WO2024120364A1 - 一种水凝胶复合物及其应用 - Google Patents

一种水凝胶复合物及其应用 Download PDF

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WO2024120364A1
WO2024120364A1 PCT/CN2023/136305 CN2023136305W WO2024120364A1 WO 2024120364 A1 WO2024120364 A1 WO 2024120364A1 CN 2023136305 W CN2023136305 W CN 2023136305W WO 2024120364 A1 WO2024120364 A1 WO 2024120364A1
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hyaluronic acid
hydrogel
aldehyde
carboxymethyl chitosan
cells
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PCT/CN2023/136305
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English (en)
French (fr)
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宿烽
许青良
孔韶文
刘苏
张士璀
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青岛蓝谷多肽生物医药科技有限公司
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Publication of WO2024120364A1 publication Critical patent/WO2024120364A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention relates to the fields of biomaterials and biomedicine, and in particular to a hydrogel composite.
  • Hydrogel is a type of three-dimensional network structure polymer that contains a large number of hydrophilic groups but is insoluble in water.
  • 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.
  • hydrogels 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.
  • CB-MNCs Cord Blood Mononuclear Cells
  • TGF-b, IL-10, IL-6 secreting cytokines
  • CCL2, CCL5, CXCL12 chemokines
  • VEGF VEGF, IGF, bFGF, SDF, HGF
  • CB-MNCs Another interesting property of CB-MNCs is immunomodulation, which may directly or indirectly affect different components of the immune system, thereby controlling inflammation.
  • 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.
  • cord blood mononuclear cells can successfully heal acute and chronic wounds in experimental models and patients.
  • 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.
  • umbilical cord blood mononuclear cells 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.
  • 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.
  • 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.
  • 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;
  • the cells are selected from fibroblasts, muscle cells, epithelial cells, mucosal cells and/or stem cells;
  • 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;
  • volume ratio of the carboxymethyl chitosan and hyaluronic acid is preferably 2:1, 4:1, 6:1 or 8:1;
  • 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.
  • FBS fetal bovine serum
  • ABS adult bovine serum
  • BSA bovine serum albumin
  • PBS PBS
  • balanced salt solution BSS
  • 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;
  • 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.
  • the present invention provides a use of a hydrogel complex in preparing a skin wound material.
  • 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;
  • 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.
  • 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.
  • 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 substance for repairing wounds may be a drug or a medical device.
  • the present invention provides a method for preparing a hydrogel composite, the preparation method comprising the following steps:
  • the ratio of the carboxymethyl chitosan to the hyaluronic acid is 1-10:1.
  • 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.
  • 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.
  • FBS fetal bovine serum
  • ABS adult bovine serum
  • BSA bovine serum albumin
  • PBS PBS
  • balanced salt solution BSS
  • FIG. 1 Schematic diagram of the oxidation of hyaluronic acid to aldehyde hyaluronic acid.
  • FIG. 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 6 Rheological properties of NOCC/A-HA hydrogel.
  • Figure 10 (a) Fluorescence staining of CB-MNCs surviving in PBS hydrogel for 1-21 days;
  • FIG11 (a) Live and dead cell staining of stem cells cultured in vitro in DMEM medium for 0-96 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.
  • 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.
  • 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.
  • HA hyaluronic acid
  • NOCC carboxymethyl chitosan
  • 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.
  • NOCC and A-HA were dissolved in phosphate buffered saline (PBS) at a concentration of 30 mg/mL;
  • NOCC/A-HA hydrogel 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.
  • 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.
  • the swelling rate of freeze-dried hydrogels was determined by gravimetric analysis.
  • 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
  • Md is the dry mass of the hydrogel after swelling and then freeze-drying.
  • 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.
  • 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 aldeh
  • 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
  • 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.
  • 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.
  • 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 100 ⁇ m. 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • CB-MNC umbilical cord blood mononuclear cells
  • A-HA solution and the umbilical cord blood mononuclear cell (CB-MNC) solution were mixed in equal volumes.
  • 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.
  • CB-MNCs cord blood mononuclear cells
  • AO-PI stain live/dead cells.
  • Live cells are green fluorescence
  • dead cells are red fluorescence.
  • the CB-MNC system is loaded with fluorescent live and dead cell staining to detect cell survival rate.
  • CB-MNC cytocompatibility testing was performed by live/dead cell staining to determine cell viability.
  • 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.
  • 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 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).
  • 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.

