CN115463083A - Injectable hydrogel material and preparation and application methods thereof - Google Patents

Abstract

The invention provides an injectable hydrogel material and its preparation and application method. The hydrogel material comprises hydrogel component A and hydrogel component B; hydrogel component A includes fibrinogen and skin Stem cells, hydrogel component B includes thrombin solution; hydrogel component A is a solution, containing 50‑100 mg of fibrinogen per milliliter of hydrogel component A solution, per milliliter of hydrogel component A solution contains 10 ⁶ ‑10 ⁹ skin stem cells; the concentration of the thrombin solution is 10‑100 mg/mL. The preparation method is that the skin stem cell component is uniformly mixed with the fibrinogen solution in proportion to prepare the hydrogel component A; the thrombin is dissolved in the calcium chloride solution to prepare the hydrogel component B containing the thrombin solution. The injectable hydrogel material of the present invention is used in the preparation of repair materials for promoting hair follicle regeneration. The fibrinogen in the hydrogel material component can cooperate with skin stem cells to promote the dryness of skin stem cells, which is more conducive to skin repair and hair follicle regeneration.

Description

A kind of injectable hydrogel material and its preparation and application method

technical field

The invention belongs to the fields of regenerative medicine and biomaterials, and in particular relates to an injectable hydrogel material and its preparation method and application.

Background technique

Hair follicles are important appendages of the skin that are formed in response to signals from the dermis during skin morphogenesis. Hair originates from hair follicles and is very important to the human body, including functions such as temperature regulation, body protection, sensory activity, and social interaction. Hair loss (Alopecia) caused by various reasons such as environment, heredity, and stress is becoming more and more common in the world, affecting the physical, psychological and social well-being of individuals. The main methods for treating hair loss are drug therapy and hair follicle transplantation. Hair follicle transplantation is an effective treatment that changes the distribution of hair follicles in the scalp without increasing the number of hairs. However, hair follicle transplantation also has some problems. 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 is necessary to continuously obtain hair follicle donations to achieve the final therapeutic effect. Based on the contradictions in reality, hair follicle transplantation at this stage often cannot achieve the optimal therapeutic effect. Therefore, the most fundamental solution is to realize the regeneration of hair follicles in patients with hair loss.

Due to its good biocompatibility, hydrogel materials are widely used in the field of skin repair as a biorepair material. However, most of the existing hydrogel materials are combined with drugs for skin healing, and skin healing does not necessarily regenerate damaged hair follicles. Especially 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. In the prior art, there is also a plan to promote hair follicle regeneration by adding hair follicle regeneration factors into the hydrogel, but since the hair follicle regeneration factors need certain biological activity after all, the selection and combination of hydrogel types is very important. However, the existing hydrogels added with hair follicle regeneration factor either have poor hair follicle regeneration effect due to component mismatch, or are inconvenient to use due to the nature of the material. Therefore, the hydrogel material prepared by the prior art scheme still cannot meet the requirements of human hair follicle regeneration. Therefore, it is of great practical significance to develop a hydrogel material that can be applied to human hair follicle regeneration.

Contents of the invention

The object of the present invention is to provide an injectable hydrogel material and its preparation and application method in view of the technical problems existing in the background technology.

The technical scheme that the present invention takes is as follows:

An injectable hydrogel material, comprising a hydrogel component A and a hydrogel component B; the hydrogel component A includes fibrinogen and skin stem cells, and the hydrogel component B includes Thrombin solution; the hydrogel component A is a solution, containing 50-100 mg of fibrinogen per milliliter of hydrogel component A solution, and containing 10 ⁶ - skin stem cells per milliliter of hydrogel component A solution ¹⁰⁹ ; the concentration of the thrombin solution is 5-25U/mL.

Further preferably, the above-mentioned skin stem cells include epidermal stem cells and dermal stem cells; the number content ratio of the epidermal stem cells and dermal stem cells is 1: (1-2).

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

Further preferably, the above-mentioned hydrogel component A also includes a sodium chloride solution with a concentration of 0.9%. The liquid environment in the hydrogel component A is set to be similar to the saline environment, which is more conducive to the survival of skin stem cells and is beneficial to prolong the service life of the injectable hydrogel material.

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

Further preferably, the above-mentioned hydrogel component A further includes an active factor that promotes hair follicle proliferation; the active factor is at least one of BMP4, BMP6 and FGF7.

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:

(1) Cut and separate epidermal stem cells and dermal stem cells from skin tissue for culture, resuspend in PBS after separation and culture, and collect cells after centrifugation; discard the supernatant, and take the centrifuged cell pellet to obtain the skin stem cell group Minute;

(2) Dissolving fibrinogen in sodium chloride solution to obtain fibrinogen solution, mixing the skin stem cell components with the fibrinogen solution in proportion to obtain hydrogel component A; dissolving thrombin in chloride Hydrogel component B containing thrombin solution was prepared in calcium solution.

