CN114732938A - Functional sponge for promoting wound healing and reducing scar formation of wound surface and preparation method and application thereof - Google Patents

Abstract

The invention discloses a functional sponge for promoting wound healing and reducing scar formation on a wound surface, and a preparation method and application thereof. The method of the invention comprises the following steps: dissolving hyaluronic acid in water with pH of 4.5-5.5, and stirring to obtain hyaluronic acid glue solution; adding EDC and NHS, stirring and reacting in a dark place, then adding the chitosan oligosaccharide-europium complex, continuing to react, injecting the reaction solution into a mould, and continuing to react in a shaking table to obtain cross-linked hyaluronic acid gel; and (3) freeze-drying to obtain the hyaluronic acid/chitosan oligosaccharide-europium functional sponge, namely the functional sponge for promoting wound healing and reducing scar formation of the wound surface. The method HAs mild experimental conditions and simple and convenient steps, uses the chitosan oligosaccharide-europium complex as a cross-linking agent, introduces the chitosan oligosaccharide with anti-inflammatory effect and europium element with vascularization promotion, and HAs obvious effects of oxidation resistance, anti-inflammatory, vascularization promotion and fibrosis reduction of the obtained HA-CE and good application prospect.

Description

A functional sponge for promoting wound healing and reducing the formation of wound scars and its preparation method and application

technical field

The invention belongs to the technical field of biomedical engineering, and in particular relates to a functional sponge for promoting wound healing and reducing the formation of scars on wound surfaces, and a preparation method and application thereof.

Background technique

For many skin damage diseases, skin burns are a common type of damage, mainly caused by high temperature, or skin damage caused by radiation, radiation, electricity, friction or exposure to chemicals, causing varying degrees of skin function and appearance. damage, which may be life-threatening. Although early burn wound excision and skin grafting can significantly improve the outcome of severe burn patients, however, slow wound healing, infection, pain and scarring remain major problems in burn research and clinical management, with the greatest challenge being the prevention of skin scarring and treatment. The incidence of scarring after skin burns is about 70%, which can cause neuropathic pain, surface irregularities, stiffness and contractures, causing great trauma to patients and their families, and the burden is not only physical but also psychological. and economical. Therefore, on the basis of promoting the healing of burn wounds, reducing the formation of scars on wounds is very important to improve the physical and psychological recovery of patients. More basic and clinical research is needed to reduce the formation of scars in the healing of burn wounds, and even achieve no scarring. repair.

The treatment of scars caused by excessive repair of skin wounds is a worldwide problem. At present, there is no effective treatment and intervention for treating scars. In adults, wound healing aims to rapidly restore the skin's barrier function, but results in scarring. Although there is currently no therapy to support scar-free wound healing in adult skin, since the discovery of the phenomenon of scar-free healing in mammalian embryos, it is suggested that the wound environment is one of the most basic guarantees for scar-free healing after skin trauma. There is no possibility of scar healing after skin trauma. The key to the phenomenon of scar-free healing in mammalian embryos lies in the fact that the wound site in the early embryo is in an anti-inflammatory environment, and maintaining an anti-inflammatory microenvironment is expected to promote scar-free wound healing. Therefore, the development of functional biomaterials that can both promote tissue regeneration and regulate tissue inflammatory microenvironment has important theoretical significance and clinical value, which can achieve rapid repair of burned skin and reduce scar formation.

Hyaluronic acid is a linear polysaccharide composed of repeating alternating disaccharide units, namely D-glucuronic acid and N-acetylglucosamine; it is also an immunoneutral polysaccharide, which is ubiquitous in the human body and affects many cells and cells. Organizational functions are critical. Meanwhile, hyaluronic acid is an important component of the extracellular matrix (ECM), and its structural and biological properties mediate its activity in cell signaling, wound repair, morphogenesis, and stromal tissue. Hyaluronic acid can be modified in a number of ways to alter the properties of the material, including modifications that result in hydrophobicity and bioactivity. After chemical modification, hyaluronic acid can be converted into a variety of physical forms, such as viscoelastic solutions, soft or hard hydrogels, electrospun fibers, nonwoven webs, macroporous and fibrous porous sponges, flexible sheets and nanomaterials. liquid etc.

SUMMARY OF THE INVENTION

In order to solve the related problems, the primary purpose of the present invention is to provide a preparation method of a functional sponge for promoting wound healing and reducing scarring on wound surfaces.

