CN116077717B - Bioburden system for wound healing and application thereof - Google Patents

Abstract

The invention relates to the field of biomedical materials, in particular to a bioburden system for wound healing and application thereof. The invention provides a bioburden system for wound healing, which comprises at least two gel layers and a tissue particle layer positioned between the at least two gel layers, wherein the gel layers comprise a mixture of giant salamander skin secretion freeze-dried powder and a solvent; the tissue microparticles comprise amniotic membrane microparticles.

Description

Bioburden system for wound healing and application thereof

Priority application

The present application claims priority to the chinese patent application filed 11/05 of 2021 [ CN2021113053740 ], entitled bioburden system of giant salamander-containing skin secretions, preparation method and use thereof, which is incorporated by reference in its entirety.

Technical Field

The invention relates to the field of biomedical materials, in particular to a bioburden system for wound healing and application thereof.

Background

The skin is the largest organ of the human body and its main function is to protect internal organs and tissues from external wounds and pathogen infection, sense and regulate body temperature. The skin comprises epidermis, dermis, subcutaneous tissue. The outermost layer of the skin, the epidermis, is avascular and is largely divided into basal, acanthal, granular, transparent and stratum corneum. Since the epidermis epithelium is avascular, the nutrient supply of the epidermis is dependent on highly vascularized dermis. The dermis is mainly composed of fibroblasts and macrophages. In addition, dermis also contains nerve endings, lymphatic vessels, collagen and connective tissue. Depending on the extent (or depth) to which the skin is damaged, wounds can be divided into partial cortical wounds and full-thickness wounds. Partial cortical wounds involve only the epidermis and/or the superficial dermis, with no damage to the dermal blood vessels. Whereas a full-thickness wound involves disruption of the dermis and extends to deeper layers of tissue, involving disruption of dermal blood vessels. Healing of partial cortical wounds occurs through simple regeneration of epithelial tissue, whereas wound healing of wounds of full-thickness skin defects is more complex.

Burns and chronic wounds affect over 1100 and 650 tens of thousands of people, respectively, each year; it was estimated that medical costs could rise to $224 billion by 2024. Unfortunately, large-area skin wounds or burns often face scarring after treatment, and disordered collagen fibers can impede regeneration of hair follicles, sebaceous glands, and sweat glands. This fact not only affects the appearance and leads to mental diseases, but also to serious physiological dysfunctions such as impeding joint movement, sweat secretion and skin temperature regulation. For wounds (traumatic) or burn wounds, the clinically usual treatment is autologous skin grafting, which further gives secondary damage to the patient, making it especially unsuitable for large-area wounds. On the other hand, xenografts (xenograft) are always at risk of immune rejection and disease transmission.

Over the past several decades, many wound dressings have emerged to improve the outcome of skin repair, such as dressings that prevent infection and inflammation and dressings that deliver bioactive substances (e.g., growth factors). However, the general success that these dressings have reported is to accelerate wound closure, rarely involving regeneration of the skin appendages. But the skin appendages play an important role in skin function, and their growth is also one of the hallmarks of functional, scar-free skin regeneration.

Cell therapy appears to be a good choice for regeneration of skin appendages. Cell therapy refers to the use of the characteristics of certain cells with specific functions, and the cells have the therapeutic effects of promoting tissue and organ regeneration, organism rehabilitation and the like after being obtained by a bioengineering method and/or being treated by in vitro amplification, special culture and the like. For regeneration engineering applications, cells are typically used in combination with programmable biological materials, or as whole cultured cell sheets without scaffolds (complete cultured cell sheets). However, scaffolds loaded with cells or cell patches involve isolation and culture of cells. In addition, it often takes several weeks to culture and expand cells tailored for each patient, which can be extremely costly (including time and money).

Thus, an ideal skin repair material should not only be cost-effective and readily available, but also be able to promote complete skin healing in a scar-free manner while achieving regeneration of the skin appendages.

Disclosure of Invention

In view of this, in one aspect, the present invention provides a bioburden system for wound healing comprising at least two gel layers and a layer of tissue microparticles positioned between the at least two gel layers, the gel layers comprising a mixture of giant salamander skin secretion lyophilized powder and a solvent; the tissue microparticles comprise amniotic membrane microparticles.

In one embodiment of the invention, the granularity of the giant salamander skin secretion lyophilized powder is 20-300 meshes.

In one embodiment of the invention, the granularity of the giant salamander skin secretion lyophilized powder is 60 meshes.

In one embodiment of the invention, the tissue microparticles range in size from about 50 to 300 μm.

In one embodiment of the invention, the tissue microparticles are about 200 μm in size.

In one embodiment of the invention, the giant salamander comprises one or more of the genera giant salamander, cryptobranchia, mangrove, andrias, and polar giant salamander.

On the other hand, the invention also provides application of the bioburden system in wound repair.

In one embodiment of the invention, the wound is a skin wound.

In one embodiment of the invention, the wound is a full-thickness wound.

In one embodiment of the invention, the bioburden system promotes regeneration and/or vascularization of skin appendages and/or remodeling of extracellular matrix at the wound site.

In one embodiment of the invention, the bioburden system promotes recruitment of interfollicular epithelial stem cells to the wound.

In one embodiment of the invention, the bioburden system promotes proliferation and/or migration of keratinocytes.

In one embodiment of the invention, the bioburden system promotes healing of the wound.

In one embodiment of the invention, the healing is scar-free healing.

As used herein, "bioburden system" includes hydrogels of biological origin and tissue microparticles of biological origin. The hydrogel of biological origin is a hydrogel generated by freeze-dried powder of skin secretion of giant salamander and a solvent, and is preferably giant salamander Skin Secretion (SSAD) hydrogel. The tissue microparticles comprise living cells, preferably amniotic membrane microparticles. The bioburden system can be applied to the wound in the following modes: wound-biogenic hydrogel-biogenic tissue microparticles-biogenic hydrogel (in order from the wound end). In one embodiment of the invention, SSAD hydrogels can form strong interactions with wounds via hydrogen bonding, van der waals forces, pi-pi electrons, or cation-pi interactions, and can interact with amniotic particles to form strong adhesion, thereby acting as carrier scaffolds to support the activity of the amniotic particles. The giant salamander skin secretion freeze-dried powder can be any liquid which can enable the giant salamander skin secretion freeze-dried powder to be stably glued and is harmless to tissue particles because the giant salamander skin secretion freeze-dried powder is glued when meeting water. Specifically, the solvent should be a reagent that does not contain a strong reducing agent and a strong acid, that is, reagents commonly used in clinic can gel the lyophilized powder of the skin secretion of the giant salamander. Exemplary solvents may be buffers such as physiological saline, purified water, fluids such as wound fluids, and cell culture media. The solvents used for the different gel layers may be the same or different.

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 major components of the bioburden system of the present invention, namely the bioderived hydrogel and the bioderived tissue microparticles, are not obtained by genetic recombination techniques. However, the bioburden system may include a polypeptide obtained by gene recombination.

As used herein, "wound" refers to a physical disruption of continuity or integrity of a tissue structure. "wound healing" refers to the partial or complete restoration of tissue integrity. In one embodiment, 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.

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

As used herein, "scar" refers to dermal tissue, typically including fibrous tissue, formed as a result of wound healing. Scars may be caused by "wounds". "scar-free healing" refers to less, significantly less or no scar formed by natural healing of a wound.

As used herein, "synergistic effect" is a result that indicates significantly greater than the effect expected from each component applied to a wound alone. In one embodiment of the invention, the SSAD hydrogel in the bioburden system provided by the invention has a synergistic effect with amniotic membrane microparticles.

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, "promoting" refers to a further increase in the described subject at existing levels, including one or more of quantitative levels, expression levels, functional levels, and competence levels.

