WO2024120364A1

WO2024120364A1 - Hydrogel compound and use thereof

Info

Publication number: WO2024120364A1
Application number: PCT/CN2023/136305
Authority: WO
Inventors: 宿烽; 许青良; 孔韶文; 刘苏; 张士璀
Original assignee: 青岛蓝谷多肽生物医药科技有限公司
Priority date: 2022-12-07
Filing date: 2023-12-05
Publication date: 2024-06-13
Also published as: CN118141976A

Abstract

A hydrogel compound. The hydrogel compound comprises carboxymethyl chitosan, aldehyde hyaluronic acid, and a cell. The carboxymethyl chitosan and the aldehyde hyaluronic acid undergo a Schiff base reaction to form a hydrogel. The hydrogel can provide a growth and differentiation environment for the cell, can meet the requirements for local retention, growth, differentiation, and exosome permeation of the cell, is degradable and injectable, facilitates clinical minimally invasive operations, and better exerts a relatively good effect in the treatment of skin wounds caused by burning, mechanical damage, and inflammation.

Description

A hydrogel composite and its application

Technical Field

The invention relates to the fields of biomaterials and biomedicine, and in particular to a hydrogel composite.

Background technique

Hydrogel is a type of three-dimensional network structure polymer that contains a large number of hydrophilic groups but is insoluble in water. Among them, bio-based hydrogel is a hydrogel made of natural polymer materials, including cellulose, starch, chitin, sodium alginate, and hyaluronic acid, through physical or chemical cross-linking. The composition structure and physical and chemical properties of bio-based hydrogels are similar to those of stem cell extracellular matrix, with good biocompatibility, degradability, and stimulus responsiveness. Combined with its inherent porous structure and water absorption and swelling properties, it has been widely studied and applied in the fields of drug delivery, tissue engineering, biosensing, and environmental sanitation. In recent years, based on the fact that the structure of hydrogels is similar to that of natural extracellular matrix, this type of hydrogel has the advantages of being conducive to cell survival, good biocompatibility, low implantation trauma, and easy filling. It has become the most promising tissue engineering scaffold material and is widely used in the biomedical field.

According to statistics, 26 million people are burned and scalded in my country every year, and an average of nearly 70,000 people face the treatment of burn wounds every day. Severe and extensive burns require specialized medical care and long-term hospitalization, resulting in high social costs. In severe cases, treatment options are limited and the mortality rate may reach 100%; although fluid and electrolyte balance, respiratory support and early debridement can be restored in time during treatment, adequate treatment of the wound itself is essential to improve patient prognosis. Proper coverage of the wound not only reduces fluid loss, but also reduces the risk of subsequent infection; autologous skin grafting is considered the standard of care treatment for these wounds because it provides rapid coverage and improves wound healing. However, patients with large-area burns lack sufficient healthy skin for transplantation, and for these people, treatment alternatives are advantageous. When autologous transplantation is not possible, skin substitutes with dermal matrix can be used as a treatment option for large-area wounds. These skin substitutes may be acellular or contain differentiated cells, usually autologous keratinocytes and fibroblasts. Although this approach has been successful, the culture of autologous cells is very time-consuming, and allogeneic fibroblasts or keratinocytes may be rejected shortly after placement. On the other hand, when using bioactive substance secreting cell scaffolds for treatment, the distance between cells and capillaries is required to be within 300-400μm to ensure that the cells loaded in the scaffold survive and fully function. However, it is difficult to find an implantation site that meets this condition in the treatment of burns and trauma, and other possible suitable implantation sites cannot achieve the expected ideal effect. This results in the implanted scaffold only partially attached to the surface of the human body's own capillaries, while the other parts that are not attached to the capillaries cannot be exchanged in time due to the low concentration of oxygen and nutrients, causing the loaded cells to fail to function normally, reduce survival rate or even die, and the product has a low persistence.

This urgently requires a new matrix as a carrier of in vitro cells to solve the key problem of cell survival in current wound dressings, achieve wound treatment such as burns, and save patients from the mental and physical pain caused by repeated replacement of traditional dressings and stem cell injection treatments. Physical burden.

