Hemostatic effect and safety evaluation of the absorbable macroporous polysaccharides composite hemostatic material prepared by a green fabrication approach

Abstract

Carboxymethyl chitosan is widely used in the medical field such as wound healing and other medical fields. We previously fabricated the absorbable macroporous polysaccharides composite hemostatics (AMPCs) mainly composed of carboxymethyl chitosan which possess excellent hemostatic effect. To further elucidate the impact of CMCTs on the hemostatic effect and biosafety of AMPCs, carboxymethyl chitosan with different properties were used to prepare AMPCs. By comparing the physical and chemical properties, AMPCs performed high water absorption ability, especially Group 1 (swelling ratio reached 5792%), which facilitated the rapid formation of blood clots. It was confirmed by blood clotting index (BCI) and blood coagulation tests in vitro that Group 1 showed a slightly higher coagulation capacity than groups 2 and 3, which may be due to the positive charge on the surface of the cations in the salts attaches to the negative charge on the surface of the red blood cells, an electrostatic neutralization reaction occurs. The biosafety was a preliminary evaluation by implanted AMPCs into the back of Sprague-Dawley rats and the tissue was harvested after feeding for 28 days. The AMPCs exhibited good biosafety for whole blood and major organs during the degradation in vivo: during the degradation of AMPCs, excluding changes in some serum indicators, no tissue necrosis or inflammatory cell infiltration was observed in these organs, either by gross observation or histological analysis. These findings demonstrate that expecting to develop a highly functional and safe hemostatic agent based on Group 1 for rapid hemostasis applications in emergencies.

Keywords

Hemostatic absorbable polysaccharide biosafety carboxymethyl chitosan

Introduction

Effective hemostasis is one of the key measures of reducing mortality and blood loss after injury.¹ Absorbable materials that can be biodegraded have been widely used in surgical procedures, such as cardiovascular, hepatic and orthopedic surgeries.^2,3 So far, various absorbable hemostatic materials, such as collagen, oxidized cellulose, absorbable gelatin sponge, chitosan hemostatic dressings, thrombin and so on, have been developed to be applied in vivo.⁴ Aiming to be applied for clinical application as a hemostatic agent, effectiveness, that is hemostatic effect and biosafety is essential to be investigated.

As the only bio-sourced cationic polysaccharide in nature, chitosan can promote the aggregation of red blood cells by reacting with the negative charge, which can activate platelets and the fibrinolysis system to play a role in rapid coagulation.⁵ However, chitosan is almost insoluble in water, which limits its applications in the hemostatic agent. As a water-soluble derivative of chitosan, carboxymethyl chitosan (CMCTs) has excellent water solubility, antibacterial ability and biocompatibility, which facilitate its biomedical application such in wound dressing, anti-adhesion, anti-microbial and controlled drug release.^6–10 Carboxymethyl chitosan hydrogel has been explored as a promising hemostatic material for its three dimensions (3D) network and excellent water (blood) absorption capacity, as well as above mentioned advantages.¹¹ Several commercial hemostatic materials based on chitosan and its derivatives have been developed, for instance, Celox Rapid (UK), Arista (USA) and the absorbable macroporous polysaccharides composite hemostatic materials (CMPHP) (China).^12–14 However, their application in severe blood bleeding is still weak.

To form 3D networks, conventional cross-linking methods have been divided into two categories: chemical and physical cross-linking. Radiation cross-linking is considered an ideal technique for preparing medical appliances without any cytotoxic additives.¹⁵ High-energy electron beams can break chemical bonds in polymer chains to initiate free radicals more efficiently than other methods. Thereafter, free radical recombination can occur to form a 3D network structure in ambient temperature with any initiator, crosslinking agent and organic solvents. In our previous study, the absorbable macroporous polysaccharides composite hemostatics (AMPCs) composed of CMCTs were fabricated by the green approach, for instance, radiation crosslinking. AMPCs possessed excellent hemostatic ability in the small-artery severed injury model and thyroid surgery clinical study and were better than Celox Rapid, Arista and CMPHP in terms of water absorption capacity.^16,17 To further elucidate the impacts of CMCTs on the hemostatic effects and mechanism of AMPCs, as well as their biosafety, AMPCs were prepared from three CMCTs with different properties. Furthermore, properties of AMPCs such as swelling behavior, gel fraction, in vitro blood coagulation test, etc. were investigated to evaluate their potential as hemostatic material. Furthermore, the biosafety evaluation of the AMPCs was preliminarily studied in vivo in Sprague-Dawley rats. The AMPCs is expected to be a novel type of hemostatic material that can be applied in the treatment of severe bleeding.