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 an experimental graph of the characteristics of a material and the effect of SSAD on MA bioactivity;

FIG. 2 is an experimental graph of the effect of SSAD on HaCaT cell activity;

FIG. 3 is an experimental graph of the effect of SA on skin wound healing in vivo;

FIG. 4 is an experimental plot of immunofluorescent staining and quantification results of regenerated skin tissue;

FIG. 5 is an experimental diagram summarizing single cell transcriptome analysis;

FIG. 6 is an experimental plot of additional analysis of IFECs;

fig. 7 is a schematic diagram showing the proposed SA-promoted skin wound healing process;

FIG. 8 is a SEM image showing the adhesion interface between SSAD hydrogels derived from different mesh powders loaded with MA (blue pentagram for SSAD hydrogels, red triangle for MA);

FIG. 9 is an experimental plot of TUNEL detection of MA and SA (nuclei stained blue, apoptotic cells stained green);

FIG. 10 is an experimental plot of the effect of SSAD on AMSCs in vitro;

FIG. 11 is an experimental view of the therapeutic mechanism of SA for in vivo wound healing 35 days after injury;

FIG. 12 is a graph showing the results of flow cytometric analysis of integrin- β1 positive cell rates for SSAD and blank;

FIG. 13 is an experimental plot of quality control of scRNAseq data;

FIG. 14 is an experimental plot of cell division into 20 populations using unsupervised clustering of semat based on global gene expression patterns;

FIG. 15 is an experimental plot of cell division into 20 populations using unsupervised clustering of semat based on global gene expression patterns;

FIG. 16 is a violin diagram showing the expression level and distribution of marker genes of IFECs;

FIG. 17 is an experimental plot of GO and KEGG analysis of IFESCs compared to other cell populations of the control and SSAD groups;

FIG. 18 is a graph of an experimental GO and KEGG analysis of IFESCs of the SSAD group;

FIG. 19 is an experimental plot of En-1 immunofluorescent staining for reticulocytes on day 35 post-surgery;

FIG. 20 is an experimental plot of regeneration of skin appendages at various time points of the SA group;

FIG. 21 is a graph of experiments assessing in vivo toxic effects of different groups on heart, liver, spleen, lung and kidney on day 35 post-injury;

FIG. 22 is an experimental diagram for preparing MA;

FIG. 23 is an experimental chart showing the processing procedure of each experimental group;

fig. 24 is a graph showing a method for quantifying the percent scar per sample (black arrow indicates wound boundary at baseline, black vertical line indicates fully regenerated skin tissue).

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, a range The description of (c) should be taken as having specifically disclosed sub-ranges 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 such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.

Detailed description of the drawings

Fig. 1: a is SSAD powder. B is SSAD hydrogel. C is fresh amniotic membrane. D and E are HE stained images of the longitudinal sections of (D) amniotic membrane surface and (E) MA, where blue circles represent amniotic epithelial cells and purple triangles represent basement membrane. F is an SEM image showing the adhesion interface between SSAD hydrogel and MA, where blue pentads represent SSAD hydrogel and red triangles represent MA. G is the cell viability of MA (MA cultured in normal medium or SSAD conditioned medium) measured by live/dead cell staining, where live cells are stained green and dead cells are stained red. H is the viable cell rate quantified based on the viable/dead cell staining image. TUNEL assay, where I is MA and SA, where nuclei are stained blue and apoptotic cells are stained green. J is the number of apoptotic cells quantified based on TUNEL detection images.

Fig. 2: a and B are the scratch test (A) and the transwell migration test (B) of HaCaT cells. C is a quantification of the wound healing rate of HaCaT cells in the scratch test. D is a quantification of the relative cell number of HaCaT cells in the transwell assay. E is a CCK8 assay showing proliferation of HaCaT cells at 24 h. F is the venn plot of transcriptome profile between control and SSAD groups. G is a volcanic chart showing the identified up-and down-regulated genes induced by SSAD conditioned medium. H is a heat map of genes involved in significant upregulation of proliferation, migration and apoptosis of cells in both groups (fold change. Gtoreq.2, P < 0.05). I is a KEGG pathway enrichment analysis of the identified DEGs, the panel (panel) shows the 20 most significantly enriched pathways.

Fig. 3: a is a representative graph showing the wound healing of different groups, with blue scale (scales) diameter: 20mm. B is a quantification of wound closure rate for the different groups (n=4; < 0.001). C is a representative HE staining of skin tissue at day 35 post-injury to observe overall morphology, where black triangles represent wound boundaries, BV: vascular, HF: hair follicle, SG: sebaceous glands. D is Masson's trichromatic staining of skin tissue at day 35 post-injury to observe collagen fiber neogenesis, alignment, and maturity (reorganization). E is a quantification of the relative skin thickness at the wound center for the different groups on day 35 after injury. F is a quantification of the number of hair follicles in the different groups on day 35 after injury. G is a quantification of the number of sebaceous glands of the different groups on day 35 after injury. H is a quantification of scar rate for the different groups on day 35 after injury (n=3; < 0.001).

Fig. 4: a is IFESC staining (brown) on days 7 and 14. B is staining of new blood vessels (red circles) on day 14 and day 35. C is staining of Sebaceous Glands (SG) and Hair Follicles (HF) on day 35. D is staining of adipocytes (green) on day 35, in all cases nuclei were counter stained blue. E is a quantification of the density of IFESCs on days 7 and 14. F is a quantification of the number of vascular rings on day 14 and day 35. G is a quantification of the number of hair follicles on day 35. H is a quantification of the number of sebaceous glands on day 35. Adipocytes (PLNI 1) at day 35 ⁺ ) Is a percentage of (n=3; * P:<0.001)。

fig. 5: a is the workflow of a single cell experiment (post-wound, PW). B is the division of cells from the wound into 8 main categories, the middle column indicates that the percentages of cells of the 8 categories are average out of all samples analyzed, and the composition (%) of cells in the control and SSAD groups are listed on the right. C is a heat map showing marker gene expression of cells in 20 clusters and 8 categories. D is an up-and down-regulated gene in the IFECs population, IFESC: interfollicular epidermal stem cells, BC: basal cells, SC: a spine cell; KERs: keratinocytes.

Fig. 6: a is a sub-population (subspectors) of IFECs showing 5 sub-populations. B is the cell distribution of IFECs in the control and SSAD groups. C is the fraction (fractions) of the different subgroups of IFECs in the control and SSAD groups. D is the number of different subgroups of IFECs in the SSAD group. E is a correlation analysis of the IFEC subpopulation. F is the expression level of the selection genes (selection genes) which are enriched in IFESCs, BCs, SCs, KERs and KERs 2, respectively. G is a violin plot showing marker gene expression for different IFEC subpopulations. H is the distribution of cells on a pseudo-trajectory, the darker the color, the earlier the period of cell differentiation. I is the pseudo-temporal order of the IFECs (pseudotemporal ordering) and the distribution of 5 subsets along the trajectory. J is a heat map of gene expression of the first 50 DEGs (organized into 4 clusters) in pseudo-temporal order, KERs shown at the bottom and SCs and BCs shown at the top.

Fig. 7: SA can recruit IFESCs and enhance regeneration of blood vessels, hair follicles, and sebaceous glands; regeneration of skin appendages is closely related to scar-free skin regeneration.

Fig. 10: a is SSAD hydrogel of different meshes for detecting MA and loading MA by a TUNEL method, cell nuclei are blue, and apoptotic cells are green. B and C are scratch experiments and transwell migration experiments of AMSCs. D is a quantification of wound healing rate of AMSCs in scratch experiments. E is the quantification of the relative cell number of AMSCs in the transwell method. F is CCK8 assay showing proliferation of AMSCs on different days. G is a transcriptome analysis that shows genes in the MAPK signaling pathway are up-and down-regulated.

