CN117209800A

CN117209800A - Cell-loaded hydrogel microsphere based on giant salamander skin secretion and application thereof

Info

Publication number: CN117209800A
Application number: CN202310361959.7A
Authority: CN
Inventors: 张曦木; 郑荔文; 陈丽玲; 刘亚娴; 韦峰
Original assignee: Stomatological Hospital of Chongqing Medical University
Current assignee: Stomatological Hospital of Chongqing Medical University
Priority date: 2022-06-10
Filing date: 2023-04-06
Publication date: 2023-12-12

Abstract

The application relates to the field of biological materials, in particular to a cell-loaded hydrogel microsphere based on giant salamander skin secretion and application thereof. The application provides a preparation method of hydrogel microspheres, which is characterized by comprising the following steps: s1, dispersing methacryloylated gelatin and a photoinitiator in water and heating to obtain a first gel solution; s2, adding the giant salamander skin secretion hydrolysate into the first gel solution to obtain a second gel solution; s3, respectively injecting the second gel solution and the oil phase solution into a water phase channel and an oil phase channel of the microfluidic device, wherein the second gel solution is cut into monodisperse microspheres by the oil phase solution; s4, carrying out photo-curing crosslinking on the monodisperse microspheres to obtain hydrogel microspheres. Experiments prove that the hydrogel microsphere provided by the application has good biocompatibility and degradability, can be used as a good injectable micro biological bracket, and can be accurately conveyed into a living body by a minimally invasive method.

Description

Cell-loaded hydrogel microsphere based on giant salamander skin secretion and application thereof

The present application claims priority to "a cell-loaded hydrogel microsphere and its use" of chinese patent application CN202210657192.8 filed on 6/10 of 2022, which is incorporated herein by reference in its entirety.

Technical Field

The invention relates to the field of biological materials, in particular to a cell-loaded hydrogel microsphere based on giant salamander skin secretion and application thereof.

Background

Wound repair is a complex dynamic process involving a variety of intracellular and intercellular pathways, as well as restoring tissue integrity and self-stability. This dynamic process includes inflammation, proliferation, epithelialization, angiogenesis and collagen matrix formation, and in summary, wound repair can be divided into three phases: inflammatory response, cell proliferation and differentiation, and tissue remodeling. Large-area full-layer wound surfaces are often difficult to repair, so that the human health is seriously influenced, and even the life is threatened. Currently, the standard method of clinically treating skin defects is autologous skin grafting. However, since the transplanted tissue is taken from the patient, this method causes secondary damage to the patient, which not only increases the pain of the patient, but also easily creates a risk of complications such as donor site infection.

Disclosure of Invention

In a first aspect, the present invention provides a method for preparing hydrogel microspheres, which is characterized in that the method comprises:

s1, dispersing methacryloylated gelatin and a photoinitiator in water and heating to obtain a first gel solution;

S2, adding the giant salamander skin secretion hydrolysate into the first gel solution to obtain a second gel solution, wherein the volume ratio of the first gel solution to the giant salamander skin secretion hydrolysate comprises 9:1-2:3;

s3, respectively injecting the second gel solution and the oil phase solution into a water phase channel and an oil phase channel of the microfluidic device, wherein the second gel solution is cut into monodisperse microspheres by the oil phase solution;

s4, carrying out photo-curing crosslinking on the monodisperse microspheres to obtain hydrogel microspheres.

In some embodiments, the concentration of the methacryloylated gelatin comprises 10% to 12.5% (w/v).

The choice of biological material is critical for the regulation of cellular functions of tissue engineering. Natural hydrogels (e.g., alginate, hyaluronic acid, agarose, chitosan, collagen, fibrin, polyethylene glycol) have attracted considerable attention in tissue engineering. However, most hydrogels are limited by poor mechanical properties and limited cell attachment. The methacryloylated gelatin adopted by the invention is prepared by the reaction of gelatin and Methacrylic Anhydride (MA), and has a series of advantages of good biocompatibility (including biosafety and biofunctionality), strong cell adhesiveness, adjustable physicochemical properties and the like.

In some embodiments, the oil phase solution comprises mineral oil and sorbitan oleate.

In some embodiments, the volume ratio of the mineral oil to the sorbitan oleate comprises 9:1.

In some embodiments, the concentration of the giant salamander skin secretion hydrolysate comprises 5% to 10% (w/v).

In some embodiments, the sample injection rate of the second gel solution in S3 includes 20 μl/min, and the sample injection rate of the oil phase solution includes 400-700 μl/min.

Microfluidic technology can rapidly produce monodisperse microgels of tunable size by manipulating and controlling the flow of a variety of immiscible liquids. The invention prepares the monodisperse hydrogel microsphere with adjustable size and uniform size in a high-flux mode by utilizing a microfluidic technology. The hydrogel microsphere prepared by the invention is a three-dimensional crosslinked polymer network, has good structural characteristics, comprises high moisture, high elasticity and a porous structure, has good permeability to oxygen and metabolites, and can well simulate a natural extracellular matrix. In addition, the hydrogel microsphere prepared by the method has the advantages of high structural stability, uniform size and high injectability, and is suitable for various scenes. In addition, the hydrogel microspheres of the present invention are small in size, can be injected through small needles and catheters, and imbibe particles, which facilitates minimally invasive delivery of cells and biologics.

In some embodiments, the photoinitiator comprises lithium phenyl-2, 4, 6-trimethylbenzoyl phosphite or ruthenium (Ru).

In some embodiments, the concentration of the photoinitiator comprises 0.25% (w/v).

In some embodiments, the heating time of the heating is greater than or equal to 30 minutes and the time of the photocuring crosslinking is greater than or equal to 3 minutes.

In a second aspect, the present invention provides the hydrogel microspheres prepared by the above method.

In some embodiments, the hydrogel microspheres comprise a diameter of 150 to 300 μm.

In some embodiments, the hydrogel microspheres comprise a diameter of 200 to 250 μm.

In a third aspect, the present invention provides the use of the hydrogel microspheres described above for the preparation of an injectable formulation, characterized in that the injectable formulation is used for promoting tissue regeneration.

In some embodiments, the tissue comprises one or more of skin, bone, and dental pulp.

In some embodiments, the injectable formulation is for one or more of subcutaneous injection, intra-articular injection, and intra-dental intramedullary injection.

In a fourth aspect, the present invention provides a method for preparing a cell-loaded hydrogel microsphere, comprising:

s4, carrying out photocuring crosslinking on the monodisperse microspheres to obtain hydrogel microspheres;

s5, co-culturing the stem cells and the hydrogel microspheres to obtain the cell-carrying hydrogel microspheres.

In some embodiments, the method further comprises a step of removing the oil phase solution remaining in the hydrogel microspheres, the step performed after S4 and before S5.

In some embodiments, the stem cells include one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose stem cells, and dental pulp stem cells.

Stem cells have the potential to proliferate continuously and differentiate in multiple directions. In the invention, umbilical cord mesenchymal stem cells (UC-MSCs) are taken as an example to prepare the cell-carrying hydrogel microsphere with the capability of promoting tissue repair and regeneration. In addition, other stem cells having self-renewing and multipotent potential, such as bone marrow mesenchymal stem cells (BMSCs), adipose stem cells (ADSCs), and dental pulp stem cells, may be used.

In some embodiments, the co-cultivation time comprises 3-7 days.

In a fifth aspect, the present invention provides cell-loaded hydrogel microspheres prepared by the above method.

In a sixth aspect, the present invention provides the use of the cell-loaded hydrogel microspheres described above for the preparation of a subcutaneous injection for injection at a point of site, characterized in that the subcutaneous injection is used to promote wound healing in skin.

In some embodiments, the acupuncture points include Xinshu, pishu, shenshu and Zusanli.

In some embodiments, the wound comprises a full layer wound.

In some embodiments, the hydrogel microsphere loaded with cells is injected through a point site (not other point injection or common acupuncture), so that the stem cells can be slowly released at the point site and play a role of local long-term stimulation, and the healing of full-layer skin defect wounds is promoted.

Advantageous effects

According to the invention, the giant salamander skin secretion hydrolysate is mixed with GelMA to form a novel hydrogel, and the hydrogel is cut into uniform hydrogel microspheres by using microfluidic equipment. Experiments prove that the hydrogel microsphere provided by the invention has good biocompatibility and degradability, can be used as a good injectable micro biological bracket, and can be accurately conveyed into a living body by a minimally invasive method. The hydrogel microsphere provided by the invention can provide a good three-dimensional platform for cell adhesion and proliferation and promote tissue regeneration. The hydrogel microsphere loaded with the cells formed by stem cells (such as human umbilical cord mesenchymal stem cells) can promote the repair and regeneration of skin wound surface and improve the wound surface repair efficiency (including earliest realization of wound surface closure and re-epithelialization, more mature newly-generated collagen fibers, more regeneration of skin appendages in dermis layers, smaller formed scar and the like). Similarly, experiments prove that the hydrogel microsphere provided by the invention can also promote bone and dental pulp regeneration.

In some embodiments, the cell-loaded hydrogel microsphere is injected into the acupoint of the SD rat full-layer skin defect model, and as a result, the cell-loaded hydrogel microsphere provided by the invention can be delivered to the acupoint by subcutaneous injection (for example, an injection needle can be used for stimulating the acupoint for a long time), so that the wound repair can be remarkably promoted, the wound repair efficiency is higher, and the repair effect is better (the repair effect is better than that of a conventional acupoint catgut implantation (a conventional absorbable collagen thread).

As used herein, the term "hydrogel" refers to a natural or synthetic polymer network that is highly absorbent (e.g., can absorb and/or retain a substantial volume of water).

As used herein, the term "hydrogel microsphere" refers to a substance having a three-dimensional crosslinked polymer network of high moisture and having the form of microspheres, which have a diameter of about 100-400 μm.

As used herein, the term "biological tissue" or "tissue" refers to any tissue of or derived from a living or dead organism. Biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues that make up an organ or portion or region of an organism. Biological tissue includes connective tissue (e.g., reticulocyte connective tissue, dense connective tissue, elastic tissue, loose connective tissue, and adipose tissue), muscle tissue (e.g., skeletal muscle, smooth muscle, and cardiac muscle), genitourinary tissue, gastrointestinal tissue, lung tissue, bone tissue, neural tissue, and epithelial tissue (e.g., single-layer epithelium and multiple-layer epithelium), endodermal-derived tissue, mesodermal-derived tissue, and ectodermal-derived tissue. Different biological tissues may form organs of specific functions. The organism (i.e., subject or individual) to which the biological tissue is related or derived may be any animal, including mammals and non-mammals (e.g., invertebrates). The biological tissue may be intact or may have one or more incisions, cracks, defects, or other types of wounds. In some embodiments, the biological tissue is mammalian tissue.

As used herein, "wound," "wound" or "wound bed" are used interchangeably to refer to a physical disruption of continuity or integrity of a tissue structure. Wounds may be due to cuts, abrasions, avulsions, lacerations, punctures, cancers, diabetic ulcers or lesions, burns, surgery, or other injuries. In some embodiments, the wound may result in bleeding.