Summary of the invention

Cord Blood Mononuclear Cells (CB-MNCs) have been investigated as a therapeutic alternative for burn wounds. In the past few years, CB-MNCs have been effectively used to treat a variety of diseases, such as blood and immune-mediated diseases, cardiac dysfunction, bone injuries, and skin ulcers, both in experimental models and in humans. CB-MNCs are considered suitable for the treatment of skin ulcers and burn wounds, not only because they are able to generate different types of cells, but also because of their paracrine potential, secreting cytokines (TGF-b, IL-10, IL-6), chemokines (CCL2, CCL5, CXCL12), and growth factors (VEGF, IGF, bFGF, SDF, HGF), which may contribute to the healing process. Another interesting property of CB-MNCs is immunomodulation, which may directly or indirectly affect different components of the immune system, thereby controlling inflammation. In addition, cord blood mononuclear cells have little immunogenicity, so readily available allogeneic cells can be used. Studies have shown that the injection of green fluorescent protein-labeled CB-MNCs around the ulcer can accelerate epithelialization, promote angiogenesis, and increase the expression of vascular and endothelial growth factors. Studies have also found that cord blood mononuclear cells can successfully heal acute and chronic wounds in experimental models and patients. Recent studies have shown that CB-MNC treatment can promote the healing of burns in rats and pigs; in addition, CB-MNC promotes angiogenesis and accelerates the development of granulation tissue. Cord blood mononuclear cells have also been shown to be effective for patients with burns who are unresponsive to conventional treatment. Rasulov verified in clinical treatment that repeated local application of autologous cord blood mononuclear cells can promote wound healing and normalize plasma levels in patients with burns. Clinical and experimental support for the use of autologous and allogeneic cord blood mononuclear cells to treat burn wounds. When using umbilical cord blood mononuclear cells to treat allogeneic and xenogeneic burn wounds, it takes a long time to expand umbilical cord blood mononuclear cells, and autologous cells may not be available for treatment immediately; based on the low expression of MHC molecules by umbilical cord blood mononuclear cells, allogeneic and xenogeneic applications are possible. Therefore, allogeneic umbilical cord blood mononuclear cells from healthy donors may be a more suitable alternative for the initial and acute care of patients with extensive burns.

Taking into account the advantages of cord blood mononuclear cells in trauma treatment and the constraints in using cord blood mononuclear cells to treat homologous and heterologous burn wounds, this research team prepared and screened hydrogels with good biosafety, and further co-cultured fibroblasts and cord blood mononuclear cells with hydrogels (NOCC/A-HA) respectively. It was found that the survival rate of surviving cells co-cultured with hydrogels in this study was greatly improved; at the same time, the study also confirmed that the hydrogel (NOCC/A-HA) of the present invention can prevent tissue adhesion when wound tissue is damaged and improve the efficiency of wound healing; in the in vitro mouse burn model treatment experiment, the hydrogel complex loaded with cord blood mononuclear cells healed the mouse burn wound faster, without scars after healing, and after 21 days of treatment, it was observed that the wound was completely closed, and the hair regeneration of the treated mice in the wound area was more obvious than that of other groups; there was no significant difference between the new skin and the surrounding skin;

The present invention was completed based on the data of hydrogel (NOCC/A-HA) co-cultured cells.

In a first aspect, the present invention provides a hydrogel composite, comprising carboxymethyl chitosan, aldehyde hyaluronic acid and cells, wherein the carboxymethyl chitosan and the aldehyde hyaluronic acid form a hydrogel through a Schiff base reaction, wherein the volume ratio of the carboxymethyl chitosan to the aldehyde hyaluronic acid is 1-10:1; the aldehyde hyaluronic acid is obtained by modifying hyaluronic acid through functional group aldehyde modification, The hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid;

Further, the cells are selected from fibroblasts, muscle cells, epithelial cells, mucosal cells and/or stem cells;

Furthermore, the stem cells are selected from one or more of umbilical cord stem cells, subtotipotent stem cells, neural stem cells, umbilical cord mesenchymal stem cells, liver stem cells, myocardial stem cells, endothelial progenitor cells, epidermal fibroblast stem cells and cartilage stem cells;

Further, the volume ratio of the carboxymethyl chitosan and hyaluronic acid is preferably 2:1, 4:1, 6:1 or 8:1;

Furthermore, the hydrogel complex also includes one or more culture media selected from DMEM, MEM, RPMI1640, fetal bovine serum (FBS), adult bovine serum (ABS), bovine serum albumin (BSA), PBS, balanced salt solution (BSS), and the like.

In a second aspect, the present invention provides a cell culture medium containing a hydrogel, wherein the cell culture medium contains carboxymethyl chitosan, aldehyde hyaluronic acid and a cell culture fluid, wherein the carboxymethyl chitosan and the aldehyde hyaluronic acid form a hydrogel through a Schiff base reaction, wherein the volume ratio of the carboxymethyl chitosan to the aldehyde hyaluronic acid is 1-10:1; the aldehyde hyaluronic acid is obtained by modifying hyaluronic acid through functional group aldehyde modification, and the hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid;

Furthermore, the culture medium is selected from one or more of DMEM, MEM, RPMI1640, fetal bovine serum (FBS), adult bovine serum (ABS), bovine serum albumin (BSA), PBS, balanced salt solution (BSS), etc.