Fig. 11: a is a comparison of the number of genes up-regulated and down-regulated between the different groups. B is a Venn diagram comparing transcript spectra between different groups. C is a heat map of genes involved in significant upregulation of wound healing processes following SA treatment (fold change. Gtoreq.2, P < 0.05). D is KEGG pathway enrichment analysis shows 20 pathways with important biological significance. E is GO term that the genes co-expressed in the control, MA, and SSAD groups, vs. SA groups, are enriched in biological processes.

Fig. 12: se:Sub>A and E are gating of all events using side scatter regions (side scatter arese:Sub>A, SSC-se:Sub>A) and side scatter heights (side scatterheight, SSC-H) to eliminate the double P1. B and F are based on size and internal complexity, forward scattered light region (forward scatter arese:Sub>A, FSC-se:Sub>A) and side scattered light region (SSC-se:Sub>A) gating to select viable cell objects (gate P2). C and G are nuclear single cells (gate P3) expressing CD29 using phycoerythrin channel region (PE-A) and SSC-A gating. Gating on the groups D and H (Gating hierarchy ofpopulations) showed a control CD29 positive cell rate of 17.2% and SSAD of 27.1% and sorted CD29+ cells for downstream scRNA-Seq.

Fig. 17: a is GO enrichment analysis of the DEGs of the IFESC population. B is KEGG enrichment analysis of the IFESC population.

Fig. 18: a is GO enrichment analysis of the DEGs of the IFESC population of the SSAD group. B is the KEGG path of the ifec population of SSAD groups.

Fig. 22: a is fresh placenta. B is the amniotic membrane peeled from the placenta. C is fresh MA.

Fig. 23: i is 1mLMA for each wound surface of MA group. ii and iii each wound surface of the SSAD group was covered with 100mg SSAD powder and mixed with 300 μl of physiological saline (iii). iv-vi each wound surface of the SA group was covered with 50mg of SSAD powder, (v) mixed with 150. Mu.L of physiological saline, (vi) mixed with 1mLMA, vi was covered with 50mg of SSAD powder and finally mixed with 150. Mu.L of physiological saline.

Embodiment one: materials and methods

1.1 summary of the experiments of the invention: one of the objects of the present invention is to prepare SSAD hydrogels of different mesh sizes of loaded amniotic membrane Microparticles (MA) for scar-free healing of skin tissue. The invention uses live dead cell staining and TUNEL experiments to detect toxicity of SSAD hydrogels of different mesh sizes to amniotic membrane Microparticles (MA). The scratch and Transwell experiments prove that the SSAD hydrogel can promote the proliferation of HaCat cells, and the CCK8 experiments prove that the SSAD hydrogel can promote the migration of HaCat cells. A full-thickness skin defect model (full-thickness skin defect model) was created on the back of the rat and the skin tissue was observed for scar-free healing by staining tissue sections. Finally, the mechanisms of SSAD hydrogels to promote scar-free healing were explored using ex vivo transcriptome sequencing of HaCat cells, transcriptome sequencing of rat skin tissue, and single cell sequencing. The sample size (including the number of rats per group) was selected based on historical data to ensure that the data was adequate. All rats were male to eliminate any potential confounding effects of sex differences. All rats were randomly assigned to groups.

1.2 Preparation of SSAD hydrogels: washing Chinese giant salamander with clear water to remove molt. By gently scratching the back skin, the animals secrete mucus. Collecting mucus, purifying, freeze drying, grinding to obtain SSAD powder. The SSAD powder was prepared by milling into different mesh sizes, namely 20 mesh, 60 mesh and 300 mesh. The SSAD powder was stored at-20 ℃ until further use. The SSAD powder forms a hydrogel upon exposure to water.

1.3 Preparation of MA: after informed consent was obtained, fresh human placenta (fig. 22A) was obtained directly from the parturient receiving caesarean section. Serological tests showed that all donors had human beingsImmunodeficiency virus type I or II, human hepatitis b or c, or syphilis are seronegative. All procedures were performed under sterile conditions. After the amniotic membrane was removed from the placenta (FIG. 22B), the placenta was sterilized with a kit containing 100U mL ^-1 The amniotic membrane was thoroughly washed with sterile phosphate buffered saline of penicillin and streptomycin to remove blood residues. The amniotic membrane was minced with a four-leaf electric pulverizer, and then filtered through a metal mesh filter, to obtain MA fragments having a fineness of about 200 μm as shown in FIG. 22C. Of course, amniotic membrane particles having a size in the range of about 50-300 μm are suitable. After loading the SSAD hydrogel with MA, the samples were fixed with 4% glutaraldehyde for 24 hours, dehydrated with gradient ethanol, and then observed at 10kV with a scanning electron microscope SEM (FEI/Philips).

1.4 Cell viability test of MA: fresh MA was cultured in normal medium or SSAD conditioned medium. Cell viability of MA was measured using a fluorescence live/dead detection kit (Solarbic) according to the manufacturer's instructions. Living cells were stained green with calcein-AM and dead cells were stained red with propidium iodide. The numbers of dead and living cells were counted under a fluorescence microscope (Carl-Zeiss). Three fields of view are selected for each sample. Fresh MA served as control. Cell viability was calculated according to equation (1).

Wherein N is _{Living body} Is the number of living cells, N _{Total (S)} Is the total number of cells.

1.5TUNEL detection: to observe early apoptosis of cells in living MA, an in vitro model was used to simulate the treatment of skin wound defects. The samples were divided into control MA group (no special treatment) and experimental SA group (SSAD hydrogel loaded with MA). Sample culture at 37℃in 5% CO ₂ . At various time points, frozen sections were obtained and stained with TUNEL detection kit (Roche) according to the manufacturer's instructions. The experimental results were observed under a fluorescence microscope (Olympus) and photographed. Count the number of apoptotic cells and select for each sample4 fields of view.

1.6 cell proliferation assay: at various time points after inoculation of AMSCs cells (Shaanxi Stem cell engineering Co., ltd.) and HaCaT cells (Mengbio), the cells were inoculated with 5X 10 cells ³ Individual cells/well AMSCs or HaCaT cell suspensions, covered 96-well plates. After cell adhesion, 100. Mu.L of SSAD conditioned medium (0.5 mg. Multidot.mL) was transferred to each well using a 96-well plate with a 100. Mu.L suction nozzle ^-1 ) Or normal medium. At various time points, they were incubated for 3h in fresh medium containing 10. Mu.L/100. Mu.L of cell counting kit-8 (Beyotime). Proliferation was quantified by measuring absorbance at 450nm using a microplate reader (PerkinElmer).

1.7 scratch and transwell test：

1.7.1 scratch test: AMSCs cells and HaCaT cells were individually seeded into 6-well cell culture plates (Solarbio) at a density of 5X 10 per well ⁵ Individual cells. Once the cells reached confluence, the center of each well was streaked with 200. Mu.L of sterile pipette tips and the medium was replaced with fresh SSAD conditioned medium (0.5 mg mL-1). Normal medium served as control. After 24h incubation, closure of the scratches was captured with an inverted microscope (Carl-Zeiss) and 3 representative images of each scratch area were captured and analyzed. The result is expressed as a wound healing rate according to formula (2).

Wherein S is _{Initial initiation} Is the initial wound size, S _{Currently, the method is that} Is the current wound size. Each test was performed 3 times.

1.7.2transwell test : transwell chambers were used according to the manufacturer's protocol. AMSCs and HaCaT cells were inoculated into the upper chamber, respectively, and the lower chamber was filled with normal medium or SSAD conditioned medium (0.5 mgmL ^-1 ). After 24h of incubation, the migrated cells were fixed and stained with 0.1wt% crystal violet. The result of transwell is expressed as relative cell number according to equation (3).

Wherein N is _SSAD Is the average number of migrating cells of the SSAD group, N _Control Is the average number of migrating cells in the control group.

