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Uncontrolled bleeding is one of the leading causes of death. Achieving rapid hemostasis ensures the survival of the subject as a first aid during combat, traffic accidents, and death reduction operations. Nanoporous fiber-reinforced composite scaffold (NFRCS) derived from a simple hemostatic film-forming composition (HFFC) as a continuous phase can trigger and enhance hemostasis. The development of the NFRCS is based on the design of the dragonfly’s wing. The dragonfly wing structure consists of transverse and longitudinal wings, and the wing membranes are connected to each other to maintain the integrity of the microstructure. HFFC uniformly coats the surface of the fiber with a film of nanometer thickness and connects the randomly distributed cotton thickness (Ct) (dispersed phase) to form a nanoporous structure. The combination of continuous and dispersed phases reduces the cost of the product by ten times compared to commercially available products. Modified NFRCS (tampons or wristbands) can be used in a variety of biomedical applications. In vivo studies have concluded that the developed Cp NFRCS triggers and enhances the coagulation process at the site of application. NFRCS can modulate the microenvironment and act at the cellular level due to its nanoporous structure resulting in better wound healing in the excision wound model.
Uncontrolled bleeding during combat, intraoperative and emergency situations can pose a serious threat to the life of the wounded1. These conditions further lead to an overall increase in peripheral vascular resistance, leading to hemorrhagic shock. Appropriate measures to control bleeding during and after surgery are considered potentially life-threatening2,3. Damage to large vessels leads to massive blood loss, resulting in a mortality rate of ≤ 50% in combat and 31% during surgery1. Massive blood loss leads to a decrease in body volume, which reduces cardiac output. An increase in total peripheral vascular resistance and a progressive impairment of microcirculation lead to hypoxia in the life-support organs. Hemorrhagic shock may occur if the condition continues without effective intervention1,4,5. Other complications include the progression of hypothermia and metabolic acidosis, as well as a coagulation disorder that impedes the coagulation process. Severe hemorrhagic shock is associated with a higher risk of death6,7,8. In grade III (progressive) shock, blood transfusion is essential for patient survival during intraoperative and postoperative morbidity and mortality. To overcome all of the above life-threatening situations, we have developed a nanoporous fiber-reinforced composite scaffold (NFRCS) that utilizes a minimal polymer concentration (0.5%) using a combination of water-soluble hemostatic polymers.
With the use of fiber reinforcement, cost-effective products can be developed. The randomly arranged fibers resemble the structure of a dragonfly’s wing, balanced by the horizontal and vertical stripes on the wings. The transverse and longitudinal veins of the wing communicate with the wing membrane (Fig. 1). NFRCS consists of reinforced Ct as a scaffold system with better physical and mechanical strength (Figure 1). Due to the affordability and craftsmanship, surgeons prefer to use cotton thread gauges (Ct) during operations and dressings. Hence, considering its multiple benefits, including > 90% crystalline cellulose (imparts in the enhancement of hemostatic activity), Ct was used as a skeletal system of NFRCS9,10. Hence, considering its multiple benefits, including > 90% crystalline cellulose (imparts in the enhancement of hemostatic activity), Ct was used as a skeletal system of NFRCS9,10. Следовательно, учитывая его многочисленные преимущества, в том числе > 90% кристаллической целлюлозы (участвует в повышении гемостатической активности), Ct использовали в качестве скелетной системы NFRCS9,10. Therefore, given its many benefits, including >90% crystalline cellulose (involved in increased hemostatic activity), Ct was used as the NFRCS skeletal system9,10.因此，考虑到它的多重益处，包括> 90% 的结晶纤维素（有助于增强止血活性），Ct 被用作NFRCS9,10 的骨架系统。因此，考虑到它的多重益处，包括> 90% Therefore, given its many benefits, including over 90% crystalline cellulose (helps enhance hemostatic activity), Ct was used as a scaffold for NFRCS9,10. Ct was superficially coated (nano-thick film formation was observed) and interconnected with a hemostatic film-forming composition (HFFC). HFFC acts like a matrigel, holding randomly placed Ct together. The developed design transmits stress within the dispersed phase (reinforcing fibers). It is difficult to obtain nanoporous structures with good mechanical strength using minimal polymer concentrations. In addition, it is not easy to customize different molds for different biomedical applications.
The figure shows a diagram of the NFRCS design based on the dragonfly wing structure (A). This image shows a comparative analogy of the wing structure of a dragonfly (the intersecting and longitudinal veins of the wing are interconnected) and a cross-sectional photomicrograph of Cp NFRCS (B). Schematic representation of NFRCS.
