CN115678048A - Injectable composite hydrogel capable of promoting wound healing and reducing scar formation and preparation method and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶及其制备方法和应用。以细胞外基质主要成分为原料进行化学改性，并负载间充质干细胞和维替泊芬药物，通过席夫碱反应原位交联形成水凝胶。本发明的可注射复合水凝胶负载的间充质干细胞能够分泌抗纤维化细胞因子抑制组织纤维化，而维替泊芬能够抑制YAP基因表达防止伤口愈合过程中Engrailed‑1（En1）基因的激活，两者协同预防和减少疤痕形成。此外，复合水凝胶中的细胞外基质成分结合干细胞的免疫调节和改善血管生成作用能够有效促进创面愈合。本发明可应用于预防和减少疤痕形成、创伤修复和组织工程等领域。The invention discloses an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, a preparation method and application thereof. The main components of the extracellular matrix are chemically modified, loaded with mesenchymal stem cells and verteporfin, and cross-linked in situ by Schiff base reaction to form a hydrogel. The mesenchymal stem cells supported by the injectable composite hydrogel of the present invention can secrete anti-fibrotic cytokines to inhibit tissue fibrosis, and verteporfin can inhibit the expression of YAP gene to prevent the expression of Engrailed-1 (En1) gene in the process of wound healing activated, both synergistically prevent and reduce scarring. In addition, the extracellular matrix components in the composite hydrogel combined with the immune regulation and improved angiogenesis of stem cells can effectively promote wound healing. The invention can be applied in the fields of preventing and reducing scar formation, wound repair, tissue engineering and the like.

Description

Translated fromChinese

一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶及其制备方法和应用An injectable composite hydrogel that promotes wound healing and reduces scar formation andIts preparation method and application

技术领域technical field

本发明涉及生物医学材料和组织工程领域，具体涉及一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶及其制备方法和应用。The invention relates to the fields of biomedical materials and tissue engineering, in particular to an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, as well as its preparation method and application.

背景技术Background technique

目前医学上治疗疤痕的主要方法可分为手术治疗方法和非手术治疗方法。其中，非手术治疗方法由于侵入性低、损伤小、易操作和恢复时间短等优点，应用更广泛。非手术治疗方法包括物理疗法和药物法。物理疗法主要有激光治疗法、压迫治疗法、放射疗法、冷冻疗法和康复治疗法等。但物理治疗法时间较长，其中激光治疗法只能去除突起的皮肤，不能填平凹下的地方，且激光使用不当还会产生新疤痕或不均匀色斑等问题。在药物法中，一方面是往疤痕内注射曲安奈德、复方倍他米松、泼尼松龙等糖皮质激素类药物。但是，这类药物易引起注射部位皮肤萎缩、毛细血管扩张、局部皮肤色素沉着或脱失等问题。此外，激素类药物会干扰体内色素的正常代谢。另一方面是采用疤痕贴和祛疤药膏，如硅酮凝胶、多磺酸粘多糖乳膏、复方肝素钠尿囊素药膏，减少疤痕形成。而这类药物通常见效慢，容易引发皮肤红肿和疼痛以及过敏现象，有时还会加重疤痕症状。At present, the main methods of treating scars in medicine can be divided into surgical treatment methods and non-surgical treatment methods. Among them, non-surgical treatment methods are more widely used due to the advantages of low invasiveness, less damage, easy operation and short recovery time. Nonsurgical treatments include physical therapy and medication. Physical therapy mainly includes laser therapy, compression therapy, radiation therapy, cryotherapy and rehabilitation therapy. However, physical therapy takes a long time. Among them, laser therapy can only remove the protruding skin, but cannot fill in the sunken place, and improper use of laser will also cause problems such as new scars or uneven pigmentation. In the drug method, on the one hand, triamcinolone acetonide, compound betamethasone, prednisolone and other glucocorticoid drugs are injected into the scar. However, such drugs are likely to cause problems such as skin atrophy at the injection site, telangiectasia, and local skin pigmentation or loss. In addition, hormone drugs can interfere with the normal metabolism of pigments in the body. On the other hand, scar plasters and scar-removing ointments, such as silicone gel, polysulfonic acid mucopolysaccharide cream, and compound heparin sodium allantoin ointment, are used to reduce scar formation. These drugs are usually slow to take effect, can easily cause skin redness and pain, as well as allergies, and sometimes aggravate scarring symptoms.

间充质干细胞（MSCs）能够分泌抗纤维化细胞因子，降低α-SMA和I 型胶原基因表达，具有一定的抑制组织纤维化效果，因而具有减少疤痕形成的潜力。然而，已有研究报道表明MSCs治疗疤痕的效果并不显著。背后原因主要是伤口处的微环境不利于干细胞存活并且组织能够清除移植的干细胞，从而不利于干细胞因子的分泌以及作用发挥。此外，单纯依靠干细胞分泌的抗纤维化细胞因子难以完全预防或者减少疤痕的形成。然而，目前尚无相关研究报道和专利申请。Mesenchymal stem cells (MSCs) can secrete anti-fibrotic cytokines, reduce the expression of α-SMA and type I collagen genes, and have a certain effect on inhibiting tissue fibrosis, so they have the potential to reduce scar formation. However, previous research reports have shown that the effect of MSCs in treating scars is not significant. The main reason behind it is that the microenvironment of the wound is not conducive to the survival of stem cells and the tissue can clear the transplanted stem cells, which is not conducive to the secretion and function of stem cell factors. In addition, relying solely on the anti-fibrotic cytokines secreted by stem cells is difficult to completely prevent or reduce the formation of scars. However, there are no relevant research reports and patent applications at present.

