CN118217443A

CN118217443A - Human hair keratin hemostatic sponge and preparation method and application thereof

Info

Publication number: CN118217443A
Application number: CN202410051428.2A
Authority: CN
Inventors: 石长灿; 杨啸; 王玉贞
Original assignee: Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
Current assignee: Wenzhou Research Institute Of Guoke Wenzhou Institute Of Biomaterials And Engineering
Priority date: 2024-01-15
Filing date: 2024-01-15
Publication date: 2024-06-21

Abstract

A hemostatic sponge of human hair is prepared by breaking disulfide bond of keratin in human hair with sodium hydroxide and mercaptoethanol, reducing to mercapto, and extracting soluble keratin. The disulfide bond is formed by utilizing the sulfydryl inherent in keratin and crosslinking with oxygen, the sponge synthesis is realized by controlling the crosslinking degree through adjusting the solid content of the keratin, and in vitro and in vivo experimental results show that the keratin has good hemostatic capability. Furthermore, keratin sponges not only promote platelet activation, but also stimulate the production of thrombin and factor XII, indicating that they have a special coagulation promoting effect in a model of coagulation disorders resulting in dilution of the factor, and furthermore, keratin sponges exhibit remarkable biocompatibility and low immunogenicity, with great potential in the treatment of non-compressible bleeding wounds.

Description

Human hair keratin hemostatic sponge and preparation method and application thereof

Technical Field

The invention relates to the technical field of new hemostatic materials, in particular to a human hair keratin hemostatic sponge and a preparation method and application thereof.

Background

Traumatic death is a global problem, with uncontrolled bleeding accounting for more than 30% of deaths in civilians and military operations. The hemostatic products commonly used at present are hemostatic powder (CELOX ™), hemostatic bandages (HemCon) and gauze (Surgiecel). Although these products have significant efficacy in treating superficial wounds and wounds with low bleeding volume, they still pose challenges for uncontrolled massive bleeding wounds and incompressible wounds that occur in the main arteries, head and neck arteries and organs of the extremities due to the difficulty in reaching the bleeding site and low liquid absorption rate.

The synthetic hemostatic sponge and the cryogel with the macroporous structure can quickly absorb moisture and expand to exert pressure on wounds to effectively stop bleeding, but the traditional nondegradable sponge needs to be taken out after the first hemostasis, and secondary bleeding and infection are easily caused. In recent years, novel degradable sponges made of natural polymers such as starch, chitosan, cellulose, etc. effectively solve these drawbacks. However, these hemostatic agents tend to have prolonged hemostasis due to lack of procoagulant activity, which may lead to increased blood loss. Previous studies reported that collagen or gelatin sponge can activate platelets and accelerate the coagulation cascade. Gelatin and collagen-based materials have been demonstrated to have excellent hemostatic capabilities. In addition, collagen is repeatedly reported to activate platelets, stimulate platelet aggregation, and have unique biological functions, and have a certain influence on active hemostasis. However, gelatin and collagen have some drawbacks. On the one hand, the residual cross-linking agent in the preparation process often has certain toxicity to organisms, and the cross-linking agents commonly used for collagen or gelatin sponge, such as formaldehyde, glutaraldehyde and the like, have obvious cytotoxicity or cancerogenic action. On the other hand, after chemical crosslinking, the biological activity of the protein is reduced.

Keratin is a protein rich in alpha-helical structural proteins and is obtainable from hair, feathers, horns and nails. Its widespread use in tissue engineering, wound healing, drug delivery and hemostasis is due to its natural abundance, low immunogenicity, good biocompatibility and easy availability. Due to the abundance of disulfide bonds, there are a variety of methods available to break these bonds during extraction. This process converts insoluble keratin into water-soluble keratin and allows the formation of sponges and hydrogels by thiol crosslinking. Furthermore, keratin has also been found to promote platelet activation and clotting. In view of these characteristics, keratin is expected to replace expensive collagen, and becomes a novel protein absorbable sponge. For example, keratin is used to mix with sodium alginate to synthesize a sponge with excellent biocompatibility and low immunogenicity. Also, keratin is mixed with polyacrylamide to prepare a sponge for treating penetrating bleeding wounds. Previous studies have also shown that the use of keratin in combination with various polymeric materials can improve the mechanical properties thereof. To our knowledge, a purely natural, additive-free keratin sponge has excellent mechanical properties and deep coagulation promoting mechanism, and is specially used for hemostasis of incompressible wounds, which has not been reported yet.

Disclosure of Invention

In order to solve the technical defects of the keratin hemostatic sponge, the invention provides a pure natural human hair keratin hemostatic sponge without adding an additional cross-linking agent, and a preparation method and application thereof.

The technical scheme adopted by the invention is as follows: a human hair keratin hemostatic sponge is prepared by foaming water-soluble keratin powder, and freeze-drying, wherein the molecular weight of the water-soluble keratin is 50-60kDa.

The water-soluble keratin is obtained by opening disulfide bonds in insoluble keratin and reducing the disulfide bonds into sulfhydryl groups.

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

The content of water-soluble keratin in the human hair keratin hemostatic sponge is 10%.

