阅读说明：本技术 结合表皮葡萄球菌的水凝胶及其在治疗创面中的应用 (Hydrogel combined with staphylococcus epidermidis and application of hydrogel in treatment of wound surfaces ) 是由 张宏宇 肖健 李春波 许可 于 2021-09-06 设计创作，主要内容包括：本发明涉及一种结合表皮葡萄球菌的水凝胶及其在治疗创面中的应用,所述水凝胶包括表皮葡萄球菌、1.5～2.5wt％环糊精、3～5wt％聚丙烯酸、1～2wt％琼脂、适量pH调节剂和余量的去离子水,所述表皮葡萄球菌的量为3-7×10～(6)个/g水凝胶。本发明的结合表皮葡萄球菌的水凝胶能够长期稳定的保持表皮葡萄球菌的活性,该水凝胶能够快速减轻创面瘢痕。(The invention relates to a staphylococcus epidermidis-combined hydrogel and application thereof in treating wound surfaces, wherein the hydrogel comprises staphylococcus epidermidis, 1.5-2.5 wt% of cyclodextrin, 3-5 wt% of polyacrylic acid, 1-2 wt% of agar, a proper amount of pH regulator and the balance of deionized water, and the amount of the staphylococcus epidermidis is 3-7 multiplied by 10 6 Per gram of hydrogel. The hydrogel combined with the staphylococcus epidermidis can stably maintain the activity of the staphylococcus epidermidis for a long time, and the hydrogel can quickly reduce scars of wound surfaces.)

1. A staphylococcus epidermidis-binding hydrogel, comprising: comprises staphylococcus epidermidis, 1.5-2.5 wt% of cyclodextrin, 3-5 wt% of polyacrylic acid, 1-2 wt% of agar, a proper amount of pH regulator and the balance of deionized water, wherein the amount of the staphylococcus epidermidis is 3-7 multiplied by 10⁶Per gram of hydrogel.

2. The hydrogel of claim 1, wherein the staphylococcus epidermidis-binding hydrogel is a film.

3. The hydrogel according to claim 1, wherein the pH of the Staphylococcus epidermidis-binding hydrogel is 6.5-7.5.

4. The hydrogel of claim 1, the hydrogel being located in a sealed pouch.

5. The method for preparing the hydrogel according to any one of claims 1 to 4, which comprises the following steps:

(1) adding deionized water accounting for 40-60% of the total amount of the preparation into a preparation tank, adding cyclodextrin, uniformly stirring, adding a staphylococcus epidermidis suspension, and stirring for 10-20 min;

(2) dissolving polyacrylic acid in the rest deionized water, adding agar, heating to 45-60 ℃, and stirring for 15-20 min;

(3) and (3) mixing the dispersions obtained in the step (1) and the step (2), uniformly stirring at the temperature of 30-40 ℃, adjusting the pH, placing the mixture in a container with a plane bottom surface, and cooling at room temperature to obtain the gel film.

6. Use of a staphylococcus epidermidis-binding hydrogel according to any one of claims 1 to 4 for the rapid repair of superficial skin lesions.

7. Use of a staphylococcus epidermidis-binding hydrogel according to any one of claims 1 to 4 for wound scar repair.

Technical Field

The invention belongs to the field of biomedical materials, and particularly relates to a staphylococcus epidermidis-combined hydrogel and application thereof in treatment of wound surfaces.

Background

For many years, emergency treatment of sudden accidents in daily life, wound hemostasis during operation of doctors on patients and rescue and hemostasis measures of injured soldiers in war are mostly realized by physical compression, such as using bandages, gauze, rubber strips and the like, but the effect is limited. In clinical medicine, the requirements of bandaging and hemostasis of a full chain of a treatment ladder are met, particularly for acute severe wound bleeding in an emergency, due to the moisture state diversity and the dynamic complexity of an emergent bleeding environment, the existing commercially available hemostatic products cannot achieve the effects of quickly and effectively stopping bleeding and resisting bacteria, and hemorrhagic shock is very easy to occur to endanger life. The hemostatic materials in the market at present mainly comprise collagen, oxidized regenerated cellulose, alginates, zeolite, chitosan and the like.

