CN115678048A - Injectable composite hydrogel capable of promoting wound healing and reducing scar formation and preparation method and application thereof - Google Patents
Injectable composite hydrogel capable of promoting wound healing and reducing scar formation and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and a preparation method and application thereof. The main component of the extracellular matrix is used as a raw material to carry out chemical modification, and the mesenchymal stem cells and the verteporfin drug are loaded to form hydrogel through Schiff base reaction in-situ crosslinking. The mesenchymal stem cells loaded by the injectable composite hydrogel can secrete anti-fibrosis cytokine to inhibit tissue fibrosis, and the verteporfin can inhibit YAP gene expression to prevent activation of Engrailed-1 (En 1) genes in the wound healing process, so that the two cooperate to prevent and reduce scar formation. In addition, the extracellular matrix component in the composite hydrogel can effectively promote wound healing by combining the immunoregulation and angiogenesis improvement effects of stem cells. The invention can be applied to the fields of scar formation prevention and reduction, wound repair, tissue engineering and the like.
Description
Technical Field
The invention relates to the field of biomedical materials and tissue engineering, in particular to injectable composite hydrogel capable of promoting wound healing and reducing scar formation and a preparation method and application thereof.
Background
The current major medical methods for treating scars can be divided into surgical and non-surgical treatment methods. Among them, the non-surgical treatment method is more widely used due to the advantages of low invasiveness, small injury, easy operation, short recovery time, and the like. Non-surgical treatment methods include physical therapy and pharmaceutical methods. The physical therapy mainly includes laser therapy, compression therapy, radiotherapy, cryotherapy, rehabilitation therapy, etc. However, the physical therapy is long, wherein the laser therapy only can remove the raised skin and can not fill the depressed area, and the improper use of the laser can generate new scars or uneven color spots. In the pharmaceutical method, on one hand, glucocorticoid drugs such as triamcinolone acetonide, compound betamethasone, prednisolone and the like are injected into scars. However, such drugs tend to cause problems such as skin atrophy, telangiectasia, local skin pigmentation or loss at the site of injection. In addition, hormonal drugs interfere with the normal metabolism of pigments in the body. On the other hand, scar patches and scar-removing ointments, such as silicone gel, mucopolysaccharide polysulfonate creams and compound heparin sodium urocystine ointments, are adopted to reduce scar formation. However, these drugs usually take effect slowly, easily cause skin inflammation and pain, and allergy, and sometimes aggravate scar symptoms.
Mesenchymal Stem Cells (MSCs) can secrete anti-fibrotic cytokines, reduce the gene expression of alpha-SMA and type I collagen, and have a certain effect of inhibiting tissue fibrosis, so that the MSCs have the potential of reducing scar formation. However, there have been reports of studies that show that the effect of MSCs in treating scars is not significant. The reasons behind this are mainly that the microenvironment at the wound site is not conducive to stem cell survival and the tissue is able to clear the transplanted stem cells, thereby not conducive to the secretion and functioning of stem cell factors. Furthermore, anti-fibrotic cytokines that rely solely on stem cell secretion are difficult to completely prevent or reduce scar formation. However, no relevant research report and patent application exists at present.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, a preparation method and application thereof. The injectable composite hydrogel is formed by compounding a bionic extracellular matrix hydrogel support, stem cells and a verteporfin drug. The bionic extracellular matrix hydrogel scaffold is prepared by using main components of an extracellular matrix and derivatives (hyaluronic acid, collagen or gelatin) thereof as raw materials, and respectively preparing oxidized hyaluronic acid, carbohydrazide collagen or carbohydrazide gelatin through oxidation reaction and amino modification. The bionic extracellular matrix hydrogel support has good mechanical properties, is not easy to be subjected to enzymolysis, can provide a compatible environment for stem cells, and meets the requirements of stem cell growth, proliferation, metabolism and the like. And has the effect of reducing scar formation. And because the verteporfin can be slowly released in the hydrogel, the concentration of the medicine contacting with the wound is avoided from being too high, so that the medicine effect is prolonged, and the administration frequency and the clinical burden are reduced. In addition, the injectable composite hydrogel can provide a mild oxygen-isolating condition for the wound and a closed and moist environment for the scar, so that collagen rearrangement is facilitated. Therefore, the invention can exert the functions of the bionic hydrogel scaffold, the stem cells and the verteporfin to the maximum extent, synergistically prevent and reduce scar formation and promote wound healing.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides a preparation method of injectable composite hydrogel capable of promoting wound healing and reducing scar formation, which comprises the following steps:
