effectiveness of NaOH extraction in dissolving the amorphous hemicellulose contained in these fibers. The results obtained are similar to those for Triumfetta cordifolia (TC) fibers (8.1–12 %) [23], and lower than those for sisal (13.6 %), jute (12 %), and flax (12 %) fibers used in the production of textile yarns [23,40,43], and luffa cylindrica fibers from Cameroon (9–15 %) [6], sida rhombifolia fibers (11.73–13.28 %) [1], plantain pseudostem fibers (11.6–13 %) [21], and plantain leaf fibers (11.73–13.28 %) [2]. The emphasis is therefore on strengthening biocomposites for use in dry and humid environments.

The density (Fig. 8) is 0.633 ± 0.06 g cm⁻³ for fibers extracted by still water retting, 1.14 ± 0.03 g cm⁻³, 1.396 ± 0.06 g cm⁻³, and 1.522 ± 0.08 g cm⁻³ for fibers extracted with 2.5 %, 5 %, and 7.5 % NaOH, respectively. The values increase with the percentage of NaOH. The increase in density is due to the reduction in non-cellulosic elements, the narrowing of diameters, and the porous nature of the fibers [19,23]. Several studies have shown fiber densification with increasing NaOH percentage [23,23,42,44]. Although the density of the water-extracted fiber in this study is close to that obtained in the work of Kempe et al. [45], the results show that the location where the Carica papaya fiber is planted influences these characteristics. The low density of Carica papaya fiber from Cameroon makes it possible to produce composite materials of lower weight than those made from synthetic fibers (the density of synthetic fibers varies between 1.8 and 2.54 g cm⁻³). The results obtained are lower than those for cotton (1.5–1.6 g cm⁻³), sisal

Table 1 compares the physical characteristics of a number of plant fibers. The diameter of the extracted fibers is comparable to that of coir, jute and sisal fibers, which are marketed and exploited for biocomposite reinforcement. These extracted fibers are denser than coir, jute and papaya fibers when extracted with NaOH, and in the same order of magnitude when extracted by water retting. Carica papaya pseudostem fibers are less hydrophilic than jute, sisal, TC and Comus pineapple fibers [19,47], so applications have been

(1.33–1.5 g cm⁻³), and flax (1.52 g cm⁻³) fibers [6,23,46] for fibers extracted with water, 2.5 %, and 5 % NaOH, and of the same order of magnitude for fibers extracted with 7.5 % NaOH.

directed towards biocomposite reinforcement and yarn production. Carica papaya fibers can be oriented in this direction, making them an alternative material for composite reinforcement.

## 3.2. Chemical and microstructural results for Carica papaya fibers

### 3.2.1. Fourier transform infrared spectrometry (FTIR)

Fig. 9 shows the FTIR spectrum of papaya fibers extracted with water and NaOH. The different group identification parameters are shown in Table 2. A similarity is observed in the spectra of Fig. 8, with a broad band at 3342 cm⁻¹, 3339 cm⁻¹, 3324 cm⁻¹, and 3334 cm⁻¹ for fibers extracted with water, 2.5% NaOH, 5% NaOH, and 7.5% NaOH, respectively, attributed to the hydroxyl group -OH and the O-H hydrogen bonds of hemicellulose and cellulose [44]. A decrease in these peaks is observed as the percentage of NaOH increases. This leads to a decrease in the hydrophilicity of these fibers and corroborates the results obtained on water absorption [23].

The peak near 3342-3334 cm⁻¹ that was observed in the specimens is impacted by the presence of water in the fiber and is characteristic of the elongation of -OH from hemicellulose and O-H from cellulose [19,50]. The bands between 2920 and 2900 cm⁻¹ show stretching of C-H and CH₂ [20]. However, the characteristic peak at 1731 cm⁻¹, which corresponds to the symmetrical extension of the C=O of hemicelluloses and pectins [20], is highlighted by the decrease in signal. These results confirm that hemicellulose is also eliminated from papaya trunk fiber surfaces only after alkaline pre-treatment with 2.5% NaOH. A peak was also observed at 1625-1593 cm⁻¹ corresponding to the shearing of the -OH bond in free water [19,44]. The position of this peak remained unchanged for both water-extracted and NaOH-extracted fibers. However, the intensity of the peaks decreased after the fibers were treated. As an additional indication of the presence of lignins in our fibers, we observed a peak at 1507-1506 cm⁻¹ which corresponds to a symmetrical C=C stretching of the lignin [18,20]. We also observed a peak at 1317 cm⁻¹ corresponding to the stretching vibrations of CH and C-O in the ester (cellulose and hemicellulose) [20]. The band between 1029 and 1027 cm⁻¹ corresponds to the vibrations of the OH and C=O bonds in cellulose [19]. We also observed stretching of the CH and OH bonds in cellulose at the peak located at 898-897 cm⁻¹ [1,18]. Similar observations have been made on the extraction of fibers using alkaline treatments [19,23].

### 3.2.2. Thermogravimetric analysis (TGA)

The thermal behavior of treated and untreated papaya fibers analyzed by TGA and DTG is presented in Fig. 10. The thermograms are similar to those of other plant fibers observed in the literature [20,23]. A mass loss of 7.76%, 9.07%, 8.69%, and 13.27% respectively for fibers extracted by water retting, 2.5% NaOH, 5% NaOH, and 7.5% NaOH is observed around 30°C and 150°C. This is attributed to moisture loss and decomposition of the low molar masses of the fibers [22,30]. After this dehydration phase, thermal stability is observed around 150°C and 225°C, indicating that these fibers are suitable for use in the production of biocomposites with polymers. The onset of thermal degradation between 250°C and 352°C is attributed to depolymerization of hemicellulose and cleavage of glycosidic bonds in amorphous cellulose, cellulose, pectin, and lignin. In general, the thermal depolymerization of glycosidic bonds in cellulose occurs between 220°C and 310°C and that of α-cellulose between 310°C and 390°C, while that of lignin occurs slowly over the entire decomposition temperature range (210°C–450°C) [22,30,51]. From 352°C to 600°C corresponds to the thermal decomposition of the different elements to form volatile products [19], which are 13.8%, 18.24%, 24.59, and 16.22%, respectively, for fibers extracted with water, 2.5% NaOH, 5% NaOH, 7.5% NaOH.

The analysis in Table 3 compares the extracted fibers with other fibers in the literature. The extraction technique and the percentage of NaOH have an influence on thermal stability. The thermal stabilization temperature is higher for fibers extracted with 5% NaOH (200°C–332.66°C) than for fibers extracted with 2.5% NaOH (215°C–351.72°C), 7.5% NaOH (175°C–322.9°C), and close to that extracted with water (220°C–352.95°C). This result may be associated with the partial dissolution of non-cellulosic matter and other impurities contained in the fibers during the retting extraction. In general, the removal of non-cellulosic matter is accompanied by an improvement in the thermal degradation properties of natural fibers [23,52].