Materials and methods

Characteristics of CMCTs

To study the effect of different carboxymethyl chitosan on AMPC, properties of CMCTs, which were produced by three different manufacturers (Group1: Qingdao Headful Biotechnology Co., Ltd, Group2: Qingdao Honghai Biotechnology Co., Ltd, Group3: Nantong Lvshen Biological Engineering Co., Ltd) with different characteristics, such as water content, pH, protein content, heavy metal content (Pb), Fe content, chloroacetic acid residue, diglycolic acid residue, ash content and endotoxin were determined by the method of Chinese pharmaceutical standard: Medical carboxymethyl chitosan (YY/T 0953-2020).¹⁸

Synthesis of the AMPCs

A series of CMCTs/gelatin composite hydrogels were fabricated according to the previous study,¹⁹ on this basis, electron beam irradiation was utilized instead of gamma-irradiation. Performed in a GMP manufacturer, CMCTs/gelatin composite hydrogels were lyophilized and pulverized to obtain hemostatic AMPCs. In addition, AMPCs were prepared from different CMCTs, which were referred to as HDF-210622F (Group 1), HH-210812 (Group 2) and LS-210926 (Group 3) respectively.

Physical and chemical properties of the AMPCs

Swelling ratio

Swelling ratios were performed by immersing AMPCs in an excess of distilled water at room temperature. In the measurements, the samples were removed from distilled water and weighed after removing the excess distilled water from their surfaces. The swelling ratio was expressed as a ratio of the weight of the swollen hydrogel to the weight of its corresponding dried gel.

Swelling dynamics

The samples of AMPCs with known mass were dried in a vacuum until the mass was constant and soaked in deionized water to swell. The gel was taken out at certain intervals and the excess water on the surface was gently absorbed with filter paper. The gel quality at different times was weighed and recorded. The swelling ratio SR of the gel was calculated according to

S R = (m_{t} - m_{0}) / m_{0}

(1)

In formula (1), m_t is the mass of the gel at time t, and m₀ is the dry mass of the gel. When the swelling mass of the gel does not change anymore, SR is the equilibrium swelling ratio.

Gel fraction

A certain amount of AMPCs was weighed and placed in excess distilled water at room temperature. In the measurement, the samples were removed from distilled water and then dried to constant weight in the oven at 60°C for 12 h.

The gel fraction was calculated according to

g e l f r a c t i o n = m / m_{0} {(1 - a)}^{*} 100 %

(2)

where m means the weight of AMPCs after drying, m₀ means the initial weight of AMPCs and a means loss factor of AMPCs on drying.

Loss on drying

Accurately weigh the specified amount of the substance to be examined (based on the method reference). Take a pre-dried glass-stoppered LOD bottle (dried under the same conditions as the test sample for 30 min). Transfer the test sample to the LOD and accurately weigh the overall. Dry the substance to constant mass under vacuum conditions at 60°C.

pH

Absorbable macroporous polysaccharides composite hemostatics was formulated to a 3 mg/mL concentration. The test was carried out according to the pH determination method 0631, Part 4, Pharmacopoeia of the People’s Republic of China 2020 Edition.

Hemostatic properties of the AMPCs

Blood clotting index (BCI)

0.1 g of each AMPCs sample was taken, and 10 mL of normal saline was added respectively to form 10 g/L sample solution. The experimental groups were divided into the AMPCs group and the control group. The blank control group was added with 1 mL normal saline, and the AMPCs group was added with 1 mL sample solution, respectively. 100 μL of fresh anticoagulant rabbit whole blood was added to each group, then 10 μL of calcium chloride aqueous solution (0.2 mol/L) was added to each tube to inhibit the thrombin activity, and incubated in 37°C constant temperature water bath for 2 min. After incubation, 10 mL of deionized water was added to the centrifuge tube to destroy/dissolve the uncoagulated blood cells, and the cells were further incubated for 3 min at 37°C constant temperature water bath. The suspensions were centrifuged at 3500 r/min for 1 min and 200 μL of supernatant was collected. The absorbance value was measured at 540 nm wavelength.

In vitro blood coagulation test

Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB) were measured by the coagulation method. All data were measured by Prolong PUN-2048B semi-automatic coagulation analyzer and automated blood coagulation analyzer. Reagent kits were from Shanghai Sun Biotechnology Co. LTD.

Compressive strength of blood clots

The AMPCs were mixed with fresh whole blood containing an anticoagulant in a certain proportion. All the groups were kept at 37°C until the blood fluidity disappeared. The blood clots were taken out with care and tested by a TA. XT.plus texture analyzer (STABLE MICROSYSTEMS LTD, UK) for their compressive strength. Testing speed = 1 mm/s and press distance = 0.3 mm.

