CN113663132B

CN113663132B - Adipose tissue regeneration hydrogel and preparation method and application thereof

Info

Publication number: CN113663132B
Application number: CN202110819423.6A
Authority: CN
Inventors: 肖锷; 魏强; 王鹏
Original assignee: Hunan Meibai Biomedical Co ltd
Current assignee: Hunan Meibai Biomedical Co ltd
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2022-10-18
Anticipated expiration: 2041-07-20
Also published as: CN113663132A

Abstract

The invention discloses an adipose tissue regeneration hydrogel which comprises sodium hyaluronate, polyethylene glycol and extracellular matrix, wherein the extracellular matrix is formed by inducing adipose stem cells in vitro through recombinant cytokines and comprises 60-70wt% of collagen, 25-35wt% of glycoprotein and 1-3wt% of mucopolysaccharide. The adipose tissue regeneration hydrogel provided by the invention has stable physical properties and good histocompatibility, and can be integrated with the tissues of a receiving area without surgical operation by only injection when implanted.

Description

Adipose tissue regeneration hydrogel and preparation method and application thereof

Technical Field

The invention relates to the technical field of regeneration engineering, in particular to adipose tissue regeneration hydrogel based on an adipose-derived stem cell extracellular matrix, and a preparation method and application thereof.

Background

Autologous fat transplantation refers to the steps of sucking redundant fat from some parts (such as waist, abdomen, thighs and the like), centrifuging, purifying, selecting complete fat cell particles, improving the survival rate of the fat cells, and filling other parts (such as temples, apple muscles, cheeks and the like) in a multi-level and multi-point manner by adopting a fine combined injection technology to achieve the effects of wrinkle removal and shaping, so that the autologous fat transplantation is an important means for filling and recovering the facial contour and the volume and is an important method for correcting asymmetric deformity of the face.

The autologous fat transplantation, as the tissue of a patient, has biological characteristics far superior to those of any prosthesis material, so that the autologous fat transplantation is non-toxic and harmless to the patient and cannot generate immune response and rejection response. However, the technology has a high operation requirement, the survival rate of fat transplantation is low, and surgical complications may also exist in an adipose tissue supply area, for example, currently, a research is known in which a directional differentiation drug is combined with a three-dimensional culture system to induce the directional differentiation of stem cells, that is, a drug is added into a culture solution and enters the three-dimensional culture system through diffusion to promote the differentiation of the stem cells in the three-dimensional culture system, but the method requires continuous replacement of the culture solution to maintain the concentration of the drug, and is cumbersome to operate and unsuitable for the research and application of the directional differentiation of the stem cells in a fit manner. The research also has been carried out on mixing the medicines in the three-dimensional culture system and promoting the differentiation of the stem cells in the three-dimensional culture system by utilizing the slow release effect of the hydrogel system, but the medicines, particularly macromolecular medicines, are released quickly and are easy to be eliminated by being released in a medium outside the three-dimensional culture system, so that the effective medicine concentration in the three-dimensional culture system can only be maintained for 2-3 days, and the long-term directional differentiation of the stem cells in the three-dimensional culture system is not suitable.

Therefore, the above methods, which promote differentiation of stem cells in a three-dimensional culture system based on a drug-containing culture solution or a hydrogel, have problems of low stem cell transplantation survival rate, unfavorable storage, and sale, and poor stability.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, and provides the adipose tissue regeneration hydrogel which has stable physical properties and good histocompatibility, only needs injection when being implanted, and can be fused with the receiving area tissue without surgical operation.

In order to achieve the above purpose, the invention provides the following technical scheme:

an adipose tissue regeneration hydrogel comprises sodium hyaluronate, polyethylene glycol and extracellular matrix, wherein the extracellular matrix is induced by adipose-derived stem cells in vitro through recombinant cytokines and comprises 60-70wt% of collagen, 25-35wt% of glycoprotein and 1-3wt% of mucopolysaccharide.

