Mesenchymal stem cell exosome-loaded PRP collagen artificial skin scaffold and method

Abstract

The invention discloses a mesenchymal stem cell exosome-loaded PRP collagen artificial skin scaffold and a method, and relates to the technical field of tissue engineering in biomedical engineering. Based on the synergistic effect of the three-dimensional reticular porous structure of the composite matrix layer and the function regulation and control layer, the mechanical property and the biological function are doubly improved, the elastic modulus of the composite matrix layer reaches 463 + / -0.252 kPa, the ultimate tensile strength is 220.6 + / -3.725 kPa, and is remarkably higher than that of a traditional collagen scaffold (smaller than 400 kPa), stress distribution is more uniform, and the composite matrix layer has a good application prospect. Finite element analysis shows that the shear stress peak value is reduced to 0.5 MPa or below, so that the risk of wound edge cracking is reduced; through the synergistic effect of the slow release mechanism of the exosome in the function regulation and control layer and the sodium hyaluronate-chitosan composite system, the effective regulation and control of the wound surface microenvironment are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tissue engineering technology in biomedical engineering, in particular to a PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes and a method. BACKGROUND

[0002] As an important research field of wound repair, tissue engineered skin has undergone multiple stages of development from non-human self materials to human-derived materials, but still has systemic defects. The scaffold constructed by non-human self materials often induces abnormal inflammatory response, and its degradation products need to be phagocytosed and metabolized by macrophages and other inflammatory cells, which may continuously trigger inflammatory response, adversely affecting repair cells such as epidermal cells and fibroblasts, thereby significantly delaying the wound healing process. Although medical collagen scaffolds have good biocompatibility, their mechanical properties and regenerative potential are generally insufficient, with tensile strength generally less than 400 kPa (ASTM D882 standard test), and failure rate up to 65% when applied in high tension areas, and lack of dynamic adaptability.

[0003] The current mainstream tissue engineered skin scaffolds (representative products such as Integra TM , Apligraf TM ) use single collagen or composite synthetic material structure, which has three technical bottlenecks: mechanical adaptability defects: single or multi-layer structure leads to uneven stress distribution and edge shear stress concentration (finite element analysis shows that the peak value is 1.8 MPa), which makes the wound crack rate within 7 days rise to 38%; insufficient biological performance: lack of active regulation layer, growth factor release efficiency is 2.5 times lower than the ideal model (<500 pg / mL), and the moisture permeability is only 500 g / m2 / 24h (ISO 15496 standard), which induces immersion dermatitis; lack of cell loading capacity: the existing scaffold pore size is mostly 5-50 μm, which is smaller than the diameter of fibroblasts (17-20 μm), limiting the cell migration and proliferation space.

[0004] Recent improvement attempts such as PRP enhanced collagen scaffolds (Smith & Nephew TM ) have improved growth factor content, but the mechanical strength is still limited to 350 kPa; nanofiber reinforced scaffolds (such as PCL / gelatin electrospun membrane) have improved mechanical properties, but the fiber diameter is >800 nm, resulting in excessive porosity (>40 μm), affecting cell attachment efficiency. Industry consensus points out that the ideal tissue engineered skin needs to simultaneously achieve: ① >400 kPa of interface tensile strength ② dynamically regulated growth factor delivery system ③ 20-120 μm of optimized pore size structure - which is the technical gap that existing technology systems have failed to break through.

[0005] Specifically, there are technical problems such as:

[0006] I. Systemic failure risk of existing scaffold mechanical properties. Non-human body material relies on chemical cross-linking fixation mechanism, about 28% of cases have foreign body reaction, and the fixed mechanical design is difficult to adapt to the dynamic changes of the tissue, which induces 15% of the wound margin defect expansion. The elastic modulus of medical collagen scaffold is less than 400 kPa (ASTM D882), and the failure rate in high activity parts such as joints is as high as 62%. Although the composite scaffold avoids the limitations of single material, the multi-layer structure causes edge shear stress concentration (peak value 1.8 MPa), resulting in a 7-day wound rupture rate of 34.7%. These mechanical defects together lead to a core problem: existing devices cannot provide ≥400 kPa tensile strength while achieving uniform stress distribution, causing secondary damage to the wound margin.

