Application of acellular extracellular matrix in skin photoaging resistance and medical instrument of acellular extracellular matrix

Abstract

The invention relates to an application of an acellular extracellular matrix as an active ingredient in preparation of a medicine or a medical instrument for treating skin aging. The acellular extracellular matrix comprises collagen and glycosaminoglycan, and the DNA content is lower than 50 ng / mg dry weight. Through verification, the technical scheme provided by the invention not only can recover the dermis volume, but also can reverse degenerative vascular changes. And the polypeptide has low immunogenicity, retains a plurality of bioactive components, has regeneration potential in a plurality of tissues, and can be effectively applied to the field of skin repair and regeneration when being used as an active component to prepare medicines or medical instruments.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a decellularized extracellular matrix and its application in the preparation of drugs and medical devices for promoting skin rejuvenation. Background Art

[0002] As the largest organ of the human body, the skin is first affected by various internal and external environmental factors, making it the earliest and most obvious sign of body aging. Skin aging and other skin disease states account for approximately 1.79% of the global disease burden and are the fourth leading cause of disability worldwide. Therefore, preventing and treating skin aging has become an urgent clinical problem. Among various factors, ultraviolet radiation (UVR), as an environmental oxidant and mutagen, has a particularly profound impact. It can accelerate skin aging through oxidative stress, DNA damage, and activation of matrix metalloproteinases (MMPs). This process is called skin photoaging, and its clinical features include the appearance of deep wrinkles, dryness, roughness, uneven pigmentation, reduced repair ability, and increased risk of malignant transformation. More importantly, photoaging accounts for approximately 80% of facial skin aging, significantly affecting the physical and mental health of patients.

[0003] Pathological changes occur throughout the entire thickness of photoaged skin, with the dermis being the most severely affected. Long-term UVR exposure induces fibroblast senescence, reduces collagen synthesis, and promotes the overproduction of the senescence-associated secretory phenotype (SASPs). These factors together result in excessive degradation of the extracellular matrix (ECM). In addition, UVR also disrupts the skin's immune system, leading to persistent low-grade inflammation and an immunosuppressed state. It is worth noting that the microvascular system of photoaged skin also undergoes significant changes. Although short-term UVR may cause acute skin damage, leading to vasodilation and neovascularization, long-term UVR exposure usually results in degenerative changes, including reduced vascular density and diameter, and decreased pericyte coverage on the vessel wall. The homeostasis of the microcirculation system is crucial for overall skin health. It not only facilitates the effective delivery and exchange of nutrients, signaling molecules, metabolic wastes, and thermal energy between tissues and blood but also plays an indispensable role in regulating immune responses and the processes of tissue repair and regeneration. Therefore, the state of the microvascular system is crucial during skin aging, and it is necessary to target the remodeling of the microvascular system as a key point for skin rejuvenation and regeneration.

[0004] In recent years, many innovative biomaterials for treating photoaged skin have emerged, including recombinant collagen, mesenchymal stem cells, extracellular vesicles, and nanoparticles, etc. These materials greatly complement traditional interventions such as sunscreen, laser therapy, dermal fillers, and topical retinoids. However, most current treatment strategies mainly focus on simple physical filling of the skin or functional regulation of major skin cells such as keratinocytes or dermal fibroblasts, and pay no attention to the remodeling of the microvascular system. Considering its important role in tissue regeneration, it is necessary to develop more targeted biomaterials that can not only effectively restore the dermal volume but also reverse the degenerative vascular changes caused by UVR radiation. Summary of the Invention

[0005] Decellularized Extracellular Matrix (dECM) is a biomaterial that retains bioactive components after removing cellular components during the decellularization process. It is well-known for its low immunogenicity and regenerative potential in various tissues, but its clinical application is limited due to the relatively scarce donor sources. With the progress of tissue engineering and regenerative medicine, the concept of "biobank" has gradually gained attention in fields such as medical research, clinical treatment, and drug development.

[0006] The present invention provides an application of decellularized extracellular matrix in the preparation of drugs for promoting skin repair. The application includes, but is not limited to, the application in the preparation of drugs for repairing aging skin, the application in the preparation of drugs for anti-aging and aging-related skin regeneration, and / or the application in the preparation of drugs for promoting the repair of skin defects.

[0007] The present invention also provides an application of decellularized extracellular matrix in the preparation of medical devices for promoting skin repair. The application includes, but is not limited to, the application in the preparation of medical devices for repairing aging skin, the application in the preparation of medical devices for anti-aging and aging-related skin regeneration, and / or the application in the preparation of medical devices for promoting the repair of skin defects.

[0008] The application includes, but is not limited to, the application of decellularized extracellular matrix in the preparation of drugs for rejuvenating aging skin by regulating the remodeling of the microvascular system.

[0009] The application also includes, but is not limited to, the application of the acellular extracellular matrix in the preparation of drugs for promoting the function of senescent endothelial cells, the application in the preparation of drugs for improving the proliferation ability of senescent vascular endothelial cells, and / or the application in the preparation of drugs for enhancing the tube formation ability and migration ability of senescent vascular endothelial cells, the application in the preparation of drugs for inhibiting the secretion of SASP in senescent fibroblasts, the application in the preparation of drugs for alleviating the senescent phenotype of senescent fibroblasts, the application in the preparation of drugs for improving the oxidative stress of senescent fibroblasts, and the application in the preparation of drugs for enhancing the migration ability of senescent fibroblasts.