Recombinant cytokines are cytokine products produced by genetic engineering techniques, and cytokines are a class of highly active, multifunctional polypeptide molecules that regulate cell function produced by immune cells and related cells, excluding immunoglobulins, complements and general physiological cell products, and are generally produced by related cells such as lymphocytes, monocytes, macrophages, fibroblasts, endothelial cells, and the like.

The extracellular matrix is a network with a fine structure formed by assembling various biological macromolecules secreted by cells to extracellular spaces in the development process of an organism, is distributed between the cells and tissues, surrounds the cells or forms a basement membrane of epithelial cells, and the cells are mutually connected or the cells and the basement membrane to form tissues and organs which are connected into an organic whole.

As a practical way, the recombinant cytokine comprises the following components in percentage by volume:

30-100ug/mL of carrageenan and/or 0.03-0.1mg/mL of dextran sulfate; and the number of the first and second electrodes,

the recombinant cytokine also comprises 0.5-1.5v% of insulin-transferrin-selenium supplement.

As a practical way, the weight ratio of the extracellular matrix, the sodium hyaluronate and the polyethylene glycol is 2-20:5-30:2-20.

As a practical mode, the molecular weight of the sodium hyaluronate is 50-150KD, and the molecular weight of the polyethylene glycol is 1-10KD.

As a practical manner, the adipose tissue regeneration hydrogel further comprises a pharmaceutically acceptable carrier;

preferably, the formulation of the adipose tissue-derived hydrogel is preferably selected from a lyophilized powder or an injection.

When the adipose tissue-regenerating hydrogel is applied, the adipose tissue-regenerating hydrogel may be optionally administered by intramuscular injection, subcutaneous injection, or the like.

A preparation method of adipose tissue regeneration hydrogel comprises the following steps:

s1, adding recombinant cell factors into adipose-derived stem cells, and inducing extracellular matrix with collagen content of 60-70wt%, glycoprotein content of 25-35wt% and mucopolysaccharide content of 1-3wt% in vitro;

s2, freeze-drying the extracellular matrix, and adding sodium hyaluronate and polyethylene glycol for mixing to obtain a mixture;

and S3, dissolving the mixture in water, and then adding 4- (4,6-dimethoxytriazine-2-yl) -4-methylmorpholine hydrochloride to perform an activation reaction to obtain the adipose tissue regeneration hydrogel.

As a practical way, the recombinant cytokine contains 0-10ng epidermal growth factor, 2-30ng PDGF-BB, 10-60ng insulin-like growth factor-1, 1-100ng transforming growth factor beta 1, 1-30ng dexamethasone, 30-100ug carrageenan and/or 0.03-0.1mg dextran sulfate, and 0.005-0.015mL insulin-transferrin-selenium supplement per mL.

As a practical mode, the temperature of the activation reaction is 25-37 ℃ and the time is 3-6h.

It is another object of the present invention to provide use of the adipose tissue-regenerating hydrogel for wrinkle removal and volume filling of the skin.

Compared with the prior art, the invention has the following beneficial effects:

the method adopts an adipose-derived stem cell in-vitro tissue engineering method, a natural tissue material extracellular matrix with the component composition ratio similar to that of adipose tissue is formed through induction of recombinant cytokines, the formed extracellular matrix is combined with a natural polymer material sodium hyaluronate and the like, and an artificially synthesized polymer material, namely a polymer hydrogel, is formed through crosslinking.

