Umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells as well as preparation method and application of umbilical cord hydrogel dressing

Abstract

The invention belongs to the technical field of medical auxiliary materials, and particularly relates to an immortalized human umbilical cord mesenchymal stem cell-loaded umbilical cord hydrogel dressing and a preparation method and application thereof, and the preparation method comprises the following steps: freeze-drying and crushing an umbilical cord acellular matrix to obtain umbilical cord acellular matrix powder; weighing umbilical cord decellularized extracellular matrix powder, putting the umbilical cord decellularized extracellular matrix powder into a pepsin-containing HCl solution, and stirring to form a pre-gel solution; preparing the pregel solution into an isotonic pregel solution; fully stirring and uniformly mixing the isotonic pregel solution and the immortalized human umbilical cord mesenchymal stem cells, and standing to obtain the umbilical cord hydrogel dressing; the content of the umbilical cord acellular epimatrix in the umbilical cord hydrogel dressing is 6-10 mg / mL; the content of the immortalized human umbilical cord mesenchymal stem cells in the umbilical cord hydrogel dressing is (3-4) * 10 < 6 > / mL. The umbilical cord hydrogel dressing loaded with the immortalized human umbilical cord mesenchymal stem cells, provided by the invention, has good biocompatibility and can accelerate healing of pressure sores.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical dressings, and particularly relates to a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] Pressure ulcers, also known as bedsores or pressure ulcers, are caused by long-term pressure on local tissues, resulting in blood circulation disorders, ischemia, hypoxia, nutritional deficiency of the skin, and accumulation of metabolites, causing subcutaneous tissue damage or ulceration and necrosis. It also includes local injuries to subcutaneous tissues caused by external forces such as friction and shear force, usually occurring at bony prominences (such as the sacrum, calcaneus, and ischium). In developed countries such as the United States, up to 3 million people suffer from pressure ulcers, and the total annual treatment cost is as high as $11 billion. Pressure ulcers impose a huge economic burden not only on patients and their families but also on the healthcare system and society.

[0003] According to the NPIAP staging system, pressure injuries are divided into six types, namely: stage 1, stage 2, stage 3, stage 4, unstageable pressure injuries, and deep tissue pressure injuries. The specific treatment of pressure ulcers depends on the stage of skin damage. Skin damage in stage 1 needs to cover the affected area to avoid continued pressure. Stage 2 pressure injuries usually use dressings to keep the wound moist. Stage 3 and stage 4 pressure injuries or deeper injuries generally require debridement of necrotic tissue, wound negative pressure treatment, autologous or artificial skin grafting. Other treatments include a variety of adjuvant therapies, such as electrical stimulation, hyperbaric oxygen therapy, therapeutic ultrasound, and topical oxygen therapy, but their indications are not yet clear. These therapies can cause adverse reactions to the skin when treating pressure ulcers. For example, wound dressings, topical medications, and electrical stimulation may cause skin-related complications, including inflammation, erythema, pain, etc. In addition, the failure rate of skin grafting is 12% - 24%, and repeated or multiple surgeries are required. Despite the large amount of resources spent on prevention and treatment, the incidence and mortality of pressure ulcers remain high. Therefore, it is crucial to explore new and effective methods for treating pressure ulcers.

[0004] Biological scaffolds can provide a 3D growth space for stem cells to adhere and proliferate, as well as a suitable extracellular microenvironment, enabling the formation of interactions between cells and between cells and the matrix, providing important mechanical and chemical information for cells, better maintaining cell activities and functions, and contributing to matrix remodeling, cell migration, and cell adhesion. They are used as carriers for encapsulating cells, drugs, and bioactive molecules. Currently, skin tissue engineering scaffolds are divided into two major categories: synthetic and natural. Synthetic materials (such as polylactic acid and polyglycolic acid) have controllable mechanical properties and can be customized in shape and pore size, but they cannot simulate the complex in vivo microenvironment. Natural materials (such as collagen, chitosan, and decellularized extracellular matrix) have the advantages of biocompatibility, biodegradability, and low immunogenicity. In particular, decellularized extracellular matrix (dECM) has become a research hotspot. dECM retains bioactive signals by removing tissue cell components and provides a three-dimensional cell culture carrier. It is divided into three categories according to its source: autologous, allogeneic, and xenogeneic. Autologous dECM lacks corresponding tissue sources and has limitations such as surgical complications, making it difficult to meet clinical needs. In xenogeneic-derived decellularized extracellular matrix, there may still be some immunogenic substances remaining. After these residual antigens enter the human body, they may trigger an immune response, which may seriously affect the survival and function of the implant and even lead to implant failure. Considering the above reasons, dECM derived from human umbilical cord, as a natural tissue-specific biomaterial from human neonatal tissue, may be an ideal material for loading cells.