As used herein, "wound healing" or "wound repair" refers to the partial or complete restoration of tissue integrity. In some embodiments, wound healing includes temporal and spatial healing procedures including wound closure and processes involving wound closure. In some embodiments, the tissue is skin, i.e., the wound is a skin wound, such as a dermal or epidermal wound. "promoting wound healing" is understood to mean the restoration of skin tissue from a disruption of continuity or integrity, in particular to restoration or partial restoration of one or more of the skin (dermis and epidermis), connective tissue thereunder, and appendages. In some embodiments, promoting wound healing includes achieving the same degree of healing, with less time required for healing. In some embodiments, promoting wound healing includes achieving a higher degree of healing within the same healing time. In particular, it may involve: neovascularization; fibroblast, endothelial, and epithelial cells ; extracellular matrix deposition; re-planting the epidermis; and one or more of remodeling.

As used herein, "full-thickness wound" refers to the destruction of the epidermis and dermis and deeper structures (e.g., dermal blood vessels).

As used herein, the term "wound closure" refers to the closure of a wound, including closing the wound and not exposing internal tissue. In some embodiments, the wound closure includes the sides of the wound re-engaging to form a continuous barrier (e.g., intact skin). In some embodiments, the wound closure further comprises hemostasis.

As used herein, "scar" refers to the morphological and histopathological changes in the appearance of tissue caused by post-traumatic injury, which is a necessary product in the repair of body wounds. When the scar grows beyond a certain limit, various complications (such as damage to the appearance, dysfunction and the like) may occur, and huge physical and mental pains (especially scars left after burns, scalds and severe trauma) are brought to the patient. "scar-free healing" refers to less, significantly less or no scar formed by natural healing of a wound.

As used herein, the term "biocompatible" refers to a material that is substantially non-toxic in the in vivo environment in which it is intended to be used, and that is not substantially rejected by the physiological system of the individual. The evaluation of biological material biocompatibility should follow both principles of biosafety and biofunctionality, requiring that the biological material have very low toxicity, while at the same time requiring that the biological material be able to properly excite the corresponding function of the body (e.g., material interaction with the body's environment) in a particular application. In some embodiments, the biocompatibility meets FDA requirements. The immune response and tissue repair processes in vivo are quite complex, and it is often not sufficient to determine the biocompatibility of a material by a cell or tissue. The hydrogel microsphere provided by the invention has higher biocompatibility to organisms (especially wound environment) by a series of in vitro and in vivo experiments with reference to the requirements of International standards organization (International Standards Organization, ISO) 10993 and national standard GB/T16886.

As used herein, "regeneration" is a repair process in which a tissue or organ is partially lost by external action, and on the basis of the remainder, a structure morphologically and/or functionally identical to the lost portion is grown.

As used herein, "biologically derived" refers to organisms and parts of organisms that are derived or obtained from naturally occurring organisms. In other words, the giant salamander skin secretion lyophilized powder and the giant salamander skin secretion hydrolysate according to the present invention are not obtained by a genetic recombination technique.

Abbreviations involved in the present invention:

h-UcMSCs: human umbilical cordmesenchymal stem cells human umbilical cord mesenchymal stem cells; SSAD: skin secretion ofAndrias davidianus giant salamander skin secretions; BSA: bovine serum albumin, bovine serum albumin; ECM: extracellular matrix, extracellular matrix; PBS: phosphate buffer saline phosphate buffered saline; SEM: scanning electron microscope, scanning electron microscope; alpha-MEM: minimum essential medium-alpha, alpha-basal medium; CCK8: cell Counting Kit-8, cell proliferation count kit; h & E: hematoxylin-eosin staining; gelMA: methacrylate Gelatin, methacryloylated gelatin; LAP: lithium phenyl-2,4,6-trimethylbenzoyl phosphite; MA: methacrylic anhydride methacrylic anhydride; FBS: fetal bovine serum, fetal bovine serum; HRP: horseradish Peroxidase horseradish peroxidase; DMSO: dimethyl sulfoxide, dimethyl sulfoxide.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.

FIG. 1 is a photograph of GelMA and SSAD hydrogel microspheres;

FIG. 2 is a scanning electron microscope image of GelMA (G) and SSAD (GS) hydrogel microspheres;

FIG. 3 shows the backbone staining of h-UcMSCs on gel microspheres (cytoskeleton stained red, nuclei stained blue; a is h-UcMSCs-GelMA loaded hydrogel microsphere, b is h-UcMSCs-SSAD loaded hydrogel microsphere);

FIG. 4 shows the results of a CCK8 cell proliferation assay for the biocompatibility of GelMA/SSAD hydrogel microspheres;

FIG. 5 shows staining of live dead cells (live cells stained green, dead cells stained red) on days 3 and 7 of h-UcMSCs GelMA/SSAD-loaded hydrogel microspheres;

FIG. 6 shows migration of h-UcMSCs cells in SSAD hydrogel microspheres (left: cell migration at day 2 after h-UcMSCs-loaded SSAD hydrogel microspheres are seeded in 6-well plates; right: cell migration at day 4 after h-UcMSCs-loaded SSAD hydrogel microspheres are seeded in 6-well plates);

FIG. 7 shows the effect of each experimental group (A-GSC, A-GC, A-C, A-GS, A-CE, W-GSC, W-C and Cont (control group)) on the repair of defective skin;

FIG. 8 shows in vivo biodegradation of GelMA hydrogel microspheres and SSAD hydrogel microspheres (days 5, 7 and 9);

FIG. 9 shows in vivo biocompatibility evaluation of SSAD hydrogel microspheres (GS) in rats 9 days after in vivo injection;

FIG. 10 shows experimental results of the adhesion of different SSAD hydrogel microspheres to articular surfaces;

fig. 11 shows experimental results of SSAD hydrogel microsphere implantation into the pulp cavity.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.

In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of ranges 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Embodiment one: preparation and characterization of SSAD hydrogel microspheres

An exemplary preparation method of 1.1.1SSAD hydrolysate:

S1, mixing a disulfide bond reducing agent with the first solvent to obtain a second solvent.

Suitable first solvents should be substantially free of ions, such as ultrapure water, deionized water, water for injection, pure water, and the like.

S2, mixing the giant salamander skin secretion freeze-dried powder with the second solvent to obtain a first mixed solution.

In practice, the mixing order of the disulfide bond reducing agent, the first solvent and the lyophilized powder of the giant salamander skin secretion (i.e., the step of obtaining the first mixed solution) is not particularly limited. For example, the first mixed solution may be obtained by adding and mixing the disulfide bond reducing agent, the first solvent and the giant salamander skin secretion lyophilized powder together, or may be obtained by mixing the disulfide bond reducing agent with the first solvent and then adding the giant salamander skin secretion lyophilized powder therein, or may be obtained by mixing the giant salamander skin secretion lyophilized powder with the first solvent and then adding the disulfide bond reducing agent therein, or may be obtained by mixing the disulfide bond reducing agent with the giant salamander skin secretion lyophilized powder and then adding the first solvent therein. The proportions of the disulfide bond reducing agent, the first solvent and the giant salamander skin secretion lyophilized powder can be correspondingly adjusted according to actual needs, and the invention is not limited to the above. As an example, the mass ratio of the giant salamander skin secretion lyophilized powder to the disulfide bond reducing agent may be 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. The mass ratio of the first solvent to the giant salamander skin secretion lyophilized powder may be 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1 or 4:1. The concentration of the second solvent may be about 1% -5% (a specific value may be selected to be about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, w/w%) according to practical circumstances. The lower limit (minimum) of the mass ratio of the giant salamander skin secretion lyophilized powder and the disulfide bond reducing agent may be 3:1. The lower limit (minimum) of the mass ratio of the first solvent to the giant salamander skin secretion lyophilized powder may be 4:1.

In some embodiments, the giant salamander comprises one or more of the genera giant salamander, cryptobranchia, mangrove, andrias, and polar. Skin is an important respiratory organ of giant salamander, and is distributed with mucous glands and granular glands. The skin secretion of the Chinese giant salamander is selected because the skin secretion can represent mucus secreted by amphibians such as giant salamander by stimulation. The manner of electrical stimulation and skin scraping can promote mucous secretion.

And S3, fully hydrolyzing the first mixed solution to obtain a second mixed solution.

The temperature conditions for the hydrolysis include 0-8deg.C (preferably 0-4deg.C). The hydrolysis time is related to the specific hydrolysis temperature, the concentration of the second solvent, the concentration of the giant salamander skin secretion lyophilized powder and the like. In the temperature range of 0-8 ℃, the higher the temperature, the shorter the hydrolysis time is relatively; the higher the concentration of the second solvent, the shorter the hydrolysis time is relatively; the higher the concentration of the giant salamander skin secretion lyophilized powder, the longer the hydrolysis time is relatively. The hydrolysis time may also depend on the particular disulfide reducing agent species used. Therefore, the hydrolysis time can be correspondingly adjusted according to actual needs, and the invention is not limited to the above. Exemplary hydrolysis times include 2 to 168 hours, preferably 12 to 48 hours. Mixing may be performed in a mixing manner commonly used in the art, such as shaking, vortexing, and the like. Preferably, S3 can be carried out in a low-temperature vacuum reaction kettle, and the vacuum degree of the vacuum reaction kettle is less than 5kPa.

S4, centrifuging the second mixed solution and collecting supernatant.

The specific conditions of centrifugation can be adjusted correspondingly according to actual needs, and the invention is not limited to the specific conditions. Exemplary centrifugation conditions include centrifugation at 4500rpm for 10min at 4 ℃.

S5, placing the supernatant into a dialysis bag with the molecular weight cut-off less than or equal to 1000, and placing the dialysis bag into the first solvent for full dialysis to obtain the giant salamander skin secretion hydrolysate. The concentration of the giant salamander skin secretion hydrolysate can comprise 5-7%.

The temperature conditions for the dialysis include 0-8deg.C (preferably 0-4deg.C). Preferably, a dialysis bag with a molecular weight cut-off of 500 or 1000 is used. The dialysis time is actually related to the way the treatment is performed during the dialysis, and can be shortened, for example, by increasing the number of times the first solvent in which the dialysis bag is replaced. Therefore, the dialysis time can be correspondingly adjusted according to actual needs, and the invention is not limited to the above.

Wherein the disulfide bond reducing agent comprises a specific disulfide bond reducing agent. In the present invention, the "specific disulfide bond reducing agent" means a disulfide bond reducing agent capable of reducing intermolecular disulfide bonds in a protein or polypeptide to be reduced, while retaining intramolecular disulfide bonds as much as possible without reduction. The specific disulfide bond reducing agent includes tris (2-carboxyethyl) phosphine hydrochloride (TCEP-HCl). Of course, in the preparation method of the giant salamander skin secretion hydrolysate provided by the invention, common protein denaturants/organic solvents known in the art can be used in combination, such as vitamin C, acetic acid, hydrochloric acid, beta-mercaptoethanol, dithiothreitol, EDTA, urea, ethanol, acetone, guanidine hydrochloride, sodium dodecyl sulfate and sodium hydroxide, and proteases (such as pepsin, trypsin, papain and subtilisin).

