Injectable hydrogel material and preparation and application method thereof

Abstract

The present invention provides an injectable hydrogel material and a preparation and application method thereof. The hydrogel material comprises a hydrogel component A and a hydrogel component B; the hydrogel component A comprises fibrinogen and skin stem cells, and the hydrogel component B comprises a thrombin solution; the hydrogel component A is a solution, and each milliliter of the hydrogel component A solution contains 50-100 milligrams of fibrinogen and each milliliter of the hydrogel component A solution contains 10 6 ‑10 9 The concentration of the thrombin solution is 10-100 mg / mL. The preparation method is to mix the skin stem cell component with the fibrinogen solution in proportion to obtain a hydrogel component A; and dissolve the thrombin in the calcium chloride solution to obtain a hydrogel component B containing the thrombin solution. The injectable hydrogel material of the present invention is used in the preparation of a material for promoting hair follicle regeneration and repair. The fibrinogen in the hydrogel material component can cooperate with the skin stem cells to promote the improvement of the dryness of the skin stem cells, which is more conducive to the repair of the skin and the regeneration of the hair follicles.

Description

Technical Field

[0001] The invention belongs to the field of regenerative medicine and biomaterials, and in particular relates to an injectable hydrogel material and a preparation method and application thereof. Background Art

[0002] Hair follicles are important appendages of the skin. They are formed during the process of skin morphogenesis according to signals from the dermis. Hair originates from hair follicles and is very important to the human body. It includes functions such as temperature regulation, body protection, sensory activity, and social interaction. Alopecia caused by various reasons such as environment, genetics, and stress is becoming more and more common worldwide, affecting individuals' physical, psychological, and social well-being. The main methods for treating hair loss are drug therapy and hair follicle transplantation. Hair follicle transplantation is an effective treatment method that changes the distribution of scalp hair follicles without increasing the number of hair. However, there are also some problems with hair follicle transplantation. For example, hair follicle transplantation requires hair follicle donors, but the reality is that there are very few hair follicle donors; and because hair loss treatment is often gradual, it requires continuous hair follicle donations to achieve the final treatment effect. Based on the contradiction in reality, hair follicle transplantation at this stage often cannot achieve the optimal treatment effect. Therefore, achieving the regeneration of hair follicles in hair loss patients themselves is the most fundamental solution.

[0003] Hydrogel materials are widely used in the field of skin repair as a biological repair material due to their good biocompatibility. However, most of the existing hydrogel materials are used for skin healing after being combined with drugs, and skin healing does not necessarily regenerate damaged hair follicles. In particular, in areas rich in hair follicles such as the human scalp, conventional skin healing drugs plus hydrogels cannot effectively restore hair follicle regeneration to the original level. There are also schemes in the prior art to promote hair follicle regeneration by adding hair follicle regeneration factors to hydrogels, but since hair follicle regeneration factors require certain biological activity after all, the selection and combination of hydrogel types is very important. The existing hydrogels added with hair follicle regeneration factors either have poor hair follicle regeneration effects due to component mismatch or are inconvenient to use due to material properties. Therefore, the hydrogel materials prepared by the prior art solutions still cannot meet the requirements of human hair follicle regeneration. Therefore, it is of significant practical significance to develop a hydrogel material that can be used for human hair follicle regeneration. Summary of the invention

[0004] The purpose of the present invention is to provide an injectable hydrogel material and a preparation and application method thereof in view of the technical problems existing in the background technology.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An injectable hydrogel material comprises a hydrogel component A and a hydrogel component B; the hydrogel component A comprises fibrinogen and skin stem cells, the hydrogel component B comprises a thrombin solution; the hydrogel component A is a solution, each milliliter of the hydrogel component A solution contains 50-100 mg of fibrinogen, each milliliter of the hydrogel component A solution contains 10 6 -10 9 The concentration of the thrombin solution is 5-25U / mL.

[0007] Further preferably, the skin stem cells include epidermal stem cells and dermal stem cells; the ratio of the number of epidermal stem cells to dermal stem cells is 1:(1-2).

[0008] Further preferably, the thrombin solution also includes free calcium ions in a concentration range of 35-50 mM. Adding an appropriate concentration of calcium ions to the hydrogel component is beneficial to the final formation of the hydrogel.