In a third aspect, the present invention provides a use of a hydrogel complex in preparing a skin wound material.

Further, the hydrogel complex comprises carboxymethyl chitosan, aldehyde hyaluronic acid and cells, carboxymethyl chitosan and aldehyde hyaluronic acid form a hydrogel through Schiff base reaction, wherein the volume ratio of carboxymethyl chitosan to aldehyde hyaluronic acid is selected from 1-10:1; the aldehyde hyaluronic acid is obtained by hyaluronic acid through functional group aldehyde modification, and the hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid;

Furthermore, the hydrogel complex also includes one or more culture media such as DMEM, MEM, RPMI1640, fetal bovine serum (FBS), adult bovine serum (ABS), bovine serum albumin (BSA), PBS, balanced salt solution (BSS), etc.

Furthermore, the trauma refers to trauma to the skin or mucous membrane caused by burns, mechanical damage or inflammation, and the mucous membrane includes oral mucosa or nasal mucosa, etc.

Furthermore, the substance for repairing wounds may be a drug or a medical device.

In a fourth aspect, the present invention provides a method for preparing a hydrogel composite, the preparation method comprising the following steps:

Synthesis of S01 aldehyde hyaluronic acid: Sodium hyaluronate is aldehyde-modified;

S02 Synthesis of hydrogel complex: First, the required cell culture medium is injected into the sterilized aldehyde hyaluronic acid solution, and then the cells are injected into the aldehyde hyaluronic acid solution containing the cell culture medium, and finally carboxymethyl chitosan is added in proportion to form a hydrogel complex.

Furthermore, the ratio of the carboxymethyl chitosan to the hyaluronic acid is 1-10:1.

In a fifth aspect, the present invention provides a method for preparing a culture medium containing a hydrogel, the preparation method comprising the following steps:

S02 hydrogel synthesis: inject the cell culture medium into the sterilized hyaluronic acid solution, and then add carboxymethyl chitosan in proportion, wherein the ratio of carboxymethyl chitosan to hyaluronic acid is selected from 1-10:1, and finally form a hydrogel culture medium.

Furthermore, the cell culture medium includes one or more of DMEM, MEM, RPMI1640, fetal bovine serum (FBS), adult bovine serum (ABS), bovine serum albumin (BSA), PBS, balanced salt solution (BSS), etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Schematic diagram of the oxidation of hyaluronic acid to aldehyde hyaluronic acid.

Figure 2 1HNMR spectrum of A-HA.

Figure 3 Schematic diagram of the preparation of NOCC/A-HA hydrogel via Schiff base reaction.

Figure 4 SEM image of the morphology of NOCC/A-HA hydrogel.

Figure 5 Injectability and self-healing properties of NOCC/A-HA hydrogel.

Figure 6 Rheological properties of NOCC/A-HA hydrogel.

Figure 7 Equilibrium swelling property of NOCC/A-HA hydrogel.

Figure 8 Compatibility of NOCC/A-HA hydrogel with cells

Figure 9 Hemolytic activity of NOCC/A-HA hydrogel

Figure 10 (a) Fluorescence staining of CB-MNCs surviving in PBS hydrogel for 1-21 days;

(b) Trend graph of the average survival rate of CB-MNCs in PBS hydrogels for 1-21 days;

(c) Fluorescence staining of CB-MNCs that survived in hydrogels prepared with 1% human albumin saline for 1-21 days;

(d) Trend graph of the average survival rate of CB-MNCs in hydrogels prepared with 1% human albumin saline for 1-21 days;

(e) Fluorescence staining of CB-MNCs that survived in DMEM hydrogels for 1-21 days;

(f) Trend graph of the average survival rate of CB-MNCs in DMEM hydrogels from 1 to 21 days.

FIG11 (a) Live and dead cell staining of stem cells cultured in vitro in DMEM medium for 0-96 h;

(b) Trend graph of the average survival rate of stem cells in DMEM medium from 0 to 96 h;

(c) Live and dead cell staining of stem cells cultured in vitro in saline containing 1% human albumin for 0-96 h;

(d) Trend graph of the average survival rate of stem cells in saline containing 1% human albumin from 0 to 96 h.

Figure 12 Wound healing after 21 days of treatment with hydrogel alone and wound healing after 21 days of treatment with hydrogel-loaded CB-MNC system.

Fig. 13 HE staining sections of the skin of mice in the Control group, Gel group, and Gel+CB-MNC group at 0-21 days.