Another object of the present invention is to provide a functional sponge for promoting wound healing and reducing wound scarring obtained by the above preparation method.

Another object of the present invention is to provide the application of the above-mentioned functional sponge for promoting wound healing and reducing wound scarring.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A preparation method of a functional sponge for promoting wound healing and reducing wound scarring, comprising the following steps:

S1. Preparation of chitosan oligosaccharide-europium complex (COS-Eu):

Dissolving the chitosan oligosaccharide in water, adjusting the pH of the solution to 5-6, adding europium nitrate under stirring, and stirring the reaction; collecting the obtained sample by centrifugation, freeze-drying, to obtain a chitosan oligosaccharide-europium complex;

S2. Preparation of hyaluronic acid/chitooligosaccharide-europium functionalized sponge (HA-CE):

Dissolving hyaluronic acid in pH=4.5-5.5 water, stirring to obtain hyaluronic acid glue; adding EDC and NHS, stirring the reaction in the dark, then adding the chitosan oligosaccharide-europium complex prepared in step S1, and continuing the reaction, The reaction solution is injected into the mold, and the reaction is continued in a shaking table to obtain a cross-linked hyaluronic acid gel; freeze-drying to obtain a hyaluronic acid/chitooligosaccharide-europium functionalized sponge, which is used to promote wound healing. A functional sponge that reduces scarring.

Further, the water in the preparation method refers to deionized water.

Further, the polymerization degree of the chitosan oligosaccharide described in step S1 is 2-6, and the molecular weight is ≤1000.

Further, the ratio of the chitosan oligosaccharide and water described in step S1 is 1-2 g: 100 mL; more preferably, it is 1.2 g: 100 mL.

Further, the reagent used for adjusting the pH of the solution described in step S1 is a hydrochloric acid solution; more preferably, a 1 mol/L hydrochloric acid solution.

Further, the pH of the solution described in step S1 is 5.5.

Further, the added amount of europium nitrate described in step S1 is calculated according to the final concentration of the europium nitrate in the system of 0.01-0.03 mol/L; more preferably, it is calculated according to 0.01 mol/L.

Further, the conditions of the stirring reaction in step S1 are: temperature 25-100°C, time 5-24h; more preferably: temperature 50°C, time 5h.

Further, the rotational speed of the centrifugation described in step S1 is preferably 8000-12000 rpm; more preferably 10000 rpm.

Further, the average molecular weight of the hyaluronic acid described in step S2 is 80-130 KDA.

Further, the ratio of the hyaluronic acid and water described in step S2 is 1-3 g: 100 mL; more preferably, it is 3 g: 100 mL.

Further, the pH of the water described in step S2 is 5.

Further, the stirring time in step S2 is 20-30h; preferably 24h.

Further, the addition amounts of EDC and NHS described in step S2 are calculated according to their final concentration in the system of 20-50 mmol/L; more preferably, it is calculated according to 50 mmol/L.

Further, the conditions of the light-proof stirring reaction described in step S2 are: temperature 20-30°C (room temperature), time 10-15min; preferably: temperature 20-30°C, time 15min.

Further, the addition amount of the chitosan oligosaccharide-europium complex described in step S2 is calculated according to the chitooligosaccharide-europium complex: hyaluronic acid=mass ratio of 0.1-1:1-3; more preferably, it is calculated as 1:3 .

Further, the time for continuing the reaction described in step S2 is 25-35 min; more preferably 30 min.

Further, the time for continuing the reaction in the shaker described in step S2 is 20-30 h; more preferably 24 h.

A functional sponge for promoting wound healing and reducing wound scarring is obtained by the above preparation method.

Application of the above-mentioned functional sponge for promoting wound healing and reducing the formation of wound scars in the preparation of skin wound dressings.

Further, the skin wounds are skin wounds such as skin burns and scalds.

Mechanism of the present invention: The present invention introduces hyaluronic acid (HA) under the premise of preparing chitosan oligosaccharide-europium complex (COS-Eu) to form hyaluronic acid/chitooligosaccharide-europium that can regulate the microenvironment of wound inflammation Functionalized sponge (HA-CE). COS-Eu can improve the stretching and antioxidant properties of hyaluronic acid. In addition, the sponge can regulate the wound microenvironment in stages, polarize macrophages to form M2 type, have anti-inflammatory effects, and can promote vascularization and reduce Scar formation, allowing the wound to heal quickly and well.