Further preferably, the above-mentioned epidermal stem cells are obtained by shearing and separating the skin tissue, digesting them with dispase and collagenase, and then sieving to collect the cells, and then using CNT-07 or Keratinocyte-SFM epidermal keratinocyte medium for adherent culture epidermal stem cells.

Further preferably, the above-mentioned dermal stem cells are obtained by cutting and separating the skin tissue, digesting with dispase and collagenase, and then sieving to collect the cells, and culturing them in DMEM/F12 medium containing B27, EGF and bFGF.

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

Further preferably, the hydrogel material is used to prepare hypodermic injections, ointments or skin patches.

Compared with prior art, the beneficial effect of the present invention is:

(1) The injectable hydrogel material of the present invention selects fibrinogen and thrombin as matrix materials, has high biocompatibility, has injectability, and can be used as a support 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 dryness of skin stem cells, which is more conducive to skin repair and hair follicle regeneration. The present invention can prepare materials for skin repair and hair follicle regeneration by mixing skin stem cells and hydrogel; it can repair skin and realize hair follicle regeneration at the same time, and has the characteristics of fast gelation and convenient operation, and is suitable for skin Rapid Repair is especially applied in the regeneration of hair follicles. At the same time, because the hydrogel is mixed with components A and B, it is fluid before mixing, which can not only mix well with cells, but also fit well with skin wounds. The front end can be quickly cross-linked when it reaches the wound site for mixing.

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

(3) In the present invention, calcium ions and sodium ions are respectively added to different hydrogel components, which not only simulates the equilibrium environment of body fluids to maintain the activity of biological materials, but also further enhances the cementation effect of the hydrogel.

(4) The present invention prepares component A and component B of the hydrogel separately, and then mixes the two components during use, which avoids premature cementation caused by premature mixing of the hydrogel components and is not conducive to injection applications .

Description of drawings

Figure 1: Schematic diagram of the fibrin hydrogel gelation performance test process, (a) the gelation diagram of the A/B component of the fibrin hydrogel, (b, c) the electron microscope images of the fibrin hydrogel with different concentrations;

Figure 2: The test results of cell growth in injectable fibrin hydrogel, (a) the growth diagram of dermal stem cells cultured in fibrin hydrogel for 1 day and 4 days, (b) the growth of dermal stem cells in fibrin hydrogel Live-death staining diagrams after 1 day and 4 days cultured in protein hydrogel, (c) Statistical graph of cell viability of dermal stem cells in fibrin hydrogel, (d) graph of dermal stem cells in fibrin hydrogel The graph of cell proliferation experiment results in ;

Figure 3: Detection results of cell gene expression in injectable fibrin hydrogel; (a, b) detection results of characteristic proteins of dermal stem cells, (c) AP staining results of dermal stem cells, (d) Real-time fluorescent quantitative PCR result graph, (e) real-time fluorescent quantitative PCR result graph;

Figure 4: (a) Hair follicle regeneration after fibrin hydrogel was used for wound hair follicle regeneration in mice, (b, c, d) HE staining and immunofluorescence staining results;

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

Figure 6: (a) Topography of hair follicle regeneration in mice 6 months after cell transplantation, (b) and (c) are anatomical images of hair follicle regeneration in mice at 4 weeks and 6 months after cell transplantation, (d) HE staining results of mouse hair follicle regeneration tissue 6 months after cell transplantation.

detailed description

In order to facilitate the understanding of the present invention, the present invention will be described more fully and in detail below, but the protection scope of the present invention is not limited to the following specific examples.

Unless otherwise defined, all technical terms used hereinafter have the same meanings as commonly understood by those skilled in the art. The terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the protection scope of the present invention.

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.

Example 1:

1. Culture of skin stem cells:

Extraction, cultivation and collection of epidermal stem cells:

After 1-3 days after birth, the C57 newborn mice were disinfected with iodine and alcohol, and the back skin was cut out, and then the skin tissue was cut into small pieces of ^6-10mm2 size, and digested with Dispase II at 37°C 60-90min. Separate the epidermis from the dermis with sterile forceps. The epidermis was fully minced with sterile scissors, digested with collagenase (Collagenase I) at 37°C for 60min, and passed through a 40μm cell sieve. Centrifuge at 1300rpm for 5min to collect cells, use CNT-07 or Keratinocyte-SFM epidermal keratinocyte culture medium for adherent culture, culture in 37℃, 5% _CO2 incubator for 1h, remove non-adherent cells, and adherent cells are epidermal stem cells.

