Application of decellularized extracellular matrix in skin anti-photoaging and its drug and device

Abstract

The application of a kind of decellular extracellular matrix as active ingredient in the preparation of drug or medical device for treating skin aging. The decellular extracellular matrix includes collagen and glycosaminoglycan, and the DNA content is less than 50 ng / mg dry weight. It is verified that the technical scheme of the application can not only restore the volume of dermis, but also reverse the degenerative vascular changes. And it has low immunogenicity, retains a variety of bioactive ingredients, has a regeneration potential in a variety of tissues, and the drug or medical device made of it as active ingredient can be effectively applied in the field of skin repair and regeneration.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a decellularized extracellular matrix and application thereof in preparation of medicines and medical devices for promoting skin rejuvenation. BACKGROUND

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

[0003] Pathological changes occur throughout the layers of photoaged skin, with the dermis being the most severely affected. Long-term UVR exposure induces fibroblast senescence, reduces collagen synthesis, and promotes excessive production of the senescence-associated secretory phenotype (SASPs), all of which contribute to excessive degradation of the extracellular matrix (ECM). In addition, UVR also damages the skin's immune system, leading to a state of persistent low-level inflammation and immune suppression. Notably, the microvascular system of photoaged skin also undergoes significant changes. While short-term UVR can cause acute skin damage, leading to vasodilation and neovascularization, long-term UVR exposure typically results in regressive changes, including reduced blood vessel density and diameter, and decreased pericyte coverage on the vessel wall. The homeostasis of the microcirculatory system is crucial for overall skin health, as it not only facilitates the efficient transport and exchange of nutrients, signaling molecules, metabolic waste and thermal energy between tissues and blood, but also plays an indispensable role in regulating immune responses and processes of tissue repair and regeneration. Therefore, the status of the microvascular system is crucial in the process of skin aging, and it is necessary to target the remodeling of the microvascular system as a key target for skin rejuvenation and regeneration.

[0004] In recent years, a number of innovative biomaterials have emerged for the treatment of photoaged skin, including recombinant collagens, mesenchymal stem cells, extracellular vesicles, and nanoparticles, which greatly complement traditional interventions such as sunscreens, laser therapy, dermal fillers, and topical retinoids. However, most current therapeutic strategies mainly focus on simple physical filling of the skin or functional regulation of skin primary cells such as keratinocytes or dermal fibroblasts, with little attention paid to the remodeling of the microvascular system. Given its important role in tissue regeneration, it is necessary to develop more targeted biomaterials that not only effectively restore dermal volume but also reverse the degenerative vascular changes caused by UVR radiation. SUMMARY

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

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

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

[0008] The use includes but is not limited to use of a decellularized extracellular matrix in the preparation of a medicament for rejuvenating photoaged skin by regulating microvascular remodeling.

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

[0010] The application provides a decellularized extracellular matrix microneedle, which delivers the decellularized extracellular matrix to the dermis to promote skin repair (including but not limited to rejuvenation of photoaged skin) and use as a medical device in the application of skin anti-photoaging. The so-called light is 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 of 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, adipose tissue decellularized extracellular matrix DAM can promote neovascularization, and has the potential to treat photoaged skin by remodeling the microvascular system.

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

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

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

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

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

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

[0018] The use includes, but is not limited to, the use of the adipose tissue decellular extracellular matrix DAM in the preparation of drugs for reducing the aging marker SA-β-Gal and the inflammation marker MMP1, the preparation of drugs for eliminating aging-induced ROS production, and the preparation of drugs for promoting the production of Collagen I and Collagen III in aged fibroblasts.

[0019] The DAM of the application is obtained by treating fresh adipose tissue by freeze-thaw cycles, mechanical emulsification, hypertonic saline, Triton X-100 solution and isopropyl alcohol for decellularization. Not only can the decellular extracellular matrix be efficiently obtained while reducing the risk of rejection caused by residual biological enzymes, but also the formation of new blood vessels is effectively promoted, so that DAM is effectively combined with remodeling of the microcirculation system to promote skin repair.