发明内容Contents of the invention

本发明的目的在于克服现有技术中存在的不足，而提供一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶及其制备方法和应用。所述可注射复合水凝胶由仿生细胞外基质水凝胶支架、干细胞及维替泊芬药物复合而成。其中，仿生细胞外基质水凝胶支架选用细胞外基质主要成分及其衍生物（透明质酸、胶原蛋白或明胶）为原料，经氧化反应和氨基修饰后分别制得氧化透明质酸、碳酰肼胶原蛋白或碳酰肼明胶。该仿生细胞外基质水凝胶支架拥有力学性能好且不易被酶解，能够为干细胞提供相容性环境，满足干细胞生长、增殖以及代谢等需求。同时具有减少疤痕形成的作用。且由于维替泊芬在水凝胶内能够缓慢释放，避免药物与伤口接触的浓度过高，从而延长药效，降低给药频率和临床负担。此外，可注射复合水凝胶能够为伤口提供轻度隔氧条件，为疤痕提供封闭湿润环境有利于胶原重新排列。因此，本发明可最大程度发挥仿生水凝胶支架-干细胞-维替泊芬三者的作用，协同预防和减少疤痕形成并且促进伤口愈合。The purpose of the present invention is to overcome the deficiencies in the prior art, and provide an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, as well as its preparation method and application. The injectable composite hydrogel is composed of a biomimetic extracellular matrix hydrogel scaffold, stem cells and verteporfin. Among them, the biomimetic extracellular matrix hydrogel scaffold selects the main components of the extracellular matrix and its derivatives (hyaluronic acid, collagen or gelatin) as raw materials, and prepares oxidized hyaluronic acid, carbonyl Hydrazine collagen or carbohydrazide gelatin. The biomimetic extracellular matrix hydrogel scaffold has good mechanical properties and is not easy to be degraded by enzymes, and can provide a compatible environment for stem cells to meet the needs of stem cell growth, proliferation and metabolism. It also has the effect of reducing scar formation. And because verteporfin can be released slowly in the hydrogel, the concentration of the drug in contact with the wound is avoided from being too high, thereby prolonging the drug effect, reducing the frequency of administration and clinical burden. In addition, the injectable composite hydrogel can provide a mild oxygen barrier condition for the wound, and provide a closed and moist environment for the scar to facilitate the rearrangement of collagen. Therefore, the present invention can maximize the effects of the biomimetic hydrogel scaffold-stem cells-verteporfin, synergistically prevent and reduce scar formation and promote wound healing.

为实现上述目的，本发明采用如下技术方案：To achieve the above object, the present invention adopts the following technical solutions:

本发明首先提供了一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶的制备方法，包括以下步骤：The present invention firstly provides a preparation method of an injectable composite hydrogel that can promote wound healing and reduce scar formation, comprising the following steps:

1）将透明质酸溶解于超纯水中，向其中加入高碘酸钠，室温氧化反应2~3h，再加入0.5mL乙二醇终止反应1h；反应结束后，以超纯水为透析液，将反应液使用3500Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体，转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得氧化透明质酸海绵材料；1) Dissolve hyaluronic acid in ultrapure water, add sodium periodate to it, oxidize at room temperature for 2~3h, then add 0.5mL ethylene glycol to terminate the reaction for 1h; after the reaction, use ultrapure water as dialysate , the reaction solution was dialyzed for 72 hours using a dialysis bag with a molecular weight of 3500Da; after the dialysis, the liquid in the dialysis bag was collected, transferred to a polytetrafluoroethylene container, placed in a -80°C refrigerator for 5 hours, and then kept at -50°C for 1~ Freeze-drying under the condition of 20Pa for 72h, the oxidized hyaluronic acid sponge material is obtained;

2）将天然高聚物溶解于超纯水中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺，室温活化反应2h，再加入碳酰肼粉体，室温反应12h；反应结束后，以超纯水为透析液，将反应液使用8000Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体，转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得碳酰肼天然高聚物材料；2) Dissolve the natural polymer in ultrapure water, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and activate the reaction at room temperature 2h, then add carbohydrazide powder, and react at room temperature for 12h; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction liquid with a dialysis bag with a molecular weight of 8000Da for 72h; after the dialysis, collect the liquid in the dialysis bag and transfer it to Place in a polytetrafluoroethylene container, freeze in a -80°C refrigerator for 5 hours, and then freeze-dry at -50°C and 1-20Pa for 72 hours to obtain carbohydrazide natural polymer materials;

3）将氧化透明质酸海绵材料用超纯水配制为溶液，将碳酰肼天然高聚物材料用超纯水配制为溶液；向氧化透明质酸海绵材料溶液中加入间充质干细胞，得到水凝胶前体溶液1；向碳酰肼天然高聚物材料溶液中加入维替泊芬，得到水凝胶前体溶液2；将水凝胶前体溶液1和水凝胶前体溶液2混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到所述可注射复合水凝胶。3) The oxidized hyaluronic acid sponge material was prepared as a solution with ultrapure water, and the carbohydrazide natural polymer material was prepared as a solution with ultrapure water; mesenchymal stem cells were added to the oxidized hyaluronic acid sponge material solution to obtainHydrogel precursor solution 1; add verteporfin to carbohydrazide natural polymer material solution to obtainhydrogel precursor solution 2;hydrogel precursor solution 1 andhydrogel precursor solution 2 Mix evenly, and stir at room temperature for 10-20 seconds until the mixed solution becomes gelatinous, and the injectable composite hydrogel is obtained.

进一步的，上述步骤1）中，所述透明质酸的用量为1g，所述高碘酸钠的用量为0.25g。Further, in the above step 1), the dosage of the hyaluronic acid is 1 g, and the dosage of the sodium periodate is 0.25 g.

进一步的，上述步骤2）中，所述天然高聚物选自胶原蛋白、明胶中的任意一种。Further, in the above step 2), the natural high polymer is selected from any one of collagen and gelatin.

进一步的，上述步骤2）中，所述天然高聚物的用量为9g，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的用量为8.64g，所述N-羟基琥珀酰亚胺的用量为0.58g，所述碳酰肼粉体的用量为6.6g。Further, in the above step 2), the dosage of the natural high polymer is 9g, and the dosage of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 8.64g , the dosage of the N-hydroxysuccinimide is 0.58g, and the dosage of the carbohydrazide powder is 6.6g.

进一步的，上述步骤3）中，所述氧化透明质酸海绵材料溶液的浓度为2%~8%，所述碳酰肼天然高聚物材料溶液的浓度为4%~12%。Further, in the above step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2%-8%, and the concentration of the carbohydrazide natural high polymer material solution is 4%-12%.

进一步的，上述步骤3）中，所述间充质干细胞选自人脂肪间充质干细胞、人骨髓间充质干细胞中的任意一种。Further, in the above step 3), the mesenchymal stem cells are selected from any one of human adipose-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells.

进一步的，上述步骤3）中，所述水凝胶前体溶液1和水凝胶前体溶液2的体积比为1：1。Further, in the above step 3), the volume ratio of thehydrogel precursor solution 1 and thehydrogel precursor solution 2 is 1:1.

进一步的，上述步骤3）中，所述可注射复合水凝胶中间充质干细胞的浓度为1×10⁶~1×10⁷个/mL、维替泊芬的浓度为0.01~10mg/mL。Further, in the above step 3), the concentration of the injectable composite hydrogel mesenchymal stem cells is 1×10⁶ -1×10⁷ cells/mL, and the concentration of verteporfin is 0.01-10 mg/mL.

本发明还提供了一种利用上述的制备方法制得的可注射复合水凝胶。The present invention also provides an injectable composite hydrogel prepared by the above preparation method.

本发明还提供了上述一种可注射复合水凝胶在制备促进创面愈合和/或减少疤痕形成的药物中的应用。The present invention also provides the application of the above-mentioned injectable composite hydrogel in the preparation of medicines for promoting wound healing and/or reducing scar formation.