Detailed ways

Aldehyde hyaluronic acid (A-HA): Aldehyde modified hyaluronic acid, namely, aldehyde hyaluronic acid, has the characteristics of self-crosslinking and in-situ crosslinking to form hydrogels, and can also achieve crosslinking when a small molecule crosslinking agent is added. This aldehyde hyaluronic acid and the hydrogel formed after crosslinking can be used in the fields of biomedicine and medical cosmetology. In the present invention, aldehyde hyaluronic acid is formed by the ortho-hydroxyl group in hyaluronic acid (HA) under the oxidation conditions of a strong oxidant before it can react with carboxymethyl chitosan (NOCC) to react with a Schiff base to synthesize a hydrogel.

The terms "culture medium" and "culture fluid" described herein may be a solid culture medium or a liquid culture fluid, depending on the needs of cell culture. The same culture fluid or culture medium has the same ingredients except for different forms.

Example 1 Synthesis of carboxymethyl chitosan aldehyde hyaluronic acid (NOCC/A-HA) hydrogel

1.1 Synthesis of aldehyde hyaluronic acid (A-HA)

(1) Dissolve 1.0 g of sodium hyaluronate (2.5 mmol) in 100 mL of double distilled water to a concentration of 10 mg/mL;

(2) When HA was completely dissolved, 2.5 mmol, 5 mL of sodium periodate aqueous solution was added and reacted at room temperature in the dark for 24 h;

(3) Add 1 mL of ethylene glycol to quench the unreacted sodium periodate. Stir the reaction at room temperature for 1 hour, and purify the solution by thorough dialyzing against distilled water (MWCO 10000) for 3 days, changing the water at least three times a day during the dialysis process;

(4) The dry product was obtained by freeze-drying and its structure was characterized by NMR.

1.2 Preparation of carboxymethyl chitosan aldehyde hyaluronic acid NOCC/A-HA hydrogel

(1) NOCC and A-HA were dissolved in phosphate buffered saline (PBS) at a concentration of 30 mg/mL;

(2) In situ cross-linked hydrogels were prepared by mixing NOCC and A-HA solutions in volume ratios of 2:1, 4:1, 6:1, and 8:1, and their structures were characterized by FTIR near-infrared spectroscopy.

1.3 Morphological studies

The morphology of NOCC/A-HA hydrogel was characterized by scanning electron microscopy (SEM). NOCC and A-HA were cross-linked at room temperature to form a hydrogel, and then the freeze-dried product of the hydrogel was cooled in liquid nitrogen and quickly brittle after being taken out. A thin layer of gold was applied on the cross section before observation. The test was performed on a VEGA3TESCAN electron microscope, and the surface and cross-sectional morphology of the hydrogel were observed at an accelerating voltage of 20KV.

1.4 Rheological properties study

The rheological properties of NOCC/A-HA hydrogels were measured by an ARES-G2 rotational rheometer with a 25 mm plate. The hydrogels prepared in PBS were placed on a plate for in-situ gelation and tested to analyze the changes in storage modulus G′ and loss modulus G″ with time, strain and frequency.

1.5 Equilibrium swelling properties

The swelling rate of freeze-dried hydrogels was determined by gravimetric analysis. The freeze-dried hydrogels were weighed and placed in 37°C (PBS, pH = 7.4). At set time intervals, the hydrogel samples were taken out and the surface water was wiped with filter paper and weighed. When the weight of the sample remained constant, swelling equilibrium was reached, and all experiments were repeated three times. The equilibrium swelling rate and mass loss rate were calculated using the following formula.

_M0 is the initial mass of the xerogel, _Ms is the wet mass of the swollen hydrogel, and _Md is the dry mass of the hydrogel after swelling and then freeze-drying.

1.6 Injectability and self-healing properties of NOCC/A-HA

The NOCC/A-HA hydrogel precursor solution was uniformly mixed with the neutral red solution and then loaded into a syringe for gelation, and the formed hydrogel was injected into water or written on a culture dish to test its injectability.

The self-healing behavior of the hydrogels was tested by preparing hydrogel samples in PBS, some of which were dyed red with neutral red, dividing the samples into two pieces, and then immediately placing the red gel piece and the transparent gel piece together at 37 °C to test their self-healing behavior.

1.7 Experimental Results

(1) Synthesis flow chart of aldehyde hyaluronic acid (A-HA) (see Figure 1);

(2) The obtained aldehyde hyaluronic acid was characterized by NMR (see Figures 2a-2b);

H NMR spectrum of NOCC (Figure 2a); H NMR spectrum of A-HA (Figure 2b); NOCC presents characteristic peaks at 3.1ppm (H-2), 3.5-4.0ppm (H-3 to H-6), and a small single peak at 2.2ppm, which is assigned to the methyl proton of the N-acetyl group; two small peaks are detected at 4.2 and 4.4ppm, which are assigned to the -CH2COO- protons at the N position of C2 and the O position of C6 of NOCC, respectively, which are consistent with the carboxymethyl substitution on the amino group (N position) and the primary hydroxyl group (O position); the broad signal between 3.2 and 3.7ppm of A-HA corresponds to the protons in the sugar ring; the methyl proton of the N-acetyl group of HA is detected at 2.0ppm; the signals observed at 4.9, 5.0 and 5.1ppm are aldehyde groups; the degree of oxidation is quantified by comparing the integrals of the aldehyde and methyl groups of the N-acetyl group in the HA backbone, and the degree of oxidation is 56%, and the degree of oxidation = (A aldehyde group/2)/(methyl group/3)A: peak area.

