A human hair keratin hemostatic sponge, its preparation method and application

Abstract

A human hair keratin hemostatic sponge, its preparation method, and its applications are disclosed. Sodium hydroxide and mercaptoethanol are used to break the disulfide bonds in keratin of human hair, reducing it to thiol groups, and soluble keratin is extracted from it. The sponge synthesis is achieved by utilizing the inherent thiol groups in keratin and cross-linking with oxygen to form disulfide bonds, and by adjusting the solid content of keratin to control the degree of cross-linking. In vitro and in vivo experimental results show that keratin has good hemostatic ability. Furthermore, the keratin sponge not only promotes platelet activation but also stimulates the production of thrombin and coagulation factor XII, indicating a special coagulation-promoting effect in a coagulation disorder model leading to coagulation factor dilution. In addition, the keratin sponge exhibits significant biocompatibility and low immunogenicity, showing great potential in treating uncontrollable bleeding wounds.

Description

Technical Field

[0001] This invention relates to the field of new hemostatic materials, specifically to a human hair keratin hemostatic sponge, its preparation method, and its application. Background Technology

[0002] Currently used hemostatic products include hemostatic powder (CELOX™), hemostatic bandages (HemCon®), and gauze (Surgiecel). Although these products are effective in treating superficial wounds and wounds with low bleeding, they still pose a challenge for uncontrolled large bleeding wounds and non-compressible wounds occurring in the major arteries of the limbs, the arteries of the head and neck, and organs due to their difficulty in reaching the bleeding site and low fluid absorption rate.

[0003] Synthetic hemostatic sponges and cryogels with macroporous structures can rapidly absorb water and expand quickly, applying pressure to the wound for effective hemostasis. However, traditional non-degradable sponges need to be removed after the first hemostasis, easily causing secondary bleeding and infection. In recent years, novel biodegradable sponges made from natural polymers such as starch, chitosan, and cellulose have effectively solved these shortcomings. However, due to the lack of procoagulant activity, the hemostatic time of these hemostatic agents is often prolonged, which may lead to increased blood loss. Previous studies have reported that collagen or gelatin sponges can activate platelets and accelerate the coagulation cascade reaction. Gelatin and collagen-based materials have been shown to have excellent hemostatic capabilities. In addition, collagen has been repeatedly reported to activate platelets, stimulate platelet aggregation, and have unique biological functions, which have a certain impact on active hemostasis. However, gelatin and collagen still have some drawbacks. On the one hand, the cross-linking agents remaining during the preparation process often have certain toxicity to organisms. Commonly used cross-linking agents for collagen or gelatin sponges, such as formaldehyde and glutaraldehyde, have significant cytotoxic or carcinogenic effects. On the other hand, the biological activity of proteins is reduced after chemical cross-linking.

[0004] Keratin is a protein rich in α-helical structures, obtainable from hair, feathers, horns, and nails. Its widespread use in tissue engineering, wound healing, drug delivery, and hemostasis is attributed to its natural abundance, low immunogenicity, good biocompatibility, and easy availability. Due to the presence of abundant disulfide bonds, various methods can be used to break these bonds during extraction. This process converts insoluble keratin into water-soluble keratin, allowing for the formation of sponges and hydrogels through thiol cross-linking. Furthermore, keratin has been found to promote platelet activation and coagulation. Given these properties, keratin holds promise as a novel protein-absorbable sponge, potentially replacing expensive collagen. For example, keratin has been used in combination with sodium alginate to synthesize sponges with excellent biocompatibility and low immunogenicity. Similarly, keratin has been mixed with polyacrylamide to prepare sponges for treating penetrating bleeding wounds. Previous studies have also shown that combining keratin with various polymer materials can improve its mechanical properties. To our knowledge, there is no known alternative to a purely natural, additive-free keratin sponge with superior mechanical properties and a deep coagulation mechanism, specifically designed for hemostasis of incompressible wounds. Summary of the Invention

[0005] To address the technical shortcomings of keratin hemostatic sponges, this invention provides a pure, natural human hair keratin hemostatic sponge without added cross-linking agents, its preparation method, and its applications.

[0006] The technical solution adopted in this invention is: a human hair keratin hemostatic sponge, wherein the human hair keratin hemostatic sponge is obtained by foaming water-soluble keratin powder and then freeze-drying it, wherein the molecular weight of the water-soluble keratin is 50-60kDa.

[0007] The water-soluble keratin is obtained by breaking the disulfide bonds in the insoluble keratin and reducing it to thiol groups.

[0008] The water-soluble keratin content in the human hair keratin hemostatic sponge is 5-15%.

[0009] The human hair keratin hemostatic sponge contains 10% water-soluble keratin.

[0010] A method for preparing a human hair keratin hemostatic sponge includes the following steps:

[0011] (1) Preparation of water-soluble keratin powder: human hair was washed with 0.5% (w / v) SDS solution to remove surface dust and oil, and then dried. Subsequently, a thioglycolic acid (TGA) solution with pH adjusted to 11 using NaOH solution was added to the dried hair to break the disulfide bonds of cystine in the hair at 37°C, thereby extracting soluble keratin. The resulting keratin-containing reduced solution was collected, and the remaining soluble keratin was fully dissolved by stirring and washing with 100 mM Tris solution. Then, a second cleaning was performed with deionized water. The filtrate was collected and centrifuged to remove the residue. Subsequently, hydrochloric acid was added to the solution to precipitate keratin. The obtained keratin precipitate was then dissolved in sodium hydroxide solution and filtered and centrifuged using different ultrafiltration centrifuge tubes to obtain water-soluble keratin solutions with different molecular weights. The obtained water-soluble keratin solutions were frozen at -80°C overnight and then dried to obtain water-soluble keratin powder.

