阅读说明：本技术 一种组织工程化周围神经组织及其制备方法 (Tissue engineering peripheral nerve tissue and preparation method thereof ) 是由 那思家 唐安群 屠军波 张舟 崔浩 于 2020-12-31 设计创作，主要内容包括：本发明公开了一种组织工程化周围神经组织及其制备方法,属于组织工程技术领域,制备方法如下：将种子细胞分离和培养、纯化和扩增后,体外诱导种子细胞分化成雪旺细胞,然后制备干细胞聚合体,再将干细胞聚合体植入丝素蛋白导管支架材料,形成组织工程化周围神经组织。采用上述方法制备的组织工程化周围神经组织可用于面神经及三叉神经分支等周围神经组织缺损再生修复,与目前存在的其他类型组织工程化神经相比,具有种子细胞获取容易,创伤小,体外诱导分化雪旺细胞分泌成神经相关因子提高神经再生效率,干细胞聚合体内包含充足的细胞量和丰富的细胞外基质,同时具有更好的生物活性和良好的生物安全性,并可显著缩短神经修复时间。(The invention discloses a tissue engineering peripheral nerve tissue and a preparation method thereof, belonging to the technical field of tissue engineering, wherein the preparation method comprises the following steps: after the seed cells are separated, cultured, purified and amplified, the seed cells are induced to differentiate into Schwann cells in vitro, then stem cell polymers are prepared, and then the stem cell polymers are implanted into a silk fibroin catheter stent material to form the tissue engineering peripheral nerve tissue. Compared with other existing tissue engineering nerves, the tissue engineering peripheral nerve tissue prepared by the method has the advantages that the seed cells are easy to obtain, the wound is small, the Schwann cells are induced and differentiated in vitro to secrete nerve-related factors to improve the nerve regeneration efficiency, the stem cell polymer contains sufficient cell amount and abundant extracellular matrix, and meanwhile, the tissue engineering peripheral nerve tissue has better biological activity and good biological safety, and the nerve repair time can be obviously shortened.)

1. A preparation method of tissue engineering peripheral nerve tissue is characterized by comprising the following steps: after the seed cells are separated, cultured, purified and amplified, the seed cells are induced to differentiate into Schwann cells in vitro, then stem cell polymers are prepared, and then the stem cell polymers are implanted into a silk fibroin catheter stent material to form the tissue engineering peripheral nerve tissue.

2. The method for preparing tissue-engineered peripheral nerve tissue according to claim 1, wherein: the seed cell comprises dedifferentiated fat cell, fat stem cell or bone marrow mesenchymal stem cell.

3. The method for preparing tissue-engineered peripheral nerve tissue according to claim 2, wherein: the steps of the separation and culture, purification and expansion of the dedifferentiated adipocytes are as follows:

a. extracting adipose tissue from buccal fat pad, thigh or abdomen subcutaneous adipose tissue, soaking in PBS containing penicillin and streptomycin 50-150U/mL for 3-5min for 3 times, and cutting to 1-3mm³Adding collagenase I with the volume 2-4 times of that of the adipose tissue and the concentration 0.1-0.4%, shaking, digesting for 0.5-1.5h at 37 ℃, and adding a stem cell culture medium to stop digestion; sequentially filtering with 200 μm and 100 μm filter screens, centrifuging, and collecting upper layer fat cells; washing with PBS for 3 times, centrifuging, removing supernatant, adding stem cell culture medium for resuspension, inoculating in culture flask, adding stem cell culture medium, turning over the culture flask for 180 deg., and placing in 5% CO₂In an incubator at 37 ℃, removing a culture medium after 7 days, washing with PBS for 2 times, adding a stem cell culture medium, turning over a 180-degree culture bottle for culture, changing the culture medium once every 2-3 days, carrying out passage when the cells grow to 60-80%, and marking first-generation dedifferentiation fat cells;

b. inoculating the first generation dedifferentiated adipocytes at a cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

4. The method for preparing tissue-engineered peripheral nerve tissue according to claim 2, wherein: the steps of the separation and culture, purification and amplification of the adipose-derived stem cells are as follows:

a. from the buccal fat pad, the subcutaneous adipose tissue of the thigh or the abdomenExtracting adipose tissue, placing into PBS containing penicillin and streptomycin 50-150U/mL, soaking and cleaning in culture dish for 3 times (3-5 min each time), and cutting adipose tissue into pieces of 1-3mm³Adding collagenase I with the volume 2-4 times of that of the adipose tissue and the concentration 0.1-0.4%, shaking, digesting for 0.5-1.5h at 37 ℃, and adding a stem cell culture medium to stop digestion; sequentially filtering with 200 μm and 100 μm filter screens, centrifuging to collect lower layer matrix vascular cell mixture, resuspending with PBS, filtering with 70 μm filter screen, centrifuging, discarding supernatant, adding stem cell culture medium, resuspending, inoculating into culture flask, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 2-3 days, carrying out passage when the cell growth reaches 60-80%, and marking the first generation of fat stem cells;

b. inoculating the first generation adipose-derived stem cells at a cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