[0020] The application also provides a method for preparing DAM. Fresh fat is washed with phosphate buffered saline (PBS) buffer, then subjected to three freeze-thaw cycles (for example, -80°C to 37°C), and then subjected to mechanical emulsification to destroy fat tissue. After treatment with hypertonic saline, Triton X-100 solution and isopropanol, DAM is extracted.

[0021] In a specific embodiment, fresh fat tissue obtained from liposuction is washed in PBS solution, subjected to three freeze-thaw cycles (for example, -80°C to 37°C), and subjected to mechanical emulsification using a 1.2 mm nanofat converter. The tissue is then sequentially immersed in two concentrations of hypertonic saline solution (0.5M and 1.0M) for 4 hours each, followed by overnight incubation in ddH2O. Subsequently, the tissue is treated in a 1% Triton X-100 solution for 48 hours, with the solution being replaced every 8 hours to facilitate the elution process. Subsequently, the tissue is immersed in isopropanol for 8 hours to remove lipids. Between each detergent change, the mixture is centrifuged at a speed of 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 this, a 1 mg / ml solution of porcine pepsin is added to the freeze-dried DAM, and the solution is magnetically stirred at 37°C for 24 hours. Subsequently, an equal amount of NaOH solution is added to terminate the digestion reaction, and the solution is then sterile-filtered through a 0.22 μm filter to obtain a solution containing DAM.

[0022] In the present application, DAM can be obtained by decellularizing fat tissue. Specifically, the DNA content of the decellularized DAM is reduced by more than 99%, reaching a level of less than 50 ng / mg of dry weight, which meets the recommended standard in the literature. Under the same dry weight, the collagen and glycosaminoglycans (GAGs) content in the freeze-dried DAM is about 4.5 times that in the freeze-dried fat tissue of the same weight, and plays a role in promoting repair of photoaged skin.

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

[0024] The present application also provides a DAM functionalized microneedle patch, which comprises DAMs, and can further comprise any suitable carrier, such as porous structure sodium alginate, chitosan, hyaluronic acid, gelatin, collagen, sodium carboxymethyl cellulose, polyvinylpyrrolidone and polyvinyl alcohol, and the like natural and / or synthetic carriers that can be used as a loading carrier for DAMs. In a specific embodiment, the DAM-loaded functionalized microneedle patch comprises DAMs, and hyaluronic acid is used as a loading carrier for the DAMs.

[0025] In another specific embodiment, the DAM-loaded functionalized microneedle patch can be a sodium alginate carrier, or a chitosan microneedle patch, a gelatin microneedle patch, a collagen microneedle patch, a sodium carboxymethyl cellulose microneedle patch, a polyvinylpyrrolidone microneedle patch, and a polyvinyl alcohol microneedle patch, and the like natural microneedle patches and synthetic microneedle patches.

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

[0027] Preferably, the use in the preparation of a drug for reducing senescence markers senescence-associated β-galactosidase (SA-β-Gal) and inflammation marker matrix metalloproteinase 1 (MMP 1), the use in the preparation of a drug for eliminating senescence-induced reactive oxygen species (ROS) production, the use in the preparation of a drug for promoting the production of collagen I and collagen III in senescent fibroblasts.

[0028] The present application also provides the use of the DAM functionalized microneedle patch as an anti-aging and senescence-related skin regeneration drug, preferably as a drug for rejuvenating photoaged skin.

[0029] The present application also provides a medicine for skin repair, for example, a medicine for rejuvenation of photoaged skin, comprising the DAM functionalized microneedle patch.