[0012] (2) Synthesis of human hair keratin hemostatic sponge: High molecular weight water-soluble keratin powder with a molecular weight of 50-60kDa was selected and dissolved in cysteine ​​solution. SDS was added for rapid foaming, and then poured into a mold for making sponges for freeze drying. After oxidation in air for 48-72 h, the residual compound was washed with distilled water and then freeze-dried again to obtain human hair keratin hemostatic sponge.

[0013] In step (1), the concentration of the mercaptoacetic acid (TGA) solution is 1M.

[0014] In step (1), 30KD and 50KD ultrafiltration centrifuge tubes are used.

[0015] In step (1), the water-soluble keratin solution needs to be hydrolyzed within 48 hours.

[0016] In step (2), the content of high molecular weight water-soluble keratin powder is 10%.

[0017] Application of a human hair keratin hemostatic sponge in the preparation of procoagulant materials.

[0018] The beneficial effects of this invention are as follows: This invention provides a human hair keratin hemostatic sponge, its preparation method, and its application. Sodium hydroxide and mercaptoethanol are used to break the disulfide bonds in keratin of human hair, reducing it to thiol groups, and soluble keratin is extracted from it. By utilizing the inherent thiol groups in keratin and cross-linking with oxygen to form disulfide bonds, the degree of cross-linking is controlled by adjusting the solid content of keratin to achieve sponge synthesis. In vitro and in vivo experimental results show that keratin has good hemostatic ability. Furthermore, the keratin sponge not only promotes platelet activation but also stimulates the production of thrombin and coagulation factor XII, indicating that it has a special coagulation-promoting effect in a coagulation disorder model leading to coagulation factor dilution. In addition, the keratin sponge exhibits significant biocompatibility and low immunogenicity, showing great potential in treating uncontrollable bleeding wounds. Attached Figure Description

[0019] Figure 1 Electrophoretic images of two keratins with different molecular weights.

[0020] Figure 2 These are circular dichroism chromatograms of two different keratins.

[0021] Figure 3 These are FTIR spectra: (A) FTIR spectra of keratin with different molecular weights. (B) FTIR spectra of different keratin sponges.

[0022] Figure 4 This is an SEM image of a keratin sponge.

[0023] Figure 5 These are the porosities of HK5, HK10, and HK15.

[0024] Figure 6 The following are the liquid absorption properties of sponges: (A) Average water absorption rate of three types of keratin sponges within 9 seconds. (B) Average blood absorption rate of three types of keratin sponges within 9 seconds. (C) Water absorption rate of keratin sponges at 3s, 6s, 9s, and 12s. (D) Blood absorption rate of keratin sponges at 3s, 6s, 9s, and 12s. ns P>0.05, *** P<0.001).

[0025] Figure 7 It is the inflation ratio of HK5, HK10, and HK15. ns P>0.05, * P<0.05, ** P<0.01).

[0026] Figure 8 (A) The compressive stress-strain curves of all sponges in the expanded state. (B) The maximum compressive stress of all sponges.

[0027] Figure 9 It represents the hemolysis rate of all sponges.

[0028] Figure 10 These are the in vitro coagulation results of keratin sponges. (A) Coagulation time of HK5, HK10, and HK15 keratin sponges, collagen sponges, and gelatin sponges in whole blood. (B) Coagulation time of HK10 keratin sponges, collagen sponges, and gelatin sponges in 40% diluted blood. (C) Coagulation index of HK5, HK10, and HK15 keratin sponges, collagen sponges, and gelatin sponges at 2 min, 5 min, and 10 min. (D) Images of the coagulation index of all sponges.

[0029] Figure 11 This is a diagram showing platelets and red blood cells adhering to the surface of a sponge.

[0030] Figure 12 It represents the platelet activation rate of all sponges.

[0031] Figure 13 It is coagulation factor XII produced after incubating all sponges and diluted 30% platelet-rich plasma for 2, 5 and 10 minutes.

[0032] Figure 14 It is the thrombin produced after incubating all sponges and diluted 30% platelet-rich plasma for 5, 10, 15, and 20 minutes.

[0033] Figure 15 This refers to the in vivo hemostatic properties of sponges (rat liver puncture model). (A) Hemostasis time of all sponges in the rat liver puncture model. (B) Blood loss of all sponges in the rat liver puncture model.

[0034] Figure 16 This refers to the in vivo hemostatic performance of sponges (rat liver incision model). (A) Hemostasis time of all sponges. (B) Blood loss of all sponges in the rat liver incision model.

[0035] Figure 17 This describes the degradation of all sponges in vitro in proteinase K and chymotrypsin solutions.

[0036] Figure 18 This describes the degradation of the subcutaneous sponge on the back of rats over 21 days. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Material

[0039] The human hair was obtained from a local barbershop in Wenzhou, China. Thioglycolic acid (TGA) and cysteine ​​were purchased from Shanghai Mailian Biochemical Technology Co., Ltd. (China). Sodium dodecyl sulfate (SDS), hydrochloric acid, and sodium hydroxide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). Tris(hydroxymethyl)methylaminomethane (Tris) was purchased from Sigma-Aldrich Ltd. (USA).