Further, the preparation method of the giant salamander skin secretion hydrolysate provided by the invention can further comprise the step of S6 concentrating the giant salamander skin secretion hydrolysate to obtain the concentrated giant salamander skin secretion hydrolysate. The concentration of the concentrated giant salamander skin secretion hydrolysate is more than or equal to 10%.

Exemplary concentration methods include centrifugation of the giant salamander skin exudate hydrolysate in a concentration centrifuge tube having a molecular weight cut-off of 3K or evaporative concentration of the giant salamander skin exudate hydrolysate.

Preferably, any of the above steps are performed under vacuum or an inert atmosphere.

In some embodiments, the method of making comprises: the collected skin secretion of the fresh giant salamander is put into a sterilizing centrifuge tube, washed and centrifuged for 3 times by PBS at 4 ℃ and 1 time by 5% acetic acid, and then placed into a refrigerator at-20 ℃ to be pre-frozen for 4 hours. After freeze drying for 24 hours by a freeze dryer at the low temperature of 80 ℃ below zero, the powder is crushed to 300 meshes by a freeze ball mill, and the powder is packaged and stored in a refrigerator at the temperature of 20 ℃ below zero for subsequent experiments. Giant salamander skin secretion lyophilized powder, vitamin C, tris (2-carboxyethyl) phosphine hydrochloride (Tris (2-carboxyyl) phosphine hydrochloride, TCEP) and distilled water are mixed and reacted for 24 hours according to the mass ratio of 10:5:5:100. Centrifuging at 4000rpm for 15 min to obtain supernatant, and dialyzing for 24 hr to obtain SSAD hydrolysate.

1.1.2 preparation of GelMA: gelatin (10 g) was dissolved in 100mL PBS buffer. The stirring was continued on a magnetic stirrer at 50℃and 1000rpm for 1 hour until the gelatin crystals were completely dissolved, at which point the solution was pale yellow. 5mL of MA was slowly added dropwise to the gelatin solution with a syringe under light-shielding conditions, stirring was continued at 1000rpm for 2 hours until the reaction was completed. The dialysis bag with the molecular weight of 8-12kDA is boiled and softened in advance. Transferring the completely reacted solution into a dialysis bag, removing floating foam, and tightly closing the two ends by using a dialysis bag clamp. The dialysis bag was placed in ultrapure water and stirred at 40℃and 300rpm for 5 days. During which the ultrapure water was replaced every 12 hours to remove unreacted complete MA and deleterious products. After the dialysis was completed, the solution in the dialysis bag was filtered through a 0.22 μm filter membrane and packed into 50mL centrifuge tubes. The solution was frozen in a-20 ℃ refrigerator for 24 hours. Transferred to a freeze dryer. And (5) freeze-drying to obtain white sponge-like solid, namely the GelMA sponge. Storing in-20deg.C refrigerator for long term storage.

1.1.3 preparation of GelMA hydrogel microspheres: aqueous phase: 100mg of GelMA sponge was weighed by a balance, mixed with 1mL of 0.25% (w/v) LAP (lithium phenyl-2, 4, 6-trimethylbenzoyl phosphonate) solution, and heated and dissolved in a metal bath at 60℃for 30min or more, and 10% (w/v) GelMA was in a pale yellow gel. Transfer to 1mL screw syringe for use. An oil phase: 90% by volume of mineral oil+10% by volume of sorbitan oleate. Transfer to 10mL screw syringe for use. The water phase is connected with the inner layer solution inlet of the T-shaped welding coaxial needle head, and the oil phase is connected with the outer layer solution inlet. The coaxial electrospinning nozzle had an inner needle diameter of 25G and an outer needle diameter of 18G. Screw syringes each containing a water phase and an oil phase were connected to two microinjection pumps, respectively. Wherein, the sample injection speed of the water phase is adjusted to 20 mu L/min, and the sample injection speed of the oil phase is adjusted to 400-700 mu L/min. The GelMA hydrogel solution was oil-phase-cut into monodisperse microspheres. Microspheres contained in the oil phase were collected at the sample outlet using a 50mL centrifuge tube. Meanwhile, a light curing lamp is placed at the position 1cm in front of the centrifuge tube, and the microspheres are photocrosslinked and cured for at least 3min under the light curing lamp. After the solidified GelMA hydrogel microspheres were collected, they were centrifuged at 3000rpm for 3min in a centrifuge. Microspheres were pelleted at the bottom of the centrifuge tube. Removing the upper oil solution, washing and dispersing the microspheres by using a prepared PBS buffer solution, and centrifuging at 8000rpm for 3min; the washing was repeated 3 times. Adding isopropanol for quick washing, centrifuging, removing isopropanol, adding PBS solution, and standing for 10min; the isopropanol wash was repeated 2 times. Adding 75% alcohol for washing, centrifuging, and washing with PBS solution after removing alcohol to remove residual alcohol; the wash was repeated 2 times with 75% alcohol. After centrifugation of the washed microspheres, the upper PBS solution was removed and the microspheres were retained in a microcentrifuge tube and stored in a refrigerator at 4 ℃.

1.1.4 preparation of SSAD-containing hydrogel microspheres: aqueous phase: 100mg of GelMA sponge is weighed by a balance, mixed with 0.8mL of 0.25% (w/v) LAP solution, heated and dissolved in a metal heater at 60 ℃ for more than 30min, and 10% -12.5% (w/v) GelMA is in a light yellow gel state, added with 0.2mL of 5-10% SSAD hydrolysate, fully mixed and transferred into a 1mL screw syringe for standby. An oil phase: 90% by volume of mineral oil+10% by volume of sorbitan oleate. Transfer to 10mL screw syringe for use. SSAD-containing hydrogel microspheres (table 1, table 2) were prepared in the same manner as described above.

TABLE 1 SSAD hydrogel microspheres prepared from GelMA solution and SSAD solution in different ratios

Group of	Volume ratio of GelMA solution to SSAD solution	SSAD hydrogel microsphere formation (diameter)
			1	9 (10% concentration) to 1 (5% concentration)	200-300μm
2	8 (concentration 10%): 2 (concentration 5%)	200-300μm
			3	7 (10% concentration) to 3 (10% concentration)	200-300μm
4	6 (concentration 10%): 4 (concentration 10%)	250-350μm
			5	5 (concentration 10%): 5 (concentration 10%)	250-350μm
6	4 (10% concentration) to 6 (10% concentration)	300-400μm
			7	3 (concentration 10%): 7 (concentration 10%)	Failure to generate
8	2 (10% concentration) to 8 (10% concentration)	Failure to generate
			9	1 (10% concentration) to 9 (10% concentration)	Failure to generate

TABLE 2 SSAD hydrogel microspheres prepared from aqueous and oil phase solutions at different sample injection rates

1.1.5 Scanning Electron Microscope (SEM) observation of GelMA/SSAD gel microspheres: to observe the microsphere surface morphology, gelMA hydrogel microspheres and were examined using scanning electron microscopy (scanning electron microscope, SEM)Morphology of SSAD hydrogel microspheres. Use of GelMA/SSAD hydrogel microspheres with CO ₂ And removing excessive water on the surface of the sample by a critical point drying method. After the microspheres were subjected to a metal spraying treatment, they were observed with SEM at 5 kV.

1.2 experimental results

GelMA/SSAD hydrogel microspheres

As can be seen under an optical microscope, the GelMA (left in figure 1)/SSAD (right in figure 1) hydrogel microsphere (taking 12.5% GelMA solution and 5% SSAD hydrolysate with volume ratio of 4:1 as an example) prepared under the microfluidic technology has relatively uniform size, diameter of 200-300 μm and good dispersibility.

Scanning electron microscope observation of GelMA/SSAD hydrogel microspheres

As shown in FIG. 2, the GelMA (G) hydrogel microspheres were smooth in surface as observed under a Scanning Electron Microscope (SEM). Because the added SSAD hydrolysate has certain viscosity, the surface of the microsphere containing the SSAD (GS) hydrogel microsphere is rough and wrinkled, but the spherical shape of the microsphere is not lost. In summary, after the SSAD hydrolysate and the GelMA hydrogel are mixed according to the proportion of 2:8, the SSAD hydrogel microsphere is successfully prepared through photocrosslinking under a microfluidic system.

To summarize, in this example, a large number of uniform-sized regular-morphology GelMA hydrogel microspheres were prepared by a "T" joint microfluidic device, and SSAD hydrogel microspheres were formed after addition of SSAD hydrolysate. The sphere under the light mirror keeps good uniform size and has a diameter of about 150-300 mu m (preferably 200-250 mu m); SSAD microspheres form a rough spherical surface under electron microscopy, increasing the microsphere surface area. This shows that this example, after addition of SSAD hydrolysate, newly synthesizes a hydrogel microsphere with a rougher surface by the microfluidic device, which is more favorable for cell adhesion on the microsphere surface.

Example two preparation and characterization of h-UcMSCs-loaded gel microspheres

In order to explore whether the newly synthesized SSAD hydrogel microsphere can be a suitable drug-carrying/cell-carrying microcarrier, the embodiment loads the human umbilical cord mesenchymal stem cells on the hydrogel microsphere, and evaluates the activity and proliferation characteristics of the human umbilical cord mesenchymal stem cells on the SSAD hydrogel microsphere through a CCK8 cell proliferation test and living/dead cell staining; observing the growth and adhesion condition of human umbilical cord mesenchymal stem cells on the microspheres through cytoskeletal/cell nucleus staining; the migration performance of human umbilical cord mesenchymal stem cells on the microspheres was observed by seeding the microspheres with the cells on a tissue culture plate.

2.1.1 human umbilical cord mesenchymal Stem cell culture: human umbilical cord mesenchymal stem cells (h-UcMSCs) were purchased from Yinfeng biological group (China).Cell resuscitation:(1) taking out the freezing tube from the liquid nitrogen tank; (2) immersing the freezing tube into a water bath kettle at 37 ℃, and shaking the tube body to enable the tube body to be melted rapidly; (3) after the surface of the frozen storage tube is disinfected by 75% alcohol, opening a frozen storage tube cover in an ultra-clean workbench, and sucking out the cell suspension to a 100mm culture dish containing 8mL of fresh alpha-MEM complete culture medium; (4) placing the mixture at 37deg.C and containing 5% CO ₂ Is cultured in a cell incubator; (5) after 12 hours, the solution was changed.Cell culture:(1) cells were cultured in a complete medium of α -MEM containing 10% fbs, and the medium was changed every 3 days, and when the cells were fused to 80% or more, passage was performed. The cells of 2 to 5 passages were used in subsequent experiments. (2) Washing the cells with PBS buffer for 2 times; sucking the PBS buffer solution, adding a proper amount of digestive solution containing 0.25% of pancreatin to cover cells, digesting for 2min in a cell incubator, observing the cells under an inverted microscope, retracting cytoplasm, and stopping digestion when the cells fall off from the bottom of the dish; (3) adding a proper amount of alpha-MEM complete culture medium to stop trypsin digestion, transferring into a 15mL centrifuge tube, and centrifuging at 1000rpm for 4min; (4) adding alpha-MEM complete culture medium, gently stirring, sucking 10 microliters, counting, inoculating onto microsphere or passaging according to required amount, and mixing with 5% CO ₂ Is cultured in a cell incubator.