5. The method for preparing tissue-engineered peripheral nerve tissue according to claim 2, wherein: the steps of the separation and culture, purification and amplification of the bone marrow mesenchymal stem cells are as follows:

a. extracting bone marrow from an ilium, adding the bone marrow into physiological saline containing 800-1200U/mL heparin sodium, shaking, adding 1-1.2 g/mL lymphocyte separation fluid, centrifuging, removing a white membranous layer of mononuclear cells, washing by PBS (phosphate buffer solution), centrifuging, removing supernatant, adding a stem cell culture medium for resuspension, placing in 5% CO (carbon monoxide), and placing in a container₂Culturing at 37 ℃, changing the liquid for 1 time every 1 day, and marking the first generation of mesenchymal stem cells after the cells grow to 75-85 percent and passage;

b. inoculating the first generation of bone marrow mesenchymal stem cells at the cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

6. The method for preparing tissue-engineered peripheral nerve tissue according to claim 1, wherein: in vitro induction of speciesThe steps for the differentiation of the daughter cells into schwann cells are as follows: adjusting the cell density of the screened seed to 1 × 10⁵~5×10⁵Inoculating the cells/mL into a culture dish, washing the cells with PBS for 2 times after 24-48 hours of cell adherence, adding a neurogenesis pre-induction liquid A, and placing the cells in 5% CO₂Culturing for 24 hours in an incubator at 37 ℃, removing the culture medium, washing for 2 times by PBS, adding the adult neural pre-inducing solution B, continuing culturing for 72 hours, removing the culture medium, washing for 2 times by PBS, finally adding the adult neural inducing solution, changing the solution once every 2-3 days, and continuously culturing for 1-2 weeks to finish the schwann cell induced differentiation.

7. The method for preparing tissue-engineered peripheral nerve tissue according to claim 6, wherein: the neurogenesis pre-induction liquid A is a 10% FBS DMEM culture medium containing 1mol/L beta-ME; the neurogenesis pre-induction liquid B is a 10% FBS DMEM culture medium containing 35ng/mL A-TRA; the adult nerve inducing liquid is 10% FBS DMEM culture medium containing 14 mu mol/L FSK, 10ng/mL bFGF, 5ng/mL PDGF-AA and 200ng/mL HRG-beta.

8. The method for preparing tissue-engineered peripheral nerve tissue according to claim 1, wherein: the preparation steps of the stem cell aggregate are as follows:

a. abandoning the neural pre-induction culture medium in the culture dish for inducing differentiation into Schwann cells, washing with PBS 2 times, adding stem cell polymer culture medium, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 2-3 days, and continuously culturing for 1-2 weeks to form a white velvet polymer, namely a stem cell membrane, at the bottom of the culture dish;

b. folding and plasticizing 2-8 stem cell membranes, adding a stem cell polymer culture medium, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid half a day, and continuously culturing for 3-5 days, wherein pink velvet polymers, namely stem cell polymers, are formed in the culture dish.

9. The method for preparing tissue-engineered peripheral nerve tissue according to claim 1, wherein: the preparation steps of the tissue engineering peripheral nerve tissue are as follows: stem cell polymer is implanted into the silk fibroin duct stent material and is placed in an incubator at 37 ℃ for incubation for 30 minutes, and then the tissue engineering peripheral nerve tissue is obtained.

10. The method for preparing tissue-engineered peripheral nerve tissue according to any one of claims 3 to 5, wherein: the stem cell culture medium is 10% FBS and a-MEM culture solution containing 100U/mL cyan and streptomycin.

11. The method for preparing tissue-engineered peripheral nerve tissue according to claim 8, wherein: the stem cell aggregate culture medium is a stem cell culture medium added with 50-100 mu g/mL ascorbic acid.

12. A tissue-engineered peripheral nerve tissue prepared by the method for preparing a tissue-engineered peripheral nerve tissue according to any one of claims 1 to 11.

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a tissue-engineered peripheral nerve tissue and a preparation method thereof.

Background

Nerve block destruction caused by trauma, tumor invasion or surgery is a common clinical problem. The nerve tissue damage is accompanied with nerve dysfunction, such as facial expression muscle dyskinesia caused by facial nerve branch damage, which brings great harm and pain to the body and mind of patients and family members. Therefore, exploring the method for repairing the nerve tissue defect and regenerating the nerve tissue with bioactivity has great clinical significance for restoring the tissue function of a patient and improving the life quality.