(3) The structure of NOCC/A-HA hydrogel was characterized by FTIR near-infrared spectroscopy (see Figure 2c);

The NOCC/A-HA hydrogel is cross-linked by Schiff base reaction between the amino groups of NOCC and the aldehyde groups of A-HA; HA exhibits characteristic peaks at 3400, 1616, and 1078 cm ^-1 , which are related to -OH absorption, antisymmetric stretching vibration of -COOH, and stretching absorption of CO bonds, respectively; the spectrum of A-HA is very similar to that of HA, except for a small band belonging to the aldehyde group at 1730 cm ^-1 ; in the spectrum of NOCC, the bands at 1599 and 1411 cm ^-1 are related to asymmetric and symmetric stretching of carboxylates (-COO), respectively; the spectrum of the hydrogel shows all the characteristic bands of NOCC and A-HA; 3200-3500 and The bands at 1100 cm ^-1 are attributed to free OH and _NH2 groups and CO stretching, respectively; the small band at 1730 cm ^-1 of aldehyde groups disappears, and the band at 1640 cm ^-1 belongs to carboxylate and imine groups, which proves the formation of hydrogel.

(4) Preparation of hydrogel (NOCC/A-HA) by Schiff base reaction (see Figure 3);

The aldehyde groups on hyaluronic acid react with the amino groups on carboxymethyl chitosan to form imine bonds (Figure 3 spheres). The cross-linking between the imine bonds forms a network cross-linking structure.

(5) The morphology of NOCC/A-HA hydrogel was characterized by scanning electron microscopy (SEM). The surface and cross-sectional morphologies of the hydrogel were observed at an accelerating voltage of 20 KV (see Figure 4 ), and its application scenarios were evaluated.

The volume ratio of NOCC and A-HA directly affects the rheological properties, uniformity and application range of the gel. When the volume of NOCC increases, the pore wall will be strengthened, while if the volume of NOCC is too large, there will not be enough cross-linking sites, and its internal structure will also be uneven. The surface morphology of hydrogels with different ratios was photographed by scanning electron microscopy, and all scales are 100μm. Specifically, the 2:1 ratio hydrogel has a larger pore size, fewer interconnected pores, and thinner and more fragile connections between pores (Figure 4a). The fluidity is stronger than that of other ratios, and it can be used for dressings on complex wounds; the 4:1 ratio hydrogel has a smaller pore size, uniform pore distribution, high porosity, and stronger and thicker pore walls (Figure 4b). The gel has strong viscoelasticity and has a uniform internal structure of interconnected pores. At the same time, the gel material can provide nutrients for cells and support cell attachment and proliferation, which is most conducive to application in wound dressings or cell attachment support. The 6:1 ratio hydrogel has an uneven pore size distribution and thicker pore walls (Figure 4c). The gel has a higher water absorption rate and strong viscoelasticity, which can be used to prevent postoperative adhesion. The 8:1 ratio hydrogel has a smaller pore size, high porosity and tightly connected pores (Figure 4d). The gel has weak water absorption and relatively stable viscoelasticity, and can be used as a matrix for local injection.

In summary, the NOCC/A-HA hydrogel with a ratio of 4:1 was demonstrated to be a suitable hydrogel as a bioscaffold, which has superior morphology with high uniformity, favorable pore size and appropriate density, and appropriate wettability. It has suitable physical properties and good biocompatibility. Finally, the NOCC/A-HA gel matrix was used as an effective bioscaffold material in tissue engineering applications to evaluate the survival rate of stem cells in vitro.

(6) Injectability and self-healing properties of NOCC/A-HA (see Figure 5 );

The self-healing properties of the hydrogel were evaluated by self-healing tests, and the hydrogel (NOCC/A-HA) was selected as 4:1. The dyed red hydrogel and the transparent hydrogel were cut into two pieces, and the two cut pieces were placed together at 37°C. The sample was fully integrated after 30 seconds. The healed hydrogel can be peeled off and support its own weight (Figure 5a). The self-healing ability of the hydrogel can be attributed to the reconstruction of the reversible imine bond cross-linking and the migration of components or component exchange between the two parts of the hydrogel. Rheological experiments were performed on the self-healing hydrogel. As shown in Figure 5b, the self-healing hydrogel showed G' and G" values (800, 500) very close to those of the original hydrogel, confirming the excellent self-healing ability. The self-healing ability of the hydrogel can ensure its structural integrity when applied to wounds in clinical treatment, thereby promoting the repair and treatment of complex wounds.