NFRCs were developed using HFFC as a continuous phase to address the above limitations. HFFC is composed of various film-forming hemostatic polymers including chitosan (as the main hemostatic polymer) with methylcellulose (MC), hydroxypropyl methylcellulose (HPMC 50 cp) and polyvinyl alcohol (PVA)) (125 kDa) as a support polymer that promotes thrombus formation. formation. The addition of polyvinylpyrrolidine K30 (PVP K30) improved the moisture absorption capacity of the NFRCS. Polyethylene glycol 400 (PEG 400) was added to improve polymer crosslinking in bonded polymer blends. Three different HFFC hemostatic compositions (Cm HFFC, Ch HFFC and Cp HFFC), namely chitosan with MC (Cm), chitosan with HPMC (Ch), and chitosan with PVA (Cp), were applied to Ct. Various in vitro and in vivo characterization studies have confirmed the hemostatic and wound healing activity of NFRCS. Composite materials offered by NFRCS can be used to customize various forms of scaffolding to meet specific needs.
In addition, NFRCS can be modified as a bandage or roll to cover the entire injury area of the lower extremities and other parts of the body. Specifically for combat limb injuries, the designed NFRCS design can be changed to a half arm or full leg (Supplementary Figure S11). The NFRCS can be made into a wristband with tissue glue, which can be used to stop bleeding from severe suicidal wrist injuries. Our main goal is to develop an NFRCS with as little polymer as possible that can be delivered to a large population (below the poverty line) and that can be placed in a first aid kit. Simple, efficient, and economical in design, NFRCS benefits local communities and can have a global impact.
Chitosan (molecular weight 80 kDa) and amaranth were purchased from Merck, India. Hydroxypropyl methylcellulose 50 Cp, polyethylene glycol 400 and methylcellulose were purchased from Loba Chemie Pvt. LLC, Mumbai. Polyvinyl alcohol (molecular weight 125 kDa) (87-90% hydrolysed) was purchased from National Chemicals, Gujarat. Polyvinylpyrrolidine K30 was purchased from Molychem, Mumbai, sterile swabs were purchased from Ramaraju Surgery Cotton Mills Ltd., Tamil Nadu, with Milli Q water (Direct-Q3 water purification system, Merck, India) as the carrier.
NFRCS was developed using a lyophilization method11,12. All HFFC compositions (Table 1) were prepared using a mechanical stirrer. Prepare a 0.5% solution of chitosan using 1% acetic acid in water by continuous stirring at 800 rpm on a mechanical stirrer. The exact weight of the loaded polymer indicated in Table 1 was added to the chitosan solution and stirred until a clear polymer solution was obtained. PVP K30 and PEG 400 were added to the resulting mixture in the amounts indicated in Table 1, and stirring was continued until a clear viscous polymer solution was obtained. The resulting bath of polymer solution was sonicated for 60 minutes to remove trapped air bubbles from the polymer mixture. As shown in Supplementary Figure S1(b), Ct was evenly distributed in each well of a 6-well plate (mold) supplemented with 5 ml of HFFC.
The six-well plate was sonicated for 60 min to achieve uniform wetting and distribution of HFFC in the Ct network. Then freeze the six-well plate at -20°C for 8-12 hours. Freeze plates were lyophilized for 48 hours to obtain various formulations of NFRCS. The same procedure is used to produce different shapes and structures, such as tampons or cylindrical tampons, or any other shape for different applications.
Accurately weighed chitosan (80 kDa) (3%) is dissolved in 1% acetic acid using a magnetic stirrer. To the resulting solution of chitosan was added 1% PEG 400 and stirred for 30 minutes. Pour the resulting solution into a square or rectangular container and freeze at -80°C for 12 hours. Frozen samples were lyophilized for 48 hours to obtain porous Cs13.
The developed NFRCS was subjected to experiments using Fourier transform infrared spectroscopy (FTIR) (Shimadzu 8400 s FTIR, Tokyo, Japan) to confirm the chemical compatibility of chitosan with other polymers14,15. The FTIR spectra (width of the spectral range from 400 to 4000 cm-1) of all tested samples were obtained by performing 32 scans.
