Bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix and preparation method of bio-based composite material

Abstract

The invention relates to the field of biological materials, and discloses a bio-based composite material composed of genetically modified mesenchymal stem cells and a mammalian extracellular matrix, and the bio-based composite material is prepared from mesenchymal stem cells obtained by modifying specific targets such as mTOR, FAP-alpha, COL3A1 and DPP4 genes by using a gene silencing technology. The mammals comprise pigs, sheep, cows and humans, the gene silencing technology is an s i RNA technology or shRNA technology, the mTOR and FAP-alpha gene targets are subjected to low expression, the COL3A1 gene targets are subjected to high expression, the DPP4 gene targets are subjected to low expression, the expression of the DPP4 gene targets is reduced, the expression of the DPP4 gene targets is reduced, and the expression of the DPP4 gene targets is reduced. A stabilizer is added in the extracellular matrix extraction process. The performance of the composite material is improved by precisely regulating and controlling genes, and tissue regeneration and repair are promoted; the extracellular matrix extraction method has multiple advantages and good biological activity; the biocompatibility and the mechanical property of the material are improved by adopting various innovative means, and the application prospect in the fields of orthopedics and plastic surgery is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a bio-based composite material composed of gene-modified mesenchymal stem cells and mammalian extracellular matrix and a preparation method thereof. Background Art

[0002] With the continuous development of medicine and biotechnology, the field of tissue engineering has become a hot topic of research. Tissue engineering aims to use biomaterials, cells and bioactive molecules to construct tissue or organ substitutes with specific functions to repair or regenerate damaged tissues. Mesenchymal stem cells have broad application prospects in tissue engineering due to their multidirectional differentiation potential, immunomodulatory effects and paracrine functions. At the same time, the umbilical cord and placental amniotic matrix of mammals are rich in extracellular matrix components, have good biocompatibility and biological activity, and are also widely used in the field of tissue engineering.

[0003] However, the current gene regulation methods for mesenchymal stem cells are often not precise enough, and it is difficult to achieve efficient gene silencing or expression regulation for specific targets, thus affecting the function and differentiation direction of mesenchymal stem cells. Existing tissue engineering composite materials have deficiencies in biocompatibility, mechanical properties, and promotion of tissue regeneration and repair, and are difficult to meet the needs of clinical applications. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a tissue engineering composite material composed of gene-modified mesenchymal stem cell extracellular vesicles and mammalian extracellular matrix, which solves the problems of insufficient gene regulation accuracy, poor extracellular matrix extraction process and the need to improve the performance of tissue engineering composite materials.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] A bio-based composite material composed of gene-modified mesenchymal stem cells and mammalian extracellular matrix, comprising: mesenchymal stem cells modified with gene silencing technology for specific targets such as mTOR, FAP-α, COL3A1 and DPP4 genes, and extracellular matrix extracted and processed from mammalian umbilical cord and placental amniotic matrix, wherein the mammals include pigs, sheep, cattle and humans, and the gene silencing technology is siRNA technology or shRNA technology, which can make the mTOR and FAP-α gene targets lowly expressed, the COL3A1 gene target highly expressed, and the DPP4 gene target lowly expressed.

[0007] Preferably, a stabilizer is added during the extracellular matrix extraction process.

[0008] Preferably, hydroxyapatite and a cross-linking agent are also included, and static ultrahigh pressure technology is used for mixing and sterilization.

[0009] A method for preparing a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix comprises the following steps:

[0010] S1. Gene modification of mesenchymal stem cells at specific targets using gene silencing technology;

[0011] ① Design and selection of gene silencing technology:

[0012] Design specific gene silencing sequences based on the sequence information of the target gene, and select siRNA technology or shRNA technology;

[0013] Prepare the corresponding molecules by chemical synthesis or biosynthesis to ensure their high purity and correct sequence structure;

[0014] ②Transfection of mesenchymal stem cells:

[0015] Select an appropriate transfection method to introduce gene silencing molecules into mesenchymal stem cells;

[0016] Optimize transfection conditions, including transfection reagent concentration, transfection time, and cell density to improve transfection efficiency and cell survival rate;