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Abstract

一种水凝胶复合物，所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，所述的水凝胶可为细胞提供生长分化环境，可满足细胞的局部留存、生长、分化及外泌体的渗透、可降解、可注射、方便临床的微创操作，更好为烫伤和机械损伤及炎症导致的皮肤创伤治疗中发挥较好的效果。

Description

一种水凝胶复合物及其应用 技术领域
本发明涉及生物材料和生物医学领域，具体涉及一种水凝胶复合物。
背景技术
水凝胶，是一类含有大量亲水基团，但不溶于水的三维网络结构聚合物。其中，生物基水凝胶是以天然高分子材料，包括纤维素、淀粉、甲壳素、海藻酸钠、透明质酸为主体通过物理或化学交联制成的水凝胶。生物基水凝胶的成分结构及理化性质类似干细胞外基质，具有良好的生物相容性、可降解性、刺激响应性等，结合其固有的多孔结构和吸水溶胀等特性，使其在药物传递、组织工程、生物传感、环境卫生等领域得到了广泛的研究和应用。近年来，基于水凝胶结构与天然细胞外基质相近，该类水凝胶具有利于细胞存活、生物相容性好、植入创伤小、便于填充等优点，成为最具潜力的组织工程支架材料，被广泛应用于生物医学领域。
据统计，我国每年有2600万人被烧烫伤，平均每天近7万人面对烫伤创面的治疗，严重和广泛的烫伤需要专门的医疗保健和长期住院，导致高昂的社会成本。在严重的病例中，治疗选择是有限的，死亡率可能达到100％；尽管治疗中可以及时恢复体液和电解质平衡、呼吸支持和早期清创，但充分处理伤口本身对于改善患者预后至关重要。适当覆盖伤口不仅可以减少液体流失，还可以降低随后感染的风险；自体皮肤移植被认为是这些伤口的标准护理治疗，因为它提供了快速的覆盖和改善伤口愈合。然而，大面积烫伤的患者缺乏足够的健康皮肤进行移植，对于这些人来说，治疗替代品是有利的。当无法进行自体移植时，带有真皮基质的皮肤替代物可作为大面积创伤的治疗选择。这些皮肤替代物可能是无细胞的或含有分化细胞，通常是自体角质形成细胞和成纤维细胞。尽管这种方法已经取得了成功，但自体细胞的培养非常耗时，异体成纤维细胞或角质形成细胞在放置后不久可能会被排斥。另一方面，采用生物活性物质分泌细胞支架进行治疗时，要求细胞与毛细血管间距在300～400μm以内，以保证支架内装载的细胞存活并完全发挥功能。然而，在烫伤创伤治疗中很难满足此条件的植入部位，其它各种可能的适合植入的部位又都无法达到预期的理想效果，这就导致植入的支架只能部分贴附在人体自身毛细血管表面，而另外未贴附毛细血管的部分由于氧气、营养物质等浓度较低，无法及时交换，致使装载的细胞不能发挥正常功能、存活率降低甚至死亡，产品的持效性较低。
这就急需一种新的基质来作为体外细胞的载体，解决目前伤口敷料中细胞存活的关键问题，实现烫伤等创面治疗，免除患者由于传统敷料反复更换和干细胞注射治疗带来的精神、 肉体负担。
发明内容
脐血单核细胞(CordBloodMononuclearCell，CB-MNC)已被研究作为烫伤创面的治疗替代物。在过去几年中，在实验模型和人类中，脐血单核细胞已被有效地用于治疗多种疾病，如血液和免疫介导疾病、心脏功能障碍、骨损伤和皮肤溃疡。脐血单核细胞被认为适合于治疗皮肤溃疡和烫伤创面，不仅因为它们能够产生不同类型的细胞，还因为它们的旁分泌潜能、分泌细胞因子(TGF-b、IL-10、IL-6)、趋化因子(CCL2、CCL5、CXCL12)和生长因子(VEGF、IGF、bFGF、SDF、HGF)，这可能有助于愈合过程。脐血单核细胞的另一个有趣特性是免疫调节，它可能直接或间接影响免疫***的不同组成部分，从而控制炎症。此外，脐血单核细胞几乎没有免疫原性，因此可以使用现成的同种异体细胞，研究指出沿着溃疡周围注射绿色荧光蛋白标记的CB-MNC能够加速上皮化，促进血管生成，并增加血管和内皮生长因子的表达。研究还发现脐血单核细胞可以成功治愈实验模型和患者的急性和慢性伤口。