[0010] The present invention provides a microneedle loaded with acellular extracellular matrix, which delivers the acellular extracellular matrix to the dermis to promote skin repair (including but not limited to the rejuvenation of photoaged skin) and its application as a medical device in skin anti-photoaging. The light mentioned herein refers to light with a wavelength of 280 nm to 400 nm, such as ultraviolet light.

[0011] Adipose tissue is rich in stem cells, easy to obtain and has ethical advantages, making it one of the ideal choices for tissue banks. One of the main pathological changes in the dermis during skin aging is the degradation of ECM. Using decellularized adipose-derived matrix (DAM) as a dermal filler can effectively supplement ECM components and counteract the adverse effects of skin aging. More importantly, the acellular extracellular matrix of adipose tissue, DAM, has the potential to promote neovascularization and treat photoaged skin by remodeling the microvascular system.

[0012] The present invention provides a DAM, which has a loose and porous white appearance, removes lipid droplets and nuclear structures, and the DNA content is lower than the literature-recommended standard of 50 ng / mg dry weight, retaining the effective biological components such as collagen and glycosaminoglycan to the greatest extent. The acellular extracellular matrix of adipose tissue is derived from liposuction, with relatively sufficient sources and significant ethical advantages, which is more conducive to clinical translational application. Active factors include, but are not limited to, collagen, glycosaminoglycan, growth factors, and cytokines, etc., which play active functions in regulating the remodeling of the microvascular system and promoting the repair of senescent skin.

[0013] The present invention also provides the application of DAM in the preparation of drugs for promoting skin repair. The application includes, but is not limited to, the application in the preparation of drugs for promoting the repair of senescent skin, the application in the preparation of drugs for anti-aging and promoting senescence-related skin regeneration, and / or the application in the preparation of drugs for promoting the repair of skin defects.

[0014] The present invention also provides an application of DAM in the preparation of medical devices for promoting skin repair. The application includes, but is not limited to, the application in the preparation of medical devices for repairing aging skin, the application in the preparation of medical devices for anti-aging and promoting skin regeneration related to aging, and / or the application in the preparation of medical devices for promoting the repair of skin defects.

[0015] The application includes, but is not limited to, the application of DAM in the preparation of drugs for rejuvenating aging skin by regulating the remodeling of the microvascular system.

[0016] The application also includes, but is not limited to, the application of DAM in the preparation of drugs for promoting the function of senescent endothelial cells, the application of drugs for improving the proliferation ability of senescent vascular endothelial cells, and / or the application of drugs for enhancing the tube formation ability and migration ability of senescent vascular endothelial cells, the application of drugs for inhibiting the secretion of SASP in senescent fibroblasts, the application of drugs for alleviating the senescent phenotype of senescent fibroblasts, the application of drugs for improving the oxidative stress of senescent fibroblasts, and the application of drugs for enhancing the migration ability of senescent fibroblasts.

[0017] The present invention provides a microneedle loaded with DAM, which delivers DAM to the dermis to promote skin repair (including but not limited to the rejuvenation of photoaged skin) and its application as a medical device in skin anti-photoaging. The light referred to herein is light with a wavelength of 280 nm to 400 nm, such as ultraviolet light.

[0018] The application includes, but is not limited to, the application of the adipose tissue acellular extracellular matrix DAM in the preparation of drugs for reducing the senescence marker SA-β-Gal and the inflammatory marker MMP1, the application of drugs for scavenging ROS production induced by senescence, and the application of drugs for promoting the production of Collagen I and Collagen III in senescent fibroblasts.

[0019] The DAM of the present invention is obtained by treating fresh adipose tissue through freeze-thaw cycles, mechanical emulsification, hypertonic saline, polyethylene glycol octylphenyl ether (Triton X-100) solution, and isopropanol for decellularization treatment. It can not only efficiently obtain acellular extracellular matrix while reducing the rejection risk caused by residual bioenzymes, but also effectively promote neovascularization, thus effectively combining DAM with promoting skin repair by targeting the remodeling of the microcirculation system.

[0020] The present invention also provides a method for preparing DAM. Fresh fat is washed with phosphate buffered saline (PBS), then undergoes 3 freeze-thaw cycles (for example, from -80°C to 37°C), and then the adipose tissue is disrupted by mechanical emulsification. Subsequently, it is treated with hypertonic saline, Triton X-100 solution, and isopropanol, and DAM is extracted.

[0021] In a specific embodiment, fresh adipose tissue obtained from liposuction is washed in PBS solution, undergoes 3 freeze-thaw cycles (for example, from -80°C to 37°C), and mechanical emulsification is carried out using a 1.2 mm nano-fat converter. Then the tissue is successively immersed in hypertonic saline solutions of 2 concentrations (0.5 M and 1.0 M) for 4 hours each, and then incubated overnight in ddH2O. After that, the tissue is treated in 1% Triton X-100 solution for 48 hours, and the solution is changed every 8 hours to facilitate the elution process. Subsequently, it is immersed in isopropanol for 8 hours to remove lipids. Between each detergent change, the mixture is centrifuged at 1000 rpm / min for 5 min, and the resulting precipitate is rinsed with ddH2O. Finally, the decellularized tissue is thoroughly rinsed with ddH2O (30 min, 3 times) and 75% ethanol (30 min, 3 times), and then freeze-dried in a vacuum freeze dryer. After that, 1 mg / ml porcine pepsin solution is added to the freeze-dried DAM, and it is magnetically stirred at 37°C for 24 hours. Then, an equal amount of NaOH solution is added to terminate the digestion reaction, and the solution is subsequently sterile filtered through a 0.22 μm filter to obtain a solution containing DAM.