[0017] ③Screening and verification:

[0018] After transfection, successfully transfected mesenchymal stem cells were screened by fluorescence labeling, real-time quantitative PCR, and Western blotting methods;

[0019] Verify the inhibitory effect of gene silencing on the target gene and ensure that the gene expression level meets the requirements;

[0020] S2. Cultivating modified mesenchymal stem cells;

[0021] Cultivating the modified mesenchymal stem cells under appropriate culture conditions, including using a specific culture medium, culture temperature, and carbon dioxide concentration;

[0022] Change the culture medium regularly to maintain cell growth and metabolism;

[0023] S3. Extraction of extracellular matrix from mammalian umbilical cord and placental amniotic matrix;

[0024] After cleaning and disinfecting the tissue, cut it into pieces of appropriate size and put it into a container containing buffer, enzyme inhibitors, antioxidants and stabilizers for extraction. The extraction temperature is set at 37℃±2℃ and the extraction time is set at 48 hours±4 hours;

[0025] S4. Combining mesenchymal stem cells with extracellular matrix;

[0026] The cultured mesenchymal stem cells are combined with the extracted extracellular matrix, hydroxyapatite and a cross-linking agent are added, and the mixture is mixed and sterilized by using a static ultra-high pressure technology to obtain a bio-based composite material.

[0027] Preferably, the gene silencing technology accurately regulates the expression of specific genes in mesenchymal stem cells during the gene modification process to achieve precise control of cell function and differentiation direction.

[0028] Preferably, during the extracellular matrix extraction process, enzyme inhibitors and antioxidants are added to protect the activity and stability of the extracellular matrix components.

[0029] A bio-based composite material composed of gene-modified mesenchymal stem cells and mammalian extracellular matrix is ​​used for plastic surgery implantation.

[0030] The present invention provides a tissue engineering composite material composed of gene-modified mesenchymal stem cell extracellular vesicles and mammalian extracellular matrix. It has the following beneficial effects:

[0031] 1. The present invention precisely regulates the specific gene expression of mesenchymal stem cells to produce exosomes with specific biological characteristics, thereby improving the performance of tissue engineering composite materials and having better effects in tissue regeneration and repair.

[0032] 2. The method of extracting extracellular matrix from mammalian umbilical cord and placental amniotic matrix of the present invention has the advantages of not destroying the natural extracellular matrix structure, not introducing exogenous chemicals and proteins, good biocompatibility, high safety, simple production process, low cost, etc., and the extracted extracellular matrix has better biological activity.

[0033] 3. The bio-based composite material of the present invention is added with hydroxyapatite and a cross-linking agent, and is mixed and sterilized by static ultrahigh pressure technology. It also adopts innovative means such as nano-scale materials, porous structures, and simulated mineral mineralization processes in body fluid SBF to synthesize repair materials close to natural bones, explore the application of biopolymers and bio-based polymer composites, and carry out bionic composite material structure design, thereby improving the biocompatibility and mechanical properties of the composite material. It has broad application prospects in the fields of orthopedics and plastic surgery, such as good safety, tissue integration, and durability of plastic surgery effects in facial plastic surgery implants. DETAILED DESCRIPTION

[0034] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Embodiment 1:

[0036] The embodiment of the present invention provides a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix, including exosomes produced by mesenchymal stem cells modified with gene silencing technology for specific targets such as mTOR, FAP-α, COL3A1 and DPP4 genes, and extracellular matrix extracted and processed from the umbilical cord and placental amniotic matrix of mammals, wherein the mammals include pigs, sheep, cattle and humans;

[0037] Gene silencing technology designs gene silencing sequences for specific targets:

[0038] For mTOR and FAP-α gene targets:

[0039] The sequence of the positive strand is 5'-GAUCCAGCUCAACUUUGAATT-3'

[0040] The antisense strand sequence is 5'-UUCAAAGUUGAGCUGGAUCTT-3'

[0041] For COL3A1 gene target:

[0042] The sequence of the positive strand is 5'-GGAUCCAGCUUUGAAGAATT-3'