本发明的有益效果为：The beneficial effects of the present invention are:

1）本发明提供了一种生物医用材料，以生物相容性良好的高分子材料为基材制备生物降解的可注射复合水凝胶，能实现细胞因子和药物的可控释放。1) The present invention provides a biomedical material. A biodegradable injectable composite hydrogel is prepared from a polymer material with good biocompatibility as a base material, which can realize the controllable release of cytokines and drugs.

2）本发明的可注射复合水凝胶能够保持间充质干细胞在体外生长和增殖5天以上，间充质干细胞所分泌的细胞因子能持续释放，促进创面愈合。2) The injectable composite hydrogel of the present invention can maintain the growth and proliferation of mesenchymal stem cells in vitro for more than 5 days, and the cytokines secreted by mesenchymal stem cells can be continuously released to promote wound healing.

3）本发明的可注射复合水凝胶支持维替泊芬缓慢释放7天以上，缩小药物的扩散面积，降低给药频率，达到更好的疤痕治疗效果并减轻临床负担。3) The injectable composite hydrogel of the present invention supports the slow release of verteporfin for more than 7 days, reduces the diffusion area of the drug, reduces the frequency of administration, achieves better scar treatment effect and reduces clinical burden.

4）本发明的可注射复合水凝胶可用于伤口愈合及预防和减少疤痕形成。4) The injectable composite hydrogel of the present invention can be used for wound healing and prevention and reduction of scar formation.

附图说明Description of drawings

图1：实施例1中制备的仿生水凝胶支架通过30G针头注射器的示意图。Figure 1: Schematic diagram of the biomimetic hydrogel scaffold prepared in Example 1 passing through a 30G needle syringe.

图2：实施例1中制得的仿生水凝胶支架的微观结构。Figure 2: Microstructure of the biomimetic hydrogel scaffold prepared in Example 1.

图3：维替泊芬在实施例1制备的仿生水凝胶支架中的释放曲线。Figure 3: Release curve of verteporfin in the biomimetic hydrogel scaffold prepared in Example 1.

图4：人骨髓间充质干细胞在实施例2中制备的仿生水凝胶支架中的释放曲线。Figure 4: The release curve of human bone marrow mesenchymal stem cells in the biomimetic hydrogel scaffold prepared in Example 2.

图5：实施例2中制备的仿生水凝胶支架的剪切稀化性能。Figure 5: Shear thinning performance of the biomimetic hydrogel scaffold prepared in Example 2.

图6：仿生水凝胶支架中的间充质干细胞Live/Dead染色图。Figure 6: Live/Dead staining of mesenchymal stem cells in biomimetic hydrogel scaffolds.

图7：人骨髓间充质干细胞在仿生水凝胶中的生长分布情况。Figure 7: Growth and distribution of human bone marrow mesenchymal stem cells in biomimetic hydrogels.

具体实施方式Detailed ways

为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

实施例1Example 1

步骤1）：称取1g透明质酸（30kDa）溶解于100mL超纯水中，向其中加入0.25g高碘酸钠，室温氧化反应2~3h，再加入0.5mL乙二醇室温终止反应1h；反应结束后，以超纯水为透析液，将反应液使用3500Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得氧化透明质酸海绵材料。Step 1): Weigh 1g hyaluronic acid (30kDa) and dissolve it in 100mL ultrapure water, add 0.25g sodium periodate to it, oxidize at room temperature for 2~3h, then add 0.5mL ethylene glycol to terminate the reaction at room temperature for 1h; After the reaction, use ultrapure water as the dialysate, and dialyze the reaction liquid with a 3500Da molecular weight dialysis bag for 72 hours; after the dialysis, collect the liquid in the dialysis bag and transfer it to a polytetrafluoroethylene container, and freeze it in a -80°C refrigerator 5h, and then freeze-dried at -50°C and 1-20Pa for 72h to obtain the oxidized hyaluronic acid sponge material.

步骤2）：称取9g胶原蛋白（牛来源，I型，30kDa）溶解于500mL超纯水中，加入8.64g1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）和0.58g (N-羟基琥珀酰亚胺（NHS），室温活化反应2h，再加入6.6g碳酰肼粉体，室温反应12h；反应结束后，以超纯水为透析液，将反应液使用8000Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得碳酰肼胶原蛋白材料。Step 2): Weigh 9g collagen (bovine source, type I, 30kDa) and dissolve it in 500mL ultrapure water, add 8.64g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC) and 0.58g (N-hydroxysuccinimide (NHS), activated at room temperature for 2 hours, then added 6.6g carbohydrazide powder, and reacted at room temperature for 12 hours; after the reaction, ultrapure water was used as the dialysate, The reaction solution was dialyzed for 72 hours using a dialysis bag with a molecular weight of 8000 Da; after the dialysis, the liquid in the dialysis bag was collected and transferred to a polytetrafluoroethylene container, and placed in a -80°C refrigerator for 5 hours, and then placed in a -50°C, 1-20Pa The carbohydrazide collagen material was obtained by freeze-drying under the conditions for 72 hours.

步骤3）：将氧化透明质酸海绵材料用超纯水配置成浓度为5wt%的溶液，将碳酰肼胶原蛋白材料用超纯水配置成浓度为7wt%的溶液；将5wt%的氧化透明质酸海绵材料溶液与7wt%的碳酰肼胶原蛋白材料溶液按照1:1的体积比混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到仿生水凝胶支架。Step 3): Prepare the oxidized hyaluronic acid sponge material into a solution with a concentration of 5wt% with ultrapure water, and prepare the carbohydrazide collagen material with ultrapure water into a solution with a concentration of 7wt%; The hyaluronic acid sponge material solution and the 7wt% carbohydrazide collagen material solution were mixed evenly at a volume ratio of 1:1, and stirred at room temperature for 10-20 s until the mixed solution was gelatinous, and the biomimetic hydrogel scaffold was obtained.

实施例2Example 2

步骤1）：参照实施例1步骤1）制备氧化透明质酸海绵材料。Step 1): Refer to step 1) of Example 1 to prepare the oxidized hyaluronic acid sponge material.

步骤2）：称取9g明胶（猪来源，A型，300g bloom）溶解于500mL超纯水中，加入8.64g1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐（EDC）和0.58g (N-羟基琥珀酰亚胺（NHS），室温活化反应2h，再加入6.6g碳酰肼粉体，室温反应12h；反应结束后，以超纯水为透析液，将反应液使用 8000Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得碳酰肼明胶材料。Step 2): Weigh 9g gelatin (porcine source, type A, 300g bloom) and dissolve it in 500mL ultrapure water, add 8.64g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride salt (EDC) and 0.58g (N-hydroxysuccinimide (NHS), activated at room temperature for 2 hours, then added 6.6g carbohydrazide powder, and reacted at room temperature for 12 hours; after the reaction, ultrapure water was used as the dialysate, The reaction solution was dialyzed for 72 hours using a dialysis bag with a molecular weight of 8000 Da; after the dialysis, the liquid in the dialysis bag was collected and transferred to a polytetrafluoroethylene container, and placed in a -80°C refrigerator for 5 hours, and then placed in a -50°C, 1-20Pa Freeze-drying for 72 hours under the condition to obtain carbohydrazide gelatin material.