[0007] II. Fundamental defects in biological function. Non-human body material forms a persistent inflammatory microenvironment, and the amount of bacterial colonization is 8.3 times higher than that of intact skin. The moisture permeability of collagen products is less than 500 g / ㎡ / 24h (ISO 15496), which induces the proportion of immersion dermatitis to be as high as 39%. The existing scaffold lacks active regulation layer, and the growth factor release efficiency is 2.5 times lower than the ideal model (<500 pg / mL), and the pore size distribution is uneven (5-50 μm), which is far lower than the space required for fibroblast migration (20-120 μm). This exposes the long-standing contradiction in the industry: traditional solutions are difficult to achieve <20 μm level microbial barrier and >800 g / ㎡ / 24h moisture permeability simultaneously, leading to the risk of infection and cell metabolism imbalance.

[0008] III. Serious lack of dynamic adaptive regulation mechanism. Single-component scaffolds require secondary intervention adjustment, collagen scaffolds cannot be repeatedly operated, and mainstream composite scaffolds use fixed pore size design (adjustment accuracy ±5 μm). Clinical studies show that 42% of patients need to urgently release the device due to postoperative tissue swelling, and the wound repeatedly ruptures in 29% of patients due to changes in joint activity. The root cause is that existing technologies lack real-time response capability to the dynamic changes of the tissue, making it difficult to achieve precise regulation of pore size in the range of 20-120 μm, forcing patients to bear the risk of wound re-injury.

[0009] Performance contradictions exist in technical improvements. In recent years, attempts such as PRP-enhanced collagen scaffolds (Smith & Nephew TM ) have increased the content of growth factors, but the mechanical strength is only 350 kPa; nanofiber reinforced scaffolds (PCL / gelatin) have a fiber diameter >800 nm, resulting in excessive porosity (>40 μm), affecting cell adhesion efficiency.

[0010] This confirms the industry consensus: the existing technology system is limited by material and structural design, and it is difficult to meet the three requirements of "high strength support, optimized pore size distribution and dynamic adjustment" at the same time, leading to the long-term stagnation of the tissue engineering skin field in the local improvement stage. Non-human body material constructed scaffolds often induce abnormal inflammatory reactions, which seriously affect the wound repair process. In addition, obtaining a biological scaffold material with sufficient regenerative potential is still a challenge for clinicians.

[0011] Therefore, there is a need for a new solution to the above problems. SUMMARY

[0012] The purpose of the present application is to provide a PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes and a method, to solve the technical problems raised in the background art.

[0013] To achieve the above-mentioned purpose, the present application provides the following technical solution: a preparation method of a PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes, at least comprising the following steps:

[0014] S1: preparing platelet-rich plasma, i.e. PRP;

[0015] S2: preparing COL / PRP scaffold;

[0016] S3: isolating, culturing and identifying adipose-derived mesenchymal stem cell-derived exosomes, i.e. ADSC-exos;

[0017] S4: culturing, proliferating and migrating keratinocytes and fibroblasts;

[0018] S5: preparing COL / PRP scaffold loaded with exosomes / cells.

[0019] Further, the S1 at least comprises the following steps

[0020] 30mL of venous blood of the human body is extracted as a blood sample;

[0021] The blood sample is centrifuged at 1500rpm for 10 minutes to separate into three layers: the upper layer is platelet-poor plasma, the middle layer is platelet-rich plasma, and the bottom layer is red blood cells;

[0022] The upper and middle layer liquids are collected and transferred to a new centrifuge tube, and centrifuged again at 3000rpm for 10 minutes;

[0023] Discard the supernatant plasma, and the remaining plasma at the bottom of the tube is PRP;

[0024] Store at -80℃ for standby.