Extraction, culture and collection of dermal stem cells:

The isolation of dermal stem cells is to separate the dermis from the epidermis with dispase II, then take the dermal tissue and use sterile scissors to fully chop it, digest it with collagenase (Collagenase I) at 37°C for 40-60min, and pass through a 40μm cell sieve . Cells were collected by centrifugation at 1300 rpm for 5 min, washed once with DMEM/F12, and counted with a cell counting plate. Sow the isolated dermal cells in a 10cm untreated cell culture dish, add 10mL of DMEM/F12 medium containing B27, add EGF and bFGF, and culture in suspension at 37°C and 5% _CO2 in an incubator; B27 It was added with the medium at a mass ratio of 1:50, the final concentration of EGF was 20ng/mL, and the final concentration of bFGF was 40ng/mL.

2. Preparation of injectable hydrogel:

(1) Digest the cultured epidermal stem cells and dermal stem cells and resuspend them in PBS, collect the cells after centrifugation; take the centrifuged cell pellets, discard the supernatant to obtain the skin stem cell components;

(2) Dissolve 100 mg of fibrinogen in 1 mL of 0.9% sodium chloride solution to form a 100 mg/mL fibrinogen solution, and mix the skin stem cell components obtained in step (1) with the fibrinogen solution evenly to prepare Hydrogel component A; Regulate the concentration of hydrogel component A with 0.9% sodium chloride solution, so that the fibrinogen concentration in hydrogel component A is 80mg/mL; Regulate the concentration so that the hydrogel Each milliliter of solution in component A contains 10 ⁶ -10 ⁹ epidermal stem cells and 10 ⁶ -10 ⁹ dermal stem cells, and the ratio of epidermal stem cells to dermal stem cells is 1:2.

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

Before preparing the injectable hydrogel containing skin stem cells in the present invention, a gel-forming performance test is also carried out. The fibrinogen solution with the concentration of 20mg/mL, 40mg/mL and 80mg/mL was mixed with the thrombin solution of the same concentration, and the corresponding calcium chloride and sodium chloride were added to observe the obtained fibrin hydrocoagulation. The structure of colloids. Two groups of tests were done in this test, and the test results are shown in Figure 1. From the electron microscope images of fibrin hydrogel, it can be seen that the raw materials used in this test can form colloids, and the colloids have holes to facilitate the attachment of skin stem cells. Provide support for cells. In addition, the pore uniformity of the fibrin hydrogel prepared under the condition of 80mg/mL concentration of fibrinogen solution is obviously better. The properties of the hair follicle regeneration material are more helpful. In the gelation performance test, the present invention finds that the fibrinogen solution and the thrombin solution will form a gel in a short period of time after being mixed, so there are two operating methods when using it: one is to adopt very fast transplantation , and quickly transplanted to the wound before the gel is formed. Another method is to form a gel in vitro and transplant it in the form of gel. Due to its certain shape, this kind of gel cannot fit the wound very well. Therefore, the method of forming the A/B solution in the present invention does not require a very fast speed, and at the same time, the wound can be well covered during application.

The present invention detects the cell growth in the prepared injectable hydrogel, and the results are shown in FIG. 2 . Figure (a) is the growth chart of dermal stem cells cultured in fibrin hydrogel for 1 day and 4 days. It can be seen from the figure that dermal stem cells can be stretched in fibrin hydrogel, and the cell activity is better. Figure (b) is a live-dead staining image of dermal stem cells cultured in fibrin hydrogel for 1 day and 4 days, which shows that dermal stem cells can have a good cell viability in fibrin hydrogel. Figure (c) shows that the cell viability of dermal stem cells in the fibrin hydrogel is about 80%. Figure (d) is the cell proliferation experiment of dermal stem cells in fibrin hydrogel, showing that dermal stem cells can proliferate in fibrin hydrogel. The various test results in Figure 2 indicate that the fibrin hydrogel has good biocompatibility and is suitable for the growth and proliferation of dermal stem cells.

The present invention detects the expression of cell genes in the prepared injectable hydrogel, and the results are shown in Figure 3, in which SKP represents normal cultured dermal stem cells and SKP-F represents dermal stem cells cultured on fibrinogen hydrogel. Figures (a, b) show that dermal stem cells still highly express their characteristic proteins nestin, bmp6 and fibronectin after being cultured in fibrin hydrogel; Figure (c) AP staining results of dermal stem cells show that fibrinogen hydrogel can be well maintain the stemness of dermal stem cells; in the figure (d) the real-time fluorescent quantitative PCR results show that fibrin hydrogel can enhance the stemness of dermal stem cells. The expressions of Sox2, nanog, and c-myc all increased, and only the expression of oct4 decreased slightly, but there was no significant difference. In the figure (e) the real-time fluorescent quantitative PCR results showed that after the dermal stem cells were cultured in fibrin hydrogel, the expression of hair follicle-induced genes a-sma and col-II decreased, akp2 and nestin increased, and other genes did not change significantly.