1.8 Transcriptome analysis of HaCaT cells: to analyze the effect of SSAD on the most important cells in the skin, transcriptome analysis was performed on SSAD treated HaCaT cells in vitro. Will be 5X 10 ⁶ Each HaCaT cell was seeded on a 6cm cell culture plate and cultured under SSAD conditions (0.5 mg mL ^-1 ) Or high sugar DMEM treatment, and collecting cells after 24 hours. Total RNA of HaCaT cells was prepared using Trizol reagent (Takala) according to the manufacturer's instructions. RNA concentration and purity were measured using NanoDrop 2000 (Thermo Fisher Scientific). RNA integrity was assessed using the RNANano 6000assay kit of Agilent Bioanalyzer 2100 System (Agilent Technologies). Index encoded samples were clustered at cBot ClusterGeneration System using TruSeqPE ClusterKitv-cBaot-HS (Illumina) according to manufacturer's instructions. After cluster generation, library preparations were sequenced on the Illumina platform and double-ended reads were generated.

1.9 in vivo wound healing study:32 male SD rats (6-8 weeks old; 200±20 g) were randomly divided into control group (n=8), fresh MA group (n=8), SSAD hydrogel group (n=8) and SA group (n=8). After anesthesia with 1% pentobarbital sodium (intraperitoneal injection), two full-thickness resected skin wounds (diameter: 20 mm) were created on the back of each rat. As shown in fig. 23, each wound of the control group was covered with gauze; each wound of the MA group was covered with a layer of MA (1 mL) and petrolatum gauze; each wound of the SSAD group was covered with one layer of SSAD hydrogel (100 mg); each wound of the SA group was covered with one layer of SSAD hydrogel (50 mg), one layer of MA (1 mL) and the other layer of SSAD hydrogel (50 mg). To observe the wound healing process, the wound was monitored and images were captured by digital cameras on days 0, 7, 14, 21 and 35. Using ImageJ software (National Institutes ofHealth), the wound closure rate was calculated according to the following equation (4).

Wherein S is _{Initial initiation} Is the initial wound size, S _{Currently, the method is that} Is the current wound size.

Skin samples were harvested on day 35 post-surgery. Images of HE staining and Masson's trichromatic staining were obtained using a frontal microscope (Caro-Zeiss). All quantification was performed using ImageJ and OlyVIA software (Olympus). First, the relative thicknesses of all experimental groups were counted by OlyVIA software according to the following formula (5), and calculated by HE staining image of the whole slide. Fig. 24 (black arrow indicates wound border at baseline, black vertical line indicates fully regenerated skin tissue) visually shows the calculation of scar percentage. Scoring was also confirmed by researchers not having knowledge of the experimental study design of the present invention.

Wherein S is _{Base line} Is bounded by broken muscles S _{Currently, the method is that} Is bordered by the skin that is currently being regenerated. Newly formed skin appendages on the wound surface were also counted and their densities calculated.

To characterize stem cell recruitment in different groups, IFESCs cells were labeled with integrin- α2 and integrin- β1. To assess the number of new blood vessels during wound healing, anti-alpha-SMA staining was used for myofibroblasts. To assess the number of skin appendages in the wound healing model, sebaceous gland cells were stained with rabbit polyclonal anti-CK 14 primary antibody (Abcam), hair follicle stem cells were stained with mouse monoclonal anti-CK 19 primary antibody (Abcam). To assess the regeneration of adipocytes around the neonatal follicle, adipocytes were stained with anti-PLIN 1 primary antibody (Abcam). Then, goat anti-rabbit IgG-Alexa Fluor 647 secondary antibody was applied. The samples were further counter-stained for nuclei with 4', 6-diamidino-2-phenylindole (Vector Laboratories). Cells stained positively were counted. Histological images (3 fields per section) were examined with a fluorescence microscope (Carl-Zeiss).

1.10 transcriptome analysis of wound-healing SD rats: SD rats with established skin defects were randomly divided into four groups: control, MA, SSAD, and SA. 35 days after the wound, the rats were sacrificed to collect skin. Total RNA of skin tissue was prepared using Trizol reagent (Takala) according to the manufacturer's instructions. Total RNA was then identified using Nano Drop and Agilent 2100bioanalyzer (Thermo Fisher Scientific). After reverse transcription, cDNA is synthesized. The product was checked for quality control on an Agilent 2100 bioanalyzer. The double-stranded PCR product obtained in the previous step is heat denatured and circularized by a splint oligonucleotide sequence to obtain the final library. The single stranded circular DNA is formatted into a final library. The final library was amplified with phi29 to generate DNA Nanospheres (DNBs) with more than 300 copies of one molecule. DNB was loaded onto the patterned nanoarray to produce single ended 50 base reads on the BGIseq500 platform (BGI-Shenzhen).

1.11 Single cell transcriptome analysis: skin samples were obtained by cutting off the skin at the wound (circular, diameter = 20 mm). Subcutaneous tissue was removed and 3 samples were harvested per group. Using a collagen-containing enzyme I (1 mg. ML) ^-1 ) Collagenase IV (1 mg. ML) ^-1 ) Disperse enzyme (1 mg. ML) ^-1 ) And trypsin-EDTA (0.125%) (Sigma-Aldrich) and digested skin tissue at 37℃for 1h. Positive cells were then enriched with integrin- β1 antibodies. Finally, 10×genomics Single-Cell Protocol was performed. Single cell FASTQ sequencing reads for each sample were processed and compared to the reference genome and converted to a digital gene expression matrix using Cell RangerSingle Cell Software Suite provided by the 10 x Genomics website. Using the cellrange aggregation command, single datasets were aggregated without subsampling normalization. The aggregated dataset was then analyzed with an R-pack setup. The dataset of cells expressing less than 200 genes was removed.

1.12 statistical analysis: all experimental results were analyzed using SPSS 23.0 software (IBM) and expressed as mean ± standard deviation. Using one-way analysis of variance and post hoc Tukey's test,the differences between groups were calculated. P value <0.05, the difference was considered as a statistical difference. All figures are drawn through GraphPad (Insight Science).

Embodiment two: loading MA with SSAD hydrogels

200mg of SSAD powder per 1mL of giant salamander (Andrias davidianus) mucus was obtained after purification, lyophilization, milling, and sieving (FIG. 1A). When mixed with water, the SSAD powder swelled into a homogeneous SSAD hydrogel within 1 minute (fig. 1B). When SSAD powder is mixed with an aqueous medium, the polypeptide chains swell, producing a porous network in the form of a hydrogel with a high water content. The fresh amniotic membrane was pale and translucent (fig. 1C), and after micronization, it became microtablets of about less than 200 μm (fig. 1D). Hematoxylin-eosin (HE) staining showed that amniotic epithelial cells were attached to the basal membrane and that the amniotic structure was still intact (fig. 1E). The loading of MA into SSAD hydrogels was confirmed under Scanning Electron Microscopy (SEM), where MA fragments were tightly packed by the hydrogel matrix, forming a tight binding interface (fig. 1F). Figure 8 shows SSAD hydrogels loaded with MA of different mesh. Preferably, a minimal amount of apoptosis is observed in hydrogels obtained from 60 mesh SSAD powder loaded with amniotic cells.

To further verify whether SSAD hydrogels are viable systems for loading MA in vitro, MA was cultured with normal medium (control group) or SSAD conditioned medium (SSAD group) according to 1.4, wherein qualitative and quantitative detection was performed with live/dead cell staining. Referring to fig. 1G, the number of living cells was gradually decreased with the increase of the culture time, regardless of the control group or SSAD group. However, the survival trends of the two appear to be comparable: cell viability values of the control group and SSAD group on day 3, day 5 and day 7 were calculated according to formula (1), respectively, and the results are shown in table 1 below, from which fig. 1H can be obtained.

Table 1 cell viability values of MA (P < 0.05)

	Day 3	Day 5	Day 7
				Control group	82.53±1.81％	74.75±3.71％	63.37±4.21％
SSAD group	96.43±0.60％	91.62±2.56％	76.65±2.09％

As can be seen from table 1 above, the cell viability values of the control group were significantly lower than those of the SSAD group, except for day 0 when the cells were all alive.