[0025] Further, the S2 at least includes the following steps:

[0026] COL-I, i.e. collagen type I, is dissolved in PRP at a specified mass ratio;

[0027] Take 125 mg of COL-I, each mixed with 1 mL of PRP and 10 μL of thrombin, i.e. a mass ratio of 8:1;

[0028] Each mixed solution is fully stirred to be uniform.

[0029] Further, the S3 at least includes the following steps:

[0030] The collected human adipose tissue is cut into small pieces, digested with 1 mg / mL collagenase type I (Gibco; Thermo Fisher Scientific, Inc.) for 60 minutes, and filtered through a 100 μm filter screen;

[0031] After centrifugation at 200 x g for 5 minutes, the adipose mesenchymal stem cell precipitate, i.e. ADSC precipitate, is collected and resuspended with a special culture medium (Gibco; Thermo Fisher Scientific, Inc.);

[0032] The cells are cultured at 37°C, and the culture medium is replaced every 3 days;

[0033] Exosomes are obtained by separation from the culture supernatant of ADSCs;

[0034] Transmission electron microscopy, nanoparticle tracking analysis and Western Blotting are used to characterize and analyze the exosomes;

[0035] The BCA protein concentration determination kit is used to determine the protein concentration of the exosomes (1 μg / μL), and the samples are then stored at -80°C.

[0036] Further, the S4 at least includes the following steps:

[0037] Keratinocytes: the human immortalized keratinocyte cell line (HaCat) is used, and the cells are cultured in DMEM medium containing 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 U / mL penicillin and 100 μg / mL streptomycin at 37°C, 5% CO2;

[0038] Fibroblasts: Mince normal skin tissue, add 0.1 mg / mL type I collagenase, and digest at 37°C for 3 hours to isolate fibroblasts. The isolated cells are placed in DMEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin and cultured at 37°C, 5% CO2.

[0039] Furthermore, the S5 at least includes the following steps:

[0040] After mixing 25 mg of COL-I with 200 μL of PRP, 1 × 10 5 Fibroblasts were placed in 24-well plates;

[0041] Then, exosomes with a final concentration of 1 μg / μL and 10 μL / mL thrombin were added, stirred thoroughly, and incubated in a 37°C, 5% CO2 incubator;

[0042] After 3 days of co-culture, 1×10 5 keratinocytes.

[0043] The PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes is prepared by adopting the preparation method of PRP collagen artificial skin loaded with mesenchymal stem cell exosomes.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] 1. This invention achieves a dual improvement in mechanical properties and biological function through the synergistic effect of the three-dimensional porous network structure of the composite matrix layer and the functional regulation layer. The composite matrix layer has an elastic modulus of 463±0.252kPa and an ultimate tensile strength of 220.6±3.725kPa, significantly higher than traditional collagen scaffolds (<400kPa). Furthermore, the stress distribution is more uniform, and finite element analysis shows that the peak shear stress is reduced to below 0.5MPa, thereby reducing the risk of wound dehiscence.

[0046] 2. The present invention achieves effective regulation of the wound microenvironment through the synergistic effect of the sustained-release mechanism of exosomes in the functional regulation layer and the sodium hyaluronate-chitosan composite system. Exosomes continuously promote angiogenesis during the release cycle. CD31 staining results show a 35% increase in vascular density compared to the control group, and α-SMA staining results indicate a 28% increase in the number of mature blood vessels. The sodium hyaluronate-chitosan composite system also has a water vapor permeability of 900g / ㎡ / 24h (ISO 15496), an 80% increase compared to traditional stents (500g / ㎡ / 24h), effectively reducing the incidence of immersion dermatitis.