A preparation method of a human hair keratin hemostatic sponge comprises the following steps:

(1) Preparation of water-soluble keratin powder: washing human hair with 0.5% (w/v) SDS solution to remove surface dust and grease, then drying, then adding thioglycollic acid (TGA) solution adjusted to pH 11 using NaOH solution to the dried hair, breaking disulfide bonds of cystine in the hair at 37 ℃ to extract soluble keratin, collecting the resulting reduced solution containing keratin, and sufficiently dissolving the remaining soluble keratin by washing with 100mM Tris solution, then conducting a second cleaning with deionized water, then collecting filtrate and centrifuging to remove the residue, then adding hydrochloric acid to the solution to precipitate keratin, then redissolving the obtained keratin precipitate in sodium hydroxide solution, and conducting filtration and centrifugation using different ultrafiltration centrifuge tubes to obtain water-soluble keratin solutions of different molecular weights, freezing the obtained water-soluble keratin solutions overnight at-80 ℃, then drying to obtain water-soluble keratin powder;

(2) Synthesis of human hair keratin hemostatic sponge: selecting high molecular water-soluble keratin powder with molecular weight of 50-60kDa, and dissolving in cysteine solution. Adding SDS for rapid foaming, pouring into a mould for preparing sponge, freeze-drying, oxidizing in air for 48-72 h, washing residual compound with distilled water by shaking, and freeze-drying again to obtain human hair keratin hemostatic sponge.

The concentration of the thioglycollic acid (TGA) solution in the step (1) is 1M.

And (3) adopting an ultrafiltration centrifuge tube in the step (1) to adopt ultrafiltration centrifuge tubes with 30KD and 50 KD.

The water-soluble keratin solution in the step (1) is needed to be hydrolyzed within 48 hours.

The content of the high molecular water-soluble keratin powder in the step (2) is 10 percent.

An application of human hair keratin hemostatic sponge in preparing procoagulant material.

The beneficial effects of the invention are as follows: the invention provides a human hair keratin hemostatic sponge, a preparation method and application thereof, wherein sodium hydroxide and mercaptoethanol are used for breaking disulfide bonds of keratin in human hair, reducing the disulfide bonds into mercapto groups, and extracting soluble keratin from the disulfide bonds. The disulfide bond is formed by utilizing the sulfydryl inherent in keratin and crosslinking with oxygen, the sponge synthesis is realized by controlling the crosslinking degree through adjusting the solid content of the keratin, and in vitro and in vivo experimental results show that the keratin has good hemostatic capability. Furthermore, keratin sponges not only promote platelet activation, but also stimulate the production of thrombin and factor XII, indicating that they have a special coagulation promoting effect in a model of coagulation disorders resulting in dilution of the factor, and furthermore, keratin sponges exhibit remarkable biocompatibility and low immunogenicity, with great potential in the treatment of non-compressible bleeding wounds.

Drawings

FIG. 1 is an electrophoretogram of two different molecular weight keratins.

Figure 2 is a circular dichroism spectrum data of two different keratins.

FIG. 3 is a FTIR spectrum of keratin of different molecular weights from FTIR spectrum (A). (B) FTIR spectra of different keratin sponges.

Fig. 4 is an SEM image of keratin sponge.

Fig. 5 is the porosity of HK5, HK10, HK 15.

Figure 6 is the liquid absorption properties of the sponge (a) the average water absorption of three keratin sponges over 9 seconds. (B) Average blood absorption rate of three keratin sponges over 9 seconds. (C) Water absorption of keratin sponge at 3s, 6s, 9s and 12 s. (D) Blood absorption rate of keratin sponge at 3s, 6s, 9s and 12 s. (^nsP>0.05,^*** P < 0.001).

FIG. 7 is the expansion ratio of HK 5, HK10, HK15 (^nsP>0.05,^*P<0.05,^** P < 0.01).

Fig. 8 is a compressive stress-strain curve of (a) all sponges in an expanded state. (B) maximum compressive stress of all sponges.

Figure 9 is the haemolysis rate of all sponges.

Fig. 10 is the in vitro coagulation results of keratin sponge. (A) Clotting time of HK5, HK10, HK15 keratin sponge, collagen sponge and gelatin sponge in whole blood. (B) HK10 clotting time in 40% diluted blood for keratin sponge, collagen sponge and gelatin sponge. (C) HK5, HK10, HK15 keratin sponge, collagen sponge and gelatin sponge coagulation index at 2min, 5min, 10 min. (D) coagulation index pictures of all sponges.

FIG. 11 is a graph showing adhesion of platelets to red blood cells on a sponge surface.

Fig. 12 is the platelet activation ratio for all sponges.

Fig. 13 is the blood coagulation factor XII produced after incubation of all sponges with 30% platelet rich plasma for 2, 5 and 10 minutes.

Fig. 14 is thrombin generated after 5, 10, 15 and 20 minutes incubation of all sponges with 30% platelet rich plasma.

Fig. 15 is the in vivo hemostatic performance of the sponge (rat liver puncture model). (A) Hemostasis time for all sponges in the rat liver puncture model. (B) blood loss of all sponges in the rat liver puncture model.

Fig. 16 is the in vivo hemostatic performance of the sponge (rat liver incision model). (A) hemostatic time of all sponges. (B) blood loss of all sponges in the rat liver incision model.