2.1.2 preparation of h-UcMSCs gel microspheres: gelMA/SSAD hydrogel microspheres were prepared as described above. A layer of agar which is subjected to high-temperature high-pressure sterilization treatment is uniformly paved at the bottom of the 6-pore plate in advance so as to prevent cells from growing in the pore plate in an adherent manner. The microsphere suspension was transferred to a 2mL microcentrifuge tube and centrifuged at 8000rpm for 3min, and the supernatant was removed. Mixing the remaining microsphere sediment with h-UcMSCs cell suspension with alphaMEM complete medium mix, cell seeding number of 8X 10 ⁴ cells/wells. The mixture was then transferred to a 6-well plate containing a coagulated agar bottom prepared in advance, and the cells and microspheres were thoroughly mixed at 37℃with 5% CO ₂ Incubate under incubator.

2.1.3h-UcMSCs backbone staining on gel microspheres: in order to determine the adherent growth of cells on microspheres, h-UcMSCs-carrying microspheres were stained for scaffold proteins. After co-culturing h-UcMSCs with GelMA/SSAD hydrogel microspheres for 7 days, the cell-loaded microspheres were collected and cytoskeletal/nuclear staining was performed on h-UcMSCs on the microspheres. GelMA hydrogel microspheres loaded with h-UcMSCs are abbreviated as GC; gelMA-containing SSAD hydrogel microspheres carrying h-UcMSCs are abbreviated as GSC, as follows.

Preparing a working solution:10mg of BSA powder was weighed and dissolved in 1mL of BSA buffer to obtain 1% BSA buffer. 1 microliter of 1000 XiFluor dissolved in DMSO was pipetted ^TM The 555 phalloidin was added to 1mL of a buffer containing 1% BSA to obtain a 1 Xworking solution.

Dyeing:(1) the cells mostly adhere to the hydrogel microsphere surface when viewed under a microscope. Sucking the culture solution, and washing the cell-carrying microspheres twice with PBS buffer solution; (2) fixing cells with 4% paraformaldehyde solution for 10min at room temperature; (3) washing the cell-carrying microspheres with PBS buffer solution at room temperature for 2 times, each time for 10min; (4) permeabilizing with 0.1% Triton X-100 solution in PBS buffer for 5min to increase its permeability; (5) washing the cell-carrying microspheres with PBS buffer solution for 2 times, each time for 10min; (6) taking a proper amount of freshly prepared phalloidin working solution to completely cover the cell-carrying hydrogel microspheres, and dyeing the microspheres for 30 minutes in a dark place at room temperature; (7) washing the cell-carrying microspheres with PBS buffer solution for 3 times, each time for 5min; (8) adding a sufficient amount of ready-to-use DAPI solution to counterstain the cell nuclei, and incubating for 3min at room temperature in a dark place; (9) washing the cell-carrying microspheres for 2 times with PBS buffer solution for 5min each time; and (c) were photographed under fluorescent confocal microscopy and analyzed by observation with LAS X software.

2.1.4 CCK8 assay of GelMA/SSAD hydrogel microspheres: to assess the biocompatibility of GelMA/SSAD microspheresThe proliferation of h-UcMSCs attached to the hydrogel microspheres was detected using the CCK-8 kit, and the OD was measured at 450 nm. (1) h-UcMSCs cell fusion is over 80 percent, and cell counting is carried out after digestion, centrifugation and resuspension; (2) inoculating h-UcMSCs into a 96-well plate at 5000 cells/well density, adding hydrogel microspheres, mixing the microspheres with cells thoroughly, culturing in a cell incubator at a volume of about 100 microliters per well, and measuring OD values on days 1, 3, 5, and 7, respectively; (3) the cells were inoculated into 96-well plates at a density of 5000 cells/well for adherent culture as a control group. (4) After 24 hours of co-culture of the cells and hydrogel microspheres, a working solution containing 10 microliter volume of CCK8 solution per 100 microliters was prepared; the old medium in the 96-well plate is sucked off, and 100 microliters of CCK8 working solution is added to each well; (5) wrapping the 96-well plate with tinfoil paper to avoid light, and placing the 96-well plate into a cell incubator for incubation for 2 hours; (6) after 2 hours, the absorbance value was measured at 450nm using a multifunctional microplate reader; (1) 0, returning to an ultra-clean workbench, removing CCK8 working solution in the hole, and washing with PBS buffer solution, wherein no loss of cell-carrying microspheres is required in the process; adding 100 microliters of fresh alpha-MEM complete culture medium into each well, and placing the culture medium into a cell culture box for continuous incubation; (8) similarly, the steps (4) to (7) were repeated on days 3, 5 and 7, and the OD values were measured on different days.

2.1.5 staining of live and dead cells of h-UcMSCs-loaded hydrogel microspheres: to further evaluate the biocompatibility of GelMA/SSAD microspheres, live and dead cells on cell-loaded microspheres were stained using the Calcein-AM/PI kit.

Preparing a dyeing working solution:the Calcein-AM stock solution and PI stock solution were returned to room temperature for use. To 5mL of PBS buffer, 10. Mu.L of Calcein-AM stock solution and 15. Mu.L of PI stock solution were added, and the mixture was mixed to prepare a working solution.

Dyeing:(1) washing the cell-carrying microspheres with PBS buffer for 2 times; (2) mixing a proper amount of dyeing working solution with the cell-carrying microspheres, covering the surfaces of the microspheres with the dyeing solution, and dyeing for 15min at 37 ℃ in a dark place; (3) fluorescence observation photographing was performed under a confocal microscope.

2.1.6 cell migration in h-UcMSCs hydrogel microspheres: to further evaluate the ability of cells to migrate, the cell-loaded microspheres were re-seeded onto 6-well plates without agar bottom and cells migrating from the microspheres were observed. (1) Collecting the h-UcMSCs hydrogel microspheres obtained by the method, gently washing twice with PBS buffer solution, and removing non-adherent cells and cells falling off from the microspheres; (2) spreading the cell-carrying microspheres in a 6-well plate, and adding a small amount of alpha-MEM complete medium to wet the cell-carrying microspheres; (3) after 24 hours, fresh complete medium was slowly and gently replenished along the well plate walls in a 6-well plate; (4) at 48 and 96 hours, the outward migration of cells on the microspheres was observed by an optical microscope and photographed.

2.2 experimental results

Framework staining of h-UcMSCs on gel microspheres

h-UcMSCs are loaded on the two hydrogel microspheres of GelMA/SSAD, and the two h-UcMSCs-GelMA hydrogel microspheres and the h-UcMSCs-SSAD hydrogel microspheres are respectively prepared. In order to observe the adhesion and growth of cells on both hydrogel microspheres, h-UcMSCs-carrying microspheres were stained for scaffold proteins. Cytoskeletal staining after 7 days of culture showed (FIG. 3) that h-UcMSCs adhered to the microsphere surface, the cell dispersion morphology was normal, the cells were fully extended, and the hydrogel microspheres were encapsulated. The two hydrogel microspheres of GelMA/SSAD are shown to provide a good platform for cell adhesion and proliferation.

CCK8 assay of GelMA/SSAD hydrogel microspheres

GelMA and SSAD biocompatibility was first verified by h-UcMSCs cells using a cell counting kit-8 (CCK-8) assay. On the first day after h-UcMSCs were seeded on microspheres, there was no statistical difference in cell proliferation between GSC, GC and monolayer cultured h-UcMSCs cell C groups; on the third day after inoculation, the GSC group cells proliferate fastest, the OD value is 0.84+/-0.01, the OD value of the GC group is 0.78+/-0.01, the GSC group and the GC group have obvious differences (P < 0.01), the OD value of the control group C group is 0.75+/-0.02, and the difference between the GSC group and the control group is obvious (P < 0.001); on day 5 post inoculation, there was a statistical difference between the OD value of GSC group of 1.21±0.02, gc group of 1.13±0.01, and control group C (1.08±0.01), P < 0.01 or P < 0.001; on day 7 post inoculation, the OD values of GSC groups increased to 1.97±0.08, higher than GC groups (1.82±0.04) and control groups (1.64±0.06), the differences were statistically significant (P < 0.05 or P < 0.01) (fig. 4). The experiment shows that the h-UcMSCs-SSAD hydrogel microsphere prepared by the invention can be used as an injectable cell-carrying bracket, and has good biocompatibility in the aspects of cell adhesion, proliferation and the like.

Live and dead cell staining of h-UcMSCs-carrying hydrogel microspheres

The biocompatibility of the hydrogel microspheres was further verified by cell live/dead staining. Living cells on the microspheres were stained with green fluorescence and dead cells with red fluorescence by Calcein-AM/PI cell cream staining kit. After h-UcMSCs cells were grown on GelMA/SSAD hydrogel microspheres for 3 and 7 days, cell-loaded microspheres were collected, cells on the microspheres were stained for viable and dead cells, and observed under confocal microscopy. The result of staining living and dead cells after 3 days of cell culture on the hydrogel microspheres shows that h-UcMSCs adhere and grow on the GelMA hydrogel microspheres and the SSAD hydrogel microspheres, the cell viability condition is good, and the number of dead cells represented by red fluorescence is small. The staining results of live dead cells after 7 days of cell culture on hydrogel microspheres showed that cells proliferated on the microsphere surface at day 7, the number of cells increased, and the number of dead cells was small at day 7, compared to cells cultured on hydrogel microspheres for 3 days (fig. 5). The experimental result is consistent with the CCK8 result, and shows that both hydrogel microspheres have no cytotoxicity and good biocompatibility.

Migration of h-UcMSCs cells in GelMA+SSAD hydrogel microspheres

In addition, the migration of cells from the microspheres after 7 days of culture of h-UcMSCs-carrying hydrogel microspheres was also observed. Transferring the cell-carrying microspheres cultured for 7 days into a 6-well plate, and after 2 days, as shown in the left graph of FIG. 6, h-UcMSCs cells on the surface of the SSAD hydrogel microspheres are migrated to the periphery of the well plate in a radial manner by taking the microspheres as the centers, the number of the migrated cells is large, and the stem cells maintain the original normal form; after 4 days (right panel of FIG. 6), the range of "irradiation" around the microspheres was expanded, and the number density of stem cells was increased, indicating that h-UcMSCs migrating from the microspheres still maintained good activity and proliferation properties.