3. Establish hair follicle regeneration model:

Take hair follicle regeneration experiments on mice as an example. Make a full-thickness wound (2mm, 5mm, 10mm in diameter) on the back of the nude mouse, and inject the prepared hydrogel component A and hydrogel component B into the mouse at a ratio of 1:1 through a double-barrel syringe damage. After injection, cover with 3M film and bandage. Then, the wound skin was fixed and stained at different times to detect wound healing and hair follicle regeneration. The detection results are shown in Fig. 4, Fig. 5 and Fig. 6 .

Figure 4: (a) Hair follicle regeneration after fibrin hydrogel was used for wound hair follicle regeneration in mice, showing that skin hair follicles with different wound degrees can achieve skin healing and hair follicle regeneration; (b, c, d) HE staining and The results of immunofluorescence staining showed that the wound was well repaired, with not only the epidermis and dermis, but also accessory organ structures such as hair follicles and sebaceous glands.

Figure 5: (a, b) Biotin immunofluorescence results show that regenerated skin contains sebaceous glands (c, d) CD31 immunofluorescence results show that regenerated skin contains blood vessels.

Figure 6: (a) Topography of mouse hair follicle regeneration 6 months after cell transplantation. (b) and (c) are the anatomical diagrams of mouse hair follicle regeneration at 4 weeks and 6 months after cell transplantation, respectively. (d) The HE staining results of mouse hair follicle regeneration tissues 6 months after cell transplantation showed that the hair follicles still existed, and no teratomas were formed, and the biocompatibility was good.

Comparative example 1:

Other steps are the same as in Example 1, but only epidermal stem cells are added to the hydrogel component A during the preparation of the injectable hydrogel. A hair follicle regeneration model was established for the prepared injectable hydrogel, and wound healing and hair follicle regeneration were detected. The test results showed that the prepared injectable hydrogel could heal wounds, but could not regenerate hair follicles.

Comparative example 2:

Other steps are the same as in Example 1, but only dermal stem cells are added to the hydrogel component A during the preparation of the injectable hydrogel. A hair follicle regeneration model was established for the prepared injectable hydrogel, and wound healing and hair follicle regeneration were detected. The test results showed that the prepared injectable hydrogel could heal wounds, but could not regenerate hair follicles.

The foregoing are only preferred implementations of the present invention, and the scope of protection of the present invention is not limited to the foregoing examples. For those skilled in the art, improvements and transformations obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims (10)

1. An injectable hydrogel material comprising 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 thrombin solution; the hydrogel component A is a solution, each milliliter of the hydrogel component A contains 50 to 100 milligrams of fibrinogen, and each milliliter of the hydrogel component A contains 10 skin stem cells ⁶ -10 ⁹ A plurality of; the concentration of the thrombin solution is 5-25U/mL.

2. The injectable hydrogel material of claim 1, wherein the skin stem cells comprise epidermal stem cells and dermal stem cells; the number content ratio of the epidermal stem cells to the dermal stem cells is 1: (1-2).

3. The injectable hydrogel material of claim 1 or 2, further comprising free calcium ions in a concentration ranging from 35 to 50mM in the thrombin solution.

4. Injectable hydrogel material according to claim 1 or 2, wherein the hydrogel component a further comprises a 0.9% sodium chloride solution.

5. Injectable hydrogel material according to claim 1 or 2, wherein the volume ratio of hydrogel component a to hydrogel component B is (1-2): 1.

6. a method for preparing an injectable hydrogel material according to any one of claims 1 to 5, comprising the steps of:

(1) Shearing and separating epidermal stem cells and dermal stem cells from skin tissues for culture, suspending the separated and cultured epidermal stem cells and dermal stem cells in PBS (phosphate buffer solution), centrifuging and collecting the cells; discarding the supernatant, and taking the centrifuged cell precipitate to obtain a skin stem cell component;

(2) Dissolving fibrinogen in sodium chloride solution to obtain fibrinogen solution, and mixing the skin stem cell component and the fibrinogen solution uniformly according to a proportion to obtain a hydrogel component A; the thrombin was dissolved in the calcium chloride solution to prepare a hydrogel component B comprising the thrombin solution.

7. The method of claim 6, wherein the epidermal stem cells are obtained by shearing and separating skin tissue, digesting with protein dispase and collagenase, sieving, collecting cells, and culturing them in the culture medium of CNT-07 or Keratinocyte-SFM epidermal keratinocytes in an adherent manner.

8. The method according to claim 6, wherein the dermal stem cells are obtained by cutting and separating skin tissue, digesting the skin tissue with protein dispase and collagenase, sieving the cells, and culturing the cells in a DMEM/F12 medium containing B27, EGF and bFGF.

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

10. Use according to claim 9, wherein the hydrogel material is used for the preparation of a subcutaneous injection, an ointment or a skin patch.

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