1) Dissolving hyaluronic acid in ultrapure water, adding sodium periodate into the ultrapure water, carrying out oxidation reaction for 2 to 3 hours at room temperature, and adding 0.5mL of ethylene glycol to terminate the reaction for 1 hour; after the reaction is finished, taking ultrapure water as dialysate, and dialyzing the reaction liquid for 72 hours by using a dialysis bag with 3500Da molecular weight; after the dialysis is finished, collecting the liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the liquid in the polytetrafluoroethylene container for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freezing and drying the liquid for 72 hours under the conditions of minus 50 ℃ and 1 to 20Pa to obtain an oxidized hyaluronic acid sponge material;
2) Dissolving natural high polymer in ultrapure water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, carrying out activation reaction for 2h at room temperature, adding carbohydrazide powder, and carrying out reaction for 12h at room temperature; after the reaction is finished, taking ultrapure water as dialysate, and dialyzing the reaction liquid for 72 hours by using a dialysis bag with the molecular weight of 8000 Da; after dialysis, collecting liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the liquid in the polytetrafluoroethylene container for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freezing and drying the liquid for 72 hours under the conditions of minus 50 ℃ and 1 to 20Pa to obtain the carbohydrazide natural polymer material;
3) Preparing oxidized hyaluronic acid sponge material into solution by using ultrapure water, and preparing carbohydrazide natural high polymer material into solution by using ultrapure water; adding mesenchymal stem cells into the oxidized hyaluronic acid sponge material solution to obtain a hydrogel precursor solution 1; adding verteporfin into carbohydrazide natural high polymer material solution to obtain hydrogel precursor solution 2; and (3) uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2, and stirring at room temperature for 10-20s until the mixed solution is colloidal, thus obtaining the injectable composite hydrogel.
Further, in the step 1), the amount of the hyaluronic acid is 1g, and the amount of the sodium periodate is 0.25g.
Further, in the step 2), the natural polymer may be any one selected from collagen and gelatin.
In step 2), the amount of the natural polymer is 9g, the amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 8.64g, the amount of the N-hydroxysuccinimide is 0.58g, and the amount of the carbohydrazide powder is 6.6g.
Further, in the step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2% -8%, and the concentration of the carbohydrazide natural polymer material solution is 4% -12%.
Further, in the step 3), the mesenchymal stem cell is selected from any one of a human adipose mesenchymal stem cell and a human bone marrow mesenchymal stem cell.
Further, in the step 3), the volume ratio of the hydrogel precursor solution 1 to the hydrogel precursor solution 2 is 1:1.
further, in the step 3), the concentration of the mesenchymal stem cells in the injectable composite hydrogel is 1 × 10 6 ~1×10 7 The concentration of the single drug per mL and the concentration of the verteporfin is 0.01-10mg/mL.
The invention also provides the injectable composite hydrogel prepared by the preparation method.
The invention also provides application of the injectable composite hydrogel in preparation of a medicine for promoting wound healing and/or reducing scar formation.
The beneficial effects of the invention are as follows:
1) The invention provides a biomedical material, which is a biodegradable injectable composite hydrogel prepared by taking a high polymer material with good biocompatibility as a base material, and can realize the controllable release of cytokines and drugs.
2) The injectable composite hydrogel can keep the growth and proliferation of the mesenchymal stem cells in vitro for more than 5 days, and cytokines secreted by the mesenchymal stem cells can be continuously released to promote wound healing.
3) The injectable composite hydrogel disclosed by the invention supports the sustained release of verteporfin for more than 7 days, reduces the diffusion area of the drug, reduces the administration frequency, achieves a better scar treatment effect and reduces the clinical burden.
4) The injectable composite hydrogel can be used for wound healing and preventing and reducing scar formation.
Drawings
FIG. 1: schematic representation of the biomimetic hydrogel scaffold prepared in example 1 through a 30G needle syringe.
FIG. 2: the microstructure of the biomimetic hydrogel scaffold prepared in example 1.