In order to investigate whether the SSAD hydrogel microspheres prepared in the above examples can be a suitable drug-carrying/cell-carrying microcarrier, the present example loads human umbilical cord mesenchymal stem cells on the hydrogel microspheres, and after the human umbilical cord mesenchymal stem cells are inoculated on the SSAD microspheres for 7 days through cytoskeletal/cell nucleus staining, the cells adhere and fully stretch on the surfaces of the hydrogel microspheres, so that the original morphological characteristics of the stem cells are maintained, which indicates that the surfaces of the hydrogel microspheres of the present invention are suitable for cell adhesion, and can be a carrier for transporting cells. This example uses CCK8 cell proliferation assay and live/dead cell staining to evaluate the activity and proliferation properties of human umbilical mesenchymal stem cells on gelma+ssad hydrogel microspheres. The results demonstrate that h-UcMSCs planted on the surface of GelMA+SSAD hydrogel microspheres maintained good proliferation properties and activity even at day 7. It is emphasized that the GelMA+SSAD hydrogel microsphere has good biocompatibility, and the rough surface increases the adhesion of cells on the microsphere, increases the contact surface area of cells, improves the proliferation space of cells, and has more advantages than the GelMA hydrogel microsphere and the traditional two-dimensional culture. In addition, after the h-UcMSCs are cultured on the GelMA+SSAD hydrogel microsphere for 7 days, good migration performance is still maintained, and stem cells can be widely migrated from the surface of the microsphere and continuously proliferated. The results show that the SSAD hydrogel microsphere has good biocompatibility, provides a good platform for cell adhesion and proliferation, and indicates that the SSAD hydrogel microsphere loaded with cells can be used as a tissue regeneration bracket and provides strong support for local tissue regeneration.

In conclusion, the embodiment loads the stem cells taking the human umbilical cord mesenchymal stem cells as an example on the hydrogel microspheres, and verifies that the SSAD hydrogel microspheres can be used as a potential injectable cell-carrying scaffold, have good biocompatibility and can provide a good three-dimensional platform for cell adhesion and proliferation.

Embodiment III: research on repairing effect of h-UcMSCs cell-loaded GelMA+SSAD gel microsphere acupoint injection on wounded skin

The present example further explores the therapeutic potential and application prospects of the cell-loaded SSAD hydrogel microspheres prepared in the above examples in combination with point stimulation. In this example, a model of a full-thickness defect of the skin with a diameter of 1.5cm was created on the back of SD rats, acupoints such as "Xinshu", "Pishu", "Shenshu" and "Zusanli" were selected and located by referring to the teaching materials of national higher agricultural institutions, and SSAD hydrogel microspheres carrying h-UcMSCs were injected into these acupoints of rats to evaluate the repair effect of the skin defect of rats after injection of SSAD hydrogel microsphere acupoints carrying h-UcMSCs by calculating the healing rate, relative epidermis thickness and relative scar rate, the number of skin appendages, collagen and vascularization, and the like. And the biosafety of SSAD hydrogel microspheres was evaluated by in vivo biodegradation and in vivo toxicity assays.

3.1.1 GelMA/GelMA+SSAD hydrogel microspheres, h-UcMSCs cell suspension, h-UcMSCs cell-loaded hydrogel Preparation of glue microsphere and collagen buried wire: (1) cells were cultured in a complete medium of α -MEM containing 10% fbs, and the medium was changed every 3 days, and when the cells were fused to 80% or more, passage was performed. The 2-5 generation cells are used for animal experiments. (2) GelMA/SSAD hydrogel microspheres: preparing GelMA/SSAD hydrogel microspheres by using a microfluidic device according to the method, adding a proper amount of PBS buffer solution, and preparing a suspension with the total volume of 1mL for animal experiments; (3) h-UcMSCs cell suspension: when the cells are fused to more than 80%, 0.25% pancreatin digestion solution digests the cells, centrifugates, resuspensions and counts the cells, and the cell quantity is 8 multiplied by 10 ⁴ Inoculating h-UcMSCs of cells into a 6-well plate, culturing in alpha-MEM complete medium for 12 hours, collecting cells, suspending with PBS buffer solution to prepare cell suspension with total volume of 1mL, and storing on ice for animal experiments; (4) h-UcMSCs cell-loaded hydrogel microspheres: when the cells are fused to more than 80%, 0.25% pancreatin digestive juice digests the cells, centrifugates, resuspensions and counts the cells, fully mixes the h-UcMSCs with the GelMA/SSAD hydrogel microspheres, then co-cultures the mixture for 12 hours, and collects the loaded carriers after the h-UcMSCs are loaded to the GelMA/SSAD hydrogel microspheres Adding a proper amount of PBS buffer solution into the cell hydrogel microsphere to prepare a suspension with the total volume of 1mL, and placing the suspension on ice to preserve the cell viability for animal experiments; (5) the absorbable collagen line No. 4 (Ipomoea dulcis) is trimmed with an ophthalmic scissors, each segment has a length of about 3-4 mm, and is soaked in PBS buffer solution for standby.

3.1.2 Establishment of SD rat full-layer skin defect model: 56 male SD rats were selected and weighing 180-250g (6-8 weeks old). Each rat was intraperitoneally injected with 10g/L sodium pentobarbital at an anesthetic dose of 30 mg/kg. The hair on the back of the rat was removed with an epilator and depilatory cream, and the iodophor skin surface was sterilized. A circular biopsy skin punch was used to create a circular full-thickness hole-through defect with a diameter of 15mm in the back of the rat. 56 SD rats (male, 180-250 g) were classified according to the random number table method: SSAD h-UcMSCs cell microsphere point site injection group (A-GSC group); gelMA h-UcMSCs cell microsphere cavity site injection group (A-GC group); h-UcMSCs cell suspension hole site injection group (group C); SSAD microsphere point site injection group (GS group); collagen line acupoint buried line group (CE group); SSAD h-UcMSCs cell-loaded microsphere peri-invasive injection group (W-GSC group); h-UcMSCs cell suspension peri-invasive injection group (W-C group); blank (Cont).

The acupoint injection group refers to the "comparative acupuncture" of the teaching materials of the national higher agricultural institutions, selects and locates the "Xinshu", "Pishu", "Shenshu" and "Zusanli" acupoints, and marks them on the body surface with a Mark pen. Xinshu: the intercostal space at the rear two sides of the fifth thoracic vertebra is provided with a point at the left and right sides; pishu: in the intercostal space at the rear two sides of the eleventh thoracic vertebra, a point is arranged at each of the left and right sides; shenshu: two points at the rear and left sides of the second lumbar vertebra; zu three lining: one acupoint is located on the left and right side of the musculature 0.5cm below the small head of the fibula below the knee joint. Injecting materials into the cavity muscle according to groups respectively: transferring SSAD h-UcMSCs cell microspheres into a 1mL injector by using a No. 7 injection needle, penetrating the injection needle into the acupoint for 4-5 mm, injecting the material into the muscle of the acupoint, wherein the injection volume is 100 microliters/acupoint (the total injection amount is 100 mg/point of microsphere, and the cell is 1 multiplied by 10) ⁴ cells/dots). The GelMA h-UcMSCs cell microspheres are injected into the muscles of the acupoint by the same method according to the same dosage. Groups A-C are identicalCell mass h-UcMSCs cell suspension was injected into the cavity muscle as described above. The A-GS group injected the same amount of microspheres into the muscles of the acupoints. The A-CE group uses a No. 7 buried wire needle to implant No. 4 absorbable collagen wires with the size of 3-4 mm into the muscles of the acupoint.

The periwound injection group is characterized in that an injection needle is inserted into an intradermal muscle layer at a position 0.5cm away from the periphery of a circular wound at four points of the upper, lower, left and right of the periwound, about 4-5 mm is penetrated, and SSAD loaded h-UcMSCs cell microsphere materials (W-GSC group) or h-UcMSCs cell suspensions (W-C group) with the same cell quantity are injected into the muscle layer.

The wound surface is naturally exposed after operation, so that the wound is kept clean and dry, and infection is prevented. The rats were fed in a clean cage and fed freely. Rats were treated once every 5 days until the wound healed. The rats in the blank group were not treated at all.

The rat skin full-layer defect model is built and treated, and the back skin wound surface of the rat is photographed by using a digital camera on days 0, 5, 8, 10, 15 and 18, and the wound surface healing condition is recorded by taking a black circular disc with the diameter of 1.5cm as a scale. The non-healing wound area of the rats was measured using Image J software and the wound healing rate was calculated. The calculation formula of the wound healing rate is as follows:

rats were sacrificed on day 18 and skin defects and surrounding normal skin were taken for subsequent histological analysis.

3.1.3 histological section observations:

paraffin wax slicing step: (1) the above collected rat skin tissues were fixed in 4% paraformaldehyde solution for 24 hours. (2) The solution was rinsed overnight under running water and fixed in 70% ethanol for a long period of time. (3) The tissue is dehydrated and transparent in gradient in a full-automatic dehydrator and is immersed in wax. (4) Embedding by using paraffin embedding machine, placing on a refrigerator, and demoulding after the paraffin is solidified. (5) Preserving in a refrigerator at 4 ℃. (6) The skin tissue pieces were cut into skin tissue sections with a thickness of 7 microns under a paraffin microtome.

H&E dyeingThe color step: (1) the skin tissue sections were baked in a 60 ℃ oven for 2 hours. (2) Dewaxing was performed in xylene I and II for 8min each. (3) Hydrating in ethanol according to concentration gradient, sequentially obtaining absolute ethanol I and absolute ethanol II for 5min respectively; 95% ethanol for 2min;90% ethanol for 2min;85% ethanol for 2min;75% ethanol for 2min. (4) Rehydrating in ultrapure water for 2min. (5) Hematoxylin dye solution is used for dying cell nuclei for 5min. (6) Washing with running water for 2min. (7) The eosin dye solution dyes the cytoplasm for 2min. (8) Washing with running water for 2min. (9) Dehydrating in ethanol according to ascending concentration gradient, wherein the ethanol is 80% ethanol for 15s in turn; 90% ethanol for 15s;100% absolute ethanol for 15s. Transparent in xylene for 2min.Neutral resin seals, after complete drying, sections were scanned using Olympus VS 200. />Skin section staining was observed using software Olympus Image Viewer and skin relative epidermis thickness and relative scar rate were calculated, counting skin appendages. The calculation formulas of the relative epidermis thickness (%) and the relative scar rate (%) are respectively:

the length of the skin scar is the length of the new epithelium after the wound heals, and the length of the wound is the distance between muscle faults at the baseline.