[0040] Example 1: Extraction of human hair keratin

[0041] The method for extracting keratin from human hair involves washing human hair for 6 hours with a 0.5% (w / v) SDS solution to remove surface dirt and oil, followed by overnight drying. Subsequently, a 1M thioglycolic acid (TGA) solution, adjusted to pH 11 using NaOH, is added to the dried hair to break the disulfide bonds of cystine in the hair at 37°C, thereby extracting soluble keratin within 15 hours. The resulting keratin-containing reduced solution is collected and thoroughly dissolved by stirring with a 100 mM Tris solution for 2 hours, followed by a second cleaning with deionized water. The filtrate is then collected and centrifuged at 6000 rpm for 30 minutes to remove any remaining residue. Subsequently, 1 mol / L hydrochloric acid is added to the solution to precipitate the keratin. The resulting keratin precipitate is then redissolved in a 0.1 mol / L sodium hydroxide solution and filtered and centrifuged using 30K and 50K ultrafiltration centrifuge tubes to obtain keratin solutions of different molecular weights. Importantly, this process must be completed within 48 hours. Finally, the obtained keratin solution was frozen overnight at -80°C and then dried to obtain keratin powder.

[0042] Example 2 Characterization of human hair extract

[0043] SDS-PAGE analysis.

[0044] The purity and molecular weight of keratin powder were determined using SDS-PAGE. To separate the keratin powder, a gel electrophoresis system consisting of a 10% (w / v) polyacrylamide separation gel and a 5% (w / v) polyacrylamide stacking gel was used. Subsequently, human hair extract was dissolved in ultrapure water, and 20 μL of protein solution was mixed with 5 μL of 5× loading buffer. The mixture was then boiled in the mixed solution for 10 min to denature the protein. The denaturing solution (10 μL) and protein markers (5 μL) were then loaded into the gel wells, and the gels were subjected to 80 V for 1 hour, followed by 120 V for 2 hours. The gel strips were then gently shaken and soaked with disposable Coomassie Brilliant Blue dye for 30 min, followed by soaking in deionized water for another 30 min to clean the dye before observation.

[0045] Circular dichroism spectroscopy is used to determine the secondary structure of proteins.

[0046] The secondary structures of keratin with different molecular weights were measured using circular dichroism spectroscopy (Chirascan Plus, Britain). For sample preparation, a 2 mg / ml keratin solution was used. Data were collected at 25°C with a spectral resolution of 1 nm, an averaging time of 1 second, a scan rate of 100 nm / min, and a scan range of 300–190 nm. Each sample was measured three times to ensure the accuracy and reliability of the results. The data were analyzed using BestSel for further interpretation and comparison.

[0047] Example 3: Study on the properties of keratin sponges

[0048] Morphological observation

[0049] The internal structure and blood cell adhesion of the HK sponge were observed using a scanning electron microscope (SU8010, Japan). Before imaging, each sample was dried in a vacuum drying oven and then cut with a thin blade to expose a clean cross-section. Each HK sponge cross-section was fixed to a support with a carbon ribbon facing upwards. Subsequently, the samples were sprayed with gold for 20 seconds using a high-vacuum ion sputtering instrument (EMACE600, Germany). Finally, they were observed under a scanning electron microscope at a current of 5 kV and 8 μA. These procedures provided a clear and detailed examination of the internal structure and blood cell adhesion of the HK sponge.

[0050] FTIR analysis.

[0051] The infrared absorption spectra of keratin extracted before and after cross-linking were measured using FTIR (Tensor II, Germany). The samples were ground and pulverized with dry potassium bromide at a ratio of 1:99, then compressed into thin sheets. Measurements were performed in the wavelength range of 600–4000 cm⁻¹, with a spectral recording resolution of 4 cm⁻¹, for a total of 50 scans.

[0052] Porosity Measurement

[0053] The porosity of the sponge was determined based on a previously reported method. In short, a pre-weighed sponge was soaked in ethanol for 30 minutes, then removed and weighed again. The formula for calculating porosity (P) is as follows:

[0054]

[0055] Where N0 represents the initial weight, N1 represents the weight of the sponge after soaking, ρ represents the density of ethanol (0.785 g / cm3), and V0 represents the initial volume of the sponge.

[0056] Compression measurement.

[0057] The physical properties of the sponge were determined by compression tests at room temperature using an electronic general-purpose materials testing machine (5944, USA). In the compression test, a cylindrical water-containing sponge (5 mm in height and 8 mm in diameter) was compressed at a speed of 5 mm / min, with a maximum deformation of 80%. Each test was performed three times.

[0058] Water absorption / blood ratio.

[0059] The absorption rate of the sponge in water and whole blood was measured. The sponge was weighed beforehand, immersed in water / whole blood, and removed and weighed after 3, 6, 9, and 12 seconds. The absorption rate (A) was calculated using the following formula:

[0060] W t Let W0 represent the weight at a given time, t represent the soaking time, and W0 represent the weight before absorption. The formula for the average absorption rate (AA) over 9 seconds is as follows:

[0061]

[0062] Volume expansion performance.

[0063] Soak the pre-weighed, fixed sponge in water for 20 minutes, then remove it and measure its volume again. The formula for calculating the sponge's expansion rate (E) is as follows:

[0064]

[0065] V0 and V1 represent the volume of the sponge before and after it absorbs water and expands, respectively.

[0066] Blood cell adhesion test.

[0067] Based on previously reported literature, the amount of platelets adhering to the surface of a sponge was quantified by measuring the content of lactate dehydrogenase (LDH) released from lysed platelets. Fresh rabbit blood anticoagulated with sodium citrate was centrifuged at 1000 rpm and 4°C for 15 minutes to obtain platelet-rich plasma (PRP). An equal mass of sponges was incubated in PRP at 37°C for 30 minutes, then washed three times with PBS. The sponges incubated with PRP were fixed overnight at 4°C with 2.5% glutaraldehyde solution, followed by dehydration with different ethanol gradient solutions (50%, 75%, 85%, 95%, 100%). After freeze-drying, a layer of metal was sputtered onto the surface for SEM characterization.