[0030] The benefits of the present application include but are not limited to: most existing interventions for treating aged skin repair mainly focus on simple dermal filling or functional regulation of keratinocytes / skin fibroblasts, and limited attention is paid to the remodeling of the microvascular system. Given the key role of the microvascular system in tissue regeneration, the DAM used in the present application can not only restore the dermal volume, but also reverse the degenerative vascular changes. And the adipose tissue decellularized extracellular matrix DAM proposed in the present application has low immunogenicity, retains a variety of bioactive ingredients, has regeneration potential in multiple tissues, and can be effectively applied in the field of skin repair and regeneration. BRIEF DESCRIPTION OF DRAWINGS

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

[0032] Figure 2 A result diagram showing the effect of DAM at various concentrations on the proliferation of HUVECs (1 day and 3 days), wherein the abscissa Culture time represents the culture days, and the ordinate OD value represents the absorbance reading at 450 nm using an enzyme marker, representing the cell viability; the Control group is the control group, which is unstimulated HUVECs, and the rest of the groups are photoaged HUVECs after ultraviolet B (UVB) stimulation and co-incubation with different concentrations (0.5, 1.0, 2.0, 4.0 mg / ml) of DAM.

[0033] Figure 3Figure for DAM anti-photoaging cell level verification results, Control group is the control group, which is unstimulated HUVECs, and photoaged HUVECs after UVB stimulation and each experimental group co-incubated with each concentration (0.5, 1.0, 2.0, 4.0 mg / ml) DAM; among them, A is the result figure of scratch test for detecting the migration ability of photoaged HUVECs of each concentration of DAM, B is the result figure of Transwell cell migration experiment for detecting the migration ability of photoaged HUVECs of each concentration of DAM, C is the result figure of tube formation experiment for detecting the migration ability of photoaged HUVECs of different concentrations of DAM, D is the statistical chart of scratch test for detecting the migration ability of photoaged HUVECs of each concentration of DAM, E is the statistical chart of Transwell cell migration experiment for detecting the migration ability of photoaged HUVECs of each concentration of DAM, F is the statistical chart of tube formation experiment for detecting the tube length of photoaged HUVECs of each concentration of DAM, G is the statistical chart of tube formation experiment for detecting the branch number of photoaged HUVECs of each concentration of DAM.

[0034] Figure 4 Figure for RT-qPCR analysis results of hemangiogenesis related markers (bFGF, HIF-1α, TGFβ, VEGFA) in photoaged HUVECs; Control group is the control group, which is unstimulated HUVECs; UVB group is UVB induced photoaged HUVECs; UVB / DAM is UVB induced photoaged HUVECs co-incubated with 1.0 mg / ml DAM.

[0035] Figure 5 Figure for the effect of DAM treated HUVECs or not on HDFs; among them, A is the result figure of senescence associated SA-β-Gal staining of photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h, B is the result figure of DCFH-DA probe for detecting intracellular ROS of photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h, C is the result figure of migration ability of photoaged fibroblasts co-incubated with photoaged HUVECs for 72 h by scratch test, D is the RT-qPCR statistical chart of collagen degradation and generation related markers (MMP1, MMP3, TIMP1, TIMP1 / MMP1, COL1A1, COL3A1) in photoaged fibroblasts (UVB is the control group, which is fibroblasts co-cultured with photoaged HUVECs in Transwell chamber, and photoaged HUVECs are not specially stimulated; UVB / DAM group is photoaged HUVECs cultured in medium containing 1 mg / ml DAM).

[0036] Figure 6Figure 1 is a morphological chart of DAM-loaded microneedle patches; wherein A is a digital camera chart of hyaluronic acid microneedle patches, B is an optical microscope chart of hyaluronic acid microneedle patches, and C is a SEM chart of hyaluronic acid microneedle patches.

[0037] Figure 7 Figure 2 is a characterization result chart of DAM-loaded microneedle patches; wherein A is a Fourier infrared spectrogram of hyaluronic acid (HA), adipose tissue decellularized extracellular matrix DAM, and DAM-functionalized hyaluronic acid microneedle patches (HA / DAM), B is a mechanical test chart of hyaluronic acid microneedle patches (HA) and DAM-functionalized hyaluronic acid microneedle patches (HA / DAM) under vertical force, C is a digital photo of micropore arrays formed after microneedle patches loaded with trypan blue were inserted into pig skin, and D is a hematoxylin and eosin (H&E) staining chart of mouse skin tissue sections after microneedle patch insertion.