Masson trichromatic staining step: (1) paraffin sections were dewaxed to water, as before. (2) The Weibert iron hematoxylin dye solution is prepared and used at present, and is dyed for 5min. (3) Hydrochloric acid alcohol is differentiated for 15s, and distilled water is used for washing for 2 times. (4) Masson blue-dissolving solution is dyed for 3min, and distilled water is used for washing for 1-2min. (5) Ponceau dye liquor is used for dyeing for 5min. (6) Distilled water and weak acid solution are mixed with weak acid working solution in a ratio of 2:1, and the mixture is washed for 2min. (7) Phosphomolybdic acid solution Washed with water for 2min and observed under a microscope for differentiation. (8) Washing with weak acid working solution for 1min. (9) 95% ethanol was rapidly dehydrated for 10s. Dehydrated in absolute ethanol for 2 times and 10s each time.After the neutral resin slides were completely dried, the slides were scanned using Olympus VS 200. />Skin collagen new growth was scanned and analyzed using the software Olympus VS200 software.

Double-label immunofluorescence staining: (1) paraffin sections dewaxed to water: sequentially placing the slices into xylene I for 15min, xylene II for 15min, absolute ethanol I for 5min, absolute ethanol II for 5min,85% ethanol for 5min and 75% ethanol for 5min, and washing with distilled water. (2) Antigen retrieval: the tissue sections are placed in a repair box filled with citric acid (PH 6.0) antigen retrieval liquid, and antigen retrieval is carried out in a microwave oven. Stopping the medium fire for 8min, and turning the medium fire from the low fire for 7min, wherein excessive evaporation of buffer solution should be prevented during the process, and the tablet is not dried. After natural cooling, the slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. (repair liquid and repair conditions are determined according to tissues) (3) circling, and hydrogen peroxide is used for sealing: and (3) after the sections are slightly dried, circling around the tissues by using a histochemical pen (preventing antibodies from flowing away), placing the sections into a 3% hydrogen peroxide solution, incubating the sections for 25 minutes at room temperature and in a dark place, sealing endogenous peroxidase, placing the glass slides into PBS (PH 7.4), and shaking and washing the glass slides on a decolorizing shaking table for 3 times, wherein each time is 5 minutes. (4) Serum blocking: BSA was added dropwise to the circles and incubated for 30min. (5) Adding CD3 primary antibody: the blocking solution is gently thrown away, PBS is dripped on the slice, the slice is horizontally placed in a wet box for incubation overnight. (small amount of water added to wet cartridge to prevent antibody evaporation) (6) corresponding HRP-labeled secondary antibody: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping HRP-labeled secondary antibody covering tissues of the corresponding species with the primary antibody into the circle after the sections are slightly dried, and incubating for 50 minutes at room temperature. (7) FITC-TSA was added: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. The slices are slightly dried, TSA is dripped into the rings after the slices are slightly dried, and the rings are protected from light Incubate at room temperature for 10min. After incubation, the slides were washed 3 times with shaking on a decolorizing shaker in TBST for 5min each. (8) Microwave treatment: the tissue slice is placed in an antigen retrieval liquid box filled with citric acid (PH 6.0) and heated in a microwave oven, the medium fire is stopped for 8min, the medium fire is stopped for 7min, the low fire is stopped, the primary antibody which is combined with the tissue is removed, the process should prevent the buffer solution from evaporating excessively, and the slice is not dried. (9) Adding CD68 primary antibody: the PBS-prepared primary antibody is dripped on the slice, the slice is placed in a wet box and incubated overnight at 4 ℃. (the addition of a small amount of water in the wet box prevents the antibody from evaporating). Adding a corresponding secondary antibody: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (3) dripping fluorescent secondary antibodies which cover tissues with the corresponding species of the primary antibodies into the rings after the sections are slightly dried, and incubating for 50min at room temperature in a dark place.DAPI counterstaining nuclei: and (3) dripping DAPI dye solution into the ring after the slices are slightly dried, and incubating for 10min at room temperature in a dark place. />Spontaneous fluorescence quenching: the slide was washed with shaking 3 times, 5min each time, in PBS (pH 7.4) on a decolorizing shaker. And (5) after the slices are slightly dried, adding an autofluorescence quenching agent into the rings for 5min, and washing with running water for 10min. />Sealing piece: and (5) after the slices are slightly dried, sealing the slices by using an anti-fluorescence quenching sealing tablet. / >And (5) microscopic examination and photographing: sections were observed under a fluorescence microscope and images were acquired. DAPI ultraviolet excitation wavelength is 330-380nm, emission wavelength is 420nm, and blue light is emitted; FITC excitation wavelength 465-495nm, emission wavelength 515-555nm, green light emission; CY3 excitation wavelength is 510-560nm, emission wavelength is 590nm, and red light is emitted.

3.1.4 in vivo degradation experiments: rat subcutaneous injection of GelMA/gelma+ssad hydrogel microspheres: (1) rats were intraperitoneally injected with 10g/L pentoba at an anesthetic dose of 30mg/kgSodium biturate. (2) The hair on the back of the rat was removed with an epilator and depilatory cream, and the iodophor skin surface was sterilized. (3) Transferring the GelMA/SSAD hydrogel microspheres into a 1mL syringe, selecting a No. 7 injection needle, penetrating the injection needle into subcutaneous tissue at the left back of the rat, injecting the hydrogel microspheres with an injection volume of 100 microliters/point, injecting the SSAD hydrogel microspheres with a total injection volume of 100 mg/point in the subcutaneous tissue region at the right back of the rat, and injecting the GelMA hydrogel microspheres with the injection volume identical to that of the GelMA hydrogel microspheres. (4) The degradation of the material in vivo was observed daily after the operation, and rats were sacrificed on days 5, 7, and 9, respectively, after microsphere injection, and the material at the implantation site and the corresponding skin tissue were taken for histological analysis. Histological staining analysis: h &E staining and double-label immunofluorescence staining steps are the same.

3.1.5 in vivo biocompatibility assay: (1) the specific method for subcutaneously implanting the hydrogel microspheres into rats is the same as the previous method. (2) On day 9 post-surgery, after the rats were sacrificed, the heart, liver, spleen, lung, kidney of the rats were collected for subsequent H & E staining. (3) The heart, liver, spleen, lung, kidney of normal rats were collected and used as controls for subsequent H & E staining.

3.1.6 statistical analysis: data were analyzed using SPSS24.0 software (IBM, USA), all quantitative data were independently repeated at least 3 times and expressed as mean ± Standard Deviation (SD). Statistical analysis was represented by one-way analysis of variance (ANOVA) using Tukey post hoc test. A value of P < 0.05 is considered statistically significant.

3.2 experimental results

SD rat full-thickness skin defect healing

A full-thickness skin defect model with a diameter of 1.5cm was created on the back of the rats and the general healing of the wound surface of each group of rats was recorded using a digital camera (fig. 7 a). In order to observe the effect of the injection of cell-loaded microspheres at the acupoints on wound repair, gelMA+SSAD hydrogel microspheres (A-GSC group) loaded with h-UcMSCs cells were injected at the acupoints of Xinshu, pishu, shenshu and Zusanli in the present example, and compared with a blank control group (Cont). The wound area was calculated for two groups at different time points by ImageJ software and the wound healing rate was calculated (fig. 7 b). The wound healing rate of the A-GSC group at the 5 th day is 42.85+/-3.64%, the wound healing rate of the Cont group at the 5 th day is 14.9+/-4.51%, and the wound healing rate of the A-GSC group is almost 3 times that of the Cont group, and the A-GSC group and the Cont group have obvious statistical difference (P is less than 0.001). On day 8, the wound healing rate was 76.43.+ -. 2.08% for the A-GSC group, while the wound healing rate was 50.53.+ -. 10.9% for the Cont group, with significant statistical differences (P < 0.001) between the two groups. On postoperative day 10, the wound healing rate was 88.14.+ -. 0.83% for the A-GSC group, while the wound healing rate was 66.38.+ -. 9.19% for the Cont group, with significant statistical differences (P < 0.001) between the two groups. 15 days after operation, the wound surface of the A-GSC group is mostly healed, the wound healing rate of the A-GSC group is 97.46+/-0.99%, the wound healing rate of the Cont group is 91.48 +/-2.85%, and the two groups have obvious statistical difference (P is less than 0.01). 18 days after surgery, the A-GSC group is basically completely healed, the wound healing rate of the A-GSC group is 99.5+/-0.57 percent, the wound healing rate of the Cont group is 94.33+/-0.97 percent, and the two groups have obvious statistical difference (P is less than 0.001). Therefore, compared with a blank control group, the cell-carrying hydrogel microsphere injected into the acupoint can obviously promote the healing of the full-thickness defect skin of the rat.

In order to investigate the advantage of the acupoint injection cell-loaded microspheres in repairing the wound surface of the rat full-thickness defect skin compared with the non-acupoint injection cell-loaded microspheres, the embodiment provides a GelMA+SSAD hydrogel microsphere group (A-GSC) of acupoint injection h-UcMSCs cells and a GelMA+SSAD hydrogel microsphere group (W-GSC) of injection h-UcMSCs cells at 0.5cm of the wound periphery. On postoperative day 5, the wound healing rate of the W-GSC group was 29.59.+ -. 3.97% compared to the A-GSC group (42.85.+ -. 3.64%), with significant statistical differences (P < 0.001) between the two groups. The wound healing rates of the W-GSC groups are 65.36 +/-4.47%, 80.69 +/-2.84% and 93.88+/-1.21% on the 8 th, 10 th and 15 th days after operation, and no statistical difference exists between the two groups. On postoperative day 18, the wound healing rate of the W-GSC group was 96.22.+ -. 0.33% compared to the A-GSC group (99.5.+ -. 0.57%), with statistical differences (P < 0.05) between the two groups. Thus, while injecting the cell-loaded hydrogel material at 0.5cm around the wound can also promote wound repair, it does not function as well as injecting the cell-loaded hydrogel microsphere material at the point of the wound. The wound repair effect of the full-layer defective skin is quickened by injecting the cell-carrying hydrogel microsphere material at the acupoint.