Biosafety evaluation

The systemic safety evaluation was performed following ISO 10993 part 11, to evaluate the systemic response of rats samples. Healthy rats weighing 150–200 g were procured from the animal room and randomly grouped (10 animals in each/group). Seven days before the test, animals were acclimated to laboratory conditions. Based on weight conversion of implant dose, 50 times the proposed dose for a human clinical back implant. 2.5 g/kg body weight AMPCs were implanted in five rat groups and the response was observed, immediately after implant and at 24, 48 and 72 h for evidence of any abnormalities such as respiratory, motor, reflexes, ocular signs, cardiovascular signs, salivation, convulsions, piloerection, analgesia, muscle tone, gastrointestinal, skin and loss in body weight or death. According to its clinical reactions, body weight changes, clinical examination results and pathological examination results were used to determine whether the test sample caused systemic toxicity to the rats.

Animals model

Forty SPF Sprague-Dawley rats with 20 /20 male and female (6–8 weeks, weighing 150–200 g) were selected for the experimental studies. At the beginning of the experiments, the difference in animal body weight (same-sex) should not exceed ±20% of the mean weight. Marking method: ear tag marking, feeding room words: SPF-class housing of laboratory. Drinking water: purified water after autoclave sterilization, water supply mode: free access to water intake through drinking bottles.

Clinical observation

The observations included but were not limited to animal appearance and physical signs, behavioral activities, glandular secretion, breathing and fecal traits, etc., and were recorded on time, an autopsy should be conducted in case of dead or dying animals. Weight: once a day before the test, once a day for the first 3 days, once a week after 3 days and once a week before euthanasia.

Body weight and food intake

Individual body weight was recorded once every 7 days. The first recording day was the third day before the initial study. Animals were allowed ad libitum access to food throughout the study.

Haematological and biochemical analysis

After intramuscular anesthesia with 1% pentobarbital sodium at 0.4mL/100 g*bw and blood sampling from the abdominal aorta. The following indexes were detected.

Blood without anticoagulant was collected in tubes for blood chemical analysis, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), Albumin (ALB), Uric acid (UA) and UREA using a BS-220 Fully Automatic Chemistry Analyzer Used Mindray Machine.

Whole blood count

The whole blood count was used Mindray BC-1800 Hematology Analyzer.

Necropsy

Rats were sacrificed and the relative weights were calculated as an organ-to-body weight ratio. A part of tissues was dissected and fixed in 10% formalin. The paraffin sections were prepared and stained with hematoxylin and eosin and examined with light microscopy (Olympus CX33).

Statistical analysis

Statistical analysis was performed using The Independent Samples T-Test for comparison between different groups using SPSS 25.0, and p < 0.05 was considered statistically significant.

Results and discussion

Being a hemostatic agent that is expected to be used within the bionet, properties concerning the hemostat’s properties as well as the risk of causing toxicity and adverse drug reaction should be studied in all aspects. Firstly, properties of AMPCs which correlated to their hemostasis effects were investigated, for instance, APTT, TT, PT and FIB. Moreover, the hemostatic behavior of AMPCs was studied in vitro. Finally, AMPCs were implanted into model animals to study the biosafety of the materials.

Physical and chemical properties of the CMCTs

In this study, the properties of CMCTs from different manufacturers are demonstrated in Table 1 by the method of Chinese pharmaceutical standard: Medical carboxymethyl chitosan (YY/T 0953-2020).¹⁸ The content determination of water content, pH, and impurities such as protein content, heavy metal content (Pb), Fe content, chloroacetic acid residue, diglycolic acid residue, ash content and endotoxin were investigated to estimate the influence of CMCTs on the biosafety of AMPCs.

Table 1.

Characteristics of CMCTs from different manufacturers.

Specifications	Group 1	Group 2	Group 3
Water content (%)	10.97	13.87	7.09
pH	7.52	7.7	6.88
Protein content (%)	0.16	0.21	0.06
Heavy metal content (pb)	<10 μg/g	<10 μg/g	<10 μg/g
Fe content (μg/g)	107.99	3532.89	88.9
Chloroacetic acid residue (%)	ND	ND	ND
Diglycolic acid residue (%)	ND	ND	ND
Ash content (%)	10.49	12.86	15.97
Endotoxin (EU/mg)	<0.05	<0.05	<0.05

