Extracellular vesicles of antler bud progenitor cells derived from ectoderm lineage and their application in the preparation of scarless skin regeneration drugs or medical devices

Abstract

This invention discloses extracellular vesicles (EVs) derived from ectodermal lineage progenitor cells of deer antler buds and their application in the preparation of scarless skin regeneration drugs or medical devices, belonging to the field of biomedical technology. This invention uses ABPCs (alternary blastocysts) derived from ectodermal lineages, which are highly stem and resistant to aging, as cells for producing EVs. The resulting ABPC-EVs can synergistically induce in-situ hair follicle regeneration, rapid microvascularization, and restoration of nerve innervation, exhibiting high biocompatibility and controllability. By constructing a multidimensional repair network, it improves tissue integration quality, achieving comprehensive and scarless functional skin regeneration and repair. This solves the technical problems of existing bioactive substances in the complex wound microenvironment, such as slow repair speed, inability to inhibit scar formation, poor repair quality, and difficulty in achieving functional wound regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to extracellular vesicles of antler bud progenitor cells derived from ectoderm lineage and their application in the preparation of scarless skin regeneration drugs or medical devices. Background Technology

[0002] Skin wound repair has long been a challenge in regenerative medicine. Current clinical treatments mainly rely on biological agents such as recombinant human basic fibroblast growth factor (rh-bFGF) and traditional medical dressings. In recent years, mesodermal stem cell technology, represented by adipose-derived mesenchymal stem cell extracellular vesicles (ADSC-EVs), has shown some potential in promoting healing. However, most existing technologies can only accelerate the physical closure of the wound and are difficult to achieve true tissue function reconstruction. Studies have shown that wounds treated with existing technologies are often accompanied by excessive fibrosis, leading to disordered collagen fiber arrangement and the formation of obvious hypertrophic scars. Furthermore, they generally lack the regeneration of skin appendages such as hair follicles, sebaceous glands, and nerve endings, resulting in repair effects that are significantly different from those of native skin.

[0003] The reason for this is that, in embryonic development, the skin and its appendages primarily originate from the ectoderm lineage, while most widely used stem cells and their derivatives (such as ADSC-EVs, BMSC-EVs, and UcMSC-EVs) originate from the mesoderm. This developmental lineage barrier limits their ability to precisely regulate the skin microenvironment, making it difficult to activate the endogenous developmental programs required for complete skin regeneration. Furthermore, existing bioactive substances are easily inactivated in the complex wound microenvironment, have short biological half-lives, and lack heterogeneous regulatory mechanisms targeting different stages of wound repair (inflammation, proliferation, and remodeling), leading to key technical bottlenecks in clinical translation such as uncontrollable repair quality and low regeneration efficiency. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide extracellular vesicles of antler bud progenitor cells derived from ectoderm lineage and their application in the preparation of scarless skin regeneration drugs or medical devices. This addresses the issues of existing bioactive substances being easily inactivated in the complex wound microenvironment, having short biological half-lives, lacking heterogeneous regulatory mechanisms, resulting in uncontrollable repair quality and low regeneration efficiency in clinical translation.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the use of the above-mentioned extracellular vesicles of antler bud progenitor cells derived from the ectoderm lineage in the preparation of skin scarless regeneration drugs or medical devices.

[0006] The beneficial effects of this invention are as follows: The specific regeneration-promoting factor combination carried by the outer vesicles of ABPCs extracted by this invention induces the regeneration of skin appendages by reshaping the immune microenvironment of the wound, inhibits scar formation at the molecular level, and ultimately achieves scarless skin regeneration and physiological function reconstruction. This overcomes the shortcomings of existing technologies that cannot balance repair speed and regeneration quality, and can be effectively applied to the preparation of scarless regeneration drugs or medical devices, with broad application prospects.

[0007] Furthermore, extracellular vesicles from ectodermal lineage-derived antler bud progenitor cells were prepared by the following method: First, CX43+ and FGFR2+ were used as markers to sort the cells in the antler embryonic base to obtain antler bud progenitor cells. Then, the outer vesicles were extracted to obtain antler bud progenitor cell outer vesicles derived from the ectoderm lineage.