[0009] Further preferably, the hydrogel component A also includes a sodium chloride solution with a concentration of 0.9%. Setting the liquid environment in the hydrogel component A to be similar to a physiological saline environment is more conducive to the survival of skin stem cells and to extending the service life of the injectable hydrogel material.

[0010] Further preferably, the volume ratio of the hydrogel component A to the hydrogel component B is (1-2): 1. By optimizing the ratio of the hydrogel component A to the hydrogel component B, a hydrogel material with good pores can be prepared, which is conducive to the attachment of skin stem cells.

[0011] Further preferably, the hydrogel component A further comprises an active factor that promotes hair follicle proliferation; the active factor is at least one of BMP4, BMP6 and FGF7.

[0012] Based on the same technical idea, the present invention also provides a method for preparing the above-mentioned injectable hydrogel material, comprising the following steps:

[0013] (1) epidermal stem cells and dermal stem cells are sheared and separated from skin tissue for culture, and then resuspended in PBS after separation and culture, and the cells are collected after centrifugation; the supernatant is discarded, and the cell pellet after centrifugation is obtained to obtain the skin stem cell component;

[0014] (2) Dissolving fibrinogen in sodium chloride solution to obtain a fibrinogen solution, and uniformly mixing the skin stem cell component with the fibrinogen solution in proportion to obtain a hydrogel component A; and dissolving thrombin in calcium chloride solution to obtain a hydrogel component B containing the thrombin solution.

[0015] Further preferably, the epidermal stem cells are obtained by shearing and separating skin tissue, digesting with protein dispersase and collagenase, collecting cells through a sieve, and then culturing them adherently using CNT-07 or Keratinocyte-SFM epidermal keratinocyte culture medium.

[0016] Further preferably, the dermal stem cells are obtained by shearing and separating skin tissue, digesting with protein dispersase and collagenase, collecting cells through a sieve, and culturing them in a DMEM / F12 medium containing B27, EGF and bFGF.

[0017] Based on the same technical idea, the present invention also provides an application method of the above-mentioned injectable hydrogel material, and the above-mentioned injectable hydrogel material is applied to the preparation of a material for promoting hair follicle regeneration and repair.

[0018] Further preferably, the hydrogel material is used to prepare a subcutaneous injection, an ointment or a skin patch.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The injectable hydrogel material of the present invention selects fibrinogen and thrombin as the matrix material, which has high biocompatibility and is injectable, and can be used as a supporting material for skin damage repair and hair follicle regeneration. The fibrinogen in the hydrogel material component can cooperate with skin stem cells to promote the improvement of the dryness of skin stem cells, which is more conducive to skin repair and hair follicle regeneration. The present invention can prepare a material for skin repair and hair follicle regeneration by mixing skin stem cells and hydrogel; it can achieve hair follicle regeneration while repairing the skin, and has the characteristics of fast gelation and easy operation, which is suitable for rapid skin repair, especially in the application of hair follicle regeneration. At the same time, because the hydrogel is a mixture of two components A and B, it is a fluid before mixing, which can not only mix well with cells, but also fit well with skin wounds, and can be quickly cross-linked when it is mixed at the front end of the double-barreled syringe and reaches the wound.

[0021] (2) A mixture of epidermal stem cells and dermal stem cells was selected as seed cells. The two types of cells can better simulate the real structure of the skin, have a wider source, are easier to separate, and can regenerate hair follicles well.

[0022] (3) The present invention adds calcium ions and sodium ions to different hydrogel components respectively, thereby simulating the biological fluid balance environment to maintain the activity of the biomaterial and further enhancing the bonding effect of the hydrogel.