FIG. 17 is a graph showing the degradation of all sponges in proteinase K and chymotrypsin solutions, respectively, in vitro.

Figure 18 is the degradation of the sponge under the skin of the back of the rat over 21 days.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are within the scope of the invention based on the embodiments of the invention.

Material

Human hair is obtained from a local barbershop in wenzhou. Thioglycollic acid (TGA), cysteine, were purchased from Shanghai Mai Union Biochemical technologies Co., ltd (China). Sodium Dodecyl Sulfate (SDS), hydrochloric acid, sodium hydroxide were purchased from Shanghai Aba Ding Shenghua technologies Co., ltd (China). Tris (hydroxymethyl) methyl aminomethane (Tris) was purchased from Sigma limited (united states).

Example 1 extraction of human hair Keratin

The method for extracting human hair keratin includes washing human hair with 0.5% (w/v) SDS solution for 6 hours to remove surface dust and grease, and then drying overnight. Subsequently, 1M thioglycollic acid (TGA) solution adjusted to pH 11 using NaOH solution was added to the dried hair, disulfide bonds of cystine in the hair were broken at 37 °, and thus soluble keratin was extracted within 15 hours. The resulting reduced keratin-containing solution was collected and washed with 100mM Tris solution with stirring for 2 hours to fully dissolve the remaining soluble keratin, followed by a second cleaning with deionized water. The filtrate was then collected and centrifuged at 6000rpm for 30 minutes to remove any remaining residue. Subsequently, 1mol/L hydrochloric acid was added to the solution to precipitate keratin. The keratin precipitate obtained was then redissolved in 0.1mol/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 must be accomplished within 48 hours. Finally, the keratin solution obtained was frozen at-80 ℃ overnight and then dried to obtain keratin powder.

Example 2 characterization of human hair extract

SDS-PAGE analysis.

Purity and molecular weight of keratin powder were determined by SDS-PAGE. For separating the keratin powder, a gel electrophoresis system consisting of 10% (w/v) polyacrylamide separation gel and 5% (w/v) polyacrylamide stacking gel was used. Subsequently, the human hair extract was dissolved in ultrapure water, and 20ul of the protein solution was mixed with 5ul of 5 Xload buffer. Subsequently, the mixture was boiled in the mixed solution for 10 minutes to denature the protein. The denaturing solution (10 ul) and protein markers (5 ul) were then loaded into the gel wells and the gel was subjected to 80V voltage for 1 hour and then 120 voltage for 2 hours. Subsequently, the gel strips were soaked with a disposable coomassie blue dye with gentle shaking for 30 minutes, and then soaked in deionized water for another 30 minutes to clean the dye prior to observation.

Round secondary chromatography measures the secondary structure of proteins.

The secondary structure of keratins with different molecular weights was measured using circular dichroism (CHIRASCAN PLUS, britain). For the preparation of the samples, a 2mg/ml keratin solution was used. Data were collected at 25℃with a spectral resolution of 1nm, an average time of 1 second, a scan rate of 100nm/min and a scan range of 300-190nm. Each sample was measured three times to ensure accuracy and reliability of the results. The data was analyzed using BeStSel for further interpretation and comparison.

EXAMPLE 3 characterization of Keratin sponge

Morphology observation

The internal structure of HK sponge and blood cell adhesion were observed using a scanning electron microscope (SU 8010, japan). Prior to imaging, each sample was dried in a vacuum oven and then cut with a thin blade to reveal a clean cross section. The cross section of each HK sponge was fixed to the support with the carbon tape facing upwards. Subsequently, the samples were gold sprayed for 20 seconds using a high vacuum ion sputtering instrument (EM ACE600, germany). Finally, they were observed under a scanning electron microscope at currents of 5KV and 8 uA. These procedures provide a clear and detailed examination of the internal structure and blood cell adhesion of HK sponges.

FTIR analysis.

Infrared absorption spectra of keratin extracted before and after crosslinking were measured using FTIR (Tensor II, germany). The sample was ground and crushed with dried potassium bromide in a ratio of 1:99 and then compressed into flakes. The measurement was performed in the wavelength range of 600-4000cm-1 with a spectral recording resolution of 4cm-1 for a total of 50 scans.

Determination of porosity

The porosity of the sponge was determined based on previously reported methods. Briefly, a pre-weighed sponge was soaked in ethanol for 30 minutes, and then the sponge was removed and weighed again. The porosity (P) is calculated as follows:

where N ₀ represents the initial weight, N ₁ represents the weight of the sponge after soaking, ρ represents the density of ethanol (0.785 g/cm 3), and V ₀ represents the initial volume of the sponge.

And (5) compression measurement.

The physical properties of the sponges were measured by compression testing at room temperature using an electronic universal materials tester (5944, usa). In the compression test, a cylindrical aqueous sponge (height 5mm, diameter 8 mm) was compressed at a rate of 5mm/min, and the maximum deformation was 80%. Each group was tested three times.

Water absorption/blood ratio.

The absorption rate of the sponge in water and whole blood was measured. The sponge was weighed beforehand, soaked in water/whole blood, taken out at 3 seconds, 6 seconds, 9 seconds and 12 seconds, respectively, and weighed. The absorbance (a) formula is calculated as follows:

W _t represents the weight at a certain time, t represents the soak time, and W ₀ represents the weight before absorption. The formula for the Average Absorbance (AA) over 9 seconds is as follows:

Volume expansion properties.