[0008] In a second aspect, the present invention provides a scarless regenerative medicine or medical device comprising extracellular vesicles of antler bud progenitor cells derived from the ectodermal lineage described above.

[0009] Furthermore, it also includes medically available carriers and / or excipients.

[0010] A third aspect of the present invention provides a method for preparing extracellular vesicles from antler bud progenitor cells derived from ectoderm lineages, comprising the following steps: S1. Donor cell acquisition: Cells within the antler embryonic base were sorted and identified using CX43+ and FGFR2+ as markers to obtain antler bud progenitor cells, which were then passaged. S2. The progenitor cells of deer antler buds obtained after passage culture are further cultured and extracted by differential centrifugation to obtain extracellular vesicles of deer antler bud progenitor cells.

[0011] The beneficial effects of this invention are as follows: This invention rapidly obtains ABPCs from ectoderm lineages by identifying and sorting cells within the antler embryonic base using markers and extracting them by differential centrifugation. The preparation method is simple, efficient, and easy to standardize, thus improving the certainty and safety of clinical applications.

[0012] Furthermore, the antler embryos were selected from the hard antlers of 2-year-old male sika deer, 5 days after their antlers fell off.

[0013] Furthermore, flow cytometry was used as the sorting method.

[0014] Furthermore, the culture medium used in S1 was DMEM medium, which was supplemented with 0.5-1.5 wt% penicillin / antibiotic (P / S), 0.05 wt%-0.15 wt% mycoplasma scavenger and 5 wt%-15 wt% fetal bovine serum (FBS).

[0015] Preferably, the culture medium used in S1 is DMEM medium, which contains 1 wt% penicillin / antibody (P / S), 0.1 wt% mycoplasma scavenger and 10 wt% fetal bovine serum (FBS).

[0016] Furthermore, S1 is propagated to the 6th-8th generation.

[0017] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: By optimizing the subculture conditions, the present invention can effectively separate and maintain ABPCs with strong regeneration capabilities for subsequent extraction of ABPCs exovesicles.

[0018] Further, the culture in S2 includes: first culturing in a complete medium containing 5 wt%-15 wt% fetal bovine serum for 20-30 h, discarding the culture medium and washing, and then culturing in a complete medium containing 5 wt%-15 wt% serum without external vesicles for 40-60 h.

[0019] Preferably, the continued culture in S2 includes: first culturing in a complete medium containing 10 wt% fetal bovine serum for 24 h, discarding the culture medium and washing, and then culturing in a complete medium containing 10 wt% serum without external vesicles for 48 h.

[0020] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: the present invention effectively prevents contamination of external vesicles by continuing to culture them in a complete culture medium without external vesicle serum.

[0021] Furthermore, when the cells in S2 are cultured until the cell viability is greater than 90%, extracellular vesicles of the deer antler bud progenitor cells are extracted.

[0022] Furthermore, the conditions for differential centrifugation of cells are as follows: first, centrifuge the culture supernatant at 500-1000 g for 10-30 min, then collect the supernatant and centrifuge at 1500-2500 g for 20-40 min, then centrifuge at 15000-17000 g for 60-80 min to collect the precipitate, and finally resuspend and centrifuge at 15000-17000 g for 60-80 min to obtain extracellular vesicles of the progenitor cells of deer antler buds.

[0023] Preferably, the conditions for differential centrifugation of cells are as follows: first, centrifuge the culture supernatant at 750 g for 20 min, then collect the supernatant and centrifuge at 2000 g for 30 min, then centrifuge at 16000 g for 70 min to collect the precipitate, and finally resuspend and centrifuge at 16000 g for 70 min to obtain extracellular vesicles of antler bud progenitor cells.

[0024] Furthermore, the differential centrifugation process was carried out at 4°C.

[0025] The beneficial effects of adopting the above-mentioned further technical solutions are as follows: the present invention removes cells by first centrifugation, removes cell debris and apoptotic bodies by second centrifugation, and precipitates extravesicles by final centrifugation.

[0026] In a fourth aspect, the present invention provides extracellular vesicles of antler bud progenitor cells derived from ectoderm lineages, prepared using the above-described preparation method.

