CN112263713A - Fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells and application thereof - Google Patents
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Abstract
The invention discloses a fibrin hydrogel bracket loaded with human umbilical cord mesenchymal stem cells and application thereof, belonging to the technical field of hydrogel. The human umbilical cord mesenchymal stem cells are cultured until the density reaches 70-80%; treating by adopting a cell digestive fluid, and resuspending the human umbilical cord mesenchymal stem cells by adopting a fibrinogen solution to obtain a human umbilical cord mesenchymal stem cell-fibrinogen solution mixed solution; and adding a thrombin solution into the mixed solution to obtain the fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells. The combined application of the fibrin hydrogel scaffold and human umbilical cord mesenchymal stem cells obviously accelerates the healing speed of wounds, promotes re-epithelialization, inhibits inflammatory reaction, promotes angiogenesis, reduces scar formation, enables the healing of the wounds to be close to normal skin tissues to a greater extent, and meets the treatment requirement.
Description
Technical Field
The invention relates to a fibrin hydrogel bracket loaded with human umbilical cord mesenchymal stem cells and application thereof, belonging to the technical field of hydrogel.
Background
Impaired wound healing has become a global public health challenge, placing a tremendous psychological, economic, and emotional burden on patients and physicians. According to the world health organization, about 18 million people die of serious burns every year worldwide. The high mortality rate in burn patients is due to increased metabolic demand caused by skin loss, severe fluid loss and increased risk of infection. Other patients, such as diabetics, have wounds that heal slowly and even do not heal, as well as a high risk of infection of the wound and other complications. Skin grafting, as a first line treatment for healing wounds, suffers from deficiencies such as limited natural skin, inconsistent skin tone, etc.
In recent years, stem cells have been widely used in the research of wound healing, and human umbilical mesenchymal stem cells (hUC-MSCs) are multifunctional, have the potential of multidirectional differentiation and paracrine effect, have strong proliferation capability, low immunogenicity, convenient material taking and no ethical dispute and are proved to be applicable to the treatment of wounds. Patent publication No. CN 107875171A: discloses an application of umbilical cord mesenchymal stem cell medicine in promoting skin wound healing, belonging to the technical field of biological medicine. Umbilical cord mesenchymal stem cells and human fibroblasts induced and differentiated by the umbilical cord mesenchymal stem cells are used as a main drug body, small molecule active peptides are bonded with the liposome carrying the drug, and magnetic particles are added into the liposome, so that the targeting property of the drug is improved, the cells can be supplemented with magnetism, the activity of the cells is improved, the senescence is delayed, and the growth of epidermal cells is facilitated; the injection is magnetized before injection, the compound electrolyte in the injection increases the magnetic susceptibility of the injection, and the magnetized injection can activate cells in the medicine and cells in the human body, thereby improving the medicine effect and the healing capability of the injection. However, the preparation process in the prior art is complicated, long in time and high in cost, and the problem that stem cells are difficult to survive at wounds cannot be solved.
Disclosure of Invention
In order to solve the problems, the invention provides a fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells.
The first purpose of the invention is to provide a preparation method of a fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells, which comprises the following steps:
s1, culturing the human umbilical cord mesenchymal stem cells until the density reaches 70-80%;
s2, treating the human umbilical cord mesenchymal stem cells obtained in the step S1 by using cell digestion solution, and re-suspending the human umbilical cord mesenchymal stem cells by using fibrinogen solution to obtain human umbilical cord mesenchymal stem cell-fibrinogen solution mixed solution;
s3, adding a thrombin solution into the mixed solution of the human umbilical cord mesenchymal stem cells and the fibrinogen solution in the step S2 to obtain the fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells.
Further, in the step S3, the volume ratio of the human umbilical cord mesenchymal stem cell-fibrinogen solution mixed solution to the thrombin solution is 0.5-2: 1.
Further, the thrombin solution is prepared by dissolving thrombin in a calcium ion solution, and the concentration of the thrombin solution is 10-25U/ml.
Further, the calcium ion solution comprises 200-400mmol/L NaCl and 30-50mmol/L CaCl2。
Further, in the step S2, the concentration of the fibrinogen solution is 5-15 mg/ml.
Further, in step S2, the cell digest is pancreatin cell digest.
Further, in the step S1, the human umbilical cord mesenchymal stem cell is a human umbilical cord mesenchymal stem cell subcultured to P1-P6 generation.
Further, in the step S1, the cultivation is carried out under the conditions of carbon dioxide concentration of 4-6%, temperature of 35-38 ℃ and saturated humidity.