[0043] The antisense strand sequence is 5'-UUCUUCAACUGGAUCCUUCTT-3'

[0044] For DPP4 gene targets:

[0045] The sequence of the positive strand is 5'-CCAAGAUGACUACGUGAAUTT-3'

[0046] The antisense strand sequence is 5'-AUUCACGUAGUCAUCUUGGTT-3'

[0047] Through WB and IF verification, the expression levels of mTOR and FAP-α in mTORLow-FAP-αLow-MSCs were significantly reduced, the expression level of COL3A1 was significantly increased, and the expression level of DPP4 was significantly reduced, which proved the effectiveness of gene silencing technology. The specific data are shown in the following table:

[0048]

[0049] Mammalian target of rapamycin (mTOR) is an atypical serine / threonine protein kinase and a member of the phosphatidylinositol kinase-related kinase (PIKK) protein family. It exists in two different complexes, namely mTORC1 and mTORC2. mTOR is relatively conservative in evolution and can integrate a variety of extracellular signals such as nutrition, energy and growth factors. It participates in biological processes such as gene transcription, protein translation, and ribosome synthesis. It plays an extremely important role in cell growth, apoptosis, autophagy and metabolism. The release of extracellular vesicles can be promoted by adding mTORC1 inhibitor rapamycin. Extracellular vesicles (EVs) are vesicle-like bodies with double membrane structures that fall off from the cell membrane or are secreted by cells. They have a diameter of 30-500nm and contain proteins, DNA / RNA and other substances. They play a role in material transfer and signal transduction between cells, especially in cell division and proliferation, and tissue regeneration and repair.

[0050] Fibroblast activation protein, al pha (FAP-al pha) is a serine protease with post-prolyl dipeptidyl peptidase activity (DPP-4 is involved in sugar metabolism) and endopeptidase activity, and is activated by cleavage at the amino-terminal Xaa-Pro sequence. It is involved in the degradation of the extracellular matrix. It is involved in a variety of cellular processes, including tissue remodeling, fibrosis, wound healing, inflammation and tumor growth. FAP-al pha is usually not present in normal tissues, but is selectively expressed in tissues that need to be remodeled and repaired.

[0051] Based on siRNA technology, the present invention constructs and invents mesenchymal stem cells with low expression of human mTOR and FAP-α genes, and at the same time achieves high expression of other targets COL3A1 and low expression of DPP4. It is verified by WB and IF that mTORLow-FAP-αLow-MSCs have stronger paracrine cytokine extracellular vesicle release and immunomodulatory effects.

[0052] Human umbilical cord tissue decellularized extracellular matrix (rUC-ECM) refers to a collection of natural biopolymer macromolecules extracted from Wharton's connective tissue after removing the amniotic membrane, umbilical vein, and umbilical artery. The main components include: collagen fibers (types I, II, III, XI, XXIV, and XXVII), mucin, hyaluronic acid (glycosaminoglycans, mucopolysaccharides, proteoglycans, glycoproteins), chondroitin sulfate, and active small molecule peptides, etc. Detailed research on the components is rare and controversial. Among them, collagen is the most important component of the skin and is called "soft gold for the skin". Hyaluronic acid is a type of polysaccharide substance that is widely present in the extracellular matrix of the human body and plays a role in protecting organs and tissues. Chondroitin sulfate is called "soft gold for joints". Human umbilical cord tissue decellularized extracellular matrix (rUC-ECM) is a natural extracellular matrix with all the characteristics of extracellular matrix. In addition, it has the characteristics of low immunogenicity and no acquired pollution. It has important application value in the field of medical beauty and plastic surgery. The present invention provides a method for extracting extracellular matrix from mammalian placenta and amniotic basal tissue. The method has the advantages of not destroying the natural extracellular matrix structure, not introducing exogenous chemicals and proteins, having good biocompatibility and safety, and having simple production process and low cost.

[0053] The bio-based composite material of the present invention is added with hydroxyapatite and a cross-linking agent, and creatively adopts static ultra-high pressure technology for mixing and sterilization, so as to ensure good biocompatibility and safety. In addition, it also has the following innovative means:

[0054] 1. The use of nano-scale materials and larger specific surface area are beneficial to increasing drug solubility and bioavailability.