The inability of skin wounds to heal is a major medical problem worldwide. Under normal circumstances, skin damage triggers a well-controlled inflammatory and tissue repair response that aids in pathogen clearance and promotes restoration of barrier integrity. However, in patients with chronic wounds, these reactions are either absent or overactive, leading to abnormal tissue formation and long-term impairment of the epidermal barrier. Therefore, understanding the mechanisms that initiate and regulate the inflammatory repair response of skin wounds is of great significance to the development of therapeutic and prophylactic strategies.

Skin microbiota is another major cause of skin inflammation and wound repair. In fact, the skin contains CD8+ T cells, which recognize staphylococcus epidermidis strains through non-classical Major Histocompatibility Complex (MHC) class I molecules and produce TH17 cytokines and epidermal growth factors, promoting wound healing. It is not clear whether the commensal bacteria are also involved in the innate immunity of type I IFN to damaged skin, but this is suggested by the description of the relationship between microbiota and expression of type I IFN on other epithelial surfaces such as the lungs and intestines.

Although neutrophil infiltration into mouse skin expressed high levels of CXCL10, blood neutrophils did not. Likewise, neutrophils in human skin blisters consistently express CXCL10, while neutrophils in blood do not. In the wound bed, neutrophils accumulate near the gram-positive flora, suggesting that they obtain CXCL10 expression upon contact with the skin microbiota. Colorimetric gram staining of 16S ribosomal RNA and Fluorescence In Situ Hybridization (FISH) showed that these bacteria were not present in the dermis prior to injury, but migrated from the epithelial surface to these sites of epidermal disruption. To determine whether these symbionts are responsible for inducing CXCL10 expression and subsequent recruitment of pDC to the injured skin, the authors injured the skin of sterile (GF) mice. Compared to specific pathogen-free (SPF) mouse skin, GF mouse injured skin failed to express CXCL10, nor did pDC be recruited into the wound. Thus, skin microbiota is a necessary and sufficient condition to induce expression of CXCL10 in skin wounds.

The discovery that skin commensals accelerate the wound healing response has many potential clinical implications. Since wound management is generally based on the principle of reducing bacterial load to prevent infection, this finding should increase the alertness for long-term use of antibiotics and disinfectants for acute skin wounds. Secondly, since chronic wounds, for whatever reason, exhibit stable growth of certain bacteria, leading to a state of healing arrest, therapeutic depletion of the skin microbiota, followed by local transplantation of beneficial co-biomass, it is possible to restart the acute type I IFN-driven wound healing response. This study found that among the skin microbiota, several commensal staphylococci, but not other bacteria, had the ability to induce CXCL10 expression and promote the formation of the full gene CXCL10-dna complex.

It is reported in the literature that many microorganisms living on the skin surface, such as staphylococcus epidermidis, are harmless to the body and cause inflammation once they invade the underlying layers of the skin, but not on the top or outer layers. Lygeping et al injected staphylococcus lipoteichoic acid (LTA) into the mouse epidermal layer, which then damaged its skin. Observations show that skin inflammation is much less in mice injected with LTA than in mice not injected. Researchers have also found that commensal microorganisms on the skin can modulate the activation process of Toll-like receptors after skin damage, thereby modulating the inflammatory response.

At present, the research on the drugs for treating the wound surface by combining staphylococcus epidermidis becomes a new hotspot. However, it is difficult to administer staphylococcus epidermidis, and hydrogel is a common effort for cell growth carriers. A hydrogel is a high molecular polymer network having a three-dimensional structure, which swells in water and retains a large amount of water without dissolving. The hydrogel has good biocompatibility and has wide application prospect in the fields of drug release systems, bionic materials, enzyme immobilization, tissue engineering and the like. Particularly environmentally sensitive hydrogels, are capable of responding to small changes in the external environment, such as physical or chemical changes, to produce corresponding changes in structural or physicochemical properties. Among them, temperature-sensitive hydrogels have been widely studied because of their easy control and applicability both in vivo and in vitro. In earlier researches of the application, the invention provides an intelligent hydrogel which comprises an antibacterial active ingredient, 1.5-2.5 wt% of cyclodextrin, 3-5 wt% of polyacrylic acid, 3-5 wt% of agar, a proper amount of pH regulator and the balance of deionized water. The hydrogel has temperature sensitivity, and can automatically adjust the release rate of the antibacterial component according to the temperature change of a wound part. The related research result has applied for Chinese invention patent with application number of 202110349023.3. The entire contents of this patent application are incorporated in the text of the present application and are part of the specification. The invention of the present application was developed based on the previous application, and the invention of the present application surprisingly found that the above-mentioned intelligent hydrogel, although directly applied to staphylococcus epidermidis, has the problem of low quantity of staphylococcus epidermidis, can be used as a carrier for staphylococcus epidermidis to bind staphylococcus epidermidis through further research and improvement, and surprisingly found that the hydrogel combined with staphylococcus epidermidis can reduce scar caused by wound surface.