The residue content of the extracted fibers ranged from 13.8% to 24.59% by mass. Chemical extraction with 5% NaOH showed a higher residue content of 24.59%, indicating that it contains higher quantities of inorganic substances. Thermal stability is considered to be the degradation threshold for the production of composite materials with thermoset and thermoplastic polymers or during

| fibers         | Diameters (μm) | Density (g.cm⁻³) | Water absorption degree (%) | References    |
| -------------- | -------------- | ---------------- | --------------------------- | ------------- |
| Coconut fiber  | 110–530        | 0.67 à 1         | 100 ±19.5                   | [1]           |
| Jute           | 40–350         | 1.1–1.5          | 281                         | [48]          |
| Sisal          | 80–300         | 0.75–1.07        | 230 ±16                     | [49]          |
| Flax           | 39.70–620      | 1.5              | 136 ±25                     | [19,47]       |
| TC             | 72.49          | 1.47             | 342.3                       | [44]          |
| Ananas comosus | 56.1–142.6     | 1.14–1.45        | 186.9–267.7                 |               |
| Agave          | 171–403        | 1.25–1.5         | –                           | [1]           |
| Papaya carica  | 309.1–356.9    | 0.84–0.97        | –                           | [9,45]        |
| Coconut        | 100–450        | 1.18             | 94–180                      | [50]          |
| Water. Ext     | 303.02         | 0.63             | 159.36                      | Present study |
| 2.5%NaOH       | 196.33         | 1.140            | 153.64                      |               |
| 5%NaOH         | 221.94         | 1.397            | 144.13                      |               |
| 7.5%NaOH       | 219.31         | 1.522            | 141.28                      |               |

J.A. Mono et al.                                                                                Heliyon 11 (2025) e41093

Fig. 9. FTIR spectrum of papaya fibers.

| Wavenumber (cm⁻¹) | Water. Ext | 2.5% NaOH | 5% NaOH | 7.5% NaOH | Designations |
|-------------------|------------|-----------|---------|------------|--------------|
| 3342              | 3339       | 3324      | 3334    | Elongation of -OH from hemicelluloses and O-H from cellulose |
| 2920              | 2915       | 2913      | 2900    | C-H and CH₂ stretching of cellulose |
| 1731              | 1731       | 1731      | 1731    | Symmetrical C=O extension of hemicelluloses and pectins |
| 1625              | 1593       | 1593      | 1593    | Shearing of the -OH bond in free water |
| 1507              | 1507       | 1506      | 1506    | Symmetrical C=C stretching of lignin |
| 1317              | 1262       | 1264      | 1317    | CH and C-O stretching vibration in the ester (cellulose and hemicellulose) |
| 1029              | 1026       | 1025      | 1027    | Elongation vibration of OH and C=O bonds in cellulose |
| 898               | 898        | 897       | 897     | Symmetrical stretching of the CH and O-H bonds in cellulose |

Fig. 10. TGA/DTGA curves for extracted fibers.

surface treatments in hot environments [2,55]. Based on Table 3 and taking into account the thermal stability criterion, two categories of fibers can be distinguished. The first category is associated with thermal stability values between 175 °C and 200 °C, which includes extraction at 5 % NaOH and 7.5 % NaOH. The second category is associated with higher thermal stability values and lies between 215 °C and 220 °C, in which we find extraction with water and 2.5 % NaOH. The thermal stability of the first category is lower than that of Carica papaya (220 °C) [9] on the same plant, which is due to the variation in the place of harvest, the conditions for obtaining the fibers and testing, and the maturity of the plant, and is also lower than that of other natural fibers [2,30]. The second category has

Table 3
Comparison of the thermal behavior of extracted fibers with other plant fibers.

| Fibers                | Dehydration (%) | Degradation temperature start (°C) | Degradation temperature end (°C) | Degradation rate (%) | Residues (%) | References    |
|-----------------------|-----------------|------------------------------------|----------------------------------|----------------------|--------------|---------------|
| Water. Ext            | 7.795           | 220                                | 352.95                           | 78.41                | 13.8         | Present study |
| 2.5%NaOH              | 9.073           | 215                                | 351.72                           | 72.74                | 18.24        |               |
| 5%NaOH                | 8.695           | 200                                | 332.66                           | 66.72                | 24.59        |               |
| 7.5%NaOH              | 13.27           | 175                                | 322.9                            | 70.47                | 16.22        |               |
| Carica Papaya         | -               | 220                                | 322.1                            | 46.13                | 36.18        | [9]           |
| Kenaf                 | -               | 219                                | 284                              | 82                   | 10.5         | [22]          |
| Banana                | -               | 263                                | 374                              | 51.5                 | 24.3         | [53]          |
| Sisal                 | -               | 220                                | 415                              | 72                   | 5.0          | [19]          |
| Triumfetta Cordifolia | -               | 235                                | 420                              | 72                   | 12.69        | [44]          |
| Okra                  | 8.4             | 220                                | 359                              | 60.6                 | 7.6          | [54]          |
| Jute                  | -               | 230                                | 442                              | 78.9                 | 11.8         | [53]          |

Values of the same order of magnitude as Carica papaya [9], kenaf (219 °C) [22], sisal and okra (220 °C) [19,30,54]. But lower than that of triumfetta cordifolia (235 °C) [44], Banana (263 °C) [19], jute (230 °C) [53], aloe vera (238 °C), sida rhombifolia (250 °C), Acacia (280 °C) [56], and pineapple fibers (240–272 °C) [30,55]. The thermal stabilities thus identified provide details on the maximum service temperatures of some of the fibers studied in the literature and in this study for the application of composites with polymers.

### 3.2.3. SEM analysis of the microstructure

Fig. 11 shows the appearance of the longitudinal surfaces of plant fibers extracted by different methods. Fig. 11a shows that water-extracted papaya fiber has deposits of impurities (non-cellulosic material) that were not removed during the water retting process [44] on the surface of the fibers. By observing the enlargement of a fiber with fewer impurities (Fig. 11b), it is possible to see that the presence of micropores could promote premature fiber breakage [23]. In addition, the presence of micropores may explain the high absorption rate highlighted above. With chemical extraction, the surfaces are cleaner but reveal rough and irregular textures that may be favorable to the fiber/matrix interface depending on the type of polymer matrix. The fiber extracted with a concentration of 2.5% NaOH is shown in Fig. 11c and its enlargement (Fig. 11d and e) highlights hollow and grooved textures on observation. The severity of the NaOH concentration above 2.5% reveals the beginnings of micro-cracks that will be detrimental to the mechanical properties (Fig. 11g), the surface finish is improved but the cracks are accentuated. Gradually, rough textures are observed (Fig. 11f–k). Extracting Carica papaya fibers with NaOH considerably reduces impurities and fiber porosity, as observed by SEM. Similar observations have been made for NaOH treatments [1,19,20].