[0027] The beneficial effects of this invention are as follows: By using ABPCs derived from ectoderm lineages that are highly stem and not prone to aging, and carrying specific ectoderm development regulatory factors, this invention can precisely activate hair follicle stem cells in situ at the wound site, thereby achieving scarless tissue regeneration.

[0028] The present invention has the following beneficial effects: 1. Overcoming lineage barriers to achieve true physiological regeneration. This invention uses ABPC derived from the ectoderm lineage, which is highly dry and resistant to aging, to prepare EVs. The resulting ABPC-EVs carry specific ectoderm development regulatory factors, which can precisely activate hair follicle stem cells in situ at the wound site. While accelerating healing, it can induce the formation of new hair follicles, making the repaired tissue highly consistent with the original skin in structure and function, rather than just a simple fibrotic cover. This solves the technical problems of existing technologies that accelerate wound closure by inducing excessive proliferation of fibroblasts, which easily leads to disordered collagen fiber arrangement and the formation of fibrotic scars, and cannot induce synchronous regeneration of skin appendages (hair follicles, sweat glands, sebaceous glands, etc.), resulting in the repaired skin lacking normal physiological functions (such as sweating, touch, hair growth, etc.).

[0029] 2. Actively inhibit fibrosis to achieve scarless repair. This invention regulates the evolution of fibroblast subpopulations in wounds through ABPC-EVs, which can specifically induce the production of regenerative fibroblasts, optimize the Type III / Type I collagen ratio, make collagen fibers neatly arranged in a network, and reduce the expression of myofibroblast markers, thus preventing scar formation from the source and solving the industry pain point of "rapid healing inevitably leaves scars".

[0030] 3. Constructing a multidimensional repair network to improve tissue integration quality. The active ingredients in ABPC-EVs of this invention can simultaneously promote the rapid regeneration of CD31 / VEGF microvessels and the directional growth of PGP9.5 nerve axons, achieving synergistic induction of vascular endothelial cells and nerve endings. Compared with the promoting effect of a single factor, through multi-target regulation, it ensures that the repaired skin not only has a good appearance, but also has normal blood perfusion and sensory feedback. This solves the technical problem in existing technologies where slow and incomplete recovery of nerve innervation and blood supply after chronic trauma repair leads to dulled sensation in the repaired tissue.

[0031] 4. High biocompatibility and controllability. The ABPC-EVs obtained in this invention are extracellular vesicles derived from non-immunogenic ABPCs. As a natural nanoscale carrier, they have extremely low immunogenicity and good biocompatibility, without the potential tumorigenic risk of cell therapy. Furthermore, ABPC-EVs are easy to standardize and preserve for a long time, have stronger permeability in the wound microenvironment, and can achieve precise drug delivery through subcutaneous injection and other methods, thus improving the certainty and safety of clinical applications. Attached Figure Description

[0032] Figure 1 Figure showing the cell proliferation activity analysis results of ABPCs and AnSCs; Figure 2 The images show the extraction and identification of ABPC-EVs and ADSC-EVs. In the images, A is a schematic diagram of the extraction process, B is a TEM image of ABPC-EVs, C is a TEM image of ADSC-EVs, D is an NTA analysis result image, E is a Zeta potential measurement image, and F is a WB detection image. Figure 3 A schematic diagram illustrating the healing-promoting capacity of EVs in vivo; Figure 4 The results of the in vivo rapid healing effect of ABPC-EVs on mouse wounds are shown in the figure. In the figure, a is a typical image of the skin wound on the back of the mouse treated with local drug on the 0th, 4th and 8th day after surgery, and b is a graph showing the change of wound area in each treatment group on the 2nd, 4th, 6th and 8th day after surgery. Figure 5 Figure 1 shows the experimental results of the effect of ABPC-EVs on the wound healing ability of mice. In this figure, a is the H&E staining image of each treatment group on the 4th and 8th day after surgery, and b is the quantitative analysis image of epidermal thickness, dermal thickness and collagen volume fraction on the 4th and 8th day after surgery. Figure 6 The images show laser speckle blood flow imaging and representative immunofluorescence staining. In the images, a is the laser speckle blood flow imaging on days 4 and 8, b is the quantitative analysis of laser speckle blood flow imaging, and c is the representative immunofluorescence staining of the wound tissue on day 8 postoperatively, showing CD34 (green), Ki67 (red), and cell nuclei (blue). Figure 7The images show the results of the ABPC-EVs-induced complete regeneration of full-thickness skin defects in mice. Image a shows optical photographs and schematic diagrams of scar area changes on postoperative days 16 and 24; image b shows the area change curve of scar tissue after treatment; image c shows the color analysis of scar tissue 24 days after treatment (higher L values ​​represent whiter color, higher A values ​​represent redder color) (n=3 biologically independent samples); images d and e show H&E staining and Masson staining of each treatment group on postoperative day 16; images f and g show H&E staining and Masson staining of each treatment group on postoperative day 24; images h and i show epidermal thickness analysis on postoperative days 16 and 24; image j shows hair follicle counting on postoperative day 24; and images kn show representative immunofluorescence staining of VEGF, CD31, PGP9.5, CD34, and Ki67 in the wound tissue of each treatment group on postoperative day 24, along with their quantification of positive staining intensity. Detailed Implementation