The blood absorption rate (BAR) for all formulations was evaluated using the method described by Chen et al. 16 with slight modifications. The developed NFRKs of all compositions were dried in a vacuum oven at 105°C overnight to remove residual solvent. 30 mg NFRCS (initial sample weight – W0) and 30 mg Ct (positive control) were placed in separate dishes containing a premix of 3.8% sodium citrate. At predetermined time intervals, i.e. 5, 10, 20, 30, 40 and 60 seconds, the NFRCS were removed and their surfaces cleaned of unabsorbed blood by placing the samples on Ct for 30 seconds. The final weight of blood absorbed by NFRCS 16 was considered (W1) at each time point. Calculate the BAR percentage using the following formula:
Blood clotting time (BCT) was determined as reported by Wang et al. 17 . The time required for whole blood (rat blood premixed with 3.8% sodium citrate) to clot in the presence of NFRCS was calculated as the BCT of the test sample. The various NFRCS components (30 mg) were placed in 10 ml screw cap vials and incubated at 37°C. Blood (0.5 ml) was added to the vial and 0.3 ml of 0.2 M CaCl2 was added to activate blood coagulation. Finally, invert the vial every 15 seconds (up to 180°) until a firm clot forms. The BCT of the sample is estimated by the number of flips vails17,18. Based on BCT, two optimal compositions from NFRCS Cm, Ch and Cp were selected for further characterization studies.
The BCT of Ch NFRCS and Cp NFRCS compositions was determined by implementing the method described by Li et al. 19 . Place 15 x 15 mm2 Ch NFRCS, Cp NFRCS, and Cs (positive control) into separate Petri dishes (37 °C). Blood containing 3.8% sodium citrate was mixed with 0.2 M CaCl2 in a 10:1 volume ratio to start the blood clotting process. 20 µl of 0.2 M CaCl2 rat blood mixture was applied to the sample surface and placed in an empty Petri dish. The control was blood poured into empty Petri dishes without Ct. At fixed intervals of 0, 3, and 5 minutes, stop clotting by adding 10 ml of deionized (DI) water to the sample containing the dish without disturbing the clot. Uncoagulated erythrocytes (erythrocytes) undergo hemolysis in the presence of deionized water and release hemoglobin. Hemoglobin at different time points (HA(t)) was measured at 540 nm (λmax hemoglobin) using a UV-Vis spectrophotometer. The absolute absorption of hemoglobin (AH(0)) in 0 min of 20 µl of blood in 10 ml of deionized water was taken as a reference standard. The relative hemoglobin uptake (RHA) of coagulated blood was calculated from the ratio HA(t)/HA(0) using the same batch of blood.
Using a texture analyzer (Texture Pro CT V1.3 Build 15, Brookfield, USA), the adhesive properties of NFRK to damaged tissue were determined. Press an open-bottomed cylindrical dish against the inside of the pork skin (without the layer of fat). Samples (Ch NFRCS and Cp NFRCS) were applied via cannula into cylindrical molds to create adhesion to the skin of the pig. After a 3 minute incubation at room temperature (RT) (25° C.), the NFRCS adhesive strength was recorded at a constant rate of 0.5 mm/sec.
The main feature of surgical sealants is to increase blood clotting while reducing blood loss. Lossless coagulation in NFRCS was evaluated using a previously published method with slight modifications 19 . Make a microcentrifuge tube (2 ml) (inner diameter 10 mm) with an 8 × 5 mm2 hole on one side of the centrifuge tube (representing an open wound). NFRCS is used to close the opening and tape is used to seal the outer edges. Add 20 µl of 0.2 M CaCl2 to the microcentrifuge tube containing the 3.8% sodium citrate premix. After 10 minutes, the microcentrifuge tubes were removed from the dishes and the increase in the mass of the dishes was determined due to the outflow of blood from the NFRK (n = 3). Blood loss Ch NFRCS and Cp NFRCS were compared with Cs.
Wet integrity of NFRCS was determined based on the method described by Mishra and Chaudhary21 with minor modifications. Place the NFRCS in a 100 ml Erlenmeyer flask with 50 ml water and swirl for 60 s without forming a top. Visual inspection and prioritization of samples for physical integrity based on collection.
The binding strength of HFFC to Ct was studied using previously published methods with minor modifications. Surface coating integrity was assessed by exposing NFRK to acoustic waves (external stimulus) in the presence of milliQ water (Ct). The developed NFRCS Ch NFRCS and Cp NFRCS were placed in a beaker filled with water and sonicated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 30 min, respectively. After drying, the percentage difference between the initial and final weight of the NFRCS was used to calculate the percent loss of material (HFFC). In vitro BCT further supported the binding strength or loss of surface materials. The efficiency of HFFC binding to Ct provides blood coagulation and an elastic coating on the surface of Ct22.
The homogeneity of the developed NFRCS was determined by the BCT of samples (30 mg) taken from randomly selected general locations of the NFRCS. Follow the previously mentioned BCT procedure to determine NFRCS compliance. Proximity between all five samples ensures uniform surface coverage and HFFC deposition in the Ct mesh.