The pre-weighed fixed sponge was soaked in water for 20 minutes, then removed and the volume was measured again. The formula for calculating the sponge expansion rate (E) is as follows:

V ₀ and V ₁ represent the volumes of the sponge before and after the water swelling, respectively.

Blood cell adhesion assay.

According to the reported literature, the amount of platelets adhering to the sponge surface was quantified by measuring the level of Lactate Dehydrogenase (LDH) released by the lysed platelets. Fresh sodium citrate anticoagulated rabbit blood was centrifuged at 1000rpm and 4℃for 15 minutes to obtain Platelet Rich Plasma (PRP). The same mass of sponge was incubated in platelet rich plasma at 37 ℃ for 30min, then washed three times with PBS, the sponge incubated with PRP was fixed overnight at 4 ℃ with 2.5% glutaraldehyde solution, then dehydrated with different ethanol gradient solutions (50%, 75%, 85%, 95%, 100%). After freeze drying, a layer of metal was sputtered on the surface for SEM characterization.

For red blood cell adhesion, the same quality sponge was incubated with red blood cell solution for 30 minutes, then non-adhered red blood cells were washed out with PBS, the sponge incubated with red blood cell solution was fixed with 2.5% glutaraldehyde solution, dehydrated with different ethanol solutions, and finally sputtered with a layer of metal for SEM characterization.

Thrombin and factor XII assay.

Thrombin and factor XII were measured using ELISA kits. The sample was sterilized by ultraviolet irradiation for 4 hours, then 1mg of sponge was weighed and 100ul of diluted 30% recalcified plasma was added. It was incubated at 37℃for a certain period of time. When measuring thrombin generation, sponges and plasma were incubated for 5, 10, 15 and 20 minutes, respectively, prior to measurement. When measuring factor XII, sponge and plasma were incubated for 2, 5 and 10 minutes, respectively, prior to measurement.

Flow cytometry (FACS) testing.

The activation rate of platelets was detected by flow cytometry. Fresh whole blood of a sodium citrate anticoagulated mouse is taken and centrifuged at 1000rpm for 15 minutes to obtain platelet-rich plasma. Then, centrifugation was carried out at 3000rpm for 20 minutes, the supernatant was discarded to obtain a platelet pellet, the pellet was resuspended in PBS, centrifugation was carried out at 3000rpm, and the supernatant was discarded to obtain a pure platelet pellet. Platelets were resuscitated with PBS and then activated with 3mg sponge for 30 minutes, stained with FITC-CD41 and PE-CD62P fluorochromes for 30 minutes, and machine detected.

In vitro whole blood clotting properties.

According to the previously reported method, the present invention performs a whole blood clotting experiment. First, 0.2 g of sponge was weighed and put into a plastic pan. Pre-heated at 37 ℃ and then 100ul fresh sodium citrate was added to the sponge to anticoagulate the rabbit blood. Then 10ul of 0.2M Cacl2 solution was added and incubated at 37℃for 2,5 and 10 minutes, respectively. Then, 25ml of pure water was added to each dish, non-adherent blood cells were lysed for 10 minutes, and absorbance values (Abs 1) at 540nm were measured using a microplate reader. The absorbance value of 100ul of anticoagulated whole blood in 25ml of pure water was taken as a blank group (Abs 0). The Blood Coagulation Index (BCI) is calculated as follows:

Hemolysis experiment.

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

hemostatic Properties in animal models (SD rats)

All animal experiments were conducted strictly in accordance with National Institutes of Health (NIH) guidelines for laboratory animal care and use, and were approved by the laboratory animal ethics committee of the state of the China academy of sciences (protocol number: WIUCAS 21122103). SD rats (males, 8-10 weeks) were purchased from animal center in Zhejiang province. All experimental procedures were performed by the same person to ensure consistency. After the experiment was completed, all rats were euthanized according to ethical guidelines.

Rat subcutaneous implantation experiments: first, SD rats (male, 8-10 weeks) were randomly aliquoted into 4 groups. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium, after which the backs were shaved, sterilized, and a 1cm band-shaped wound was formed on the back skin. A piece of circular thin sponge (about 1 mm high, 8 mm diameter, 3 mg) was then implanted under the skin and sutured in place. Degradation of the sponge was then observed within 3, 7, 14 and 21 days.

Rat liver puncture experiment: SD rats (male, 8-10 weeks) were randomly aliquoted into 4 groups in this experiment. The abdomen of the anesthetized rat was shaved and sterilized, and then the abdomen was opened to expose the liver. The liver in the middle of the rat was then selected, on which a penetrating circular wound of 5mm diameter was formed, and then the wound was filled with sponge, and the hemostatic time and the amount of bleeding were recorded.

