Introduction
The largest organ of the human body is the skin. It is an interference between the organism and the environment, which protects the organism against various threats such as viruses, bacteria, heat, cold, or damage. Being a versatile barrier against the outer environment comes with risks of damage such as abrasions, scratches, or cuts (Xu et al.
2015). These types of wounds might carry a risk of bleeding, which our organism should be able to control by activating platelets and coagulation factors through the whole coagulation cascade process. This process leads to blood clot formation, preventing further blood loss. In some situations where an intense haemorrhage is present, the coagulation cascade might not work fast enough to be able to stop the bleeding. Only in the USA, roughly 60,000 people die every year due to blood loss (Cannon
2018).Significant blood loss is the primary cause of death in combat and the second most common cause of mortality in ordinary trauma centres. In these types of situations, something which might provide help is hemostatics (Peng
2020).
The hemostatics are substances that promote blood clot formation, secure the wound against bacteria, and possibly help us with the process of healing. Hemostatic agents can be divided into several categories according to the mechanism of action by which they accelerate the stopping of bleeding: factor concentrators, procoagulants and mucoadhesives (Kinloch
1980; Wolberg et al.
2012; Behrens et al.
2014; Kaufman et al.
2015; Hong et al.
2019; Jaifu et al.
2019; Li et al.
2019; Peng
2020). The ideal hemostat should have properties such as quick and effective bleeding control, excellent biocompatibility without harmful effects on healing, ease to use, low storage requirements, long shelf-life and ability to remain in place for extended periods of time if needed (Neuffer et al.
2004; Pusateri et al.
2004; Peng
2010) A wide variety of materials are currently used in hemostatic agents, e.g., bovine collagen, oxidized cellulose, carboxymethyl cellulose or chitosan, native chitosan, kaolin, fibrin, thrombin, zeolite or cyanoacrylate(Hughes et al.
2002; Kozen et al.
2008; Martina et al.
2009; Karr et al.
2011; Cho et al.
2013; Moench et al.
2014; Renati et al.
2017).
None of the used materials provides all of the desired properties. The most commonly used hemostats are based on collagen and cellulose derivates. Collagen is one of the most widely used biomaterials. It shows excellent biocompatibility and safety thanks to its biological characteristics—biodegradability and almost no immune system responses. This makes it one of the most used materials in medical applications. The most common application forms are drug delivery systems, fundamental matrices for cell culture systems and tissue engineering (skin replacement, bone substitutes, artificial blood vessels or valves) (Lee et al.
2001; Olsen
2003; Yang et al.
2004; Liu et al.
2008; Wang et al.
2008). From the chemical point of view, collagen is a structural protein with high molecule mass involved in animal tissue. The highest collagen content is located in the skin, tendons, and other connective tissues. It has a fibrous structure made from amino acid chains that form triple helices, and these helices form fibrils that bind together to form collagen fibres (Shoulders and Raines
2009). Collagen-based hemostats support hemostasis through blood cell adsorption, activation and promotion of platelet adhesion and platelets aggregation. These aggregated active platelets further release procoagulant molecules (ADP, Ca
2+). They also allow colocalization and activation of coagulation factors on their membrane to augment thrombin production and fibrin formation to accelerate clotting. This is a direct result of contact between collagen and blood. The collagen-based hemostat does not promote biologically active coagulation or does not work in combination with thrombin (Manon-Jensen et al.
2016; Napavichayanun and Aramwit
2017; Rumbaut and Thiagarajan
2010). The risk of using collagen (especially bovine collagen) in the potential health risk regarding bovine spongiform encephalopathy (BSE), other possible diseases and religious reasons (mainly Islam and Judaism) (Nalinanon et al.
2008; Furtado et al.
2022).
Cellulose is an ideal candidate for biomaterial usage thanks to its adjustable mechanical, chemical, and physical properties. The most significant advantage is the easy availability in nature, from which a relatively low product price comes. Another advantage is cellulose properties, which meet three fundamental requirements for biomaterials – biocompatibility, bioactivity and biomechanics (Olsson and Westm
2013; Hickey and Pelling
2019). Carboxymethyl cellulose (CMC) is an anionic, water-soluble cellulose derivative. The solubility of CMC depends on the degree of substitution and the uniformity of the substitution distribution. The most used are sodium or calcium salt of CMC. Thanks to its highly hygroscopic nature, CMC hydrates rapidly, which is very convenient in applications such as hemostatic agents (Ohta et al.