[0068] For red blood cell adhesion, sponges of the same mass were incubated with red blood cell solution for 30 minutes, and then the unadhered red blood cells were washed away with PBS. The sponges incubated with red blood cell solution were fixed with 2.5% glutaraldehyde solution, dehydrated with different ethanol solutions, and finally sputtered with a layer of metal for SEM characterization.

[0069] Thrombin and coagulation factor XII test.

[0070] Thrombin and coagulation factor XII were measured using an ELISA kit. Samples were sterilized by UV irradiation for 4 hours, then 1 mg of sponge was weighed and added to 100 μL of diluted 30% recalcified plasma. The mixture was incubated at 37°C for a specified time. When measuring thrombin production, the sponge and plasma were incubated for 5, 10, 15, and 20 minutes, respectively, before measurement. When measuring coagulation factor XII, the sponge and plasma were incubated for 2, 5, and 10 minutes, respectively, before measurement.

[0071] Flow cytometry (FACS) test.

[0072] Platelet activation rate was detected by flow cytometry. Fresh whole blood from mice anticoagulated with sodium citrate was centrifuged at 1000 rpm for 15 minutes to obtain platelet-rich plasma. The plasma was then centrifuged at 3000 rpm for 20 minutes, the supernatant was discarded, and the platelet pellet was resuspended in PBS, centrifuged at 3000 rpm, and the supernatant was discarded to obtain pure platelet pellet. Platelets were resuscitated with PBS, then incubated with 3 mg sponge for 30 minutes to activate them. The pellet was then stained with FITC-CD41 and PE-CD62P fluorescent dyes for 30 minutes and analyzed by machine.

[0073] In vitro whole blood coagulation properties.

[0074] Based on previously reported methods, this invention conducted a whole blood coagulation experiment. First, 0.2 g of sponge was weighed and placed in a plastic dish. The dish was preheated at 37°C, and then 100 μL of fresh sodium citrate anticoagulated rabbit blood was added to the sponge. Next, 10 μL of 0.2 M CaCl2 solution was added, and the mixture was incubated at 37°C for 2, 5, and 10 minutes, respectively. Then, 25 ml of pure water was added to each culture dish to lyse non-adhesive blood cells for 10 minutes, and the absorbance at 540 nm was measured using a microplate reader (Abs1). The absorbance of 100 μL of anticoagulated whole blood in 25 ml of pure water was used as the blank group (Abs0). The formula for calculating the coagulation index (BCI) is as follows:

[0075]

[0076] Hemolysis experiment.

[0077] Fresh rabbit blood anticoagulated with sodium citrate was collected and diluted 10-fold with physiological saline. 1 ml of the diluted blood was incubated with 5 mg of sponge for 6 hours, centrifuged at 3000 rpm for 15 minutes, and the supernatant was collected. The absorbance at 540 nm was measured using a microplate reader. The positive control group was prepared by diluting whole blood 10-fold with pure water. The formula for calculating the hemolysis rate is as follows:

[0078]

[0079] Hemostatic performance in animal models (SD rats)

[0080] All animal experiments were conducted strictly in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Laboratory Animal Ethics Committee of the Wenzhou Institute of Laboratory Animals, Chinese Academy of Sciences (Program No.: WIUCAS21122103). SD rats (male, 8-10 weeks old) were purchased from the Zhejiang Provincial Animal Center. All experimental procedures were performed by the same person to ensure consistency. After the experiments, all rats were euthanized in accordance with ethical guidelines.

[0081] Subcutaneous implantation experiment in rats: First, male SD rats (8-10 weeks old) were randomly divided into 4 groups. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Afterwards, the backs of the rats were shaved and disinfected, and a 1 cm band-shaped wound was created on the skin. Subsequently, a thin, circular sponge (approximately 1 mm high, 8 mm in diameter, and 3 mg) was implanted under the skin and sutured in place. Degradation of the sponge was then observed at 3, 7, 14, and 21 days.

[0082] Rat liver biopsy: Male SD rats (8-10 weeks old) were randomly divided into 4 groups. The abdomen of the anesthetized rats was shaved and disinfected, and then the abdomen was opened to expose the liver. The liver in the middle of the rat was then selected, and a penetrating circular wound with a diameter of 5 mm was made on it. The wound was then filled with a sponge, and the hemostasis time and bleeding amount were recorded.

[0083] Rat liver incision model: In this experiment, male SD rats (8-10 weeks old) were randomly divided into 4 groups. Similar to the rat liver puncture experiment, the abdomen of the anesthetized rats was shaved and disinfected. The abdomen was then opened to expose the liver, where a linear wound of 0.5 cm in length and 0.2 cm in depth was created. A sponge was placed at the bleeding site, and the hemostasis time and bleeding volume were recorded.

[0084] Results and Discussion

[0085] Keratin extraction and sponge synthesis

[0086] Keratin was extracted from human hair using a reduction method. Specifically, under alkaline conditions, TGA was used to open disulfide bonds in human hair without breaking peptide bonds, while reducing them to thiol groups, thus promoting the conversion of insoluble keratin into soluble keratin. Subsequently, two keratin molecules of different molecular weights were separated by ultrafiltration and centrifugation. After freeze-drying and grinding, two keratin powders of different molecular weights were obtained: low molecular weight keratin (LK) and high molecular weight keratin (HK). Based on the initially dried hair, the average yield of keratin was 35.27% ± 0.31% (n=3), of which 31.94% represented HK and 3.33% represented LK.

[0087] Chemical characterization of human hair extract

[0088] Results of SDS-PAGE of human hair extract

[0089] This invention characterized two types of molecular weight sponges using a series of methods. First, the molecular weight of keratin was determined by SDS-PAGE. For example... Figure 1 As shown, the present invention observed two different bands at 50-60kDa and 10-30kDa, respectively, indicating that keratin was successfully separated into high molecular weight keratin and low molecular weight keratin.