[0023] (4) The present invention prepares component A and component B of the hydrogel separately and then mixes the two components when in use, thereby avoiding premature mixing of the hydrogel components leading to premature gelation, which is not conducive to injection application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Diagram of the test process of fibrin hydrogel gelation performance, (a) fibrin hydrogel A / B component gelation diagram, (b, c) electron microscopy images of fibrin hydrogels of different concentrations;

[0025] Figure 2 : Detection results of cell growth in injectable fibrin hydrogels, (a) Growth diagram of dermal stem cells after 1 day and 4 days of culture in fibrin hydrogels, (b) Live-dead staining diagram of dermal stem cells after 1 day and 4 days of culture in fibrin hydrogels, (c) Statistical results of cell viability of dermal stem cells in fibrin hydrogels, (d) Cell proliferation experimental results of dermal stem cells in fibrin hydrogels;

[0026] Figure 3 : Results of cell gene expression in injectable fibrin hydrogels; (a, b) results of protein detection of dermal stem cells, (c) results of AP staining of dermal stem cells, (d) results of real-time fluorescence quantitative PCR, (e) results of real-time fluorescence quantitative PCR;

[0027] Figure 4 :(a) Hair follicle regeneration after fibrin hydrogel was used for hair follicle regeneration in mouse wounds, (b, c, d) HE staining and immunofluorescence staining detection results;

[0028] Figure 5 :(a, b) Biotin immunofluorescence results, (c, d) CD31 immunofluorescence results;

[0029] Figure 6 :(a) The morphology of hair follicle regeneration in mice 6 months after cell transplantation, (b) and (c) are the anatomical images of hair follicle regeneration in mice 4 weeks and 6 months after cell transplantation, respectively, (d) HE staining result of hair follicle regeneration tissue in mice 6 months after cell transplantation. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention is described in more comprehensive and detailed manner below, but the protection scope of the present invention is not limited to the following specific embodiments.

[0031] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0033] Embodiment 1:

[0034] 1. Cultivation of skin stem cells:

[0035] Extraction, culture and collection of epidermal stem cells:

[0036] After the C57 newborn mice were disinfected with iodine and alcohol at 1-3 days after birth, the back skin was cut off and the skin tissue was cut into 6-10 mm 2 Small pieces of size were digested with protein dispersase (Dispase II) at 37℃ for 60-90min. Separate the epidermis from the dermis with sterile tweezers. Use sterile scissors to fully mince the epidermis, digest it with collagenase (Collagenase I) at 37℃ for 60min, and then pass it through a 40μm cell sieve. Centrifuge at 1300rpm for 5min to collect the cells, and use CNT-07 or Keratinocyte-SFM epidermal keratinocyte culture medium for adherent culture. After culturing in a 37℃, 5% CO2 incubator for 1h, remove non-adherent cells. The adherent cells are epidermal stem cells.

[0037] Extraction, culture and collection of dermal stem cells:

[0038] Dermal stem cells were isolated by separating the dermis from the epidermis with protein dispersase (Dispase II), then taking the dermal tissue and chopping it thoroughly with sterile scissors, digesting it with collagenase (Collagenase I) at 37°C for 40-60min, and then passing it through a 40μm cell sieve. The cells were collected by centrifugation at 1300rpm for 5min, washed once with DMEM / F12, and counted with a cell counting plate. The isolated dermal cells were planted in a 10cm untreated cell culture dish, 10mL of DMEM / F12 culture medium containing B27 was added, and EGF and bFGF were added, and suspended culture was carried out in an incubator at 37°C and 5% CO2; B27 and culture medium were added at a mass ratio of 1:50, the final concentration of EGF was 20ng / mL, and the final concentration of bFGF was 40ng / mL.

[0039] 2. Preparation of injectable hydrogel:

[0040] (1) digesting the cultured epidermal stem cells and dermal stem cells, resuspending them in PBS, and collecting the cells after centrifugation; taking the cell pellet after centrifugation, and discarding the supernatant to obtain the skin stem cell component;

[0041] (2) dissolving 100 mg of fibrinogen in 1 mL of 0.9% sodium chloride solution to form a 100 mg / mL fibrinogen solution, and mixing the skin stem cell component obtained in step (1) with the fibrinogen solution in proportion to obtain a hydrogel component A; adjusting the concentration of the hydrogel component A with the 0.9% sodium chloride solution to make the fibrinogen concentration in the hydrogel component A 80 mg / mL; adjusting the concentration to make the hydrogel component A contain 10 6 -10 9 Epidermal stem cells, 10 6 -10 9 The ratio of epidermal stem cells to dermal stem cells is 1:2.