[0033] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] Example 1: A method for preparing extracellular vesicles from ectodermal lineage-derived deer antler bud progenitor cells (flowchart shown in figure) Figure 2 (As shown in Figure A), the steps include: S1. Acquisition and culture of donor cells ABPCs: Hard antlers from a 2-year-old male sika deer were used to extract ABPCs. Five days after antler detachment, cells within the antler germ layer were sorted by flow cytometry, and ABPCs were identified using CX43+ and FGFR2+ as markers. The isolated cells were then prepared into a single-cell suspension in DMEM medium (11965092, Gibco), supplemented with 1 wt% P / S (15140122, Gibco), 0.1 wt% mycoplasma scavenger, and 10 wt% FBS (10099141C, Gibco). The cells were then incubated at 2 × 10⁻⁶ cells / mL. 5 pcs / cm 2 Cells were seeded at a density of 10 cm in culture dishes. After 24 h, non-adherent cells were removed, and adherent cells were cultured for further growth. Finally, when cell confluence reached 80%–90%, cells were collected and reseeded at the same density to achieve further expansion and passage to the 6th generation.

[0035] Preparation of S2 and ABPC-EVs The 6th generation ABPCs obtained from S1 were first cultured in complete medium containing 10 wt% FBS for 24 h. After discarding the culture medium, the cells were washed three times with pre-warmed PBS, and then cultured in complete medium containing 10 wt% EVs-free serum for 48 h to prevent EVs contamination. EVs were then extracted when cell viability exceeded 90% as determined by a CCK-8 assay kit (Dojindo, Kumamoto, Japan). EVs extraction was performed using differential centrifugation at 4°C, specifically including the following steps: centrifugation at 750 g for 20 min to remove cells, followed by centrifugation at 2000 g for 30 min to remove cell debris and apoptotic bodies, then centrifugation at 16000 g for 70 min to precipitate EVs. Finally, the obtained EVs were resuspended in pre-chilled PBS and centrifuged again at 16000 g for 70 min to obtain ABPC-EVs, dissolved in 100 μL of pre-chilled PBS, and stored at -80°C.