FIG. 3: release profile of verteporfin in the biomimetic hydrogel scaffold prepared in example 1.
FIG. 4 is a schematic view of: release profile of human mesenchymal stem cells in the biomimetic hydrogel scaffolds prepared in example 2.
FIG. 5: shear thinning behavior of the biomimetic hydrogel scaffolds prepared in example 2.
FIG. 6: staining pattern of mesenchymal stem cells Live/Dead in the bionic hydrogel scaffold.
FIG. 7: the growth and distribution of the human bone marrow mesenchymal stem cells in the bionic hydrogel.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Example 1
Step 1): weighing 1g of hyaluronic acid (30 kDa) and dissolving the hyaluronic acid in 100mL of ultrapure water, adding 0.25g of sodium periodate into the ultrapure water, carrying out oxidation reaction for 2 to 3 hours at room temperature, and adding 0.5mL of ethylene glycol to terminate the reaction for 1 hour at room temperature; after the reaction is finished, taking ultrapure water as dialysate, and dialyzing the reaction liquid for 72 hours by using a dialysis bag with the molecular weight of 3500 Da; after the dialysis is finished, collecting the liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the liquid in the polytetrafluoroethylene container for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freezing and drying the liquid for 72 hours under the conditions of minus 50 ℃ and 1 to 20Pa to obtain the oxidized hyaluronic acid sponge material.
Step 2): weighing 9g of collagen (bovine-derived, type I, 30 kDa) and dissolving in 500mL of ultrapure water, adding 8.64g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.58g of N-hydroxysuccinimide (NHS), carrying out activation reaction for 2h at room temperature, adding 6.6g of carbohydrazide powder, carrying out reaction for 12h at room temperature, dialyzing the reaction solution for 72h by using a dialysis bag with a molecular weight of 8000Da by using the ultrapure water as a dialyzate after the reaction is finished, collecting the liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the polytetrafluoroethylene container in a refrigerator at a temperature of-80 ℃ for 5h, and carrying out freeze drying for 72h under the conditions of-50 ℃ and 1-20Pa to obtain the carbohydrazide collagen material.
And step 3): preparing a solution with the concentration of 5wt% by using ultrapure water for the oxidized hyaluronic acid sponge material, and preparing a solution with the concentration of 7wt% by using ultrapure water for the carbohydrazide collagen material; uniformly mixing 5wt% of oxidized hyaluronic acid sponge material solution and 7wt% of carbohydrazide collagen material solution according to the volume ratio of 1.
Example 2
Step 1): reference example 1 step 1) oxidized hyaluronic acid sponge material was prepared.
Step 2): weighing 9g of gelatin (porcine origin, type A, 300g bloom) and dissolving the gelatin in 500mL of ultrapure water, adding 8.64g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.58g of N-hydroxysuccinimide (NHS), carrying out activation reaction for 2h at room temperature, adding 6.6g of carbohydrazide powder, carrying out reaction for 12h at room temperature, dialyzing the reaction liquid for 72h by using a dialysis bag with a molecular weight of 8000Da by using the ultrapure water as a dialyzate after the reaction is finished, collecting the liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the polytetrafluoroethylene container for 5h at a temperature of-80 ℃, and carrying out freeze drying for 72h at a temperature of-50 ℃ and under a pressure of 1 to 20Pa to obtain the carbohydrazide gelatin material.
3) Preparing a solution with the concentration of 5wt% by using ultrapure water for the oxidized hyaluronic acid sponge material, and preparing a solution with the concentration of 7wt% by using ultrapure water for the carbohydrazide gelatin material; uniformly mixing 5wt% of oxidized hyaluronic acid sponge material solution and 7wt% of carbohydrazide gelatin material solution according to the volume ratio of 1.
Figure 1 is a schematic representation of the biomimetic hydrogel scaffold prepared in example 1 passing through a 30G needle syringe. As can be seen from the figure, the biomimetic hydrogel scaffold prepared in example 1 can be injected by a 30G needle syringe and can rapidly gel for practical application.