Compared with the prior art, the present invention has the following advantages and effects:

1. The sponge dressing prepared by the present invention contains neither cytokines nor drugs, and starts from the effect of materials alone;

2. The experimental conditions of the present invention are mild, and the chitosan oligosaccharide-europium complex is used as the cross-linking agent, which not only introduces the anti-inflammatory effect of chitosan oligosaccharide, but also introduces the Eu element that promotes vascularization, and the steps are simple and convenient, which can realize large-scale industrialization. Batch continuous production;

3. The present invention utilizes the organic combination of -COOH and -NH ₂ and the effect of ion electrostatics to obtain the HA-CE sponge material with porous structure through drying treatment, which has anti-inflammatory effect, promotes vascularization, reduces fibrosis, and achieves the promotion of After the skin is burned, the wound heals and reduces the scarring of the wound;

4. The HA-CE functional sponge of the present invention has the advantages of simple preparation method, low cost, safety and non-toxicity, obvious effects of anti-oxidation, anti-inflammatory, promoting vascularization and reducing fibrosis, high water absorption rate and easy industrial production.

Description of drawings

Fig. 1 is the scanning electron microscope image of the COS-Eu complex prepared in Example 2;

Fig. 2 is the different sponge rheology test analysis diagram that embodiment 2 prepares;

Fig. 3 is the different sponge nuclear magnetic analysis figure that embodiment 2 prepares;

Fig. 4 is the different sponge infrared analysis figure that embodiment 2 prepares;

Fig. 5 is the scanning electron microscope picture of the HA-CE sponge that embodiment 2 prepares;

Fig. 6 is the tensile property curve diagram of the HA-CE sponge that embodiment 2 prepares;

Fig. 7 is the digital map under the wet state of the HA-CE sponge prepared in Example 2;

Figure 8 is the analysis result of the antioxidant performance of HA-CE sponge; wherein, A is the DPPH free radical scavenging rate, B is the UV-Vis spectrum of the DPPH solution after sponge treatment, C is the ABST free radical scavenging rate, HA-CE1, HA -CE5, HA-CE10 represent the HA-CE sponges obtained according to the addition amount of 0.1%, 0.5% and 1% COS-Eu respectively;

Figure 9 is a graph showing the analysis results of the anti-inflammatory performance of HA-CE sponge; wherein, A is the relative value of CD206:CD80 in each group according to the flow chart analysis, B is the expression of the pro-inflammatory factor TNF-α detected by ELISA, and C is the expression of the pro-inflammatory factor TNF-α detected by ELISA. The expression of inflammatory factor IL-10;

Figure 10 shows the results of HA-CE sponge reducing fibrosis analysis; wherein, A is the ratio of negative CD26 expression, B is the ratio of positive expression of CD26, and C is the expression of TGF-β3 gene (*p<0.05, **p<0.01 );

Figure 11 is the analysis result of HA-CE sponge promoting endothelialization; among them, A is the expression of angiogenic factor VEGF, B is the expression of angiogenic factor CD31, and C is the expression of angiogenic factor MMP91 (*p<0.05,* *p<0.01);

Figure 12 is the analysis result of HA-CE sponge to promote wound healing and scar formation; among them, A is the wound healing rate after treatment with HA, HA-COS and HA-CE10 at different times, and B is the wound surface of each group at 21d Scar Elevation Index (SEI), C is the epidermal thickness index (ETI) of the wounds in each group on 21d (*p<0.05, **p<0.01).

Detailed ways

The present invention will be described in further detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

The polymerization degree of chitosan oligosaccharide (COS) used in the following examples is 2-6, and the molecular weight is ≤1000.

The average molecular weight of the hyaluronic acid used in the following examples is 80-130 KDA.

Example 1: Optimization of the preparation process of HA-CE sponge

1. Preparation of COS-Eu complexes:

First, 1.2 g of COS was completely dissolved in 100 mL of deionized water. The pH of the COS solution was adjusted to 5.5 with 1M hydrochloric acid. The Eu( _NO3 ) ₃ was continuously stirred with the above COS solution. The obtained samples were collected by high-speed centrifugation at 10,000 rpm, frozen in liquid nitrogen, and then transferred to a freeze dryer for vacuum drying to obtain COS-Eu complexes.