[0022] In the present invention, DAM can be obtained by decellularizing adipose tissue. Specifically, after decellularization, the DNA content in DAM is reduced by more than 99%, reaching the literature-recommended standard of less than 50 ng / mg dry weight. At equal dry weights, the collagen and glycosaminoglycans (GAGs) content in freeze-dried DAM is approximately 4.5 times that of freeze-dried adipose tissue of the same weight, and it plays a role in promoting the repair of photoaged skin.

[0023] In a specific embodiment, preferably, when the concentration range of DAM is 0.5 - 2.0 mg / ml, it can rescue the cell proliferation, migration, tube formation, and angiogenesis-related gene expression of senescent human umbilical vein endothelial cells (HUVECs). The effect of the 1.0 mg / ml DAM solution is more significant.

[0024] The present invention also provides a DAM-functionalized microneedle patch, which includes DAM and may also include any suitable carrier, such as natural and / or synthetic materials that can be used as the loading carrier for DAM, like sodium alginate with porous structure, chitosan, hyaluronic acid, gelatin, collagen, sodium carboxymethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. In a specific embodiment, the DAM-functionalized microneedle patch loaded with DAM includes DAM and uses hyaluronic acid as the loading carrier for DAM.

[0025] In another specific embodiment, the DAM-functionalized microneedle patch loaded with DAM can use sodium alginate as the carrier, or be a chitosan microneedle patch, gelatin microneedle patch, collagen microneedle patch, sodium carboxymethylcellulose microneedle patch, polyvinylpyrrolidone microneedle patch, polyvinyl alcohol microneedle patch, etc., including natural microneedle patches and synthetic microneedle patches.

[0026] The present invention also provides the applications of the DAM-functionalized microneedle patch loaded with DAM, including but not limited to the application in preparing drugs for promoting the repair of aging skin, the application in preparing drugs for anti-aging and promoting skin regeneration related to anti-aging, and the application in preparing drugs for promoting the repair of skin defects. Preferably, it includes the application in preparing drugs for promoting the rejuvenation of aging skin by regulating the remodeling of the microvascular system. Preferably, it includes the application in preparing drugs for promoting the function of aging endothelial cells, improving the proliferation ability of aging vascular endothelial cells, preparing drugs for enhancing the tube formation ability and migration ability of aging vascular endothelial cells, preparing drugs for inhibiting the secretion of SASP in aging fibroblasts, preparing drugs for alleviating the aging phenotype of aging fibroblasts, preparing drugs for improving the oxidative stress of aging fibroblasts, and preparing drugs for enhancing the migration ability of aging fibroblasts.

[0027] Preferably, it includes the application in preparing drugs for reducing the aging marker senescence-associated β-galactosidase (SA-β-Gal) and the inflammatory marker matrix metalloproteinase 1 (MMP 1), the application in preparing drugs for scavenging the reactive oxygen species (ROS) generated by aging, and the application in preparing drugs for promoting the production of type I collagen (Collagen I) and type III collagen (Collagen III) in aging fibroblasts.

[0028] The present invention also provides the application of the DAM-functionalized microneedle patch as a drug for anti-aging and promoting skin regeneration related to anti-aging, preferably as a drug for promoting the rejuvenation of photoaged skin.

[0029] The present invention also provides a drug for skin repair, such as a drug for photoaging skin rejuvenation, which comprises the DAM-functionalized microneedle patch.

[0030] The beneficial effects of the present invention include but are not limited to: Most existing interventions for treating aging skin repair mainly focus on simple dermal filling or functional regulation of keratinocytes / skin fibroblasts, with limited attention paid to the remodeling of the microvascular system. Given the crucial role of the microvascular system in tissue regeneration, the DAM used in the present invention can not only restore the dermal volume but also reverse degenerative vascular changes. Moreover, the adipose tissue-derived acellular extracellular matrix DAM proposed by the present invention has low immunogenicity, retains a variety of bioactive components, has the potential for regeneration in various tissues, and can be effectively applied to the field of skin repair and regeneration. Brief Description of the Drawings

[0031] Figure 1 Characterization results of the prepared DAM; wherein, A is the optical microscope image of DAM, B is the SEM image of DAM, C is the H&E staining images of fresh adipose tissue and DAM, and D is the quantitative statistical chart of collagen, GAGs, and DNA contents before and after decellularization.

[0032] Figure 2 Results showing the effects of DAM at various concentrations on the proliferation of HUVECs (1 day and 3 days). The abscissa "Culture time" represents the number of days of culture, and the ordinate "OD value" represents the absorbance reading measured at 450 nm using an enzyme-linked immunosorbent assay reader, representing cell viability. The Control group is the control group, which is unstimulated HUVECs, and the remaining groups are photoaged HUVECs stimulated by ultraviolet B (UVB) and co-incubated with DAM at different concentrations (0.5, 1.0, 2.0, 4.0 mg / ml).