[0005] In view of this, it is very important and necessary to develop a new type of hydrogel dressing for the treatment of pressure ulcers. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a hydrogel dressing derived from umbilical cord and its preparation method and application to solve the problems in the prior art.

[0007] To achieve the above object, the present invention adopts the following technical solutions.

[0008] The present invention provides a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, including:

[0009] The umbilical cord tissue is subjected to decellularization treatment to obtain umbilical cord decellularized extracellular matrix; after the umbilical cord decellularized extracellular matrix is freeze-dried and pulverized, umbilical cord decellularized extracellular matrix powder is obtained;

[0010] The umbilical cord decellularized extracellular matrix powder is placed in an HCl solution containing pepsin and stirred to form a pre-gel solution; the pre-gel solution is prepared into an isotonic pre-gel solution;

[0011] The isotonic pre-gel solution is taken and fully stirred and mixed with immortalized human umbilical cord mesenchymal stem cells, and after standing, the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is obtained;

[0012] The content of the umbilical cord tissue decellularized extracellular matrix in the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is 6-10 mg / mL; the content of the immortalized human umbilical cord mesenchymal stem cells in the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is 3-4×10 6 Pieces / mL.

[0013] Mesenchymal stem cells (MSCs) are a type of adult stem cells with great potential. They not only have strong self-renewal and multidirectional differentiation potential, but also have unique immunomodulatory functions. They have a wide range of sources, including bone marrow, fat, umbilical cord and other tissues, and are easy to be expanded in vitro on a large scale, which makes them an ideal seed cell and gene therapy carrier in the field of cell therapy and tissue engineering, bringing new hope for the treatment of many intractable diseases.

[0014] However, although MSCs have great therapeutic potential in theory, they face many challenges in practical application. After MSCs transplantation, local tissues often have difficulty providing sufficient oxygen, nutrients, and key survival factors such as growth factors and cytokines. At the same time, the complex pathological microenvironment of the lesion site, such as the continuous activation of inflammatory response, compression of tissue edema, damage from oxidative stress, attack of immune rejection reaction, and disorder of acid-base balance, will have a serious negative impact on the survival and function of MSCs. These unfavorable factors work together to accelerate the loss of MSCs after transplantation, significantly reduce the survival rate, and severely inhibit the proliferation ability, which ultimately greatly reduces the therapeutic effect or even fails to achieve the therapeutic effect, greatly limiting the widespread application of stem cell transplantation technology in clinical practice.

[0015] In the present invention, the umbilical cord decellularized extracellular matrix is ​​prepared as a pregel, which is used as a scaffold, and immortalized human umbilical cord mesenchymal stem cells are loaded thereon, and the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is finally obtained. The decellularized extracellular matrix pregel used in the present invention is a natural hydrogel based on protein, containing complex natural extracellular matrix components and growth factors, which can promote cell adhesion and proliferation, stimulate angiogenesis and regulate immune response, induce epithelial regeneration and granulation tissue growth, and is more conducive to tissue repair. The decellularized extracellular matrix pregel is fully mixed with immortalized human umbilical cord mesenchymal stem cells to obtain an umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, which can overcome the shortcomings of MSCs used alone to a great extent. And what is used in the present invention is immortalized human umbilical cord mesenchymal stem cells, which overcomes the replication aging of natural MSCs, can be cultured for a long time, can maintain genome stability, and can ensure the continuity and stability of the experiment.

[0016] Meanwhile, the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells provided by the present invention has good biocompatibility and safety. When used for the treatment of pressure ulcers, it can provide a good healing environment for the wound and promote wound healing.

[0017] In some embodiments of the present invention, the mass-volume ratio of the umbilical cord acellular extracellular matrix powder to the HCl solution is 70-120 mg:10 mL.

[0018] In some embodiments of the present invention, the concentration of the HCl solution is 0.01 mol / L - 0.02 mol / L.

[0019] In some embodiments of the present invention, the concentration of pepsin in the HCl solution is 0.5 mg / mL - 1.5 mg / mL.

[0020] In some specific embodiments of the present invention, the pepsin is porcine pepsin.

[0021] In some embodiments of the present invention, the rotation speed of the stirring is 100 rpm - 150 rpm, and the time is 48 h - 72 h.

[0022] In some embodiments of the present invention, the pH of the pre-gel solution is adjusted to pH = 7.4, and then 10×PBS is added to make the pre-gel solution an isotonic pre-gel solution.

[0023] In some embodiments of the present invention, the pH of the pre-gel solution is adjusted using a 0.1 mol / L NaOH solution.

[0024] Specifically, in some embodiments of the present invention, the volume ratio of the pre-gel solution to the 10×PBS is 9:1.