### 3.3. Mechanical properties of Carica papaya fibers

The stress-strain curves for each extraction technique are shown in Fig. 12a. The results obtained show that the mechanical characteristics are greatly influenced by the extraction methods used for Carica papaya fibers, as has been observed with other plant fibers [6,19,23]. Firstly, identical behavior was observed for each fiber, associated with linear elastic (brittle) behavior followed by sudden rupture, probably due to the pores observed in Fig. 12b. This behavior has been observed in other fibers studied in the literature [1,6,23,24]. The Young's modulus (Fig. 12b) of water-extracted fibers is 2.142 ± 0.83 GPa with a variation rate of 39.07 % and lower than that of fibers extracted with 2.5 % NaOH is 8.271 ± 1.62 GPa with a variation rate of 19.69 %. That of fibers extracted at 5 % NaOH is 3.543 ± 0.89 GPa with a variation rate of 25.32 %. The strength of fibers extracted with 7.5 % NaOH is 2.595 ± 0.74 GPa with a variation rate of 28.86 %. The mechanical strength at break (Fig. 12c) of the fibers extracted with water (42.14 ± 10.71 MPa) and the fibers chemically extracted with a concentration of NaOH at 2.5 % NaOH (287.55 ± 56.67 MPa), 5 % NaOH (114.94 ± 31.7 MPa) and at 7.5 % NaOH (58.33 ± 16.52 MPa) and the coefficients of variation of 25.41 %, 19.7 %, 27.58 %, and 28.32 %, respectively, are given. Young's modulus and tensile strength decrease with increasing NaOH percentage. This has been observed with NA fibers [19]. The Young's modulus and mechanical strength curves for chemical extraction show linear behavior. The elongation at break (Fig. 12d) of fibers extracted by water retting (1.16 ± 0.69 %), fibers chemically extracted with a NaOH concentration of 2.5 % NaOH (3.505 ± 1.01 %), 5 % NaOH (1.905 ± 0.73 %) and 7.5 % NaOH (2.352 ± 0.69 %) for coefficients of variation of 16.02 %, 29.07 %, 38.52 %, and 29.34 %, respectively, are also given. Non-linear dispersions are observed for chemical extraction. Contrary to the fiber extracted with a concentration of 2.5 %, which has better mechanical properties overall (Fig. 12b–d), the water-retted fibers have lower values. Prolonged retting is detrimental to the fiber because, in addition to destroying non-cellulosic compounds (waxes, pectins, and fats), it also destroys the cellulosic and woody compounds that are responsible for the fiber's greater strength [6,19]. The mechanical properties of the Carica papaya fibers highlighted are close to those of other fibers (Table 4) and make them serious candidates for reinforcement of composite materials (thermoplastic and thermosetting matrices) and make it possible to envisage textile structures such as non-wovens or yarns compared with other natural plant fibers (Table 3). In the end, this study confirmed that fiber properties vary according to the extraction method used and the origin of the plant, as shown in the literature [9].

Fig. 11. Microstructure of papaya fiber; (a): Water-extracted fiber; (b): Enlargement at 300 of water-extracted fiber; (c): Extraction at 2.5% NaOH; (d): 2.5% NaOH enlargement at 300; (e): 2.5% NaOH magnification at 600; (f): Extraction at 5% NaOH; (g): Magnification at 300; (h): Magnification at 600; (i): Extraction at 7.5% NaOH; (j): 7.5% NaOH magnification at 300; (k): 7.5% NaOH magnification at 600.

3.4. Statistical analysis of variance by MANOVA

Table 5 shows the multivariate analysis of variance (MANOVA) for the concentration and extraction methods. Pillai's trace significance level is less than 0.05, rejecting the hypothesis that the extraction method has no effect on the observed properties; there are therefore significant differences due to the extraction method; similar observations have been reported in the literature [7,21,58]. A post hoc test presented in Table 6 was used to analyze the significant differences observed. The Tukey test used for this purpose shows an influence of the NaOH level compared with water extraction on all the physical and mechanical properties. Only the absorption rate showed a non-significant difference between the water method and the chemical extraction with 5% NaOH. This table also shows that the physical properties (absorption rate) are higher for water extraction than for chemical methods. On the other hand, chemical

extraction gives higher mechanical properties than water extraction.

Jean Aimé Mono: Supervision, Methodology, Conceptualization. Sandrine Emvoutou Ndongo: Writing – review & editing, Writing – original draft, Validation, Conceptualization. Odette Thérèse Adegono Assiene: Writing – review & editing, Writing – original draft, Validation, Resources. Armel Mewoli: Writing – review & editing, Writing – original draft, Validation. Rachelle Appolince Nguefack Assona: Writing – original draft, Validation, Data curation. Richard Hervé Bitete: Writing – review & editing, Visualization, Validation, Methodology, Conceptualization. Giresse Ulrich Defo Tatchum: Writing – original draft, Methodology, Formal analysis. Claude Takoumbe: Methodology, Formal analysis, Data curation, Conceptualization.

densities and reducing their diameters and hydrophilicity. The average diameter, strength, Young's modulus, and strain at break are 196.33 μm, 287.55 ± 56 MPa, 8.27 ± 1.62 GPa, and 3.50 ± 1.01 respectively. The relative humidity is stable (9%) up to a NaOH concentration of 5%. The chemical extraction method was found to reduce the hydrophilic character of Carica papaya pseudostem fibers to a water absorption rate of 141.28%. The tensile properties of Carica papaya pseudostem fibers showed a large variability and an influence of the diameter was observed, indicating the need to study the influence of the technique and the mass concentration of NaOH. Thermal analysis concluded that the water-treated fiber and the fiber treated with 2.5% NaOH exhibited better thermal stability (215–220°C) and good thermal tolerance (352°C) for use with polymers. The low density of Carica papaya fibers (0.63–1.52 g/cm³) makes them suitable for lightweight composite materials. In the light of the results presented here, the available pseudo-stem fiber from Carica papaya of Cameroonian origin has the potential to be used as a reinforcing material in the creation of composites, but also in textiles. The authors are currently working on the development of non-woven Carica papaya reinforcements obtained by carding for use in the reinforcement of polymer matrices, with a focus on the circular economy.

Multivariate analysis of variance

The data used to support the findings of this study have not been the subject of any prior study.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors would like to thank the CECAM Laboratory for carrying out the tests. The authors would like to thank Ngo Djon Pauline Sidonie and Tcheutchoua Florence Judicaelle for extracting the fibers and carrying out the tests.

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[System]
You are a **scientific‐rigor auditor**. You will receive the parsed contents of a research paper. Your job is to identify **only** those errors or flaws that directly undermine the **scientific validity** of the paper’s methods, analyses, or conclusions.

Your sole focus is to identify flaws—such as errors in experimental design, data integrity, calculations, statistical inference, or reproducibility—that directly call into question the validity of a specific claim, paragraph, or the paper as a whole.

**Do not** report issues that are purely presentational, rhetorical, stylistic, or related to citation practices.

---

After you’ve done a **detailed walkthrough** of the paper, output exactly in this format—no extra keys or commentary:

```
<analysis>
{detailed walk‑through of how you checked each section/figure and why you flagged (or did not flag) any flaw}
</analysis>

<response>
{
  "has_error": <true|false>,
  "errors": [
    {
      "location": "Section 2.1",
      "description": "Claim that ‘all X are Y’ is contradicted by citation [5], which shows examples of non‑Y X."
    },
    {
      "location": "Figure 3",
      "description": "XAxis labeled ‘Time (s)’ but units appear to be milliseconds; scale bar mismatches caption."
    }
    // …more entries…
  ]
}
</response>
```

- **Do not** include any other keys or prose outside these two tagged blocks.
- **Do not** report stylistic or citation issues.
- Be as precise as possible about **where** (section ID or figure/table) and **what** the scientific flaw is.
- Each description for the errors must be rooted in a scientific rationale explaining why it is 'wrong' (not how it could be improved).