最近的研究表明，CB-MNC治疗可以促进大鼠和猪烫伤的愈合；此外，CB-MNC促进血管生成并加速肉芽组织的发育。脐血单核细胞也被证明对常规治疗无效的烫伤患者有效。Rasulov在临床治疗中验证反复局部应用自体脐血单核细胞能够促进伤口愈合，并使烫伤患者的血浆水平正常化，临床和实验支持使用自体和异体脐血单核细胞治疗烫伤创面。采用脐血单核细胞治疗同种和异种烫伤创面时，脐血单核细胞扩增需要较长时间，自体细胞可能无法立即用于治疗；基于脐血单核细胞呈现MHC分子的低表达，可使同种和异种应用成为可能，因此，来自健康供体的异基因脐血单核细胞，可能是广泛烫伤患者初期和急性护理的更合适的替代方案。
综合上述脐血单核细胞在创伤治疗中的优势和利用脐血单核细胞治疗同种和异种烫伤创面中存在的制约因素，本研究团队通过制备、筛选出生物安全性好的水凝胶，进一步将成纤维细胞和脐血单核细胞分别与水凝胶(NOCC/A-HA)共培养，发现本研究中水凝胶共培养存活细胞的存活率得到大幅度提升；同时研究也证实本发明的水凝胶(NOCC/A-HA)可以防止伤口组织受损时发生的组织粘连，并提高伤口愈合的效率；在体外小鼠烫伤模型治疗实验中，负载脐血单核细胞的水凝胶复合物对小鼠烫伤创伤的愈合更快，愈后无疤痕，治疗21天后，观察到伤口完全闭合，同时该治疗小鼠的毛发在伤口区的再生比其他组更明显；新皮肤与周围皮肤无明显差异；
根据水凝胶(NOCC/A-HA)共培养细胞的数据，完成了本发明。
第一方面，本发明提供一种水凝胶复合物，所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比为1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得， 所述透明质酸选自天然透明质酸或者人工合成透明质酸；
进一步的，所述细胞选自成纤维细胞、肌细胞、上皮细胞、粘膜细胞和/或干细胞等；
更进一步的，所述干细胞选自脐带干细胞、亚全能干细胞、神经干细胞、脐带间充质干细胞、肝干细胞、心肌干细胞、血管内皮祖细胞、表皮成纤维干细胞和软骨干细胞中的一种或多种；
进一步的，所述羧甲基壳聚糖和醛透明质酸的体积比优选为2:1、4:1、6:1或8:1；
进一步的，所述水凝胶复合物还包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种培养液。
第二方面，本发明提供一种含有水凝胶的细胞培养基，所述细胞培养基含有羧甲基壳聚糖、醛透明质酸和细胞培养液，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比为1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得，所述透明质酸选自天然透明质酸或者人工合成透明质酸；
进一步，所述培养液选自DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种。
第三方面，本发明提供一种水凝胶复合物在制备皮肤创伤物质中的用途。
进一步的，所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得，所述透明质酸选自天然透明质酸或者人工合成透明质酸；
进一步的，所述水凝胶复合物还包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等的一种或多种培养液。
进一步的，所述创伤是指烧伤、机械损伤或炎症导致的皮肤或粘膜的创伤，所述粘膜包括口腔粘膜或鼻黏膜等。
进一步，所述修复创伤的物质可以是药物或医疗器械。
第四方面，本发明提供一种制备水凝胶复合物的方法，所述制备方法包括如下步骤：
S01醛透明质酸的合成：透明质酸钠进行醛基化修饰；
S02水凝胶复合物合成：先将需要的细胞培养液注入灭菌后的醛透明质酸溶液中，再将细胞注入含有细胞培养液的醛透明质酸溶液中，最后按比例加入羧甲基壳聚糖，形成水凝胶复合物。