Drawings

FIG. 1 shows the percentage of collagen, mucopolysaccharide and glycoprotein by mass in the extracellular matrix provided in example 3 of the present invention;

FIG. 2 is a 2500-fold enlarged view under a scanning electron microscope of the extracellular matrix obtained in example 3 after decellularization;

FIG. 3 is a 20000-fold enlargement under a scanning electron microscope after decellularization of the extracellular matrix obtained in example 3;

FIG. 4 is a 129-fold enlarged view under a scanning electron microscope of the adipose tissue-regenerating hydrogel obtained in example 4 after it is lyophilized;

FIG. 5 is a 230-fold magnified view under a scanning electron microscope of the adipose tissue-regenerated hydrogel obtained in example 4 after lyophilization;

FIG. 6 is a view at 610 times magnification under a scanning electron microscope of the adipose tissue-regenerating hydrogel obtained in example 4 after it is lyophilized;

FIG. 7 is a view showing that the adipose tissue-regenerated hydrogel obtained in example 4 is enlarged 3100 times under a scanning electron microscope after being lyophilized;

FIG. 8 is a graph showing the storage modulus and loss modulus of the adipose tissue-regenerating hydrogel synthesized in example 5;

FIG. 9 is the storage modulus and loss modulus of the adipose tissue-regenerating hydrogel synthesized in example 6;

FIG. 10 is a graph showing the storage modulus and loss modulus of the adipose tissue-regenerating hydrogel synthesized in example 7;

FIG. 11 is a graph showing the storage modulus and loss modulus of the adipose tissue-regenerating hydrogel synthesized in example 8;

FIG. 12 is a histological representation of the adipose tissue regeneration hydrogel synthesized in example 7 implanted three days subcutaneously in C57BL/6J mice;

FIG. 13 is a histological representation of the adipose tissue regeneration hydrogel synthesized in example 7 implanted subcutaneously for seven days in C57BL/6J mice;

FIG. 14 is a histological representation of the adipose tissue regeneration hydrogel synthesized in example 7 implanted subcutaneously for fourteen days in C57BL/6J mice;

FIG. 15 is a histological representation of twenty-eight days after subcutaneous implantation of the adipose tissue regeneration hydrogel synthesized in example 7 into C57BL/6J mice;

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

The Epidermal Growth Factor (EGF), also known as human oligopeptide-1, in the embodiment of the invention is an important cell Growth Factor for human endocrine, and the active polypeptide consisting of 53 amino groups has strong physiological activity. EGF is a single-chain polypeptide, can improve the proliferation and adipogenic differentiation capacity of human ADSCs after cryopreservation, and has the main function of promoting the division of skin cells. The research shows that: the trace amount of EGF can stimulate cell growth, inhibit the expression of senility gene, prevent skin senility and maintain the optimal physiological state of skin components. In addition, it can stimulate the synthesis and secretion of some macromolecules (such as hyaluronic acid, collagen, etc.) outside cells, moisten skin, and is a key factor for determining skin vitality and health.

PDGF-BB is the B subunit of platelet-derived growth factor, and platelet-derived factor (PDGF) is classified into PDGFI with a molecular weight of 31KD and containing 7% of sugar and PDGF II with a molecular weight of 28KD and containing 4% of sugar. Both consist of two highly homologous A and B chains, which give PDGF a three-form dimeric structure, PDGF-AA, PDGF-BB and PDGF-AB. Monocytes/macrophages in vivo are cells that synthesize primarily PDGF.

Insulin-like growth factors (IGFs) are multifunctional cell proliferation regulating factors and have important promotion effects on cell differentiation, proliferation and individual growth and development. Included are those found in IGF-1 and IGF-2. IGF-1 is a single-chain basic protein of 70 amino acids, 7649Da molecular weight, heat-resistant, also known as somatomedin C, a polypeptide protein substance that is similar in molecular structure to insulin. IGF-1, also known as "acid-sulfurizing factor", functions as an "uninhibited islet-like activity" (NSILA), and IGF-1 is important for infant growth and for sustained anabolic effects in adults.

Transforming growth factor beta (TGF-beta) belongs to a group of newly discovered TGF-beta super family multifunctional proteins for regulating cell growth and differentiation, and can affect the functions of growth, differentiation, apoptosis, immunoregulation and the like of various cells. Transforming growth factor-beta includes three subtypes, transforming growth factor-beta 1, transforming growth factor-beta 2 and transforming growth factor-beta 3. Human TGF-beta 1 is widely involved in various pathophysiological processes in vivo, and is closely related to the occurrence and development of various diseases such as inflammation, trauma, organ fibrosis and the like.