Wherein, the addition amount of Eu(NO ₃ ) ₃ is calculated according to its final concentration in the system as 0.01mol/L, 0.02mol/L and 0.03mol/L respectively, and the reaction temperature is set to 25, 50, 70, 100°C, The stirring time was set to 5, 24h. According to the yield and SEM morphology of the obtained samples, the final sample preparation conditions were selected as 0.01 mol/L and continuous stirring at 50 °C for 5 h.

2. Preparation of HA-CE sponge:

Weigh hyaluronic acid and dissolve it in 100 mL of deionized water with pH=5, and stir with a stirrer at low speed for 24 h to prepare a hyaluronic acid solution. EDC and NHS with a final concentration of 50 mmol/L were added, respectively, and the reaction was stirred at room temperature in the dark for 15 min to activate the carboxyl group of hyaluronic acid. Then, the COS-Eu complex was added to the reaction system, and the reaction was continued for 30 min. Immediately afterwards, an equal amount was injected into the orifice plate with a syringe, placed on a shaker, and the speed was adjusted to 300 rpm, and the reaction was continued for 24 h. Finally, it was frozen at -20°C, and then transferred to a freeze dryer for vacuum drying to obtain HA-CE sponge.

Wherein, the content of hyaluronic acid is set to 1%, 2%, and 3%, and the content of COS-Eu complex is set to be 0.1%, 0.5%, and 1%. The final screening showed that the sponge preparation conditions were 3% hyaluronic acid, 1% chitosan oligosaccharide-europium complex.

Example 2: Preparation and characterization of HA-CE sponge

Step (1) Preparation of COS-Eu complex:

First, 1.2 g of COS was completely dissolved in 100 mL of deionized water. The pH of the COS solution was adjusted to 5.5 with 1M hydrochloric acid. 0.01 mol/L Eu(NO ₃ ) ₃ and the above COS solution were continuously stirred at 50° C. for 5 h. The obtained samples were collected, frozen with liquid nitrogen, and then freeze-dried to obtain the COS-Eu complex.

Step (2) Preparation of HA-CE sponge:

Weigh 3 g of hyaluronic acid and dissolve it in 100 mL of deionized water with pH=5, and stir with a stirrer at low speed for 24 h to prepare a hyaluronic acid solution. EDC and NHS with a final concentration of 50 mmol/L were added, respectively, and the reaction was stirred at room temperature in the dark for 15 min to activate the carboxyl group of hyaluronic acid. Then, 1.0 g of COS-Eu complex was added to the reaction system, and the reaction was continued for 30 min. Immediately afterwards, an equal amount was injected into the orifice plate with a syringe, placed on a shaker, and the speed was adjusted to 300 rpm, and the reaction was continued for 24 h. Finally, it was placed in a freezer at -20°C, and then transferred to a freeze dryer for vacuum drying.

The preparation of step (3) HA sponge:

Weigh 3 g of hyaluronic acid and dissolve it in 100 mL of deionized water with pH=5, and stir with a stirrer at low speed for 24 h to prepare a hyaluronic acid solution. The same amount was injected into the orifice plate with a syringe, placed on a shaker for 24h, and the speed was adjusted to 300rpm. Finally, it was placed at -20°C, freeze-dried, and the HA sponge was obtained.

Step (4) Preparation of HA-COS sponge:

Weigh 3 g of hyaluronic acid and dissolve it in 100 mL of deionized water with pH=5, and stir with a stirrer at low speed for 24 h to prepare a hyaluronic acid solution. EDC and NHS with a final concentration of 50 mmol/L were added, respectively, and the reaction was stirred at room temperature in the dark for 15 min to activate the carboxyl group of hyaluronic acid. Then, 1.0 g of COS was added to the reaction system, and the reaction was continued for 30 min. Immediately afterwards, an equal amount was injected into the orifice plate with a syringe, placed on a shaker, and the speed was adjusted to 300 rpm, and the reaction was continued for 24 h. Finally, it was placed at -20°C, transferred to a freeze dryer for freeze drying, and the HA-COS sponge was obtained.