Water content and pH are important parts of processing CMCTs. In industrial production, CMCTs are dried, which is conducive to the subsequent drying, storage and transportation. During drying, the changes in temperature and moisture can change the activity of many structures, leading to changes in the quality and properties of materials. Therefore water content has to be kept within a certain range. The water content of Group 1 was 10.97%, Group 2 was 13.87% and Group 3 was 7.09%. The protein content is regarded as a prerequisite for safe clinical use in humans. Residual protein content can cause an immunogenic response in the body, which should be strictly controlled. The protein content of Group 1 was 0.16%, Group 2 was 0.21% and Group 3 was 0.06%. The protein content has confirmed that it was in the safe range.¹⁸

There are many sources of elemental impurities in medical devices. They may be residual catalysts added intentionally during synthesis or actual impurities.²⁰ For example, the human body has trivalent iron, but the body cannot use it directly. Instead, it is transported to the liver, where it is reduced to divalent iron. This is why excessive absorption of trivalent iron can cause iron poisoning. Iron poisoning continues to be a major toxicologic problem, with a major impact on the gastrointestinal and circulatory systems.²¹ Among them, Group 2 has the highest content, which reached about 23–40 times more than the others. Elemental impurities need to be kept to a certain limit, their entry into the body may cause acute or chronic poisoning. Considering the combination of equipment material, production process and GMP management, it is difficult for equipment, facilities, pipes, tools and other parts directly in contact with the material to enter the material and transfer to the finished product, even if there is a very small amount of iron will be removed by the subsequent process.²² However, because the mentioned above are involved in the synthesis process, it is necessary to measure and control their residues.²³

At present, the commonly used preparation process for the preparation of CMCTs requires the reaction of chloroacetic acid and chitosan under alkaline conditions. Chloroacetic acid residue, as one of the typical synthetic by-products, has attracted considerable concerns for its biological safety.²⁴ Several researchers have found that chloroacetic acid could decrease the cell viability of different cell lines. Diethylene glycol has been implicated in multiple medication-associated mass poisonings, which can result in renal and neurological toxicity. In this experiment, the content of diethylene glycol acid and chloroacetic acid is far below the tolerable exposure level of the human body, so it will not cause harm to the human body. In addition, the ash of CMCTs is 10.49, 12.86 and 15.97% (W/W) respectively. The presence of small amounts of endotoxin in the biological product can cause side effects to the host organism, such as endotoxin shock, tissue injury and even death.²⁵ Due to these reactions, it is essential to remove endotoxins from drugs, injectables, other biological and pharmaceutical products. In this experiment, all materials contained less than 0.05 EU/mg.

Characteristic of AMPCs

The properties of the AMPCs prepared from different CMCTs which were listed in Table 2. Polysaccharide polymers have the disadvantage of low stability and faster degradation in water. This could be avoided by introducing cross-linking in the system. As a hemostatic material, it is crucial to have sufficient swelling ability. The swelling ratio of the AMPCs is more than 50 times its dry weight. According to gel fraction, Group 1 was significantly higher than others, we can find that Group 1 is the best material to cross-link.

Table 2.

Characteristic of AMPCs prepared from different CMCTs.

Specifications	Group 1	Group 2	Group 3
pH	7.66	7.87	7.70
Swelling ratio (%)	5792	3967	5328
Gel fraction (%)	72.88	42.96	58.66
Loss on drying (%)	4.56	5.97	4.71

Kinetic swelling

Highly swollen hydrogels can absorb and retain large amounts of tissue fluid or blood. For hemostatic powders, the excellent water retention capacity contributes to blood enrichment and coagulation.²⁶

To explore its water retention capacity, we evaluated the swelling kinetics of different groups of AMPCs. As shown in Figure 1, the swelling rate of hemostatic powders gradually increased with the time of immersion in deionized water. After about 60 min, the three groups of AMPCs reached an equilibrium of swelling, and the swelling dynamics of Group 1 and Group 3 were significantly higher than that of Group 2.

The swelling kinetics depend not only on the degree of cross-linking but also on the relative mass fraction of polymers. In general, more cross-linking sites and more molecular chains mean higher swelling performance. According to Table 1, the water content of Group 2 was significantly higher than that of Group 1 and Group 3, which means that it possesses fewer polymer chains for the same mass. On the other hand, according to Table 2, the cross-linkage of Group 1 and Group 3 was significantly higher than that of Group 2, which means that Group 1 and Group 3 have a more complete microstructure compared to Group 2. We can find that in Group 1 and Group 3, the swelling dynamics of them are all above 65%, which indicates that they can absorb large amounts of blood before clotting.