Begin your analysis now.

[User]
International Journal of Biological Macromolecules 257 (2024) 128561

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Chitosan nonwoven fabric composited calcium alginate and adenosine diphosphate as a hemostatic bandage for acute bleeding wounds

Jianmin Yang a,b,*, Fengying Cai a, Yicheng Lv a, Ting Jiang a, Xingkai Zhao a, Xueli Hu a, Yunquan Zheng b, Xianai Shi a,b,*

a College of Biological Science and Engineering, Fuzhou University, No. 2 Xueyuan Road, Fuzhou 350108, China
b Fujian Key Laboratory of Medical Instrument and Pharmaceutical Technology, Fuzhou University, No. 2 Xueyuan Road, Fuzhou 350108, China

ARTICLE INFO

Keywords:
Chitosan nonwoven fabric
Hemostatic bandage
Synergistic hemostatic
Acute bleeding wound
Hemostatic mechanisms

ABSTRACT

Acute bleeding following accidental injury is a leading cause of mortality. However, conventional hemostatic bandages impede wound healing by inducing excessive blood loss, dehydration, and adherence to granulation tissue. Strategies such as incorporating active hemostatic agents and implementing chemical modifications can augment the properties of these bandages. Nevertheless, the presence of remote thrombosis and initiators may pose risks to human health. Here, a hemostatic bandage was developed by physically combined chitosan nonwoven fabric, calcium alginate sponge, and adenosine diphosphate. The presented hemostatic bandage not only exhibits active and passive mechanisms for promoting clotting but also demonstrates excellent mechanical properties, breathability, ease of removal without causing damage to the wound bed or surrounding tissues, as well as maintaining an optimal moist environment conducive to wound healing. In vitro evaluation results indicated that the hemostatic bandage possesses favorable cytocompatibility with low levels of hemolysis. Furthermore, it effectively aggregates various blood cells while activating platelets synergistically to promote both extrinsic and intrinsic coagulation pathways. In an in vivo rat model study involving liver laceration and femoral artery injury scenarios, our developed hemostatic bandage demonstrated rapid clot formation capabilities along with reduced blood loss compared to commercially available fabrics.

1. Introduction

Acute massive blood loss will lead to hypothermia, internal organ failure, metabolic acidosis, and other symptoms; which is a cause of death in wars, surgery, traffic accidents, and natural disasters [1,2]. Thus, using an effective hemostatic material in the early stages of bleeding is crucial for saving patients' lives and extending resuscitation time [3,4]. Although various types of hemostatic materials including sponges, powders, and hydrogels have been developed, hemostatic bandages remain the most widely employed strategy for controlling traumatic bleeding [5–7]. Hemostatic bandages have been used as effective exterior hemostatic dressings due to their breathability, safety, nonallergenicity, absorption, and ease of application [8]. However, traditional hemostatic bandages primarily depend on the hydrophilicity, porous structure, and capillary action among fibers to concentrate blood cells, thereby requiring relatively long hemostasis time and easily causing excessive blood loss [9,10]. Furthermore, these bandages fail to maintain wound moisture levels effectively while often adhering to the wound surface which can lead to dehydration and delayed wound healing [11–13]. Therefore, the development of anti-adhesive hemostatic bandages that also maintain wound moisture levels remains a significant challenge.

Recently, several studies have been conducted to improve the hemostatic properties of bandages through reduction in blood loss and hemostasis time. Consequently, various advanced hemostatic bandages have been developed [14–16]. Notably, chitosan-based hemostatic dressings such as HemCon® bandages, Celox® Rapid, and ChitoGauze®, have gained widespread utilization in military, emergency care, hospital, and other related fields [17,18]. Due to the positively charged amino groups on its skeleton, chitosan can interact electrostatically with the negatively charged erythrocyte cell membranes, thus promoting the aggregation of erythrocytes. In addition, chitosan has good biocompatibility, biodegradability, antibacterial, anti-inflammatory, and anti-tumor activities, thus it has an important application value in the field of

* Corresponding authors at: College of Biological Science and Engineering, Fuzhou University, No. 2 Xueyuan Road, Fuzhou 350108, China.
E-mail addresses: jmyang@fzu.edu.cn (J. Yang), shixa@fzu.edu.cn (X. Shi).

https://doi.org/10.1016/j.ijbiomac.2023.128561
Received 10 August 2023; Received in revised form 23 October 2023; Accepted 30 November 2023
Available online 5 December 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.

tissue engineering and regenerative medicine [19–21]. However, the erythrocyte aggregates formed by chitosan fabric during hemostasis are inherently unstable and may cause secondary bleeding upon bandage dislodgement.

To address these issues, various strategies involving chemical modifications and the incorporation of active coagulants into fabrics have been explored to enhance the hemostatic efficacy of bandages [22–24]. However, these approaches still fall short in meeting the requirements of cost-effectiveness, safety, and structural stability. For instance, the application of coagulants such as thrombin, fibrinogen, kaolin, and zeolite are hindered due to their high cost, immunogenicity, potential risk of viral transmission, and safety issues associated with distal thrombosis [25–27]. Alternatively, adenosine diphosphate (ADP) has gained significant attention as a physiologically relevant platelet agonist. ADP is the first discovered important agonist for platelet aggregation that is mainly stored in dense granules of platelets [28,29]. It can stimulate the secretion of dense particles, induce platelet aggregation and shape change, and contribute to the formation and stability of thrombosis during hemostasis process [30]. Although ADP has significant procoagulant potential, there have been limited reports on its application in hemostasis [31,32].

Fabrics are extensively utilized in the fabrication of hemostatic bandages owing to their good mechanical properties, high porosity, and extracellular matrix-like structure. Regrettably, their inadequate absorption and water retention characteristics can potentially result in wound dehydration and adhesion of the material to the injured surface during application [33–35]. Hence, the incorporation of sponges, membranes, or creams into fabric matrices can enhance hemostatic properties and cater to diverse requirements during wound healing [36–38]. Compared to other materials, sponge offers numerous advantages such as porosity, hydrophilicity, and moisture retention [39–41]. For instance, calcium alginate sponges undergo transformation from insoluble calcium alginate to sodium alginate hydrogel during hemostasis, facilitating easy removal from the wound site and reducing pain. Additionally, negatively charged alginates can expedite coagulation by activating coagulation factor XII. Furthermore, the addition of the cross-linking agent CaCl2 enhances coagulation efficiency through extrinsic and intrinsic hemostasis [42]. However, the limited mechanical strength of alginate sponges restricts their application scope [43]. Therefore, we propose a novel approach that combines fabrics with calcium alginate sponges to overcome these limitations and develop an advanced hemostatic material. The synergistic advantages of fabric and sponge in terms of their structure and composition are harnessed to achieve optimal hemostasis performance and create an ideal moist environment for wound healing.