[0038] Figure 8 Figure 3 is a macroscopic characterization chart of treated photoaged skin; wherein A is a representative macroscopic image of skin in each group on days 0, 4, 7, 14, 21, and 28, and B is a quantitative statistical chart of wrinkles in 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 node).

[0039] Figure 9 Figure 4 is a histological experimental result chart after treatment; wherein A is a Masson trichrome staining image of photoaged skin after treatment in all groups (the portion circled by a yellow dashed line is the dermis layer), B is an H&E staining image of photoaged skin after treatment in all groups, and C is a quantitative statistical chart of dermis layer thickness in each group in Figure A (the vertical coordinate Dermal thickness (μm) represents the relative dermis thickness).

[0040] Figure 10Figure 6 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue. Figure 7 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue.

[0041] Figure 11 Figure 6 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue. Figure 7 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue.

[0042] Figure 12 Figure 6 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue. Figure 7 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue.

[0043] Figure 13 Figure 6 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue. Figure 7 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue.

[0044] Figure 14 Figure 6 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue. Figure 7 is a graph showing the RT-qPCR results of the blood vessel related markers (a-SMA, CD31, ANG1, VEGFA) in the normal skin tissue of the control group mice and the UVB-induced photoaged mouse skin tissue.

[0045] Figure 15Figure of the histological experimental results of the dermis layer of photoaged skin after DAM filling treatment. DETAILED DESCRIPTION

[0046] The application will be further described with the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitation.

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

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

[0049] In order to reduce the risk of rejection while ensuring the yield of decellularized extracellular matrix, we obtained the adipose tissue-derived decellularized extracellular matrix by non-enzyme method. First, fresh adipose tissue was collected, and the adipose tissue sample was obtained from the Ninth People's Hospital of Shanghai Jiaotong University School of Medicine with the approval of the research ethics committee and in accordance with the Helsinki Declaration. After washing with PBS solution and 3 cycles of-80℃ / 37℃ freeze-thawing, the tissue was mechanically emulsified using a nano-fat converter with a diameter of 1.2 mm. Next, the tissue was soaked in 0.5M and 1.0M hypertonic saline for 4h, respectively, and then soaked in deionized water overnight. After that, we soaked the tissue in a 1% Triton X-100 solution for elution, which lasted for 48h, and the solution was replaced every 8h. Then, the obtained decellularized tissue was treated with isopropanol for 8h to remove oil. The above operations were all completed in a 37℃ constant temperature shaker, and before changing each washing agent, the mixture was centrifuged (1000rpm, 5min) and the precipitate was washed with deionized water. Finally, the decellularized extracellular matrix was washed with deionized water (30min / time x 3 times) and 75% alcohol (30min / time x 3 times), respectively, and then freeze-dried using a vacuum freeze dryer.

[0050] In order to facilitate cell culture and the preparation of dissolving microneedles, we prepared the freeze-dried adipose tissue decellularized extracellular matrix DAM into a solution form and stored it in a-80℃ refrigerator. Specifically, after adding 1mg / ml porcine pepsin to DAM and stirring magnetically at 37℃ for 24h, we added an equal volume of NaOH solution to terminate the digestion, and finally sterilized the solution through a 0.22μm filter for storage.

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

[0052] Subsequently, we characterized the obtained adipose tissue decellularized extracellular matrix DAM. After freeze-drying of the DAM obtained by the above method, it showed a loose appearance of white flocculation (Figure 1 A), and scanning electron microscope images show that it retains a three-dimensional porous fibrillar network structure Figure 1 B). Figure 1 C).

[0053] To further determine the decellularization effect, we quantified the DNA, collagen and glycosaminoglycan in DAM to determine the retention of immunogenic components and intrinsic active components Figure 1 D): DNA content achieved more than 99% elution rate, and lower than the decellularization standard of 50 ng / mg dry weight in the literature; due to the process of freeze-drying, DAM loses water and is more compact than adipose tissue structure, so the collagen and glycosaminoglycan components in DAM are about 4.5 times that of adipose tissue under the same dry weight. These results show that the method used in this embodiment efficiently removes immunogenic components in the tissue while effectively retaining active components.