[0090] Results of circular dichroism analysis of secondary structure

[0091] Subsequently, the helical structure of the extracted keratin was tested. The circular dichroism test of this invention (…) Figure 2 In this study, high molecular weight keratin also exhibits a higher α-helix structure. It has been reported that α-keratin containing more α-helix structures has been shown to promote hemostasis.

[0092] FTIR spectrum of human hair extract

[0093] Next, the present invention conducted Fourier transform infrared spectroscopy tests on the two keratin powders. Figure 3 A). A characteristic peak at 660 cm⁻¹ was observed in the keratin powder, due to the stretching vibration of CS, confirming the presence of the reduced (-SH) form of keratin. Furthermore, amide I (1600–1700 cm⁻¹), II (1480–1580 cm⁻¹), and III (1220–1330 cm⁻¹) peaks were observed in both keratin powders, which are important indicators for determining protein secondary structure. The spectra also showed the characteristic NH stretching vibration peaks of amides A and B at 3290 cm⁻¹ and 2925 cm⁻¹, respectively.

[0094] Chemical characterization of keratin sponges

[0095] Based on the series of tests conducted in this invention, high-molecular-weight keratin powder was selected to synthesize sponges for further testing and characterization. This selection was based on its high extraction rate and α-helical structure level. In previous reports, keratin was typically mixed with polymer materials to form porous sponges for hemostasis. However, to maximize the preservation of keratin's bioactivity and promote in-depth research on its coagulation mechanism, this invention focuses specifically on the thiol groups of keratin and controls the cross-linking level by adjusting the solids content. In short, it requires the re-oxidation of the thiol groups contained in the keratin powder to form disulfide bonds, and a cross-linking gradient is generated by adjusting the solids content. To produce an ideal sponge hemostatic material, this invention formulated keratin solutions of different concentrations (5%, 10%, 15%), added SDS, and rapidly stirred to obtain a keratin foam solution, which was then frozen under liquid nitrogen. After freeze-drying, this invention obtained three types of sponges: HK5, HK10, and HK15.

[0096] FTIR spectra of HK5, HK10, and HK15 keratin sponges

[0097] The chemical structures of the three sponges were characterized, and their infrared spectra were as follows: Figure 3 As shown in Figure B, by comparing the peak values ​​of the sponge and the powder, it is clear that there is no significant difference except for a significant reduction in the peak area at 660 cm⁻¹ in the sponge. The reduction in peak area is correlated with the increase in solid content, indicating effective cross-linking of thiol groups in the sponge. Furthermore, the degree of cross-linking appears to increase with increasing solid content. The results also confirm that the protein structure of the sponge remains unchanged compared to the powder, indicating that cross-linking has no effect on protein structure.

[0098] Physical characterization of keratin sponges

[0099] Morphological observation

[0100] In the synthesis of sponges, microporous structures can be formed through foaming and freeze-drying. Here, SEM images of three sponges with different solid contents clearly demonstrate the three-dimensional pore structures filled with internal interconnections. During the synthesis of the sponge, the pore structure changes due to differences in the degree of cross-linking. Figure 7 As shown, the microstructure of HK5 appears loose and disordered, while HK10 and HK15 exhibit a denser structure and smaller pores as the keratin solids content increases.

[0101] Porosity of HK5, HK10, and HK15 keratin sponges

[0102] To further analyze porosity variations, this invention quantifies porosity. Figure 8 As can be seen from the data, HK5 exhibits the highest porosity, which can be attributed to the low degree of cross-linking resulting from its low solids content. With increasing keratin content, the cross-linking strength increases accordingly, leading to a decrease in porosity.

[0103] Liquid absorption properties of HK5, HK10, and HK15 keratin sponges

[0104] Cross-linked macroporous sponges possess the ability to rapidly absorb liquids, enabling the fixed-shape sponges to quickly absorb blood and infectious fluids from wounds, thereby reducing the risk of bacterial infection. Furthermore, their rapid absorption capacity allows the sponges to quickly accumulate clotting factors at the site of bleeding wounds, promoting blood coagulation and wound healing. Keratin sponges exhibit excellent liquid absorption properties due to their porous structure and hydrophilic surface properties. To evaluate the liquid absorption performance of the sponges, experiments were conducted to determine the absorption rates of water and blood. Figure 6 A shows the average water absorption rate of the sponge within 9 seconds. The average water absorption rates of HK5 and HK10 were 5.72 ml / g / s and 5.78 ml / g / s, respectively, while the average water absorption rate of HK15 was 2.81 ml / g / s. The results indicate that there was no significant difference in the average water absorption rate between HK5 and HK10, and both showed better water absorption rates compared to HK15. However, as... Figure 6 As shown in B, the average blood absorption rates of HK5, HK10, and HK15 were 2.78 ml / g / s, 3.29 ml / g / s, and 2.23 ml / g / s, respectively. There was no significant difference in the average blood absorption rate among the three within 9 seconds, which may be attributed to the presence of more blood cells and proteins in the blood, leading to increased thickness and affecting fluidity. Conversely, the data ( Figure 6C) indicates that all sponges can absorb the vast majority of water within 5 seconds. Compared to gelatin and collagen sponges, HK sponges exhibit a higher water absorption rate, while HK5 and HK10 show comparable water absorption capacities. A slight decrease in fluid absorption rate was observed in blood. Figure 6 (D) This indicates that the macroporous structure and hydrophilicity of the sponge contribute to its excellent water absorption capacity. This is attributed to the internally cross-linked macroporous structure of HK5, which enables it to exhibit the highest water absorption rate, absorbing approximately 50 times its own weight in water after 12 seconds of contact. Furthermore, HK10 exhibits similar performance, while HK15 absorbs only about 26 times its own weight in water. In contrast, collagen sponges were found to absorb only 24 times their weight in water, and gelatin sponges only 15 times their weight in water. Notably, in blood, HK sponges still exhibit superior liquid absorption performance compared to commercial sponges. These findings highlight the potential of HK sponges in demanding liquid absorption scenarios.