At present, the method for clinically repairing the peripheral nervous tissue defect mainly adopts autologous nerve transplantation, and the method also obtains good curative effect. However, autologous nerve transplantation also has significant drawbacks: (1) the obtained self-body nerve is limited in length and diameter, so that the nerve repair curative effect is influenced due to the fact that part of the self-body nerve is not matched with the nerve of the affected area; (2) the inevitable acquisition of autologous nerves results in surgical damage to surrounding tissues, deformities and loss of nerve function in the donor area. In addition, nerve-inducing regeneration materials (such as polyglycolic acid (PGA) nerve bridging tube, fibrin glue, and the like) are adopted to repair nerve defects, which mainly play a role in guiding and supporting (CN 106730010A discloses the application of acellular nerve hydrogel in preparing the peripheral nerve injury repair composition and CN106729980B discloses the bionic nerve graft for peripheral nerve repair and the preparation method thereof), but the length of nerve regeneration is limited. Therefore, an effective method for regenerating and repairing the peripheral nervous tissue defect is clinically lacking at present.

With the rapid development of tissue engineering and regenerative medicine, more and more researchers develop tissue and organ regeneration research by using tissue engineering technology, which provides a new direction for the clinical treatment of various tissue and organ defects. A plurality of invention designs a strategy for using a seed cell/scaffold material complex for nerve tissue regeneration and repair based on nerve tissue development combined with tissue engineering and regenerative medicine technology, but the current strategy has some defects: (1) most of the early ectodermal mesenchymal stem cells or neuron cells and the like are adopted as seed cells, so that the clinical acquisition is difficult and the cell amount is insufficient; (2) the seed cells need to adopt scaffold materials such as biological glue and the like to support, transfer and fix the cells, which has certain influence on the biological activity of the cells.

In summary, most of the tissue engineering peripheral nerve tissues for peripheral nerve regeneration at present can promote nerve tissue regeneration, but only a small amount of cells remained at the nerve broken end proliferate and form myelin sheath, so that the hypomyelination hardly obtains good support and guide nerve regeneration. Therefore, at present, there is a need to develop a tissue-engineered peripheral nerve tissue which has the characteristics of easy clinical acquisition of seed cells, small wound, sufficient cell amount and rich extracellular matrix, can remarkably accelerate the parallel directional growth of nerve fibers in vivo, releases a nerve-related factor to promote the transplantation of a myelination-promoted nerve scaffold catheter structure at a nerve defect position of a receiving area, and can more effectively regenerate and repair the nerve defect.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a tissue-engineered peripheral nerve tissue and a method for preparing the same.

The invention is realized according to the following technical scheme:

a method for preparing tissue engineering peripheral nerve tissue includes such steps as separating seed cells, culturing, purifying, amplifying, inducing the seed cells to differentiate into Xuewang cells, preparing stem cell aggregate, and implanting the stem cell aggregate in silk fibroin catheter scaffold.

Further, the seed cell comprises a dedifferentiated adipocyte, an adipose-derived stem cell or a bone marrow mesenchymal stem cell.

Further, the steps of the isolation and culture, purification and expansion of the dedifferentiated adipocytes are as follows:

a. extracting 3-10 mL adipose tissue from fat tissue under buccal pad, thigh or abdomen, soaking in PBS containing penicillin and streptomycin 50-150U/mL in culture dish, cleaning for 3 times (3-5 min each time), and cutting to 1-3mm³Adding collagenase I with the volume 2-4 times of that of the adipose tissue and the concentration 0.1-0.4%, shaking, digesting for 0.5-1.5h at 37 ℃, and adding a stem cell culture medium to stop digestion; sequentially filtering with 200 μm and 100 μm filter screens, centrifuging, and collecting upper layer fat cells; washing with PBS 3 times, centrifuging, removing supernatant, adding stem cell culture medium, resuspending, and inoculating to 25cm²Adding stem cell culture medium into the culture bottle, turning the culture bottle over 180 deg., and placing in 5% CO₂In an incubator at 37 ℃, removing a culture medium after 7 days, washing with PBS for 2 times, adding a stem cell culture medium, turning over a 180-degree culture bottle for culture, changing the culture medium once every 2-3 days, carrying out passage when the cells grow to 60-80%, and marking first-generation dedifferentiation fat cells;

b. inoculating the first generation dedifferentiated adipocytes at a cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

The stem cell culture medium is a commercial a-MEM culture medium of 10% FBS and 100U/mL streptomycin.