In this study, we developed a hemostatic bandage (CS/SA/ADP) with synergistic advantages for the management of acute bleeding wounds (Scheme 1). In preparing the hemostatic bandages, chitosan nonwoven

The chitosan nonwoven fabric was initially treated with a 1% acetic acid solution (pH = 5.5) and subsequently immersed in a 0.75% sodium alginate solution. After a duration of 20 min, the samples were removed from the sodium alginate solution and rinsed with distilled water. Freeze drying was then conducted, followed by soaking the sample in a 0.2 M CaCl₂. After another round of freeze drying, the CS/SA composite hemostatic dressing was obtained. Additionally, the sodium alginate sponge underwent soaking in CaCl₂ followed by subsequent freeze drying to yield an SA hemostatic sponge. To prepare SA/ADP and CS/SA/ADP hemostatic bandages, incorporation of 200 mM ADP into the sodium alginate solution was carried out using the aforementioned method.

Freshly collected SD rat blood containing anticoagulant was centrifuged at 1500 rpm for 10 min. The supernatant was discarded, and the red blood cells were washed with saline and subsequently diluted into a 5% erythrocyte suspension. Subsequently, pre-warmed sample (10 mg) was added to 5% erythrocyte suspension (1 mL). After incubation at 37°C for 1 h, the samples were centrifuged at 1000 rpm for 10 min to precipitate the erythrocytes. The supernatant, containing the released hemoglobin (HA) from lysed erythrocytes, was collected. The absorbance of the supernatant at a wavelength of 540 nm was measured, with saline solution as a blank control and 0.1% Triton X-100 as a positive control. The hemolysis rate was calculated using the following equation: Hemolysis rate (%) = (OD_sample – OD_control) / (OD_positive – OD_control) × 100%.

A polyethylene hose with an inner diameter of 7.8 mm was incised to measure 5 mm × 8 mm, and a hemostatic bandage was applied to cover the wound. Wound covered with a chitosan nonwoven fabric served as a control. The vascular model was weighed prior to the experiment. Coagulation was initiated by mixing citrated blood with CaCl₂ (0.2 M), and 2 mL of blood was added to the polyethylene hose placed in a Petri dish. After 10 min, the polyethylene hose was removed from the Petri dish and the increase in weight of the dish due to blood leakage as well as the increase in weight of the material was measured as blood loss.

fabric was initially immersed in a mixed solution of sodium alginate and ADP, followed by cross-linking using Ca²⁺ to obtain the desired product. Given its unique fabric mesh structure and spongy properties, the hemostatic bandage not only efficiently absorbs wound exudate and provides a moist microenvironment, but also exhibits good mechanical strength and breathability. Moreover, the chitosan nonwoven fabric in the hemostatic bandage facilitates primary hemostasis, blood absorption, and concentration of clotting components. Additionally, sodium alginate, ADP, and Ca²⁺ play crucial roles in promoting platelet adsorption and accelerate coagulation. Here, the air permeability, absorbency, and mechanical properties of hemostatic bandages were characterized. In vitro coagulation properties of the hemostatic bandage were investigated by examining the erythrocyte/platelet aggregation activation, coagulation pathways, and whole blood coagulation capacity. The peeling behavior of the hemostatic bandage was evaluated in rat back wounds, while its hemostatic effect was verified through measurements of hemostasis time and blood loss in a rat liver laceration and femoral artery injury model. Furthermore, the biocompatibility of the hemostatic bandage was assessed through subcutaneous implantation and blood routine analysis.

Chitosan nonwoven fabric (120 g/m², degree of deacetylation = 95.3%, Mw ~1000 kDa) was purchased from Hismer Biotechnology Co., Ltd. (Shandong, China). Glacial acetic acid and calcium chloride (CaCl₂) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium alginate (viscosity = 200 ± 20 cps, Mw = 32–250 kDa) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China). ADP was obtained from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Mouse embryonic fibroblasts (NIH-3T3) were purchased from Dingguo Biotechnology Co., Ltd. (Beijing, China). Human umbilical vein endothelial cells (HUVECs) were obtained from Enzyme Research Biotechnology Co., Ltd. (Shanghai, China). Live/dead assay kit and cell counting kit-8 were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Lactate dehydrogenase (LDH) activity assay kit, prothrombin time (PT) assay kit, and activated partial thromboplastin time (APTT) assay kit were purchased from Solarbio Science & Technology Co., Ltd. Glutaric dialdehyde (GA), ethanol, and paraformaldehyde were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Male Sprague Dawley (SD) rats with weight of 230–250 g was obtained from Slack Experimental Animal Co., Ltd. (Shanghai, China). ChitoGauze XR® Pro and ChitoSAM™ 100 were purchased from Sibaike Biotechnology Co., Ltd. (Nanjing, China). Sorbalgon® was obtained from Bohmann Trading Co., Ltd. (Shanghai, China). The reagents used in the experiments were of analytical grade, and they need not to be purified once again.

The morphology of the hemostatic bandage was observed using a scanning electron microscope (Nova NanoSEM 230, Japan) at 20 kv. Elemental mapping of the hemostatic bandages was characterized by energy dispersive spectrometer (XFlash Detector 6|30, Germany). The chemical structure of the hemostatic bandages was characterized by Fourier transform infrared spectrometer (FT-IR, Nicolet iS50, USA). Thermal stability of hemostatic bandages was analyzed using thermogravimetric analysis on a simultaneous thermal analyzer (STA449C, Germany). Tensile strength and elongation characteristics were measured using a universal material testing machine (TA. XT PLUS, SMS, U.K.), with a 1 × 3 cm hemostatic bandages tested at a speed of 10 mm/min. The blood/water contact angle of the samples was assessed using an Easy Drop water contact angle meter (HVS430W, China) by adding drops of blood/water (5 μL) to the sample surface and measuring both the contact angle and penetration effect.

The in vitro coagulation rate of different samples was compared by performing all tests in tubes at 37°C. The pre-warmed sample (10 mg) was added to 100 μL of recalcified blood (0.2 M CaCl₂: blood = 1:10) and incubated for specific time intervals (30, 60, 90, 120, and 180 s). Afterward, 1 mL of deionized water was gently added to terminate the reaction without disturbing the clot. The released HA from free erythrocytes was measured as the method mentioned before. The unclotted state was observed at time point zero seconds (HA₀), subsequently, the amount of hemoglobin in the unclotted erythrocytes was measured at t seconds (HAt). The relative hemoglobin (RHA) value can be calculated using the following formula: RHA (%) = HAt / HA₀ × 100%.

Similarly, the pre-warmed sample (10 mg) was supplemented with 200 μL of recalcified blood and subjected to shaken. Subsequently, the centrifuge tube was periodically tilted every 10 s to monitor coagulation. The duration from the moment when samples came into contact with blood until complete cessation of flow was recorded, while employing 200 μL of recalcified blood as a blank control.