Dexamethasone (DXMS), also known as Desamasone, cortisone, and meflososol, with chemical formula C ₂₂ H ₂₉ FO ₅ . Dexamethasone is an artificial corticosteroid used to treat a variety of conditions including rheumatic diseases, certain skin disorders, severe allergies, asthma, chronic obstructive pulmonary disease, laryngitis pseudomembranous, cerebral edema, and possibly in combination with antibiotics in tuberculosis patients. The use of dexamethasone at the volume percentage concentrations described herein can induce adipogenic differentiation of adipose stem cells.

Insulin-transferrin-selenium supplement (ITS for short) is used for cell culture and can reduce the concentration of Fetal Bovine Serum (FBS) required for routine maintenance and low-density adherence of various cells.

Carrageen (CAS 9000-07-1), also known as carrageenin, carrageenan and Irish moss gum, is a general name of polysaccharide extracted from marine red algae (including carrageenin, eucheuma, gigartina, salicornia, etc.), is a mixture of multiple substances, and is prepared by alternately connecting sulfated or non-sulfated galactose and 3,6-anhydrogalactose through alpha-1,3 glycosidic bond and beta-1,4 bond, and has 1 sulfate group on D galactose unit C4 connected with 1,3. The molecular weight is more than 20 ten thousand. Carrageenan is a good coagulator and can replace common agar, gelatin, pectin and the like.

Dextran Sulfate (Dextran Sulfate Sodium), also known as Dextran Sulfate Sodium salt, dextran Sulfate Sodium; dextran sulfate, sodium dextran sulfate. Dextran sulfate accelerates VLDL and TG metabolism and competitively inhibits oxidized LDL binding to macrophage receptors, preventing foam cell formation and atherosclerotic lesion development.

SODIUM HYALURONATE (SODIUM HYALURONATE), also known as SODIUM HYALURONATE, SODIUM furfural, hyaluronic acid, avium, alice, etc. Is white or white-like granule or powder, has no odor, and has nitrogen content of 2.8-4.0% and glucuronic acid content of 37.0-51.0% when dried. Physiologically active substances widely present in animals and humans are distributed in human skin, synovial fluid of joints, umbilical cord, aqueous humor and vitreous humor. The molecular weight is 500000-730000 dalton, the solution has high viscoelasticity and profiling property, and the sodium hyaluronate is one of the constituents of human skin, is an acidic mucose which is most widely distributed in human bodies, exists in a matrix of connective tissues, and has good moisturizing effect.

The general chemical names of the polyethylene glycol are polyethylene glycol PEG, ethylene glycol polyoxyethylene ether, polymers of alpha-hydrogen-omega-hydroxyl (oxygen-1,2-ethanediyl) and polyethylene oxide (PEO-LS). No toxicity, no irritation, slightly bitter taste, good water solubility and good compatibility with many organic components. They are excellent in lubricity, moisture retention, dispersibility, adhesive, antistatic agent, softening agent and the like.

MTMM is a reagent with a molecular weight of 276.72 and a purity of 98% or more (HPLC). Chinese name: 4- (4,6-dimethoxytriazin-2-yl) -4-methylmorpholine hydrochloride (DMTMM), a reagent that activates carboxylic acids in solution or solid phase peptide synthesis, performs better than or equivalent to the most popular coupling reagents of the current day.

The word "comprising" or "comprises" in this application is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding other elements. When used to define compositions and methods, "consisting essentially of … …" is meant to exclude other elements that have any significance to the combination of purposes described. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transitional terms are within the scope of the present invention.