To observe apoptosis in living MA, terminal deoxynucleotidyl transferase dUTP notch terminal marker (TUNEL) assays were performed on MA (control) and MA-loaded SSAD hydrogels (SA). According to the TUNEL assay of 1.5, nuclei were stained blue and apoptotic cells were stained green by TUNEL assay kit (Roche). After 12h, apoptosis of the control group was initially seen to increase gradually after 24h (fig. 9). Interestingly, even after 36h, apoptosis was almost undetectable in the SA group (fig. 1I). Based on TUNEL detection images shown in fig. 9 and 1I, the number of apoptotic cells can be quantified, resulting in fig. 1J. The quantitative number of apoptotic cells in each group at the different time points shown in fig. 1J can further confirm that cells in MA-loaded SSAD hydrogels can survive longer during detection without showing any obvious signs of apoptosis. Fig. 10A illustrates apoptosis of SSAD hydrogel loading MA of different mesh sizes (20, 60 and 300 mesh).

Embodiment III: effects of SSAD on AMSCs and HaCaT cell behavior

Amniotic membrane contains amniotic mesenchymal stem cells (amniotic mesenchymal stem cells, AMSCs), which have the potential to differentiate into a variety of cell types, including keratinocytes and endothelial cells. Keratinocytes are critical for re-epithelialization and extracellular matrix formation, and therefore, skin healing is often accompanied by an increase in proliferation and migration of keratinocytes. To evaluate the effect of SSAD conditioned medium on AMSCs migration and proliferation, 1.7.1 scratch test (fig. 10B) and 1.7.2transwell test (fig. 10C) were performed, which were complementary to each other in horizontal and vertical directions. After 24h incubation, the wound healing rate measured in the scratch test was 13.03±4.07% for the control group and 68.38 ±7.83% for the SSAD group (fig. 10D). Relative cell number in transwell assay, control group was 100±12.01% and SSAD group was 186.53 ±10.92% (fig. 10E). The results from the 1.6 cell proliferation assay also revealed that the cell proliferation activity of SSAD group was significantly higher than that of control group (P < 0.001) on both day 5 and day 7 (fig. 10F).

For HaCaT cells (human immortalized keratinocytes), the scratch test results shown in fig. 2A and the transwell test results shown in fig. 2B also demonstrate that SSAD can promote cell proliferation and migration. From the results of analysis of the quantitative data graph of the scratch test shown in fig. 2C, the wound healing rate of the control group was 30.00±5.00% and the SSAD group was 64.37±8.27%. The relative cell number of the control group was 100.+ -. 8.27% and the SSAD group was 227.14.+ -. 24.57% (FIG. 2D). Furthermore, SSAD had significantly enhanced proliferation (P < 0.05) only at 24h of culture (fig. 2E).

To elucidate the mechanism by which SSAD promotes skin healing, haCaT cells were selected as representative cell types, subjected to additional transcriptomic analysis, and cultured in SSAD conditioned medium for 24h. Venn diagram showed 11,014 genes were co-expressed in the control and SSAD groups and 353 genes were specifically expressed in the SSAD group (FIG. 2F). Volcanic patterns of identified up-and down-regulated genes induced by SSAD conditioned medium showed 393 significant Differentially Expressed Genes (DEGs), 212 of which were up-regulated and 181 of which were down-regulated (fig. 2G). The present inventors also studied the effect of SSAD on the expression of genes involved in promoting proliferation and migration and inhibiting apoptosis (fig. 2H). According to the thermogram analysis of genes involved in significant upregulation of proliferation, migration and apoptosis of cells (fold change. Gtoreq.2, P < 0.05) in the control and SSAD groups, some important genes, such as LCK, IGF1, which promote proliferation, such as PRSS3, CASS4, which promote migration, and NR1H4, FGG, which inhibit apoptosis, were significantly upregulated after treatment with SSAD conditioned medium. The first 20 kyoto genes and genomic encyclopedia (KEGG) pathway enrichment assays (fig. 2I) showed that MAPK, VEGF, ras, tight junction (Tightjunction), jak-STAT and apoptosis signaling pathways are closely related to the mechanisms by which SSAD promotes wound healing. Among these, the MAPK signaling pathway is a crossover point or pathway that transmits information among cells, and plays an important regulatory role in tissue fibrosis, cell proliferation, cell differentiation, apoptosis, and inflammation. Notably, the MAPK signaling pathway was significantly activated after SSAD treatment (fig. 10G), suggesting that SSAD activated the MAPK signaling pathway by increasing Ras-related genes, thereby promoting cell proliferation and migration.

Taken together, in vitro scarification, transwell and cell proliferation assays demonstrate that SSAD has the effect of promoting migration and proliferation of both AMSCs and keratinocytes (HaCaT cells). Transcriptome analysis of SSAD-treated HaCaT cells indicated that SSAD could activate MAPK pathways, up-regulate genes that promote proliferation and migration, and down-regulate genes that promote apoptosis to promote proliferation and migration of keratinocytes.

Embodiment four: effects of SA on in vivo skin wound healing

Sprague-Dawley (SD) rats were selected as experimental models in the present invention, and full-thickness resected skin defects (diameter=20 mm) were created on the backs of SD rats, treated with MA, SSAD, SA or no (control) and examined daily (FIG. 3A). Full-thickness resected defects are the most difficult wounds to heal among a variety of wounds, and are suitable for evaluating the effect of the bioburden system provided by the present invention on wound healing. 7 days after surgery, the wound closure rate (48.95.+ -. 12.64%) was significantly higher for the SA group than for the control group (15.11.+ -. 9.50%), the MA group (20.2.+ -. 11.20%) and the SSAD group (32.21.+ -. 8.08%). 21 days after injury, the wound of the SA group healed almost completely; the average wound closure rate (98.07±1.11%) was also significantly higher for the SA group than for the control group (75.76±5.95%), the MA group (79.32 ±3.92%) and the SSAD group (87.21 ±3.67%) (fig. 3B). Thus, SA can greatly promote effective healing of large-area skin defects in a short time.

35 days post-surgery, skin samples were collected and stained with HE (to observe overall morphology) and Masson's trichromatography (to observe collagen fiber reorganization). The results demonstrate that the thickness of the neo-skin is greater for SSAD and SA groups compared to control and MA groups, similar to normal skin thickness. In addition, the SA group and SSAD treated group had more mature neo-tissues, with characteristics of normal skin tissue structures such as hair follicles, sebaceous glands and blood vessels (as shown in fig. 3C, black triangles in fig. 3C indicate the boundaries of wounds). Referring to fig. 3d, the morphology of collagen of the sa group takes the form of basket, indicating that collagen deposition is sufficient. In contrast, the regenerated skin of the control and MA groups was incomplete, the collagen layer was immature and thinner and collagen deposition was insufficient. Referring to fig. 3e, the skin thickness (expressed as relative thickness to normal skin) was 97.18 ±7.26% for the sa group, 31.43±15.15% for the control group, 50.45± 32.32% for the MA group, and 76.63 ±2.36% for the SSAD group. Among them, the SA group had the highest number of new hair follicles (15.62±2.08) (fig. 3F) and sebaceous glands (14.17±1.85) (fig. 3G) among regenerated neoskin tissues, compared to the other groups. In regenerated neoskin tissue, the number of hair follicles in the control group was 2.65±0.43 per microscopic field, and the number of sebaceous glands was 2.3±0.46; in regenerated neoskin tissue, the number of MA groups was 2.86±0.23 and 2.91±0.70, respectively; in regenerated neoskin tissue, the number of SSAD groups was 7.77±1.32 and 6.75±1.40, respectively. In general, the production of disordered extracellular matrix in regenerated skin tissue may lead to fibrosis, followed by scar tissue. In fact, similar to the above results, the scar rate was very low in the SA group of only 5.72.+ -. 5.55%, while the scar rates in the other groups were 66.93.+ -. 8.50% (control group), 56.97.+ -. 1.47% (MA group) and 19.47.+ -. 9.66% (SSAD group) (FIG. 3H).