The nominal blood contact area (NBCA) was determined as previously reported with some modifications. Coagulate the blood by clamping 20 µl of blood between the two surfaces of Ct, Ch NFRCS, Cp NFRCS and Cs. After 1 hour, the two parts of the stent were separated and manually measured the area of the clot. The average value of three repetitions was considered NBCA NFRCS19.
Dynamic Vapor Sorption (DVS) analysis was used to evaluate the effectiveness of NFRCS to absorb water from the external environment or from the injury site responsible for initiating coagulation. The DVS evaluates or records the vapor uptake and loss in a sample gravimetrically using an ultra-sensitive balance with a mass resolution of ±0.1 µg. A partial vapor pressure (relative humidity) is generated by an electronic mass flow controller around the sample by mixing saturated and dry carrier gases. As per the European Pharmacopeia guidelines, based on the percentage of moisture uptake by the samples, samples were categorized into 4 categories (0–0.012% w/w− non-hygroscopic, 0.2–2% w/w slightly hygroscopic, 2–15% moderately hygroscopic, and > 15% very hygroscopic)23. As per the European Pharmacopeia guidelines, based on the percentage of moisture uptake by the samples, samples were categorized into 4 categories (0–0.012% w/w− non-hygroscopic, 0.2–2% w/w slightly hygroscopic, 2–15 % moderately hygroscopic, and > 15% very hygroscopic)23. In accordance with the recommendations of the European Pharmacopoeia, depending on the percentage of moisture absorption by the samples, the samples were divided into 4 categories (0–0.012% w/w – non-hygroscopic, 0.2–2% w/w slightly hygroscopic, 2– fifteen %). % умеренно гигроскопичен и > 15% очень гигроскопичен)23. % moderately hygroscopic and > 15% very hygroscopic)23.根据欧洲药典指南，根据样品吸收水分的百分比，样品分为4 类（0-0.012% w/w- 非吸湿性、0.2-2% w/w 轻微吸湿性、2-15 % 适度吸湿，> 15% 非常吸湿）23。根据 欧洲 药典 指南 ， 根据 吸收 水分 的 百分比 样品 分为 分为 分为 类 （（0-0.012% W/w- 吸湿 性 、 、 、 、 0.2-2% W/w 轻微 、 2-15% 适度 吸湿 ，> 15 %非常吸湿）23。 In accordance with the recommendations of the European Pharmacopoeia, samples are divided into 4 classes depending on the percentage of moisture absorbed by the sample (0-0.012% by weight – non-hygroscopic, 0.2-2% by weight slightly hygroscopic, 2-15% by weight). % умеренно гигроскопичен, > 15 % очень гигроскопичен) 23. % moderately hygroscopic, > 15% very hygroscopic) 23. The hygroscopic efficiency of NFCS X NFCS and TsN NFCS was determined on an analyzer DVS TA TGA Q5000 SA. During this process, run time, relative humidity (RH), and real-time sample weight at 25°C24 were obtained. Moisture content is calculated by accurate NFRCS mass analysis using the following equation:
MC is NFRCS humidity. m1 – dry weight of NSAIDs. m2 is the real-time NFRCS mass at a given RH.
The total surface area was estimated using a nitrogen adsorption experiment with liquid nitrogen after emptying the samples at 25 °C for 10 h (< 7 × 10–3 Torr). The total surface area was estimated using a nitrogen adsorption experiment with liquid nitrogen after emptying the samples at 25 °C for 10 h (< 7 × 10–3 Torr). Общая площадь поверхности оценивалась с помощью эксперимента по адсорбции азота жидким азотом после опорожнения образцов при 25 °С в течение 10 ч (< 7 × 10–3 Торр). The total surface area was estimated using a nitrogen adsorption experiment with liquid nitrogen after samples were emptied at 25°C for 10 h (< 7 × 10–3 Torr).在25°C 清空样品10 小时（< 7 × 10-3 Torr）后，使用液氮的氮吸附实验估计总表面积。在 25°C Общая площадь поверхности оценивалась с использованием экспериментов по адсорбции азота жидким азотом после опорожнения образцов в течение 10 часов при 25°C (< 7 × 10-3 торр). The total surface area was estimated using nitrogen adsorption experiments with liquid nitrogen after samples were emptied for 10 hours at 25°C (< 7 x 10-3 torr). Total surface area, pore volume and NFRCS pore size were determined with a Quantachrome from NOVA 1000e, Austria using RS 232 software.