[0025] In some embodiments of the present invention, the standing time is 5 min - 10 min.

[0026] In some embodiments of the present invention, in S1, the decellularization process includes: washing the umbilical cord tissue after removing the umbilical artery and vein, placing it in trypsin for digestion, and then performing the first rinse to terminate the digestion reaction and remove trypsin, cell debris, and digestion products; after the rinse is completed, placing the umbilical cord in Triton X-100 for treatment, and then performing the second rinse to terminate the action of Triton X-100, remove the remaining Triton X-100, and remove cell debris and soluble substances; after the second rinse is completed, placing the tissue in a sodium deoxycholate solution for treatment, and then performing the third rinse to terminate the action of sodium deoxycholate, remove sodium deoxycholate, and remove cell components and debris; placing the umbilical cord tissue after the third rinse in an incubation solution containing deoxyribonuclease I (DNase I) and ribonuclease (RNase) for incubation, and after the incubation is completed, performing the fourth rinse to terminate the enzyme reaction and remove nucleic acid residues, enzyme proteins, and reaction products; after the fourth rinse is completed, placing the umbilical cord tissue in a culture dish and storing it in a -80°C refrigerator overnight to finally obtain the umbilical cord acellular extracellular matrix.

[0027] In some embodiments of the present invention, the mass percentage concentration of trypsin is 0.1% - 0.3%; the conditions for trypsin digestion are 140 rpm - 155 rpm for 1.5 h - 2.5 h.

[0028] In some embodiments of the present invention, the mass percentage concentration of Triton X-100 is 2.5% - 3.5%, and the treatment conditions for Triton X-100 are 140 rpm - 155 rpm for 1.5 h - 2.5 h.

[0029] In some embodiments of the present invention, the mass percentage concentration of the sodium deoxycholate solution is 3.5% - 4.5%, and the treatment conditions for the sodium deoxycholate solution are 140 rpm - 155 rpm for 0.5 h - 1.5 h.

[0030] In some embodiments of the present invention, in the incubation solution, the concentration of DNase I is 155 U / mL - 200 U / mL, and the concentration of RNase is 0.5 U / mL - 1 U / mL; the incubation conditions are 140 rpm - 155 rpm for 10 h - 13 h.

[0031] In some embodiments of the present invention, the first rinse, the second rinse, the third rinse, and the fourth rinse are all performed using deionized water.

[0032] In some embodiments of the present invention, the time for the first rinse is 1.5 h - 2 h; the time for the second rinse is 1.5 h - 2 h; the time for the third rinse is 1.5 h - 2 h; the time for the fourth rinse is 5.5 h - 6.5 h.

[0033] The present invention also provides a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared by the said preparation method.

[0034] The present invention also provides the application of the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells in the preparation of a drug for promoting skin wound healing.

[0035] In some technical solutions of the present invention, the drug for promoting skin wound healing includes: a drug for treating pressure sores, a drug for treating diabetic ulcers, a dressing for treating deep burn wounds or a drug for treating scald wounds.

[0036] Based on the technical solutions of the present invention, compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) In the present invention, the acellular extracellular matrix pre-gel and immortalized human umbilical cord mesenchymal stem cells are fully mixed to prepare a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells. The prepared umbilical cord hydrogel dressing has good biocompatibility and safety. The results of the hydrogel skin irritation study show that the umbilical cord-derived hydrogel dressing provided by the present invention has no irritating effect on the skin, and there is no erythema, edema, bleeding, necrosis, etc. on the skin. When it is used for the treatment of pressure sores, it can provide a good healing environment for the wound and promote wound healing. Moreover, the preparation raw materials of the present invention are rich in sources and convenient to prepare, and a natural hydrogel dressing with good biocompatibility and safety can be prepared, which has the prospects of industrialization and commercialization.

[0038] (2) In the present invention, the use of human umbilical cord-derived acellular extracellular matrix (dECM) can avoid the risks of xenogeneic immunity and disease transmission and is more favored, and it has low immunogenicity. In addition, under the influence of age, the cross-linking degree of the matrix caused by protein glycosylation and fat deposition in adult tissues will significantly affect the characteristics of dECM. However, the dECM collagen from fetal / newborn sources is immature and has a low cross-linking degree, which can reduce fibrosis and oxidative stress and is more conducive to tissue repair and remodeling.

[0039] (3) In the present invention, immortalized human umbilical cord mesenchymal stem cells are used to prepare a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells. The sources of immortalized human umbilical cord mesenchymal stem cells are rich, easy to extract, and can be amplified infinitely; the decellularization method is relatively simple, has the prospects of industrialization and commercialization, and can be mass-produced.

