CN104324417A - Tissue engineering neural restoration material constructed by autologous plasma and preparation method thereof - Google Patents

Abstract

The invention relates to a tissue engineering medical material, especially a tissue engineering nerve repair material constructed from autologous plasma, which includes a carrier and seed cells, and the carrier is constructed from autologous plasma and condensates containing fibrinogen and cell growth factors The seed cells are autologous bone marrow mesenchymal stem cells. The present invention uses autologous plasma and condensate containing fibrinogen and cell growth factors to form a carrier for supporting and cultivating seed cells, so that the seed cells and the carrier are injected into the infarcted area of the nerve tissue, and a gel-like scaffold is formed according to the shape of the infarcted area , the seed cells are attached to the gel, and grow under the support and nutrient supply of the scaffold to repair the damaged nerve and restore its biological function. The nerve repair material overcomes the problem of insufficient mechanical strength of natural materials and solves the problem of stent-graft resistance to the host.

Description

Tissue engineered nerve repair material constructed from autologous plasma and preparation method thereof

technical field

The present invention relates to medical materials, in particular to a tissue engineering nerve repair material constructed from autologous plasma and a preparation method thereof. the

Background technique

Stroke is listed by the medical profession as one of the three major diseases that threaten human health alongside coronary heart disease and cancer. Stroke is a disease with high morbidity, high mortality, high disability rate, high recurrence rate and many complications. For stroke patient survivors, 90% of patients will suffer from permanent neurological damage due to neuron loss. Functional impairment. To date, there are no effective treatments to repair brain damage caused by stroke. For most stroke patients, they will face a long recovery period and subsequent life-long clinical support life. However, even after rehabilitation treatment, 50%-95% of stroke patients still cannot fully return to normal life. Therefore, there is a great need to develop new treatments for the brain damage caused by stroke. the

In post-stroke rehabilitation, it may be possible to regenerate brain function through tissue engineering methods. Tissue engineering is the application of the principles of cell biology, biomaterials and engineering to repair and improve human diseased tissues or organs by combining cells, biomaterials and active molecules with biological functions. Although neural stem cells may be able to replace dead or injured nerve cells after acute stroke, neural stem cells may not have the ability to rebuild lost neural tissue due to lack of biomechanical support and biochemical signals. Therefore, the scaffold constructed of biomaterials in tissue engineering can provide biomechanical support for the above-mentioned neural stem cells, and at the same time guide the general shape and size of the regenerated tissue after three-dimensional growth, so the tissue engineering scaffold provides a favorable environment for the growth of neural stem cells. The environment provides the possibility for neural stem cells to rebuild lost neural tissue. the

An ideal tissue engineering scaffold should have biocompatibility and no foreign body reaction, and must also be resistant to certain pressure and pulling force, and can be sterilized, and can match the biomechanical properties of the tissue, and should be easy to replace; in addition, Materials for constructing tissue engineering scaffolds also need to have ideal biodegradable properties, and biodegraded products must be non-toxic and easily excreted from the human body. Generally, such tissue engineering scaffolds can be divided into natural materials obtained from animals and artificially synthesized synthetic materials. Natural materials include proteins of the extracellular matrix and their derivatives, eg, laminin, collagen, fibronectin, and gelatin. Although natural materials have ideal biocompatibility and cell adhesion, they also do not have sufficient mechanical strength to support the growth of neural stem cells. Such natural materials are prone to rapid degradation in the human body if they are not chemically cross-linked. Synthetic materials such as poly-D/L-lactic acid, polyethylene glycol polymers, poly[N-(2-hydroxypropyl)methacrylamide gel {poly-[N-(2-hydroxypropyl)] -methacrylamide hydrogels} etc. play an important role in axon development and repair in vitro. Although synthetic materials have advantages in controllable degradation rate, porosity, and mechanical strength, as biomaterial scaffolds, their compatibility with cells, Cell adhesion is poor and cannot interact with cells in an ideal manner. the