In order to compare the effect of injecting different materials at the acupoints on the wound repair efficiency of rats, the embodiment sets an SSAD hydrogel microsphere group (A-GSC) of injecting h-UcMSCs cells at the acupoints, a GelMA hydrogel microsphere group (A-GC) of injecting h-UcMSCs cells at the acupoints, an h-UcMSCs cell group (A-C) of injecting the acupoints, an SSAD hydrogel microsphere group (A-GS) of injecting the acupoints, and a buried line group (A-CE) of acupoints. On postoperative day 5, the wound healing rate of the A-GC group was 36.8.+ -. 0.13%, the wound healing rate of the A-C group was 34.23.+ -. 2.57%, the wound healing rate of the A-GS group was 28.23.+ -. 5.76% (P < 0.01), and the wound healing rate of the A-CE group was 25.94.+ -. 8.73% (P < 0.01) as compared to the A-GSC group (42.85.+ -. 3.64%). On day 8 after the operation, the wound healing rate of the A-GC group was 76.24.+ -. 1.47%, the wound healing rate of the A-C group was 65.91.+ -. 4.08%, the wound healing rate of the A-GS group was 61.67.+ -. 5.51% (P < 0.05), and the wound healing rate of the A-CE group was 55.81.+ -. 8.56% (P < 0.01) as compared with the A-GSC group (76.43.+ -. 2.08%). On the 10 th day after operation, the wound healing rate of the A-GC group was 86.31 + -1.86%, the wound healing rate of the A-C group was 82.44 + -1.92%, the wound healing rate of the A-GS group was 85.33+ -3.38%, and the wound healing rate of the A-CE group was 76.74+ -4.22% (P < 0.05) as compared with 88.14 + -0.83% of the A-GSC group. On day 15 after the operation, the wound healing rate of the A-GC group was 96.45.+ -. 1.26%, the wound healing rate of the A-C group was 95.97.+ -. 0.73%, the wound healing rate of the A-GS group was 94.51.+ -. 2.39%, and the wound healing rate of the A-CE group was 89.8.+ -. 3.31% (P < 0.001) as compared with the A-GSC group (97.46.+ -. 0.99%). 18 days after operation, the wound surface of the acupoint injection group is basically healed, compared with the A-GSC group (99.5+/-0.57%), the wound healing rate of the A-GC group is 98.57 +/-1.25%, the wound healing rate of the A-C group is 97.35+/-1.56%, the wound healing rate of the A-GS group is 99.02+/-0.6%, the wound healing rate of the A-CE group is 97.21 +/-0.22%, and no statistical difference exists among groups. By comparing injection of different materials at acupuncture points, the A-GSC group has the most remarkable effect of promoting wound repair, and the fastest wound closure is observed on a rat full-layer defect model; the A-GC group can also obviously promote wound healing, the healing speed is slightly lower than that of the A-GSC group (without statistical difference), and compared with the blank control group, the A-GC group has obvious difference in healing speed (P is less than 0.001, P is less than 0.01 or P is less than 0.05); the A-C group healing rate was not as good as that of the A-GSC group and the A-GC group (no statistical difference) using hydrogel microspheres as carriers, and the healing rate was significantly different (P < 0.001, P < 0.01 or P < 0.05) compared with the blank control group. Compared with a blank control group, the simple injection of the acellular hydrogel microspheres at the acupoint and the acupoint buried line also quicken the wound closure rate (P is less than 0.001, P is less than 0.01 or P is less than 0.05), but the effect is inferior to that of the group added with stem cells.

The present example also compares the wound healing rates of the W-GSC and W-C groups at different time points. In comparison to the W-GSC group (29.59+ -3.97%), the wound healing rates of W-C were 30.18+ -5.36%, 67.58+ -5.48%, 80.29 + -2.48%, 93.73 + -1.43%, 96.2+ -1.87 at postoperative days 5, 8, 10, 15 and 18, respectively. There were no statistical differences between the groups. The hydrogel microsphere has a large surface area to volume ratio, can effectively promote nutrition and moisture transfer, and improve the interaction between cells and a matrix, so that the long-term activity of the cells is maintained. As shown by in vitro experiments in the second embodiment, the hydrogel microsphere is used as a good biological scaffold, and provides a good platform for cell adhesion, growth and proliferation, so that the survival efficiency of cells in a complex in vivo microenvironment is improved.

Normal skin tissue samples at and around the wound healing site of the rat were collected on day 18 post-surgery, paraffin sections were prepared and subjected to H & E staining, masson trichromatic staining and CD 31/a-SMA immunofluorescence double staining. The wound surface of the A-GSC group and the A-GC group is completely healed on the 18 th day through H & E staining observation, the degree of re-epithelialization of the skin is high, mature fibroblasts are arranged below the epithelium, the newly-grown skin tissues are more mature, skin appendages such as hair follicles, sebaceous glands and the like are formed (fig. 7d and 7 f), wherein the A-GSC group forms 9+/-2.83 skin appendages, and the A-GC group forms 8+/-4.24 skin appendages; the rest group of wound surfaces are not completely healed at 18 days, the epithelium is discontinuous, a large amount of inflammatory cells infiltrate below the wound surfaces, granulation tissues at two sides continuously migrate to the center of the wound, and the number of newly-generated micro-blood vessels is large. The A-C groups had 4.5.+ -. 2.12 skin appendages, the A-GS, A-CE, W-GSC, W-C groups formed 1.5.+ -. 0.71, 2.5.+ -. 0.71, 1.5.+ -. 2.12, 2.5.+ -. 0.71 skin appendages, respectively, and no skin appendages were observed in the blank group. The relative epidermis thickness of each group of wound skin was measured and compared (fig. 7 c), and the results showed that the epidermis thickness was closest to the normal skin epidermis thickness (101.94 ±2.92%) at the newborn skin of the a-GSC group, and the epidermis of the blank group was significantly thickened (312.08 ±31.42%); in addition, the A-GC group skin thickness was 132.48.+ -. 17.34%, the A-C group skin thickness was 204.80.+ -. 39.30%, the A-GS group skin thickness was 186.57.+ -. 23.91%, the A-CE group skin thickness was 176.74.+ -. 6.7%, the W-GSC group skin thickness was 252.89.+ -. 46.4%, and the W-C group skin thickness was 260.73.+ -. 43.95%. Masson trichromatic staining of skin showed collagen neogenesis in each group of neogenesis skin, with blue collagen fibers and red muscle fibers (FIG. 7 g). The results show that the cell-loaded hydrogel microsphere acupoint injection groups (A-GSC group and A-GC group) are used for generating collagen deposition in a woven form, and the number of the novel collagen fibers in the groups A-GS, A-CE, W-GSC, W-C and Cont is small, so that the arrangement of the collagen fibers is disordered. This suggests that the a-GSC and a-GC groups have entered the remodeling stage of tissue repair at day 18 post-surgery, with the new skin tissue more mature, and the remaining groups of skin still in the proliferative phase of repair regeneration. Similarly, the relative scar rate results for each group of wound skin (fig. 7 e) show that the scar rate for the a-GSC group is the lowest, 25.64±1.32%; cont group was 40.46 + -2.24%, and scar rates of other groups were 33.45+ -1.18% (A-GC group), 34.02+ -2.76% (A-C group), 38.67+ -7.35% (A-GS group), 41.1+ -10.47% (A-CE group), 43.21 + -9.23% (W-GSC group), and 42.45+ -0.49% (W-C group), respectively.

CD31 is a vascular endothelial cell marker, a-SMA-labeled vascular wall cells, and newly generated blood vessels were localized using CD 31/a-SMA immunofluorescence double staining to analyze angiogenesis in regenerating skin tissue (fig. 7 h). The skin immunofluorescence staining result of the rat at 18 days after injury shows that a large number of positive cells are arranged below the wound surface of the blank group, which indicates that the wound surface of the blank group is not completely healed at 18 days after injury, a large number of blood vessels below the wound surface are regenerated to bring nutrition for tissue proliferation and repair, and granulation tissue is regenerated to replace the missing skin tissue; the wound surface of the cell-carrying hydrogel microsphere group injected at the acupoint is completely healed, and the least positive cells are observed in the visual field, because the tissue is completely re-epithelialized, the blood vessels and the cells gradually degenerate and disappear, and the skin repair enters the remodelling stage of the extracellular matrix. In the remaining groups, both the A-GS and A-CE groups observed less positive cells than the blank, and also less than the two groups injected periinvasively. The conditions of W-GSC and W-C angiogenesis were similar to those of the blank group. In other words, the A-GSC group and the A-GC group inject the cell-loaded hydrogel microsphere through the acupoint points, so that wound surface closure is quickened, skin enters a tissue remodelling stage earlier, a mature dermis structure is formed, and full-layer defect wound repair of rat skin is promoted.

SSAD hydrogel microspheres were implanted subcutaneously into SD rat skin to observe the biodegradation rate of the hydrogel microspheres. Hydrogel microspheres at 100 mg/injection site were implanted into the subcutaneous tissue of the left back of the rat, and an equal amount of SSAD hydrogel microspheres was injected into the subcutaneous tissue area of the right back of the same rat as the positive control. The degradation condition of the hydrogel microsphere in the body is observed after operation, and rats and materials are killed on the 5 th, 7 th and 9 th days after injection respectively. H & E staining results showed (fig. 8) that SSAD hydrogel microspheres were gradually hydrolyzed and broken over time until day 9, and that GelMA hydrogel microspheres were substantially absorbed and degraded, whereas GelMA hydrogel microspheres were visually observed to degrade at a slower rate than SSAD hydrogel microspheres, and by day 9 microspheres were still able to subcutaneously observe broken microspheres that were not absorbed completely, and the microspheres were not degraded completely. The experimental result shows that the SSAD hydrogel microsphere has better degradation capability.

In vivo biocompatibility detection

Assessing the long-term toxicity of SSAD hydrogel microspheres in vivo is an important factor in assessing the biocompatibility of the hydrogel microspheres. The SSAD hydrogel microsphere is implanted into the skin of an SD rat subcutaneously, no abnormal symptoms of the rat are observed during injection, and the phenomena of death of the rat and the like are avoided. Rats were sacrificed 9 days after injection and important organs such as heart, liver, spleen, lung, kidney, etc. were taken for histological observation. Compared with H & E stained sections of heart, liver, spleen, lung, kidney, etc. organs of normal rats, important organs of rats were not damaged organically nor systemically after SSAD hydrogel microsphere injection (fig. 9). The result is consistent with the result of the in-vitro biocompatibility detection of the SSAD hydrogel microsphere, and the SSAD hydrogel microsphere has good biosafety performance and is a suitable microcarrier for tissue regeneration.

Skin wound healing is an important step in completing wound closure as an important function of physical, chemical and bacterial barriers. Full-thickness wounds are characterized by complete destruction of the epidermis and dermis and deeper structures, often difficult to repair, and thus threatening the health and even life safety of the patient. Wound healing relies on a complex dynamic process involving interactions of many cell types, growth factors, cytokines and chemokines. Normal wound healing is divided into several phases: hemostasis, inflammation, proliferation and remodeling. Hemostasis is the first stage, starting immediately after the initial injury, to prevent blood loss and initiate clot formation. In the second phase, inflammatory cells are recruited to the site of injury to clear cell debris and initiate a cell signaling cascade to further heal the wound. During the proliferation phase, keratinocytes proliferate to close the wound, while myofibroblasts contract to reduce the wound size. Endothelial cells proliferate simultaneously throughout the stages of wound healing to re-vascularize damaged tissue. The last weeks to years, the extracellular matrix (ECM) remodels and forms scars. The proliferation phase generally begins on day 3 after injury for about 2 weeks. This stage is characterized by fibroblast migration and deposition of newly synthesized ECM to replace the temporary fibrin network. Fibroblasts and myofibroblasts in the surrounding tissue were stimulated to proliferate in the first 3 days, after which these cells migrated into the wound. Once in the wound, these cells proliferate in large quantities and produce the matrix proteins fibronectin, hyaluronic acid, and proteoglycans. By the end of the first week, a rich ECM accumulates, providing support for the wound and further supporting cell migration. The fibroblasts then transform into myofibroblasts, which initiate wound contraction during repair, helping to access the wound edges. Neovascularization is an important component of wound healing because it has a fundamental impact from the beginning after skin injury to the end of wound remodeling. The (micro) vascular system contributes to initial hemostasis, reduces blood loss and establishes a temporary wound matrix. Blood clot-derived cytokines and growth factors drive the recruitment of critical cells, which are critical in the healing process. This temporary wound microenvironment ensures nutrient perfusion of the wound and transport of immune cells, thereby removing cell debris. In the final stage of wound healing, the remodelling stage is responsible for the development of new epithelium and scar tissue formation, at which stage the ECM begins to mature. The diameter of the collagen bundles increases compared to the intact tissue, and as the wound matures, the density of fibroblasts and macrophages decreases due to apoptosis. Over time, capillary growth ceases, blood flow to the region decreases, and metabolic activity at the wound site decreases. The end result is a fully mature scar, reduced cell count, reduced blood vessels, and high tensile strength.