BCI

Blood clotting index refers to the ratio of the absorbance value at 540 nm of the test sample to the absorbance value at 540 nm of the aqueous solution of whole blood. If the coagulation process occurs in the organism, it leads to the transformation of fibrinogen into coagulation insoluble fibrin, and a lower BCI indicates a higher coagulation rate.

To verify the effect of CMCTs on fibrinogen conversion, BCI measurements were performed on each group. Figure 2 shows that both Group 1 and Group 3 showed significantly lower BCIs than the control group. In addition, Group 1 showed a slightly higher coagulation capacity than Groups 2 and 3 within 180 s of testing.²⁷

The mechanism may be because CMCTS carries a certain amount of free amino acids, which tend to form salts in acidic solutions and have cationic properties. While the positive charge on the surface of the cation adheres to the negative charge on the surface of the red blood cells, an electrostatic neutralization reaction occurs, which releases the repulsive effect between them and causes the blood to coagulate. At the same time, CMCTs can neutralize platelets and after activation, the surface produces a negative charge and rapidly forms clots.

In addition to the above reasons, chitosan can bind to plasma proteins and some important coagulation factors in vivo due to its specific molecular deconjugation, thus enhancing the coagulation effect, which is also supported by the crosslinking degree in Table 2.

In vitro blood coagulation test

Being a hemostatic agent, the influence of biomaterials on the blood coagulation system is important to the hemostatic effect and the biosafety of the materials.²⁸ Herein, the four parameters of coagulation, that is APTT, TT, PT and FIB, were determined in vitro to evaluate the ability of AMPCs to coagulate blood as well as their impact on the blood coagulation system of biont.

The four parameters of coagulation provide a reference value for experiments and are of great significance for the safety toxicological evaluation of SD rats. By comparison, Group 3 significantly prolonged teratesla and higher FIB than the control group (p < 0.05). Group 3 and Group 1 had significantly higher FIB compared with the control group (p < 0.05) (Figure 3). Theoretically, within a certain range, the shorter APTT, teratesla and PT index time, the higher the FIB content, indicating that the coagulation energy of the sample is stronger, which can promote the coagulation rate through exogenous coagulation and endogenous coagulation.^29,30

Our previous prospective, randomized, controlled, non-inferior clinical study found that rates of hemostasis within 5 min were 100% in both groups. Hemostasis took 91.04 ± 72.07 s in the experimental group and 75.83 ± 53.98 s in the commercial group with a mean difference of 15.21 s (upper limit of 95% CI 54.91, p < 0.05). It hinted at a non-inferiority of the experimental group to the control group. Hemostatic time, coagulation function, routine blood tests and adverse events were comparable between the two groups (p > 0.05).³¹

Compressive strength of blood clots

Blood clots play an essential role in the human organism. They must be able to stem a flow of blood in a case of bleeding, blood clots require an appropriate degree of mechanical, chemical and biological properties to block blood flow and the origin and understanding of these mechanical properties remain unclear.³² At the microstructural level, the clot consists of a 3D network of fibrin fibers that are stabilized by transglutaminase (factor XIIIa) connections and form a 3D network on the whole. In particular, the thickness and nodal density of the fibrin have a direct impact on the mechanical properties of the clot.

As is shown in Figure 4, the compressive strength of blood clots in Group 1 was significantly higher than in Group 2 and Group 3, which illustrates that while intervening at the site of bleeding, Group 1 was able to absorb blood and enrich platelets more quickly, which in turn forms clots. This is also consistent with Table 2 and Figure 3.

Safety evaluation

According to ISO 10993-1: 2009, the safety test is an important part of the work to evaluate the hemostats which aim to be applied as implants contacting the blood system. The safety of AMPCs was evaluated in the rat model after subcutaneous implantation at approximately 2.5 g/kg • bw.

Effect of the AMPCs on organ/body weight ratio

In safety studies, changes in organ weight are a sensitive indicator of toxicity to enzymes, physiological disturbances and target organ injury.³³ An increase in organ weight indicates the onset of hypertrophy, while a decrease indicates necrosis in the target organ.^34,35 There was no significant difference in mean organ and relative organ weights between the control and extraction treatment groups, except for Group 3 male kidney and Group liver. Table 3 shows the effect of CMCTs on primary organ weights relative to body weight, with no significant differences in organ weight changes. The results indicate that the vital organs were not adversely affected throughout the treatment extraction process. The mean and relative organ weights of the tested CMCTs and control groups showed statistically non-significant differences (p > 0.05).

Table 3.

Comparison of organ/body weight ratio in each group after implantation.