Further, the steps of the isolation and culture, purification and amplification of the adipose-derived stem cells are as follows:

a. extracting 3-10 mL adipose tissue from fat tissue under buccal pad, thigh or abdomen, soaking in PBS containing penicillin and streptomycin 50-150U/mL in culture dish, cleaning for 3 times (3-5 min each time), and cutting to 1-3mm³Adding collagenase I0.1-0.4% 2-4 times of adipose tissue, shaking, 37 deg.CDigesting for 0.5-1.5h, and adding a stem cell culture medium to stop digestion; sequentially filtering with 200 μm and 100 μm filter screens, centrifuging to collect lower layer matrix vascular cell mixture, resuspending with PBS, filtering with 70 μm filter screen, centrifuging, discarding supernatant, adding stem cell culture medium, resuspending, and inoculating to 25cm²In a culture flask, and placed in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 2-3 days, carrying out passage when the cell growth reaches 60-80%, and marking the first generation of fat stem cells;

b. inoculating the first generation adipose-derived stem cells at a cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

The stem cell culture medium is a commercial a-MEM culture medium of 10% FBS and 100U/mL streptomycin.

Further, the steps of the isolation and culture, purification and amplification of the mesenchymal stem cells are as follows:

a. extracting 3mL of bone marrow from an ilium, adding the bone marrow into physiological saline containing 800-1200U/mL of heparin sodium, shaking, adding 1-1.2 g/mL of lymphocyte separation liquid, centrifuging, removing a white membranous layer of a monocyte, washing by PBS (phosphate buffer solution), centrifuging, removing a supernatant, adding a stem cell culture medium for heavy suspension, placing in 5% CO (carbon monoxide), and placing in a container₂Culturing at 37 ℃, changing the liquid for 1 time every 1 day, and marking the first generation of mesenchymal stem cells after the cells grow to 75-85 percent and passage;

b. inoculating the first generation of bone marrow mesenchymal stem cells at the cell density of 100-200/mL, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid for 1 time every 1 day, and carrying out passage when the cell grows to 60-80%, and marking the screened seed cells.

Further, the step of inducing the seed cells to differentiate into the Schwann cells in vitro is as follows: adjusting the cell density of the screened seed to 1 × 10⁵~5×10⁵Inoculating the cells/mL into a culture dish, washing the cells with PBS for 2 times after 24-48 hours of cell adherence, adding 2-4mL of adult nerve pre-inducing liquid A, and placing the cells in 5% CO₂Culturing at 37 deg.C for 24 hr, removing culture medium, washing with PBS for 2 times, adding 2-4mL adult nerve pre-inducing solution B, and culturing for 72 hrAnd h, removing the culture medium, washing with PBS for 2 times, adding 2-4mL of the adult neural inducing solution, changing the solution every 2-3 days, and continuously culturing for 1-2 weeks to complete the induced differentiation of the Schwann cells.

By adopting the technical scheme, on one hand, the schwann cells can differentiate to form myelin sheath tissues to protect damaged axons and guiding shaft axon growth directions, and on the other hand, the schwann cells secrete nerve formation related factors such as NGF, GDF and FGF to induce axon growth, so that the nerve regeneration efficiency can be obviously improved by acquiring a large number of schwann cells in vitro.

Further, the neurogenesis pre-inducing solution A is a 10% FBS DMEM medium containing 1mol/L beta-ME; the neurogenesis pre-induction liquid B is a 10% FBS DMEM culture medium containing 35ng/mL A-TRA; the adult nerve inducing liquid is 10% FBS DMEM culture medium containing 14 mu mol/L FSK, 10ng/mL bFGF, 5ng/mL PDGF-AA and 200ng/mL HRG-beta.

Further, the stem cell aggregate is prepared by the following steps:

a. abandoning the neural pre-induction culture medium in the culture dish for inducing differentiation into Schwann cells, washing with PBS 2 times, adding stem cell polymer culture medium, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 2-3 days, and continuously culturing for 1-2 weeks to form a white velvet polymer, namely a stem cell membrane, at the bottom of the culture dish;

b. folding and plasticizing 2-8 stem cell membranes, adding a stem cell polymer culture medium, and placing in 5% CO₂And culturing in an incubator at 37 ℃, changing the liquid half a day, and continuously culturing for 3-5 days, wherein pink velvet polymers, namely stem cell polymers, are formed in the culture dish.

The stem cell aggregate culture medium is a stem cell culture medium added with 50-100 mu g/mL ascorbic acid.

By adopting the technical scheme, the stem cell polymer with plasticity, sufficient seed cells and abundant extracellular matrix is prepared, the extracellular matrix can ensure the transmission of nutrition and signals of the seed cells, and the stem cell polymer contains I-type collagen and growth factors and can promote the neural differentiation of the seed cells.