[0033] Figure 3It is a figure showing the results of the anti-photoaging cell-level verification of DAM. The Control group is the control group, which consists of unstimulated HUVECs, as well as each experimental group where photoaged HUVECs after UVB stimulation are co-incubated with DAM at various concentrations (0.5, 1.0, 2.0, 4.0 mg / ml). Among them, A is the figure showing the results of the scratch assay to detect the migration ability of photoaged HUVECs by DAM at various concentrations, B is the figure showing the results of the Transwell cell migration assay to detect the migration ability of photoaged HUVECs by DAM at various concentrations, C is the figure showing the results of the tube formation assay to detect the migration ability of photoaged HUVECs by different concentrations of DAM, D is the statistical chart of the scratch assay to detect the migration ability of photoaged HUVECs by DAM at various concentrations, E is the statistical chart of the Transwell cell migration assay to detect the migration ability of photoaged HUVECs by DAM at various concentrations, F is the statistical chart of the tube formation length of photoaged HUVECs by DAM at various concentrations in the tube formation assay, and G is the statistical chart of the branch number of photoaged HUVECs by DAM at various concentrations in the tube formation assay.

[0034] Figure 4 It is a heatmap of the RT-qPCR analysis results of angiogenesis-related markers (bFGF, HIF-1α, TGFβ, VEGFA) in photoaged HUVECs. The Control group is the control group, which consists of unstimulated HUVECs. The UVB group is photoaged HUVECs induced by UVB. The UVB / DAM group is photoaged HUVECs induced by UVB and co-incubated with 1.0 mg / ml DAM.

[0035] Figure 5 It is a figure showing the results of the effect of DAM treatment on HUVECs on HDFs. Among them, A is the figure showing the results of the senescence-related SA-β-Gal staining after photoaged fibroblasts are co-incubated with photoaged HUVECs for 72 h, B is the figure showing the intracellular ROS results detected by the DCFH-DA probe after photoaged fibroblasts are co-incubated with photoaged HUVECs for 72 h, C is the figure showing the results of the scratch assay to detect the migration ability after photoaged fibroblasts are co-incubated with photoaged HUVECs for 72 h, and D is the RT-qPCR statistical chart of markers related to collagen degradation and production (MMP1, MMP3, TIMP1, TIMP1 / MMP1, COL1A1, COL3A1) in photoaged fibroblasts (UVB is the control group, which is fibroblasts co-cultured with photoaged HUVECs through a Transwell chamber, and the photoaged HUVECs are not specially stimulated; the UVB / DAM group is photoaged HUVECs cultured in a medium containing 1 mg / ml DAM).

[0036] Figure 6Morphology diagram of DAM-loaded microneedle patch; among them, A is the digital camera image of the hyaluronic acid microneedle patch, B is the optical microscope image of the hyaluronic acid microneedle patch, and C is the SEM image of the hyaluronic acid microneedle patch.

[0037] Figure 7 Characterization result diagram of DAM-loaded microneedle patch; among them, A is the Fourier transform infrared spectroscopy diagram of hyaluronic acid (HA), decellularized extracellular matrix DAM of adipose tissue, and DAM-functionalized hyaluronic acid microneedle patch (HA / DAM), B is the mechanical test diagram of the hyaluronic acid microneedle patch (Hyaluronic acid, HA) and DAM-functionalized hyaluronic acid microneedle patch (HA / DAM) under vertical force, C is the digital photo of the micropore array formed after the microneedle patch loaded with trypan blue is inserted into porcine skin, and D is the hematoxylin-eosin (Hematoxylin and Eosin, H&E) staining diagram of the mouse skin tissue section after being inserted into the microneedle patch.

[0038] Figure 8 Macroscopic characterization diagram of photoaged skin after treatment; among them, A is the representative macroscopic images of each group of skin at days 0, 4, 7, 14, 21, and 28, and B is the quantitative statistical diagram of the wrinkles of each group of skin in Figure A over time (the vertical coordinate Relative wrinkle number (%) represents the relative wrinkle number (%), and the horizontal coordinate Time represents the relative observation time points).

[0039] Figure 9 Histological experimental result diagram after treatment; among them, A is the Masson trichrome staining image of photoaged skin of all groups after treatment (the part circled by the yellow dotted line is the dermis), B is the H&E staining image of photoaged skin of all groups after treatment, and C is the quantitative statistical diagram of the dermis thickness of each group in Figure A (the vertical coordinate Dermal thickness (μm) represents the relative dermis thickness).

[0040] Figure 10It is the histological staining diagram after treatment; among them, A is the immunofluorescence images of vascular markers CD31 (green) and α-SMA (red) in photoaged skin of all groups after treatment, B is the immunohistochemical diagram of type I collagen (COL1A1), C is the immunohistochemical image of type III collagen (COL3A1), D is the sirius red staining images of each group, E is the immunofluorescence image of matrix metalloproteinase MMP1 (green), F is the statistical chart of the immunofluorescence area of vascular markers CD31 (green) and α-SMA (red) in photoaged skin of all groups after treatment, G is the statistical chart of the immunohistochemical staining intensity of COL1A1, H is the statistical chart of the immunohistochemical staining intensity of COL3A1, I is the statistical chart of the ratio of type III / type I collagen, and J is the statistical chart of the fluorescence area of matrix metalloproteinase MMP1.