3）将氧化透明质酸海绵材料用超纯水配置成浓度为5wt%的溶液，将碳酰肼明胶材料用超纯水配置成浓度为7wt%的溶液；将5wt%的氧化透明质酸海绵材料溶液与 7wt%的碳酰肼明胶材料溶液按照1:1的体积比混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到仿生水凝胶支架。3) Prepare the oxidized hyaluronic acid sponge material with ultrapure water into a solution with a concentration of 5wt%, and prepare the carbohydrazide gelatin material with ultrapure water into a solution with a concentration of 7wt%; The material solution and 7wt% carbohydrazide gelatin material solution were mixed evenly at a volume ratio of 1:1, and stirred at room temperature for 10-20 s until the mixed solution was gelatinous, and the biomimetic hydrogel scaffold was obtained.

图1所示的是实施例1中制备的仿生水凝胶支架通过30G针头注射器的示意图。从该图中可以看出，实施例1中制备的仿生水凝胶支架可通过30G针头注射器注射出来，且能快速成胶便于实际应用。Figure 1 is a schematic diagram of the biomimetic hydrogel scaffold prepared in Example 1 passing through a 30G needle syringe. It can be seen from the figure that the biomimetic hydrogel scaffold prepared in Example 1 can be injected through a 30G needle syringe, and can be quickly gelled for practical application.

图2所示的是实施例1中制得的仿生水凝胶支架的微观结构。电子扫描显微镜观察发现，仿生水凝胶支架具有多孔结构并且孔径大小均一，可吸收伤口组织渗液、有利于间充质干细胞在其中的生长增殖和产生的细胞因子的分泌以及维替泊芬的缓慢释放。Figure 2 shows the microstructure of the biomimetic hydrogel scaffold prepared in Example 1. Scanning electron microscope observation found that the biomimetic hydrogel scaffold has a porous structure and uniform pore size, which can absorb wound tissue exudate, facilitate the growth and proliferation of mesenchymal stem cells in it, and the secretion of cytokines produced by it and the activity of verteporfin. Release slowly.

图3所示的是维替泊芬在实施例1制备的仿生水凝胶支架中的释放曲线。取200μL实施例1中制备的仿生水凝胶支架，与1mg/mL维替泊芬溶液均匀混合，浸没在37℃的1×PBS（PH=7.4）中，在固定时间间隔检测药物释放量。结果表明，仿生水凝胶支架具有多孔结构，支持药物的缓慢释放，从而降低给药频次。Figure 3 shows the release curve of verteporfin in the biomimetic hydrogel scaffold prepared in Example 1. Take 200 μL of the biomimetic hydrogel scaffold prepared in Example 1, mix it evenly with 1 mg/mL verteporfin solution, immerse it in 1×PBS (PH=7.4) at 37°C, and detect the drug release at regular intervals. The results showed that the porous structure of the biomimetic hydrogel scaffold supports the slow release of drugs, thereby reducing the frequency of drug administration.

图4所示的是人骨髓间充质干细胞在实施例2中制备的仿生水凝胶支架中的释放曲线。向仿生水凝胶支架中加入人骨髓间充质干细胞至其终浓度为3×10⁶个/mL，置于37℃培养箱中培养，在固定时间节点吸取培养基检测干细胞所分泌细胞因子的总含量。结果表明，仿生水凝胶支架支持间充质干细胞在其内生长时，所分泌细胞因子的释放。Figure 4 shows the release curve of human bone marrow mesenchymal stem cells in the biomimetic hydrogel scaffold prepared in Example 2. Human bone marrow mesenchymal stem cells were added to the biomimetic hydrogel scaffold to a final concentration of 3×10⁶ cells/mL, cultured in a 37°C incubator, and the culture medium was absorbed at fixed time points to detect the cytokines secreted by the stem cells. total content. The results showed that the biomimetic hydrogel scaffold supported the release of cytokines secreted by mesenchymal stem cells growing within them.

图5所示的是实施例2中制备的仿生水凝胶支架的剪切稀化性能。将仿生水凝胶支架成胶（5mm的半径和高度），放置在流变仪载物台上，以10 rad/s角频率，探究粘度随剪切速率（0.01-100/s）的变化，可观察到仿生水凝胶支架具有剪切稀化特性，随着剪切力增大，仿生水凝胶支架的粘度下降，表明其具有可注射性。Figure 5 shows the shear thinning performance of the biomimetic hydrogel scaffold prepared in Example 2. The biomimetic hydrogel scaffold was gelled (radius and height of 5mm), placed on the stage of the rheometer, and the change of viscosity with the shear rate (0.01-100/s) was explored at an angular frequency of 10 rad/s. It can be observed that the biomimetic hydrogel scaffold has shear-thinning properties, and as the shear force increases, the viscosity of the biomimetic hydrogel scaffold decreases, indicating that it is injectable.

图6所示的是仿生水凝胶支架中的间充质干细胞Live/Dead染色图。用无血清培养基浸提仿生水凝胶支架，从而得到浸提液；将间充质干细胞种植于96孔板中，加入仿生水凝胶支架浸提液，37℃培养，72h后以Calcein-AM/PI 试剂盒染色10~20min，在荧光倒置显微镜下观察细胞的形态和生长情况（使实施例1制备的仿生水凝胶支架负载人脂肪间充质干细胞，使实施例2制备的仿生水凝胶支架负载人骨髓间充质干细胞）。从图中可以看出，实施例1~2制备的仿生水凝胶支架均有良好的细胞相容性，间充质干细胞具有细长的梭状形态，并且红色的死细胞数很少。Figure 6 shows the live/dead staining of mesenchymal stem cells in the biomimetic hydrogel scaffold. The biomimetic hydrogel scaffold was leached with serum-free medium to obtain the extract; the mesenchymal stem cells were planted in a 96-well plate, added with the biomimetic hydrogel scaffold extract, cultured at 37°C, and after 72 hours, Calcein- The AM/PI kit was stained for 10-20 minutes, and the morphology and growth of the cells were observed under a fluorescent inverted microscope (the biomimetic hydrogel scaffold prepared in Example 1 was loaded with human adipose-derived mesenchymal stem cells, and the biomimetic hydrogel scaffold prepared in Example 2 was Gel scaffold loaded with human bone marrow mesenchymal stem cells). It can be seen from the figure that the biomimetic hydrogel scaffolds prepared in Examples 1-2 all have good cytocompatibility, the mesenchymal stem cells have an elongated spindle shape, and the number of red dead cells is very small.