Next, the present experimenter studied the interfollicular epithelial stem cells (interfollicular epidermis stemcells, IFESCs). Following injury, IFESCs will be recruited to the wound site to aid in tissue repair and remain in place for approximately 35 days. In the long term, such a population of cells will only exist in its original location in the interfollicular epithelium, rather than in the area to which it migrates, after wound healing. During scar-free skin healing, a key prerequisite is regeneration of the appendages. Hammond et al found that the new hair follicle was derived from IFESCs (i.e., not existing hair follicles) by lineage analysis (linear analyses). The follicular epithelium consists of a monolayer of proliferative basal cells and multiple layers of differentiated non-proliferative cells. The migration of basal stem cells to the skin surface takes about 1 week. Key biomarkers (integrin- α2 and integrin- β1) of IFESCs were stained on day 7 (fig. 4A) and day 14 (fig. 4A). On day 7, positive cell rate was 8.03±1.27% per field in SA group, which was far higher than control group (2.02±0.25%), MA group (2.80±1.23%) and SSAD group (4.73±0.66%). On day 14, the positive cell rate (4.25±1.25%) for each field in the SA group was still much higher than for the control group (0.85±0.25%), the MA group (1.57±1.23%) and the SSAD group (2.70±1.27%) (fig. 4E). More IFESCs were recruited to the wound center to aid wound healing in the SA group (fig. 4A), while the number of IFESCs on day 14 was less than on day 7. This fact indicates that IFESCs in the wound have a phenotype of hair follicle stem cells and can promote hair follicle formation.

Fibroblasts can differentiate into different lineages, with reticulocytes responsible for secreting collagen that forms scar tissue, once this occurs, the follicle cannot regenerate. This observation may explain why new hair follicles rarely form during uncontrolled wound healing. Reticulocytes can be identified based on embryo expression of engrailed-1 (EN-1). The results of the invention show that SA group has almost no EN-1 expression and SSAD group has lower EN-1 content; in contrast, both control and MA groups showed high expression of EN-1 (FIG. 19), consistent with quantitative analysis of HE staining (FIG. 3H). Thus, new hair follicles were absent from the healed skin of the control and MA groups, whereas newly regenerated hair follicles could be easily observed in the SA and SSAD groups, with a greater number of SA groups. HE staining images of the SA group at various time points (fig. 20) indicated that, 3 weeks after injury, the epithelial pouch grew down and developed into embryo hair follicles in the center of the wound, which were the early bud phase follicles. As new hair follicles mature, their shape more and more resembles that of normal hair follicles (fig. 20). Furthermore, no necrosis, hemorrhage or congestion was found in HE examination of major organs (heart, liver, spleen, lung and kidney), indicating good biocompatibility of SA (fig. 21).

Angiogenesis and remodeling of extracellular matrix are of great significance in wound healing. The new blood vessel can transport nutrients and maintain oxygen homeostasis to ensure cell proliferation and tissue regeneration. The neovasculature at the wound center was detected by positive staining for alpha-smooth muscle actin (alpha-SMA) (fig. 4B) and further quantified (fig. 4F). On day 14, the SA group showed the highest number of new blood vessels (41.33.+ -. 3.21 per field of view) compared to the control group (9.33.+ -. 1.53), MA group (16.00.+ -. 4.00) and SSAD group (24.00.+ -. 2.65). On day 35, the SA group also had the highest number of new blood vessels (36.66 ±2.08 per field) compared to the control group (5.67±0.58), MA group (9.67±1.53) and SSAD group (20.00±3.00). The early stages of angiogenesis involve the formation of excessive primary networks, which need to be reorganized into secondary vascular networks with higher primary organization. During remodeling, new blood vessels may be trimmed and abnormal blood vessels may be removed by apoptosis to create stable, well perfused blood vessels, which may resume homeostasis and form quiescent endothelial cells. This may be why the number of vessels observed on day 35 was less than on day 14 (fig. 4B).

Furthermore, cytokeratin 14 and cytokeratin 19 are selected as specific markers of hair follicles and sebaceous glands, respectively. As shown in fig. 4C, significantly more hair follicles and sebaceous glands are present in the regenerated skin tissue of the SA group, while some regenerated hair follicles and sebaceous glands can be found in the SSAD group. However, regenerated hair follicles or sebaceous glands were hardly seen in the control group and MA group. By quantification, the number of new hair follicles per field in regenerated skin tissue of the SA group was 31.00±4.58, while the SSAD group was 10.33±1.53 (fig. 4G). The number of neosebaceous glands per field in regenerated skin tissue of the SA group was 18.00±3.21, while the SSAD group was 6.67±1.52 (fig. 4H). The improved regeneration of the skin appendages indicates that the regenerated skin tissue is more mature and can heal almost without scarring.

Next, the presence of adipocytes, a cell known to guide hair follicle regeneration, was examined by immunostaining of perilipin 1 (PLIN 1) (fig. 4D). On day 35, there was little adipocyte regeneration in the control or MA group; interestingly, SSAD group showed 5.88±0.60% expression per field, whereas SA group showed 8.46±1.32% expression (fig. 4I).

Recently, studies have shown that myofibroblasts surrounding newly formed hair follicles can form dermal adipocytes after injury, and that this conversion of myofibroblasts to adipocytes can reduce scar formation. In addition, skin adipocytes can prevent scarring by increasing hair follicle regeneration and activating surrounding fibroblasts. PDGF expressed by immature adipocytes is involved in regulating the activity of hair follicle stem cells. Furthermore, during hair growth, the layer of adipocytes in the dermis of the skin expands and the thickness of the skin increases, which makes the skin soft and warm. This also explains that more new adipocytes were observed in the SA group than in the other groups, as the hair follicle content of the SA group was higher than in the other groups.

Fifth embodiment: study of therapeutic mechanism of SA on wound healing of rats

To further illustrate the therapeutic mechanism of SA in vivo, a rat skin defect model was selected for further transcriptome analysis, as detailed in the experimental procedure 1.10. Referring to fig. 11A, the SA group up-regulated 2,050 genes and down-regulated 1,229 genes compared to the control group; compared with the MA group, the SA group has up-regulated 1,278 genes and down-regulated 804 genes; compared to SSAD group, SA group up-regulated 1,003 genes and down-regulated 493 genes. The gene sets of the SA group and the other experimental groups were compared by venn diagram (fig. 11B), and it was found that 414 genes were highly expressed in the SA group and co-expressed in the other experimental groups. The experimenter performed a cluster thermogram analysis of the expression levels of these 414 genes (fold change ≡2, p < 0.05), and found that 304 of them were significantly up-regulated in the SA group and relatively down-regulated in the other groups (fig. 11C). This result suggests that the expression profile of the SA group is significantly different from the other groups. The experimenter further performed a functional enrichment analysis of the genes for these DEGs. KEGG pathway enrichment analysis revealed that the therapeutic mechanism of SA was highly correlated with extracellular matrix-receptor interactions, cell Adhesion Molecules (CAMs), MAPK signaling pathways, NK- κb signaling pathways, wnt signaling pathways, jak-STA signaling pathways, ras signaling pathways, VEGF signaling pathways, and signaling pathways that regulate pluripotency of stem cells (fig. 11D). The GO term for the enrichment of genes co-expressed in the biological process in the control vs.sa, MA, vs.sa and SSAD vs.sa groups is shown in fig. 11E and includes positive regulation of cell migration, positive regulation of keratinocyte differentiation, positive regulation of epithelial cell differentiation, stem cell development, epithelial development, positive regulation of stem cell division and epithelial cell proliferation, etc.