[0042] 500 U of thrombin was dissolved in 50 mL of 42.1 mM calcium chloride solution to form hydrogel component B.

[0043] Before preparing the injectable hydrogel containing skin stem cells, the present invention also conducted a gelation performance test. Fibrinogen solutions with concentrations of 20 mg / mL, 40 mg / mL, and 80 mg / mL were mixed with thrombin solutions of the same concentration, and corresponding calcium chloride and sodium chloride were added to observe the structure of the resulting fibrin hydrogel colloid. Two groups of tests were conducted in this experiment, and the test results are as follows: Figure 1 As shown, it can be seen from the electron microscope image of the fibrin hydrogel that the raw materials used in this experiment can form a colloid, and the colloid has pores, which is convenient for the attachment of skin stem cells and provides support for the cells. In addition, the pore uniformity of the fibrin hydrogel prepared in the case of a fibrinogen solution with a concentration of 80 mg / mL is significantly better. At this concentration, after adding skin stem cells, the skin stem cells are evenly distributed, which is more helpful for the performance of the prepared hair follicle regeneration material. In the gel performance test, the present invention found that the fibrinogen solution and the thrombin solution will form a gel in a relatively short time after mixing. Therefore, there are two operating methods when using it: one is to adopt a very fast transplantation, and quickly transplant it to the wound before the gel is formed. Another method is to form a gel in vitro and transplant it in the form of a gel. Due to a certain shape, this cannot be very well matched with the wound. Therefore, the method of forming A / B liquid in the present invention does not require a very fast speed, and at the same time, the wound can be well covered when applied.

[0044] The present invention tested the cell growth in the prepared injectable hydrogel, and the results were as follows: Figure 2As shown. In the figure (a), it is a growth diagram of dermal stem cells after 1 day and 4 days of culture in fibrin hydrogel. It can be seen from the figure that dermal stem cells can stretch in fibrin hydrogel and the cell activity is good. In the figure (b), it is a live-dead staining diagram of dermal stem cells after 1 day and 4 days of culture in fibrin hydrogel. The figure shows that dermal stem cells can have good cell viability in fibrin hydrogel. In the figure (c), the cell viability of dermal stem cells in fibrin hydrogel is statistically about 80%. In the figure (d), the cell proliferation experiment of dermal stem cells in fibrin hydrogel shows that dermal stem cells can proliferate in fibrin hydrogel. Figure 2 The various test results show that fibrin hydrogel has good biocompatibility and is suitable for the growth and proliferation of dermal stem cells.

[0045] The present invention detects the cell gene expression in the prepared injectable hydrogel, and the results are as follows Figure 3 As shown in the figure, SKP represents the dermal stem cells cultured normally, and SKP-F represents the dermal stem cells cultured in fibrinogen hydrogel. The figure (a, b) shows that the dermal stem cells still highly express their characteristic proteins nestin, bmp6 and fibronectin after being cultured in fibrin hydrogel; the figure (c) shows that the AP staining results of dermal stem cells show that fibrinogen hydrogel can well maintain the stemness of dermal stem cells; the figure (d) shows that the real-time fluorescence quantitative PCR results of fibrin hydrogel can enhance the stemness of dermal stem cells. The expression of Sox2, nanog, and c-myc increased, and only the expression of oct4 decreased slightly, but there was no significant difference. The figure (e) shows that the expression of hair follicle induction genes a-sma and col-II of dermal stem cells decreased after being cultured in fibrin hydrogel, and akp2 and nestin increased, and the changes of other genes were not obvious.

[0046] 3. Establishing hair follicle regeneration model:

[0047] Take the hair follicle regeneration experiment on mice as an example. Make full-thickness wounds on the back of nude mice (diameters are 2mm, 5mm, and 10mm respectively), and inject the prepared hydrogel component A and hydrogel component B into the injured part of the mouse at a ratio of 1:1 through a double-barreled syringe. After the injection, cover it with 3M film and bandage it. Then take the wound skin at different times for fixation and staining to detect wound healing and hair follicle regeneration. The test results are as follows: Figure 4 , Figure 5 as well as Figure 6 shown.