The second purpose of the invention is to provide the fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells, which is prepared by the preparation method.
The third purpose of the invention is to provide the application of the fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells in the preparation of products for promoting the healing of skin wounds.
The invention has the beneficial effects that:
(1) the fibrin hydrogel scaffold provided by the invention is of a three-dimensional network structure, has good biosafety and degradability, is beneficial to survival, adhesion and proliferation of human umbilical cord mesenchymal stem cells, and prolongs the survival time of the cells at wounds. And the gel can be formed within ten seconds, the blood coagulation is simulated, and the preparation is simple.
(2) The human umbilical cord mesenchymal stem cells used in the invention are used as pluripotent stem cells with multidirectional differentiation potential and strong paracrine effect, and have the advantages of strong proliferation capacity, low immunogenicity, convenient material acquisition and no ethical dispute. It can directly provide repairing cells for healing skin wounds, accelerate the healing rate, paracrine various cytokines, inhibit inflammatory reaction, promote the proliferation of capillaries, enhance microcirculation, and inhibit the generation of fibroblasts to reduce the scar formation of wound surfaces.
(3) The combined application of the fibrin hydrogel scaffold and human umbilical cord mesenchymal stem cells obviously accelerates the healing speed of wounds, promotes re-epithelialization, inhibits inflammatory reaction, promotes angiogenesis, reduces scar formation, enables the healing of the wounds to be close to normal skin tissues to a greater extent, and meets the treatment requirement.
Drawings
FIG. 1 shows the healing of skin wounds in mice;
FIG. 2 is a graph of the wound healing rates for four groups;
FIG. 3 shows paraffin section HE staining of mouse skin full-thickness tissues;
FIG. 4 is QCPR quantitative evaluation of vascular endothelial growth factors VEGF, VEGFA levels.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1: primary isolation and culture of human umbilical cord mesenchymal stem cells
After the human umbilical cord is obtained by an operating table, the human umbilical cord is immersed in PBS (phosphate buffer solution) containing 1% double antibody (streptomycin) under the aseptic condition, a 50ml centrifugal tube is sealed and aseptic, and the human umbilical cord is transported to a laboratory in an ice box at 4 ℃. Taking out the umbilical cord matrix in a clean bench, washing with 0.9% normal saline for 2 times, removing the part with blood stasis at two ends, fully washing the periphery of the umbilical cord and the inner cavity of the umbilical vein with 1% double-antibody-containing PBS buffer solution, removing arteriovenous vessels, taking the umbilical cord matrix, and shearing into pieces of 4-5 mm3The tissue blocks of the size were inoculated into T25 flasks containing 1ml of DMEM/F12(1:1) medium (containing 10% FBS), 5 blocks per flask, 10 flasks in total, and 5% CO at 37 deg.C2Culturing in a saturated humidity cell culture box, changing the culture medium for the first time after 24h, then changing the culture medium every 2-3d, removing tissue blocks when the cells grow to 70-80% of fusion, and digesting and subculturing by 0.25% trypsin (containing 0.02% EDTA).
Example 2: preparation of fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells
A preparation method of a fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells specifically comprises the following steps:
(1) after primary isolation culture, carrying out subculture on the human umbilical cord mesenchymal stem cells, taking subcultured P1-P6 generation human umbilical cord mesenchymal stem cells, and culturing in a carbon dioxide incubator by using a culture bottle to ensure that the density of the human umbilical cord mesenchymal stem cells reaches 70-80%.
(2) Fibrinogen was dissolved in sterile water for injection to prepare a fibrinogen solution (10 mg/ml).
(3) The thrombin solution (25U/ml) was prepared by dissolving thrombin in a calcium ion solution (containing 300mmol/L NaCl,40mmol/L CaCl 2).
(4) Human umbilical cord mesenchymal stem cells were digested with 0.25% Trypsin-EDTA and the resulting cells were resuspended in fibrinogen solution.
(5) And adding an isovolumetric thrombin solution into the mixed solution of the human umbilical cord mesenchymal stem cells and the fibrinogen solution obtained by resuspension, and obtaining the fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells within a few seconds.
Example 3: mouse full-thickness skin wound experiment
A full-thickness skin wound model is constructed by utilizing SPF-grade BALB/C mice, a fibrin hydrogel bracket loaded with human umbilical cord mesenchymal stem cells is locally injected into a wound, and the effect of the fibrin hydrogel bracket on skin wound healing is evaluated.