实施例3Example 3

步骤1）：参照实施例1步骤1）制备氧化透明质酸海绵材料。Step 1): Refer to step 1) of Example 1 to prepare the oxidized hyaluronic acid sponge material.

步骤2）：参照实施例2步骤2）制备碳酰肼胶原蛋白材料碳酰肼明胶材料。Step 2): Refer to step 2) of Example 2 to prepare a carbohydrazide collagen material and a carbohydrazide gelatin material.

步骤3）：将氧化透明质酸海绵材料用超纯水配置成浓度为5wt%的溶液，将碳酰肼明胶材料用超纯水配置成浓度为7wt%的溶液；向100μL 5wt%的氧化透明质酸海绵材料溶液中加入浓度为6×10⁶个/mL的人骨髓间充质干细胞悬液，再与100μL 7wt%的碳酰肼明胶材料溶液混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到可注射复合水凝胶，所述可注射复合水凝胶中间充质干细胞的浓度为3×10⁶个/mL。Step 3): Prepare the oxidized hyaluronic acid sponge material with ultrapure water into a solution with a concentration of 5wt%, and prepare the carbohydrazide gelatin material with ultrapure water into a solution with a concentration of 7wt%; Add human bone marrow mesenchymal stem cell suspension with a concentration of 6×¹⁰⁶ cells/mL to the acid sponge material solution, then mix with 100 μL 7wt% carbohydrazide gelatin material solution, and stir at room temperature for 10-20 seconds until the mixed solution In gel form, an injectable composite hydrogel is obtained, and the concentration of mesenchymal stem cells in the injectable composite hydrogel is 3×10⁶ cells/mL.

图7所示的是本实施例人骨髓间充质干细胞在可注射复合水凝胶中的生长分布情况。分别于成胶后第一天和第五天对负载间充质干细胞的水凝胶进行Live/Dead染色，以激光扫描共聚焦显微镜观察干细胞在水凝胶内的3D分布图。从图中可以看出，从第一天到第五天，人骨髓间充质干细胞在可注射复合水凝胶内数目增多。Figure 7 shows the growth and distribution of human bone marrow mesenchymal stem cells in the injectable composite hydrogel of this example. Live/Dead staining was performed on the hydrogel loaded with mesenchymal stem cells on the first day and the fifth day after gel formation, and the 3D distribution of stem cells in the hydrogel was observed with a laser scanning confocal microscope. It can be seen from the figure that from the first day to the fifth day, the number of human bone marrow mesenchymal stem cells in the injectable composite hydrogel increased.

实施例4Example 4

步骤1）：参照实施例1步骤1）制备氧化透明质酸海绵材料。Step 1): Refer to step 1) of Example 1 to prepare the oxidized hyaluronic acid sponge material.

步骤2）：参照实施例1步骤2）制备碳酰肼胶原蛋白材料。Step 2): Refer to step 2) of Example 1 to prepare carbohydrazide collagen material.

步骤3）：将氧化透明质酸海绵材料用超纯水配置成浓度为5wt%的溶液，将碳酰肼胶原蛋白材料用超纯水配置成浓度为7wt%的溶液；向100μL 5wt%的氧化透明质酸海绵材料溶液中加入浓度为6×10⁶个/mL的人脂肪间充质干细胞悬液，得到水凝胶前体溶液1；向100μL 7wt%的碳酰肼胶原蛋白材料溶液中加入2mg/mL的维替泊芬溶液，得到水凝胶前体溶液2；将水凝胶前体溶液1和水凝胶前体溶液2按照1:1的体积比混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到可注射复合水凝胶，所述可注射复合水凝胶中间充质干细胞的浓度为3×10⁶个/mL、维替泊芬的浓度为1mg/mL。Step 3): Prepare the oxidized hyaluronic acid sponge material into a solution with a concentration of 5wt% with ultrapure water, and prepare the carbohydrazide collagen material with ultrapure water into a solution with a concentration of 7wt%; Add human adipose-derived mesenchymal stem cell suspension at a concentration of 6×¹⁰⁶ cells/mL to the hyaluronic acid sponge material solution to obtainhydrogel precursor solution 1; add to 100 μL 7wt% carbohydrazide collagen material solution 2mg/mL verteporfin solution to obtainhydrogel precursor solution 2; mixhydrogel precursor solution 1 andhydrogel precursor solution 2 according to the volume ratio of 1:1, and stir at room temperature for 10~20s , until the mixed solution is colloidal, an injectable composite hydrogel is obtained, the concentration of the injectable composite hydrogel mesenchymal stem cells is 3×10⁶ cells/mL, and the concentration of verteporfin is 1 mg/mL .

实施例5Example 5

步骤1）：参照实施例1步骤1）制备氧化透明质酸海绵材料。Step 1): Refer to step 1) of Example 1 to prepare the oxidized hyaluronic acid sponge material.

步骤2）：参照实施例2步骤2）制备碳酰肼胶原蛋白材料碳酰肼明胶材料。Step 2): Refer to step 2) of Example 2 to prepare a carbohydrazide collagen material and a carbohydrazide gelatin material.

步骤3）：将氧化透明质酸海绵材料用超纯水配置成浓度为5wt%的溶液，将碳酰肼明胶材料用超纯水配置成浓度为7wt%的溶液；向100μL 5wt%的氧化透明质酸海绵材料溶液中加入浓度为6×10⁶个/mL的人骨髓间充质干细胞悬液，得到水凝胶前体溶液1；向100μL7wt%的碳酰肼明胶材料溶液中加入2mg/mL的维替泊芬溶液，得到水凝胶前体溶液2；将水凝胶前体溶液1和水凝胶前体溶液2按照1:1的体积比混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到可注射复合水凝胶，所述可注射复合水凝胶中间充质干细胞的浓度为3×10⁶个/mL、维替泊芬的浓度为1mg/mL。Step 3): Prepare the oxidized hyaluronic acid sponge material with ultrapure water into a solution with a concentration of 5wt%, and prepare the carbohydrazide gelatin material with ultrapure water into a solution with a concentration of 7wt%; Add human bone marrow mesenchymal stem cell suspension at a concentration of 6×¹⁰⁶ cells/mL to the acid sponge material solution to obtainhydrogel precursor solution 1; add 2 mg/mL to 100μL 7 wt% carbohydrazide gelatin material solution verteporfin solution to obtainhydrogel precursor solution 2; mixhydrogel precursor solution 1 andhydrogel precursor solution 2 according to the volume ratio of 1:1, stir at room temperature for 10-20s, until mixed The solution is gel-like, and an injectable composite hydrogel is obtained, the concentration of the injectable composite hydrogel is 3×10⁶ mesenchymal stem cells/mL, and the concentration of verteporfin is 1 mg/mL.