[0055] 2. Design a porous structure to facilitate cell attachment and growth.

[0056] 3. Simulate the mineralization process in body fluid SBF to synthesize repair materials that are close to natural bone.

[0057] 4. Explore the application of biopolymers and bio-based polymer composites to improve the performance of composites.

[0058] 5. Design bionic composite materials to imitate the structure and function of organisms and improve the biocompatibility and mechanical properties of composite materials.

[0059] The bio-based composite material of the present invention has broad application prospects in the fields of orthopedics and plastic surgery.

[0060] Example 2

[0061] A method for preparing a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix, comprising the following steps:

[0062] S1. Gene modification of mesenchymal stem cells at specific targets using gene silencing technology;

[0063] According to the sequence information of the target gene, specific gene silencing sequences are designed. These sequences are usually 19-25 nucleotides in length, with high specificity and effectiveness. Gene silencing molecules are prepared by chemical synthesis or biosynthesis. The synthesized molecules should have high purity and correct sequence structure. Liposome transfection or electroporation transfection is selected as the transfection method to introduce the gene silencing molecules into mesenchymal stem cells. The transfection conditions, including the concentration of the transfection reagent, transfection time, and cell density, are optimized to improve the transfection efficiency and cell survival rate.

[0064] The transfection method uses liposome transfection and electroporation transfection, and the transfection conditions are optimized to improve the transfection efficiency and cell survival rate. After transfection, the gene silencing molecule binds to the mRNA of the target gene, inducing the gene silencing mechanism, resulting in the inhibition of the expression of the target gene.

[0065] After transfection, successfully transfected mesenchymal stem cells were screened by fluorescence labeling, real-time quantitative PCR, and Western blotting methods to verify the inhibitory effect of gene silencing on the target gene and ensure that the gene expression level met the requirements.

[0066] S2. Cultivating the modified mesenchymal stem cells and producing extracellular vesicles;

[0067] The modified mesenchymal stem cells are cultured under appropriate culture conditions, including the use of specific culture medium, culture temperature, carbon dioxide concentration, and regular replacement of the culture medium to maintain cell growth and metabolism.

[0068] S3. Extraction of extracellular matrix from mammalian umbilical cord and placental amniotic matrix using water extraction process;

[0069] After the tissue is cleaned and disinfected, it is cut into pieces of appropriate size and placed in a container containing a buffer, enzyme inhibitors, antioxidants and stabilizers for extraction. The extraction temperature is set at 37°C ± 2°C and the extraction time is set at 48 hours ± 4 hours.

[0070] S4. Combining mesenchymal stem cells with extracellular matrix;

[0071] The cultured mesenchymal stem cells are combined with the extracted extracellular matrix, hydroxyapatite and a cross-linking agent are added, and the mixture is mixed and sterilized by using a static ultrahigh pressure technology to obtain a bio-based composite material;

[0072] Implementation Benefit 3:

[0073] Targeting the mTOR gene target, gene silencing technology is used to modify the genes of mesenchymal stem cells. The corresponding gene silencing sequence is designed and synthesized, and the gene silencing molecules are introduced into mesenchymal stem cells using liposome transfection method, and the transfection conditions are optimized to ensure transfection efficiency and cell survival rate.

[0074] The modified mesenchymal stem cells were cultured using a specific culture medium at 37°C and 5% carbon dioxide concentration, and the culture medium was replaced regularly.

[0075] When the cells grew to a certain density, the culture supernatant was collected, and after removing impurities by centrifugation and filtration, the extracellular vesicles were purified by ultracentrifugation and characterized.

[0076] The extracellular matrix is ​​extracted from the human umbilical cord and placental amniotic matrix. The tissue is cleaned, disinfected, cut into small pieces, placed in a container containing a buffer solution, and extracted at 37°C for 48 hours. Enzyme inhibitors, antioxidants and stabilizers are added during the extraction process. Mesenchymal stem cells and extracellular matrix are combined in a specific ratio to obtain a bio-based composite material.