[0048] Figure 4:(a) The hair follicle regeneration image after fibrin hydrogel was used to regenerate hair follicles in mouse wounds, showing that skin follicles with different degrees of wounds can achieve skin healing and hair follicle regeneration; (b, c, d) HE staining and immunofluorescence staining test results show that the wounds have been well repaired, with not only the epidermis and dermis, but also the accessory organ structures such as hair follicles and sebaceous glands.

[0049] Figure 5 :(a, b) Biotin immunofluorescence results showed that the regenerated skin contained sebaceous glands. (c, d) CD31 immunofluorescence results showed that the regenerated skin contained blood vessels.

[0050] Figure 6 :(a) The morphology of mouse hair follicle regeneration 6 months after cell transplantation. (b) and (c) are the anatomical images of mouse hair follicle regeneration 4 weeks and 6 months after cell transplantation, respectively. (d) HE staining of mouse hair follicle regeneration tissue 6 months after cell transplantation shows that hair follicles still exist and no teratoma is formed, indicating good biocompatibility.

[0051] Comparative Example 1:

[0052] The other steps were the same as those in Example 1, but in the preparation of the injectable hydrogel, only epidermal stem cells were added to the hydrogel component A. A hair follicle regeneration model was established for the prepared injectable hydrogel to detect wound healing and hair follicle regeneration. The test results showed that the prepared injectable hydrogel could heal the wound, but could not achieve hair follicle regeneration.

[0053] Comparative Example 2:

[0054] The other steps were the same as those in Example 1, but during the preparation of the injectable hydrogel, only dermal stem cells were added to the hydrogel component A. A hair follicle regeneration model was established for the prepared injectable hydrogel to detect wound healing and hair follicle regeneration. The test results showed that the prepared injectable hydrogel could heal the wound, but could not achieve hair follicle regeneration.

[0055] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. An injectable hydrogel material, characterized in that: The invention comprises a hydrogel component A and a hydrogel component B; the hydrogel component A comprises fibrinogen and skin stem cells, and the hydrogel component B comprises a thrombin solution; the hydrogel component A is a solution, and each milliliter of the hydrogel component A solution contains 50-100 mg of fibrinogen and each milliliter of the hydrogel component A solution contains 10 6 -10 9 The concentration of the thrombin solution is 5-25U / mL; The volume ratio of the hydrogel component A to the hydrogel component B is (1-2): 1; The method for preparing the injectable hydrogel material comprises the following steps: (1) Epidermal stem cells and dermal stem cells are sheared and separated from skin tissue for culture. After separation and culture, they are resuspended in PBS and collected after centrifugation. The supernatant is discarded and the cell pellet is collected after centrifugation to obtain the skin stem cell component. (2) dissolving fibrinogen in a sodium chloride solution to obtain a fibrinogen solution, and uniformly mixing the skin stem cell component with the fibrinogen solution in proportion to obtain a hydrogel component A; dissolving thrombin in a calcium chloride solution to obtain a hydrogel component B containing the thrombin solution; The epidermal stem cells are obtained by shearing and separating the skin tissue, digesting it with protein dispersase and collagenase, collecting the cells by sieving, and then culturing them with CNT-07 or Keratinocyte-SFM epidermal keratinocyte culture medium to obtain epidermal stem cells; The dermal stem cells are obtained by shearing and separating skin tissue, digesting with protein dispersase and collagenase, collecting cells through sieving, and culturing in a DMEM / F12 culture medium containing B27, EGF and bFGF.

2. The injectable hydrogel material according to claim 1, characterized in that: The skin stem cells include epidermal stem cells and dermal stem cells; the number ratio of the epidermal stem cells to the dermal stem cells is 1:(1-2).

3. The injectable hydrogel material according to claim 1 or 2, characterized in that: The thrombin solution also includes free calcium ions in a concentration range of 35-50 mM.

4. The injectable hydrogel material according to claim 1 or 2, characterized in that: The hydrogel component A also includes a sodium chloride solution with a concentration of 0.9%.

5. Use of the injectable hydrogel material according to any one of claims 1 to 4 in the preparation of a material for promoting hair follicle regeneration and repair.

6. The use according to claim 5, characterized in that: The hydrogel material is used to prepare subcutaneous injections, ointments or skin patches.

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