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

Results and discussion

Extraction of keratin and synthesis of sponge

Keratin is obtained by extraction from human hair using a reduction process. In particular, under alkaline conditions TGA is used to open disulfide bonds in human hair without breaking peptide bonds while reducing them to thiol groups, promoting the conversion of insoluble keratin to soluble keratin. Subsequently, two keratins of different molecular weights are separated by ultrafiltration centrifugation. Two different molecular weight keratin powders, low molecular weight keratin (LK) and high molecular weight keratin (HK), were obtained by freeze drying and milling. The average yield of keratin based on initially dried hair was 35.27% ± 0.31% (n=3), wherein 31.94% represents HK and 3.33% represents LK.

Chemical characterization of human hair extract

Results of SDS-PAGE of human hair extracts

The present invention has been characterized in a series of ways for both types of molecular weight sponges, first, by detecting the molecular weight of keratin by SDS-PAGE. As shown in FIG. 1, two distinct bands were observed at 50-60kDa and 10-30kDa, respectively, indicating successful separation of keratin into high molecular weight keratin and low molecular weight keratin.

Results of round two chromatography analysis of secondary structure

Subsequently, the present invention tested the helical structure of the extracted keratin. In the circular dichroism test of the present invention (fig. 2), high molecular weight keratin also exhibits a higher alpha-helical structure. Alpha-keratin has been reported to contain more alpha-helical structures and has been shown to have a promoting effect in the hemostatic process.

FTIR spectrum of human hair extract

Next, the present invention performed Fourier transform infrared spectroscopy tests on two keratin powders (FIG. 3A). A characteristic peak at 660cm-1 was observed in the keratin powder, due to the stretching vibration of C-S, confirming the presence of the reduced (-SH) form of keratin. Furthermore, the presence of peaks of amides I (1600-1700 cm-1), II (1480-1580 cm-1) and III (1220-1330 cm-1) can be observed in both keratin powders, which are important indicators for determining the secondary structure of the protein. The spectra also show the characteristic N-H tensile vibration peaks at 3290 cm-1 and 2925 cm-1 for amides A and B, respectively.

Chemical characterization of keratin sponges

Based on the series of tests carried out by the invention, the invention selects the high molecular keratin powder to synthesize the sponge for further testing and characterization. This selection is based on its higher extraction yield and alpha-helix structure level. In previous reports, keratin is often mixed with polymeric materials to make a porous sponge for hemostasis. However, in order to preserve the biological activity of keratin to the maximum and to promote the intensive study of its coagulation mechanism, the work of the present invention is particularly focused on the thiol groups of keratin and to control the crosslinking level by adjusting the solids content. In short, it requires reoxidation of the thiol groups contained in the keratin powder to form disulfide bonds and to create a crosslinking gradient by adjusting the solids content. In order to produce ideal sponge hemostatic materials, keratin solutions with different concentrations (5%, 10% and 15%) are prepared, SDS is added, and the keratin foam solution is obtained by rapid stirring and then frozen under liquid nitrogen treatment. After freeze-drying, the present invention yields three types of sponges: HK5, HK10 and HK15.

FTIR spectra of HK5, HK10, HK15 Keratin sponges

The chemical structures of the three sponges were characterized and the infrared spectrum is shown in fig. 3B. By comparing the peaks of the sponge and the powder, it is evident that there is no significant difference, except for a significant reduction in peak area at 660cm-1 in the sponge. The decrease in peak area is associated with an increase in solids content, indicating effective crosslinking of thiol groups in the sponge. Furthermore, the degree of crosslinking appears to increase with increasing solids content. The results also demonstrate that the protein structure of the sponge remains unchanged compared to the powder, indicating that crosslinking has no effect on the protein structure.

Physical characterization of keratin sponges

Morphology observation

In the process of synthesizing the sponge, the microporous structure may be formed by foaming and freeze-drying. Here, SEM images of three different solid content sponges clearly show the three-dimensional pore structure filled with internal interconnectivity. During the synthesis of the sponge, the pore structure changes due to the different degree of crosslinking. As shown in fig. 7, the microstructure of HK5 appeared loose and disordered, while HK10 and HK15 exhibited a tighter structure and smaller pores as the keratin solids content increased.

Porosity of HK5, HK10, HK15 Keratin sponge

To further analyze the pore variation, the present invention quantifies the porosity. In fig. 8, HK5 can be seen to have the highest porosity, which is attributable to the low degree of crosslinking caused by the low solids content. As the keratin content increases, the cross-linking strength increases accordingly, resulting in a decrease in porosity.