The preparation of step (5) HA-Eu sponge:

Weigh 3 g of hyaluronic acid and dissolve it in 100 mL of deionized water with pH=5, and stir with a stirrer at low speed for 24 h to prepare a hyaluronic acid solution. EDC and NHS with a final concentration of 50 mmol/L were added, respectively, and the reaction was stirred at room temperature in the dark for 15 min to activate the carboxyl group of hyaluronic acid. Then, the final concentration of 0.01mol/LEu(NO ₃ ) ₃ was added into the reaction system, and the reaction was continued for 30 min. Immediately afterwards, an equal amount was injected into the orifice plate with a syringe, placed on a shaker, and the speed was adjusted to 300 rpm, and the reaction was continued for 24 h. Finally, it was placed at -20°C, freeze-dried, and the HA-Eu sponge was obtained.

Figure 1 is a scanning electron microscope image of the prepared COS-Eu complexes.

Figure 2 is the rheological test analysis diagram of different sponges prepared.

Figure 3 is the NMR analysis chart of different sponges prepared.

Figure 4 is an infrared analysis chart of different sponges prepared.

Figure 5 is a scanning electron microscope image of the prepared HA-CE sponge.

Figure 6 is a graph showing the tensile properties of the prepared HA-CE sponge.

Figure 7 is a digital image of the prepared HA-CE sponge in wet state.

According to the rheological test and analysis results in Figure 2, HA, HA-COS and HA-CE were screened out for nuclear magnetic test analysis and infrared spectrum analysis. From the results of NMR analysis in Fig. 3, it can be seen that the new chemical shift at δ=2.89ppm is the proton on -HN-C=O, indicating that in HA-COS and HA-CE, the -NH ₂ on the COS structure and the - in HA COOH organically combined.

It can be seen from the infrared spectrum of Fig. 4 that HA-COS and HA-CE have an obvious characteristic absorption peak at 3296.17cm ^-1 , which is offset from the -NH ₂ characteristic absorption peak of COS (3697.3cm ^-1 ). shift, indicating that the -COOH carboxyl group of HA reacts with _-NH2 of COS, which affects the position of _-NH2 . In addition, HA-COS and HA-CE have characteristic absorption peaks at 1618.79 cm ^-1 and 1415.30 cm ^-1 , which are antisymmetric and symmetric stretching vibration peaks of -COOH in HA (respectively at 1618.79 cm ^-1 and 1408.12 cm ^{-1 )} . ) showed a slight change in peak intensity or position, further indicating that -COOH of HA was organically combined with -NH ₂ of COS. The absorption peaks of all groups around 1034.66cm ^-1 are characteristic absorption peaks of sugar, indicating that the organic combination between HA and COS or the introduction of Eu ions does not affect the sugar ring structure of HA or COS.

It can be seen from the tensile properties curve in Figure 6 that the COS-Eu complex can significantly improve the tensile properties of hyaluronic acid.

Example 3: Performance analysis of HA-CE sponge

1. Analysis of antioxidant properties of HA-CE sponge

1,1-Diphenyl-2-picrylhydrazine radical (DPPH) is widely used in the quantitative determination of the antioxidant capacity of samples. The DPPH radical has a single electron and has a strong absorption at 517nm, and its alcohol solution is purple. When there is a free radical scavenger, its absorption gradually disappears due to its single electron pairing, and the lighter the color, the lower the absorbance value.

The sponge was added to the DPPH solution (0.1 mM, 4 mL, using methanol as a solvent), and after shaking in the dark for 30 min, the color changes in each group were recorded by photographing, and the supernatant was collected and transferred to a glass cuvette. The absorbance of the sample supernatant at 517 nm was recorded using a UV spectrophotometer, and each group of samples was tested 3 times. Finally, the DPPH scavenging rate was used to characterize the scavenging effect of the sponge on DPPH free radicals, and the DPPH scavenging ability was evaluated by the following formula:

Among them, Ac and As are the absorbance values of blank group and experimental group, respectively.

The results are shown in Figure 8. It can be seen from the detection results that the addition of COS or COS-Eu complexes greatly improves the scavenging ability of the sponge to DPPH free radicals, and at the same time, the UV-Vis spectrum shows that its characteristic absorption peak at 517 nm is correspondingly reduced (Fig. 8 A and B). This is mainly because the _-NH2 or -OH structure of COS enables it to release protons, which can pair with the DPPH radical with a single electron, thus possessing antioxidant activity. On the other hand, the introduction of COS-Eu complex, Eu ³⁺ can enhance its antioxidant capacity. In addition, COS-Eu also improved the scavenging ability of the HA sponge against 2,2-azido-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt radical (ABST ⁺ ) (Fig. 8). C).