[0105] Expansion characteristics

[0106] like Figure 7 As shown, when a fixed-shape sponge comes into contact with water, HK5 can expand to 800%, while HK10 and HK15 can expand to 600% and 200%, respectively. This expansion may be related to the pore size formed during the sponge's cross-linking process. The morphology of the sponge pores was observed during compression and recovery after contact with liquid. Before contact with liquid, the sponge is compressed and fixed, resulting in smaller pores. After absorbing liquid, the sponge's superior ability to recover its shape exceeds the viscosity of the liquid, causing the pores to recover and enlarge, thus absorbing more liquid. When applied to hemostatic wounds, it can rapidly accumulate blood and promote the coagulation process.

[0107] Mechanical properties

[0108] Stable mechanical properties of sponges are crucial for effective wound sealing and filling. Previous studies have typically involved synthesizing sponges from natural keratin and polymer materials; however, simple keratin sponges have been reported to generally exhibit weak mechanical properties and limited hemostatic performance. In this study, pure keratin sponges with strong mechanical properties were obtained through the re-oxidative crosslinking of thiol groups and controlled crosslinking degree. The axial stress-strain of three sponges was tested, revealing that the maximum stress of the sponge increased with increasing crosslinking degree. Here, HK10 exhibited moderate mechanical properties and good recyclability, while HK15 sponge showed significantly stronger mechanical properties. Figure 8 A) Higher compressive stress is required to achieve 80% deformation, and HK15 is more prone to damage due to its high cross-linking and low porosity. On the other hand, this invention provides a quantitative analysis of the maximum stress, with HK5, HK10, and HK15 at 12.08 kPa, 60.88 kPa, and 205.76 kPa, respectively. Figure 8 B). Furthermore, compared to collagen and gelatin sponges, keratin sponges exhibited superior mechanical strength in the tests conducted (n=5), while gelatin sponges showed the weakest mechanical properties.

[0109] Biocompatibility and in vitro coagulation of keratin sponges

[0110] Results of hemolysis experiment of keratin sponge

[0111] This invention employs an in vitro hemolysis method to assess the blood compatibility of sponges, a widely used approach. A saline group served as the negative control, and a deionized water group as the positive control. The hemolysis rates of HK5, HK10, and HK15 sponges were measured. Images of the centrifuged supernatant and final hemolysis rates for all sponges and the control group are shown below. Figure 9 As shown, the hemolysis rates of the HK5, HK10, and HK15 sponge groups were only 0.46%, 0.42%, and 1.45%, respectively, all within the acceptable range for biomaterials (less than 5%), and these values ​​are superior to those reported in other studies. This indicates that the hemostatic sponge exhibits excellent blood compatibility.

[0112] In vitro coagulation test results of keratin sponge

[0113] In vitro coagulation assays were performed using clotting time (BCT) and clotting index (BCI). In this study, commercially available gelatin sponges and collagen sponges were selected as controls. The control group consisted of two sets of commercially available hemostatic sponges: collagen sponges and gelatin sponges. BCT results showed that the clotting time of all sponges, including HK sponges, collagen sponges, and gelatin sponges, was shorter than that of the control group, but there was no significant difference in clotting time in whole blood among the different sponges. Figure 10 A). However, it is important to consider that during massive hemorrhage or fluid resuscitation, the coagulation status may deteriorate due to the consumption or dilution of coagulation factors. To assess whether this material can maintain high coagulation capacity under such conditions, a blood dilution model was developed. In this model, blood was diluted 40% with physiological saline, and HK10 sponge, known for its excellent mechanical and fluid absorption properties, along with commercial sponges, were selected for blood dilution experiments. Surprisingly, in diluted whole blood, HK10 exhibited better hemostatic properties than collagen and gelatin sponges ( Figure 10 (B) Based on this phenomenon, the present invention hypothesizes that the sponge may affect certain clotting factors in the blood, thus demonstrating its advantage in diluted blood with relatively low levels of clotting factors. This finding can provide a reference for further research on keratin sponges in patients with coagulation disorders. In the BCI experiment, a higher absorbance value of hemoglobin was observed, indicating a slower clotting rate. To further investigate this, whole blood and sponges were incubated for 2, 5, and 10 minutes, respectively, and then the absorbance of hemoglobin was tested. Figure 10 C indicates that the absorbance of hemoglobin decreases with increasing incubation time, suggesting that the coagulation-promoting effect of HK sponge is significantly better than that of gelatin sponge, but slightly worse than that of collagen sponge. No significant difference among the three only appeared after an incubation time of 10 minutes. See the attached images for details. Figure 10 As shown in D.

[0114] Research on coagulation mechanism

[0115] Previous reports have shown that sponges, due to their three-dimensional scaffold structure, can rapidly enrich blood components through their liquid absorption properties, thereby promoting the coagulation process. During coagulation, the enriched red blood cells promote platelet activation by releasing ADP. Subsequently, activated platelets can promote thrombin formation, activate coagulation factors XI and XII, and accelerate the intrinsic coagulation process. Therefore, the adhesion behavior of sponges to platelets and red blood cells in blood is the focus of this invention.