2015; Ergun et al.
2016). CMC is widely used in the food industry for many applications, such as thickeners or stabilizers. In the healthcare industry, it is used in several drug deliveries (time-release, nasal release, gastrointestinal delivery, mucosal delivery), lubricant in eye drops, as a scaffold in tissue engineering, wound healing, and hemostatic agents (Ugwoke et al.
2000; Schwarz
2003; Chen et al.
2010; Aravamudhan et al.
2014). CMC-based hemostatics work on the principle of dissolving in the blood, leading to increased blood viscosity and accelerating the development of clots. Some researchers suggest that CMC acts as a bridge for fibrin polymerization, leading to the formation of thick fibrin formation; on the other hand, other researchers indicate that dissolved CMC activated platelet coagulation.
Using a mixture of these commonly used materials could provide enhanced properties of prepared hemostatics thanks to the fact that there will be two mechanisms of action on fastening the hemostasis. Both help in the improvement of blood clotting in the bleeding wound (Wang et al.
2007; AOSHIMA et al.
2012). Combining collagen and CMC might result in hemostatic agents with superior properties, decreasing the time needed to stop bleeding and thus saving many lives, as we preliminary proved in our previous paper by combining bovine collagen and CMC (Paprskářová et al.
2021). We also proved in one of our previous papers(Babrnáková et al.
2019) that combining collagen with polysaccharides to create wound dressings enhances biocompatibility. The hypothesis of our research was to combine equine or porcine collagen, as a subsidiary type of commonly used bovine collagen, and fibrous carboxymethyl cellulose to form a highly functional freeze-dried hemostatic wound dressing which will have better hemostatic properties than commonly used hemostat on the market—Tachosil. Tachosil is an equine collagen sponge with coated fibrinogen and thrombin on the surface, being cost-ineffective. The usage is mainly to stop minor local internal bleeding (Di Carlo et al.
2011). We have found out that adding fibrous CMC into the equine or porcine collagen creates a synergy effect by enhancing the final hemostatic properties of freeze-dried sponge wound dressings.
Experimental
Materials and methods
The fibrous sodium salt of carboxymethyl cellulose (Holzbecher, Zlíč, Czech Republic) with a degree of substitution DS = 0.602, pH = 4.958 and Mn of 300 kDa (measured by GPC/SEC). Equine and bovine collagen were obtained from Collado s.r.o., Brno, Czech Republic. Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin/EDTA, XTT ((2,3-bis-(2- methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide) were purchased from Sigma Aldrich (St. Louis, MO, USA). Porcine heparinized blood was obtained from Veterinary Research Institute (Brno, Czech Republic).
Fabrication of wound dressings
Hemostatic wound dressings were prepared either from 1% equine or porcine collagen, 1% CMC or by mixing 1% water-solutions of collagen and CMC. Final solutions were poured into moulds and placed into a lyophilizer (Martin Christ, Epsilon 2 10D LSCPlus, Osterode am Hartz, Germany) and freeze-dried (− 35° C, 15 Pa, 48 h). The grammage of prepared samples was 50 g·m
−2. All prepared samples are in Table
1.
Table 1
The description of samples prepared in our experiments
Equine collagen | 1% Equine collagen freeze-dried sponge |
Porcine collagen | 1% Porcine collagen freeze-dried sponge |
CMC | 1% Fibrous CMC freeze-dried sponge |
Eq+CMC | 1% (Equine collagen + fibrous CMC) freeze-dried sponge |
Por+CMC | 1% (Porcine collagen + fibrous CMC freeze-dried sponge) |
Infrared spectroscopy
Fourier-transformed infrared spectroscopy (FTIR) with attenuated total reflectance (ATR-FTIR, Vertex 70/70v, Bruker, Billerica, MA, USA) was performed to characterize the chemical composition of porous foams. Presented ATR-FTIR spectra were taken from averaging 32 scans with a spectral resolution of 4 cm−1. The displayed spectra in the wavenumber range 4000–500 cm−1 were normalized using min–max normalization (OPUS software, Bruker, Billerica, MA, USA). The ATR-FTIR spectra were measured in an evacuated condition on a diamond ATR crystal.