[0040] (4) The umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells provided by the present invention is a honeycomb-structured, water-absorbent, biodegradable viscoelastic hydrogel, which has good biocompatibility and safety, and has no skin irritation such as skin redness and stinging after use, and can meet the requirements of being a tissue engineering scaffold.

[0041] (5) The umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells provided in the present invention can provide a moist environment and accelerate the healing of mouse pressure ulcers; therefore, the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells provided in the present invention is well applicable to the treatment of pressure ulcers. Description of the Drawings

[0042] Figure 1 It is a process diagram for the preparation of the umbilical cord acellular extracellular matrix hydrogel provided in the present invention.

[0043] Figure 2 It is a scanning electron microscope image of umbilical cord acellular extracellular matrix hydrogels with different concentrations; among them, the first row represents the scanning electron microscope image with a scale bar of 500 μm, and the second row represents the scanning electron microscope image with a scale bar of 100 μm.

[0044] Figure 3 It is the swelling ratio of umbilical cord acellular extracellular matrix hydrogels with different concentrations in PBS (pH = 7.4).

[0045] Figure 4 It is the frequency scan and time scan diagrams of the elastic modulus (G') and viscous modulus (G") of umbilical cord acellular extracellular matrix hydrogels with different concentrations; among them, Figure 4 A in it represents the frequency scan diagram of G' and G" of umbilical cord acellular extracellular matrix hydrogels with different concentrations; Figure 4 B in it represents the time scan diagram of G' and G" of umbilical cord acellular extracellular matrix hydrogels with different concentrations.

[0046] Figure 5 It is the degradation rate of umbilical cord acellular extracellular matrix hydrogels with different concentrations in different degradation media; among them, Figure 5 A in it represents the degradation rate of the umbilical cord acellular extracellular matrix hydrogel in the presence of 2000 U / mL lysozyme; Figure 5 B in it represents the degradation rate of the umbilical cord acellular extracellular matrix hydrogel in PBS.

[0047] Figure 6 It is a diagram showing the results of the cell compatibility test of umbilical cord acellular extracellular matrix hydrogels with different concentrations in the present invention.

[0048] Figure 7 It is a diagram showing the results of the biosafety test of the umbilical cord acellular extracellular matrix hydrogel; among them, Figure 7 represents the images of the back skin of mice at different time points after treatment; Figure 7 B in it represents the HE staining image of the skin 72 hours after treatment.

[0049] Figure 8 It is a representative image of the pressure ulcer healing process with different treatment methods.

[0050] Figure 9 The healing rate curve graph of pressure ulcers for different treatment methods. Detailed implementation manners

[0051] Next, in combination with the embodiments in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0052] The following will be described in combination with specific embodiments.

[0053] I. Experimental materials:

[0054] 1. The umbilical cord is of human origin, but is usually treated as medical waste. It has a wide source and is an excellent tissue source for obtaining a large number of mesenchymal stem cells in clinical and research.

[0055] 2. Immortalized human umbilical cord mesenchymal stem cells are disclosed in the article "Establishing an hTERT-driven immortalized umbilical cord-derived mesenchymal stem cell line and its therapeutic application in mice with liver failure" by Qi Chen, Meixian Jin, Simin Wang, Kexin Wang, Liqin Chen, Xiaojuan Zhu, Ying Zhang, Shao Li, Yang Li, Youmin Zeng1, Rui Hu, Shuqin Zhou* and Qing Peng*, Journal of Tissue Engineering, September 19, 2023.

[0056] Example 1

[0057] A preparation method of an umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, comprising:

[0058] (1) Obtain the umbilical cord, remove the umbilical artery and vein, and wash with cold phosphate buffered saline for 1 day;

[0059] (2) Place the washed umbilical cord in 0.1% trypsin and digest at a rotation speed of 150 rpm for 2 hours, and then rinse with deionized water for 2 hours;

[0060] (3) Place the umbilical cord in a 3% Triton X-100 (w / v) solution and treat it at 150 rpm for 2 h, followed by rinsing with deionized water for 2 h;

[0061] (4) Place the umbilical cord in a 4% sodium deoxycholate solution and treat it at 150 rpm for 1 h, followed by rinsing with deionized water for 2 h;

[0062] (5) Place the umbilical cord in 200 U / mL DNase I and 1 U / mL RNase and incubate it at 150 rpm for 12 h, followed by rinsing with deionized water for 6 h;

[0063] (6) Place the umbilical cord in a petri dish and store it in a -80 °C refrigerator overnight; Place the umbilical cord in a vacuum freeze concentrator and freeze-dry it for 96 h;

[0064] (7) Place the umbilical cord in a cryogenic grinder and grind it into a dry powder to form umbilical cord acellular extracellular matrix powder;