Further, the preparation steps of the tissue engineering peripheral nerve tissue are as follows: stem cell polymer is implanted into the silk fibroin duct stent material and is placed in an incubator at 37 ℃ for incubation for 30 minutes, and then the tissue engineering peripheral nerve tissue is obtained.

The silk fibroin catheter scaffold material is a commercial silk fibroin membrane and is prepared according to the diameter of damaged peripheral nerves.

A tissue engineering peripheral nerve tissue prepared by the preparation method of the tissue engineering peripheral nerve tissue.

The invention has the advantages and beneficial effects that:

(1) the tissue engineering peripheral nerve tissue has components and structures similar to those of natural nerve tissue, so that the tissue engineering peripheral nerve tissue can play a normal function of the nerve tissue after being transplanted into an organism, and differentiated Schwann cells in the tissue engineering peripheral nerve tissue can continuously secrete neurotrophic factors to promote the regeneration of the nerve tissue with normal biological activity and finally repair peripheral nerve defects;

(2) the invention takes dedifferentiated fat cells and fat stem cells as seed cells, is convenient for clinical acquisition and has enough cell quantity, and can differentiate Schwann cells through in vitro induction; the use of the culture technology of the stem cell polymer breaks through the concepts of seed cells, scaffold materials and microenvironment in the traditional tissue engineering, and the stem cell polymer not only ensures enough seed cells, but also contains rich extracellular matrix, can remarkably accelerate the parallel directional growth of nerve fibers in vivo, releases nerve-related factors to promote myelination, and more effectively regeneratively repairs the nerve defects. The autocrine adult nerve related growth factor and type I collagen of the mesenchymal stem cell are combined with extracellular matrix protein and stored in the extracellular matrix, and the extracellular matrix ensures the function of transmitting signals and nutrition among cells and provides a microenvironment for proliferation and differentiation of the stem cell. In addition, the used silk fibroin conduit stent material has good physical characteristics, no biological potential safety hazard and good bacteriostatic action;

(3) the preparation method of the tissue engineering peripheral nerve tissue has the advantages of simple operation, proper strength, strong plasticity, good cell density and cell distribution, abundant extracellular matrix, avoidance of cell loss during cell inoculation of materials and the like.

Drawings

FIG. 1 is a diagram of a culture of dedifferentiated adipocytes, adipose-derived stem cells and bone marrow mesenchymal stem cells of the present invention and a diagram of Schwann cells induced to differentiate in vitro;

FIG. 2 is a view showing the preparation process and histological observation of the cell aggregate of the present invention;

FIG. 3 is a graph showing the results of histological examination 8 weeks after the tissue-engineered peripheral nerve tissue of the present invention was transplanted into the buccal branch of facial nerve of rat at 10mm defect.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and examples.

Example 1

The tissue engineering peripheral nerve tissue prepared by the embodiment adopts dedifferentiated adipocytes as seed cells, is separated, cultured, amplified and induced in vitro into Schwann cells, then prepares dedifferentiated adipocyte polymers, and finally compounds the cell polymers with the silk fibroin conduit stent material to form the tissue engineering peripheral nerve tissue, wherein the preparation steps are as follows:

(1) extracting 8 mL of adipose tissue from fat tissue of buccal pad, thigh or abdomen subcutaneous adipose tissue, soaking in PBS containing penicillin and streptomycin 100U/mL for 3 times, cleaning for 4min each time, and cutting adipose tissue to 1mm³Adding 0.25% collagenase I with the volume 3 times that of the adipose tissues, shaking, digesting for 1 hour at 37 ℃, and adding a stem cell culture medium to stop digestion. Filtering with 200 μm and 100 μm filter screens, centrifuging, and collecting upper layer fat cells. Washing with PBS 3 times, centrifuging, removing supernatant, adding stem cell culture medium, resuspending, and inoculating to 25cm²Adding stem cell culture medium into the culture bottle, turning the culture bottle over 180 deg., and placing in 5% CO₂In an incubator at 37 ℃, removing a culture medium after 7 days, washing with PBS for 2 times, adding a stem cell culture medium, turning over a 180-degree culture bottle for culture, changing the culture medium once every 2 days, carrying out passage when the cells grow to 80%, and marking first-generation dedifferentiated adipocytes;

the stem cell culture medium: 10% FBS and 100U/mL of a commercial a-MEM culture solution of penicillin and streptomycin;

(2) screening dedifferentiated adipocytes: taking the first generation of dedifferentiated adipocytes, seeding at a cell density of 200/mL, and placing in 5% CO₂Culturing at 37 deg.C, changing liquid 1 time every 1 day, subculturing when cell growth reaches 80%, and marking screened dedifferentiated fat cell;