Figure 2 shows the microstructure of the biomimetic hydrogel scaffold prepared in example 1. The observation of an electron scanning microscope shows that the bionic hydrogel scaffold has a porous structure and uniform pore size, can absorb wound tissue exudate, and is beneficial to growth and proliferation of mesenchymal stem cells in the bionic hydrogel scaffold, secretion of generated cytokines and slow release of verteporfin.
Figure 3 shows the release profile of verteporfin in the biomimetic hydrogel scaffold prepared in example 1. 200 μ L of the biomimetic hydrogel scaffold prepared in example 1 was taken, mixed uniformly with 1mg/mL verteporfin solution, immersed in 1 × PBS (PH = 7.4) at 37 ℃, and the amount of drug released was detected at fixed time intervals. The result shows that the bionic hydrogel stent has a porous structure and supports the slow release of the drug, thereby reducing the drug administration frequency.
Fig. 4 shows a release profile of human mesenchymal stem cells in the biomimetic hydrogel scaffolds prepared in example 2. Adding human mesenchymal stem cells into the bionic hydrogel scaffold until the final concentration is 3 multiplied by 10 6 And culturing the cells per mL in an incubator at 37 ℃, and sucking a culture medium at fixed time nodes to detect the total content of the cell factors secreted by the stem cells. The result shows that the bionic hydrogel scaffold supports the release of secreted cytokines when the mesenchymal stem cells grow in the bionic hydrogel scaffold.
Figure 5 shows the shear-thinning behavior of the biomimetic hydrogel scaffolds prepared in example 2. The bionic hydrogel bracket is gelatinized (with the radius and the height of 5 mm), placed on a rheometer objective table, and the change of viscosity along with the shear rate (0.01-100/s) is researched at the angular frequency of 10 rad/s, so that the bionic hydrogel bracket can be observed to have the shear thinning characteristic, and the viscosity of the bionic hydrogel bracket is reduced along with the increase of the shear force, which shows that the bionic hydrogel bracket has injectability.
FIG. 6 shows Live/Dead staining pattern of mesenchymal stem cells in a biomimetic hydrogel scaffold. Leaching the bionic hydrogel support by using a serum-free culture medium to obtain a leaching solution; the mesenchymal stem cells are planted in a 96-well plate, a bionic hydrogel scaffold leaching solution is added, the culture is carried out at 37 ℃, the cells are dyed for 10 to 20min by a Calcein-AM/PI kit after 72h, and the morphology and the growth condition of the cells are observed under a fluorescence inverted microscope (the bionic hydrogel scaffold prepared in example 1 is loaded with the human adipose-derived mesenchymal stem cells, and the bionic hydrogel scaffold prepared in example 2 is loaded with the human bone marrow mesenchymal stem cells). As can be seen from the figure, the bionic hydrogel scaffolds prepared in examples 1-2 have good cell compatibility, the mesenchymal stem cells have a slender spindle-shaped form, and the number of red dead cells is small.
Example 3
Step 1): reference example 1 step 1) an oxidized hyaluronic acid sponge material was prepared.
Step 2): reference example 2, step 2) preparation of carbohydrazide collagen material carbohydrazide gelatin material.
And step 3): preparing a solution with the concentration of 5wt% by using ultrapure water for the oxidized hyaluronic acid sponge material, and preparing a solution with the concentration of 7wt% by using ultrapure water for the carbohydrazide gelatin material; to 100. Mu.L of a 5wt% solution of oxidized hyaluronic acid sponge material was added a concentration of 6X 10 6 Uniformly mixing the suspension of human mesenchymal stem cells per mL with 100 mu L of 7wt% carbohydrazide gelatin material solution, stirring at room temperature for 10-20 s until the mixed solution is colloidal, and obtaining the injectable composite hydrogel, wherein the concentration of the mesenchymal stem cells in the injectable composite hydrogel is 3 x 10 6 one/mL.
Fig. 7 shows the growth distribution of the human mesenchymal stem cells in the injectable composite hydrogel according to the embodiment. Live/Dead staining is carried out on the hydrogel loaded with the mesenchymal stem cells on the first day and the fifth day after gelling, and a laser scanning confocal microscope is used for observing a 3D distribution diagram of the stem cells in the hydrogel. As can be seen from the figure, the number of human bone marrow mesenchymal stem cells increased within the injectable composite hydrogel from the first day to the fifth day.