Group	Control	Control	Group 1	Group 1	Group 2	Group 2	Group 3	Group 3
Group	♂	♀	♂	♀	♂	♀	♂	♀
Heart	0.0032 ± 0.0002	0.0032 ± 0.0003	0.0034 ± 0.0003	0.0032 ± 0.0001	0.0031 ± 0.0003	0.0041 ± 0.0015	0.0033 ± 0.0001	0.0035 ± 0.0003
Liver	0.038 ± 0.0019	0.0359 ± 0.0014	0.0377 ± 0.0021	0.0381 ± 0.0027	0.0357 ± 0.0021	0.0418 ± 0.0029	0.0434 ± 0.0094	0.0389 ± 0.0028
Spleen	0.0024 ± 0.0006	0.0025 ± 0.0003	0.0023 ± 0.0003	0.0024 ± 0.0002	0.0028 ± 0.0004	0.0029 ± 0.0007	0.0025 ± 0.0003	0.0028 ± 0.0006
Lung	0.0043 ± 0.0003	0.0053 ± 0.0003	0.0045 ± 0.0006	0.0054 ± 0.0004	0.0045 ± 0.0005	0.0054 ± 0.0005	0.0046 ± 0.0003	0.0053 ± 0.0005
Kidney	0.0077 ± 0.0004	0.0081 ± 0.0006	0.0078 ± 0.0009	0.0074 ± 0.0005	0.0077 ± 0.0007	0.0081 ± 0.0004	0.0087 ± 0.0003^a	0.0085 ± 0.0005
Adrenal gland	0.0003 ± 0	0.0004 ± 0.0001	0.0003 ± 0	0.0005 ± 0.0001	0.0003 ± 0.0001	0.0004 ± 0	0.0003 ± 0.0001	0.0004 ± 0
Thymus	0.0015 ± 0.0001	0.0019 ± 0.0002	0.0014 ± 0.0002	0.0018 ± 0.0003	0.0015 ± 0.0002	0.0017 ± 0.0002	0.0016 ± 0.0003	0.0021 ± 0.0002
Testis	0.0094 ± 0.0006	/	0.0095 ± 0.0005	/	0.0093 ± 0.0008	/	0.01 ± 0.0011	/
Epididymis	0.0032 ± 0.0004	/	0.0029 ± 0.0002	/	0.0035 ± 0.0007	/	0.0033 ± 0.0002	/
Ovary	/	0.001 ± 0.0002	/	0.001 ± 0.0002	/	0.0011 ± 0.0001	/	0.0008 ± 0.0002
Uterus	/	0.0021 ± 0.0004	/	0.0019 ± 0.0003	/	0.0023 ± 0.0004	/	0.002 ± 0.0003

^aMeans the difference is statistically significant, p < 0.05.

The relative weights of vital organs such as the liver, kidney and heart were normal indicating no toxic effects in the control and the experimental groups which were not statistically significant (p > 0.05). No significant differences were observed in liver, kidney, heart and small intestine weights, supporting the safety of CMCTs, which is also in line with the previous studies.³⁶

Haematological and biochemical analysis

Liver and kidney functions are essential for the maintenance of the body’s internal environment to measure their normal function by serum biochemical analysis, which is essential in the toxicological assessment of xenobiotics.³⁷ Serum liver function tests reflect the activity status of the liver. Liver enzymes (ALT and AST) describe the integrity of the cells involved, while albumin and total protein levels describe their function.³⁸ ALT and AST are mainly produced by hepatocytes and any factor that predisposes to liver damage may lead to increased serum levels of these enzymes.³⁹ High levels of liver enzymes are a sign of hepatocyte toxicity,⁴⁰ while a decrease may indicate enzyme inhibition.⁴¹ Since AST is also present in large numbers in the kidney, testis, heart and other organs, ALT becomes the most sensitive and specific marker of liver injury or toxicity.

In this study, the estimation of serum biochemical parameters in the experimental animals showed no significance compared to controls (p > 0.05). However, the transaminases ALT/AST showed an overall decrease in the animals implanted with AMPCs compared to the respective controls. Many studies have confirmed that elevated serum levels of liver enzymes and transaminases (ALT and AST) are not directly related to liver injury, but their elevated levels lead to inflammation, cell leakage and damage to liver cell membranes. The main target organ of a drug or bioactive compound is the liver, where it is exposed to foreign substances absorbed by the intestine and metabolized to other compounds that may or may not be hepatotoxic to mice.³⁶ Thus, the reduction of liver enzymes (ALT and AST) in the liver after in vivo implantation of AMPCs may be due to the inhibitory effect of enzymes, or to hepatic metabolic dysfunction with the intervention of certain compounds. However, these changes may not be toxicologically significant because they were not confirmed by biochemical results (ALT, AST) (Table 4).