Example 1 Synthesis of extracellular matrix of adipose-derived stem cells

Mixing 0.1ng/mL EGF, 2ng/mL PDGF-bb, 160ng/mL IGF-1, 1ng/mL TGF-beta 1, 1ng/mL dexamethasone and 100ug/mL carrageenan to obtain a first mixture, and adding ITS into the first mixture to form recombinant cytokine, wherein the addition amount of ITS is 0.5v% of the recombinant cytokine;

the adipose-derived stem cells are induced by the recombinant cytokine in vitro to form extracellular matrix proteins.

Example 2

Unlike example 1, the recombinant cytokine in this example comprises 10ng/mL EGF, 30ng/mL PDGF-bb, 160ng/mL IGF-1, 100ng/mL TGF- β 1, 30ng/mL dexamethasone, and 0.03mg/mL dextran sulfate mixed to form a first mixture, and ITS added to the first mixture to form the recombinant cytokine, wherein the amount of ITS added is 1.5v% of the recombinant cytokine.

Example 3

Unlike example 1, the recombinant cytokine in this example comprises 8ng/mL EGF, 16ng/mL PDGF-bb, 160ng/mL IGF-1, 75ng/mL TGF-beta 1, 18ng/mL dexamethasone, 0.03mg/mL dextran sulfate and 60ug/mL carrageenan mixed to form a first mixture, and ITS is added to the first mixture to form the recombinant cytokine, wherein the amount of ITS added is 1.3v% of the recombinant cytokine.

The proportion of each main component of the extracellular matrix in the present example was determined to be similar to that of the natural fat extracellular matrix, wherein the proportions of collagen (collagen), mucopolysaccharides (sGAGs) and glycoproteins (glycoprotens) in the fat Tissue are shown in the following table, as described in the literature (Tissue Eng Part C methods.2009 Sep;15 (3): 309-21.Doi:

TABLE 1 summary of the proportion of the dry weight components of the dermal and adipose extracellular matrices

^a Indicates a statistically significant (p＜0.05)difference from Matrigel.

The extracellular matrix formed by the induction scheme of this example has the components shown in fig. 1, the collagen content is 60-70wt%, the glycoprotein content is 25-35wt%, and the polysaccharide content is 1-3wt%, and fig. 2 and fig. 3 show the extracellular matrix obtained in example 1 after decellularization under scanning electron microscope images at different magnifications, and a large amount of extracellular matrix proteins can be seen. Therefore, the proportion of each main component in the extracellular matrix generated by in vitro induction of the recombinant cell factor is similar to that of the natural fat extracellular matrix, the problem of the survival rate of transplantation is solved compared with the fat cells used in autologous fat transplantation, and the fat cells are convenient to store, store and sell.

Example 4 Synthesis of hydrogel for adipose tissue regeneration

Freeze-drying the extracellular matrix obtained in example 3, mixing 2-20mg/ml of extracellular matrix with 5-30mg/ml of sodium hyaluronate with a molecular weight of 50-150KD and 2-20mg/ml of polyethylene glycol with a molecular weight of 1KD, dissolving the mixture in water, and reacting at 25-37 ℃ for 3-6 hours under the activation of DMTMM to form the adipose tissue regeneration hydrogel.

The morphology of the adipose tissue-regenerated hydrogel obtained in this example is shown in fig. 4, and fig. 5 to 7 show views of the adipose tissue-regenerated hydrogel after being enlarged under a scanning electron microscope at different magnifications, from which it can be seen that the lyophilized adipose tissue-regenerated hydrogel is in a porous state.

Example 5

Unlike example 4, the molecular weight of the polyethylene glycol in this example was 2.5kD.

Example 6

Unlike example 4, the molecular weight of the polyethylene glycol in this example was 5KD.

Example 7

Unlike example 4, the molecular weight of the polyethylene glycol in this example was 7.5kD.

Example 8

Unlike example 4, the molecular weight of polyethylene glycol in this example was 10kD.

FIGS. 8 to 11 show the hardness and elasticity of the adipose tissue-regenerated hydrogels obtained in examples 5 to 8 in sequence, and it can be seen that the adipose tissue-regenerated hydrogels of the present application can obtain hydrogel materials with different moduli, all of which have three-dimensional cross-linked networks and better fluidity, through different synthesis schemes.