(1) An SPF-grade BALB/C nude mouse (8 weeks old, 18-20g in weight and female) is randomly divided into a human umbilical cord mesenchymal stem cell + fibrin hydrogel group, a human umbilical cord mesenchymal stem cell group, a fibrin hydrogel group and a normal saline group.
(2) The isoflurane gas anesthesia machine is used for carrying out high-concentration high-oxygen flow induction anesthesia on the mouse, the prone position is fixed on an operation table after satisfactory anesthesia, low-concentration low-oxygen flow is maintained for anesthesia, and the back skin is disinfected by an iodine disinfectant for three times.
(3) After the sterile hole towel was spread, the skin was perforated (diameter: 8mm) at the lower part of the center of the back of the nude mouse by a sterilized skin punch to prepare a model of the full-thickness skin wound of the mouse.
(4) Respectively injecting physiological saline, fibrin hydrogel, human umbilical cord mesenchymal stem cells, fibrin hydrogel and human umbilical cord mesenchymal stem cells into the wound, feeding 30ul of each mouse, sealing the wound with a 3M antibacterial operation membrane, and repeatedly adding the medicine once on the seventh day.
(5) The skin wound healing of each mouse was recorded by taking pictures with a digital camera on days 1, 4, 7, 10, and 14 after the operation, as shown in fig. 1.
On the 7 th day after operation, the wounds of the mice locally applying the fibrin hydrogel and the human umbilical cord mesenchymal stem cells are observed to be obviously smaller than those of the other three groups, and the wound healing condition of the fibrin hydrogel and the human umbilical cord mesenchymal stem cells group on the 14 th day is also better than that of the other groups and is basically and completely healed. Fig. 2 counts the wound healing rates of the four groups, and it can be seen that the fibrin hydrogel + human umbilical cord mesenchymal stem cell group starts from day 7, the wound healing speed is obviously faster than that of the other three groups, and the wound healing speed approaches to complete healing by day 14. The fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells on the animal level is proved to have better curative effect on wound repair and can accelerate the healing of the wound.
Example 4: HE staining and QPCR evaluation index
(1) After anesthesia of each group of mice on day 7 and 14, a skin punch was used to take a full-thickness tissue of a circular area 8mm in diameter from the original wound, and half-cut was made into paraffin sections for HE staining, as shown in FIG. 3. It can be seen that the number of the microvascular cells of the fibrin hydrogel + human umbilical cord mesenchymal stem cell group is obviously more than that of the other three groups, and the skin repair of the fibrin hydrogel + human umbilical cord mesenchymal stem cell group is closer to the normal tissue at the 14 th day.
(2) The other half of the tissues were quantitatively evaluated for the levels of vascular endothelial growth factors VEGF, VEGFA and epidermal growth factor EGF by QCPR, as shown in FIG. 4. It can be seen that the fibrin hydrogel + human umbilical cord mesenchymal stem cell groups on day 7 and day 14 had higher levels of VEGF, VEGFA than the other three groups. The fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells on the animal level is proved to have better curative effect on wound repair, can promote re-epithelialization and angiogenesis of wounds and enhance microcirculation.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A preparation method of a fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
s1, culturing the human umbilical cord mesenchymal stem cells until the density reaches 70-80%;
s2, treating the human umbilical cord mesenchymal stem cells obtained in the step S1 by using cell digestion solution, and re-suspending the human umbilical cord mesenchymal stem cells by using fibrinogen solution to obtain human umbilical cord mesenchymal stem cell-fibrinogen solution mixed solution;
s3, adding a thrombin solution into the mixed solution of the human umbilical cord mesenchymal stem cells and the fibrinogen solution in the step S2 to obtain the fibrin hydrogel scaffold loaded with the human umbilical cord mesenchymal stem cells.
2. The method according to claim 1, wherein in the step S3, the volume ratio of the human umbilical cord mesenchymal stem cell-fibrinogen solution mixed solution to the thrombin solution is 0.5-2: 1.
3. The method according to claim 2, wherein the thrombin solution is prepared by dissolving thrombin in a calcium ion solution, and the concentration of the thrombin solution is 10 to 25U/ml.
4. The method as claimed in claim 3, wherein the calcium ion solution comprises 200-400mmol/L NaCl and 30-50mmol/L CaCl2。
5. The method according to claim 1, wherein the fibrinogen solution is contained in a concentration of 5 to 15mg/ml in the step of S2.
6. The method according to claim 1, wherein in step S2, the cell digest is pancreatin cell digest.
7. The method of claim 1, wherein the human umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells subcultured to generation P1-P6 in step S1.
8. The method according to claim 1, wherein the culturing is carried out under a condition of a carbon dioxide concentration of 5%, a temperature of 37 ℃ and a saturated humidity in step S1.