实施例6Example 6

将2.5~3kg/只的新西兰兔，应用戊巴比妥钠与异氟烷复合麻醉，在兔耳内侧无毛区域建立每只耳朵4个直径10mm的伤口。组别设置为：对照组（向伤口处注射200μL生理盐水，每三天注射一次）、单纯间充质干细胞组（向伤口处注射200μL浓度为3×10⁶个/mL的人脂肪间充质干细胞悬液，每三天注射一次）、单纯维替泊芬组（向伤口处注射200μL浓度为1mg/mL的维替泊芬溶液，每三天注射一次）、载间充质干细胞水凝胶组（将200μL实施例3制备的可注射复合水凝胶成胶后贴附于伤口处，每3天更换一次新的与上一次相同的可注射复合水凝胶）、载间充质干细胞和维替泊芬的水凝胶组（将200μL实施例4制备的可注射复合水凝胶成胶后贴附于伤口处，每3天更换一次新的与上一次相同的可注射复合水凝胶），商业3M敷料组（将商业3M敷料贴附于伤口处，每3天更换一次新的敷料），硅酮凝胶（将硅酮凝胶涂敷于伤口处，每3天涂敷一次）。在治疗第15天后，将兔子安乐死，统计对比伤口闭合率及SEI（疤痕升高指数）。伤口愈合计算公式为：伤口闭合率（%）= （A₀-A_t）/A₀×100% ， 其中，A₀ 表示第0天创面面积；A_t 表示第 t 天创面面积。疤痕升高指数（SEI）计算公式：SEI=（A+B）/B，其中A为高于正常表皮皮肤的疤痕组织厚度，B为正常表皮皮肤厚度。结果如下表所示。2.5~3kg/New Zealand rabbits were anesthetized with pentobarbital sodium and isoflurane, and four wounds with a diameter of 10mm were established on the hairless area inside the rabbit ears. The groups were set as follows: control group (inject 200 μL of normal saline into the wound, once every three days), simple mesenchymal stem cell group (inject 200 μL of human adipose-derived mesenchymal cells at a concentration of 3×10⁶ cells/mL into the wound Stem cell suspension, injected once every three days), simple verteporfin group (inject 200 μL of verteporfin solution with a concentration of 1 mg/mL into the wound, injected once every three days), mesenchymal stem cell-loaded hydrogel Group (200 μL of the injectable composite hydrogel prepared in Example 3 was gelled and attached to the wound, and replaced with a new injectable composite hydrogel every 3 days), containing mesenchymal stem cells and Verteporfin hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 4 was gelled and attached to the wound, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days ), commercial 3M dressing set (apply commercial 3M dressing to the wound and replace it with a new dressing every 3 days), silicone gel (apply silicone gel to the wound and apply it every 3 days) . After the 15th day of treatment, the rabbits were euthanized, and the wound closure rate and SEI (Scar Elevation Index) were compared statistically. The calculation formula for wound healing is: wound closure rate (%) = (A₀ -A_t )/A₀ ×100%, where A₀ represents the wound area onday 0; A_t represents the wound area on day t. Scar Elevation Index (SEI) calculation formula: SEI=(A+B)/B, where A is the thickness of scar tissue higher than normal epidermal skin, and B is the thickness of normal epidermal skin. The results are shown in the table below.

表1 伤口闭合率统计结果Table 1 Statistical results of wound closure rate

表2 伤口愈合中的CD31(+)血管数统计结果Table 2 Statistical results of CD31(+) blood vessels in wound healing

表3 SEI（疤痕升高指数）统计结果Table 3 SEI (Scar Elevation Index) statistical results

注：SEI = 1，表示疤痕与周围未受伤的真皮等高；SEI > 1，表示疤痕升高；Note: SEI = 1, indicating that the scar is at the same height as the surrounding uninjured dermis; SEI > 1, indicating that the scar is elevated;

SEI = 2，表示疤痕比正常组织真皮厚度增加100%。SEI = 2, indicating that the scar has a 100% increase in dermal thickness compared to normal tissue.

SEI>2，表示疤痕比正常组织真皮厚度增加100%以上。SEI>2 means that the thickness of the dermis of the scar is increased by more than 100% compared with the normal tissue.

从伤口闭合率可以看出（表1），与商业3M敷料相比，载间充质干细胞水凝胶组、载间充质干细胞和维替泊芬的水凝胶组的伤口闭合率有明显提高，由于水凝胶基材选自天然高分子材料，本身具有良好的生物相容性并且水凝胶内负载了间充质干细胞，水凝胶的多孔结构支持干细胞因子的分泌，促进创面修复。并且组织学染色中，在第15天，载间充质干细胞水凝胶组、载间充质干细胞和维替泊芬的水凝胶组的伤口上皮再生完整率高于商业3M敷料组。伤口愈合过程中需要生成血管，促进恢复皮肤结构和功能，而表2中CD（+）血管数量的统计结果表明，在第10天载间充质干细胞水凝胶组的CD31（+）血管生成数量为22.38%±0.69%，高于商业3M敷料组11.41%±1.00%。结果显示，载间充质干细胞水凝胶组能够更好地促进伤口血管生成和再上皮化。As can be seen from the wound closure rate (Table 1), compared with the commercial 3M dressing, the wound closure rate of the hydrogel group loaded with mesenchymal stem cells, the hydrogel group loaded with mesenchymal stem cells and verteporfin significantly improved. Improvement, since the hydrogel base material is selected from natural polymer materials, it has good biocompatibility and the hydrogel is loaded with mesenchymal stem cells. The porous structure of the hydrogel supports the secretion of stem cell factors and promotes wound repair. . And in histological staining, on the 15th day, the complete rate of wound epithelial regeneration in the hydrogel group loaded with mesenchymal stem cells, the hydrogel group loaded with mesenchymal stem cells and verteporfin was higher than that of the commercial 3M dressing group. Blood vessels need to be generated during wound healing to promote the restoration of skin structure and function, and the statistical results of the number of CD (+) blood vessels in Table 2 show that the CD31 (+) angiogenesis of the mesenchymal stem cell-loaded hydrogel group on the 10th day The number was 22.38%±0.69%, which was higher than the commercial 3M dressing group of 11.41%±1.00%. The results showed that the mesenchymal stem cell-loaded hydrogel group could better promote wound angiogenesis and re-epithelialization.