(3) the density of the dedifferentiated adipocytes after screening was adjusted to 3X 10⁵Inoculating the cells/mL in a culture dish, washing the cells after 24 hours of cell adherence for 2 times by PBS, adding 4mL of neurogenesis pre-induction liquid A, and placing the cells in 5% CO₂Culturing for 24 hours in an incubator at 37 ℃, removing the culture medium, washing with PBS for 2 times, adding 4mL of adult nerve pre-inducing liquid B, continuing culturing for 72 hours, removing the culture medium, washing with PBS for 2 times, finally adding 4mL of adult nerve inducing liquid, changing the liquid every 2 days, and continuously culturing for 10 days to complete the schwann cell induced differentiation;

the neurogenesis pre-induction liquid A is a 10% FBS DMEM culture medium containing 1mol/L beta-ME; the neurogenesis pre-induction liquid B is a 10% FBS DMEM culture medium containing 35ng/mL A-TRA; the adult nerve inducing liquid is 10% FBS DMEM culture medium containing 14 mu mol/L FSK, 10ng/mL bFGF, 5ng/mL PDGF-AA and 200ng/mL HRG-beta.

(4) Abandoning the neural pre-induction culture medium in the culture dish for inducing differentiation into Schwann cells, washing with PBS for 2 times, adding the culture medium containing 100 mug/mL ascorbic acid stem cells, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 3 days, and continuously culturing for 2 weeks to form a white velvet polymer, namely a dedifferentiation adipocyte diaphragm, at the bottom of the culture dish; folding 3 cell sheets, adding cell polymer culture medium, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution half a day, and continuously culturing for 5 days, wherein pink velvet polymers, namely de-differentiated adipocyte polymers, are formed in the culture dish.

(5) According to the diameter of the peripheral nerve defect of the receptor area, silk fibroin catheter stent material is adopted to wrap de-differentiated fat cell polymer for in vitro compounding, 100 microgram/mL ascorbic acid stem cell culture medium is added, and 5% CO is placed₂Culturing in 37 deg.C incubator, changing half amount of liquid every day, and continuously culturing for 3 days to complete de-differentiation of fat cell aggregate andand (3) compounding the silk fibroin conduit scaffold material to obtain the tissue-engineered peripheral nerve tissue.

The silk fibroin conduit stent material is prepared from a silk fibroin membrane according to the diameter of damaged peripheral nerves, wherein the silk fibroin membrane is purchased from Suzhou Simulter Biotechnology GmbH.

For example, fig. 1A shows primary dedifferentiated adipocytes under an inverted microscope, and fig. 1D shows schwann cells induced to differentiate in vitro under an inverted microscope.

Example 2

The tissue-engineered peripheral nerve tissue prepared in this embodiment adopts mesenchymal stem cells as seed cells, is separated, cultured, amplified and induced in vitro to form schwann cells, then prepares a mesenchymal stem cell polymer, and finally compounds the mesenchymal stem cell polymer and a scaffold material in vitro to form the tissue-engineered peripheral nerve tissue, which comprises the following specific steps:

(1) separating, culturing and screening the bone marrow mesenchymal stem cells: extracting 3mL from iliac bone, injecting into a centrifuge tube containing 0.3mL heparin sodium physiological saline (1000U/mL), shaking, adding 6mL lymphocyte separation solution (1.2 g/mL), centrifuging, removing white membranous layer of monocyte, washing with PBS for 2 times, centrifuging, removing supernatant, adding dry cell culture medium, resuspending, and placing in 5% CO₂Culturing at 37 deg.C, changing liquid 1 time every 1 day, and marking the first generation of bone marrow mesenchymal stem cells after the cells grow to 80% passage.

The stem cell culture medium: 10% FBS and 100U/mL of a commercial a-MEM culture of penicillin and streptomycin.

(2) Bone marrow stromal cell screening: taking first generation bone marrow mesenchymal stem cells, inoculating the first generation bone marrow mesenchymal stem cells at the cell density of 100/mL, and placing the first generation bone marrow mesenchymal stem cells in 5% CO₂Culturing at 37 deg.C, changing liquid 1 time every 1 day, and marking screened bone marrow mesenchymal stem cells after passage when the cells grow to 80%.