Disclosure of Invention

Based on the above background art, the technical problem to be solved by the present invention is to provide an epidermal staphylococcus-binding hydrogel and its application in wound treatment. In order to realize the purpose of the invention, the following technical scheme is adopted:

one aspect of the present invention relates to a staphylococcus epidermidis-binding hydrogel comprising an epidermisStaphylococcus, 1.5-2.5 wt% of cyclodextrin, 3-5 wt% of polyacrylic acid, 1-2 wt% of agar, a proper amount of pH regulator and the balance of deionized water, wherein the amount of the staphylococcus epidermidis is 3-7 multiplied by 10⁶Per gram of hydrogel. The present invention helps to increase the load of staphylococcus epidermidis by adjusting the content of agar, and presumably helps to reduce the viscosity of the system after the content of agar is reduced, thereby helping to load staphylococcus epidermidis.

In a preferred embodiment of the present invention, the staphylococcus epidermidis-binding hydrogel is a film. The hydrogel is arranged into a film shape, so that the hydrogel can be directly used for dressing of a wound scar part.

In a preferred embodiment of the present invention, the pH of the Staphylococcus epidermidis-binding hydrogel is 6.5-7.5.

In a preferred embodiment of the invention, the hydrogel is located in a sealed bag.

The invention also relates to a preparation method of the staphylococcus epidermidis-combined hydrogel, which comprises the following preparation steps:

1. adding deionized water accounting for 40-60% of the total amount of the preparation into a preparation tank, adding cyclodextrin, uniformly stirring, adding a staphylococcus epidermidis suspension, and stirring for 10-20 min;

2. dissolving polyacrylic acid in the rest deionized water, adding agar, heating to 45-60 ℃, and stirring for 15-20 min;

3. and (3) mixing the dispersions obtained in the step (1) and the step (2), uniformly stirring at the temperature of 30-40 ℃, adjusting the pH, placing the mixture in a container with a plane bottom surface, and cooling at room temperature to obtain the gel film.

The invention also relates to application of the staphylococcus epidermidis-binding hydrogel in rapid repair of superficial skin injuries.

The invention also relates to application of the staphylococcus epidermidis-binding hydrogel in repairing wound scars.

Advantageous effects

The hydrogel combined with the staphylococcus epidermidis can stably maintain the activity of the staphylococcus epidermidis for a long time, and the hydrogel can quickly reduce scars of wound surfaces.

Detailed Description

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.

Example 1: a preparation method of a hydrogel combined with staphylococcus epidermidis comprises the following steps:

the concentration is 3X 10⁷Per g of staphylococcus epidermidis suspension 10g

Cyclodextrin 1g

Polyacrylic acid 2g

Agar 0.75g

proper amount of pH regulator

50g of deionized water

The preparation method comprises the following steps:

1. adding deionized water of which the total amount is half of the total amount into a preparation tank, adding cyclodextrin, uniformly stirring, uniformly mixing the cyclodextrin and a staphylococcus epidermidis suspension, and stirring for 10-20 min;

2. dissolving polyacrylic acid in the rest deionized water, adding agar, heating to 50 ℃, and stirring for 15-20 min;

3. mixing the dispersions obtained in step 1 and step 2, stirring uniformly at 37 ℃, adjusting the pH to 7.0, placing the mixture in a culture dish, and cooling at room temperature to obtain a gel film with the thickness of 1 mm.