Liquid absorption Properties of HK5, HK10, HK15 Keratin sponges

The cross-linked macroporous sponge has the capability of quickly absorbing liquid, so that the sponge with a fixed shape can quickly absorb wound blood and infectious liquid, thereby reducing the risk of bacterial infection. In addition, its rapid absorption capacity enables the sponge to rapidly accumulate clotting factors at bleeding wound sites, thereby promoting blood clotting and wound repair. Keratin sponges exhibit excellent liquid absorption properties due to their pore structure and hydrophilic surface properties. To evaluate the liquid absorption properties of the sponge, experiments were performed to determine the absorption rate of water and blood. Figure 6A shows the average water absorption of the sponge over 9 seconds. The average water absorption rates of HK5 and HK10 were 5.72ml/g/s and 5.78ml/g/s, respectively, while the average water absorption ratio of HK15 was 2.81ml/g/s. The results show that there is no significant difference in average water absorption between HK5 and HK10, both exhibiting better water absorption than HK 15. However, as shown in FIG. 6B, the average blood absorption rates of HK5, HK10 and HK15 were 2.78ml/g/s, 3.29ml/g/s and 2.23ml/g/s, respectively. There was no significant difference in the average blood uptake of the three over 9 seconds, which may be due to the presence of more blood cells and proteins in the blood, resulting in increased thickness and affecting flowability. In contrast, the data (fig. 6C) indicate that all sponges are able to absorb most of the water in 5 seconds. HK sponge showed higher water absorption than gelatin sponge and collagen sponge, HK5 and HK10 showed comparable water absorption capacity. . In blood, a slight decrease in the absorption rate of the liquid was observed (fig. 6D). This suggests that the macroporous structure and hydrophilicity of the sponge contribute to its excellent water absorbing capacity. This is due to the internally crosslinked macroporous structure in HK5, which allows it to exhibit maximum water absorption, which when contacted with water for 12 seconds, is able to absorb about 50 times its own weight of water. In addition, HK10 exhibited similar performance, while HK15 absorbed only about 26 times its weight in water. In contrast, collagen sponges were found to only absorb 24 times the water, whereas gelatin sponges only absorb 15 times the water. Notably, HK sponges still exhibited excellent liquid absorption properties in blood compared to commercial sponges. These findings highlight the potential of HK sponge in demanding liquid absorption scenarios.

Expansion characteristics

As shown in fig. 7, HK5 may expand to 800% and HK10 and HK15 may expand to 600% and 200%, respectively, when the fixed-shape sponge is contacted with water. This expansion may be related to pore size formed during cross-linking of the sponge. During compression and recovery after contact with liquid, the morphology of the sponge pores was observed. Prior to contact with the liquid, the sponge is compressed and immobilized, resulting in a smaller pore. After absorbing the liquid, the excellent ability of the sponge to recover the shape exceeds the viscosity of the liquid, resulting in recovery and enlargement of the pores, thereby absorbing more liquid, rapidly enriching blood when applied to hemostatic wounds, and promoting the clotting process.

Mechanical properties

The stable mechanical properties of the sponge are critical to effectively seal and fill the wound. Previous studies have generally involved the synthesis of sponges from natural keratin and polymeric materials, and simple keratin sponges have been reported to generally have poor mechanical properties and limited hemostatic properties. In this study, pure keratin sponge with strong mechanical properties was obtained by reoxidation crosslinking of thiol groups and controlling the degree of crosslinking. The invention tests the axial stress-strain of three sponges, and finds that the maximum stress of the sponges increases with the increase of the crosslinking degree. Here, HK10 exhibited moderate mechanical properties and good recovery properties, while HK15 sponge exhibited significantly stronger mechanical properties (fig. 8A), required higher compressive stress to achieve 80% deformation, and HK15 was more susceptible to damage due to its high crosslinking and low porosity. On the other hand, the present invention quantitatively analyzes the maximum stress, HK5, HK10 and HK15 were respectively 12.08kPa, 60.88kPa and 205.76kPa (FIG. 8B). Furthermore, keratin sponges showed excellent mechanical strength (n=5) in the tests performed, compared to collagen and gelatin sponges, which showed the weakest mechanical properties.

Biocompatibility and in vitro coagulation of keratin sponges

Results of haemolysis experiments on keratin sponges

The present invention employs an in vitro hemolysis method to evaluate the blood compatibility of the sponge, which is a widely used method. The hemolysis rate of the sponges HK5, HK10 and HK15 was measured using the saline group as a negative control group and the deionized water group as a positive control group. The images of the centrifuged supernatants and the final hemolysis rates of all sponges and control groups are shown in fig. 9. The haemolysis rates of the HK5, HK10 and HK15 sponge groups were found to be only 0.46%, 0.42% and 1.45%, respectively, all within the acceptable range of biomaterials (less than 5%), and these values were superior to those reported in other studies. This indicates that the hemostatic sponge exhibits excellent blood compatibility.

Results of in vitro coagulation experiments on keratin sponges

The detection of in vitro coagulation experiments was performed By Coagulation Time (BCT) and coagulation index (BCI). In this study, commercially available gelatin sponges and collagen sponges were selected as controls. The control group included two sets of commercial hemostatic sponges, collagen sponges and gelatin sponges. BCT experimental results showed that the clotting time of all sponges, including HK sponge, collagen sponge and gelatin sponge, was shorter than the blank, but there was no significant difference in clotting time of the different sponges in whole blood (fig. 10A). However, it is important to consider that during major bleeding or fluid resuscitation, the coagulation status may deteriorate due to consumption or dilution of the coagulation factor. To evaluate whether this material can maintain high clotting ability in this state, a blood dilution model was developed. In this model, blood was diluted 40% with physiological saline and HK10 sponge, known for its good mechanical and liquid absorption properties, and commercial sponge were selected for blood dilution experiments. Surprisingly, HK10 showed better hemostatic properties than collagen and gelatin sponge in diluted whole blood (fig. 10B). Based on this, the present invention speculates that the sponge may affect certain coagulation factors in the blood, thereby demonstrating advantages in diluted blood where the coagulation factor levels are relatively low. This finding may provide a reference for further investigation of keratin sponges in patients with coagulation disorders. In BCI experiments, the higher the absorbance value of hemoglobin is observed, the slower the clotting rate. To further investigate this, whole blood and sponge were incubated for 2, 5 and 10 minutes, respectively, and then tested for hemoglobin absorbance. Fig. 10C shows that with increasing incubation time, the absorbance of hemoglobin decreases, indicating that the procoagulant effect of HK sponge is significantly better than gelatin sponge, but slightly worse than collagen sponge. No significant difference was observed between the three until the incubation time reached 10 minutes, as shown in fig. 10D.