2. Anti-inflammatory properties of HA-CE sponge

The expression of macrophage markers CD206 (M2) and CD80 (M1) in RAW264.7 cells cultured with HA-CE sponge was detected by flow cytometry. RAW264.7 cells were seeded on a 24-well plate at a density of 2 × 10 ⁵ cells per well, and sponges cut into appropriate sizes were added, and 2 mL of normal cell culture medium was added to culture for 1, 2, and 3 days. Cells were washed twice with PBS and collected. Cells were then incubated with antibody solutions containing FITC-CD206 (1:200, Invitrogen) or PE-CD80 (1:200, Biolegend) for 30 min at 4°C. The cells were resuspended in PBS, and the ratio of FITC and PE was detected using a flow cytometer (FACSCalibur ^™ , BD Bioscience) before analysis. At the same time, the cell supernatant was collected at each culture time point, and ELISA was used to analyze the expression of inflammation-related factors (IL-10, TNF-α).

As shown in A in Figure 9, the values of CD206:CD80 in the sponge group and the IL-4 group were higher than those in the blank group, indicating that the sponge can promote the polarization of macrophages to form M2 type. In addition, the value of CD206:CD80 in the HA sponge group was also higher than that in the blank group. After consulting the literature, it was known that small molecules of HA also had anti-inflammatory effects, but after the addition of COS-Eu, the value of CD206:CD80 was greatly improved ( Days 1 and 3), indicating that COS-Eu can enhance the ability of sponges to stimulate macrophage polarization to form M2. As can be seen from B and C in Figure 9, for the pro-inflammatory factor TNF-α, the expression levels of TNF-α in the sponge and IL-4 groups were lower than those in the blank group, and on days 2 and 3, the TNF-α expression in the HA-CE group was lower than that in the blank group. -α expression level was lower than that of the HA group. For the anti-inflammatory factor IL-10, on the 1st and 3rd day, the expression levels of IL-10 in the sponge group and the IL-4 group were higher than those in the blank group; on the 3rd day, the IL-10 expression in the HA-CE10 group The expression level was higher than that of the other groups. The reason for this may be that the HA-CE sponge is gradually degraded slowly over time, and COS also acts as a small molecule on macrophages, thereby exerting its anti-inflammatory effect.

3. In vitro HA-CE sponge reduces fibrosis analysis

L929 cells were seeded on a 24-well plate at a density of 2×10 ⁵ cells per well, and then an appropriate size of HA-CE sponge was carefully placed into the well plate, and 1.5 mL of normal cell culture medium was added to culture for 24 h. The HA-CE sponge was then removed, and the cells were trypsinized and collected by centrifugation. Cells were collected by washing twice with phosphate buffered saline (PBS). Cells were then incubated with an antibody solution containing FITC-CD26 (1:200, Raybiotech) for 30 minutes at 4°C. Cells were resuspended in PBS and the proportion of FITC was detected using a flow cytometer (FACS Calibur ^™ , BD Bioscience) prior to analysis. In addition, the expression of fibrosis-related gene (TGF-β3) was detected by qRT-PCR.

As shown in A and B in Figure 10, the CD26- cells in the HA ^- CE10 sponge group increased, while the CD26 ⁺ cells decreased, indicating that the sponge can inhibit the fibrosis of L929 cells. In addition, the reduction of CD26 ⁺ cells in the HA-CE10 group was significantly higher than that in the HA and HA-COS groups, indicating that the introduction of COS-Eu can increase the ability of the sponge to inhibit fibrosis, which is expected to reduce scars in the late stage of skin damage repair. Play an important role. It has been reported in the literature that TGF-β3 inhibits scar formation, and TGF-β3 gene expression is lower in less scarred skin tissue. The potential of HA-CE10 sponge to inhibit scarring was evaluated by measuring the mRNA expression level of TGF-β3 gene after L929 cells were co-cultured with sponge. It can be seen from C in Figure 10 that HA-CE10 can significantly up-regulate the expression of TGF-β3 gene, indicating that the introduction of COS-Eu makes HA-CE10 have the potential to reduce scarring.