Prepare 5% RBCs (saline as diluent) from whole blood. Then transfer an aliquot of HFFC (0.25 ml) to a 96-well plate and 5% RBC mass (0.1 ml). Incubate the mixture at 37°C for 40 minutes. A mixture of red blood cells and serum was considered as a positive control, and a mixture of saline and red blood cells as a negative control. Hemagglutination was determined according to the Stajitzky scale. The proposed scales are as follows: + + + + dense granular aggregates; + + + smooth bottom pads with curved edges; + + smooth bottom pads with torn edges; + narrow red rings around the edges of the smooth pads; – (negative) discrete red button 12 in the center of the lower well.
The hemocompatibility of NFRCS was studied according to the method of the International Organization for Standardization (ISO) (ISO10993-4, 1999)26,27. The gravimetric method described by Singh et al. Minor modifications were made to assess thrombus formation in the presence of or on the surface of NFRCS. 500 mg of Cs, Ch NFRCS and Cp NFRCS were incubated in phosphate buffered saline (PBS) for 24 hours at 37°C. After 24 hours, PBS was removed and NFRCS was treated with 2 ml of blood containing 3.8% sodium citrate. On the surface of the NFRCS, add 0.04 ml of 0.1 M CaCl2 to the incubated samples. After 45 minutes, 5 ml of distilled water was added to stop coagulation. Coagulated blood on the surface of NFRK was treated with 36-38% formaldehyde solution. The clots fixed with formaldehyde were dried and weighed. The percentage of thrombosis was estimated by calculating the weight of the glass without blood and sample (negative control) and the glass with blood (positive control).
As an initial confirmation, the samples were visualized under an optical microscope to understand the ability of the HFFC surface coating, Ct interconnected, and Ct network to form pores. Thin sections of Ch and Cp from NFRCS were trimmed with a scalpel blade. The resulting section was placed on a glass slide, covered with a coverslip, and the edges were fixed with glue. The prepared slides were viewed under an optical microscope and photographs were taken at different magnifications.
Polymer deposition in Ct networks was visualized using fluorescence microscopy based on the method described by Rice et al.29. The HFFC composition used for the formulation was mixed with a fluorescent dye (amaranth), and NFRCS (Ch & Cp) were prepared as per the method mentioned previously. The HFFC composition used for the formulation was mixed with a fluorescent dye (amaranth), and NFRCS (Ch & Cp) were prepared as per the method mentioned previously. The HFFC composition used for formulation was mixed with a fluorescent dye (amaranth) and NFRCS (Ch and Cp) was obtained according to the previously mentioned method.将用于配方的HFFC 组合物与荧光染料（苋菜）混合，并按照前面提到的方法制备NFRCS（Ch & Cp）。将用于配方的HFFC 组合物与荧光染料（苋菜）混合，并按照前面提到的方法制备NFRCS（Ch & Cp）。 The HFFC composition used in the formulation was mixed with a fluorescent dye (Amaranth) and received NFRCS (Ch and Cp), as mentioned earlier. Thin sections of NFRK were cut from the obtained samples, placed on glass slides, and covered with cover slips. Observe the prepared slides under a fluorescent microscope using a green filter (310-380 nm). Images were taken at 4x magnification to understand Ct relationships and excess polymer deposition in the Ct network.
The surface topography of NFRCS Ch and Cp was determined using an atomic force microscope (AFM) with an ultra-sharp TESP cantilever in tapping mode: 42 N/m, 320 kHz, ROC 2-5 nm, Bruker, Taiwan. Surface roughness was determined by root mean square (RMS) using software (Scanning Probe Image Processor). Various NFRCS locations were rendered on 3D images to check for surface uniformity. The standard deviation of the score for a given area is defined as the surface roughness. The RMS equation was used to quantify the surface roughness of NFRCS31.
FESEM-based studies were performed using FESEM, SU8000, HI-0876-0003, Hitachi, Tokyo, to understand the surface morphology of Ch NFRCS and Cp NFRCS, which showed better BCT than Cm NFRCS. The FESEM study was performed according to the method described by Zhao et al. 32 with minor modifications. NFRCS 20 to 30 mg Ch NFRCS and Cp NFRCS were premixed with 20 µl of 3.8% sodium citrate premixed with rat blood. 20 μl of 0.2 M CaCl2 was added to the blood-treated samples to initiate coagulation and the samples were incubated at room temperature for 10 minutes. In addition, excess erythrocytes were removed from the NFRCS surface by rinsing with saline.
Subsequent samples were treated with 0.1% glutaraldehyde and then dried in a hot air oven at 37°C to remove moisture. The dried samples were coated and analyzed 32 . Other images obtained during the analysis were clot formation on the surface of individual cotton fibers, polymer deposition between Ct, erythrocyte morphology (shape), clot integrity, and erythrocyte morphology in the presence of NFRCS. Untreated NFRCS areas and Ch and Cp treated NFRCS areas incubated with blood were scanned for elemental ions (sodium, potassium, nitrogen, calcium, magnesium, zinc, copper and selenium)33. Compare elemental ion percentages between treated and untreated samples to understand elemental ion accumulation during clot formation and clot homogeneity.