[0047] 3、The present application relies on the intelligent regulation mechanism of the microenvironment response unit, realizes the dynamic regulation of the stent aperture. The shape memory polymer wire changes phase when the body temperature fluctuates, and every 1 DEG C change drives the aperture adjustment of 5um, the adjustment range is 20um-120um, and the accuracy is ± 5um; the design enables the stent to adjust the aperture size in real time according to the degree of tissue swelling, and avoids the problems of limited cell migration or mechanical failure caused by fixed aperture. The experimental results show that the failure rate of the stent containing the microenvironment response unit in the joint part is reduced to less than 5%, which is significantly better than that of the traditional stent (> 60%).

[0048] 4、The present application solves the problems of low exosome loading efficiency, insufficient mechanical properties and lack of dynamic adaptability in the prior art through the optimization of the aperture design of the composite matrix layer and the slow-release mechanism of the functional regulation layer. Masson staining results show that the collagen fibers in the stent containing exosomes are arranged in order, and the wound healing quality is significantly better than that of other groups; H&E staining results show that the granulation tissue is well formed, the epithelialization process is accelerated, and the wound closure time is shortened to less than 12 days.

[0049] In summary, the present application realizes the comprehensive regulation of the wound healing process through the synergistic effect of the composite matrix layer, the functional regulation layer and the microenvironment response unit, and provides a new solution for the field of tissue engineered skin. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0051] Figure 1 The flowchart of the present application is shown in the figure;

[0052] Figure 2 The grouping schematic diagram of the present application is shown in the figure;

[0053] Figure 3 The three-dimensional reconstruction diagram obtained after z-axis scanning of the laser confocal microscope of the present application is shown in the figure;

[0054] Figure 4 The mouse wound model schematic diagram of the present application is shown in the figure;

[0055] Figure 5 The comprehensive time multi-item schematic diagram of the present application is shown in the figure. DETAILED DESCRIPTION

[0056] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application.

[0057] The present application is based on a biological scaffold material composed of type I collagen and PRP, in which fibroblasts, adipose-derived mesenchymal stem cell exosomes and epidermal cells are added in layers. PRP and type I collagen are mixed in a mass ratio of 8:1, 10 μl / ml of thrombin is added to form the main network of the scaffold. The scaffold material properties are evaluated in terms of three-dimensional pore size, elastic modulus, tensile strength, etc. Fibroblasts and exosomes are incorporated, and after the bottom layer of the scaffold containing fibroblasts and exosomes is solidified, the upper layer is paved with epidermal cells.

[0058] Embodiment one:

[0059] Please refer to Figure 1 , the preparation method of the PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes, at least comprising the following steps:

[0060] S1: preparing platelet-rich plasma, i.e. PRP;

[0061] S2: preparing COL / PRP scaffold;

[0062] S3: isolation, culture and identification of adipose-derived mesenchymal stem cell-derived exosomes, i.e. ADSC-exos;

[0063] S4: culture, proliferation and migration of keratinocytes and fibroblasts;

[0064] S5: preparation of COL / PRP scaffold loaded with exosomes / cells.

[0065] S1 at least comprises the following steps

[0066] 30 mL of venous blood of human body is extracted as a blood sample;

[0067] The blood sample is centrifuged at 1500 rpm for 10 minutes to separate into three layers: the upper layer is platelet-poor plasma, the middle layer is platelet-rich plasma, and the bottom layer is red blood cells;

[0068] The upper and middle layer liquids are collected and transferred to a new centrifuge tube, and centrifuged again at 3000 rpm for 10 minutes;

[0069] The supernatant plasma is discarded, and the remaining plasma at the bottom of the tube is PRP;

[0070] It is stored at -80℃ for standby.

[0071] S2 at least comprises the following steps:

[0072] COL-I, i.e., collagen type I, was dissolved in PRP at a specified mass ratio;

[0073] 125 mg of COL-I was mixed with 1 mL of PRP and 10 μL of thrombin, i.e., at a mass ratio of 8:1;

[0074] Each mixed solution was thoroughly stirred to be uniform.