[0054] As shown in Figure 1 (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-eosin (H&E) staining images of fresh adipose tissue and adipose tissue decellularized 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 function of photoaging endothelial cells

[0056] The normal functioning of vascular endothelial cells is essential for the maintenance of vascular formation and 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 photoaging endothelial cells for in vitro experiments to detect the regulatory effect of DAM on photoaging HUVECs. The cell proliferation detected by CCK-8 experiment showed that DAM with a concentration of 0.5-2.0 mg / ml had the ability to promote cell proliferation Figure 2 ). As shown in Figure 2 the effect of different concentrations of DAM on HUVECs proliferation (1 day and 3 days). The abscissa Culture time represents the culture days, and the ordinate OD value represents the absorbance reading at 450 nm using a microplate reader, representing cell viability. The Control group is the control group, which is unstimulated HUVECs, and the rest are photoaging HUVECs after UVB stimulation with different concentrations of DAM. The ordinate OD value represents the absorbance reading at 450 nm using a microplate reader, representing cell viability.

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

[0058] To evaluate the regulatory effect of DAM on cell migration ability, scratch assays 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). Figure 3 A, Figure 3 D) and Transwell cell migration assay ( Figure 3 Experiments (B, E) showed that 0.5-2.0 mg / ml DAM significantly rescued cell migration. Furthermore, the angiogenesis capacity of endothelial cells was assessed using a tube-forming assay. Figure 3 C, Figure 3 F, Figure 3 G), the results showed that 0.5-2.0 mg / ml DAM enhanced the inhibited angiogenesis capacity and had a salvage effect on the formation of tubular structures and branching points.

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

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

[0061] As one of the most abundant cell types in the dermis, fibroblasts play a crucial role in maintaining the structure and function of the skin, and their functional state greatly influences tissue regeneration and rejuvenation. Therefore, a co-culture system of HUVECs and human dermal fibroblasts (HDFs) was constructed. Photoaged HDFs were co-cultured with HUVECs pretreated / not pretreated with 1.0 mg / ml DAM and compared to investigate the interaction between the two.

[0062] like Figure 5As shown in Figure A, DAM-treated HUVECs reduced the activity of senescence-related β-galactosidase SA-β-Gal in HDFs, indicating a reduction in the cellular senescence phenotype. Furthermore, DCFH-DA probe staining revealed that DAM-treated HUVECs significantly scavenged UV-induced ROS production, suggesting improved oxidative metabolism in photoaged fibroblasts. Figure 5 B). Scratch assay results showed that DAM-treated HUVECs enhanced the cell migration ability of HDFs through intercellular communication. Figure 5 C). Furthermore, the expression levels of relevant biomarkers were assessed by qRT-PCR, including ECM remodeling and degradation balance-related factors: MMPs (MMP1 and MMP3) and tissue metalloproteinase inhibitor-1 (TIMP-1); and synthesis and secretion-related factors: 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 exhibited a stronger tendency for ECM synthesis, decreased expression of MMPs, increased expression of the antagonistic TIMP-1, and significantly increased expression levels of collagen synthesis-related factors. This suggests that the restoration of HUVECs cell function can be achieved through cell-cell interactions, thereby restoring HDF function and further contributing to the regeneration and rejuvenation of the dermis.