[0065] (8) Weigh 122 mg of umbilical cord acellular extracellular matrix powder, irradiate it with ultraviolet light for 2 h, and then place it in 9 mL of 0.01 mol / L HCl solution containing porcine pepsin (the concentration of porcine pepsin is 1 mg / mL), and stir it at 150 rpm for 48 h to form a pre-gel solution; In the prepared pre-gel solution, the content of umbilical cord acellular extracellular matrix powder is 10 mg / mL;

[0066] (9) Add the pre-gel solution to 900 μL of 0.1 mol / L NaOH solution and mix well, neutralize it to pH 7.4, add 1 mL of 10×PBS to make the neutralized pre-gel into an isotonic pre-gel solution, and store it in a 4 °C refrigerator. The volume ratio of the pre-gel solution adjusted to pH 7.4 to 10×PBS is 9:1.

[0067] (10) Take the isotonic pre-gel and immortalized human umbilical cord mesenchymal stem cells (hTERT-UCMSCs) and stir them well. Incubate them at 37 °C for 5 min to obtain a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells. In each milliliter of the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared, it contains 10 mg of umbilical cord acellular extracellular matrix hydrogel and 4×10 6 immortalized human umbilical cord mesenchymal stem cells.

[0068] Example 2

[0069] A preparation method of a umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, comprising:

[0070] (1) Obtain the umbilical cord, remove the umbilical arteries and veins, and wash it with cold phosphate buffered saline for 1 d;

[0071] (2) Place the washed umbilical cord in 0.2% trypsin and digest it at a rotation speed of 140 rpm for 2.5 h, and then rinse it with deionized water for 2.5 h;

[0072] (3) Place the umbilical cord in 2.5% Triton X-100 (w / v) solution and process it at a rotation speed of 140 rpm for 2 h, and then rinse it with deionized water for 1.5 h;

[0073] (4) Place the umbilical cord in 3.5% sodium deoxycholate solution and process it at a rotation speed of 140 rpm for 1 h, and then rinse it with deionized water for 1.5 h;

[0074] (5) Place the umbilical cord in 155 U / mL DNase I and 0.5 U / mL RNase and incubate it at a rotation speed of 150 rpm for 10 h, and then rinse it with deionized water for 5.5 h;

[0075] (6) Place the umbilical cord in a petri dish and store it in a -80 °C refrigerator overnight; place the umbilical cord in a vacuum freeze concentrator and freeze-dry it for 96 h;

[0076] (7) Place the umbilical cord in a cryogenic grinder and grind it into dry powder to form umbilical cord acellular extracellular matrix powder;

[0077] (8) Weigh 100 mg of umbilical cord acellular extracellular matrix powder, irradiate it with ultraviolet light for 2 h, and then place it in 9 mL of a solution containing porcine pepsin with a concentration of 0.02 mol / L HCl (the concentration of porcine pepsin is 0.5 mg / mL), and stir it at a rotation speed of 100 rpm for 50 h to form a pre-gel solution; in the prepared pre-gel solution, the content of umbilical cord acellular extracellular matrix powder is 8 mg / mL;

[0078] (9) Add 1.8 mL of 0.1 mol / L NaOH solution to the pre-gel solution and mix it well, neutralize it to pH 7.4, add 1 mL of 10×PBS to make the neutralized pre-gel into an isotonic pre-gel solution, and store it in a 4 °C refrigerator. The volume ratio of the pre-gel solution adjusted to pH 7.4 to 10×PBS is 9:1.

[0079] (10) Take the isotonic pre-gel and immortalized human umbilical cord mesenchymal stem cells and stir them well. Incubate them at 37 °C for 10 min to obtain an umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells. In each milliliter of the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared, it contains 8 mg of umbilical cord acellular extracellular matrix hydrogel and 3.5×10 6 immortalized human umbilical cord mesenchymal stem cells.

[0080] Example 3

[0081] A preparation method of an umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, comprising:

[0082] (1) Obtain the umbilical cord, remove the umbilical artery and vein, and wash it with cold phosphate-buffered saline for 1 day;

[0083] (2) Place the washed umbilical cord in 0.1% trypsin and digest it at a speed of 150 rpm for 2 h, then rinse it with deionized water for 2 h;

[0084] (3) Place the umbilical cord in a 3% Triton X-100 (w / v) solution and treat it at a speed of 150 rpm for 2 h, then rinse it with deionized water for 2 h;

[0085] (4) Place the umbilical cord in a 4% sodium deoxycholate solution and treat it at a speed of 150 rpm for 1 h, then rinse it with deionized water for 2 h;

[0086] (5) Place the umbilical cord in 170 U / mL DNase I and 1 U / mL RNase and incubate it at a speed of 150 rpm for 12 h, then rinse it with deionized water for 6.5 h;