Morphological analysis and porosity
Scanning electron microscopy (SEM) was performed for all foam scaffolds to evaluate their morphology. The surfaces of the intact collagen and CMC scaffolds and the cross sections of these scaffolds were all coated with 15 nm thick gold using Coater Leica EM ACE600 (Wetzlar, Germany). SEM was carried out using Tescan MIRA3-XMU (Brno, Czech Republic) to observe the surface characteristics, pore sizes, and pore distribution of the porous materials. Images were taken in a secondary electron emission mode, scan mode was RESOLUTION, beam density was 10, and high voltage was 5 kV.
The porosity and pore sizes from the images obtained through the SEM analysis were evaluated by ImageJ 2 software.
Absorptive capacity
The samples were cut into pieces of 5 × 5 cm and weighted precisely to the nearest 0.01 g (w
dry). The weight of the samples was multiplied by a coefficient of 40. That gave us a weight of a solution which was then slowly added to the sample in a glass container. Physiological saline solution tempered to 37 °C was used as a solution. The container with a wet sample was placed into an incubator at 37 °C for 30 min. Then the sample was mounted with tweezers by a corner and let drip for 30 s. After that, the sample was weighted to the nearest 0.01 g (wet). The absorptive capacity was then calculated according to Eq. (
1):
$$Absorptive\,capacity= \frac{{w}_{wet}-{w}_{dry}}{{w}_{dry}}$$
(1)
Blood sorption
Enhancing knowledge about how the wound dressings behave in contact with blood and blood absorption was tested. The method was adopted from Ong et al. (
2008) and modified. The absorption efficiency of wound dressings was determined in whole heparinized blood. The dressings were weighted (w
ini) and placed inside a plastic cup, and 1.5mL of blood was added. The cups were closed and put into an incubator at 37 °C for 2 h. After that time, the dressings were dried off any excess blood and weighed (w
wet). The capacity was calculated according to Eq. (
2):
$$Blood\,sorption\,capacity= \frac{{w}_{wet}-{w}_{ini}}{{w}_{ini}}$$
(2)
Clotting time
Clotting time is the time needed for the hemostatic to clot the blood. Testing was done according to Barba et al.
2018. Fresh whole blood was collected from animals, and heparin (10 mg·mL
−1) was immediately added to prevent spontaneous clotting. 100 mg or 1 cm
3 of the sample was put in an Eppendorf tube, and 1 mL of blood was added. After the addition of blood, the tubes were rotated several times and then every other 30 s. The time was measured until the clot was formed.
Cytotoxicity assay
NIH-3T3 (fibroblast cell line that was isolated from a mouse NIH/Swiss embryo cells, 3-day transfer, inoculum 3×105 cells) were cultured in DMEM (Dulbecco’s Modified Eagle Medium) medium supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were harvested by trypsinization in a 0.25% solution of trypsin/EDTA in PBS at 80% confluence. The extracted test was used to evaluate the cytotoxic effect of samples. Each material was incubated with complete DMEM culture media at a concentration of 33 mg·mL−1 for 24 h. NIH-3T3 cells were seeded at the concentration of 104 cells/well into a 96-well plate prior to initiating the assay. After 24 h of cell growth, the cell culture media was removed and replaced with the extract media. Cells were then incubated at 37° C and 5% CO2 for 24 h prior to evaluation with XTT assay. XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide) was used according to the manufacturer’s protocols. Briefly, the extract medium was removed from the well plate, and cells were gently washed in PBS buffer solution. Then the 100 µl of fresh DMEM medium and 50 µl XTT (XTT, 1 mg·mL−1 in PBS, pH 7.4) labelling mixture was added per well. Absorbance was measured after 4 h of incubation at 37 °C with a plate reader at 450 nm. The cytotoxic effect of materials was evaluated as the percentage of viable cells. As a positive control the empty plastic well plate was used.
In vivo partial nephrectomy
The whole method was provided according to the published procedure (Paprskářová et al.
2021). Sixty male Wistar laboratory rats (AnLab, Czech Republic) weighing 265 ± 62 g were allocated into six groups of ten animals at random. I.m. administration of a blend of tiletamine and zolazepam was used to anaesthetize these animals (Zoletil 100, Virbac 5 1., France). It was given a dose of 65 mg·kg
−1. The peritoneum was then surgically opened, and a partial nephrectomy of the caudal pole of the left kidney was performed. Immediately after this action, 1 cm
2 pieces of hemostatics were applied to the incisions until achieving hemostasis. The time needed for hemostasis was measured. The hemostatics were left at the wound after hemostasis was achieved. The wounded kidney was returned to the peritoneal cavity. The peritoneum and skin were sutured.