[0063] like Figure 5 As shown, (A) Senescence-related SA-β-Gal staining of photoaged fibroblasts and photoaged HUVECs after 72 h of co-incubation (scale bar: 100 μm). (B) Detection of intracellular ROS production in photoaged fibroblasts and photoaged HUVECs after 72 h of co-incubation using the DCFH-DA probe (scale bar: 200 μm). (C) Detection of migration ability of photoaged fibroblasts and photoaged HUVECs after 72 h of co-incubation using a scratch assay (scale bar: 200 μm). (D) RT-qPCR analysis of markers related to collagen degradation and generation in photoaged fibroblasts (MMP1, MMP3, TIMP1, TIMP1 / MMP1, COL1A1, COL3A1). UVB served as the control group, consisting of fibroblasts co-cultured with photoaged HUVECs in Transwell chambers without any special stimulation. In the UVB / DAM group, photoaged HUVECs were cultured in a medium containing 1 mg / ml DAM.

[0064] 4. DAM promotes the repair of photo-aged skin in mice.

[0065] To investigate the effect of DAM on the repair of aging skin defects, 10-12-week-old photo-aged mice were used as experimental subjects. In the local treatment of skin-related diseases, how to penetrate the skin's keratin barrier to achieve efficient delivery of active ingredients is a key issue. Microneedles, as a minimally invasive and simple drug delivery system, have been widely used in various tissue regeneration, wound healing, skin rejuvenation, and other studies due to their controllable drug delivery and strong loading capacity.

[0066] As a commonly used dermal filler in clinical practice, hyaluronic acid (HA) has good biological safety and the ability to stimulate dermal fibroblast proliferation and promote collagen secretion. Therefore, in this example, DAM was dispersed in hyaluronic acid. The specific method is as follows:

[0067] Two sterile syringes were labeled as Syringe A and Syringe B. Syringe A was used to extract the hyaluronic acid gel filler material (HA), and Syringe B was used to extract the DAM solution. The volume ratio of the two was 7:3 (HA:DAM). The outlet ends of Syringe A and Syringe B were connected through a medical-grade three-way valve to ensure sealing. The pistons of Syringe A and Syringe B were alternately pushed at a frequency of 20-30 times per minute, and the process was repeated for 30 times. During the injection process, the three-way valve was kept open to allow the HA gel and DAM solution to be fully homogenized by shear force and convection, forming a DAM-doped composite filler material (HA / DAM).

[0068] The photo-aged mouse model was randomly divided into groups, and the corresponding intervention was performed on the 0th day after modeling: (1) HA group: subcutaneous injection of hyaluronic acid gel filler material on the back of the photo-aged mouse; (2) HA / DAM group: filling of HA / DAM composite material in the dermis of the back of the photo-aged mouse. Sampling was performed at 28 days, and histological sectioning and staining experiments were performed. The results are as follows Figure 15 As shown, compared with the HA group, the dermal layer thickness of the mouse back skin after HA / DAM filling was significantly increased, indicating that the composite filler material doped with DAM has a better effect on promoting collagen fiber synthesis in photo-aged skin tissue.

[0069] Dried hyaluronic acid has some strength, so it is also used as a matrix material for dissolving microneedles in this example.

[0070] 4.1 Preparation and characterization of DAM functionalized microneedle patches

[0071] After the hyaluronic acid powder was configured into a 15wt% solution, it was poured into the mold, and after the bubbles were eliminated in the vacuum drying oven, it was transferred to the microneedle patch obtained by dehydration and solidification at 37°C. The photos taken by the digital camera showed no obvious deformation or tip loss Figure 6 A). Microscope images and scanning electron microscopy showed that the tips of the microneedles were neat and sharp, with no visible fracturesFigure 6 B, Figure 6 C)。

[0072] Figure 7 A shows the FTIR spectra of DAM, HA and DAM-loaded functionalized microneedle patches. The characteristic peaks of DAM and HA can be seen in the functionalized microneedle spectra, indicating that DAM is successfully loaded into the microneedles and does not chemically react with HA. In clinical use, the microneedles need to have appropriate mechanical strength to ensure that they can effectively penetrate the stratum corneum barrier of the skin without significant deformation or breakage. As shown in Figure 7 B, the incorporation of DAM to some extent enhances the mechanical properties of the microneedle patch, while the load breaking force of the blank microneedle composed of 15wt% HA has already met the minimum average force required for skin penetration (0.1 N). To further verify its skin puncture ability, tests were carried out on pig skin and mouse skin. As shown in Figure 7 C, the microneedle patch loaded with trypan blue dye in the matrix material can leave a complete array of blue pinholes on fresh pig skin, indicating that the microneedles can successfully penetrate the skin. The hematoxylin-eosin staining results of the mouse skin tissue sections show that the needle tips successfully penetrate the stratum corneum and enter the dermis ( Figure 7 D), which indicates that the prepared dissolving microneedles can meet the clinical application scenarios of transdermal drug delivery.