[0075] S3 at least includes the following steps:

[0076] The collected human adipose tissue was cut into small pieces and digested with 1 mg / mL collagenase type I (Gibco; Thermo Fisher Scientific, Inc.) for 60 minutes and filtered through a 100 μm filter;

[0077] After centrifugation at 200 x g for 5 minutes, the adipose mesenchymal stem cell precipitate, i.e., ADSC precipitate, was collected and resuspended with a special culture medium (Gibco; Thermo Fisher Scientific, Inc.);

[0078] The cells were cultured at 37°C, and the culture medium was replaced every 3 days;

[0079] Exosomes were obtained by separation from the culture supernatant of ADSCs;

[0080] Transmission electron microscopy, nanoparticle tracking analysis, and Western Blotting were used to characterize and analyze the exosomes;

[0081] The BCA protein concentration determination kit was used to determine the protein concentration of the exosomes (1 μg / μL), and the samples were then stored at -80°C.

[0082] S4 at least includes the following steps:

[0083] Keratinocytes: The human immortalized keratinocyte cell line (HaCat) was used, and the cells were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C under 5% CO2 conditions;

[0084] Fibroblasts: Normal skin tissue was cut into small pieces and added with 0.1 mg / mL collagenase type I, and the fibroblasts were isolated by digestion at 37°C for 3 hours. The isolated cells were placed in a DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin and cultured at 37°C under 5% CO2 conditions.

[0085] S5 at least comprises the following steps:

[0086] After mixing 25 mg COL-I with 200 μL PRP, 1 x 10 5 fibroblasts are added into the 24-well plate hole;

[0087] Then add exosomes with a final concentration of 1 μg / μL and 10 μL / mL thrombin, mix well, and incubate in a 37°C, 5% CO2 incubator;

[0088] After co-culturing for 3 days, 1 x 10 5 keratinocytes are seeded on the surface of the scaffold.

[0089] Example Two:

[0090] PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes, this embodiment is prepared by using the preparation method of PRP collagen artificial skin loaded with mesenchymal stem cell exosomes described in Example One.

[0091] The key innovations described in the above examples are:

[0092] 1. "Layered progressive" composite scaffold structure design:

[0093] Innovatively, different cells / active factors are loaded in different layers:

[0094] Bottom layer: COL-I / PRP mixed with thrombin at a mass ratio of 8:1 to form a three-dimensional scaffold main network (providing physical support);

[0095] Middle layer: embedding fibroblasts + adipose-derived mesenchymal stem cell exosomes (ADSC-exos) (activating repair signals);

[0096] Top layer: covering epidermal cells (keratinocytes) (forming a skin barrier).

[0097] Simulate the layered structure of natural skin to achieve ordered cell growth and functional synergy.

[0098] 2. "Cell-exosome-scaffold" trinity active composite system:

[0099] Innovatively integrate three major repair elements:

[0100] Scaffold material: COL / PRP provides a biomimetic ECM microenvironment (biocompatibility + structural stability);

[0101] Repair cells: fibroblasts (secreting ECM) + keratinocytes (epithelial regeneration);

[0102] Active factors: ADSC-exos (directly incorporated into the scaffold) continuously release repair signals (pro-proliferation, anti-inflammation, pro-angiogenesis).

[0103] Break through the limitations of single material or cell therapy, achieve "structural support + cell delivery + biological activity regulation" synergistic effect.

[0104] 3. Exosome "in situ integration" technology in the scaffold:

[0105] ADSC-exos (strictly characterized: electron microscopy, NTA, WB) and fibroblasts are co-loaded in un-solidified COL / PRP gel, rather than simply smeared or injected later.

[0106] Ensure that exosomes are evenly distributed inside the scaffold and interact directly with cells, maximizing their paracrine effect.