进一步的，所述羧甲基壳聚糖和醛透明质酸的比例为1-10:1。
第五方面，本发明提供一种含有水凝胶的培养基的制备方法，所述制备方法包括如下步骤：
S01醛透明质酸的合成：透明质酸钠进行醛基化修饰；
S02水凝胶合成：将细胞培养液注入灭菌后的醛透明质酸溶液中，再按比例加入羧甲基壳聚糖，其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1，最终形成水凝胶培养基。
进一步的，所述细胞培养液包括DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS、平衡盐溶液(BSS)等中的一种或多种。
附图说明
图1透明质酸氧化为醛透明质酸的示意图。
图2 A-HA的1HNMR光谱。
图3通过席夫碱反应制备NOCC/A-HA水凝胶的示意图。
图4 NOCC/A-HA水凝胶的形态扫描电镜图。
图5 NOCC/A-HA水凝胶的可注射性和自愈合性图。
图6 NOCC/A-HA水凝胶的流变性。
图7 NOCC/A-HA水凝胶的平衡溶胀性。
图8 NOCC/A-HA水凝胶与细胞相容性
图9 NOCC/A-HA水凝胶的溶血性实验
图10(a)CB-MNC在PBS单纯水凝胶中存活1-21天的荧光染色图；
(b)CB-MNC在PBS单纯水凝胶中存活1-21天的平均存活率趋势图；
(c)CB-MNC在含1％人血白蛋白生理盐水制备的水凝胶中存活1-21天的荧光染色图；
(d)CB-MNC在含1％人血白蛋白生理盐水制备的水凝胶中存活1-21天的平均存活率趋势图；
(e)CB-MNC在DMEM水凝胶中存活1-21天的荧光染色图；
(f)CB-MNC在DMEM水凝胶中存活1-21天的平均存活率趋势图。
图11(a)体外培养干细胞在DMEM培养基中0-96h的活死细胞染色；
(b)干细胞在DMEM培养基中0-96h的平均存活率趋势图；
(c)体外培养干细胞在含1％人血白蛋白生理盐水中0-96h的活死细胞染色；
(d)干细胞在含1％人血白蛋白生理盐水中0-96h的平均存活率趋势图。
图12单纯水凝胶治疗21天后的创面愈合情况和水凝胶负载CB-MNC体系治疗21天后的创面愈合情况。
图13 Control group，Gel group，Gel+CB-MNC组小鼠0-21天皮肤的HE染色切片。
具体实施方式
醛透明质酸(A-HA)：醛基化修饰的透明质酸既醛透明质酸，具有自交联和原位交联形成水凝胶的特性，也可在加入小分子交联剂的情况下实现交联，这种醛基化透明质酸及其交联后形成的水凝胶可被用于生物医药和医学美容等领域。在本发明中，醛透明质酸由透明质酸(HA)中的邻位羟基在强氧化剂氧化条件下形成醛基后，才能与羧甲基壳聚糖(NOCC)发生席夫碱反应，合成水凝胶。
本文所述术语“培养基”和“培养液”，依据细胞培养的需求，可以选择固体形式的培养基或者液体形式的培养液，同一种培养液或培养基除形态不同外，其所含成分是一致的。
实施例1羧甲基壳聚糖醛透明质酸(NOCC/A-HA)水凝胶的合成
1.1醛透明质酸(A-HA)的合成
(1)将透明质酸钠1.0g，2.5mmol溶于100mL双重蒸馏水中，浓度为10mg/mL；
(2)当HA完全溶解后，添加高碘酸钠水溶液2.5mmol，5mL，在黑暗中室温下反应24h；
(3)加入1mL乙二醇以淬灭未反应的高碘酸钠。将反应在室温下在搅拌1小时，将所得溶液通过蒸馏水进行彻底透析(MWCO 10000)3天来纯化溶液，在透析过程中每天至少换水三次；
(4)通过冷冻干燥获得干燥产物，并通过核磁进行结构表征。
1.2羧甲基壳聚糖醛透明质酸NOCC/A-HA水凝胶的制备
(1)将NOCC和A-HA以30mg/mL的浓度溶于磷酸盐缓冲液(PBS)中；
(2)通过将NOCC和A-HA溶液以2:1、4:1、6:1、8:1的体积比混合来制备原位交联的水凝胶，并通过FTIR近红外广谱进行结构表征。
1.3形态学研究