[0041] Figure 11 It is the RT-qPCR statistical chart of vascular-related markers (α-SMA, CD31, ANG1, VEGFA) in normal skin tissues of control group mice and photoaged skin tissues of UVB-induced mice.

[0042] Figure 12 It is the transcriptome sequencing analysis diagram of normal skin tissues of control group mice and photoaged skin tissues of UVB-induced mice; among them, A is the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in the transcriptome sequencing of normal skin tissues of control group mice and photoaged skin tissues of UVB-induced mice, and B is the circular heat map of differentially expressed genes related to the VEGFA signaling pathway.

[0043] Figure 13 It is the transcriptome sequencing analysis diagram of untreated photoaged HUVECs and photoaged mouse HUVECs treated with DAM; among them, A is the result diagram of KEGG enrichment analysis, and B is the KEGG network diagram representing the interaction between enriched pathways.

[0044] Figure 14 It is the Western blotting result diagram of each group of HUVECs. Among them, GAPDH is the internal reference protein, and the results of VEGFA, VEGFR2, phosphorylated VEGFR2 (p-VEGFR2), PI3K, phosphorylated PI3K (p-PI3K), Akt, and phosphorylated Akt (p-Akt) of unstimulated normal HUVECs (Control group), untreated photoaged HUVECs (UVB group), and photoaged mouse HUVECs treated with DAM (UVB / DAM group) are shown.

[0045] Figure 15This is a histological experimental result diagram of the dermis of photoaged skin after DAM filling treatment. Detailed implementation manners

[0046] In combination with the following specific embodiments and the accompanying drawings, the invention will be further described in detail. The processes, conditions, experimental methods, etc. for implementing the present invention, except for the specifically mentioned content below, are all common knowledge and well-known common sense in the art, and the present invention has no particularly restricted content.

[0047] 1. Preparation and characterization of adipose tissue-derived decellularized extracellular matrix DAM

[0048] 1.1 Preparation of adipose tissue-derived decellularized extracellular matrix DAM

[0049] In order to reduce the risk of rejection while ensuring the yield of decellularized extracellular matrix, we obtained decellularized extracellular matrix from adipose tissue by an enzyme-free method. First, fresh adipose tissue was collected. The adipose tissue samples were obtained from the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine and approved by the Research Ethics Committee, in line with the Helsinki Declaration. After washing with PBS solution and three cycles of -80°C / 37°C freeze-thawing, it was mechanically emulsified using a nano-fat converter with a diameter of 1.2 mm. Next, the tissue was soaked in 0.5 M and 1.0 M hypertonic saline for 4 h respectively, and then overnight in deionized water. After that, the tissue was soaked in a 1% Triton X-100 solution for elution, and this process lasted for 48 h, with the solution changed every 8 h. Then, the obtained decellularized tissue was treated with isopropanol for 8 h to remove grease. The above operations were all completed in a 37°C constant temperature shaker, and before changing each detergent, the mixture was centrifuged (1000 rpm, 5 min), and the precipitate was washed with deionized water. Finally, the decellularized extracellular matrix was washed with deionized water (30 min / time × 3 times) and 75% alcohol (30 min / time × 3 times) respectively, and then freeze-dried using a vacuum freeze dryer.

[0050] For the convenience of cell culture and the preparation of dissolving microneedles, we prepared the freeze-dried adipose tissue-derived decellularized extracellular matrix DAM into a solution form and stored it in a -80°C refrigerator. Specifically, 1 mg / ml of porcine pepsin was added to DAM and magnetically stirred at 37°C for 24 h, then an equal volume of NaOH solution was added to terminate digestion, and finally the solution was sterilized through a 0.22 μm filter and stored for later use.

[0051] 1.2 Characterization of adipose tissue-derived decellularized extracellular matrix DAM

[0052] Subsequently, we characterized the obtained adipose tissue-derived decellularized extracellular matrix DAM. After freeze-drying the DAM obtained by the above method, it presented a loose white flocculent appearance (Figure 1 A), and scanning electron microscopy images showed that it retained the three-dimensional porous fibrillar network structure ( Figure 1 B). Histological staining confirmed the effective decellularization of DAM, and no lipid droplets or cell nuclei were seen in the field of view ( Figure 1 C).

[0053] To further determine the decellularization effect, we quantified DNA, collagen, and glycosaminoglycans in DAM to judge the retention of immunogenic components and inherent active components ( Figure 1 D): The DNA content achieved an elution rate higher than 99% and was lower than the decellularization standard of 50 ng / mg dry weight in the literature; due to the loss of water from DAM during freeze-drying and its denser structure compared to adipose tissue, the collagen and glycosaminoglycan components in DAM were approximately 4.5 times that of adipose tissue under the same dry weight. These results indicate that the method used in this example efficiently removed the immunogenic components in the tissue while achieving the retention of active components.

[0054] As Figure 1 shown, (A) Optical microscope image of DAM (scale bar: 1 mm). (B) Scanning electron microscope (SEM) image of DAM (left scale bar: 100 μm, right scale bar: 50 μm). (C) Hematoxylin and eosin (H&E) staining images of fresh adipose tissue and decellularized adipose tissue extracellular matrix DAM (scale bar: 50 μm). (D) Quantitative analysis of collagen, GAGs, and DNA content before and after decellularization.