Tissue engineering scaffolds have been successfully applied to the regeneration of skin, bone, cartilage and peripheral nerves. For example, the patent document CN1168494C discloses "an injection-type multi-biological factor composite bone repair with animal fibrin as a carrier". Materials", the composite bone repair material uses fibrin in mammalian blood as a carrier, and uses genetically recombined bone morphogenetic protein, fibroblast growth factor and β-transforming growth factor in the damaged area. With the absorption and degradation of the carrier, the bone Biological factors such as morphogenetic proteins are slowly released, rapidly solidified in the body, and can be arbitrarily molded into the shape of the injured part, so that the new bone shape can completely restore the original physiological shape, so as to promote new bone growth, accelerate wound repair and promote fracture Healing effect. In addition, as the patent document of Chinese Patent Publication No. CN102114268A discloses "A Tissue Engineering Nerve Scaffold and Its Preparation Method and Application", the patent document uses chemical extraction to remove the main antigens that cause rejection - cells, axons and marrow and use chondroitinase ABC to remove the chondroitin sulfate proteoglycan in the allogeneic nerve, and obtain the decellularized allogeneic nerve with the basement membrane tube of the extracellular matrix intact as the tissue engineering nerve scaffold, which is suitable for repairing rats, For peripheral nerve defects such as rabbits, dogs, and humans, it has a significant effect on promoting axon regeneration, and can increase the length of repairing peripheral nerve defects. Bioartificial livers, pancreas, and kidneys are also in clinical development, while research on regeneration of central nervous system injuries is also under development. However, since the natural materials used in the tissue engineering scaffolds in the prior art are generally derived from animals such as pigs and sheep, such natural materials will have the problem of scaffold graft versus host after transplanted into the human body, and there are different degrees of immunity. Rejection. However, when synthetic materials are transplanted into the human body, they will cause rejection and cannot allow the body to form new interconnected and functional tissues. the

Contents of the invention

In order to overcome the deficiencies in the prior art, the first object of the present invention is to provide a tissue engineered nerve repair material constructed from autologous plasma, which overcomes the problem of insufficient mechanical strength of natural materials and solves the problem of stent grafting. Anti-host problem. the

In order to achieve the above-mentioned object one, the technical solution adopted by the present invention is: a tissue engineering nerve repair material constructed from autologous plasma, characterized in that: it includes a carrier and a seed cell, and the carrier is composed of fibrinogen and cell growth factor It is constructed from autologous plasma and condensate, and the seed cells are autologous bone marrow mesenchymal stem cells. the

In the above structure, autologous plasma contains a variety of cell growth factors, such as PDGF, TGF-β, VEGF, etc., all from autologous, which can promote the growth of seed cells without immune rejection. The above-mentioned nerve repair material has good plasticity, and can be injected into the stroke part of a stroke patient to shape it into the shape of the damaged part arbitrarily, so as to reduce the cerebral infarction area of the stroke patient and restore the biological function of the damaged nerve cells. the

As a further configuration of the present invention, the plasma containing fibrinogen and aggregates are mixed at a volume ratio of 1:1-3:7. the

In the above structure, the fibrin/condensate carrier is prepared by mixing blood plasma and condensate, which is used for culturing and supporting the growth of seed cells. The average fibrinogen content is 2.48g/L (the normal range of human fibrinogen content is 2-4g/L) by measuring the average of the three blood types of A, B, and AB, and the volume content of plasma is 30%. ~50%, that is, when the volume ratio of plasma and coagulum is 1:1~3:7, the plasma and coagulum are mixed together with seed cells and injected into the brain infarct area of stroke patients, and the coagulum can be formed according to the shape of the infarct area. The gel-like scaffold allows the seed cells attached to the gel to evenly distribute the infarcted area and repair the damaged nerves in the infarcted area in a larger range. The gel can support and provide nutrients for the seed cells. the

As a further configuration of the present invention, the condensate is one of calcium ion solution or calcium chloride-thrombin mixed solution. the

In the above structure, the gel formation time and the strength of the gel can be controlled by adjusting the concentration of calcium ions or thrombin, so that the most suitable carrier for seed cell growth can be obtained.

As a further configuration of the present invention, the calcium ion concentration is 10-50 mmol/L; the thrombin concentration is 0-2 U/ml. the

In the above structure, the calcium ion concentration will be diluted to a concentration of 5-25mmol/L after adding the plasma containing fibrinogen. And when the calcium ion concentration is 5mmol/L, the time for the gel to reach the plateau is about 40 minutes, and when the calcium ion concentration is 10mmol/L or greater than 10mmol/L, the gel can be formed and the plateau reached The duration is about 20 minutes. The concentration of thrombin is diluted to 0-1mmol/L after adding the plasma containing fibrinogen. Although thrombin can make the plasma containing fibrinogen form a gel at all concentrations, when the concentration of thrombin is too high, the formation of The speed of the gel is too fast, and a stable gel may have been formed during the process of loading the solution into a 96-well plate; when the gel concentration is 0-1 mmol/L, it takes 10- 16 minutes or so. Therefore, the concentration of thrombin mixed with plasma is more suitable between 0-1mmol/L. the

The second objective of the present invention is to provide a method for preparing tissue engineering nerve repair materials constructed from autologous plasma. the