[0077] Embodiment 4:

[0078] Targeting the FAP-α gene target, gene silencing molecules were introduced into mesenchymal stem cells by electroporation transfection, and synthetic gene silencing sequences were designed and the transfection effect was verified.

[0079] The modified mesenchymal stem cells are cultured under specific culture conditions (eg, 36° C., 4% carbon dioxide concentration), and the culture medium is regularly replaced to maintain cell growth and metabolism.

[0080] The cell culture supernatant was collected, and the extracellular vesicles were purified and characterized by size exclusion chromatography.

[0081] The extracellular matrix was extracted from the umbilical cord and placental amniotic matrix of pigs. After cleaning and disinfecting the tissue, water extraction was performed at a temperature of 39°C and a time of 52 hours. Enzyme inhibitors, antioxidants and stabilizers were added, and mesenchymal stem cells and extracellular matrix were combined to prepare a bio-based composite material.

[0082] Experimental example

[0083] Purpose:

[0084] To study the effects of different extraction temperatures and extraction times on the extracellular matrix components extracted from mammalian umbilical cord and placental amniotic matrix.

[0085] Experimental Materials:

[0086] Mammalian (swine, sheep, cow and human umbilical cord and placental amniotic matrix), buffer, enzyme inhibitors, antioxidants, stabilizers.

[0087] Experimental methods:

[0088] The umbilical cord and placental amniotic matrix of mammals from different sources were cleaned, disinfected and cut into small pieces.

[0089] Divide into different experimental groups, and set different extraction temperatures (35°C, 37°C, 39°C) and extraction times (44 hours, 48 ​​hours, 52 hours).

[0090] Aqueous extractions were performed in vessels containing buffer, enzyme inhibitors, antioxidants, and stabilizers.

[0091] After extraction, the components of the extracellular matrix in different experimental groups were analyzed, including the content determination of collagen, elastin, glycosaminoglycans and other components.

[0092]

[0093]

[0094] 1. Analysis of collagen content

[0095] Effect of extraction time:

[0096] At the same extraction temperature, analyze the changes in collagen content as the extraction time increases. Taking 37°C as an example, compare the collagen content when the extraction time is 44 hours (X4), 48 hours (X5), and 52 hours (X6). If within a certain time range, the collagen content increases with time, but after a certain time point, the content tends to stabilize or decrease, indicating that there is an optimal value for the extraction time.

[0097] Consider the effect of long-term extraction on collagen degradation. As the extraction time increases, the action of some enzymes will cause the collagen to gradually degrade, so that the content will no longer increase or even decrease.

[0098] 2. Analysis of elastin content

[0099] Effect of extraction temperature:

[0100] Similar to collagen, observe the changes in elastin content at different temperatures. For example, when the extraction time is 52 hours, compare the elastin content at different temperatures (Y3, Y6, Y9). If the elastin content reaches a maximum at a certain temperature, it means that the temperature is more favorable for the extraction of elastin.

[0101] Analyze the effect of temperature on the structure and function of elastin. Different temperatures will affect the elasticity and stability of elastin, and too high a temperature will cause elastin to lose its elasticity.

[0102] Effect of extraction time:

[0103] At a fixed temperature, analyze the effect of extraction time on elastin content. Taking 39°C as an example, compare the elastin content at different extraction times (Y7, Y8, Y9). If the elastin content increases with time within a certain period of time and then tends to stabilize or decrease, it means that the extraction time needs to be controlled within a reasonable range.

[0104] Consider the possibility of prolonged extractions causing degradation and structural changes to elastin. Prolonged extractions can cause degradation or structural changes to elastin, which can affect its content and properties.

[0105] 3. Glycosaminoglycan content analysis

[0106] Effect of extraction temperature:

[0107] Observe the changes in glycosaminoglycan content at different temperatures. For example, when the extraction time is 44 hours, compare the glycosaminoglycan content at different temperatures (Z1, Z4, Z7). If the glycosaminoglycan content is higher at a specific temperature, it means that the temperature is more conducive to the extraction of glycosaminoglycans.