9. A fibrin hydrogel scaffold loaded with human umbilical cord mesenchymal stem cells prepared by the preparation method of any one of claims 1 to 8.
10. Use of the human umbilical cord mesenchymal stem cell-loaded fibrin hydrogel scaffold of claim 9 in the preparation of a product for promoting healing of a skin wound.
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Cited By (10)
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CN112891632A (en) * | 2021-02-06 | 2021-06-04 | 万美蓉 | Preparation method of human umbilical cord intima-fibrin composite skin scaffold with immunogen removed |
CN113174065A (en) * | 2021-06-09 | 2021-07-27 | 西北农林科技大学 | Preparation method of bacteriostatic hydrogel containing human umbilical cord mesenchymal stem cell freeze-dried powder |
CN113197917A (en) * | 2021-04-26 | 2021-08-03 | 苏州京赛诺生物科技有限公司 | Human umbilical cord mesenchymal stem cell preparation and application thereof |
CN113430164A (en) * | 2021-05-30 | 2021-09-24 | 华中科技大学同济医学院附属协和医院 | Cell culture system, culture method and application |
CN113502261A (en) * | 2021-07-16 | 2021-10-15 | 上海市东方医院(同济大学附属东方医院) | Large-scale 3D (three-dimensional) hypoxia mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
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CN114796611A (en) * | 2021-01-27 | 2022-07-29 | 华东理工大学 | Implantable antibacterial coating and preparation method and application thereof |
CN115678048A (en) * | 2022-11-27 | 2023-02-03 | 福州大学 | Injectable composite hydrogel capable of promoting wound healing and reducing scar formation and preparation method and application thereof |
CN117547554A (en) * | 2024-01-12 | 2024-02-13 | 山东康根源生物集团有限公司 | Mesenchymal stem cell repair preparation and preparation method thereof |
CN115678048B (en) * | 2022-11-27 | 2024-05-31 | 福州大学 | Injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and preparation method and application thereof |
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CN114796611A (en) * | 2021-01-27 | 2022-07-29 | 华东理工大学 | Implantable antibacterial coating and preparation method and application thereof |
CN114796611B (en) * | 2021-01-27 | 2023-06-30 | 华东理工大学 | Implantable antibacterial coating, preparation method and application thereof |
CN112891632A (en) * | 2021-02-06 | 2021-06-04 | 万美蓉 | Preparation method of human umbilical cord intima-fibrin composite skin scaffold with immunogen removed |
CN113197917A (en) * | 2021-04-26 | 2021-08-03 | 苏州京赛诺生物科技有限公司 | Human umbilical cord mesenchymal stem cell preparation and application thereof |
CN113430164A (en) * | 2021-05-30 | 2021-09-24 | 华中科技大学同济医学院附属协和医院 | Cell culture system, culture method and application |
CN113174065A (en) * | 2021-06-09 | 2021-07-27 | 西北农林科技大学 | Preparation method of bacteriostatic hydrogel containing human umbilical cord mesenchymal stem cell freeze-dried powder |
CN113174065B (en) * | 2021-06-09 | 2023-07-25 | 西北农林科技大学 | Preparation method of antibacterial hydrogel containing human umbilical mesenchymal stem cell freeze-dried powder |
CN113502261A (en) * | 2021-07-16 | 2021-10-15 | 上海市东方医院(同济大学附属东方医院) | Large-scale 3D (three-dimensional) hypoxia mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
CN113502261B (en) * | 2021-07-16 | 2023-08-29 | 上海东方星际干细胞科技有限公司 | Large-scale 3D low-oxygen mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
CN114557956A (en) * | 2021-12-30 | 2022-05-31 | 江苏拓弘生物科技有限公司 | Temperature-sensitive hydrogel loaded with umbilical cord mesenchymal stem cells and preparation method thereof |
CN115678048A (en) * | 2022-11-27 | 2023-02-03 | 福州大学 | Injectable composite hydrogel capable of promoting wound healing and reducing scar formation and preparation method and application thereof |
CN115678048B (en) * | 2022-11-27 | 2024-05-31 | 福州大学 | Injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and preparation method and application thereof |
CN117547554A (en) * | 2024-01-12 | 2024-02-13 | 山东康根源生物集团有限公司 | Mesenchymal stem cell repair preparation and preparation method thereof |
CN117547554B (en) * | 2024-01-12 | 2024-05-14 | 山东康根源生物集团有限公司 | Mesenchymal stem cell repair preparation and preparation method thereof |
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