Example 4
Step 1): reference example 1 step 1) oxidized hyaluronic acid sponge material was prepared.
Step 2): reference example 1, step 2) carbohydrazide collagen material was prepared.
Step 3): preparing a hyaluronic acid oxide sponge material into a solution with the concentration of 5wt% by using ultrapure water, and preparing a carbohydrazide collagen material into a solution with the concentration of 7wt% by using ultrapure water; to 100. Mu.L of a 5wt% solution of oxidized hyaluronic acid sponge material was added a concentration of 6X 10 6 Obtaining a hydrogel precursor solution 1 by using per mL of human adipose mesenchymal stem cell suspension; adding 100 μ L of 7wt% carbohydrazide collagen material solution2mg/mL verteporfin solution to obtain hydrogel precursor solution 2; uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2 according to the volume ratio of 1 6 The concentration of the verteporfin is 1mg/mL.
Example 5
Step 1): reference example 1 step 1) an oxidized hyaluronic acid sponge material was prepared.
Step 2): reference example 2 step 2) carbohydrazide collagen material carbohydrazide gelatin material was prepared.
Step 3): preparing a solution with the concentration of 5wt% by using ultrapure water for the oxidized hyaluronic acid sponge material, and preparing a solution with the concentration of 7wt% by using ultrapure water for the carbohydrazide gelatin material; to 100. Mu.L of a 5wt% solution of oxidized hyaluronic acid sponge material was added a concentration of 6X 10 6 The individual/mL of human mesenchymal stem cell suspension is used for obtaining hydrogel precursor solution 1; adding 2mg/mL verteporfin solution into 100 mu L of 7wt% carbohydrazide gelatin material solution to obtain hydrogel precursor solution 2; uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2 according to the volume ratio of 1 6 The concentration of the verteporfin is 1mg/mL.
Example 6
Applying pentobarbital sodium and isoflurane to 2.5-3 kg/New Zealand rabbit for composite anesthesia, and establishing 4 wounds with the diameter of 10mm in each ear in the hairless area on the inner side of the rabbit ear. The group is set as follows: control group (injecting 200 μ L physiological saline to wound, once every three days), and simple mesenchymal stem cell group (injecting 200 μ L concentration 3 × 10 to wound 6 one/mL of human adipose-derived mesenchymal stem cell suspension, which is injected once every three days), a pure Verteporfin group (200 muL of Verteporfin solution with the concentration of 1mg/mL is injected into a wound and is injected once every three days), and a mesenchymal stem cell-loaded hydrogel group (200 pieces of Verteporfin solution are injected into the wound and are injected once every three days)μ L of the injectable composite hydrogel prepared in example 3 was attached to the wound after gelling, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), a mesenchymal stem cell and verteporfin-loaded hydrogel group (200 μ L of the injectable composite hydrogel prepared in example 4 was attached to the wound after gelling, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), a commercial 3M dressing group (a commercial 3M dressing was attached to the wound, and a new dressing was replaced every 3 days), and a silicone gel (silicone gel was applied to the wound, and applied every 3 days). After day 15 of treatment, rabbits were euthanized and wound closure rate and SEI (scar elevation index) were statistically compared. The calculation formula for wound healing is: wound closure rate (%) = (a) 0 -A t ）/A 0 X 100%, wherein A 0 Represents the wound area on day 0; a. The t Represents the wound area on day t. Scar Elevation Index (SEI) calculation formula: SEI = (a + B)/B, where a is scar tissue thickness above normal epidermal skin and B is normal epidermal skin thickness. The results are shown in the following table.
TABLE 1 statistical results of wound closure Rate
TABLE 2 statistics of CD31 (+) number of vessels in wound healing
TABLE 3 SEI (scar elevation index) statistics
Note: SEI = 1, indicating the height of the scar and the surrounding intact dermis; SEI > 1, indicating elevated scar;
SEI = 2, indicating 100% increase in scar thickness over normal tissue dermis.
SEI >2, indicating more than 100% increase in scar thickness over normal tissue dermis.