[0108] Analyze the effect of temperature on the activity and stability of glycosaminoglycans. Too high or too low temperature will affect the biological activity and structural stability of glycosaminoglycans.

[0109] Effect of extraction time:

[0110] At the same temperature, analyze the effect of extraction time on glycosaminoglycan content. Taking 35°C as an example, compare the glycosaminoglycan content at different extraction times (Z1, Z2, Z3). If the glycosaminoglycan content increases first and then tends to stabilize or decrease with the extension of extraction time, it means that the extraction time needs to be optimized.

[0111] Consider the effects of prolonged extraction on the degradation and structural changes of glycosaminoglycans. Prolonged extraction can lead to degradation or structural changes of glycosaminoglycans, thus affecting their content and function.

[0112] In general, the extraction temperature and extraction time have a significant effect on the content of collagen, elastin and glycosaminoglycans in the extracellular matrix. Through the analysis of experimental data, a more suitable combination of extraction temperature and extraction time is determined to maximize the extraction efficiency and quality of extracellular matrix components, thereby providing better raw materials for the preparation of tissue engineering composite materials.

[0113] Test example:

[0114] Testing of the implantation effect of the bio-based composite material prepared by the present invention in facial plastic surgery:

[0115] 1. Test Purpose

[0116] To evaluate the implantation effects of bio-based composite materials in facial plastic surgery, including safety, tissue integration, and durability of plastic surgery effects.

[0117] 2. Test Materials and Methods

[0118] Test Materials:

[0119] A bio-based composite material composed of mesenchymal stem cells modified at specific targets using gene silencing technology and extracellular matrix extracted from mammalian umbilical cord and placental amniotic matrix.

[0120] The control group was implanted with traditional silicone gel for facial plastic surgery.

[0121] Test subjects:

[0122] Several patients who needed facial plastic surgery were selected and randomly divided into an experimental group and a control group.

[0123] Surgical method:

[0124] Experimental group: Bio-based composite materials were used for facial plastic surgery. Customized implant shapes and sizes were designed according to the patient's specific needs and facial structure, and the composite materials were implanted in the corresponding parts.

[0125] Control group: Facial plastic surgery was performed on the same part using traditional implant materials.

[0126] Postoperative observation and evaluation:

[0127] Safety Assessment:

[0128] Regular physical examinations are performed after surgery, including routine blood tests and liver and kidney function tests, to observe for any adverse events such as infection and rejection reactions.

[0129] Use imaging examinations (such as ultrasound, CT) to observe whether the position and shape of the implant are normal.

[0130] Tissue Integration Assessment:

[0131] Tissue biopsies were taken at different time points after surgery (e.g., 1 week, 1 month, 3 months, and 6 months) to observe the fusion of the composite material with the surrounding tissues. Histological staining was used to analyze the cell infiltration and angiogenesis around the implants.

[0132] Immunohistochemistry was used to detect the expression levels of inflammatory factors in the tissues surrounding the implants and to evaluate the degree of immune response.

[0133] Evaluation of the durability of plastic surgery effects:

[0134] Professional doctors and patients will conduct subjective evaluations of the plastic surgery results, using a scoring scale to score facial contour, symmetry, and naturalness.

[0135] Facial photographs are taken regularly, and image analysis software is used to measure changes in the dimensions and angles of key facial parts to assess the durability of the cosmetic effects.

[0136] 3. Test Results and Analysis

[0137] Security:

[0138] The patients in the experimental group did not experience any adverse events such as infection or rejection after surgery, and their blood routine and liver and kidney function indicators were basically normal. Imaging examinations showed that the implant was in a stable position and had a normal morphology.

[0139] Some patients in the control group experienced mild inflammatory reactions and rejection, but these symptoms were relieved after treatment.

[0140] Organizational integration:

[0141] Tissue biopsy results showed that the composite material in the experimental group began to fuse with the surrounding tissue early after surgery, and the degree of fusion gradually increased over time. There was abundant cell infiltration and angiogenesis around the implant, and the expression level of inflammatory factors was low.