Table 4.

Blood biochemical indexes at 28th days after implantation.

Group	Control	Control	Group 1	Group 1	Group 2	Group 2	Group 3	Group 3
Group	♂	♀	♂	♀	♂	♀	♂	♀
ALT (U/L)	55.08 ± 7.33	65.27 ± 8.51	59.4 ± 6.94	52.24 ± 10.3	60.3 ± 8.39	45.32 ± 8.02	66.18 ± 26.44	45.83 ± 11.71
AST (U/L)	153.54 ± 33.33	299.13 ± 108.64	164.32 ± 74.38	185.68 ± 86.85	269.68 ± 70.78	152.74 ± 33.18	211.4 ± 128.13	177.55 ± 55.75
ALP (U/L)	378.16 ± 93.26	383.03 ± 87.98	249.08 ± 87.78	214.14 ± 42.31	347.32 ± 53.19	225.08 ± 53.51	353.8 ± 132.64	142.75 ± 22.25^a
TP (g/L)	1.32 ± 1.11	3.87 ± 2.11	1.8 ± 1.1	0.98 ± 1.77	2.22 ± 1.85	1.02 ± 0.78	1.95 ± 0.81	1.35 ± 0.29
ALB (g/L)	32.92 ± 0.82	34.27 ± 11.29	35.06 ± 0.68	31.14 ± 3.53	30.82 ± 2.52	36.3 ± 3.66	32.65 ± 1.99	34.25 ± 0.79
UA (umol/L)	316.72 ± 69.02	445.4 ± 190.62	206.5 ± 49.74	181.38 ± 55.52	297.06 ± 55.71	152.4 ± 21.49	474 ± 253.51	230.6 ± 51.36
UREA (mmol/L)	9.26 ± 1.4	12.22 ± 3.17	9.36 ± 0.79	10.55 ± 2.24	12.09 ± 1.99	23.37 ± 5.94*	8.77 ± 1.18	13.72 ± 1.77

^ameans the difference is statistically significant, p < 0.05.

Whole blood count

The results of the hematological tests are summarized in Table 5. All hematological parameters tested including total blood cells, hemoglobin, erythrocytes, total leukocytes, neutrophils, monocytes, lymphocytes, filling cell volume and platelet count were within the normal range compared to the controls. No significant toxicological differences were found between AMPCs in the experimental and control animals (p > 0.05). For the measured hematological parameters, there were no significant differences between the control and the experimental groups.

Table 5.

Whole blood count at 28th days after implantation.

Group	Control	Control	Group 1	Group 1	Group 2	Group 2	Group 3	Group 3
Group	♂	♀	♂	♀	♂	♀	♂	♀
WBC^a (10^9/L)	6.52 ± 1	6.35 ± 3.79	9.15 ± 1.59^a	6.82 ± 1.67	8.45 ± 2.93	6.38 ± 2.07	3.72 ± 2.26	5.58 ± 1.85
HGB (g/L)	175.2 ± 10.08	156.5 ± 46.41	161.25 ± 6.8	176.4 ± 14.59	181.5 ± 11.7	147 ± 57.79	171.8 ± 15.77	175.6 ± 17.7
Rbc^a (10^12/L)	8.02 ± 0.56	6.63 ± 2.04	7.195 ± 0.17^a	7.89 ± 0.57	8.32 ± 0.62	6.44 ± 2.2	7.7 ± 0.75	7.74 ± 0.7
HCT (%)	45.26 ± 2.52	41.7 ± 10.4	41.35 ± 1.4	45.4 ± 3.49	47.95 ± 4.47	37.83 ± 14.12	45.28 ± 4.28	45 ± 3.96
MCV (fL)	56.58 ± 1.93	63.95 ± 5.52	57.55 ± 1	57.6 ± 1.86	57.73 ± 3.87	58.2 ± 2.6	58.86 ± 2.36	58.26 ± 0.88
MCH (pg)	21.84 ± 0.63	23.63 ± 0.84	22.35 ± 0.61	22.28 ± 0.88	21.8 ± 0.78	22.43 ± 1.65	22.26 ± 0.68	22.62 ± 0.34^a
Mchc (g/L)	386.6 ± 8.23	371.75 ± 21.11	389.25 ± 4.79	388 ± 3.61	379 ± 13.64	386 ± 12.38	379.2 ± 5.07	389.4 ± 6.31
RDW-CV (%)	12.16 ± 0.32	14.75 ± 1.47	12.15 ± 0.17	13.56 ± 0.3	14.25 ± 0.31	13.15 ± 0.33^a	13.98 ± 0.45	12.82 ± 0.54
RDW-SD (fL)	25.18 ± 0.38	35.2 ± 7.16	25.7 ± 0.92	28.32 ±1.17	29.58 ± 2.63	28.03 ± 1.66^a	29.98 ± 2.12	27.62 ± 0.74^a
Plt^a (10^9/L)	537.2 ± 184.5	549.25 ± 108.48	751.25 ± 116.4	684 ± 242.8	441 ± 194.29	622.75 ± 310.74	767.8 ± 133.81	565.2 ± 306.13
Mpv (fL)	6.42 ± 0.67	6.65 ± 0.4	6.2 ± 0.18	6.58 ± 0.34	7.98 ± 1.55	6.85 ± 0.34	6.8 ± 0.22	6.82 ± 0.59
Pdw	15.18 ± 0.54	15.85 ± 0.57	14.95 ± 0.26	15.18 ± 0.32	15.85 ± 0.17	15.25 ± 0.54	15.26 ± 0.11	15.5 ± 0.68
Pct (%)	0.34 ± 0.12	0.36 ± 0.07	0.465 ± 0.07	0.45 ± 0.16	0.33 ± 0.11	0.42 ± 0.22	0.52 ± 0.1	0.37 ± 0.18
P-LCR Q-(%)	10.5 ± 3.95	12.5 ± 2.03	9.05 ± 1.26	11.04 ± 2.14	21.45 ± 12.72	12.9 ± 2.76	12.36 ± 1.42	13.04 ± 4.17