Experimental example in vivo fat regeneration-inducing function of adipose tissue-regenerating hydrogel

After the adipose tissue regeneration hydrogel synthesized in example 7 was implanted into subcutaneous tissues of C57BL/6J mice (purchased from lakeshoda laboratory animals ltd, han shang in the absence of specific pathogenic microorganisms, kept at a constant temperature and humidity and maintained at a circadian (12) rhythm), tissue specimens were taken out at 3 days, 7 days, 14 days, and 21 days after the operation, respectively, and subjected to H & E staining (hematoxylin-eosin staining method).

As a result, as shown in FIGS. 12 to 15, in FIG. 12, tissues obtained 3 days after the implantation of the adipose tissue-regenerating hydrogel synthesized in example 7 under the skin of C57BL/6J mice were shown, and H & E staining of a portion indicated by an arrow in the figure was bluish purple, indicating that the adipose tissue-regenerating hydrogel was implanted; the tissue is shown 7 days after implantation in fig. 13, with partially degraded hydrogel material indicated by the arrows, indicating that the material has begun to degrade; FIG. 14 is a view showing a portion indicated by an arrow indicating that fat cells begin to form 14 days after the adipose tissue-regenerating hydrogel is implanted under the skin, and blood vessels containing red blood cells are visible from a star-shaped marking portion; in FIG. 15, the arrows indicate the formation of vacuolated adipocytes 21 days after the implantation of the adipose tissue-regenerating hydrogel under the skin, and the stars indicate the formation of new vascular tissue. The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. An adipose tissue regeneration hydrogel, which is characterized by comprising sodium hyaluronate, polyethylene glycol and extracellular matrix; wherein the extracellular matrix is induced by adipose-derived stem cells in vitro by recombinant cytokines and comprises 60-70wt% of collagen, 25-35wt% of glycoprotein and 1-3wt% of mucopolysaccharide;

the recombinant cytokine comprises the following components in percentage by volume:

0.1-10ng/mL of epidermal growth factor;

PDGF-BB 2-30ng/mL；

insulin-like growth factor-1-160 ng/mL;

transforming growth factor beta 1-100ng/mL;

dexamethasone 1-30ng/mL; and the number of the first and second groups,

2. The adipose tissue regeneration hydrogel according to claim 1, wherein the mass ratio of the extracellular matrix, the sodium hyaluronate and the polyethylene glycol is 2-20:5-30:2-20.

3. The adipose tissue regeneration hydrogel of claim 2, wherein the molecular weight of the sodium hyaluronate is 50-150KD, and the molecular weight of the polyethylene glycol is 1-10KD.

4. The adipose tissue-regenerating hydrogel according to any one of claims 1 to 3, wherein the adipose tissue-regenerating hydrogel further comprises a pharmaceutically acceptable carrier.

5. The adipose tissue-derived hydrogel according to any one of claims 1 to 3, wherein the adipose tissue-derived hydrogel is in a dosage form selected from injections.

6. The adipose tissue-derived hydrogel according to any of claims 1 to 3, wherein the adipose tissue-derived hydrogel is in a dosage form selected from a lyophilized powder injection.

7. A method for preparing the adipose tissue-regenerating hydrogel according to any one of claims 1 to 3, comprising the steps of:

8. The method of claim 7, wherein the recombinant cytokine comprises 0.1-10ng epidermal growth factor, 2-30ng PDGF-BB, 10-160ng insulin-like growth factor-1, 1-100ng transforming growth factor β 1, 1-30ng dexamethasone, 30-100ug carrageenan and/or 0.03-0.1mg dextran sulfate, and 0.005-0.015mL insulin-transferrin-selenium supplement per mL.

9. The method according to claim 7, wherein the activation reaction is carried out at a temperature of 25 to 37 ℃ for 3 to 6 hours.