Ethic
All in vivo testing was done at Veterinary and Pharmaceutical University (currently Faculty of Pharmacy at Masaryk University, Brno) in accordance with the guidelines of SUKL (State Institute for Drug Control) and the Ministry of Health of the Czech Republic. The Scientific Committee for the Protection of Animals at the university approved the in vivo testing. After approval, a partial nephrectomy model in rats was carried out.
Statistical analysis
Origin lab software was used to carry out statistical analysis of obtained results. All of the data was evaluated using Tukey’s test, using five samples of each type (ten samples for in vivo testing), which uses pairwise post-hocptesting. The level of significance was set at 0.001, 0.01 and 0.05. Data from cytotoxicity testing were analyzed using Student’s t-test with a confidence level of 95%. The data was presented as mean ± standard error (n = 3).
Discussion
Hemostatic wound dressings are a versatile aid in stopping bleeding and achieving hemostasis of the cause of the bleeding. The wound dressing might come in various forms and application designs. The collagen-based and carboxymethyl cellulose-based hemostatics are nowadays commonly used in medicine to stop bleeding. The idea of our research was to combine equine or porcine collagen, as a subsidiary type of collagen to mostly used bovine collagen, and fibrous carboxymethyl cellulose to form a highly functional freeze-dried hemostatic wound dressing which will have better hemostatic properties than commonly used hemostatic on the market (Tachosil).
Overall characterization experiments were conducted to support the hypothesis that using mixtures of collagens with fibrous CMC creates hemostatic wound dressings with improved properties, which could lead to faster hemostasis and, thus, lower blood loss. SEM morphological analysis showed that the freeze-dried sponges have a highly porous structure with interconnected pores of average size 42.03-161.71 μm. The samples consisting of equine collagen and CMC, which showed the best in vivo properties, have a pore size of 95.76±88.67 mm, which might imply that this is the right pore size for interaction with blood. The size of pores supports the finding of Shou et al. (
2020), who made chitosan/DOPA-based hydrogel with a pores size range of 100 to 150 μm. The porous structure is an important parameter that allows liquid/blood to be absorbed into the structure and interact with as much of the wound dressing material as possible. The high inner surface allows the collagen and CMC to interact with the coagulation cascade faster and thus increase the cascade speed of action. The absorption capacity is an important characteristic of primary wound dressings, as it implies the ability to concentrate clotting factors and thus improve the hemostasis speed. (Huang et al.
2019). For hemostatic wound dressing, it is important to find the perfect ratio of absorption. When absorption is too high, and swelling of the wound dressing occurs, it can potentially create a localized pressure that is undesirable (Ranjbar et al.
2021). On the other hand, when the absorption is low, the concentration of clotting factor might not occur or can happen at a slower pace, resulting in uncontrolled bleeding. The best overall abortion capacity was measured for the fibrous CMC, which in contact with saline solution, creates a transparent rigid sort of hydrogel that locks a large amount of liquid into its structure but maintains excellent mechanical properties. Equine and porcine collagen did not perform so well when tested alone, but the mixture of them with fibrous CMC strongly enhanced their absorption properties and made them hold a large amount of saline solution, which might help the overall hemostatic activity, as it will be more likely to concentrate the clotting factors. After the absorption capacity according to Czech CSN EN 13726-1, the blood sorption was also tested to assess how the wound dressings behave in interaction with blood. The fibrous CMC also showed identical sorption as when it interacted with the saline solution. The CMC was able to hold a large amount of blood in its structure and lock it inside of it and did not lose shape or mechanical properties. This also allows wound dressings to absorb exudate from the wound and keep the wound clean. The equine collagen performs worse in blood than in saline solution, which might be caused by the fact that the whole blood started to clot on the surface of the collagen wound dressing, and thus it prevented the penetration of the aqueous part of the blood deeper into the sample. On the other hand, porcine collagen has a slightly elevated value, which might indicate that the interaction between porcine collagen and blood is not as strong as in the case of equine collagen. This might be due to a protein interaction between the wound dressing and blood. The addition of CMC to collagen has again improved the effect on the sorption. These results lead to the conclusion that CMC enhances the sorption properties of collagen and might be able to promote hemostasis in synergy with collagens, as with the in vitro hemostatic testing, a clotting time test was performed. The results showed superior clotting properties of fibrous CMC, but CMC does not necessarily create a clot due to its hemostatic properties but because of the ability to absorb a large amount of liquid and create a sort of hydrogel that acts like a clot. This creates a great synergy in collagen mixtures when there are two mechanisms of action – creating a clot made from CMC and then collagen interacting with blood proteins and supporting platelet adhesion and complete activation of the coagulation cascade. This property could be beneficial for hemostatics function in our mixture of CMC with collagen, as it could quickly seal the wound, and then the collagen component could engage and accelerate the body’s response to bleeding and significantly reduce the time needed for hemostasis. Porcine collagen showed poor clotting time, and this could be due to the fact that the porcine collagen could have contained pig fat residues, whereas the horse is a lean animal, and the obtained collagen might have a higher purity of proteins. If we compare the results of the clotting time with those of Schroeder et al. (
2021), our results show the lower time needed for hemostasis.