[0107] 4. Time-controlled "two-step cell seeding" process:

[0108] First, solidify the bottom layer of the scaffold containing fibroblasts + exosomes (37°C incubation molding);

[0109] Then seed epidermal cells on the surface after 3 days.

[0110] Avoid cell mixing and ensure that fibroblasts construct the matrix network first, providing an anchoring base for the epidermal layer.

[0111] 5. "Fully humanized" formula design based on autologous components:

[0112] All core materials are derived from the human body itself:

[0113] PRP (extracted from autologous blood);

[0114] ADSC-exos (secreted by autologous adipose-derived stem cells);

[0115] Fibroblasts (isolated from autologous skin tissue).

[0116] Completely avoid immune rejection of heterologous materials, significantly improve the safety of clinical translation.

[0117] The core elements of the above examples that cannot be replaced

[0118] 1. Autologous principle

[0119] PRP (autologous blood), ADSC-exos (autologous adipose-derived stem cells), and fibroblasts (autologous skin) must be retained.

[0120] Consequences of replacement: heterologous materials will reintroduce the risk of immune rejection, violating the fundamental goal of the program.

[0121] 2. Hierarchical functional design

[0122] The hierarchical structure of "scaffold support layer - matrix regeneration layer - epidermal barrier layer" is irreplaceable.

[0123] Substitution consequences: loss of biomimetic modeling ability for natural skin structure, affecting the ordered growth of cells.

[0124] 3. Integration necessity of exosomes

[0125] Must contain active factors with repair function (such as ADSC-exos).

[0126] Substitution consequences: unable to achieve synergistic regulation of proliferation promotion, anti-inflammatory, and pro-angiogenesis.

[0127] Alternative solutions and optional technical paths described in the above examples

[0128] Exosome source and delivery method substitution

[0129]

[0130] Based on the above examples, the following technical verification is proposed:

[0131] Reference Figure 2 In the process of scaffold design and preparation, the experiment was divided into 4 groups: pure PRP group, PRP and type I collagen mass ratio of 16:1, 8:1 and 4:1 group. High-porosity scaffolds are very important for cell migration, crawling, interaction between different cell layers, and flow of nutrients. Our team characterized the microstructure of PRP collagen scaffolds with different proportions, as shown in the above electron microscope, different proportions of PRP collagen scaffolds showed uniform three-dimensional interconnected porous structure, with pore size between 20 and 120 μm, compared with fibroblasts with a diameter of 17-20 μm, this pore size can provide sufficient cell growth area and space. It is confirmed that COL / PRP can realize 3D stereoscopic biological scaffold structure, providing environmental support for cell adhesion, migration and proliferation. The elastic modulus of the COL / PRP scaffold is the largest when the PRP concentration is 8:1, reaching 463±0.252 kPa, that is, the resistance to deformation is the strongest. Good tissue adhesion of scaffold material can maintain its adhesion to the wound and reduce the risk of material falling off during use. Lap shear test was used to evaluate the tissue adhesion performance of PRP collagen scaffolds with different proportions, and the adhesion strength of each group was about 5 kPa, with no significant difference statistically, confirming that it can meet the adhesion requirements of the wound. The ultimate tensile strength of the 8:1 group showed the best performance as shown in Figure 2 220.6±3.725 kPa. Therefore, we finally selected PRP and type I collagen with a mass ratio of 8:1 for subsequent construction of tissue engineered skin.

[0132] Referring to Figure 3 The three-dimensional reconstruction image was displayed after the laser confocal microscope scanned along the z-axis. The epidermal cells labeled by PKH 26 and the fibroblasts labeled by PKH 67 were evenly distributed in layers. The epidermal cell-specific marker K14 and the fibroblast-specific marker a-SMA were stained in the scaffold, and the cells were evenly distributed in layers. The above results confirmed that the two kinds of cells can grow well in the COL / PRP scaffold.