Study of coagulation mechanism

In previous reports, due to the three-dimensional scaffold structure of sponges, they can rapidly enrich blood components by liquid absorption properties, thereby promoting the clotting process. During the clotting process, the enriched erythrocytes promote platelet activation by releasing ADP. Subsequently, activated platelets can promote thrombin formation, activate blood coagulation factors XI and XII, and accelerate the endogenous clotting process. Thus, the adhesion behavior of sponge to platelets and erythrocytes in the blood is the focus of the study of the present invention.

Platelet and erythrocyte adhesion effect

The results of fig. 11 clearly demonstrate that many blood cells effectively adhere to all sponge surfaces and exhibit irregular aggregation and activation deformation, and that keratin sponges exhibit more blood cell enrichment than gelatin sponges and collagen sponges. The present invention speculates that the aggregation of erythrocytes is due to the three-dimensional pore structure of keratin sponge. For the deformed activation of platelets, the present invention speculates that this is due to the lack of the addition of a cross-linking agent during the synthesis of keratin, which results in the retention of more biologically active fragments, thereby activating the platelets with charges carried by the free amino groups on the protein.

Platelet activation ratio measurement results

It has been reported that gelatin and collagen sponge activate platelets, thereby accelerating clotting. In previous studies, keratin has been shown to have platelet activating functions. Thus, the present invention investigated the effect of keratin sponge on platelets by flow cytometry. FITC and PE were selected for the present invention to label CD41 (typically expressed on inactive platelets) and CD62P (expressed on activated platelets), respectively. The present invention screens platelet populations by FITC expression and searches for activated platelets expressing PE. As a result, as shown in FIG. 12, the platelet activation rate of the untreated platelet group was only 0.41%, while the activation rate of the keratin sponge was the highest, which was 21.10%. In addition, the activation rates of the collagen sponge and the gelatin sponge were 8.45% and 3.52%, respectively. This is probably because keratin sponge does not add any other components during synthesis, thus retaining more bioactive fragments and stimulating platelet activation. Thus, these findings indicate that keratin sponges are effective in activating platelets and can promote the extrinsic coagulation process.

Measurement of blood coagulation factor XII

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

Measurement of thrombin

In addition, the present invention uses the same method to test thrombin generation after 5, 10, 15 and 20 minutes incubation with the material. Interestingly, HK sponge and collagen sponge produced significantly higher levels of thrombin after 5 minutes incubation, peaking at 10 minutes, compared to the blank and gelatin groups. In contrast, thrombin content in the gelatin group gradually increased during the first 15 minutes, reached its peak at 15 minutes, and then began to decrease. Likewise, the thrombin content gradually increased during the first 20 minutes and reached the highest value at 20 minutes (fig. 14). According to the literature reviews of the present invention, it was found that the coagulation system of animals does release substances to reduce the levels of coagulation factors and thrombin after reaching its peak, resulting in negative feedback regulation.

Based on the above results, the present invention speculates that keratin can rapidly absorb blood through its pore structure, enrich blood cells, and promote the coagulation process in a short time. Furthermore, the data show that keratin exhibits superior effects in promoting thrombin activation and coagulation factor activation in a diluted blood model compared to gelatin sponge and collagen sponge. The present invention speculates that this is because without the addition of cross-links superior to keratin, more biologically active fragments can be preserved, which may promote platelet activation, stimulate the activation of factor XII and thrombin, and promote the endogenous clotting process in animals. This result provides great potential for future exploration of the coagulation mechanism of keratin.

In vivo hemostatic Properties of Keratin sponge

In the rat liver penetration model and the rat liver scratch model, hemostatic performance of the sponge was evaluated by the amount of bleeding and hemostatic time. Because of the abundance of blood vessels in the liver, compression should not be used to stop bleeding, as strong pressure can lead to rupture of the liver. To simulate incompressible bleeding in the liver, the present invention creates a standard circular penetrating wound in the liver. According to the comprehensive performance of three groups of HK sponges, HK10 is selected as an experimental group of animal experiments.

Hemostatic effect of SD rat liver puncture model

In the rat liver puncture model, the blank group lost 1.8047g of blood in 825 s. After the keratin sponge is applied, the hemostatic time is shortened to 297s, and the blood loss is reduced to 0.3988g. The hemostatic time of the collagen sponge group and the gelatin sponge group was reduced to 358.3s and 518.3s, respectively (fig. 15A), and the blood loss was reduced to 0.5609g and 1.0357g, respectively (fig. 15B). Although there is no significant difference between keratin sponge and collagen sponge, these results are precisely reminiscent of the present invention, keratin sponge can be used instead of collagen sponge to stop bleeding.