1. For platelet adhesion analysis, 200 μL of PRP was co-incubated with each sample at 37 °C for 30 min and subsequently washed with PBS to remove unadhered platelets. Afterward, 1 mL of 1% Triton X-100 was added and lysed at 37 °C for 1 h. Finally, the activity of platelet LDH was evaluated using an LDH assay kit at a wavelength 450 nm. Platelet adhesion was calculated by plotting a standard curve. PRP without any material was used as a control. The platelet adhesion rate is calculated as follows: Platelet adhesion rate (%) = ODSample/ODControl × 100%. Furthermore, platelet aggregation on the hemostatic bandages was also observed by SEM.

2. The coagulation activation pathway of the hemostatic bandages was assessed using APTT and PT assay kits. Whole blood from healthy SD rats was centrifuged at 3000 rpm for 10 min, and the supernatant was removed to obtain platelet-poor plasma (PPP). For the APTT assay, 10 mg of the sample was incubated with 100 μL of PPP at 37 °C for 3 min, followed by the addition of 100 μL of APTT reagent. After incubation 5 min at 37 °C with gentle mixing, 100 μL of warmed 25 mM CaCl2 solution was added, and the blood clotting time was recorded. For the PT assay, the samples were incubated with 100 μL of PPP at 37 °C for 3 min, followed by the addition of 100 μL of PT reagent, and the time required for PPP clotting was recorded.

3. The hair on the back of the anesthetized SD rats was removed, and the area was disinfected with iodophor. A surgical blade was used to cut 1 cm on each side of the midline of the rat’s back, followed by application of a hemostatic bandage measuring 35 mm × 15 mm to cover the wound site. After allowing approximately 1 h for clot formation and maturation, the peak force required for peeling off the bandage was

4. Freshly collected SD rat blood containing anticoagulant was centrifuged at 1500 rpm for 10 min to isolate red blood cell (RBC) and platelet-rich plasma (PRP). RBC were diluted with PBS at a volume ratio of 1:1, and 150 μL of RBC was incubated with each sample for 30 min at 37 °C, and 150 μL RBC was used as control. Afterward, the unadhered erythrocytes were removed by rinsing with PBS, followed by lysis of the adhered erythrocytes on the material with 1 mL of deionized water. The absorbance of the lysate at a wavelength of 540 nm was measured. The erythrocyte adhesion rate is calculated as follows: RBC adhesion rate (%) = ODSample/ODControl × 100%.

5. All animal experiments followed strictly the requirements of the National Research Council’s Guide for the Care and Use of Laboratory Animals (8th edition) and were formally approved by the Animal Ethics Committee of Fuzhou University. During the experimental period, all animals had free access to food and water.

6. Additionally, erythrocytes were fixed with 2.5% (v/v) GA/PBS for 3 h, followed by two rinses with deionized water and dehydration using sequential ethanol solutions (60%, 70%, 80%, 90%, and 100%) for 15 min each. After drying, the adhesion of RBC on the hemostatic bandages was observed by SEM.

measured using a digital push-pull meter.

SD rats were utilized to establish models of liver laceration and leg femoral artery hemorrhage. Rats were initially anesthetized via intraperitoneal injection of sodium pentobarbital (1%, 40 mg/kg) and immobilized. For the rat liver laceration hemorrhage model, abdominal hair was removed prior to incising the abdominal epithelial tissue to expose the liver. A wound measuring 1 cm in length and 0.5 cm in depth was then created on the left lobe of the liver. Subsequently, a pre-weighed sample measuring 3 cm × 3 cm was applied over the wound site. The hemostasis time was recorded, and blood loss was calculated by weighing the sample after achieving hemostasis. Following hemostasis, the bandage was removed and the liver repositioned. After a period of 7 days, H&E and Masson staining were performed on excised liver samples from the incision site to evaluate any potential impact of bandage residue on hepatic tissues.

The leg was prepared for the rat femoral artery model, and the femoral artery was exposed and punctured using a 20 G needle. The pre-weighed sample was covered, and gentle pressure was applied to the bleeding site. Hemostasis time and blood loss were recorded.

SD rats were anesthetized with sodium pentobarbital. After shaving the hair on the back, a 1 × 1 cm hemostatic bandage was implanted under the skin on the back of the rats, sutured immediately, and sterilized with iodophor. Blood samples were collected from rats on day 7 and day 21 for routine blood tests, and skin reactions of each group were observed. Subsequently, the rats were euthanized, and tissue samples were excised and fixed with 4% paraformaldehyde for preservation. The histocompatibility of the materials was assessed through H&E staining and immunofluorescence imaging.

The results presented in this study were obtained through at least three independent experiments and are reported as mean values ± standard deviation. One-way analysis of variance (ANOVA) and student t-test was used for the treatment comparison. Comparisons with *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered statistically significant, the NS represented no significant difference.

Chitosan nonwoven fabric is composed of randomly oriented fibers (Fig. 1A). A sponge layer can be observed on the surface, which is embedded in the nonwoven fabric in CS/SA/ADP. The EDS mapping and FT-IR results confirmed the successful binding of sodium alginate, ADP, and Ca2+ to the chitosan nonwoven fabric (Figs. S1 and S2). As shown in Fig. 1B, the hemostatic bandage has good flexibility (capable of twisting 360° in situ), which is beneficial for hemostasis at different wound sites. Moreover, CS/SA/ADP has higher tensile strength and lower elongation compared to CS alone (Fig. 1C and D). The incorporation of sodium alginate and Ca2+ into the hemostatic bandage enhances its structural compactness and mechanical properties, resulting in reduced elongation. Consequently, the prepared hemostatic bandages maintain their shape at wound sites extended periods, thereby facilitating rapid wound healing following hemostasis [44].

Hemostatic bandages should possess absorbent and breathable properties to prevent dehydration and excessive accumulation of exudate at the wound site [45,46]. As shown in Fig. 1E, SA and SA/ADP sponge exhibit a higher water vapor transmission rate, while CS/SA/ADP demonstrate reduced permeability. The reason can be attributed to the increased density of the CS/SA/ADP due to infiltration of calcium alginate into the chitosan nonwoven fabric pores, thereby impeding air flow and subsequently reducing the rate of water vapor transmission. However, CS/SA/ADP maintains good air permeability, facilitating oxygen exchange between the wound and external environment. Blood/water adsorption test showed that CS/SA/ADP adsorbed more red blood cells and platelets than water, and was also significantly higher than CS/SA blood adsorption (Fig. 1F). This suggests that ADP can effectively adsorb blood cells and promote their aggregation on the hemostatic bandage surface. Furthermore, the contact angle measurement as well as diffusion analysis demonstrated rapid absorption and diffusion of blood when added to CS/SA/ADP (Fig. 1G). However, CS maintains a certain contact angle within 5 s due to its inherent hydrophobicity. Therefore, the addition of calcium alginate improves the hydrophilicity of the hemostatic bandage.