[0087] (6) Place the umbilical cord in a petri dish and keep it in a -80 °C refrigerator overnight; Place the umbilical cord in a vacuum freeze concentrator and freeze-dry it for 96 h;

[0088] (7) Place the umbilical cord in a cryogenic grinder and grind it into dry powder to form umbilical cord acellular extracellular matrix powder;

[0089] (8) Weigh 70 mg of umbilical cord acellular extracellular matrix powder, irradiate it with ultraviolet light for 2 h, then place it in 9 mL of 0.01 mol / L HCl solution containing porcine pepsin (porcine pepsin concentration is 1 mg / mL) and stir it at a speed of 150 rpm for 48 h to form a pre-gel solution; In the prepared pre-gel solution, the content of umbilical cord acellular extracellular matrix powder is 6 mg / mL;

[0090] (9) Add the pre-gel solution to 900 μL of 0.1 mol / L NaOH solution and mix well, neutralize it to pH 7.4, add 1 mL of 10×PBS to make the neutralized pre-gel into an isotonic pre-gel solution, and store it in a 4 °C refrigerator. The volume ratio of the pre-gel solution adjusted to pH 7.4 to 10×PBS is 9:1.

[0091] (10) Take the isotonic pre-gel and immortalized human umbilical cord mesenchymal stem cells and stir them well. Incubate them at 37 °C for 8 min to obtain the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells. In each milliliter of the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared, it contains 6 mg of umbilical cord acellular extracellular matrix hydrogel and 3×10 6 immortalized human umbilical cord mesenchymal stem cells.

[0092] Performance detection:

[0093] 1. Prepare the pre-gel solutions described in Examples 1 to 3 into umbilical cord decellularized extracellular matrix hydrogels, and detect the performance of the umbilical cord decellularized extracellular matrix hydrogel scaffolds.

[0094] The preparation method of umbilical cord decellularized extracellular matrix hydrogel (UC-dECM), see the preparation flow chart in Figure 1 , including: taking the pre-gel solutions prepared in Examples 1 to 3, adding 900 μL of 0.1 mol / L NaOH solution thereto and mixing well, neutralizing to pH 7.4, adding 1 mL of 10×PBS to make the neutralized pre-gel into an isotonic pre-gel solution, and placing it at 37 °C to form umbilical cord decellularized extracellular matrix hydrogel (UC-dECM).

[0095] (1) Physicochemical property detection

[0096] Detect the appearance morphology, electron microscopy structure, swelling ratio, rheological properties and degradation properties of UC-dECM prepared using the pre-gel solutions provided in Examples 1 to 3 respectively. dECM-6, dECM-8, and dECM-10 respectively represent that the concentration of umbilical cord decellularized extracellular matrix powder in the pre-gel is 6 mg / mL, 8 mg / mL, and 10 mg / mL.

[0097] 1) Scanning electron microscopy: Place 400 μL of hydrogel of each composition type in a refrigerator at -20 °C and freeze overnight, and then dry it. After spraying gold on the surface of the hydrogel for 30 s, observe the surface morphology of the hydrogel using a scanning electron microscope.

[0098] 2) Swelling ratio detection method: Measure the dry weight (W dry ) of 400 μL of hydrogel, and then immerse the samples in PBS buffer solution (pH 7.4) at 37 °C for hydration for 1, 2, 3, 4, 5, 6, 7 h respectively. Take them out, gently dry the surface moisture with filter paper, and weigh the hydrogel to obtain its weight (W swollen ). The water absorption ratio (Q) of the hydrogel can be calculated as: Q = (W swollen - W dry ) / W dry

[0099] 3) Rheological property detection method: Use a stainless steel parallel plate rotor with a diameter of 25 mm for rheological measurement. G' characterizes the elastic modulus of the sample, and G" characterizes the viscous modulus of the sample. Perform a dynamic strain sweep from 0.1 to 10 rad / s at room temperature to determine the linear viscoelastic range of the hydrogel, and record the change curves of the storage modulus (G') and the loss modulus (G").

[0100] 4) Degradation performance detection method: First, freeze-dry the hydrogel after reaching swelling equilibrium and weigh its mass, denoted as Wo. Then, soak the initial hydrogel in PBS or PBS solution containing 2000 U / mL lysozyme, and place it in a constant temperature shaker (37 °C, 70 rpm). At the measured time points, take out the hydrogel, wash it with ultrapure water and then freeze-dry it, and accurately weigh its mass after drying, denoted as (Wt). Calculate the in vitro degradation rate of the scaffold using the formula: weight loss rate (%) = (Wt) / (Wo) × 100%

[0101] (2) Cell compatibility and blood compatibility detection

[0102] Perform biocompatibility and safety detection on the dECM hydrogel containing 10 mg / mL dECM. Specifically, use the CCK8 method and hemolysis test to detect the cell compatibility and blood compatibility of the hydrogel.