Collagens and CMC are known for their biocompatibility and low cytotoxicity. Our results from cytotoxicity testing of the extracts from the wound dressing showed higher toxicity of the extracts compared to the plastic control. This result is quite surprising and might be caused by the low pH of collagen extract. The value of the pH level of extracts could play a vital role in the cytotoxicity testing of wound dressings, as the cell viability is affected by pH (Kruse et al.
2017). Porcine collagen has higher pH than equine, which correspond with obtained results. The low level of pH could also affect the in vivo testing because the lower pH can enhance blood clot formation (Gissel et al.
2016). The lower pH in the wound caused by the wound dressings might even help with later healing of the wound, as a low pH level below four is believed to improve healing properties. (Milne and Connolly
2014; Percival et al.
2014). This corresponds with our in vivo testing, where the samples with lower pH level act as the better hemostat. For the final assessment of created hemostatic wound dressings, the in vivo partial nephrectomy was performed in rats to specify the hemostatic properties of the wound dressings in the bleeding wound. Testing validated our assumptions from the in vitro, where we stated that porcine collagen has worse properties than equine collagen. CMC works as a great hemostatic material and, in a mixture with equine collagen, even better. Collagens alone did not report as great properties as Tachosil, but in a mixture with CMC, it creates a highly functional hemostatic. The mixture of equine collagen and fibrous CMC collagen has hemostatic properties similar to Tachosil, which makes it a great protentional candidate for further research and testing. Using wound dressings based on equine collagen and CMC might provide the same hemostatic properties as Tachosil but at a lower price, thanks to the absence of expensive proteins such as thrombin and fibrinogen, which could make it more affordable and available. The interesting fact is that the consumption of the mixture of equine collagen and CMC was lower than the average of the individual substances. It might be caused by some synergy and highly functional interactions between them, where the CMC creates the hydrogel structure, seals the wound and allows the collagen to actively interact with clotting factors and blood proteins and enhances the hemostasis. Hemostatic tests in rats were also conducted by Goncharuk et al. (
2021) when they tested polyvinyl formal sponges on rats’ livers, they didn’t measure the time needed for achieving hemostasis, but they measured blood loss. (Zheng et al.
2023) tested W-HAP-PVA aerogel (composed of ultralong hydroxyapatite nanowires and poly(vinyl alcohol) on rats’ liver, and in rabbit femoral artery injury and when compared, our wound dressings can achieve faster hemostasis time. All the main results show great potential in the addition of fibrous CMC into collagen-based hemostatic wound dressings. As we look at the obtained results of absorption capacity and blood sorption, the addition of fibrous CMC improved the absorption properties of used collagen and led to a more highly functional wound dressing. The hemostatic tests consisting of clotting time and partial nephrectomy in rats support our hypothesis that the fibrous CMC does enhance the properties of collagen. When we look at the results, the enhancement of hemostasis is significant, and the mixtures of equine collagen and porcine collagen with the addition of fibrous CMC have hemostatic properties comparable to the Tachosil. The development of effective hemostatic materials is a complex and challenging task. Collagen and CMC are two natural polymers that have shown great promise for hemostatic applications. The use of collagen-CMC composites has been shown to be an effective strategy for enhancing hemostasis. However, there are still several challenges that need to be addressed in order to develop these materials for clinical use. These include optimizing the composition and structure of the composite materials, improving their mechanical properties, and ensuring their safety and efficacy in humans.
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