NOCC/A-HA水凝胶的形态通过扫描电子显微镜(SEM)表征。NOCC和A-HA在室温下交联形成水凝胶，然后将水凝胶的冻干产物在液氮中降温，取出后迅速脆断，在观察之前在横截面上涂上一层薄薄的金，测试在VEGA3TESCAN电子显微镜上进行，使用20KV的加速电压下观察水凝胶的表面和横截面的形貌。
1.4流变性质研究
NOCC/A-HA水凝胶的流变特性通过25mm平板的ARES-G2旋转流变仪测量。将在PBS中制备的水凝胶置于平板上原位成胶进行测试，分析储能模量G′和损耗模量G″随时间、应变及频率的变化。
1.5平衡溶胀性质
用重量分析法测定冻干水凝胶的溶胀率。将冷冻干燥的水凝胶称重，并在37℃(PBS，pH＝7.4)中。在设定的时间间隔，取出水凝胶样品用滤纸擦拭表面水后称重。当样品的重量保持恒定时，达到溶胀平衡，所有实验均重复三次。使用以下公式计算平衡溶胀率和质量损失率。

M0是干凝胶的初始质量，Ms是溶胀的水凝胶的湿质量，Md是溶胀后再冻干的水凝胶的干燥质量。
1.6 NOCC/A-HA的可注射性和自愈性
将NOCC/A-HA水凝胶前体溶液与中性红溶液均匀混合，然后装入注射器中进行凝胶化，将形成的水凝胶注入水中或在培养皿上书写以测试其可注射性。
水凝胶的自愈合行为是在PBS中制备水凝胶样品，其中一些用中性红染成红色，将样品分成两块，然后立即在37℃下将红色的胶块和透明的胶块放在一起，以测试其自愈合行为。
1.7实验结果
(1)醛透明质酸(A-HA)的合成流程图(见图1)；
(2)制得的醛透明质酸在通过核磁进行结构表征(见图2a-2b)；
NOCC的核磁共振氢谱(图2a)；A-HA的核磁共振氢谱(图2b)；NOCC在3.1ppm(H-2)、3.5-4.0ppm(H-3至H-6)处呈现特征峰，在2.2ppm处呈现小单峰，分配给N-乙酰基的甲基质子；在4.2和4.4ppm处检测到两个小峰，分别分配给NOCC的C2的N位和C6的O位的-CH2COO-质子，与氨基(N位)和伯羟基(O位)上的羧甲基取代一致；A-HA在3.2和3.7ppm之间的宽信号对应于糖环中的质子；HA的N-乙酰基的甲基质子在2.0ppm检测到；在4.9、5.0和5.1ppm下观察到的信号为醛基；通过比较HA主链中N-乙酰基的醛和甲基的积分来量化氧化程度得到的氧化度为56％，氧化度＝(A醛基/2)/(甲基/3)A：峰面积。
(3)NOCC/A-HA水凝胶通过FTIR近红外广谱进行结构表征(见图2c)；
NOCC/A-HA水凝胶通过NOCC的氨基和A-HA的醛基之间的席夫碱反应交联；HA在3400、1616和1078cm-1处呈现特征峰，分别与-OH吸收、-COOH的反对称拉伸振动和C-O键的拉伸吸收有关；A-HA的光谱与HA的光谱非常相似，只是在1730cm-1处出现一个属于醛基的小带；在NOCC的光谱中，1599和1411cm-1处的谱带分别与羧酸盐(-COO)的不对称和对称拉伸有关；水凝胶的光谱显示了NOCC和A-HA的所有特征谱带；3200-3500和 1100cm-1处的谱带分别归因于游离OH和NH2基团以及CO拉伸；醛基1730cm-1处的小条带消失，1640cm-1的条带属于羧酸盐和亚胺基团，这证明水凝胶的形成。
(4)通过席夫碱反应制备水凝胶(NOCC/A-HA)(见图3)；
醛透明质酸上的醛基与羧甲基壳聚糖上的氨基发生席夫碱反应形成亚胺键(图3圆球)。亚胺键之间的交联形成了网状交联结构。
(5)NOCC/A-HA水凝胶的形态通过扫描电子显微镜(SEM)表征，使用20KV的加速电压下观察水凝胶的表面和横截面的形貌(见图4)，并评估其应用场景；