[0036] Comparative Example 1: A method for preparing adipose-derived mesenchymal stem cell extravesicles (flowchart shown in figure) Figure 2 (As shown in Figure A), the steps include: S1. Acquisition and culture of donor cells ADSCs The SD rat pups (approximately 10 days old) used in this comparative example were approved by the Ethics Committee of Xijing Hospital, the First Affiliated Hospital of the Fourth Military Medical University. After euthanizing the pups by cervical dislocation, they were quickly immersed in 75% anhydrous ethanol solution for 5 minutes for initial disinfection, and then transferred to a laminar flow hood for further processing. In the laminar flow hood, the skin was incised along the groin area using sterile surgical scissors to fully expose and separate the adipose tissue. The separated adipose tissue was placed in a 10 cm culture dish containing pre-cooled sterile PBS (phosphate-buffered saline) to clean the tissue surface and remove visible blood vessels, fascia, and other impurities. After processing, the adipose tissue was minced and transferred to a 15 mL centrifuge tube, with an equal volume of 0.1% type I collagenase solution added. The centrifuge tube was placed in a 37°C incubator and digested by shaking at a frequency of once every 15 minutes for 45 minutes, until the tissue fragments were completely dissolved. After digestion, an appropriate amount of DMEM medium was added to the centrifuge tube. This medium was pre-added with 1 wt% P / S, 0.1 wt% mycoplasma scavenger, and 10 wt% FBS to terminate the digestion reaction. Then, DMEM medium was added to the centrifuge tube at a 1:1 ratio, and the tube was centrifuged at 4°C and 2000 rpm for 10 min. After centrifugation, the undigested adipose tissue was discarded, and the lower layer was transferred to a new centrifuge tube. The cells were resuspended in 5 mL of DMEM medium containing 1 wt% P / S and 0.1 wt% mycoplasma scavenger. The cells were then centrifuged again at 4°C and 2000 rpm for 10 min. After removing the supernatant, the cells were resuspended in 1 mL of DMEM medium containing 1 wt% P / S, 0.1 wt% mycoplasma scavenger, and 10 wt% FBS. The resuspended cell suspension was transferred to a 10 cm culture dish for culture. After 24 h of culture, half the medium was replaced. Subsequently, the medium was changed every 2-3 days according to the cell growth status. When the cells grew to 80% density at the bottom of the culture dish, they were digested with trypsin and passaged to the 6th generation.

[0037] Preparation of S2 and ADSC-EVs The preparation method of EVs is the same as in Example 1. The 6th generation ADSCs obtained in S1 are used to extract EVs to obtain ADSC-EVs.

[0038] Experimental Example 1: Comparison of cell proliferation capacity between AnSCs and ABPCs I. Experimental Methods In this invention, the MTS method was used to analyze the difference in proliferation kinetics between ABPCs and antler bud-based mixed stem cell populations (AnSCs) obtained by flow cytometry sorting in Example 1 S1 within 12-96 h.

[0039] II. Experimental Results and Analysis Experimental results are as follows Figure 1 As shown.

[0040] The results showed that within the first 24 hours of culture, ABPCs exhibited a significantly faster proliferation initiation rate than AnSCs, with cell viability approaching 220% at 24 hours, while AnSCs remained in a slow growth phase. This indicates that the cell population enriched through specific antibody sorting removed slow-proliferating or maturely differentiated components from the mixed population, significantly enhancing the population's proliferation potential. Simultaneously, ABPCs rapidly entered a plateau phase after 24 hours and maintained a very high level (viability remained above 220% from 24 to 96 hours), with its standard deviation showing good controllability. In contrast, the growth curve of AnSCs showed a significant lag, reflecting the heterogeneity of the cell cycle within the mixed population. Furthermore, until the 96-hour observation endpoint, the cell viability of the ABPCs group remained stable, demonstrating that the sorting operation of this invention did not damage cell viability; rather, by enriching specific cell lineages, it ensured a more robust metabolic level for this population during long-term culture.

[0041] Experimental Example 2: Physicochemical Characterization I. Experimental Methods The microstructure of ABPC-EVs and ADSC-EVs obtained in Example 1 and Comparative Example 1 was observed using transmission electron microscopy (TEM). Their particle size distribution was detected using nanoparticle tracking analysis. The absolute value of the outer vesicle potential was detected using zeta potential analysis. Protein expression was detected by Western blotting (WB).

[0042] II. Analysis of Experimental Results Experimental results are as follows Figure 2 As shown.

[0043] The results showed that ABPC-EVs were similar to ADSC-EVs in morphology, surface charge, marker expression, and particle size distribution. Specifically, TEM results showed that both exhibited typical cup-shaped or spherical morphologies. Figure 2 Figures B and C in the middle). NTA analysis results show that the particle size distributions of ABPC-EVs and ADSC-EVs are similar, with the main peak at approximately 145.8 nm (Figure B and Figure C in the middle). Figure 2 (Figure D). Zeta potential measurements show that both ABPC-EVs and ADSC-EVs exhibit significant negative surface charges (Figure D). Figure 2(See Figure E). There was no significant difference between the two groups, and the absolute values ​​of the potentials were both greater than 30 mV, indicating that the prepared nanoparticles have excellent electrostatic repulsion and colloidal stability in aqueous solution, and are not prone to aggregation, which is consistent with the physical characteristics of high-quality extracellular vesicles. Western blot results confirmed that both expressed characteristic markers of extracellular vesicles such as CD9, CD63, CD81, and HSP90, and were negative for the endoplasmic reticulum membrane protein Calnexin. Figure 2 Figure F in the middle shows the successful preparation of ABPC-EVs and ADSC-EVs.