The injectability of the hydrogels was evaluated by syringe injection experiments (Figure 5c). For injectable hydrogels for biological applications, gelation time plays an important role, as shorter gelation times may lead to needle clogging, while longer gelation times may result in better performance. The results showed that NOCC/A-HA hydrogel can be continuously injected through a 26G needle and maintain its integrity after injection.

(7) Rheological properties of NOCC/A-HA (see Figure 6 );

Rheological properties of hydrogels of different ratios: The storage modulus of hydrogels of different ratios changes at 27°C and under different oscillating strain conditions (see Figure 6a). The gelation process of the mixed NOCC and A-HA aqueous solution was monitored in situ on the rheometer plate by the change of storage modulus (G') and loss modulus (G") over time. For all samples, the storage modulus was initially lower than the loss modulus. The 2:1 ratio hydrogel had the lowest viscoelasticity under the same stress conditions. The 4:1 ratio hydrogel had a higher viscoelasticity under the same stress conditions and did not change significantly with the increase of stress, which was relatively stable. The 6:1 ratio hydrogel had a lower viscoelasticity than the 4:1 ratio under the same stress conditions. The 8:1 ratio hydrogel had a lower viscoelasticity under the same stress conditions, but did not change significantly with the increase of stress, which was relatively stable.

Rheological properties of hydrogels of different ratios under different frequency conditions: At 27°C and 0.1-100 Hz frequency, the changes of hydrogels of different ratios with strain (see Figure 6b). At the same frequency, hydrogels of 2:1, 4:1, 6:1, and 8:1 ratios can all stably maintain the gel state.

(8) Equilibrium swelling properties of NOCC/A-HA.

The swelling ratio of the hydrogels of various ratios in physiological saline (pH=7.4, 37°C) for 24 hours (see FIG. 7 ).

The swelling properties of hydrogels are of great significance for their application as cell carriers or scaffolds. Swelling rates of hydrogels with different ratios in saline at 37°C for 24 hours. The 4:1 ratio hydrogel showed the highest swelling rate, which is due to the uniform porous structure that is conducive to water absorption and water retention.

Example 2 Synthesis and evaluation of hydrogel-encapsulated cells

2.1 Hydrogel and cell compatibility test

In order to evaluate the compatibility of hydrogels and cell co-culture and to determine whether the hydrogels have cytotoxicity, hydrogels with ratios of 2:1, 4:1, 6:1, and 8:1 were co-cultured with L929 cells, and the cell survival rate was determined by MTT staining experiments.

The experiment found that the cell proliferation rate of all samples was higher than 75% within 72 hours (see Figure 8). Therefore, the hydrogels in different proportions are not toxic to L929 mouse fibroblasts and can be safely used in clinical applications in accordance with ISO 10993.

2.2 Hemolytic activity of hydrogel

Take 200 μL of resuspended human red blood cell suspension and mix it with different proportions of gel extract, mix it by blowing and place it in 37°C for 1 hour. Set up blank control PBS, negative control BSA solution (20 μM) and positive control 0.1% TritonX-100. After 1 hour, take out the incubated cells and centrifuge them at 1000g for 10 minutes at room temperature. Take a sterile and clean 96-well plate and inject the supernatant obtained by centrifugation into each well, 200 μL per well. Place it under an ELISA reader to read the absorbance value of each well, and the reading wavelength is 540nm.

The experiment found that compared with the positive control group, the hydrogels in different proportions did not have a significant hemolytic effect during co-incubation with blood cells; therefore, it can be considered that this hydrogel has no hemolytic activity on mammalian blood cells (see Figure 9).

2.3 Synthesis of hydrogel-encapsulated cells and cell survival experiments

In order to evaluate the situation of culturing cells in hydrogels, the encapsulation of umbilical cord blood mononuclear cells (CB-MNC) in hydrogels under sterile conditions was first tested. That is, NOCC and A-HA solutions were sterilized by UV, and then the A-HA solution and the umbilical cord blood mononuclear cell (CB-MNC) solution were mixed in equal volumes. Then, NOCC and A-HA solutions were mixed in a sterilized well plate at a volume ratio of 2:1, 4:1, 6:1, and 8:1 to form hydrogels in situ. The cell density in the gel was fixed at 1.0×10 ⁶ cells/ml and cultured for observation.