[0055] 2. DAM can promote the cell functions of photoaged endothelial cells

[0056] The normal exercise of vascular endothelial cell functions is crucial for angiogenesis and the maintenance of dermal microenvironment homeostasis. Different concentrations of DAM (0 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 4.0 mg / ml) were used to co-culture with photoaged endothelial cells, and in vitro experiments were conducted to detect the regulatory effect of DAM on photoaged HUVECs. The cell proliferation detected by the CCK-8 assay showed that DAM at concentrations of 0.5 - 2.0 mg / ml had the ability to promote cell proliferation ( Figure 2 ). As Figure 2 shown, the effects of different concentrations of DAM on the proliferation of HUVECs (1 day and 3 days). The abscissa Culture time represents the number of days of culture, and the ordinate OD value represents the absorbance reading at 450 nm using an enzyme-linked immunosorbent assay reader, representing cell viability. The Control group is the control group, which is unstimulated HUVECs, and the remaining groups are photoaged HUVECs stimulated by UVB and different concentrations

[0057] (0.5, 1.0, 2.0, 4.0 mg / ml) Co-incubation with DAM.

[0058] To evaluate the regulatory effect of DAM on cell migration ability, scratch assays ( Figure 3 A, Figure 3 D) and Transwell cell migration assays ( Figure 3 B, E) were performed on cells treated with different concentrations of DAM (0 mg / ml, 0.5 mg / ml, 1.0 mg / ml, 2.0 mg / ml, 4.0 mg / ml). The experimental results showed that DAM at 0.5 - 2.0 mg / ml had a significant effect on rescuing cell migration ability. In addition, tube formation assays were used to evaluate the angiogenesis ability of endothelial cells ( Figure 3 C, Figure 3 F, Figure 3 G). The results showed that DAM at 0.5 - 2.0 mg / ml enhanced the inhibited angiogenesis ability and had a rescue effect on the formation of tubular structures and branch points.

[0059] qRT-PCR analysis also showed similar results. After co-incubating photoaged HUVECs with 1.0 mg / ml DAM for 72 h, the expression levels of angiogenesis-related genes (bGFG, HIF-1α, TGFβ, VEGFA) were significantly upregulated ( Figure 4 ). As shown in Figure 4 the RT-qPCR analysis of angiogenesis-related markers (bFGF, HIF-1α, TGFβ, VEGFA) in photoaged HUVECs, which was represented by a heatmap. The Control group was the control group, which were unstimulated HUVECs; the UVB group was UVB-induced photoaged HUVECs; the UVB / DAM group was UVB-induced photoaged HUVECs co-incubated with 1.0 mg / ml DAM.

[0060] 3. HUVECs treated with DAM can alleviate the senescent phenotype of senescent fibroblasts through cell-cell communication.

[0061] As one of the most important cell types in the dermis, fibroblasts play a key role in maintaining the structure and function of the skin. Therefore, their functional state greatly affects tissue regeneration and rejuvenation. Thus, a co-culture system of HUVECs and human dermal fibroblasts (HDFs) was constructed, and photoaged HDFs were co-cultured with HUVECs pretreated with / without 1.0 mg / ml DAM for comparison to explore the interaction between the two.

[0062] As Figure 5As shown in Figure A, DAM-treated HUVECs reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in HDFs, indicating a reduced senescent phenotype in the cells. In addition, DCFH-DA probe staining showed that DAM-treated HUVECs could significantly scavenge the ROS production induced by ultraviolet irradiation, indicating an improved oxidative metabolism in photoaged fibroblasts ( Figure 5 B). The results of the scratch assay showed that DAM-treated HUVECs enhanced the cell migration ability of HDFs through cell-cell communication ( Figure 5 C). In addition, the expression levels of related biomarkers were evaluated by qRT-PCR, including factors related to the balance of ECM remodeling and degradation: MMPs (MMP1 and MMP3) and tissue inhibitor of metalloproteinase-1 (TIMP-1); and factors related to synthesis and secretion: transforming growth factor-β (TGF-β), collagen type I (Col I), and collagen type III (Col III) ( Figure 5 D). The results showed that HDFs co-cultured with DAM-treated HUVECs showed a stronger tendency for ECM synthesis, with reduced expression of MMPs and increased expression of the antagonistic TIMP-1. At the same time, the expression levels of factors related to collagen synthesis were also significantly increased, suggesting that the restoration of HUVEC cell function can restore the function of HDFs through cell-cell interactions, further contributing to the regeneration and rejuvenation of the dermis.

[0063] As Figure 5 shown, (A) Senescence-associated SA-β-Gal staining of photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h (scale bar: 100 μm). (B) Detection of intracellular ROS production in photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h by DCFH-DA probe (scale bar: 200 μm). (C) Detection of migration ability of photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h by scratch assay (scale bar: 200 μm). (D) RT-qPCR analysis of collagen degradation and production-related markers (MMP1, MMP3, TIMP1, TIMP1 / MMP1, COL1A1, COL3A1) in photoaged fibroblasts. UVB was the control group, which were fibroblasts co-cultured with photoaged HUVECs through Transwell chambers, and the photoaged HUVECs were not specially stimulated. In the UVB / DAM group, photoaged HUVECs were cultured in a medium containing 1 mg / ml DAM.

[0064] 4. DAM promotes the repair of photoaged skin in mice.