^aMeans the difference is statistically significant, p < 0.05.

Necropsy

Another perspective on biocompatibility assessment is to focus on the systemic safety of AMPCs during their degradation in vivo. Through systemic safety assessment, an attempt was made to determine the potential effects of various types of AMPCs on vital organs in vivo. Four weeks after subcutaneous dorsal implantation, all rats survived in good health and no adverse effects were observed.

A series of organs in the rats were evaluated as a whole by gross observation and important organs involved in immunity and metabolism were assessed by histological staining, including the liver, spleen, kidney and thymus. As shown in Figure 5(a), none of the tissue necrosis or inflammatory cell infiltration was observed in these organs during the degradation of AMPCs, either by gross observation or histological analysis. The histological structure was normal, indicating that the release of components of AMPCs into the body did not give rise to significant organ lesions in vivo.

On the other hand, AMPCs altered serum indicators of renal function. For example, Group 2 significantly reduced uric acid (UA) levels in male rats (Figure 5(b)) and Group 2 increased urea levels in female rats (Figure 5(c)). Thus, Group 2 may have caused some degree of impairment of renal function in both males and females in vivo.

Conclusion

In the field of biomaterials, chitosan, as a natural polymer, has attracted much attention because of its excellent physicochemical properties. Carboxymethyl chitosan-based on chitin has many excellent physical and biological properties such as high moisturizing ability and excellent biocompatibility. Meanwhile, many factors, such as the source of chitosan, production area, production batch, etc., can affect the quality of carboxymethyl chitosan. By comparison, the hemostatic material of group 1 performed better, mainly reflected in the higher water absorption rate, which was conducive to the rapid formation of blood clots. In a further study, it was confirmed by BCI, in vitro blood coagulation test, etc. That AMPCs showed an excellent hemostatic effect in multiple studies in vitro due to their good coagulation ability. The experimental according to the expected purpose of use, the way of use, the maximum clinical dose and the previous research data, in this article, we designed a scientific and reasonable experimental protocol and comprehensively evaluates the safety of AMPCs under the condition of animal tolerance. The results confirmed that AMPCs showed excellent biosafety for whole blood and major organs during the degradation process in vivo.

In brief, carboxymethyl chitosan of Group 1 is most suitable for the preparation of hemostatic materials. On these basics, we have developed a hemostatic with strong function, high safety and convenient preparation and use, which is expected to be applied to rapid hemostasis in emergencies.

Footnotes

Acknowledgments

The authors acknowledge Dr Chengyong He for his advice on experimental design and the timely help given in analyzing a large number of samples.

Declaration of conflicting interests

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

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Fujian Marine Economy Development Subsidy Fund Project (grant numbers FJHJF-L-2022–5); the Science and Technology Project of Fujian Province (grant number 2022Y4009); the Science and Technology Project of Fujian Province (grant number 2021Y4007); and the Fifth Round of Fujian Health Education Joint Key Project (grant number 2019-WJ-36).

ORCID iDs

Lihao Wu

Gong Chen

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