[0044] Experimental Example 3: In vivo experiment to induce skin regeneration I. Animal Model Establishment and Drug Administration (1) Modeling Eight-week-old male C57BL / 6 mice were selected, and a full-thickness skin defect model (approximately 8 mm in diameter) was prepared on the back.

[0045] (2) Experimental grouping The experiment was divided into four groups: Vehicle group (PBS), rh-bFGF group (positive drug control), ADSC-EVs group (endoderm mesenchymal stem cell-derived control), and ABPC-EVs experimental group.

[0046] (3) Administration method Perform subcutaneous injections at multiple points (4 symmetrical points) around the wound periphery, with a dose of 5 × 10⁻⁶. 6 particles / mL, 20μL per spot.

[0047] II. Dynamic assessment of repair quality (study diagram as shown) Figure 3 (As shown) (1) Healing rate Wound closure was recorded and healing rate was calculated on days 0, 4, 8, 12 and 16.

[0048] (2) Histological remodeling After healing, skin tissue was taken for H&E staining and Masson staining to observe the arrangement of collagen fibers.

[0049] (3) Regeneration of accessory organs The number and developmental stage of newly formed hair follicles were counted to determine whether physiological regeneration had been achieved.

[0050] (4) Functional evaluation Immunofluorescence staining was used to detect vascular markers CD31 / VEGF and neural marker PGP9.5 to assess the degree of vascularization and restoration of nerve innervation.

[0051] III. Experimental Results and Analysis (1) Effects of ABPC-EVs on rapid in vivo wound healing in mice Experimental results are as follows Figure 4 As shown.

[0052] The results showed that the wound closure rate in the ABPC-EVs group was significantly higher than that in the control groups: on the 4th day after surgery, the wounds of mice in the ABPC-EVs group were significantly closed, and the wound area was significantly smaller than that in the ADSC-EVs group and the rh-bFGF group; on the 8th day after surgery, the wounds of mice in the ABPC-EVs group were almost completely closed, with significantly better results than those in the ADSC-EVs group and the rh-bFGF group. Figure 4 (Figure a). From day 2 to 8 post-surgery, the wound area in the ABPC-EVs experimental group showed significant changes, with over 90% closure by day 6. The wound was almost completely closed and its appearance was close to normal skin, demonstrating significantly better treatment results than other groups. Figure 4 (Figure b in the middle)

[0053] (2) Effects of ABPC-EVs on wound repair quality in mice In addition to healing speed, ABPC-EVs also demonstrated outstanding performance in improving repair quality: histological evaluation results showed that the epidermal and dermal thickness of the ABPC-EVs experimental group were significantly lower than those of other control groups, having returned to normal levels. H&E staining results showed that the collagen fibers in the ABPC-EVs experimental group were neatly and densely arranged, and the collagen volume fraction was significantly higher than that of other control groups. Figure 5 Figures a and b in the paper demonstrate the effective inhibitory effect of this application on scar formation when repaired with ABPC-EVs, blocking pathological scarring, unlike the fibrosis covering scar formation in the control group.

[0054] Laser speckle flow imaging showed that the blood supply in the ABPC-EVs experimental group was rapidly restored, significantly better than that in the other control groups. Figure 6 Figures a and b show that the ABPC-EVs experimental group established abundant blood perfusion in the early postoperative period, providing sufficient oxygen and nutrients for tissue regeneration.

[0055] Immunofluorescence analysis results showed that ( Figure 6 (Figure c) In the ABPC-EVs experimental group, newly formed hair follicles and sebaceous glands were observed as early as the 8th day after surgery, while the other control group had no such structure at all. This indicates that the present invention has achieved a leap from fibrotic repair to functional regeneration of wounds through ABPC-EVs.