[0065] Using 10 - 12 - week - old photoaged mice as experimental subjects, the effect of DAM on the repair of senescent skin defects was explored. In the local treatment of skin - related diseases, how to penetrate the skin keratin barrier to achieve efficient delivery of active ingredients is a key issue. As a minimally invasive and simple drug delivery system, microneedles have the advantages of controllable drug delivery and strong loading capacity, and are widely used in various studies such as tissue regeneration, wound healing, and skin rejuvenation.

[0066] Since hyaluronic acid (HA), as a commonly used dermal filler in clinics, has good biosecurity and the ability to stimulate the proliferation of dermal fibroblasts and promote collagen secretion. Therefore, in this example, DAM was dispersed in hyaluronic acid. The specific method is as follows:

[0067] Take two sterile syringes, labeled as syringe A and syringe B respectively. Syringe A draws hyaluronic acid gel filling material (HA); syringe B draws DAM solution, and the volume ratio of the two is 7:3 (HA:DAM). Connect the outlet ends of syringe A and syringe B through a medical - grade three - way valve to ensure airtightness. Alternately push the pistons of syringe A and syringe B, and repeat the injection 30 times at a frequency of 20 - 30 times per minute. Keep the three - way valve passage open during the injection process, so that the HA gel and DAM solution are fully homogenized through shear force and convection to form a DAM - doped composite filling material (HA / DAM).

[0068] Randomly divide the photoaged model mice into groups and perform corresponding interventions on the 0th day after modeling: (1) HA group: Subcutaneously inject hyaluronic acid gel filling material into the back of photoaged mice; (2) HA / DAM group: Fill the HA / DAM composite material into the dermis of the back of photoaged mice. Sample and perform tissue section and staining experiments at 28 days. The results are as follows Figure 15 As shown, compared with the HA group, the thickness of the dermis layer of the back skin of mice filled with HA / DAM increased significantly, indicating that the composite filling material doped with DAM is more effective in promoting the synthesis of collagen fibers in photoaged skin tissue.

[0069] Dry hyaluronic acid has some strength, so this example also uses it as the matrix material for dissolving microneedles.

[0070] 4.1 Preparation and Characterization of DAM - Functionalized Microneedle Patches

[0071] After configuring hyaluronic acid powder into a 15wt% solution, pour it into a mold, remove air bubbles in a vacuum drying oven, and then transfer it to 37°C for dehydration and curing to obtain a microneedle patch. No obvious deformation or tip loss was observed in the photos taken by a digital camera ( Figure 6 A). Microscopic images and scanning electron microscopy showed that the tips of the microneedles were neat and sharp, without visible fractures (Figure 6 B, Figure 6 C).

[0072] Figure 7 Figure A shows the FTIR spectra of DAM, HA, and the functionalized microneedle patch loaded with DAM. Characteristic peaks of DAM and HA are visible in the functionalized microneedle spectrum, indicating that DAM was successfully loaded into the microneedles and did not undergo a chemical reaction with HA. In clinical use, microneedles need to have appropriate mechanical strength to ensure that they can effectively penetrate the skin stratum corneum barrier without significant deformation or fracture. As Figure 7 shown in Figure B, the incorporation of DAM enhanced the mechanical properties of the microneedle patch to a certain extent, and the load fracture force of the blank microneedles composed of 15 wt% HA already met the minimum average force (0.1 N) required for skin penetration. To further verify its skin puncture ability, tests were conducted on pig skin and mouse skin. As Figure 7 shown in Figure C, the microneedle patch loaded with trypan blue dye in the matrix material was able to leave a complete blue pinhole array on fresh pig skin, indicating that the microneedles could successfully penetrate the skin. The hematoxylin-eosin staining results of the tissue sections of mouse skin showed that the needle tips successfully penetrated the stratum corneum and entered the dermis( Figure 7 Figure D), and the above results indicate that the prepared dissolving microneedles can meet the clinical application scenario of transdermal drug delivery.

[0073] 4.2 DAM-functionalized microneedle patch promotes the repair of photoaged skin

[0074] The mice with photoaged skin models were randomly grouped and corresponding interventions were carried out on days 0, 4, 7, 14, and 21 after modeling: (1) Control group: Normal mice without ultraviolet irradiation did not receive any intervention except for hair removal at each time point; (2) UVB group: Photoaged mice did not receive any treatment; (3) RA group: 0.05% retinoic acid cream was applied to the dorsal skin of photoaged mice; (4) HA group; Blank microneedles without loaded DAM were injected into the dorsal skin of photoaged mice; (5) HA / DAM group: Functionalized microneedles loaded with DAM were injected into the dorsal skin of photoaged mice.

[0075] At each time point, photos were taken with a digital camera to monitor the changes in wrinkles on the dorsal skin of each group. Compared with the UVB group, the RA, HA, and HA / DAM groups had varying degrees of rescue effects on the deep wrinkles on the skin surface, and the effect of the HA / DAM group was the most obvious, similar to the skin appearance of the Control group( Figure 8 Figure A, Figure 8 Figure B). As Figure 8 and Figure 9 shown, the H&E and Masson staining results of the skin tissue sections were basically consistent with the macroscopic manifestations, and the HA / DAM group had the best effect on improving the dermal thickness.