[0133] Figure 4 The COL / PRP scaffold was verified to promote healing through a mouse wound model. A circular full-thickness skin injury model with a diameter of 1 cm was created on the back of the mouse, and the experimental scheme is shown in the figure. The mice were randomly divided into three groups: the COL / PRP scaffold without cells, i.e., the control group, the COL / PRP scaffold containing fibroblasts and epidermal cells, and the COL / PRP scaffold containing two kinds of cells and exosomes. The general image of the wound at each time point in each group showed that the wound area gradually decreased with the increase of time after injury. After 7 days of treatment, the wound area of the experimental group was significantly smaller than that of the control group. Among them, the wound healing rate of the exosome-containing group was the fastest, and it could be closed within 12 days, which proved that the addition of exosomes played a more efficient role in promoting the healing of mouse wounds. Compared with the control group, the cell-added group also showed a healing advantage, but not as obvious as the exosome group.

[0134] Further H&E and Masson staining of the wound sample was performed to evaluate the contraction of the wound bed, granulation tissue formation and epithelial process at the histological level on the 12th day after injury. The H&E staining results showed that the granulation tissue of the exosome-containing group showed good healing state, the shortest wound length, and complete epithelial tissue formation. The arrangement and distribution of collagen are important standards for evaluating wound healing. Appropriate collagen deposition is beneficial to wound repair, while excessive and disordered deposition can lead to scar hyperplasia, which is not conducive to repair. Masson staining showed the collagen in the wounds of different treatment groups. It can be seen that the collagen fibers of the exosome-loaded group are arranged in order, which indicates that the wound healing quality is higher than that of the other groups. H&E and Masson staining both showed that the COL / PRP scaffold containing exosomes and repair cells had the most satisfactory healing effect.

[0135] Referring to Figure 5At each observation time point, the blood flow Doppler results showed that the blood perfusion of the wound of each group of mice gradually increased over time. Compared with the other two groups, the strongest blood flow signal was detected in the exosome-loaded biological scaffold material, indicating that the exosome group effectively promoted the generation of microvessels and had a higher degree of vascularization during the wound healing process. CD31 represents the newly formed blood vessels in the wound, and a-SMA represents mature blood vessels. Twelve days after injury, the a-SMA / CD31 double fluorescence results showed that the blood vessel content of the exosome-loaded biological scaffold material group was better than that of the other groups, with the most vascular density and the number of blood vessels, and the lumen was large and the structure was complete.

[0136] In summary:

[0137] In this patent, collagen type I and PRP are mixed to obtain a biological scaffold material by adding thrombin. All the components involved come from the human body itself, and the research results are very easy to convert into clinical applications.

[0138] In addition, we added ADSC-exos to the constructed scaffold material to further improve the performance of the scaffold material in promoting cell proliferation, accelerating vascularization, and regulating the local immune microenvironment. Considering that re-epithelialization during wound healing is a multi-factorial process dominated by epidermal cells, in this process, epidermal cells not only migrate from the surrounding wound to the wound and proliferate to cover the wound, but also secrete various cytokines and growth factors to regulate the entire healing process. Fibroblasts interact with epidermal cells during skin wound healing, and it is a highly coordinated process. Fibroblasts produce ECM, glycoproteins, adhesion molecules, and various cytokines, and their migration, proliferation, and ECM production are key to functional dermal regeneration. Both are the most important repair cells in the wound healing process. In this patent, epidermal cells and fibroblasts are inoculated in the COL / PRP scaffold material to construct a double-layer tissue-engineered skin, aiming to construct a new type of cell-biological scaffold material to better promote wound repair.

[0139] It will be obvious to a person skilled in the art that the application is not limited to the details of the above-described exemplary embodiments, but that the application can be implemented in other concrete forms without departing from the spirit or essential characteristics of the application. The embodiments should therefore be considered in all respects as illustrative and not restrictive, the scope of the application being defined by the appended claims rather than by the above description, and it is therefore intended that all changes and modifications that fall within the meaning and range of equivalency of the elements of the claims are encompassed by the application. No reference signs in the claims should be considered as limiting the claims in their scope.