从表3的SEI（疤痕升高指数）可以看出载间充质干细胞和维替泊芬的水凝胶组的疤痕治疗效果最好，SEI指数为1.26±0.02，可能是由于表面有水凝胶覆盖起到低氧、保水湿润效果，含有间充质干细胞分泌细胞因子促进伤口愈合，伤口愈合后期分泌抗纤维化因子抑制组织纤维化，且含有祛疤痕有效成分维替泊芬，达到优异的祛疤痕效果。载间充质干细胞水凝胶组为1.78±0.15；单纯维替泊芬组为1.70±0.18；单纯间充质干细胞组为2.27±0.04；硅酮凝胶组为2.75±0.16，硅酮凝胶只是为疤痕组织提供一个闭合、水润的环境，促使疤痕组织中的成纤维细胞、和胶原得到部分改善，例如胶原重新排列接近正常皮肤顺序。在愈合伤口中，载间充质干细胞和维替泊芬的水凝胶组，维替泊芬缓慢释放对YAP基因表达的抑制作用会阻断En1激活并促进ENF介导的修复，在30天内使皮肤再生，功能性毛囊和皮脂腺恢复而不会发生纤维化。具体的是疤痕形成过程中皮肤深层组织特定的Engrailed-1谱系阴性成纤维细胞细胞（Engrailed-1 lineage negative fibroblasts,ENF）中Engrailed-1基因在YAP蛋白作用下激活并表达，会启动促纤维化细胞程序以响应伤口中的局部组织力学。而维替泊芬是YAP抑制剂，能阻断YAP的作用，从而抑制纤维化减少疤痕形成。From the SEI (Scar Elevation Index) in Table 3, it can be seen that the scar treatment effect of the hydrogel group loaded with mesenchymal stem cells and verteporfin is the best, and the SEI index is 1.26±0.02, which may be due to the presence of hydrogel on the surface. The glue cover has the effect of hypoxia, water retention and moistening. It contains mesenchymal stem cells that secrete cytokines to promote wound healing. In the later stage of wound healing, anti-fibrosis factors are secreted to inhibit tissue fibrosis. It also contains verteporfin, an effective ingredient for removing scars, to achieve excellent Scar removal effect. The mesenchymal stem cell-loaded hydrogel group was 1.78±0.15; the simple verteporfin group was 1.70±0.18; the simple mesenchymal stem cell group was 2.27±0.04; It just provides a closed and moist environment for scar tissue, and promotes partial improvement of fibroblasts and collagen in scar tissue, such as collagen rearrangement close to normal skin order. In healing wounds, a hydrogel group loaded with mesenchymal stem cells and verteporfin, slow release of verteporfin inhibits YAP gene expression blocks En1 activation and promotes ENF-mediated repair within 30 days Regenerates the skin and restores functional hair follicles and sebaceous glands without fibrosis. Specifically, in the process of scar formation, the Engrailed-1 gene in Engrailed-1 lineage negative fibroblasts (ENF) in deep skin tissue is activated and expressed under the action of YAP protein, which will initiate fibrosis Cellular programming to respond to local tissue mechanics in wounds. Verteporfin is a YAP inhibitor, which can block the action of YAP, thereby inhibiting fibrosis and reducing scar formation.

实施例7Example 7

取30kg/只杜洛克红毛猪，按5mL/只皮下注射浓度为100 mg/mL的盐酸二甲苯胺噻嗪溶液，随后对杜洛克红毛猪背部清洗、脱毛和消毒，接着沿猪背部中线两侧对称建立18个伤口（2cm×2cm）。组别设置为：对照组（向伤口处注射200μL生理盐水，每三天注射一次）、单纯间充质干细胞组（向伤口处注射200μL浓度为3×10⁶个/mL的人脂肪间充质干细胞悬液，每三天注射一次）、单纯维替泊芬组（向伤口处注射200μL浓度为1mg/mL的维替泊芬溶液，每三天注射一次）、载间充质干细胞水凝胶组（将200μL实施例3制备的可注射复合水凝胶成胶后贴附于伤口处，每3天更换一次新的与上一次相同的可注射复合水凝胶）、载间充质干细胞和维替泊芬的水凝胶组（将200μL实施例4制备的可注射复合水凝胶成胶后贴附于伤口处，每3天更换一次新的与上一次相同的可注射复合水凝胶），硅酮凝胶（将硅酮凝胶涂敷于伤口处，每3天涂敷一次）。在治疗第 60天后，取杜洛克红毛猪的伤口组织，统计SEI（疤痕升高指数）。Take 30kg/per Duroc red-haired pig, inject 5mL/per subcutaneously with 100 mg/mL xylazine hydrochloride solution, then wash, depilate and disinfect the back of the Duroc red-haired pig, and then along the sides of the midline of the pig's back Eighteen wounds (2cm×2cm) were established symmetrically. The groups were set as follows: control group (inject 200 μL of normal saline into the wound, once every three days), simple mesenchymal stem cell group (inject 200 μL of human adipose-derived mesenchymal cells at a concentration of 3×10⁶ cells/mL into the wound Stem cell suspension, injected once every three days), simple verteporfin group (inject 200 μL of verteporfin solution with a concentration of 1 mg/mL into the wound, injected once every three days), mesenchymal stem cell-loaded hydrogel Group (200 μL of the injectable composite hydrogel prepared in Example 3 was gelled and attached to the wound, and replaced with a new injectable composite hydrogel every 3 days), containing mesenchymal stem cells and Verteporfin hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 4 was gelled and attached to the wound, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days ), silicone gel (apply silicone gel to the wound, and apply it every 3 days). After the 60th day of treatment, the wound tissue of the Duroc red-haired pig was collected, and the SEI (Scar Elevation Index) was counted.

从SEI可以看出，载间充质干细胞和维替泊芬的水凝胶组的疤痕治疗效果最接近于正常皮肤，SEI指数为1.36±0.05，可能是由于表面有水凝胶覆盖起到低氧、保水湿润效果，含有间充质干细胞分泌细胞因子促进伤口愈合，伤口愈合后期分泌抗纤维化因子抑制组织纤维化，且含有祛疤痕有效成分维替泊芬，达到优异的祛疤痕效果。载间充质干细胞水凝胶组为1.61±0.11；单纯维替泊芬组为1.58±0.04；单纯间充质干细胞组为2.37±0.13；硅酮凝胶组为2.17±0.08，硅酮凝胶只是为疤痕组织提供一个闭合、水润的环境，促使疤痕组织中的成纤维细胞、和胶原得到部分改善，例如胶原重新排列接近正常皮肤顺序。It can be seen from the SEI that the scar treatment effect of the hydrogel group loaded with mesenchymal stem cells and verteporfin is closest to that of normal skin, and the SEI index is 1.36±0.05, which may be due to the fact that the surface is covered with hydrogel to play a low role in scar treatment. Oxygen, water retention and moisturizing effects, containing mesenchymal stem cells secrete cytokines to promote wound healing, secrete anti-fibrosis factors in the later stage of wound healing to inhibit tissue fibrosis, and contain the active ingredient verteporfin to achieve excellent scar removal effect. The mesenchymal stem cell-loaded hydrogel group was 1.61±0.11; the simple verteporfin group was 1.58±0.04; the simple mesenchymal stem cell group was 2.37±0.13; It just provides a closed and moist environment for scar tissue, and promotes partial improvement of fibroblasts and collagen in scar tissue, such as collagen rearrangement close to normal skin order.