Additionally, the degradation test results demonstrated that CS exhibited limited degradability in PBS, whereas the CS/SA/ADP

experienced a weight loss of about 23 % by day 7 (Fig. 1H). The reduction in weight of CS/SA/ADP was primarily attributed to calcium alginate dissociation. Both CS and CS/SA/ADP degraded significantly in the presence of lysozyme solution, with the latter retaining approximately 65 % of its initial weight by day 7. Furthermore, thermogravimetric analysis revealed that the CS/SA/ADP possessed good thermal stability (Fig. S3).

The cytocompatibility of hemostatic bandage was investigated by cell viability and cell migration using 3T3 and HUVEC as cell models (Fig. 2A). The extracts from the hemostatic bandage did not exhibit any cytotoxic effects on both 3T3 and HUVEC cells, with cell viability ranging from 95 % to 130 % for both models (Fig. 2B and C). Moreover, the live/dead fluorescence images showed that cells cultured in different extract groups displayed normal growth patterns, exhibiting comparable morphology and viability to those in the control group (Fig. 2D and E). These findings indicated that the good cytocompatibility of the prepared hemostatic bandages.

As depicted in Fig. 2F and G, the CS/SA/ADP treatment significantly enhanced the cell migration compared to the control group. In particular, CS/SA/ADP showed a relative scratch closure of 74 % - 80 % for both types of cells at 48 h, whereas the CS group demonstrated only about 50 % (Fig. 2H and I). Notably, it was observed that CS/SA/ADP promoted wound healing more effectively than CS/SA alone due to ADP’s ability to activate signaling pathways upon binding to cell surface receptors involved in biobehavioral processes such as cell activation, migration, and proliferation [47,48]. These results indicated that the prepared hemostatic bandage could promote wound healing. Furthermore, no rupture or lysis of red blood cells occurred after the contact between the prepared samples and blood (Fig. 2J). Moreover, the measured hemolysis rate was <5 %, indicating high hemocompatibility of hemostatic bandages.

The levels of free HA in red blood cells serve as indicators of coagulation, and the absorbance value are normalized to RHA [49]. As shown in Fig. 3A, the RHA level decreased at a faster rate in the CS/SA/ADP group compared to other groups, suggesting an accelerated coagulation process. Approximately 77.67 % ± 5.72 % of erythrocytes were

entrapped within the clot in the CS/SA/ADP group at 30 s, whereas this percentage was 57.92% ± 7.99% in the CS/SA group (Fig. 3B). Furthermore, analysis of RHA values in both SA and SA/ADP groups confirmed that ADP can enhance blood coagulation under in vitro conditions.

In order to validate the blood loss of CS/SA/ADP at the injury site in vitro, a polyethylene hose was filled with blood and punctured on the side to simulate a bleeding wound. As shown in Fig. 3C and D, CS exhibited significant blood penetration resulting in a blood loss of 1.80 ± 0.16 g, while CS/SA measured as 0.98 ± 0.22 g. Remarkably, CS/SA/ADP displayed minimal blood loss (0.55 ± 0.14 g), representing an approximate three-fold reduction compared to the CS group. In addition, the in vitro evaluation of whole blood clotting time was conducted. The untreated whole blood exhibited a clotting time conversion rate of 100% (Fig. 3E). CS, CS/SA, and CS/SA/ADP demonstrated reduced clotting times of 53.98% ± 6.40%, 39.69% ± 3.24%, and 29.44% ± 2.69%,

respectively. This reduction in clotting time can be attributed to the primary hemostatic function of CS through blood cell absorption, independent of the coagulation system [50]. However, when CS/SA/ADP absorbs blood, it releases ADP and Ca2+, triggering a cascade reaction within the coagulation pathway. The combination of active hemostasis and passive hemostasis significantly enhances hemostasis efficiency. It is noteworthy that alginate also contributes to an enhanced procoagulant effect [51].

Blood cell aggregation has been reported to be an important factor in primary hemostasis. To confirm the hemostatic mechanism of the hemostatic bandage, the interaction between erythrocytes/platelets and the hemostatic material was observed using SEM. As shown in Fig. 4A, a substantial number of blood cells adhered to hemostatic materials while maintaining their biconcave disk shape. Similarly, platelet adhesion to these hemostatic materials was also observed. Furthermore, there was a significant increase in platelet aggregation and pseudopodia formation

in the CS/SA/ADP group compared to the CS/SA group. The formation of platelet pseudopods may be attributed to ADP binding with platelet surface receptors, which stimulates signaling pathways leading to morphological changes (Fig. 4B) [52,53]. Additionally, quantitative analysis revealed that fewer erythrocytes adhered to CS while CS/SA/ADP exhibited the highest number of erythrocytes adhesion (Fig. 4C and D). This result can be attributed to CS having a relatively smooth surface with maximum porosity and poor interaction with erythrocytes. By contrast, CS/SA/ADP possesses a rough fiber-like structure along with sponge-like characteristics enabling tight attachment of numerous erythrocytes.

Intrinsic and extrinsic coagulation pathways play crucial roles in secondary hemostasis [54]. Thus, the APTT and PT were measured to assess the activation of the coagulation pathway by the CS/SA/ADP hemostatic bandage. As shown in Fig. 4E, the APTT values of CS/SA/ADP were significantly lower compared to those of CS/SA and CS. Similarly, the PT values of CS/SA/ADP were comparable to SA, CS/SA, and SA/ADP but significantly lower than those of CS (Fig. 4F). Moreover, both APTT and PT values in the control group were significantly higher than those in the CS/SA/ADP group. Hence, it can be inferred that the reduction in APTT and PT values observed with CS/SA/ADP may be attributed to synergistic hemostatic effects resulting from the presence of ADP, Ca2+, and alginate, which activate both internal and external coagulation pathways [55–57].

The challenge faced by current hemostatic materials is the potential tearing of wounds and subsequent pain and secondary bleeding upon material removal after achieving hemostasis [58]. A back skin bleeding model was fabricated on the back of rats to assess the easy of peeling for

the prepared hemostatic bandages (Fig. S4). The prepared hemostatic bandages (CS/SA/ADP) had relatively low peel forces without causing any wound tearing during removal (Figs. 5A, B and S5). The peel force was approximately 5.1, 2.5, and 2.6 times lower than that of ChitoGauze® XR Pro, ChitoSAM™ 100, and CS group, respectively. By contrast, Sorbalgon® had a lower peel force but left more residue in the wound necessitating debridement (Fig. S6). These findings indicated that the prepared hemostatic bandage possesses low adhesion strength and easy peeling properties, which mitigate pain and secondary bleeding associated with material removal post-wound treatment [59].

The hemostatic performance of hemostatic bandages in noncompressible wounds was evaluated by using a rat liver laceration hemorrhage model [60,61]. As shown in Fig. 5C, blood spread rapidly into the ChitoGauze® XR Pro and completely saturated all four sides with a blood loss of 0.83 ± 0.17 g (Fig. 5D). ChitoSAM™ 100 and CS are made of chitosan nonwoven fabric, exhibiting similar blood permeability to Sorbalgon®, as all three penetrated three layers of bandages. However, the rate of blood diffusion and extent of wetting were comparatively smaller for these three groups compared to ChitoGauze® XR Pro. This result may be due to the fact that chitosan fiber fabrics can control bleeding better than chitosan gauze for primary hemostasis. Upon application at the bleeding site, CS/SA prevented blood penetration into other layers; only a small area on one side became soaked, indicating its superior hemostatic effect relative to the CS group. Notably, CS/SA exhibited lower blood loss (0.57 ± 0.15 g) than the CS group (0.74 ± 0.04 g). Furthermore, The CS/SA/ADP significantly enhanced hemostasis by confining blood solely within the first layer of the bandage and limited to a small area, resulting in an approximate blood loss of only 0.32 ± 0.15 g.