The thickness of the Cp HFFC surface coating on the Ct surface was determined using FESEM. The cross sections of Cp NFRCS were cut from the framework and sputter coated. The resulting sputter coating samples were observed by FESEM and the thickness of the surface coating was measured 34 , 35 , 36 .
X-ray micro-CT provides high-resolution 3D non-destructive imaging and allows you to study the internal structural arrangement of NFRK. Micro-CT uses an X-ray beam passing through the sample to record the local linear attenuation coefficient of the X-rays in the sample, which helps to obtain morphological information. The internal location of Ct in Cp NFRCS and blood-treated Cp NFRCS was examined by micro-CT to understand absorption efficiency and blood clotting in the presence of NFRCS37,38,39. The 3D structures of blood-treated and untreated Cp NFRCS samples were reconstructed using micro-CT (V|tome|x S240, Phoenix, Germany). Using VG STUDIO-MAX software version 2.2, several X-ray images were taken from different angles (ideally 360° coverage) to develop 3D images for NFRCS. The collected projection data was reconstructed into 3D volumetric images using the corresponding simple 3D ScanIP Academic software.
In addition, to understand the distribution of the clot, 20 µl of premixed citrated blood and 20 µl of 0.2 M CaCl2 were added to the NFRCS to initiate blood clotting. The prepared samples are left to harden. The NFRK surface was treated with 0.5% glutaraldehyde and dried in a hot air oven at 30–40°C for 30 min. The blood clot formed on the NFRCS was scanned, reconstructed, and a 3D image of the blood clot was visualized.
Antibacterial assays were performed on Cp NFRCS (best compared to Ch NFRCS) using the previously described method with minor modifications. The antibacterial activity of Cp NFRCS and Cp HFFC was determined using three different test microorganisms [S.aureus (gram-positive bacteria), E.coli (gram-negative bacteria) and white Candida (C.albicans)] growing on agar in Petri dishes in an incubator. Uniformly inoculate 50 ml of the diluted bacterial culture suspension at a concentration of 105-106 CFU ml-1 onto the agar medium. Pour the medium into a Petri dish and let it solidify. Wells were made on the surface of the agar plate to fill with HFFC (3 wells for HFFC and 1 for negative control). Add 200 µl HFFC to 3 wells and 200 µl pH 7.4 PBS to the 4th well. On the other side of the petri dish, place a 12 mm Cp NFRCS disk on the solidified agar and moisten with PBS (pH 7.4). Ciprofloxacin, ampicillin and fluconazole tablets are considered reference standards for Staphylococcus aureus, Escherichia coli and Candida albicans. Measure the zone of inhibition manually and take a digital image of the zone of inhibition.
After institutional ethical approval, the study was conducted at the Kasturba Medical College of Education and Research in Manipal, Karnataka, in southern India. The in vitro TEG experimental protocol has been reviewed and approved by the Institutional Ethics Committee of Kasturba Medical College, Manipal, Karnataka (IEC: 674/2020). Subjects were recruited from volunteer blood donors (aged 18 to 55) from the hospital blood bank. In addition, an informed consent form was obtained from the volunteers for the collection of blood samples. Native TEG (N-TEG) was used to study the effect of the Cp HFFC formulation on whole blood premixed with sodium citrate. N-TEG is widely recognized for its role in point-of-care resuscitation, which creates problems for clinicians due to the potential for clinically significant delay in results (routine coagulation tests). N-TEG analysis was performed using whole blood. Informed consent and detailed medical history were obtained from all participants. The study did not include participants with hemostatic or thrombotic complications such as pregnancy/postpartum or liver disease. Subjects taking drugs that affect the coagulation cascade were also excluded from the study. Basic laboratory tests (hemoglobin, prothrombin time, activated thromboplastin and platelet count) were performed on all participants according to standard procedures. N-TEG determines blood clot viscoelasticity, initial clot structure, particle interaction, clot strengthening, and clot lysis. The N-TEG analysis provides graphical and numerical data on the collective effects of several cellular elements and plasma. N-TEG analysis was performed on two different volumes of Cp HFFC (10 µl and 50 µl). As a result, 1 ml of whole blood with citric acid was added to 10 μl of Cp HFFC. Add 1 ml (Cp HFFC + citrated blood), 340 µl mixed blood to 20 µl 0.2 M CaCl2 containing TEG dish. Thereafter, TEG dishes were loaded into TEG® 5000, US to measure R, K, alpha angle, MA, G, CI, TPI, EPL, LY 30% of blood samples in the presence of Cp HFFC41.