以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (10)

Translated fromChinese

1.一种可促进创面愈合并减少疤痕形成的可注射复合水凝胶的制备方法，其特征在于：包括以下步骤：1. a preparation method of an injectable composite hydrogel that can promote wound healing and reduce cicatrix formation, is characterized in that: comprise the following steps:1）将透明质酸溶解于超纯水中，向其中加入高碘酸钠，室温氧化反应2~3h，再加入0.5mL乙二醇终止反应1h；反应结束后，以超纯水为透析液，将反应液使用3500Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体，转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得氧化透明质酸海绵材料；1) Dissolve hyaluronic acid in ultrapure water, add sodium periodate to it, oxidize at room temperature for 2~3h, then add 0.5mL ethylene glycol to terminate the reaction for 1h; after the reaction, use ultrapure water as dialysate , the reaction solution was dialyzed for 72 hours using a dialysis bag with a molecular weight of 3500Da; after the dialysis, the liquid in the dialysis bag was collected, transferred to a polytetrafluoroethylene container, placed in a -80°C refrigerator for 5 hours, and then kept at -50°C for 1~ Freeze-drying under the condition of 20Pa for 72h, the oxidized hyaluronic acid sponge material is obtained;2）将天然高聚物溶解于超纯水中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺，室温活化反应2h，再加入碳酰肼粉体，室温反应12h；反应结束后，以超纯水为透析液，将反应液使用8000Da分子量的透析袋透析72h；透析结束后，收集透析袋内液体，转移至聚四氟乙烯容器中，置于-80℃冰箱中冰冻5h，再在-50 ℃、1~20Pa的条件下冷冻干燥72h，即获得碳酰肼天然高聚物材料；2) Dissolve the natural polymer in ultrapure water, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and activate the reaction at room temperature 2h, then add carbohydrazide powder, and react at room temperature for 12h; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction liquid with a dialysis bag with a molecular weight of 8000Da for 72h; after the dialysis, collect the liquid in the dialysis bag and transfer it to Place in a polytetrafluoroethylene container, freeze in a -80°C refrigerator for 5 hours, and then freeze-dry at -50°C and 1-20Pa for 72 hours to obtain carbohydrazide natural polymer materials;3）将氧化透明质酸海绵材料用超纯水配制为溶液，将碳酰肼天然高聚物材料用超纯水配制为溶液；向氧化透明质酸海绵材料溶液中加入间充质干细胞，得到水凝胶前体溶液1；向碳酰肼天然高聚物材料溶液中加入维替泊芬，得到水凝胶前体溶液2；将水凝胶前体溶液1和水凝胶前体溶液2混合均匀，室温搅拌10~20s，直至混合溶液呈胶状，即得到所述可注射复合水凝胶。3) The oxidized hyaluronic acid sponge material was prepared as a solution with ultrapure water, and the carbohydrazide natural polymer material was prepared as a solution with ultrapure water; mesenchymal stem cells were added to the oxidized hyaluronic acid sponge material solution to obtain Hydrogel precursor solution 1; add verteporfin to carbohydrazide natural polymer material solution to obtain hydrogel precursor solution 2; hydrogel precursor solution 1 and hydrogel precursor solution 2 Mix evenly, and stir at room temperature for 10-20 seconds until the mixed solution becomes gelatinous, and the injectable composite hydrogel is obtained.2.根据权利要求1所述的制备方法，其特征在于：步骤1）中，所述透明质酸的用量为1g，所述高碘酸钠的用量为0.25g。2. The preparation method according to claim 1, characterized in that: in step 1), the dosage of the hyaluronic acid is 1 g, and the dosage of the sodium periodate is 0.25 g.3.根据权利要求1所述的制备方法，其特征在于：步骤2）中，所述天然高聚物选自胶原蛋白、明胶中的任意一种。3. The preparation method according to claim 1, characterized in that: in step 2), the natural high polymer is selected from any one of collagen and gelatin.4.根据权利要求1所述的制备方法，其特征在于：步骤2）中，所述天然高聚物的用量为9g，所述1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的用量为8.64g，所述N-羟基琥珀酰亚胺的用量为0.58g，所述碳酰肼粉体的用量为6.6g。4. The preparation method according to claim 1, characterized in that: in step 2), the amount of the natural high polymer is 9 g, and the 1-(3-dimethylaminopropyl)-3-ethyl The amount of carbodiimide hydrochloride was 8.64g, the amount of N-hydroxysuccinimide was 0.58g, and the amount of carbohydrazide powder was 6.6g.5.根据权利要求1所述的制备方法，其特征在于：步骤3）中，所述氧化透明质酸海绵材料溶液的浓度为2%~8%，所述碳酰肼天然高聚物材料溶液的浓度为4%~12%。5. The preparation method according to claim 1, characterized in that: in step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2% to 8%, and the carbohydrazide natural high polymer material solution The concentration is 4%~12%.6.根据权利要求1所述的制备方法，其特征在于：步骤3）中，所述间充质干细胞选自人脂肪间充质干细胞、人骨髓间充质干细胞中的任意一种。6 . The preparation method according to claim 1 , wherein in step 3), the mesenchymal stem cells are selected from any one of human adipose-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells.7.根据权利要求1所述的制备方法，其特征在于：步骤3）中，所述水凝胶前体溶液1和水凝胶前体溶液2的体积比为1：1。7 . The preparation method according to claim 1 , wherein in step 3), the volume ratio of the hydrogel precursor solution 1 and the hydrogel precursor solution 2 is 1:1.8.根据权利要求1所述的制备方法，其特征在于：步骤3）中，所述可注射复合水凝胶中间充质干细胞的浓度为1×10⁶~1×10⁷个/mL、维替泊芬的浓度为0.01~10mg/mL。8. The preparation method according to claim 1, characterized in that: in step 3), the concentration of the injectable composite hydrogel mesenchymal stem cells is 1×10⁶ ~1×10⁷ cells/mL, dimension The concentration of tepofin is 0.01~10mg/mL.9.一种利用权利要求1所述的制备方法制得的可注射复合水凝胶。9. An injectable composite hydrogel prepared by the preparation method according to claim 1.10.如权利要求9所述的可注射复合水凝胶的应用，其用途在于：所述可注射复合水凝胶在制备促进创面愈合和/或减少疤痕形成的药物中的应用。10. The application of the injectable composite hydrogel as claimed in claim 9, wherein the application of the injectable composite hydrogel is in the preparation of medicines for promoting wound healing and/or reducing scar formation.

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