IFESCs are present in the microenvironment in which they reside and respond to normal tissue homeostasis, which is passive in repairing wounds. To more clearly explore the mechanism by which SSAD promotes skin healing, scRNA-seq analysis was also performed on sorted cells obtained from wound skin 14 days after injury, the experimental procedure is shown in fig. 5A and described in 1.11. Integrin beta 1 is one of the marker genes of IFESCs. To enrich for IFESCs, integrin- β1 positive cells were enriched using flow sorting. Flow cytometry results showed that the control group had an integrin- β1 positive cell rate of 17.2% and the SSAD group had a positive cell rate of 27.1% (fig. 12). This data indicates that more IFESCs are likely to be present after SSAD treatment.

Cells were then isolated from samples of the control and SSAD groups and applied to the 10×scrna-seq platform. A total of 7,130 cells in the control group and 11,085 cells in the SSAD group were captured. After cell filtration, 5,874 single cell transcriptomes were included in the final dataset (2,491 for control group and 3,383 for SSAD group) (fig. 13). First, cells were divided into 20 groups (fig. 14) by computationally pooling cells of control and SSAD groups to create one virtual aggregation, based on global gene expression patterns (fig. 15), using the semat for unsupervised clustering, which was then assigned to 8 major cell categories (fig. 5B): fibroblasts (FIBs), langerhans Cells (LCs), T Cells (TCs), macrophages (MACs), myoepithelial cells (myoepithelial cells, MECs), mast Cells (MCs), follicular epithelial cells (interfollicular epidermis cells, IFECs) and endothelial cells (endothelial cells, ECs). The composition of each cell population is listed in order to determine the proportion of cells from both groups in all cell populations. On day 14 post-injury, the percentage of IFECs (82.17%) was higher for SSAD than for control (17.83%). FIG. 5C is a thermal diagram showing marker genes for 8 major classes of cells. Genes associated with IFECs (Krt 73, igfbp3, tchh, krt75, hspb1, mt4 and Krt 25) were expressed substantially higher than the control samples (fig. 5D).

Differentiation of the interfollicular epithelium (IFE) is progressive, comprising the following stages: early in differentiation, basal layers of the epidermis form, associated with mitosis on the basal plate; in mid-differentiation, basal cells exit the cell cycle and form the stratum spinosum; at the end of differentiation, the cells stop transcriptional activity and die, flatten, enucleate, and watertight stratum corneum formation. Thus, the experimenter selected cells defined as IFECs in the first primary cluster and performed a second round of unsupervised clustering on them as five cell subsets (IFESCs, basal Cells (BCs), spinus Cells (SCs), keratinocytes 1 (keratinocyte 1, KER 1) and keratinocytes 2 (KER 2)) of IFECs (fig. 6A, 6B). From the experimental results, the number of IFECs in SSAD groups was generally higher than that in control groups, especially IFESCs, BCs and SCs, while the number and ratio of kes were almost the same (fig. 6C-6D). Correlation analysis showed that the 5 cell subsets of IFECs were highly correlated (fig. 6E). The Feacher plot (FIG. 6F) and violin plot (FIG. 6G) further show the level and distribution of expression of key marker genes of IFECs (FIG. 16).

To reconstruct the developmental trajectories during differentiation, pseudotime analysis of the marker gene expression of IFECs in the resulting scRNA-seq data was performed using Monocle 2. The pseudo-time path has 3 branches (fig. 6H-I), and different cell populations can be arranged relatively clearly at different branch locations of the pseudo-time path (fig. 6I). In general, different developmental processes of IFE lineage cells from IFESCs to kes can be observed (fig. 6H), as IFESCs are localized to the right at the beginning of pre-branching before the bifurcation point (fig. 6I). The distribution of IFESCs and kes in the developmental track is relatively concentrated, but BCs and SCs can be found at several time points along the developmental track (fig. 6I), suggesting that BCs and SCs may be in an intermediate stage of cellular development. For further understanding, the first 50 DEGs in the pseudo-time analysis were subjected to a thermographic analysis by the experimenter (fig. 6J). These DEGs may activate IFESCs to differentiate in two directions, respectively. Indeed, by comparing the DEGs in different clusters with the heat map, the IFESCs are split into 2 branches after the bifurcation point: KERs and BCs/SCs (FIGS. 6H-J). Since the highest proportion of IFESCs to IFECs, especially in the SSAD group, the experimenter was then concerned with enrichment analysis of the DEGs in IFESCs.

GO analysis showed that marker genes involved in biological processes related to wound healing, epidermal development, hair follicle development and stem cell migration were enriched in the IFESC population (fig. 17A). To further investigate the role of SSAD in wound healing, GO analysis was performed on the DEGs of the control group and SSAD. The regeneration and development of hair follicles, differentiation of epidermal cells, stem cell migration and proliferation associated with scar-free healing are all uniquely enriched in the SSAD group (fig. 18A), such as hair follicle development, upregulation of epithelial cell differentiation, upregulation of stem cell proliferation, hair follicle morphogenesis, and MAPK cascade. KEGG pathway enrichment analysis also showed similar results (fig. 17B), wnt pathway associated with hair follicle regeneration, ECM-receptor interaction pathway associated with maintaining skin microenvironment homeostasis, cell adhesion molecule pathway associated with cell adhesion, and MAPK signaling pathway associated with stem cell proliferation and migration were all uniquely enriched in SSAD group (fig. 18B).

In conclusion, based on transcriptome analysis data, mechanisms of SA to promote scar-free healing of skin wounds were explored. Based on the genes co-expressed in the SA and SSAD groups, the MA group and the control group, 304 up-regulated genes out of the 414 co-expressed genes were analyzed. The SA set of enriched KEGG pathways, including extracellular matrix-receptor interaction signaling pathways. The extracellular matrix is an important component of the skin microenvironment, and its synthesis and degradation are important processes to maintain the ecological balance of the skin microenvironment. Pathways involved in the regulation of multipotency, recruitment, proliferation, and migration of stem cells, including signaling pathways that regulate multipotency stem cells, MAPK signaling pathways, and Ras signaling pathways. Pathways involved in angiogenesis, including the Jak-STA signaling pathway and the VEGF signaling pathway. It was also observed that DEGs was enriched in Wnt signaling pathway. Wnt signaling in epidermal keratinocytes is essential for hair follicle regeneration, where excessive Wnt acting on the wound may promote follicle regeneration by changing cell fate and increasing the number of cells capable of producing hair. This fact explains to some extent the reason why the SA group promotes scar-free healing of the skin.

GO analysis further showed that up-regulated genes co-expressed between SA and SSAD, MA and control group are involved in epidermal development, stem cell division, stem cell development, cell proliferation, regulation of epithelial cell differentiation, regulation of vascular endothelial cell proliferation, regulation of smooth muscle cell migration, etc. These biological processes promote proliferation, migration and differentiation of epidermal cells. The epidermal cells of the wound epidermis are reported to have the phenotype of hair follicle stem cells. This observation also corresponds to the enrichment of the aforementioned SA group for DEGs in the Wnt signaling pathway. By regulating proliferation and migration of vascular endothelial cells and migration of smooth muscle cells with positive feedback, SA treatment can significantly contract wounds and promote proliferation of blood vessels. The possible mechanisms by which SA promotes skin regeneration are again illustrated in fig. 7.

IFESCs and fibroblasts are important cells essential in wound healing. IFESCs divide, proliferate and migrate rapidly to close the wound, while fibroblasts migrate to the defect area and secrete collagen to repair the wound. Rapid repair of fibroblasts often results in scarring, particularly full-thickness skin defects. In healthy skin, collagen fibers were arranged in a grid or basket, but during wound healing, fibroblasts arranged collagen fibers in parallel, resulting in hard and fragile tissue, consistent with the results of the control group (fig. 3D). Fibroblasts play a role in wound healing, but are also believed to be responsible for scar tissue formation. In the single cell sequencing results of the present invention, the SSAD group had more IFESCs and fewer fibroblasts, while the control group was the opposite. This data indicates that the wound heals in the SSAD group, unlike the control group. The unique enrichment of GO and KEGG pathways in the IFESC population of the SSAD group also further demonstrates that the use of SSAD is beneficial in promoting proliferation and recruitment of more IFESCs in skin wounds, activating Wnt pathway, and enhancing follicular regeneration and scar-free healing.