[0103] (3) Skin irritation test

[0104] Test method: Conduct a skin irritation test using 6-8-week-old male BALB / c mice. The experimental steps are as follows: Anesthetize the mice and shave the hair on their backs, and then divide the mice into three groups (n = 6 in each group).

[0105] Positive control group (Positive): Apply 0.8% (volume ratio) formalin (standard irritant) to the back skin of the mice.

[0106] Negative control group (Negative): Apply 0.9% NaCl to the back skin of the mice.

[0107] UC-dECM group: Apply 10 mg / mL UC-dECM hydrogel to the back skin of the mice.

[0108] Take pictures at 1, 24, 48, and 72 hours after treatment to record the erythema and edema conditions. Euthanize the mice 72 hours later and collect skin tissues for histopathological examination.

[0109] 2. Detection results

[0110] (1) Physicochemical property detection

[0111] The results of scanning electron microscopy observation are as Figure 2 shown. The scanning electron microscope observed the microstructures of three different concentrations of hydrogels. From Figure 2 it can be seen the scanning electron microscope images of the hydrogel material at different magnifications. All samples have a uniform and interconnected porous microstructure, which conforms to the typical hydrogel material structure.

[0112] The measured results of the swelling ratio are as Figure 3As shown, all hydrogels can basically reach the swelling equilibrium state within 6 h and maintain a stable swelling rate at subsequent times, indicating that the hydrogels have relatively excellent dimensional stability. The equilibrium swelling rates of the hydrogels with concentrations of 6 mg / mL, 8 mg / mL, and 10 mg / mL are 15.99 ± 0.61%, 21.72 ± 1.74%, and 24.95 ± 1.61% respectively. The higher the concentration of dECM, the higher the swelling rate of the corresponding hydrogel.

[0113] As Figure 4 shown in A of Figure 4 is the relationship diagram of the elastic modulus (G') and viscous modulus (G") of the umbilical cord acellular extracellular matrix hydrogel with time. It can be seen from the figure that when G' is greater than G", the hydrogel shows a gel state.

[0114] The measurement results of the degradation rate are as Figure 5 shown Figure 5 in A and B of

[0115] respectively are the degradation curves of the hydrogel under the conditions of no lysozyme and lysozyme concentration of 2000 U / mL. It can be seen that lysozyme can greatly increase the degradation rate of the hydrogel. The results show that the hydrogel gradually degrades with the extension of time, which can provide a suitable dynamic space for cell proliferation and migration. Figure 6 shown, the cell survival rates of hTERT-UCMSCs in different concentrations of UC-dECM hydrogels at 24 and 28 hours are above 98%, and the cell viability and proliferation ability are not affected. Therefore, it can be judged that the UC-dECM hydrogel is non-toxic to hTERT-UCMSCs, indicating that the UC-dECM hydrogel has good cell compatibility.

[0116] The results of the hemolysis experiment are shown in Table 1. After measuring the absorbance of each group with an enzyme-labeled instrument, the hemolysis rates of 6, 8, and 10 mg / mL UC-dECM hydrogels are 0.10%, 0.64%, and 0.77% respectively, all of which meet the requirements of this detection of biomaterials (<5%), indicating that the dECM hydrogel has good blood compatibility.

[0117] Table 1 Results of hemolysis experiment (X±S, n = 3)

[0118] Group OD Hemolysis rate (%) 6mg / mL dECM 0.051±0.001 0.10 8mg / mL dECM 0.052±0.003 0.64 10mg / mL dECM 0.054±0.002 0.77 Negative control group 0.050±0.002 0 Positive control group 0.448±0.010 100

[0119] As Figure 7 shown Figure 7 in A of Figure 7As shown in B of [Figure 0], the results of HE staining of the skin 72 hours after treatment are presented. In the positive control group of mice, inflammatory cell infiltration occurred, while in the negative control and dECM groups of mice, there was no inflammatory cell infiltration, bleeding, necrosis, etc. in the skin, indicating that the dECM hydrogel has no skin irritation.

[0120] 3. Therapeutic effect of umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells on pressure ulcers

[0121] Test method: C57BL6 male mice aged 6 - 8 weeks were used to establish a pressure ulcer model on the back skin of the mice according to the description of Stadler et al. (Stadler I, Zhang RY, Oskoui P, et al. Development ofa simple, noninvasive, clinically relevant model ofpressure ulcers in the mouse[J]. JInvest Surg, 2004, 17(4):221 - 227.). The mice were divided into four groups: (1) hTERT - UCMSCs / UC - dECM hydrogel group: The umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared in Example 1 was used to cover the wound. (2) UC - dECM group (dECM): The UC - dECM dressing (the content of acellular extracellular matrix was 10mg / mL) was used to cover the wound. (3) hTERT - UCMSCs group: 10 6 hTERT - UCMSCs were injected subcutaneously. (4) PBS wet gauze group (Ctrl): PBS wet gauze.