As can be seen from the wound closure rate (table 1), compared with the commercial 3M dressing, the wound closure rate of the mesenchymal stem cell-loaded hydrogel group, the mesenchymal stem cell-loaded hydrogel group and verteporfin-loaded hydrogel group is significantly improved, and since the hydrogel base material is selected from natural polymer materials, the hydrogel itself has good biocompatibility and the mesenchymal stem cells are loaded in the hydrogel, the porous structure of the hydrogel supports the secretion of stem cell factors, and the wound repair is promoted. And in histological staining, the wound epithelium regeneration integrity rate of the mesenchymal stem cell-loaded hydrogel group, the mesenchymal stem cell-loaded hydrogel group and the verteporfin-loaded hydrogel group was higher than that of the commercial 3M dressing group on day 15. Angiogenesis is required during the wound healing process to promote restoration of skin structure and function, and the statistical result of the number of CD (+) vessels in table 2 shows that the number of CD31 (+) vessels in the mesenchymal stem cell-loaded hydrogel group at day 10 is 22.38% ± 0.69%, which is higher than that in the commercial 3M dressing group 11.41% ± 1.00%. The results show that the mesenchymal stem cell-loaded hydrogel group can better promote wound angiogenesis and re-epithelialization.
From the SEI (scar elevation index) in table 3, it can be seen that the hydrogel group carrying mesenchymal stem cells and verteporfin has the best scar treatment effect, the SEI index is 1.26 ± 0.02, and probably because the hydrogel covers the surface to play the effects of hypoxia and moisture retention, the hydrogel contains mesenchymal stem cells to secrete cytokines to promote wound healing, and the fibrosis-resistant factors are secreted in the later stage of wound healing to inhibit tissue fibrosis, and the hydrogel contains the effective component verteporfin for removing scars, so that the excellent scar removing effect is achieved. The mesenchymal stem cell-loaded hydrogel group is 1.78 +/-0.15; the pure verteporfin group is 1.70 +/-0.18; the simple mesenchymal stem cell group is 2.27 +/-0.04; the silicone gel group is 2.75 + -0.16, and the silicone gel merely provides a closed, moist environment for the scar tissue, which promotes partial improvement of fibroblasts, and collagen in the scar tissue, such as collagen rearrangement close to normal skin order. In a healed wound, the inhibition effect of the sustained release of the verteporfin on the expression of the YAP gene can block the activation of En1 and promote the ENF-mediated repair of the hydrogel group carrying the mesenchymal stem cells and the verteporfin, the skin can be regenerated within 30 days, and the functional hair follicle and the sebaceous gland are recovered without fibrosis. Specifically, the Engrailed-1 gene in specific Engrailed-1 lineage negative fibroblast (ENF) of the skin deep tissue during scar formation is activated and expressed under the action of YAP protein, and a fibrosis cell promoting program is started to respond to local tissue mechanics in a wound. And the verteporfin is a YAP inhibitor and can block the function of YAP, thereby inhibiting fibrosis and reducing scar formation.
Example 7
30 kg/pig were subcutaneously injected with xylazine hydrochloride solution at a concentration of 100 mg/mL at a rate of 5 mL/pig, followed by washing, dehairing and sterilizing of the back of the Duroc pig, followed by symmetrically creating 18 wounds (2 cm. Times.2 cm) along both sides of the midline of the back of the pig. The group is set as follows: control group (injecting 200 μ L physiological saline to wound, once every three days), and simple mesenchymal stem cell group (injecting 200 μ L concentration 3 × 10 to wound 6 one/mL suspension of human adipose mesenchymal stem cells was injected every three days), a pure verteporfin group (200 μ L of verteporfin solution with a concentration of 1mg/mL was injected to the wound every three days), a mesenchymal stem cell-loaded hydrogel group (200 μ L of the injectable composite hydrogel prepared in example 3 was attached to the wound after gelling, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), a mesenchymal stem cell-and verteporfin-loaded hydrogel group (200 μ L of the injectable composite hydrogel prepared in example 4 was attached to the wound after gelling, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), and a silicone gel (silicone gel was applied to the wound and applied every 3 days). On day 60 after treatment, the wound tissue of Duroc red hair pigs was taken and the SEI (scar elevation index) was counted.