Summarizing:

recovery of skin wounds requires a range of procedures including hemostasis, angiogenesis, re-epithelialization and extracellular matrix remodeling. These phases are not independent, but are interleaved with each other. Skin repair is therefore one of the most complex processes in the human body, and when large areas of skin defects occur (especially defects that are also full-thickness wounds), scars tend to develop. Ideal skin healing is complete regeneration, where the new tissue has the same structural, aesthetic and functional properties as the original intact skin.

SSAD hydrogels are rich in various growth factors associated with wound healing, angiogenesis and re-epithelialization, such as stromal cell derived factor-1 (SDF-1), vascular Endothelial Growth Factor (VEGF), platelet derived factor (PDGF), EGF, HGF, and basic fibroblast growth factor (bFGF) (table 2). SDF-1 is down-regulated in the wound, which may lead to delayed wound healing. Whereas SDF-1 is the most abundant growth factor in SSAD, it plays a key role in initial wound healing and can significantly enhance stem cell migration and recruitment. Although SSAD hydrogels can promote wound healing to some extent, the administration of SSAD hydrogels alone does not achieve scar-free healing that promotes more severely damaged wounds (e.g., full-thickness wounds).

TABLE 2 quantitative content of growth factors in SSAD

Surprisingly and unexpectedly, in the bioburden system provided by the invention, a synergistic effect exists between hydrogel formed by the giant salamander skin secretion freeze-dried powder and the solvent (simply referred to as "giant salamander skin secretion hydrogel") and the amniotic membrane particles, namely the effect of the bioburden system as a whole on promoting healing of a full-layer wound is greater than that of independently applying the giant salamander skin secretion hydrogel and the amniotic membrane particles, and the bioburden system comprises the following specific steps: the bioburden system provided by the invention can promote the generation of skin appendages and the complete and scar-free healing of skin wounds (especially full-thickness wounds).

Although it has been demonstrated that both giant salamander skin exudate hydrogels (specifically SSAD hydrogels) and amniotic membrane contain a variety of growth factors that promote wound healing, the inventors have found that the application of such hydrogels or amniotic membrane microparticles alone (simply referred to as "individual application") does not achieve the practical effects of the bioburden system provided by the present invention, i.e., the individual application does not promote complete reconstitution of full-thickness skin. Application alone does not restore the wound to its intact structural, aesthetic and functional properties. The bioburden system provided by the present invention exhibits excellent ability to promote skin regeneration.

Amniotic membrane is the innermost membrane of a mammalian placenta, which is rich in proteins and other extracellular matrix molecules, which can promote wound healing and angiogenesis. The low immunogenicity of amniotic membrane makes it an ideal allograft with low risk of rejection. However, because amniotic membrane is relatively thin, highly elastic, and has extremely low mechanical strength, it is difficult to stably adhere to a wound, and it can be used only in the form of a large sheet as a temporary biological dressing. According to the invention, fresh amniotic membrane is micronized to obtain amniotic membrane particles retaining living cells (such as AMSCs) and growth factors (such as bFGF, EGF, PDGF, VEGF, HGF), and a bioburden system in the form of giant salamander skin secretion hydrogel-amniotic membrane particles-giant salamander skin secretion hydrogel is creatively formed. The giant salamander skin secretion hydrogel (preferably SSAD hydrogel) can establish (e.g. wound) strong interactions with the contact matrix and strong adhesion with the amniotic membrane particles through pi-pi electrons or cation-pi interactions, so that the amniotic membrane particles can be firmly and stably positioned between two layers of hydrogels and are prevented from falling off and being interfered by external environment. Unexpectedly, the two layers of hydrogels do not poison the amniotic particles (i.e., are highly biocompatible) and the strong adhesion of the hydrogels does not destroy the activity of living cells in the amniotic particles, even the porous network structure of the hydrogels can support the normal activity of the cells. In addition, if the amniotic membrane is not micronized (i.e., the amniotic membrane remains intact), it is not only detrimental to adhesion to the wound bed, but also cells in the amniotic membrane are difficult to obtain nutrients such as oxygen and are prone to apoptosis. That is, the amniotic membrane microparticles are instead better able to achieve nutrient/oxygen exchange and secretion of growth factors in the hydrogel environment.

The biological load system provided by the invention does not need to separate or culture special cells, does not need to manually add additional growth factors, and can effectively release the growth factors only by using the giant salamander skin secretion hydrogel and the amniotic membrane particles, thereby promoting the scar-free healing of skin wounds. Of course, according to the actual wound condition, proper growth factors, cell culture mediums and proper granularity of giant salamander skin secretion freeze-dried powder can be further added. In addition, a gel layer and an amniotic membrane microparticle layer can be further added.

For hydrogels obtained from powders of different particle sizes, the smaller the particle size of the giant salamander skin secretion lyophilized powder, the larger the pore size of the hydrogel formed. The larger the pore size of the hydrogel, the faster the content such as growth factors is released, and the faster the hydrogel itself degrades. The smaller the pore size of the hydrogel, the denser the porous network formed, and the denser network structure may be detrimental to respiration of living cells. In other words, too large or too small a pore size of the hydrogel is detrimental to cell survival, and one skilled in the art can determine the appropriate particle size based on the actual situation and pre-experiments.

In summary, as shown in fig. 7 and 1F, the bioburden system of the present invention not only maintains the biological activities of amniotic membrane and AMSCs, but the whole system can synergistically promote healing through migration of keratinocytes, recruitment of endogenous stem cells, formation of new blood vessels, and regeneration of skin appendages (hair follicles, sebaceous glands, etc.). The bioburden system of the invention brings new hopes for promoting the cicatrix-free healing of large-area skin wounds.

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 (10)

1. The application of the giant salamander skin secretion freeze-dried powder in preparing a biological load system for promoting wound healing is characterized in that the biological load system comprises two gel layers and an amniotic membrane particle layer positioned between the two gel layers, and the gel layers comprise a mixture of the giant salamander skin secretion freeze-dried powder and a solvent; the gel layer is used for supporting the activity of the amniotic membrane particles, the amniotic membrane particles are micro-plates smaller than 200 mu m, the amniotic membrane particles contain living cells, and the cells comprise amniotic mesenchymal stem cells; in one of the gel layers and one of the amniotic membrane particle layers, the ratio of the giant salamander skin secretion lyophilized powder to the amniotic membrane particle comprises 50 mg:1 mL.

2. The use of claim 1, wherein the particle size of the giant salamander skin secretion lyophilized powder comprises 20 mesh, 60 mesh and 300 mesh.

3. The use of claim 1, wherein the giant salamander comprises one or more of the genera giant salamander, cryptobranchia, mangrove, andrias, giant salamander, andrias, north giant salamander, polar giant salamander.

4. The use of claim 1, wherein the wound is a skin wound.

5. The use of claim 4, wherein the wound is a full-thickness wound.

6. Use according to claim 1, wherein the bioburden system is used to promote regeneration and/or vascularization of skin appendages and/or remodeling of extracellular matrix at the wound site.

7. The use of claim 1, wherein the bioburden system is used to promote recruitment of interfollicular epithelial stem cells to the wound.

8. The use according to claim 1, wherein the bioburden system is used to promote proliferation and/or migration of keratinocytes.

9. The use of claim 1, wherein the bioburden system is for promoting healing of the wound.

10. The use of claim 9, wherein the healing is scar-free healing.