(3) Adjusting the density of the screened bone marrow mesenchymal stem cells to 5 multiplied by 10⁵After one/mL, the cells were inoculated in a culture dish and washed 2 times with PBS after 24 hours of cell adhesionAdding 4mL adult nerve pre-inducing liquid A, and placing in 5% CO₂Culturing in an incubator at 37 ℃ for 24 hours, removing the culture medium, washing with PBS for 2 times, adding 4mL of adult nerve pre-inducing liquid B, continuing culturing for 72 hours, removing the culture medium, washing with PBS for 2 times, finally adding 4mL of adult nerve inducing liquid, changing the liquid every 3 days, and continuously culturing for 12 days to complete the schwann cell induced differentiation;

the neurogenesis pre-induction liquid A is a 10% FBS DMEM culture medium containing 1mol/L beta-ME; the neurogenesis pre-induction liquid B is a 10% FBS DMEM culture medium containing 35ng/mL A-TRA; the adult nerve inducing liquid is 10% FBS DMEM culture medium containing 14 mu mol/L FSK, 10ng/mL bFGF, 5ng/mL PDGF-AA and 200ng/mL HRG-beta.

(4) Abandoning the neural pre-induction culture medium in the culture dish for inducing differentiation into Schwann cells, washing with PBS 2 times, adding the culture medium containing 50 mug/mL ascorbic acid stem cells, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 3 days, and continuously culturing for 2 weeks to form a white velvet polymer, namely a stem cell membrane, at the bottom of the culture dish; folding and plasticizing 3 stem cell sheets, adding stem cell polymer culture medium, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the liquid half a day, and continuously culturing for 5 days, wherein pink velvet-shaped polymer, namely the bone marrow mesenchymal stem cell polymer, is formed in the culture dish.

(5) According to the diameter of the peripheral nerve defect of the receptor area, a silk fibroin catheter bracket material is adopted to wrap the bone mesenchymal stem cell polymer for in vitro compounding, 50 microgram/mL ascorbic acid stem cell culture medium is added, and the mixture is placed in 5 percent CO₂Culturing in an incubator at 37 ℃, changing liquid half a day, continuously culturing for 3 days, and finishing the compounding of the mesenchymal stem cell polymer of the marrow and the silk fibroin conduit stent material to obtain the tissue engineering peripheral nervous tissue.

The silk fibroin conduit stent material is prepared from a silk fibroin membrane according to the diameter of damaged peripheral nerves, wherein the silk fibroin membrane is purchased from Suzhou Simulter Biotechnology GmbH.

The tissue engineered peripheral nerve tissue prepared in this example, as shown in fig. 1C, is primary mesenchymal stem cells under an inverted microscope; fig. 2A is a mesenchymal stem cell membrane under a body type microscope, fig. 2B is a stem cell polymer polymerized by the mesenchymal stem cell membrane, fig. 2C is a surface structure of the mesenchymal stem cell polymer under a scanning electron microscope, fig. 2D & E are HE and Masson staining for observing the structure of the mesenchymal stem cell polymer, and fig. 2F is immunohistochemical staining for observing the expression of type I collagen of the mesenchymal stem cell polymer.

Example 3

The tissue-engineered peripheral nerve tissue prepared in this embodiment adopts adipose-derived stem cells as seed cells, is separated, cultured, amplified and induced in vitro into Schwann cells, then prepares adipose-derived stem cell polymers, and finally compounds the adipose-derived stem cell polymers and the scaffold material in vitro to form the tissue-engineered peripheral nerve tissue, wherein the preparation steps are as follows:

(1) extracting 6mL adipose tissue from buccal fat pad, thigh or abdomen subcutaneous adipose tissue, soaking in PBS containing penicillin and streptomycin 100U/mL for 3 times (3 min each time), and cutting adipose tissue to 1mm³Adding 0.3% collagenase I with the volume 3 times that of the adipose tissues, shaking, digesting for 1 hour at 37 ℃, and adding a stem cell culture medium to stop digestion. Sequentially filtering with 200 μm and 100 μm filter screens, centrifuging to collect lower layer matrix vascular cell mixture (SVF), resuspending with PBS, filtering with 70 μm filter screen, centrifuging, discarding supernatant, adding stem cell culture medium, resuspending, and inoculating to 25cm²In a culture flask, and placed in 5% CO₂Culturing in 37 deg.C incubator, changing liquid every 2 days, subculturing when cell growth reaches 75%, and labeling first generation adipose-derived stem cell

The stem cell culture medium: 10% FBS and 100U/mL of a commercial a-MEM culture of penicillin and streptomycin.

(2) Fat stem cell screening: taking first generation adipose-derived stem cells, inoculating the cells at the cell density of 200/mL, and placing the cells in 5% CO₂And culturing at 37 ℃, changing the liquid for 1 time every 1 day, carrying out passage when the cells grow to 75%, and marking the screened adipose-derived stem cells.