[0056] (3) Effect of ABPC-EVs on the healing quality of full-thickness skin injuries in mice To further evaluate the impact of ABPC-EVs on the healing quality of full-thickness skin injuries in mice, this invention characterizes the histological features 16-24 days post-surgery.

[0057] The results showed that 24 days post-surgery, the scars in the ABPC-EVs experimental group almost completely disappeared (Figures a and b). Color analysis showed that the scar tissue 24 days after ABPC-EVs treatment was whiter than that of other control groups and closer to normal skin. H&E and Masson staining results showed that by day 24, the regenerated skin in the ABPC-EVs experimental group had formed a highly ordered layered structure (Figures d-g). Histological evaluation showed that the ABPC-EVs experimental group produced a large number of structurally intact mature hair follicles and sebaceous gland units, which were highly consistent with the expression of skin appendage markers (such as CD34 and Ki67) (Figures k-n). The epidermal thickness on days 16 and 24 and the hair follicle count on day 24 visually demonstrated (Figures h-j) that the ABPC-EVs experimental group could significantly reduce the epidermal thickness of the wound while inducing hair follicle tissue regeneration, achieving high-quality, scarless, and functional regeneration of the wound.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of extracellular vesicles from ectodermal lineage-derived deer antler bud progenitor cells in the preparation of scarless skin regeneration drugs or medical devices.

2. Use according to claim 1, characterized in that, The extracellular vesicles of the antler bud progenitor cells derived from the ectoderm lineage were prepared by the following method: First, CX43+ and FGFR2+ were used as markers to sort the cells in the antler embryonic base to obtain antler bud progenitor cells. Then, the outer vesicles were extracted to obtain antler bud progenitor cell outer vesicles derived from the ectoderm lineage.

3. A scarless regeneration drug or medical device, characterized in that, Including extracellular vesicles of antler bud progenitor cells derived from the ectoderm lineage as described in claim 1 or 2.

4. The scarless regenerative drug or medical device according to claim 3, characterized in that, It also includes medically available carriers and / or excipients.

5. A method for preparing extracellular vesicles from antler bud progenitor cells of ectodermal lineage as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Donor cell acquisition: Cells within the antler embryonic base were sorted and identified using CX43+ and FGFR2+ as markers to obtain antler bud progenitor cells, which were then passaged. S2. The progenitor cells of deer antler buds obtained after passage culture are further cultured and extracted by differential centrifugation to obtain extracellular vesicles of deer antler bud progenitor cells.

6. The method for preparing extracellular vesicles from antler bud progenitor cells of ectodermal lineage according to claim 5, characterized in that, The culture medium used in S1 is DMEM medium, which contains 0.5-1.5 wt% antibiotics, 0.05 wt%-0.15 wt% mycoplasma scavenger and 5 wt%-15 wt% fetal bovine serum; and it is passaged to the 6th-8th generation.

7. The method for preparing extracellular vesicles from antler bud progenitor cells of ectodermal lineage according to claim 5, characterized in that, The S2 culture process includes: first culturing in a complete medium containing 5 wt%-15 wt% fetal bovine serum for 20-30 h, then discarding the culture medium and washing the sample, followed by culturing in a complete medium containing 5 wt%-15 wt% serum without external vesicles for 40-60 h.

8. The method for preparing extracellular vesicles from antler bud progenitor cells of ectodermal lineage according to claim 5, characterized in that, In step S2, the cells are cultured until the cell viability is greater than 90% before extracting extracellular vesicles from the progenitor cells of the deer antler bud.

9. The method for preparing extracellular vesicles from antler bud progenitor cells of ectodermal lineage according to claim 5, characterized in that, The conditions for the differential centrifugation method of cells are as follows: first, centrifuge the culture supernatant at 500-1000 g for 10-30 min, then collect the supernatant and centrifuge at 1500-2500 g for 20-40 min, then centrifuge at 15000-17000 g for 60-80 min to collect the precipitate, and finally resuspend and centrifuge at 15000-17000 g for 60-80 min to obtain extracellular vesicles of the progenitor cells of deer antler buds.

10. An extracellular vesicle from the progenitor cells of antler buds derived from an ectodermal lineage, characterized in that, It is prepared by the preparation method according to any one of claims 5-9.

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