The in vivo study protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC), Kasturba School of Medicine, Manipal Institute of Higher Education, Manipal (IAEC/KMC/69/2020). All animal experiments were performed in accordance with the recommendations of the Committee for the Control and Supervision of Animal Experimentation (CPCSEA). All in vivo NFRCS studies (2 × 2 cm2) were performed on female Wistar rats (weighing 200 to 250 g). All animals were acclimatized at a temperature of 24-26°C, the animals had free access to standard food and water ad libitum. All animals were randomly divided into different groups, each group consisted of three animals. All studies were performed in accordance with Animal Studies: Report of In Vivo Experiments 43 . Before the study, the animals were anesthetized by intraperitoneal (ip) administration of a mixture of 20-50 mg of ketamine (per 1 kg of body weight) and 2-10 mg of xylazine (per 1 kg of body weight). After the study, the bleeding volume was calculated by evaluating the difference between the initial and final weight of the samples, the average value obtained from the three tests was taken as the bleeding volume of the sample.
The rat tail amputation model was implemented to understand the potential of NFRCS to modulate bleeding in trauma, combat, or traffic accident (injury model). Cut off 50% of the tail with a scalpel blade and place in air for 15 s to ensure normal bleeding. In addition, test samples were placed on the tail of a rat by applying pressure (Ct, Cs, Ch NFRCS and Cp NFRCS). Bleeding and PCT were reported for test specimens (n = 3)17,45.
The effectiveness of NFRCS pressure control in combat was investigated on a model of the superficial femoral artery. The femoral artery is exposed, punctured with a 24G trocar, and bled within 15 seconds. After uncontrolled bleeding is observed, the test specimen is placed at the puncture site with pressure applied. Immediately after application of the test sample, the clotting time was recorded and hemostatic efficiency was observed for the next 5 minutes. The same procedure was repeated with Cs and Ct46.
Dowling et al. 47 proposed a liver injury model to assess the hemostatic potential of hemostatic materials in the context of intraoperative bleeding. BCT was recorded for Ct samples (negative control), Cs framework (positive control), Ch NFRCS samples, and Cp NFRCS samples. The suprahepatic vena cava of the rat was exposed by performing a median laparotomy. After that, the distal part of the left lobe was cut out with scissors. Make an incision in the liver with a scalpel blade and let it bleed for a few seconds. Accurately weighed Ch NFRCS and Cp NFRCS test samples were placed on the damaged surface without any positive pressure and BCT was recorded. The control group (Ct) then applied pressure followed by Cs 30 s47 without breaking the injury.
In vivo wound healing assays were performed using an excisional wound model to evaluate the wound healing properties of the developed polymer-based NFRCSs. Models of excisional wounds were selected and performed according to previously published methods with minor modifications19,32,48. All animals were anesthetized as previously described. Use a biopsy punch (12 mm) to make a circular deep incision in the skin of the back. Prepared wound sites were dressed with Cs (positive control), Ct (recognizing that cotton pads interfere with healing), Ch NFRCS and Cp NFRCS (experimental group) and a negative control without any treatment. On each day of the study, the area of the wound was measured in all rats. Use a digital camera to take a picture of the wound area and put on a new dressing. The percentage of wound closure was measured by the following formula:
Based on the percentage of wound closure on the 12th day of the study, the rat skin of the best group was excised ((Cp NFRCS) and the control group) and studied by H& E staining and Masson’s trichrome staining. Based on the percentage of wound closure on the 12th day of the study, the rat skin of the best group was excised ((Cp NFRCS) and the control group) and studied by H& E staining and Masson’s trichrome staining. Based on the percentage of wound closure on the 12th day of the study, the skin of the rats of the best group ((Cp NFRCS) and the control group) was excised and examined by staining with hematoxylin-eosin and Masson’s trichrome.根据研究第12天的伤口闭合百分比，切除最佳组((Cp NFRCS)和对照组)的大鼠皮肤，进行H&E染色和Masson三色染色研究。根据研究第12天的伤口闭合百分比，切除最佳组((Cp NFRCS)和对照组)的大鼠皮肤，进行H&E染色和眳组） Rats in the best group ((Cp NFRCS) and control groups) were excised for hematoxylin-eosin staining and Masson’s trichrome staining based on percent wound closure on day 12 of the study. The implemented staining procedure was carried out according to previously described methods49,50. Briefly, after fixation in 10% formalin, the samples were dehydrated using a series of graded alcohols. Use a microtome to obtain thin sections (5 µm thick) of the excised tissue. Thin serial sections of controls and Cp NFRCS were treated with hematoxylin and eosin to study histopathological changes. Masson’s trichrome stain was used to detect the formation of collagen fibrils. The results obtained were blindly studied by pathologists.