As shown in Fig. 5E, the hemostasis time of CS/SA/ADP was 31.2 ± 5.49 s, whereas that of ChitoGauze® XR Pro, ChitoSAM™ 100, and Sorbalgon® ranged from 50 to 105 s. Furthermore, the hemostasis time

of CS and SA was closer to each other, which was 73.0 ± 12.47 s and 69.2 ± 18.35 s, respectively. The results suggested that the inclusion of sodium alginate, Ca²⁺, and ADP in hemostatic bandages may enhance procoagulant activity and expedite hemostasis time through synergistic effects [30,62]. It is worth mentioning that the hemostatic time of this hemostatic bandage was improved compared to previously reported ADP or Ca²⁺ doped hemostatic dressings [31,63].

Fig. 6A, blood rapidly diffused to all four sides of the ChitoGauze® XR Pro, and there was blood seepage from the seams of the rat’s leg (Fig. S7). When employing ChitoSAM® 100 and CS, two layers of the dressing were saturated with blood penetration and diffusion, while only small amounts of blood remained in layers 3 and 4 (Fig. 6B). In contrast, Sorbalgon® exhibited high absorbency during hemostasis, moistening all four layers of the dressing and resulting in a higher blood loss of approximately 1.13 ± 0.20 g. Compared to CS, a minimal amount of blood was observed on the second side of the CS/SA, and blood loss

A rat femoral artery injury model was used to assess the hemostatic effect of the hemostatic bandage on a compressible wound. As shown in

was reduced to 0.70 ± 0.11 g. In CS/SA/ADP, the blood loss decreased further to 0.45 ± 0.08 g, with only the first layer of the bandage being infiltrated by blood while keeping the other layers dry. Additionally, the shortest hemostasis time was observed in CS/SA/ADP, indicating the strong hemostatic ability (Fig. 6C). Therefore, these findings demonstrated that the CS/SA/ADP hemostatic bandage possesses equivalent efficacy in severe bleeding models involving liver laceration and femoral artery injury.

Jianmin Yang: Conceptualization, Methodology, Formal analysis, Supervision, Funding acquisition, Project administration, Writing – review & editing. Fengying Cai: Conceptualization, Investigation, Methodology, Formal analysis, Writing – original draft. Yicheng Lv: Conceptualization, Investigation, Formal analysis. Ting Jiang: Investigation, Formal analysis. Xingkai Zhao: Investigation. Xueli Hu: Investigation. Yunquan Zheng: Methodology, Resources, Formal analysis. Xianai Shi: Conceptualization, Methodology, Supervision, Resources, Project administration.

In vivo biocompatibility was assessed by adhering a hemostatic bandage to the back skin of rats with a clinically approved medical sterile dressing [64]. As shown in Fig. 7A, no significant irritation, such as erythema itching or cracking (no scratches on the back), was observed in the skin covered with the hemostatic bandage after 24 h of adhesion. H&E staining was employed to assess the impact of residue from the hemostatic bandage on liver wound healing in vivo. As shown in Fig. 7B, no discernible pathological alterations were found at the wound site. Notably, the Masson staining results showed that the hemostatic bandage containing ADP promoted wound healing compared with CS nonwoven fabric (Fig. 7C).

demonstrated significantly superior hemostatic efficacy compared to commercial products such as ChitoGauze® XR Pro, ChitoSAM™ 100 and Sorbalgon® in a severe bleeding model of liver laceration and femoral artery injury in rats. Moreover, the lower peeling force of the hemostatic bandage was effective in preventing adhesion to granulation tissue after hemostasis in a rat dorsal hemorrhage model. Additionally, subcutaneous implantation and blood routine analysis confirmed good biocompatibility of the prepared hemostatic bandage. These results indicate that the prepared hemostatic bandage exhibits a straightforward synthesis process, easy peeling without residue, and good hemostatic properties suitable for emergency applications.

A hemostatic bandage with synergistic advantages was developed for the treatment of acute bleeding wounds. The results showed that the hemostatic bandage not only retained the advantages of sponge in providing a moist environment and absorptivity, but also inherited the breathability and mechanical characteristics of chitosan nonwoven fabric. In vitro cellular experiments showed that the hemostatic bandage had high cell compatibility. Moreover, under the synergistic effect of plasma absorption, large aggregation of red blood cells/platelets, and activation of internal/external coagulation cascades, hemostatic bandages can achieve faster hemostasis. The hemostatic bandage

In evaluating the biological safety of the materials, the prepared hemostatic bandage was subcutaneously implanted onto the back of rats, and blood samples were regularly collected for hematological analysis and histological examination (Fig. 7D). Blood tests were conducted to evaluate whether degradation of the hemostatic bandage posed any potential systemic toxicity [65]. As shown in Fig. 7E, the degradation products of the hemostatic bandages showed no evident signs of systemic toxicity, and the blood was similar to that of healthy animals. Histological changes in subcutaneous tissues treated with CS/SA and CS/SA/ADP were examined using H&E staining (Fig. 7F). In CS/SA-treated tissues, neutrophil aggregation around the tissue was observed on day 7 post-implantation, indicating a mild local host inflammatory response. Hemostatic bandages containing ADP exhibited reduced recruitment of inflammatory cells. At day 21 after implantation, a small number of inflammatory cells persisted in CS/SA-treated tissues; however, no inflammation was evident in CS/SA/ADP-treated tissues.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

This work was supported by the Nature Sciences Funding of Fujian Province (2022J01095) and Special Fund for Marine Development of Fujian Province (FJHJF-L-2022-4).

The immune response of the hemostatic bandage to subcutaneous implantation was further characterized by immunofluorescence staining for lymphocytes (CD3) and macrophages (CD68). At the junction of CS/SA and subcutaneous tissues, a high amount of CD3 and CD68 infiltration was observed at 7 days, whereas CS/SA/ADP had less inflammatory cell infiltration (Fig. 7G). At 21 days, some inflammatory cells were still present in CS/SA, whereas almost no cells were observed in CS/SA/ADP. These results suggested that the in vivo implantation of hemostatic bandages effectively fills both cells and the extracellular matrix within the material interstices, leading to the generation of nascent interconnected tissues rather than a mere formation of a capsule layer. Furthermore, the presence of ADP promotes inflammation resolution and reduces host’s inflammatory response. Collectively, these results demonstrated high biocompatibility of the prepared hemostatic bandages.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2023.128561.

Data will be made available on request.

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J. Yang et al.                                                                                International Journal of Biological Macromolecules 257 (2024) 128561

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https://doi.org/10.1016/j.ijbiomac.2025.139804

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