2.4 In vitro cell survival assay

In order to observe the survival time of CB-MNC in hydrogels prepared in physiological saline and saline containing 1% human albumin, cells were encapsulated using the aforementioned cell encapsulation method, and all cells were stained with AO-PI kit after culture.

To demonstrate the beneficial effects of hydrogels on cell survival, cord blood mononuclear cells (CB-MNCs) were cultured in liquid culture media, namely DMEM and saline containing 1% human albumin. After culture, all cells were stained with AO-PI kit.

Use AO-PI to stain live/dead cells. When observing fluorescence under a fluorescence microscope, you can adjust the resolution contrast to get a more beautiful picture. Live cells are green fluorescence, and dead cells are red fluorescence. The CB-MNC system is loaded with fluorescent live and dead cell staining to detect cell survival rate.

2.5 Experimental Results

CB-MNC cytocompatibility testing was performed by live/dead cell staining to determine cell viability. In hydrogels prepared in saline, the cells were round and evenly distributed in the hydrogel after 21 days of culture (Figure 10a); on the 21st day, the cell viability was 51% (Figure 10b). CB-MNCs were also cultured in hydrogels prepared in saline containing 1% human albumin (Figure 10c) and cultured in DMEM medium (see Figure 10e). The cell morphology was very similar to that of hydrogels prepared in saline. In contrast, the survival rates of cells cultured in hydrogels prepared in saline with 1% human albumin (Figure 10d) and DMEM (Figure 10f) on day 21 were 56% and 55%, respectively, which was slightly higher than the survival rate of cells prepared in saline. These findings indicate that human albumin is beneficial to cell growth.

In the absence of hydrogel, after 12 hours of culture in DMEM, the cell viability was 47%, and all cells were apoptotic after 96 hours (Figure 11a and b). When cells were cultured in saline containing 1% human albumin (Figure 11c, d), the cell viability was 55% after 12 hours, and all cells were apoptotic after 96 hours. Therefore, although both culture media can provide nutrients for cell growth, cells ultimately cannot survive due to the lack of a good living environment. Therefore, we determined that the hydrogel network can facilitate the coupling of adhesion ligands for cell attachment, thereby providing an environment conducive to cell growth.

All umbilical cord blood mononuclear stem cells died within 96 hours after being cultured in vitro. The survival rate of stem cells in hydrogel bioscaffolds was greatly improved (numerical comparison). In PBS hydrogel/hydrogel prepared with 1% human albumin saline/DMEM hydrogel, more than 55% of stem cells survived on the 15th day after encapsulation. The results showed that NOCC/A-HA hydrogel has good cell compatibility and non-toxicity; the interconnected porous structure of NOCC/A-HA hydrogel allows nutrients and oxygen to permeate, providing a suitable environment for cells.

Example 3 Establishment and treatment of in vitro mouse scald model

A mouse scald model was established, and the hydrogel-loaded CB-MNC system was used to treat the scald, and the treatment effect and scar condition after healing were observed. A hydrogel system loaded with CB-MNC was developed to evaluate its potential in treating scalds and preventing scar formation. A deep second-degree scald model was established on the dorsal skin of mice, and the wounds were treated with an untreated scald group (Control group) as a control, a simple hydrogel group (Gel group) and a hydrogel group loaded with CB-MNC (Gel+CB-MNC group) and the wound healing was observed.

The Control group and Gel group both had wound swelling on the first day after the burn, while the Gel+CB-MNC group did not have wound swelling. This may be attributed to the ability of CB-MNCs to reduce wound inflammation. The burns in the Control group healed slowly, accompanied by darkening of the epidermis, scabs on the wound surface, and subcutaneous inflammation, and the scars were larger after healing. Moreover, scabs appeared on the wound surface from the 3rd to the 7th day, the epidermis darkened and unevenly colored, and there was an inflammatory reaction under the skin. The healing speed of the Gel group was faster than that of the control group, and the scars after healing were smaller than those of the control group. The Gel+CB-MNC group healed faster than the other two groups, and there were no scars after healing. After 21 days of treatment, complete wound closure was observed. In addition, the regeneration of hair in the wound area was more obvious in this group than in the other groups. There was no significant difference between the new skin and the surrounding skin (see Figure 12a).

The wound healing area graphs and data results show that the simple hydrogel has a certain ability to promote burn repair. The hydrogel loaded with CB-MNCs can promote burn healing, promote wound hair growth, avoid inflammatory response and reduce scar formation after healing (see Figure 12b).

Example 4 Histophysiology of wound healing

The epidermis and tissue regeneration of the healing wounds were indicated by using hematoxylin and eosin (H&E) staining. The condition of the callus tissue was observed.