On the 18 th day after operation, the A-GSC and A-GC wounds are completely closed, re-epithelialization is realized, tissue repair enters a remodelling stage, blood vessels are gradually degenerated, and the expression quantity of CD31 and alpha-SMA is low; in Masson staining, the amount of collagen is high, while the amount of myofibroblasts is reduced, and collagen is deposited in a woven form. This effect appears to be closely related to stem cells, since in the cell-free injected group, the repair effect is much less than in the blank group, but not as involved as in the cell-free injected group. In addition to stem cells, stimulation of the hole sites is one of the important factors, and the wounds of the W-GSC group, the W-C group and the blank group are not completely closed at the 18 th day after operation, so that tissue repair is still in a proliferation stage, blood vessels are rich, the expression quantity of CD31 and alpha-SMA is high, the quantity of myofibroblasts is high, and collagen deposition is insufficient.

The inventor has found in previous experiments that giant salamander skin secretions (skin secretion ofAndrias davidianus, SSAD) can be made into hydrogels and have good adhesion. However, if cells are entrapped in bulk hydrogels, this tends to limit contact between cells and communication of nutrients and metabolites, and the rate of migration of cells from the hydrogel is slow, and the distance between extracellular molecules is less than optimal, resulting in poor nutrient exchange.

According to the invention, giant salamander Skin Secretion (SSAD) and GelMA are mixed to form a novel hydrogel, and the hydrogel is cut into uniform SSAD hydrogel microspheres by using microfluidic equipment. The microsphere has good biocompatibility and degradability. Based on the above, the mesenchymal stem cells are loaded on the microsphere to form the injectable cell-loaded hydrogel microsphere. The larger surface area to volume ratio of the hydrogel microspheres formed by the invention can promote effective nutrient and moisture transfer and improve the interaction between cells and a matrix, thereby maintaining the long-term activity of encapsulated cells.

The cell-carrying microsphere can promote the repair and regeneration of skin wound surfaces (especially full-layer wound defects) by injecting the cell-carrying microsphere at the acupoint points of the back and the hind limbs of a rat. The inventors have found that this effect of significantly promoting wound repair is related to two factors: first, point stimulation; secondly, the effect of cells loads stem cells on the basis of the long-term stimulation of the points of the acupoints, so that the wound surface repairing efficiency is higher, the repairing effect is better, and the repairing effect is better than that of the conventional point-buried line or periwound injection of cells/medicines. In other words, the cell-loaded hydrogel microsphere (for example, SSAD h-UcMSCs hydrogel microsphere) provided by the invention is used as an injectable micro-biological scaffold with good performance, and can be delivered to acupoints through an injection needle and stimulate the acupoints for a long time, so that the repair of skin wounds can be better promoted.

Example IV

GelMA microspheres and SSAD hydrogel microspheres containing different SSAD volume ratios (10%, 30% and 50%) are placed on the joint surface of a rabbit knee joint, the microspheres on the joint surface are subjected to constant-speed flushing by using a 1mL syringe with PBS, each flushing time is 30 seconds, the flushing amount is 1mL, and whether the microspheres can adhere to the joint surface or not is observed in the flushing process.

Experimental results

As shown in fig. 10, gelMA microspheres were completely washed away from the articular surface with PBS within 30 seconds; the microspheres containing the SSAD component could not be completely washed out by PBS, wherein 30% of the microspheres (30% ssad@gelma, volume ratio of aqueous solution of gelma (10% concentration) to SSAD hydrolysate (10% concentration) was 7:3) and hardly lost after washing, 10% of the microspheres (10% ssad@gelma, volume ratio of aqueous solution of gelma (10% concentration) to SSAD hydrolysate (10% concentration) was 9:1) and some of the microspheres lost after washing, and 50% of the microspheres (50% ssad@gelma, volume ratio of gelma aqueous solution (10% concentration) to SSAD hydrolysate (10% concentration) was 1:1), the remaining amount of the microspheres was between 10% of SSAD and 30% of microspheres.

The above experiments show that microspheres containing SSAD components can adhere to tissue surfaces (the articular surfaces are examples in this example) and can withstand a degree of fluid washout (e.g., joint fluid in the joint cavity) as compared to conventional hydrogel microspheres (e.g., gelMA microspheres). Conventional hydrogel microspheres cannot withstand the scouring of flowing liquid, and in practical application, the conventional hydrogel microspheres may cause tissue adhesion under the action of the flowing liquid.

Example five

6-week-old nude mice were selected and anesthetized with isoflurane gas. Microspheres loaded with dental pulp stem cells (for example, 30% ssad@gelma) were injected into the pulp cavity portion, and after 3 months, nude mice were sacrificed and pulp cavity tissues were extracted.

Paraffin wax slicing step: (1) the above collected tissues were fixed in a 4% paraformaldehyde solution for 24 hours. (2) The solution was rinsed overnight under running water and fixed in 70% ethanol for a long period of time. (3) The tissue is dehydrated and transparent in gradient in a full-automatic dehydrator and is immersed in wax. (4) Embedding by using paraffin embedding machine, placing on a refrigerator, and demoulding after the paraffin is solidified. (5) Preserving in a refrigerator at 4 ℃. (6) The skin tissue pieces were cut into tissue sections with a thickness of 7 microns under a paraffin microtome.

H&E, dyeing: (1) the skin tissue sections were baked in a 60 ℃ oven for 2 hours. (2) Dewaxing was performed in xylene I and II for 8min each. (3) Hydrating in ethanol according to concentration gradient, sequentially obtaining absolute ethanol I and absolute ethanol II for 5min respectively; 95% ethanol for 2min;90% ethanol for 2min;85% ethanol for 2min;75% ethanol for 2min. (4) Rehydrating in ultrapure water for 2min. (5) Hematoxylin dye solution is used for dying cell nuclei for 5min. (6) Washing with running water for 2min. (7) The eosin dye solution dyes the cytoplasm for 2min. (8) Washing with running water for 2min. (9) Dehydrating in ethanol according to ascending concentration gradient, wherein the ethanol is 80% ethanol for 15s in turn; 90% ethanol for 15s;100% absolute ethanol for 15s. Transparent in xylene for 2min.Neutral resin seals, after complete drying, sections were scanned using Olympus VS 200.

Experimental results

As shown in fig. 11, the stem cell loaded microspheres form a certain soft tissue in the pulp chamber, which is similar to pulp tissue. In addition, regenerated tissue was found to form a large amount of fresh dentin by tissue sections. The above experiments show that microspheres loaded with stem cells can be implanted into the pulp cavity for tissue regeneration.

Summarizing:

according to the invention, SSAD hydrolysate is added into GelMA hydrogel, and the synthesized SSAD hydrogel is prepared into monodisperse microspheres with uniform diameters by utilizing a microfluidic technology. After the SSAD is added, the hydrogel microspheres form a rough surface with certain viscosity, and the surface is favorable for the adhesion of cells on the surface of the hydrogel microspheres, can be stably attached on the surface of tissues and can resist the scouring of flowing liquid. In addition, compared with the conventional hydrogel microsphere, the hydrogel microsphere provided by the invention has the effect of promoting cell proliferation, and has the advantages of faster degradation rate in vivo and reduced inflammatory response caused by local part.

The hydrogel microsphere prepared by the invention has good biocompatibility and degradability. SSAD carried cell hydrogel microspheres formed by loading human umbilical cord mesenchymal stem cells on microspheres by injecting on the acupoints of the back, the spleen, the kidney, the Zusanli and the like of rats, wherein the rats of the group show the fastest wound surface closing rate, are favorable for forming skin appendages on dermis layers, promote the deposition of extracellular matrixes such as collagen and the like on damaged parts, and enable the repaired and regenerated tissues to be more approximate to normal skin tissues.

The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.

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Claims

1. A method for preparing hydrogel microspheres, the method comprising:

s1, dispersing methacryloylated gelatin and a photoinitiator in water and heating to obtain a first gel solution; s2, adding the giant salamander skin secretion hydrolysate into the first gel solution to obtain a second gel solution, wherein the volume ratio of the first gel solution to the giant salamander skin secretion hydrolysate comprises 9:1-2:3;

2. The method of claim 1, further comprising S5 co-culturing stem cells with the hydrogel microspheres to obtain the cell-loaded hydrogel microspheres.

3. The method of claim 1, wherein the concentration of the methacryloylated gelatin comprises 10% to 12.5% (w/v); the oil phase solution comprises mineral oil and sorbitan oleate.

4. The method of claim 1, wherein the photoinitiator comprises lithium or ruthenium (Ru) phenyl-2, 4, 6-trimethylbenzoyl phosphite, and the concentration of the photoinitiator comprises 0.25% (w/v).

5. The method of claim 1, wherein the concentration of giant salamander skin secretion hydrolysate comprises 5% to 10% (w/v).

6. The method of claim 2, further comprising the step of removing residual oil phase solution from the hydrogel microspheres, said step being performed after S4 and before S5.

7. The method of claim 1, wherein the second gel solution sample injection rate in S3 comprises 20 μl/min and the oil phase solution sample injection rate comprises 400-700 μl/min.

8. The method of claim 2, wherein the stem cells comprise one or more of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose stem cells, and dental pulp stem cells.

9. Hydrogel microspheres obtainable by the process according to any one of claims 1 to 8.

10. Use of the hydrogel microspheres of claim 9 in the preparation of an injectable formulation for promoting tissue regeneration.

11. The use of claim 10, wherein the tissue comprises one or more of skin, bone, and dental pulp.

12. The use according to claim 10, wherein the injectable formulation is for one or more of subcutaneous injection, intra-articular injection and intra-dental pulp injection.

13. Use of the hydrogel microspheres of claim 9 for the preparation of a subcutaneous injection for point site injection, wherein the subcutaneous injection is used to promote skin wound healing.

14. The use according to claim 13, wherein the acupoints comprise behens, behens and zu sanli.

15. The use of claim 13, wherein the wound comprises a full layer wound.