4. In vitro HA-CE sponge promotes endothelialization

VEGF, CD31 and MMP9 are important indicators of vascularization. VEGF can promote vascularization in the early stage of wound healing and provide nutrients for later tissue remodeling. CD31 and MMP9 are highly expressed genes in the post-healing tissue remodeling stage.

qRT-PCR detection of endothelialization-related genes (VEGF, CD31, MMP9) expression:

HUVEC cells were seeded on a 24-well plate at a density of 2 × 10 ⁵ cells per well, cultured overnight, and then an appropriate size of HA-CE sponge was carefully placed into the well plate, and 1.5 mL of normal cell culture medium was added to culture until 24h. The HA-CE sponge was then removed, and the cells were trypsinized and collected by centrifugation. Cells were collected by washing twice with phosphate buffered saline (PBS). Total RNA was extracted and then reverse transcribed into cDNA. The cDNA was mixed well with primers related to inflammatory factors (VEGF, CD31, MMP9). Real-time polymerase chain reaction (qRT-PCR) analysis was performed using a real-time PCR system (Applied Biosystems). The results were analyzed using the 2- ^ΔΔCt method.

It can be seen from Figure 11 that the HA-CE10 sponge up-regulates the expression of VEGF and CD31 genes, the HA sponge up-regulates the gene expression of CD31 and MMP9, and the HA-COS sponge up-regulates the expression of MMP9. Each sponge has different degrees of influence on the indicators of vascularization. However, only the HA-CE10 sponge could up-regulate the expression of VEGF, indicating that HA-CE10 has the potential to promote neovascularization in the early stage of wound healing.

5. In vivo HA-CE sponge promotes wound healing and scar formation related index analysis

In the experiment, healthy SD rats (about 300 g, 18 rats) were used for the burn wound healing experiment. Specifically, after the rats were anesthetized with isoflurane, the back hair of the rats was shaved, and the rats were randomly divided into 3 groups, with n=6 in each group. Then four standardized full-thickness skin burn wounds were established on the back of the rat with a high-temperature brass probe (1.5 cm in diameter, 100° C., 50 s) under sterile conditions. Wound sites were treated with sterile silicone membranes, HA, HA-COS, and HA-CE10, respectively. Additionally, secure the dressing with 3M Microporous Surgical Tape to prevent it from falling out. The wound healing at 7, 14 and 21 days was observed and recorded. All dressings were changed every 3 days, during which the rats were kept on a normal diet. On the 21st day after operation, the scar formation after HA-CE10 sponge intervention wound treatment was evaluated according to formulas (a) and (b) by statistical semi-quantitative scar elevation index (SEI) and epidermal thickness index (ETI).

Among them, TWD refers to the height of the dermis of the entire wound site, and ND refers to the height of the normal dermis, which is measured from the adjacent normal dermal tissue. SEI=1 or >1 means no scar repair or scarring, respectively.

Among them, EST and En represent the height of epidermis in scar tissue and the height of epidermis in normal skin, respectively. ETI=1 or >1 indicates complete healing of the scarless wound and epidermal hypertrophy after healing, respectively.

The healing rate was calculated according to the wound healing (A in Figure 12). On the 3rd and 7th days after the operation, the wound healing rate of the HA-COS and HA-CE10 groups was significantly better than that of the blank group and the HA group. On the 7th day, the wound healing rate of the HA-CE10 group was better than that of the HA-COS group, indicating that the presence of Eu ions can help wound healing, which may be related to vascularization. At 14 and 21 days after the operation, the wounds in each group were basically healed, but there were similarities and differences in the management of wound prognosis in each group. The specific performance is that, on the 21st day after surgery, compared with other groups, the values of Scar Elevation Index (SEI) (B in Figure 12) and Epidermal Thickness Index (ETI) (C in Figure 12) treated with HA-CE10 were relatively lower , were close to 1, while the SEI and ETI values of the other groups were all greater than 1, indicating that the HA component alone had little inhibitory effect on wound scar formation, while the introduction of COS and Eu could well improve the wound prognosis, gradually approaching Scar-free healing.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the described embodiments, and any other changes, modifications, substitutions, Combinations and simplifications should all be equivalent replacement modes, which are all included in the protection scope of the present invention.