[0073] 4.2 DAM functionalized microneedle patches promote repair of photoaged skin

[0074] The photoaged model mice were randomly divided into groups, and corresponding interventions were carried out on days 0, 4, 7, 14 and 21 after modeling: (1) Control group: normal mice without ultraviolet irradiation were not intervened except for hair removal at each time point; (2) UVB group: photoaged mice were not treated; (3) RA group: photoaged mice were applied with 0.05% retinoic acid cream on the back skin; (4) HA group: photoaged mice were injected with blank microneedles without DAM on the back skin; (5) HA / DAM group: photoaged mice were injected with DAM-loaded functionalized microneedles on the back skin.

[0075] At each time point, photographs were taken by a digital camera to monitor the changes in the wrinkle condition of the back skin of each group. Compared with the UVB group, the RA, HA and HA / DAM groups had different degrees of rescue effect on the deep wrinkles on the skin surface, among which the HA / DAM group had the most obvious effect, similar to the skin appearance of the Control group ( Figure 8 A, Figure 8 B). As shown in Figure 8 and Figure 9 , 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 improvement effect on the dermal thickness.

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

[0077] 5. The role of microvascular system in it

[0078] Using a photoaging model, C57 mice were subjected to 8 weeks of UVB irradiation on their back skin. qRT-PCR results showed that the expression of angiogenesis-related genes in the skin tissue of the mice decreased after UVB irradiation Figure 11 ).

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

[0080] Photoaged HUVECs (UVB group) and 1 mg / ml DAM-treated photoaged HUVECs (UVB / DAM) for 72 h were subjected to transcriptome sequencing. KEGG enrichment analysis results showed that the differentially expressed genes of DAM-treated cells were mainly enriched in the "PI3K-Akt signaling pathway", "MAPK signaling pathway", "mTOR signaling pathway" and other pathways closely related to blood vessel formation Figure 13 A). The network diagram based on the enriched pathways shows that the "PI3K-Akt signaling pathway" plays a core role in it 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 DAM-treated photoaged mouse HUVECs. Western Blot experimental results verified this pathway Figure 14 ).

[0081] The embodiment applies DAM to the remodeling of local microvascular system, and combines the microvascular remodeling effect brought by DAM with the intervention of aging-related diseases to produce significant beneficial effects. The bioactive ingredients carried in DAM can promote neovascularization, regulate the remodeling of microvascular system, and be more beneficial to the regeneration of aging skin, so that DAM has better effects in promoting the rejuvenation of aging skin.

[0082] The protection scope of the present application is not limited to the above embodiments. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. The application of an extracellular matrix-based active ingredient in the preparation of drugs or medical devices for treating photoaging of the skin, characterized in that... The decellularized extracellular matrix includes collagen and glycosaminoglycans, with a DNA content of less than 50 ng / mg dry weight; the skin is the dermis, and the photoaging is caused by exposure to light with a wavelength of 280 nm to 400 nm; the medical device is a microneedle, with the decellularized extracellular matrix as the active ingredient loaded in the microneedle and filled into the dermis. The decellularized extracellular matrix system is obtained by washing fresh fat with phosphate buffer, then subjecting it to three freeze-thaw cycles from -80°C to 37°C, followed by mechanical emulsification to destroy the adipose tissue, and then sequentially treating it with 0.5M and 1.0M hypertonic saline solutions and 1% Triton X-100 solution for 48 hours, followed by soaking in isopropanol for 8 hours, centrifugation, and washing.

Images