[0122] Preparation of hTERT - UCMSCs / UC - dECM hydrogel dressing: The pre - gel was prepared according to the method described in Example 1. 150 μL of the prepared pre - gel was mixed with 10 6 hTERT - UCMSCs and then inoculated in a 48 - well plate, and then placed in an incubator at 37°C with 5% CO2 for 5 minutes to form a hydrogel dressing about 1.5 mm thick for use in the hTERT - UCMSCs / UC - dECM hydrogel group.

[0123] Preparation of UC - dECM hydrogel dressing: The pre - gel was prepared according to the method described in Example 1. 150 μL of the prepared pre - gel was inoculated in a 48 - well plate, and then placed in an incubator at 37°C with 5% CO2 for 5 minutes to form a hydrogel dressing about 1.5 mm thick for use in the UC - dECM group.

[0124] Wound surface images were collected on the 0th, 3rd, 5th, 7th, 10th, and 14th days after treatment, and the dressings were replaced. The wound surface images collected on the 0th, 3rd, 5th, 7th, 10th, and 14th days after treatment were measured and analyzed using Image J. According to the formula, wound healing rate (%) = [A0 - A(3, 5, 7, 10, 14)) / A0] × 100%. A0 and A(3, 5, 7, 10, 14) represent the areas of the unhealed wound surface regions on the 0th, 3rd, 5th, 7th, 10th, and 14th days, respectively.

[0125] 5. Test results

[0126] The test results are as Figure 8 and Figure 9 shown. hTERT-UCMSCs, UC-dECM, and hTERT-UCMSCs / UC-dECM hydrogels all have therapeutic effects on pressure ulcers, and the therapeutic effect of the hTERT-UCMSCs / UC-dECM hydrogel is better than that of hTERT-UCMSCs and UC-dECM hydrogels.

[0127] In summary, the hydrogel dressing provided by the present invention has good biocompatibility and safety, without skin irritation such as skin redness and stinging; it provides a moist environment and can accelerate the healing of pressure ulcers in mice. Therefore, the hydrogel dressing provided by the present invention is very suitable for use as a pressure ulcer dressing.

[0128] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as these combinations of technical features do not conflict, they should be considered to be within the scope described in this specification.

[0129] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A method for preparing an umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells, characterized in that: include: Decellularizing the umbilical cord tissue to obtain an umbilical cord decellularized extracellular matrix; The umbilical cord decellularized extracellular matrix is ​​freeze-dried and crushed to obtain umbilical cord decellularized extracellular matrix powder; placing the umbilical cord decellularized extracellular matrix powder in a HCl solution containing pepsin, and stirring to form a pre-gel solution; preparing the pregel solution as an isotonic pregel solution; The isotonic pre-gel solution and the immortalized human umbilical cord mesenchymal stem cells are fully stirred and mixed, and after standing, the umbilical cord hydrogel dressing loaded with the immortalized human umbilical cord mesenchymal stem cells is obtained; The content of the umbilical cord tissue decellularized extracellular matrix in the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is 6-10 mg / mL; the content of the immortalized human umbilical cord mesenchymal stem cells in the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells is 3-4×10 6 Pieces / mL.

2. The preparation method according to claim 1, characterized in that The mass volume ratio of the umbilical cord decellularized extracellular matrix powder to the HCl solution is 70-120 mg:10 mL.

3. The preparation method according to claim 2, characterized in that: The concentration of the HCl solution is 0.01 mol / L to 0.02 mol / L.

4. The preparation method according to claim 3, characterized in that: The concentration of pepsin in the HCl solution is 0.5 mg / mL to 1.5 mg / mL.

5. The preparation method according to claim 3, characterized in that: The stirring speed is 100 rpm to 150 rpm, and the stirring time is 48 h to 72 h.

6. The preparation method according to claim 2, characterized in that: The pH of the pregel solution was adjusted to pH=7.4, and then 10×PBS was added to make the pregel solution an isotonic pregel solution.

7. The preparation method according to claim 2, characterized in that: The standing time is 5 to 10 minutes.

8. The umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the umbilical cord hydrogel dressing loaded with immortalized human umbilical cord mesenchymal stem cells as claimed in claim 8 in the preparation of a medicament for treating and promoting the healing of skin wounds.

10. The use according to claim 9, characterized in that The medicine for promoting skin wound healing includes: medicine for treating bedsores, medicine for treating diabetic ulcers, dressings for treating deep burn wounds or medicine for treating scald wounds.

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