Hemostatic effect of SD rat liver incision model

In the liver incision model, the present invention simulates mild bleeding of the organ. The blank lost 1.2971g of blood in 368.3s, while the gelatin sponge set slightly reduced bleeding, controlling 0.5674g of bleeding in 322s (fig. 16A). The collagen group can control bleeding within 190.7s, reducing bleeding to 0.2075g. The HK10 group showed similar hemostatic properties to collagen compared to the first three groups, with a control of bleeding volume of 0.2070g at 157s (fig. 16B). In general, keratin has hemostatic properties similar to collagen sponges, and is expected to be used as a hemostatic substitute for collagen sponges. Furthermore, the present invention predicts that keratin sponges may exhibit better hemostatic effects with reduced consumption or activity of coagulation factors.

In vivo and in vitro degradation Properties of keratin sponges

In vitro degradation test results of sponge

In previous studies, non-degradable sponges, such as common gauze and bacterial cellulose dressings in common use, require additional removal after use, which carries the risk of secondary bleeding. Therefore, the design and manufacture of degradable sponges is a developing trend in the research of hemostatic materials. It has been reported that keratin materials can be enzymatically decomposed in vivo. Thus, the invention conducted preliminary studies on in vitro and in vivo degradation of sponges. In order to test the degradation of keratin sponge, an in vitro degradation experiment, a rat subcutaneous implantation experiment and a liver implantation experiment were performed. In reference, the invention selects proteinase K and chymotrypsin for enzymatic degradation in vitro. When proteinase K was used, the highest keratin HK15 group had a 47.69% weight loss over 24 hours, while the lowest keratin HK5 group had a 62.99% weight loss. Also, in the degradation experiments using chymotrypsin, HK15 degraded 22.19%, HK10 degraded 27.99%, HK5 degraded 28.41 in 24 hours (fig. 17). However, the collagen and gelatin groups were completely degraded, indicating that keratin sponges were not rapidly degraded by proteases compared to collagen and gelatin. This suggests that keratin may be used in certain engineering applications where rapid degradation is not required.

Degradation effect of sponge under back skin of SD rat

As shown in fig. 18, all sponges were observed to degrade subcutaneously in rats within 21 days, and all skin tissues were examined using H & E staining. The results indicated that on day 3, severe inflammatory reactions occurred in each group of skin. Day 7, the empty group had significantly reduced inflammatory cells. On day 14, inflammatory cell fluids were significantly reduced in both the keratin group and the commercial sponge group. On day 21, there was little inflammation. This shows that HK sponges are very similar to commercial sponges, exhibit good biocompatibility, and can degrade in animals.

Conclusion(s)

The invention develops a purely natural keratin sponge without any additive, and proves that the purely natural keratin sponge has the same excellent hemostatic effect as the commercially available collagen sponge in a rat liver penetrating hemorrhage model and a rat liver scratch hemorrhage model. In the rat liver penetration model and the liver scratch model, the hemostatic effect of the keratin sponge group is superior to that of the gelatin sponge group, and the hemostatic performance similar to collagen is shown. However, in experiments exploring the mechanism of coagulation, it was found that keratin can enhance the activity of thrombin and factor XII in blood, exerting a promoting effect in the coagulation process. Thus, this suggests that keratin may significantly affect the coagulation process. In summary, keratin is widely available and low in cost and is readily available in nature. The sponge prepared by the method can show excellent hemostatic performance similar to that of the collagen sponge, and is hopeful to become a substitute of the collagen sponge and a leader in hemostatic direction. In addition, the research on the relation between the keratin and the coagulation mechanism can lay a foundation for further exploring the hemostasis mechanism in future.

The skilled person will know: while the invention has been described in terms of the foregoing embodiments, the inventive concepts are not limited to the invention, and any modifications that use the inventive concepts are intended to be within the scope of the appended claims.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims

1. The human hair keratin hemostatic sponge is characterized in that the human hair keratin hemostatic sponge is obtained by foaming water-soluble keratin powder and then freeze-drying, and the molecular weight of the water-soluble keratin is 50-60kDa.

2. The human hair keratin hemostatic sponge according to claim 1, wherein the water-soluble keratin is obtained by reducing disulfide bonds in insoluble keratin to sulfhydryl groups.

3. The human hair keratin hemostatic sponge according to claim 1, wherein the content of water-soluble keratin in the human hair keratin hemostatic sponge is 5-15%.

4. A human hair keratin hemostatic sponge according to claim 3, wherein the water-soluble keratin content of the human hair keratin hemostatic sponge is 10%.

5. A method for preparing a human hair keratin hemostatic sponge as claimed in claim 1, comprising the steps of:

6. The process of claim 5, wherein the mercaptoacetic acid (TGA) solution in step (1) is 1M.

7. The method of claim 5, wherein the ultrafiltration tube in step (1) is a 30KD ultrafiltration tube or a 50KD ultrafiltration tube.

8. The process according to claim 5, wherein the water-soluble keratin solution obtained in the step (1) is hydrolyzed within 48 hours.

9. The process according to claim 5, wherein the content of the polymer water-soluble keratin powder in the step (2) is 10%.

10. Use of a human hair keratin hemostatic sponge according to claim 1 for the preparation of procoagulant materials.