The stability of Cp NFRCS samples was studied at room temperature (25°C ± 2°C/60% RH ± 5%) for 12 months51. Cp NFRCS (surface discoloration and microbial growth) was visually inspected and tested for fold wear resistance and BCT according to the above methods outlined in the Materials and Methods section.
The scalability and reproducibility of Cp NFRCS was examined by preparing Cp NFRCS with a size of 15×15 cm2. In addition, 30 mg samples (n = 5) were excised from various Cp NFRCS fractions and the BCT of the studied samples was evaluated as described earlier in the Methods section.
We have attempted to develop various shapes and structures using Cp NFRCS compositions for various biomedical applications. Such shapes or configurations include conical swabs for nosebleeds, dental procedures, and cylindrical swabs for vaginal bleeding.
All data sets are expressed as mean ± standard deviation and were analyzed by ANOVA using Prism 5.03 (GraphPad, San Diego, CA, USA) followed by Bonferroni’s multiple comparisons test (*p<0.05).
All procedures performed in human studies were in accordance with the standards of the Institute and the National Research Council, as well as the Declaration of Helsinki 1964 and its subsequent amendments, or similar ethical standards. All participants were informed about the features of the study and its voluntary nature. Participant data remains confidential once collected. The in vitro TEG experimental protocol has been reviewed and approved by the Institutional Ethics Committee of Kasturba Medical College, Manipal, Karnataka (IEC: 674/2020). Volunteers signed informed consent to collect blood samples.
All procedures performed in animal studies were carried out in accordance with the Kastuba Faculty of Medicine, Manipal Institute of Higher Education, Manipal (IAEC/KMC/69/2020). All animal experiments designed were conducted in accordance with the guidelines of the Committee for the Control and Supervision of Animal Experimentation (CPCSEA). All authors follow the ARRIVE guidelines.
The FTIR spectra of all NFRCS were analyzed and compared with the chitosan spectrum shown in Figure 2A. Characteristic spectral peaks of chitosan (recorded) at 3437 cm-1 (OH and NH stretching, overlap), 2945 and 2897 cm-1 (CH stretching), 1660 cm-1 (NH2 strain), 1589 cm-1 (N–H bending ), 1157 cm-1 (bridge stretch O-), 1067 cm-1 (stretch C–O, secondary hydroxyl), 993 cm-1 (stretch CO, Bo-OH) 52.53.54. Supplementary Table S1 shows the FTIR NFRCS absorption spectrum values for chitosan (reporter), pure chitosan, Cm, Ch, and Cp. The FTIR spectra of all NFRCS (Cm, Ch and Cp) showed the same characteristic absorption bands as pure chitosan without any significant changes (Fig. 2A). The FTIR results confirmed the absence of chemical or physical interactions between the polymers used to develop the NFRCS, indicating that the polymers used are inert.
In vitro characterization of Cm NFRCS, Ch NFRCS, Cp NFRCS and Cs. (A) represents the combined FTIR spectra of the compositions of chitosan and Cm NFRCS, Ch NFRCS and Cp NFRCS under compression. (B) a) Whole blood uptake rate of NFRCS Cm, Ch, Cp, and Cg (n = 3); The Ct samples showed a higher BAR because the cotton swab has a higher absorption efficiency; b) Blood after blood absorption Illustration of the absorbed sample. Graphical representation of the BCT of test sample C (Cp NFRCS had the best BCT (15 s, n = 3)). Data in C, D, E, and G were shown as mean ± SD, and the error bars represent SD, ***p < 0.0001. Data in C, D, E, and G were shown as mean ± SD, and the error bars represent SD, ***p < 0.0001. Данные в C, D, E и G представлены как среднее ± стандартное отклонение, а планки погрешностей представляют стандартное отклонение, ***p <0,0001. Data in C, D, E, and G are presented as mean ± standard deviation, and error bars represent standard deviation, ***p<0.0001. C、D、E 和G 中的数据显示为平均值± SD，误差线代表SD，***p < 0.0001。 C、D、E 和G 中的数据显示为平均值± SD，误差线代表SD，***p < 0.0001。 Данные в C, D, E и G показаны как среднее значение ± стандартное отклонение, планки погрешностей представляют стандартное отклонение, ***p <0,0001. Data in C, D, E, and G are shown as mean ± standard deviation, error bars represent standard deviation, ***p<0.0001.