(3) Adjusting the density of the screened adipose-derived stem cells to 4X 10⁵After one/mL, the cells were inoculated in a culture dish, washed 2 times with PBS after 24 hours of cell adhesion, and added with 4mL of neurogenesis pretreatmentInducing liquid A, and placing in 5% CO₂Culturing for 24 hours in an incubator at 37 ℃, removing the culture medium, washing with PBS for 2 times, adding 4mL of adult nerve pre-inducing liquid B, continuing culturing for 72 hours, removing the culture medium, washing with PBS for 2 times, finally adding 4mL of adult nerve inducing liquid, changing the liquid every 2 days, and continuously culturing for 10 days to complete the schwann cell induced differentiation;

the neurogenesis pre-induction liquid A is a 10% FBS DMEM culture medium containing 1mol/L beta-ME; the neurogenesis pre-induction liquid B is a 10% FBS DMEM culture medium containing 35ng/mL A-TRA; the adult nerve inducing liquid is 10% FBS DMEM culture medium containing 14 mu mol/L FSK, 10ng/mL bFGF, 5ng/mL PDGF-AA and 200ng/mL HRG-beta.

(4) Abandoning the neural pre-induction culture medium in the culture dish for inducing differentiation into Schwann cells, washing with PBS for 2 times, adding the culture medium containing 100 mug/mL ascorbic acid stem cells, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution once every 3 days, and continuously culturing for 2 weeks to form a white velvet polymer, namely an adipose-derived stem cell membrane, at the bottom of the culture dish; folding and plasticizing 3 stem cell sheets, adding cell polymer culture medium, and placing in 5% CO₂Culturing in an incubator at 37 ℃, changing the culture solution half a day, and continuously culturing for 5 days, wherein pink velvet-shaped polymer, namely adipose-derived stem cell polymer, is formed in the culture dish.

(5) According to the diameter of the peripheral nerve defect of the receptor area, the silk fibroin catheter stent material is adopted to wrap the adipose-derived stem cell polymer for in vitro composition, 100 microgram/mL ascorbic acid stem cell culture medium is added, and 5 percent CO is placed₂Culturing in an incubator at 37 ℃, changing liquid half a day, and continuously culturing for 3 days to complete the compounding of the adipose-derived stem cell polymer and the silk fibroin conduit scaffold material, thereby obtaining the tissue-engineered peripheral nerve tissue.

The silk fibroin conduit stent material is prepared from a silk fibroin membrane according to the diameter of damaged peripheral nerves, wherein the silk fibroin membrane is purchased from Suzhou Simulter Biotechnology GmbH.

The tissue engineered peripheral nerve tissue prepared in this example, as shown in fig. 1B, is primary adipose-derived stem cells under an inverted microscope; FIG. 3A is a tissue engineered peripheral nerve tissue transplanted into a 10mm cheek branch defect of rat facial nerve; 3B-D are histological examination of the tissue-engineered peripheral nerve after 8 weeks, and it can be seen that the physiological structure of the tissue-engineered peripheral nerve tissue is very close to that of the natural peripheral nerve tissue, and has typical structures of myelin sheath, adventitia, and axon.

FIG. 1A is a primary dedifferentiated adipocytes under an inverted microscope; FIG. 1B is a primary adipose stem cell under an inverted microscope; fig. 1C is a primary mesenchymal stem cell under an inverted microscope; FIG. 1D is Schwann cells induced in vitro under an inverted microscope.

FIG. 2A shows the polymerization process of cell membrane under a body type microscope, FIG. 2B shows the polymerization process of cell aggregates observed in general, FIG. 2C shows the surface of cell aggregates under a scanning electron microscope, FIG. 2D, E shows the distribution of cells inside the cell aggregates and the amount of extracellular matrix observed by HE and Masson trichrome staining, and FIG. 2F shows the expression of type I collagen in cell polymerization observed by immunohistochemical staining.

FIG. 3A is a tissue engineered peripheral nerve tissue graft repaired rat facial nerve-buccal branch defect about 10mm in length; FIG. 3B shows toluidine blue staining of tissue engineered peripheral nerve tissue 8 weeks after reconstruction of buccal branch repair of rat facial nerves, and observed that myelin sheath number, arrangement and morphology are similar to those of native peripheral nerve tissue; FIG. 3C1-3 is NF-200 immunofluorescence staining to observe the arrangement and the configuration of nerve fibers of tissue-engineered peripheral nerve tissue, which is similar to natural peripheral nerve tissue; FIG. 3D1-4 is a photograph of GFAP (green) and β -Tubulin (red) immunofluorescent stained tissue engineered peripheral nerve tissue, with the observation that the green adventitia and purple nerve axon structure are similar to native peripheral nerve tissue. In conclusion, the tissue-engineered peripheral nerve tissue has a physiological structure very close to that of the natural peripheral nerve tissue, and has typical structures such as myelin sheaths, adventitia, axons and the like.