Example 2:

the same as in example 1 except that polyacrylic acid was used in an amount of 2.5 g.

Example 3:

the same as in example 1, except that the amount of the Staphylococcus epidermidis suspension was 6 g.

Comparative example 1:

the same as in example 1, except that no staphylococcus epidermidis suspension was added.

Comparative example 2:

the same as in example 1, except that the staphylococcus epidermidis suspension was replaced by an equivalent number of lactobacillus bulgaricus suspensions.

Example 4: bacterial Activity assay

Viable strains in the gel film were observed under a microscope and counted, the gel film was observed under a 400-fold microscope, the number of strains in a single visual field was used as a counting reference, and the survival rate of the strains at different times and under different storage conditions was calculated using the number of strains in the newly prepared gel film as a base of 100%, with the results shown in table 1.

Table 1: bacterial Activity assay

The experimental results show that the hydrogel can be used for binding the staphylococcus epidermidis and can maintain the activity of the staphylococcus epidermidis for a long time. The hydrogel of the present invention is more suitable for hermetic storage in consideration of the fact that the development environment causes the loss of water in the hydrogel.

Example 5: wound surface scar repairing experiment

The method comprises purchasing 30 big-ear white rabbits with negligible male and female parts and body weight of 1.8-2.2kg, adaptively feeding for 7 days, observing each white rabbit, and feeding water without obvious abnormality, and bringing into experiment. Establishing a scar animal model: performing intravenous injection of 30g/L pentobarbital solution into ear margin for anesthesia, making circular wound surface with diameter of 1cm on rabbit ear ventral side along long axis, cutting off whole skin layer at 2 parts per ear at interval of about 3.0cm, and performing natural healing of wound surface to form hypertrophic scar hard mass after 21 days. No unhealed or unhealed wound surface infection and formation of new scar tissue.

Grouping, 30 rabbits were randomly selected and divided into 6 groups, namely blank group, example 1 group, example 2 group, example 3 group, comparative example 1 group and comparative example 2 group, and each group had 5 rabbits and 20 wounds. The blank group was a rabbit model with scar left without any intervention, the other groups were applied with gel films prepared in examples 1, 2, 3, 1 and 2, which were slightly larger than the wound surface, and applied to the newly developed scar surface of rabbit ears and fixed, and the evaluation was performed every 3 days and day 13. And (3) evaluating the curative effect: the efficacy assessment was performed using the Vancouver Scar Scale (VSS) as shown in Table 2, with higher scores indicating more scar.

Table 2: wenggol scar scale scoring standard

Statistical treatment: statistical analysis of the data was performed using SPSS software. The results are expressed as x. + -. SD and are shown in Table 3.

Table 3: comparison of VSS values before and after intervention of local scar of rabbit ear

Group of	Number of	Before intervention	Day 13
				Blank group	20	8.50±1.92	8.29±1.72
EXAMPLE 1 group	20	8.45±1.88	5.02±1.78
				EXAMPLE 2 group	20	8.42±1.83	5.12±1.91
EXAMPLE 3 group	20	8.40±1.95	6.02±1.87
				Comparative example 1 group	20	8.39±1.79	7.28±1.89
Comparative example 2 group	20	8.51±1.81	7.38±1.95

As a result: no difference in VSS score before scar intervention was assessed in 6 groups. Statistical analysis and repeated measurement analysis of variance show that except for a blank group, the VSS value of the scar after dry prognosis of other groups is reduced, and the intervention of the scar is improved to a certain extent by using a comparative example 1 group and a comparative example 2 group which do not contain staphylococcus epidermidis through VSS mean analysis, and the hypothesis is that the hydrogel disclosed by the invention can supplement water and isolate the influence of the external environment, but the VSS mean values of the comparative example 1 group and the comparative example 2 group are not significantly different (P >0.05), and the VSS values of the dry prognosis of the example 1 group, the example 2 group and the example 3 group are all reduced (P <0.05), the VSS is obviously reduced, the significant difference is obvious, and the scar is well improved. The data also show that there is some correlation between VSS values and the amount of staphylococcus epidermidis used.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.