NOCC和A-HA的体积比直接影响到凝胶的流变性能、均匀性和应用范围。当NOCC体积增加时，孔壁会得到加强，而NOCC体积过大，没有足够的交联键位点，其内部结构也会出现不均匀的现象。不同比例的水凝胶的表面形态，由扫描电镜拍摄，所有比例尺都是100μm。具体来说，2:1比例水凝胶孔径较大，孔隙相互连接较少，孔隙之间的连接更薄，更脆弱(图4a)，流动性较其他各比例较强，可用于复杂创面的敷料；4:1比例水凝胶孔径较小，孔洞分布均匀，孔隙率高，孔壁则更强更厚(图4b)，凝胶的粘弹性强，具有均匀的相互连接的孔隙内部结构，同时凝胶材料可以为细胞提供营养物质，并支持细胞的附着和增殖，最有利于应用于伤口敷料或细胞附着支持。6:1比例水凝胶孔径分布不均匀，孔壁较厚(图4c)，凝胶吸水率较高，粘弹性较强可用于防止手术后粘连。而8:1比例水凝胶孔径较小，孔隙率高孔隙连接紧密(图4d)，凝胶的吸水性弱，粘弹性较稳定，可用于局部注射作为基质使用。
综上所述，NOCC/A-HA水凝胶的4:1比例被证明是一种适合作为生物支架的水凝胶，它具有优越的形态，具有高度的均匀性，有利的孔径大小和适当的密度，以及适当的润湿性。具有合适的物理特性和良好的生物相容性。最后，将NOCC/A-HA凝胶基质作为组织工程应用中的一种有效的生物支架材料来评价干细胞在体外的存活率。
(6)NOCC/A-HA的可注射性和自愈性(见图5)；
通过自愈合试验评估水凝胶的自修复性能，水凝胶(NOCC/A-HA)选择4:1。将染色的红色水凝胶和透明水凝胶切成两块，并在37℃下将切好的两块放在一起。样品在30秒后完全整合。愈合的水凝胶可以剥离并支撑其自身重量(图5a)。水凝胶的自修复能力可归因于可逆亚胺键交联的重建，以及水凝胶两部分之间组分或组分交换的迁移。对自修复水凝胶进行了流变学实验，如图5b所示，自愈合的水凝胶呈现出与原始水凝胶非常接近的G'和G”值(800、500)，从而证实了出色的自愈能力。水凝胶的自愈能力能够确保其在临床治疗中应用于伤口时的结构完整性，从而促进复杂伤口的修复和治疗。
通过注射器注射实验评估水凝胶的可注射性(图5c)。对于生物学应用的可注射水凝胶，胶凝时间起着重要的作用，因为较短的胶凝时间可能导致针头堵塞，而较长的胶凝时间则可 导致封装细胞不可逆转的损失。研究发现NOCC/A-HA水凝胶可以通过26G针头连续注入，并且在注入后保持其完整性。
(7)NOCC/A-HA的流变性(见图6)；
各比例水凝胶的流变特性：在27℃和不同振荡应变条件下，各比例水凝胶的储能模量变化(见图6a)。通过存储模量(G')和损失模量(G”)随时间的变化，在流变仪板上原位监测混合NOCC和A-HA水溶液的凝胶化过程。对于所有样品，储能模量最初低于损耗模量2:1比例水凝胶施加同等应力条件下粘弹性最小，4:1比例水凝胶施加同等应力条件下粘弹性较高且随着应力增加粘弹性无明显变化较为稳定，6:1比例水凝胶施加同等应力条件下粘弹性较4:1小，8:1比例水凝胶施加同等应力条件下粘弹性较小但随着应力增加粘弹性无明显变化较为稳定。
各比例水凝胶在不同频率条件下的流变特性：在27℃和0.1-100Hz频率下，各比例水凝胶随应变的变化(见图6b)。在同等频率下2:1，4:1，6:1，8:1比例水凝胶都能稳定保持凝胶状态。
(8)NOCC/A-HA的平衡溶胀性。
在生理盐水(pH＝7.4，37℃)中24小时，各比例水凝胶的溶胀率(见图7)。
水凝胶的溶胀特性对于作为细胞载体或支架的应用具有重要意义。不同比例的水凝胶在生理盐水中37℃下24小时的溶胀率。4:1比例水凝胶表现出最高的膨胀率，这是因为均匀的多孔结构有利于吸水和保水性能。
实施例2水凝胶封装细胞的合成与评估
2.1水凝胶与细胞相容性实验
为了评估水凝胶与细胞共培养的相容性，以此判断水凝胶是否拥有细胞毒性，将2:1，4:1，6:1，8:1比例水凝胶与L929细胞进行共培养，通过MTT染色实验确定细胞的存活率。
实验发现，所有样品的细胞增殖率在72小时内均高于75％(见图8)。因此，不同比例的水凝胶对L929小鼠成纤维细胞没有毒性，可以按照ISO 10993安全的在临床应用中使用。
2.2水凝胶的溶血性实验
取200μL重悬好的人血红细胞悬液与不同比例的凝胶浸提液混合，吹打混匀后置于37℃中培养1h。设置空白对照PBS、阴性对照BSA溶液(20μM)和阳性对照0.1％TritonX-100。1h后，将孵育完毕的细胞取出，室温下1000g离心10min。取无菌干净的96孔板，将离心所得上清注入各孔中，每孔200μL。置于酶标仪下读取各孔吸光值，读数波长为540nm。