[0142] In the control group, the fusion of traditional implant materials with surrounding tissues was relatively slow, and fibrous capsule formation occurred during the fusion process, and the expression level of inflammatory factors was relatively high.

[0143] Durability of plastic surgery effect:

[0144] The subjective evaluation results showed that the patients in the experimental group were more satisfied with the plastic surgery results, with natural and symmetrical facial contours, and the results were maintained well over time.

[0145] Image analysis results showed that the size and angle of key facial parts in the experimental group changed less, and the plastic surgery effect was more durable than that in the control group.

[0146] 4. Test Conclusion

[0147] Bio-based composite materials composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix have good safety, tissue integration and durability of plastic surgery effects in facial plastic surgery implants, providing a new and potential implant material option for plastic surgery.

[0148] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix, characterized in that: include: The invention is composed of mesenchymal stem cells modified by gene silencing technology for specific targets such as mTOR, FAP-α, COL3A1 and DPP4 genes, and extracellular matrix extracted and processed from the umbilical cord and placental amniotic matrix of mammals, wherein the mammals include pigs, sheep, cattle and humans. The gene silencing technology is siRNA technology or shRNA technology, which can make the mTOR and FAP-α gene targets lowly expressed, the COL3A1 gene target highly expressed, and the DPP4 gene target lowly expressed.

2. The bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix according to claim 1, characterized in that: A stabilizer is added during the extracellular matrix extraction process.

3. The bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix according to claim 1, characterized in that: It also includes hydroxyapatite and a cross-linking agent, and adopts static ultra-high pressure technology for mixing and sterilization.

4. A method for preparing a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Gene modification of mesenchymal stem cells at specific targets using gene silencing technology; ① Design and selection of gene silencing technology: Design specific gene silencing sequences based on the sequence information of the target gene, and select siRNA technology or shRNA technology; Prepare the corresponding molecules by chemical synthesis or biosynthesis to ensure their high purity and correct sequence structure; ②Transfection of mesenchymal stem cells: Select an appropriate transfection method to introduce gene silencing molecules into mesenchymal stem cells; Optimize transfection conditions, including transfection reagent concentration, transfection time, and cell density to improve transfection efficiency and cell survival rate; ③Screening and verification: After transfection, successfully transfected mesenchymal stem cells were screened by fluorescent labeling, real-time quantitative PCR, and Western blot; Verify the inhibitory effect of gene silencing on the target gene and ensure that the gene expression level meets the requirements; S2. Cultivating modified mesenchymal stem cells; Cultivating the modified mesenchymal stem cells under appropriate culture conditions, including using a specific culture medium, culture temperature, and carbon dioxide concentration; Change the culture medium regularly to maintain cell growth and metabolism; S3. Extraction of extracellular matrix from mammalian umbilical cord and placental amniotic matrix; After cleaning and disinfecting the tissue, cut it into pieces of appropriate size and put it into a container containing buffer, enzyme inhibitors, antioxidants and stabilizers for extraction. The extraction temperature is set at 37℃±2℃ and the extraction time is set at 48 hours±4 hours; S4. Combining mesenchymal stem cells with extracellular matrix; The cultured mesenchymal stem cells are combined with the extracted extracellular matrix, hydroxyapatite and a cross-linking agent are added, and the mixture is mixed and sterilized by using a static ultra-high pressure technology to obtain a bio-based composite material.

5. The method for preparing a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix according to claim 4, characterized in that: The gene silencing technology precisely regulates the expression of specific genes in mesenchymal stem cells during the gene modification process to achieve precise control of cell function and differentiation direction.

6. The method for preparing a bio-based composite material composed of genetically modified mesenchymal stem cells and mammalian extracellular matrix according to claim 4, characterized in that: During the extracellular matrix extraction process, enzyme inhibitors and antioxidants are added to protect the activity and stability of the extracellular matrix components.

7. Use of a bio-based composite material composed of the genetically modified mesenchymal stem cells and mammalian extracellular matrix as claimed in any one of claims 1 to 6 in plastic surgery implantation.

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