In order to achieve the above-mentioned purpose two, the technical solution adopted in the present invention is: a preparation method of a tissue engineering nerve repair material constructed from autologous plasma, which is characterized in that it comprises the following steps:

(1) Preparation of seed cells: obtain autologous bone marrow cells, and obtain bone marrow mesenchymal stem cells through in vitro culture, and the cell density is 10 ⁶ -10 ⁸ ml;

(2) Preparation of plasma: Obtain autologous venous blood with anticoagulant-containing blood collection tubes, centrifuge at 3000rpm for 5-10min, extract supernatant, and obtain plasma containing fibrinogen and cell growth factors;

(3) Preparation of calcium chloride-thrombin mixed solution: (3a) prepare a calcium ion solution with a concentration of 10-50mmol/L; (3b) take the calcium ion solution in (3a) and dilute thrombin to 0-1U/L ml, to obtain calcium chloride-thrombin solution;

(4) adding the calcium chloride-thrombin mixture obtained in step (3) to the plasma obtained in step (2), and mixing evenly to obtain a fibrin/aggregate carrier;

(5) Adding the bone marrow mesenchymal stem cells obtained in step (1) to the fibrin/aggregate carrier obtained in step (4) to obtain a bone marrow mesenchymal stem cell/fibrin-aggregate solution nerve repair material.

As a further setting of the present invention, the preparation of the seed cells in the step (2): culturing autologous bone marrow cells in vitro by differential culture method, and culturing and proliferating through complete medium;

As a further setting of the present invention, the preparation of the step (3) calcium chloride-thrombin mixed solution: take the medical calcium chloride with a concentration of 450mmol/L in step (3a) and dilute it with PBS diluent to prepare a concentration of 10~ 50mmol/L calcium ion solution.

The present invention will be further described below in conjunction with the accompanying drawings. the

Description of drawings

Accompanying drawing 1 shows the situation that specific embodiment of the present invention-based fibrin/aggregate carrier and plasma gel formation of blood plasma and calcium ion;

Accompanying drawing 2 shows that specific embodiment two of the present invention is based on the situation that the fibrin/condensate carrier of plasma and calcium chloride-thrombin forms a gel;

Accompanying drawing 3 shows that the specific implementation of the present invention three is based on the situation that the fibrin/condensate carrier of blood plasma and calcium chloride-thrombin forms gel;

Accompanying drawing 4 shows that specific embodiment 4 of the present invention is based on the impact of different concentrations of calcium ion concentration-fixed concentration of thrombin on the gelation time of fibrin/aggregate formation;

Accompanying drawing 5 shows that specific embodiment 4 of the present invention is based on the impact of different concentrations of calcium ion concentration-fixed concentration of thrombin on the gelation time of fibrin/aggregate formation;

Accompanying drawing 6 shows the experimental result about the calcium ion concentration of optimum seed cell growth of specific embodiment five of the present invention;

Accompanying drawing 7 shows the experimental results of the specific embodiment six of the present invention about the most suitable plasma ratio for seed cell growth;

Accompanying drawing 8 shows the experimental results that the cerebral infarction area of stroke rats is significantly reduced after the injection of bone marrow mesenchymal stem cells/fibrin-condensate repair material compared with the blank control group;

Accompanying drawing 9 shows the situation of limb repair after the injection of bone marrow mesenchymal stem cells/fibrin-condensate repair material to stroke rats according to the present invention.

Detailed ways

A specific embodiment of the present invention is a tissue engineering nerve repair material constructed from autologous plasma, including a carrier and seed cells. stem cells. Autologous plasma contains a variety of cell growth factors, such as PDGF, TGF-β, VEGF, etc., which are all from the body. These cytokines can promote the growth of bone marrow mesenchymal stem cells without causing immune rejection in the body. The resulting nerve repair material has good plasticity, and can be injected into the stroke site of a stroke patient to shape the shape of the damaged site arbitrarily, restore the biological function of the damaged nerve cells, reduce the cerebral infarction area of the stroke patient, and play a role in the treatment of stroke. patient effect. the

The above fibrinogen plasma and aggregates are mixed at a volume ratio of 1:1 to 3:7. A fibrin/aggregate carrier made by mixing blood plasma with aggregate, used to culture and support the growth of seeded cells. The average fibrinogen content is 2.48g/L (the normal range of human fibrinogen content is 2-4g/L) by measuring the average of the three blood types of A, B, and AB, and the volume content of plasma is 30%. ~50%, that is, when the volume ratio is 1:1~3:7, the plasma is mixed with the condensate and injected together with the seed cells into the brain infarction area of the stroke patient. After that, the gel-like scaffold can be formed according to the shape of the infarction area. The seed cells attached to the gel can be evenly distributed in the infarction area, and the damaged nerves in the infarction area can be repaired in a larger range. The gel can support and provide nutrients for the seed cells. the