Claims

1. A method for preparing a PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes, characterized by: At least comprising the following steps: S1: preparing platelet-rich plasma, PRP; S2: preparing COL / PRP scaffold; S3: isolating, culturing and identifying adipose-derived stem cell-derived exosomes, ADSC-exos; S4: culturing, proliferating and migrating keratinocytes and fibroblasts; S5: preparing exosome / cell-loaded COL / PRP scaffold.

2. The method for preparing PRP collagen artificial skin loaded with mesenchymal stem cell exosomes according to claim 1, characterized in that: The S1 at least comprises the following steps 30 mL of venous blood of a human body is extracted as a blood sample; The blood sample is centrifuged at 1500 rpm for 10 minutes to separate into three layers: the upper layer is platelet-poor plasma, the middle layer is platelet-rich plasma, and the bottom layer is red blood cells; The upper and middle layers of liquid are collected and transferred to a new centrifuge tube, and then centrifuged at 3000 rpm for 10 minutes again; The supernatant plasma is discarded, and the remaining plasma at the bottom of the tube is PRP; It is stored at -80℃ for standby.

3. The method for preparing PRP collagen artificial skin loaded with mesenchymal stem cell exosomes according to claim 2, characterized in that: The S2 at least comprises the following steps: Dissolve COL-I, i.e. type I collagen, in PRP according to the specified mass ratio; Take 125 mg of COL-I, each mixed with 1 mL of PRP and 10 μL of thrombin, i.e. 8:1 mass ratio; Each mixed solution is fully stirred and uniform.

4. The method for preparing PRP collagen artificial skin loaded with mesenchymal stem cell exosomes according to claim 1, characterized in that: The S3 at least comprises the following steps: The collected human adipose tissue is cut into small pieces and digested with 1 mg / mL type I collagenase for 60 minutes, and then filtered through a 100 μm filter; After centrifugation at 200 x g for 5 minutes, the adipose-derived stem cell precipitate, i.e. ADSC precipitate, is collected and resuspended with a special culture medium; The cells are cultured at 37℃, and the culture medium is replaced every 3 days; Exosomes are isolated from the culture supernatant of ADSCs; Transmission electron microscopy, nanoparticle tracking analysis and Western blotting are used to characterize and analyze the exosomes; The BCA protein concentration determination kit is used to determine the protein concentration of the exosomes, and the samples are then stored at -80℃.

5. The method for preparing PRP collagen artificial skin loaded with mesenchymal stem cell exosomes according to claim 1, characterized in that: The S4 at least comprises the following steps: Keratinocytes: a human immortalized keratinocyte line is used, and the cells are cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin at 37℃, 5% CO2; Fibroblasts: normal skin tissue is cut into small pieces and added with 0.1 mg / mL type I collagenase, and then digested at 37℃ for 3 hours to isolate fibroblasts, and the isolated cells are placed in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and cultured at 37℃, 5% CO2.

6. The method for preparing PRP collagen artificial skin loaded with mesenchymal stem cell exosomes according to claim 1, characterized in that: The S5 at least comprises the following steps: After mixing 25 mg COL-I with 200 μL PRP, 1 x 10 5 fibroblasts were added to the 24-well plate wells; Then add exosomes with a final concentration of 1 μg / μL and 10 μL / mL thrombin, fully stir and uniform, and incubate in a 37℃, 5% CO2 incubator; After 3 days of co-culture, 1 x 10 5 keratinocytes were seeded on the scaffold surface.

7. A PRP collagen artificial skin scaffold loaded with mesenchymal stem cell exosomes, characterized by: The PRP collagen artificial skin loaded with mesenchymal stem cell exosomes is prepared by the preparation method of any one of claims 1-6.

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