According to SEI, the scar treatment effect of the hydrogel group carrying the mesenchymal stem cells and the verteporfin is closest to that of normal skin, the SEI index is 1.36 +/-0.05, the hydrogel covers the surface to play the effects of hypoxia and moisture retention, the mesenchymal stem cells secrete cytokines to promote wound healing, the anti-fibrosis factors are secreted in the later period of wound healing to inhibit tissue fibrosis, and the scar-removing active ingredient verteporfin is contained, so that the excellent scar-removing effect is achieved. The mesenchymal stem cell-loaded hydrogel group is 1.61 +/-0.11; the pure verteporfin group is 1.58 plus or minus 0.04; the simple mesenchymal stem cell group is 2.37 +/-0.13; the silicone gel group is 2.17 + -0.08. The silicone gel merely provides a closed, moist environment for the scar tissue, which promotes partial improvement of the fibroblasts, and collagen in the scar tissue, e.g., collagen rearrangement close to normal skin order.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (10)
1. A preparation method of injectable composite hydrogel capable of promoting wound healing and reducing scar formation is characterized by comprising the following steps: the method comprises the following steps:
1) Dissolving hyaluronic acid in ultrapure water, adding sodium periodate into the ultrapure water, performing oxidation reaction for 2 to 3 hours at room temperature, and adding 0.5mL of ethylene glycol to terminate the reaction for 1 hour; after the reaction is finished, taking ultrapure water as dialysate, and dialyzing the reaction liquid for 72 hours by using a dialysis bag with 3500Da molecular weight; after the dialysis is finished, collecting the liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the liquid in the polytetrafluoroethylene container for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freezing and drying the liquid for 72 hours under the conditions of minus 50 ℃ and 1 to 20Pa to obtain an oxidized hyaluronic acid sponge material;
2) Dissolving natural high polymer in ultrapure water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, carrying out activation reaction for 2h at room temperature, adding carbohydrazide powder, and carrying out reaction for 12h at room temperature; after the reaction is finished, taking ultrapure water as dialysate, and dialyzing the reaction liquid for 72 hours by using a dialysis bag with the molecular weight of 8000 Da; after dialysis, collecting liquid in the dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the liquid in the polytetrafluoroethylene container for 5 hours in a refrigerator at the temperature of minus 80 ℃, and then freezing and drying the liquid for 72 hours under the conditions of minus 50 ℃ and 1 to 20Pa to obtain the carbohydrazide natural polymer material;
3) Preparing oxidized hyaluronic acid sponge material into solution by using ultrapure water, and preparing carbohydrazide natural high polymer material into solution by using ultrapure water; adding mesenchymal stem cells into the oxidized hyaluronic acid sponge material solution to obtain a hydrogel precursor solution 1; adding verteporfin into carbohydrazide natural high polymer material solution to obtain hydrogel precursor solution 2; and (3) uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2, and stirring at room temperature for 10-20s until the mixed solution is colloidal, thus obtaining the injectable composite hydrogel.
2. The method of claim 1, wherein: in the step 1), the dosage of the hyaluronic acid is 1g, and the dosage of the sodium periodate is 0.25g.
3. The method of claim 1, wherein: in the step 2), the natural high polymer is selected from any one of collagen and gelatin.
4. The method of claim 1, wherein: in the step 2), the using amount of the natural high polymer is 9g, the using amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 8.64g, the using amount of the N-hydroxysuccinimide is 0.58g, and the using amount of the carbohydrazide powder is 6.6g.
5. The method of claim 1, wherein: in the step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2% -8%, and the concentration of the carbohydrazide natural polymer material solution is 4% -12%.
6. The method of claim 1, wherein: in the step 3), the mesenchymal stem cell is selected from any one of human adipose mesenchymal stem cell and human bone mesenchymal stem cell.
7. The production method according to claim 1, characterized in that: in the step 3), the volume ratio of the hydrogel precursor solution 1 to the hydrogel precursor solution 2 is 1:1.
8. the production method according to claim 1, characterized in that: in the step 3), the concentration of the mesenchymal stem cells in the injectable composite hydrogel is 1 multiplied by 10 6 ~1×10 7 The concentration of the single drug per mL and the concentration of the verteporfin is 0.01-10mg/mL.
9. An injectable complex hydrogel prepared by the preparation method of claim 1.
10. Use of an injectable composite hydrogel according to claim 9 for: the injectable composite hydrogel is applied to preparation of medicines for promoting wound healing and/or reducing scar formation.
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