Claims (10)

1. A method of making a functional sponge for promoting wound healing and reducing scar formation on a wound surface, comprising: the method comprises the following steps:

s1, preparation of a chitosan oligosaccharide-europium complex:

dissolving chitosan oligosaccharide in water, adjusting the pH value of the solution to 5-6, adding europium nitrate under a stirring state, and stirring for reaction; centrifuging and collecting the obtained sample, and freeze-drying to obtain a chitosan oligosaccharide-europium complex;

s2, preparation of hyaluronic acid/chitosan oligosaccharide-europium functionalized sponge:

dissolving hyaluronic acid in water with pH of 4.5-5.5, and stirring to obtain hyaluronic acid glue solution; adding EDC and NHS, stirring and reacting in a dark place, adding the chitosan oligosaccharide-europium complex prepared in the step S1, continuing to react, injecting the reaction solution into a mould, and continuing to react in a shaking table to obtain cross-linked hyaluronic acid gel; and (3) freeze-drying to obtain the hyaluronic acid/chitosan oligosaccharide-europium functional sponge, namely the functional sponge for promoting wound healing and reducing scar formation.

2. The method of preparing a functional sponge for promoting wound healing and reducing scar formation on a wound as claimed in claim 1, wherein:

the polymerization degree of the chitosan oligosaccharide in the step S1 is 2-6, and the molecular weight is less than or equal to 1000;

the average molecular weight of the hyaluronic acid in the step S2 is 80-130 KDA.

3. A method of preparing a functional sponge for use in promoting wound healing and reducing scar formation on a wound as claimed in claim 1 or 2 wherein:

the ratio of the chitosan oligosaccharide to water in the step S1 is 1-2 g: 100 mL;

the addition amount of the europium nitrate in the step S1 is 0.01-0.03 mol/L of the final concentration of the europium nitrate in the system.

4. A method of preparing a functional sponge for promoting wound healing and reducing scar formation on a wound as claimed in claim 1 or 2, wherein:

the conditions of the stirring reaction described in step S1 are: the temperature is 25-100 ℃, and the time is 5-24 h.

5. A method of preparing a functional sponge for promoting wound healing and reducing scar formation on a wound as claimed in claim 1 or 2, wherein:

the ratio of the chitosan oligosaccharide to the water in the step S1 is 1.2 g: 100 mL;

the addition amount of the europium nitrate in the step S1 is 0.01mol/L according to the final concentration of the europium nitrate in the system;

the conditions of the stirring reaction described in step S1 are: the temperature is 50 ℃, and the time is 5 h;

the reagent for adjusting the pH of the solution in the step S1 is a hydrochloric acid solution;

the pH of the solution described in step S1 is 5.5;

the rotation speed of the centrifugation in the step S1 is 8000-12000 rpm.

6. A method of preparing a functional sponge for promoting wound healing and reducing scar formation on a wound as claimed in claim 1 or 2, wherein:

the ratio of the hyaluronic acid to the water in the step S2 is 1-3 g: 100 mL;

the addition amount of EDC and NHS in the step S2 is calculated according to the final concentration of EDC and NHS in the system being 20-50 mmol/L;

the addition amount of the chitosan oligosaccharide-europium complex in the step S2 is as follows: hyaluronic acid is 0.1-1: 1-3 counts.

7. A method of preparing a functional sponge for promoting wound healing and reducing scar formation on a wound as claimed in claim 1 or 2, wherein:

the ratio of the hyaluronic acid to the water in the step S2 is 3 g: 100 mL;

EDC and NHS are added in step S2 in the amount of 50mmol/L of final concentration in the system;

the addition amount of the chitosan oligosaccharide-europium complex in the step S2 is as follows: hyaluronic acid in a mass ratio of 1: 3, counting;

the pH of the water described in step S2 is 5;

the stirring time in the step S2 is 20-30 h;

the conditions for the light-shielding stirring reaction in step S2 are as follows: the temperature is 20-30 ℃, and the time is 10-15 min;

the continuous reaction time in the step S2 is 25-35 min;

the continuous reaction time in the shaking table in the step S2 is 20-30 h.

8. A functional sponge for promoting wound healing and reducing scar formation on a wound surface, which is prepared by the preparation method of any one of claims 1-7.

9. Use of a functional sponge as claimed in claim 8 for promoting wound healing and reducing scar formation on a wound bed in the manufacture of a skin wound dressing.

10. Use according to claim 9, characterized in that:

the skin wound comprises skin burn and scald.

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