On the first day after the burn, it was observed that the epidermal cell nuclei of the burned skin were condensed, the deep dermis was damaged, the collagen fibers were fused, and the hair follicles remained. These observations met the criteria for deep second-degree burns. On the 7th day, the epidermis in the Gel+CB-MNC group regenerated, the edema of the spinous cell layer was reduced, and the inflammatory infiltration was reduced; the epidermis in the Gel group regenerated, the dermis was loosely arranged, and the inflammatory infiltration was moderate; the control group showed incomplete keratinization and high inflammatory infiltration. On the 14th day, new blood vessels and regeneration appeared in the skin appendages in the Gel+CB-MNC group; the amount of blood vessel regeneration in the Gel group was small, and the inflammatory infiltration was reduced; the control group showed imperfect keratinization and reduced epidermal regeneration. On the 21st day, the epidermis and dermis in the Gel+CB-MNC group recovered well without scar formation; the epidermis thickened after the wound surface recovered in the Gel group; the skin appendage regeneration, epidermal thickening, and scar formation were less after the wound surface recovered in the control group (see Figure 13).

Claims

A hydrogel composite comprises carboxymethyl chitosan, aldehyde hyaluronic acid and cells. Carboxymethyl chitosan and aldehyde hyaluronic acid form a hydrogel through Schiff base reaction, wherein the volume ratio of carboxymethyl chitosan to aldehyde hyaluronic acid is selected from 1-10:1; the aldehyde hyaluronic acid is obtained by hyaluronic acid through functional group aldehyde modification, and the hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid.
A hydrogel composite according to claim 1, characterized in that the cells are selected from fibroblasts, muscle cells, epithelial cells, mucosal cells and/or stem cells.
The hydrogel composite according to claim 1 is characterized in that the volume ratio of the carboxymethyl chitosan and the hyaluronic acid is selected from 2:1, 4:1, 6:1 or 8:1.
A cell culture medium containing a hydrogel, the culture medium containing carboxymethyl chitosan, aldehyde hyaluronic acid and a cell culture fluid, wherein the carboxymethyl chitosan and the aldehyde hyaluronic acid form a hydrogel through a Schiff base reaction, wherein the volume ratio of the carboxymethyl chitosan to the aldehyde hyaluronic acid is selected from 1-10:1; the aldehyde hyaluronic acid is obtained by modifying hyaluronic acid through functional group aldehyde modification, and the hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid.
A cell culture medium containing a hydrogel as described in claim 4 is further characterized in that the culture fluid is selected from one or more of DMEM, MEM, RPMI1640, fetal bovine serum (FBS), adult bovine serum (ABS), bovine serum albumin (BSA), PBS and balanced salt solution (BSS).
A use of a hydrogel composite in preparing a skin wound material, the hydrogel composite comprising carboxymethyl chitosan, aldehyde hyaluronic acid and cells, wherein the carboxymethyl chitosan and the aldehyde hyaluronic acid form a hydrogel through a Schiff base reaction, wherein the volume ratio of the carboxymethyl chitosan to the aldehyde hyaluronic acid is selected from 1-10:1; the aldehyde hyaluronic acid is obtained by hyaluronic acid through functional group aldehyde modification, and the hyaluronic acid is selected from natural hyaluronic acid or artificially synthesized hyaluronic acid.
The use of a hydrogel composite in the preparation of a skin trauma material as claimed in claim 6, characterized in that the trauma refers to trauma to the skin or mucous membrane caused by burns, mechanical damage or inflammation, and the mucous membrane includes oral mucosa or nasal mucosa, etc.
The use of a hydrogel composite in preparing a skin wound substance as claimed in claim 1, characterized in that the wound repairing substance can be a drug or a medical device.
A method for preparing a hydrogel composite, the preparation method comprising the following steps:

Synthesis of S01 aldehyde hyaluronic acid: Sodium hyaluronate is aldehyde-modified;

Synthesis of S02 hydrogel complex: first inject the required cell culture fluid into the sterilized hyaluronic acid solution, then inject the cells into the hyaluronic acid solution containing the cell culture fluid, and finally add carboxymethyl chitosan according to proportion, wherein the ratio of carboxymethyl chitosan to hyaluronic acid is selected from 1-10:1.
A method for preparing a culture medium containing a hydrogel, the preparation method comprising the following steps:

Synthesis of S01 aldehyde hyaluronic acid: Sodium hyaluronate is aldehyde-modified;

S02 hydrogel synthesis: inject the cell culture medium into the sterilized hyaluronic acid solution, and then add carboxymethyl chitosan in proportion, wherein the ratio of carboxymethyl chitosan to hyaluronic acid is selected from 1-10:1, and finally form a hydrogel culture medium.