[0076] As Figure 10 shown, the most obvious microvascular remodeling phenomenon occurred in the HA / DAM group, with a significant increase in blood vessel density, and the blood vessel structure being more complete and orderly, fully demonstrating that HA / DAM can effectively promote angiogenesis in vivo. In addition, in the HA / DAM group, the contents of type I and type III collagen were significantly increased, and the ratio of type III / I collagen returned to the normal level, indicating that HA / DAM treatment can effectively activate the collagen synthesis pathway and improve the tissue structure of photoaged skin. Meanwhile, the abnormally elevated expression of MMP-1 in photoaged skin returned to the normal level after DAM treatment, indicating that DAM can effectively inhibit the overexpression of MMP-1, thereby reducing the degradation of collagen.

[0077] 5. Role of the microvascular system therein

[0078] Using a photoaging model, the dorsal skin of C57 mice was irradiated with UVB for 8 weeks. The qRT-PCR results showed that the expression of angiogenesis-related genes in the dorsal skin tissue of mice decreased after UVB irradiation ( Figure 11 ).

[0079] To better reveal the changes in angiogenesis-related genes in photoaged skin, transcriptome sequencing was performed on the dorsal skin of normal mice and photoaged mice. KEGG enrichment analysis showed that compared with normal skin, the differentially expressed genes in photoaged skin were significantly enriched in the angiogenesis-related pathways ( Figure 12 A), Figure 12 and Figure B shows significant differential expression of genes related to the VEGF signaling pathway between the two groups ( Figure 12 B).

[0080] Transcriptome sequencing was performed on photoaged HUVECs (UVB group) and photoaged HUVECs treated with 1 mg / ml DAM for 72 h (UVB / DAM). The results of KEGG enrichment analysis showed that the differentially expressed genes of the cells after DAM treatment were mainly enriched in pathways closely related to angiogenesis such as the "PI3K-Akt signaling pathway", "MAPK signaling pathway", and "mTOR signaling pathway" ( Figure 13 A). The network diagram drawn based on the enriched pathways showed that the "PI3K-Akt signaling pathway" played a core pathway role therein ( Figure 13 B). As Figure 13 shown, (A) KEGG enrichment analysis and (B) KEGG network diagram representing the interaction between enriched pathways of transcriptome sequencing of untreated photoaged HUVECs and photoaged mouse HUVECs treated with DAM. The Western Blot experimental results verified this pathway ( Figure 14 ).

[0081] In this embodiment, DAM is applied to the remodeling of the local microvascular system, and at the same time, it is proposed to combine the effect of microvascular system remodeling brought by DAM with the intervention of senescence-related diseases, resulting in significant beneficial effects. The bioactive components carried in DAM can promote neovascularization, regulate the remodeling of the microvascular system, and are more conducive to the regeneration of senescent skin, thus making DAM have better efficacy in promoting the rejuvenation of senescent skin.

[0082] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, the changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the scope of protection is defined by the appended claims.

Claims

1. Use of acellular extracellular matrix as an active ingredient in the preparation of a drug or medical device for treating skin aging, characterized in that The decellularized extracellular matrix includes collagen and glycosaminoglycan, and the DNA content is lower than 50ng / mg dry weight; the skin is the dermis, and the aging is wrinkling due to exposure to light.

2. The application according to claim 1, wherein The light is a light with a wavelength of 280nm to 400nm.

3. The application according to claim 1, characterized in that The decellularized extracellular matrix is ​​prepared by washing fresh fat with phosphate buffer, subjecting it to 3 freeze-thaw cycles from -80°C to 37°C, destroying the adipose tissue by mechanical emulsification, and then successively subjecting it to 0.5M and 1.0M hypertonic saline solutions, as well as 1% TritonX-100 solution for 48 hours, soaking it in isopropanol for 8 hours, and centrifuging and washing it to obtain it.

4. The application according to claim 1, characterized in that The medical device is a microneedle or a microneedle patch, and the decellularized extracellular matrix is ​​carried in the microneedle or the microneedle patch as an active component.

5. The application according to claim 1, wherein The medical device is a filler, which is filled in the dermis.

6. The application according to claim 1, characterized in that Used to prepare drugs for reducing the aging marker senescence-associated β-galactosidase and the inflammatory marker matrix metalloproteinase 1, or used to prepare drugs for clearing the production of reactive oxygen induced by aging, or used to prepare drugs for promoting the production of type I collagen and type III collagen in senescent fibroblasts, or used to prepare drugs for improving the proliferation ability of senescent vascular endothelial cells, or used to prepare drugs for enhancing the tube-forming ability of vascular endothelial cells, or used to prepare drugs for enhancing the migration ability of vascular endothelial cells.

7. A decellularized extracellular matrix microneedle patch, characterized in that, The acellular extracellular matrix is ​​made of active ingredients loaded in a load carrier and carried to the dermis by microneedles to promote the formation of new blood vessels in the dermis and reshape the microvascular system.

8. The microneedle patch according to claim 7, wherein The load carrier is selected from hyaluronic acid.

9. The microneedle patch according to claim 7, wherein The load carrier is selected from one or more of porous sodium alginate, chitosan, gelatin, collagen, sodium carboxymethyl cellulose, polyvinyl pyrrolidone and polyvinyl alcohol.

10. The microneedle patch according to claim 7, wherein The dermis ages due to exposure to light.

Images