[0116] Platelet-erythrocyte adhesion effect

[0117] Figure 11 The results clearly show that numerous blood cells effectively adhere to the surface of all sponges, exhibiting irregular aggregation and activated deformation. Compared to gelatin and collagen sponges, keratin sponges show greater blood cell enrichment. This invention hypothesizes that the aggregation of erythrocytes is due to the three-dimensional porous structure of the keratin sponge. Regarding the platelet deformation and activation, this invention hypothesizes that this is because no cross-linking agent was added during keratin synthesis, leading to the retention of more bioactive fragments, thereby activating platelets with the charge carried by the free amino groups on the protein.

[0118] Platelet activation ratio assay results

[0119] Research reports indicate that gelatin and collagen sponges can activate platelets, thereby accelerating blood clotting. Keratin has been shown to activate platelets in previous studies. Therefore, this invention investigated the effect of keratin sponges on platelets using flow cytometry. This invention selected FITC and PE to label CD41 (typically expressed on inactive platelets) and CD62P (expressed on activated platelets), respectively. This invention screened platelet populations by FITC expression and identified activated platelets expressing PE. Results are as follows... Figure 12 As shown, the platelet activation rate in the untreated platelet group was only 0.41%, while the activation rate of keratin sponge was the highest, at 21.10%. Furthermore, the activation rates of collagen sponge and gelatin sponge were 8.45% and 3.52%, respectively. This is likely because no other components are added during the synthesis of keratin sponge, thus retaining more bioactive fragments and stimulating platelet activation. Therefore, these findings indicate that keratin sponge is effective in activating platelets and can promote the extrinsic coagulation process.

[0120] Measurement results of coagulation factor XII

[0121] The platelet-activated coagulation process depends on the activation of coagulation factors in the coagulation cascade. In the intrinsic coagulation pathway, activation of coagulation factor XII leads to activation of coagulation factor X, which in turn induces thrombin production. To further investigate the effect of keratin sponges on the intrinsic coagulation pathway, this invention examined the interaction between keratin and coagulation factor XII. To assess the effect of keratin on coagulation factors in a diluted blood model, platelet-rich plasma was diluted 30% and then incubated with keratin sponges for 2, 5, and 10 minutes, respectively. After incubation, the production of coagulation factor XII was assessed. Interestingly, after two minutes of incubation, the coagulation factor XII content in the HK sponge was significantly higher than that in other commercial sponge groups, peaking at 5 minutes. At 10 minutes, the coagulation factor content decreased slightly, but the overall content in the HK sponge group was still higher than that in the control group and the commercial sponge group. Figure 13 ).

[0122] Thrombin measurement results

[0123] Furthermore, the same method was used to test thrombin production after incubation with the materials for 5, 10, 15, and 20 minutes. Interestingly, compared to the control group and the gelatin group, the HK sponge and collagen sponge produced significantly higher thrombin levels after 5 minutes of incubation, peaking at 10 minutes. Conversely, the thrombin content in the gelatin group gradually increased in the first 15 minutes, reaching its highest point at 15 minutes, and then began to decrease. Similarly, the thrombin content in the control group gradually increased in the first 20 minutes, reaching its highest value at 20 minutes. Figure 14 According to the literature review presented in this invention, it has been found that the animal coagulation system does indeed release substances after reaching its peak to reduce the levels of coagulation factors and thrombin, forming a negative feedback regulation.

[0124] Based on the above results, this invention hypothesizes that keratin can rapidly absorb blood through its porous structure, enriching blood cells and promoting the coagulation process in a short time. Furthermore, data show that keratin exhibits superior effects in promoting thrombin activation and coagulation factor activation in a diluted blood model compared to gelatin sponges and collagen sponges. This invention hypothesizes that this is because the absence of cross-linking agents superior to keratin allows for the preservation of more bioactive fragments, which may promote platelet activation, stimulate the activation of coagulation factor XII and thrombin, and promote the intrinsic coagulation process in animals. This result provides great potential for future exploration of the coagulation mechanism of keratin.

[0125] In vivo hemostatic properties of keratin sponges

[0126] In rat liver penetration and scratch models, the hemostatic performance of the sponge was evaluated by the amount of bleeding and the time to hemostasis. Because the liver is richly vascularized, compression should not be used for hemostasis, as strong pressure can cause liver rupture. To simulate incompressible bleeding in the liver, a standard circular penetrating wound was created on the liver. Based on the overall performance of the three groups of HK sponges, HK10 was selected as the experimental group for animal experiments.

[0127] Hemostatic effect of liver biopsy model in SD rats

[0128] In a rat liver biopsy model, the control group lost 1.8047g of blood within 825s. After applying keratin sponge, the hemostasis time was shortened to 297s, and the blood loss was reduced to 0.3988g. The hemostasis time in the collagen sponge group and the gelatin sponge group was reduced to 358.3s and 518.3s, respectively. Figure 15 A), blood loss decreased to 0.5609g and 1.0357g respectively. Figure 15 B). Although there was no significant difference between keratin sponges and collagen sponges, these results precisely remind the present invention that keratin sponges can replace collagen sponges for hemostasis.

[0129] Hemostatic effect of SD rat liver incision model

[0130] In a liver incision model, this invention simulated mild bleeding in the organ. The control group lost 1.2971g of blood within 368.3s, while the gelatin sponge group slightly reduced the amount of bleeding, controlling 0.5674g of bleeding within 322s. Figure 16 A). The collagen group controlled bleeding within 190.7 seconds, reducing the bleeding volume to 0.2075g. Compared with the first three groups, the HK10 group showed similar hemostatic properties to collagen, controlling the bleeding volume to 0.2070g at 157 seconds. Figure 16 (B) Overall, keratin possesses hemostatic properties similar to collagen sponges and holds promise as a hemostatic alternative to collagen sponges. Furthermore, this invention predicts that keratin sponges may exhibit better hemostatic effects in cases of coagulation factor depletion or reduced activity.