实验发现与阳性对照组相比，不同比例的水凝胶在与血细胞共孵育过程中不具有显著溶血作用；因此可以认为此水凝胶对哺乳动物血细胞无溶血活性(见图9)。
2.3水凝胶封装细胞的合成与细胞存活实验
为了评估水凝胶培养细胞的情况，首先测试了脐带血单个核细胞(CB-MNC)在无菌条件下在水凝胶中的封装情况。即将NOCC和A-HA溶液通过紫外灭菌，然后将A-HA溶液与脐带血单个核细胞(CB-MNC)溶液等体积混合均匀后，分别将NOCC和A-HA溶液以2:1、4:1、6:1、8:1的体积混合在灭菌后的孔板中原位形成水凝胶，凝胶中的细胞密度固定为1.0×106个细胞/ml，培养观察。
2.4体外细胞存活实验
为了观察CB-MNC在生理盐水、含1％人白蛋白的盐水中制备的水凝胶中的存活时间，用前面提到的细胞封装方法进行细胞封装，培养后所有细胞用AO-PI试剂盒染色。
为了证明水凝胶对细胞存活的有益作用，将脐带血单个核细胞(CB-MNC)培养在液体培养基中，即DMEM以及含1％人白蛋白的盐水中。培养后所有细胞用AO-PI试剂盒染色。
利用AO-PI进行活/死细胞的染色。在荧光显微镜下观察荧光时，可调节分辨率对比度等得到更美观的图片。活细胞为绿色荧光，死细胞为红色荧光。负载CB-MNC体系通过荧光活死细胞染色法，检测细胞的存活率。
2.5实验结果
通过活细胞/死细胞染色进行CB-MNC细胞相容性测试以确定细胞活力。在生理盐水制备的水凝胶中，细胞呈圆形，在培养21天后均匀分布在水凝胶中(图10a)；第21天，细胞存活率为51％(图10b)。CB-MNC也在含有1％人白蛋白的盐水中制备的水凝胶中培养(图10c)，并在DMEM培养基中培养(见图10e)。细胞形态与生理盐水中制备的水凝胶非常相似。相反，在1％人白蛋白的盐水中制备的水凝胶(图10d)和DMEM(图10f)中培养的细胞在第21天的存活率分别为56％和55％，即略高于生理盐水中制备细胞的存活率。这些发现表明，人白蛋白对细胞生长有益。
无水凝胶存在时，在DMEM中培养12小时后，细胞活力为47％，96小时后所有细胞均凋亡(图11a和b)。在含1％人白蛋白的盐水中培养细胞的情况下(图11c，d)，12小时后细胞活力为55％，96小时之后所有细胞都凋亡。因此，尽管两种培养基都可以为细胞生长提供营养，由于缺乏良好的生存环境，细胞最终都无法生存。因此，我们确定水凝胶网络可以有利于细胞附着的粘附配体偶联，从而提供有利于细胞生长的环境。
脐血单核干细胞在体外培养基培养，在96小时内全部死亡。在水凝胶生物支架中干细胞的存活率得到了大幅度提升(数值比较)，在PBS单纯水凝胶/含1％人血白蛋白生理盐水制备的水凝胶/DMEM水凝胶中，包载后的第15天还有55％以上的干细胞存活。结果表明， NOCC/A-HA水凝胶具有良好的细胞相容性和无毒性；NOCC/A-HA水凝胶相互连通的多孔结构使营养物质和氧气得以渗透，为细胞提供了适宜的环境。
实施例3体外小鼠烫伤模型的建立与治疗
建立小鼠烫伤模型，使用水凝胶负载CB-MNC体系治疗烫伤，观察治疗效果，愈后瘢痕情况。开发了载有CB-MNC的水凝胶***，以评估其在治疗烫伤并预防疤痕形成的潜力。在小鼠背侧皮肤建立深Ⅱ度烫伤模型，分别用不处理的烫伤组(Control group)作为对照，单纯水凝胶组(Gel group)和载有CB-MNC的水凝胶组(Gel+CB-MNC group)治疗伤口并观察伤口愈合情况。
Control group和Gel group在烫伤后的第一天都有伤口肿胀，而Gel+CB-MNC group没有出现伤口肿胀。这可能归因于CB-MNCs减少伤口炎症反应的能力。Control group烫伤愈合缓慢伴有表皮颜色加深，创面结痂和皮下炎症等现象，愈后疤痕较大。而且第3～7天创面出现结痂，表皮颜色加深，颜色不均匀，皮下有炎症反应。Gel组愈合速度较对照组快，愈后瘢痕较对照组小。Gel+CB-MNC组与其他两组相比愈合更快，愈后无疤痕，治疗21天后，观察到伤口完全闭合。此外，该组的毛发在伤口区的再生比其他组更明显。新皮肤与周围皮肤无明显差异(见图12a)。