[0131] In vitro and in vivo degradation properties of keratin sponges

[0132] Results of in vitro degradation experiments of sponges

[0133] Previous studies have shown that non-degradable sponges, such as commonly used gauze and bacterial cellulose dressings, require additional removal after use, which carries the risk of secondary bleeding. Therefore, the design and manufacture of biodegradable sponges is a developing trend in hemostatic material research. It has been reported that keratin can be degraded by enzymes in vivo. Therefore, this invention conducted preliminary studies on the in vitro and in vivo degradation of sponges. To test the degradation of keratin sponges, in vitro degradation experiments, subcutaneous implantation experiments in rats, and liver implantation experiments were conducted. Referring to the literature, this invention selected proteinase K and chymotrypsin for in vitro enzymatic degradation. When proteinase K was used, the HK15 group, with the highest keratin content, experienced a 47.69% weight loss within 24 hours, while the HK5 group, with the lowest keratin content, experienced a 62.99% weight loss. Similarly, in the degradation experiment using chymotrypsin, HK15 degraded by 22.19%, HK10 by 27.99%, and HK5 by 28.41% within 24 hours. Figure 17 However, the complete degradation of collagen and gelatin groups indicates that keratin sponges cannot be rapidly degraded by proteases compared to collagen and gelatin. This suggests that keratin can be used in certain engineering applications where rapid degradation is not required.

[0134] Degradation effect of sponge on the subcutaneous back of SD rats

[0135] like Figure 18 As shown, all sponges were observed to degrade in rat skin over 21 days, and all skin tissues were examined using H&E staining. Results indicated that severe inflammatory responses were observed in all groups of skin on day 3. On day 7, inflammatory cells were significantly reduced in the control group. On day 14, inflammatory cell fluid was significantly reduced in both the keratin group and the commercial sponge group. On day 21, almost no inflammation was observed. This suggests that the HK sponge is very similar to the commercial sponge, exhibits good biocompatibility, and can degrade in animals.

[0136] in conclusion

[0137] This invention develops a pure, natural keratin sponge without any additives and demonstrates that it exhibits the same excellent hemostatic effect as commercially available collagen sponges in rat liver penetrating hemorrhage and rat liver scratch hemorrhage models. In both models, the keratin sponge group showed superior hemostatic effect compared to the gelatin sponge group, exhibiting hemostatic properties similar to collagen. However, experiments exploring coagulation mechanisms revealed that keratin can enhance the activity of thrombin and coagulation factor XII in the blood, playing a promoting role in the coagulation process. Therefore, this indicates that keratin may significantly influence the coagulation process. In summary, keratin is widely available, inexpensive, and easily obtained in nature. Sponges prepared from keratin can exhibit excellent hemostatic properties similar to collagen sponges and are expected to become a substitute for collagen sponges. Furthermore, this invention's research on the relationship between keratin and coagulation mechanisms lays the foundation for further exploration of its hemostatic mechanism in the future.

[0138] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0139] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A human hair keratin hemostatic sponge, characterized in that, The human hair keratin hemostatic sponge is obtained by foaming and freeze-drying water-soluble keratin powder. The molecular weight of the water-soluble keratin is 50-60 kDa. The water-soluble keratin is obtained by breaking the disulfide bonds in insoluble keratin and reducing it to thiol groups. The content of water-soluble keratin in the human hair keratin hemostatic sponge is 10%. The human hair keratin hemostatic sponge is prepared through the following steps: (1) Preparation of water-soluble keratin powder: human hair was washed with 0.5% w / v SDS solution to remove surface dust and oil, and then dried. Subsequently, a thioglycolic acid (TGA) solution with pH adjusted to 11 using NaOH solution was added to the dried hair to break the disulfide bonds of cystine in the hair at 37°C, thereby extracting soluble keratin. The resulting keratin-containing reduced solution was collected, and the remaining soluble keratin was fully dissolved by stirring and washing with 100 mM Tris solution. Then, a second cleaning was performed with deionized water. The filtrate was collected and centrifuged to remove the residue. Subsequently, hydrochloric acid was added to the solution to precipitate keratin. The obtained keratin precipitate was then dissolved in sodium hydroxide solution and filtered and centrifuged using different ultrafiltration centrifuge tubes to obtain water-soluble keratin solutions with different molecular weights. The obtained water-soluble keratin solutions were frozen at -80°C overnight and then dried to obtain water-soluble keratin powder. (2) Synthesis of human hair keratin hemostatic sponge: Select high molecular weight water-soluble keratin powder with a molecular weight of 50-60kDa, dissolve it in cysteine ​​solution, add SDS for rapid foaming, then pour it into a mold for making sponge and freeze dry it. After oxidizing in air for 48-72 h, wash the residual compound with distilled water and then freeze dry it again to obtain human hair keratin hemostatic sponge.

2. The human hair keratin hemostatic sponge according to claim 1, characterized in that, In step (1), the concentration of the thioglycolic acid (TGA) solution is 1M.

3. The human hair keratin hemostatic sponge according to claim 1, characterized in that, In step (1), 30KD and 50KD ultrafiltration centrifuge tubes are used.

4. The human hair keratin hemostatic sponge according to claim 1, characterized in that, In step (1), the water-soluble keratin solution needs to be hydrolyzed within 48 hours.

5. The human hair keratin hemostatic sponge according to claim 1, characterized in that, In step (2), the content of high molecular weight water-soluble keratin powder is 10%.

6. The application of the human hair keratin hemostatic sponge according to claim 1 in the preparation of hemocoagulant materials.

Images