伤口愈合区域图形和数据结果表明，单纯水凝胶具有一定的促进烫伤修复的能力。负载CB-MNCs的水凝胶可促进烫伤愈合，在愈合过程中具有促进创面毛发生长，避免炎症反应和减少愈后瘢痕形成(见图12b)。
实施例4愈合伤口的组织生理学
通过使用苏木精和伊红(H&E)染色指示愈合伤口的表皮和组织再生。观察愈伤组织的情况。
烫伤后第一天，观察到烫伤皮肤表皮细胞核固缩，真皮深层受损，胶原纤维融合，毛囊残留，这些观察符合深二度烫伤的标准。第7天，Gel+CB-MNC组表皮再生，棘细胞层水肿减少，炎症浸润减少；Gel组表皮再生，真皮排列松弛，炎症浸润中度；对照组显示不完全角化和高炎症浸润。第14天，Gel+CB-MNC组皮肤附件出现新生血管和再生；Gel组血管再生量少，炎症浸润减少；对照组表现出不完美角化和表皮再生的减少。第21天，Gel+CB-MNC组表皮及真皮恢复状态良好，无瘢痕形成；Gel组创面恢复后表皮增厚；对照组创面恢复后皮肤附肢再生、表皮增厚和瘢痕形成较少(见图13)。

Claims (10)

  1. 一种水凝胶复合物，所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得，所述透明质酸选自天然透明质酸或者人工合成透明质酸。
  2. 由权利要求1所述一种水凝胶复合物，其特征在于所述细胞选自成纤维细胞、肌细胞、上皮细胞、粘膜细胞和/或干细胞。
  3. 由权利要求1所述一种水凝胶复合物，其特征在于所述羧甲基壳聚糖和醛透明质酸的体积比选自2:1、4:1、6:1或8:1。
  4. 一种含有水凝胶的细胞培养基，所述培养基含有羧甲基壳聚糖、醛透明质酸和细胞培养液，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得，所述透明质酸选自天然透明质酸或者人工合成透明质酸。
  5. 如权利要求4所述一种含有水凝胶的细胞培养基进一步，其特征在于所述培养液选自DMEM、MEM、RPMI1640、胎牛血清(FBS)、成牛血清(ABS)、牛血清白蛋白(BSA)、PBS和平衡盐溶液(BSS)中的一种或多种。
  6. 一种水凝胶复合物在制备皮肤创伤物质中的用途，所述水凝胶复合物包括羧甲基壳聚糖、醛透明质酸和细胞，羧甲基壳聚糖和醛透明质酸通过席夫碱反应形成水凝胶，其中羧甲基壳聚糖和醛透明质酸的体积比选自1-10:1；所述醛透明质酸由透明质酸通过官能团醛基化修饰获得，所述透明质酸选自天然透明质酸或者人工合成透明质酸。
  7. 如权利要求6所述一种水凝胶复合物在制备皮肤创伤物质中的用途，其特征在于所述创伤是指烧伤、机械损伤或炎症导致的皮肤或粘膜的创伤，所述粘膜包括口腔粘膜或鼻黏膜等。
  8. 如权利要求一种水凝胶复合物在制备皮肤创伤物质中的用途，其特征在于所述所述修复创伤的物质可以是药物或医疗器械。
  9. 一种制备水凝胶复合物的方法，所述制备方法包括如下步骤：
    S01醛透明质酸的合成：透明质酸钠进行醛基化修饰；
    S02水凝胶复合物合成：先将需要的细胞培养液注入灭菌后的醛透明质酸溶液中，再将细胞注入含有细胞培养液的醛透明质酸溶液中，最后按比例加入羧甲基壳聚糖，其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1。
  10. 一种含有水凝胶的培养基的制备方法，所述制备方法包括如下步骤：
    S01醛透明质酸的合成：透明质酸钠进行醛基化修饰；
    S02水凝胶合成：将细胞培养液注入灭菌后的醛透明质酸溶液中，再按比例加入羧甲基壳聚糖，其中羧甲基壳聚糖和醛透明质酸的比例选自1-10:1，最终形成水凝胶培养基。
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