The above-mentioned condensate is one of calcium ion solution or calcium chloride-thrombin mixed solution. By adjusting the concentration of calcium ions or thrombin, the gel formation time and the strength of the gel can be controlled, so that the most suitable carrier for seed cell growth can be obtained. The above-mentioned calcium ion concentration is 10-50 mmol/L. The calcium ion concentration will be diluted to a concentration of 5-25mmol/L after adding plasma containing fibrinogen. When the calcium ion concentration is 5mmol/L, the time for the gel to reach the plateau is about 40 minutes, and when the calcium ion concentration is 10mmol/L or greater than 10mmol/L, the gel can be formed and reach the plateau The period of time is about 20 minutes, as can be seen in Example 1 below. the

Specific embodiment one of the fibrin/condensate carrier based on plasma and calcium ions:

Dilute medical calcium chloride (450mmol/L) with PBS to prepare calcium ion solutions with concentrations of 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L;

First add 50ul of plasma to the 96-well plate, and then add 50ul of the above-mentioned calcium ion solutions of different concentrations (gently, do not generate air bubbles). At this time, the calcium ion concentration is diluted to 5mmol/L, 10mmol/L, 15mmol/L, 20 mmol/L, 25 mmol/L, 3 replicate wells for each concentration of the above calcium ion solution, that is, each concentration has three experimental groups.

Then, continuous testing was performed by a full-wavelength microplate reader, with an interval of 2 minutes, for a total of 60 minutes. the

The blood plasma of A, B, and AB blood types is tested according to the above method, so that there are 9 values for each concentration of calcium ions, the average value of these 9 values is taken, and the graph is drawn by Origin within the standard error range (for The picture is beautiful, and the value is taken at intervals). As shown in Figure 1, when the concentration of calcium chloride is 5mmol/L, the time for the gel to reach the plateau is about 40 minutes, and the concentration of calcium chloride is greater than or equal to 10mmol/L (10mmol/L, 15mmol/L, 20mmol/L , 25mmol/L), can form a gel, and the time to reach the plateau is about 20 minutes. the

The above-mentioned thrombin concentration is 0-2 U/ml. When the thrombin concentration is 0, it is a pure calcium ion solution. When the thrombin concentration is greater than 0, it is a calcium chloride-thrombin solution. When the thrombin is added to the plasma containing fibrinogen, its concentration will be reduced by the plasma. Diluted to 0-1mmol/L, although thrombin can make plasma containing fibrinogen form a gel at various concentrations, but when the concentration of thrombin is too high, the speed of gel formation is too fast, and it may be difficult to fill all the solutions. A stable gel has been formed before it is put into a 96-well plate; when the gel concentration is 0-1 mmol/L, it can be seen from Figure 3 that it takes about 12-18 minutes to form a stable gel. Therefore, the concentration of thrombin mixed with plasma is more suitable between 0-1mmol/L. the

Specific embodiment two of the fibrin/condensate carrier based on plasma and calcium chloride-thrombin:

Take medical calcium chloride (450mmol/L) and dilute it with PBS to prepare a 10mmol/L calcium ion solution;

Dilute thrombin with diluted 10mmol/L calcium ion solution, the concentrations are 2U/ml and 31.2U/ml respectively;

First add 50ul of plasma to the 96-well plate, then add 50ul of calcium ion solution containing thrombin (gently, do not generate air bubbles), at this time the calcium ion concentration is diluted to 5mmol/L, and the thrombin concentration is diluted to 1U/ml. 15.6U/ml, each concentration of the above-mentioned thrombin has 3 duplicate wells. Immediately after that, consecutive tests were performed by a full-wavelength microplate reader with 2-minute intervals for a total of 60 minutes.

The blood plasma of A, B, and AB blood types is tested in the same way, so there are 9 values for each thrombin concentration, and the average value of these 9 values is drawn by Origin; although after 1 hour of the experiment, by naked eyes It can be observed that both concentrations of thrombin experimental groups have formed gels, but as shown in Figure 2, the final concentration of thrombin is 1U/ml and 15.6U/ml, only the final concentration of thrombin is 1U/ml It has a rising-plateau curve, but the thrombin with a final concentration of 15.6U/ml does not have this curve, and there is almost no rising process, which may be because the gel formation speed of the experimental group with high concentration of thrombin is too fast, A stable gel is formed before all the components are loaded into the 96-well plate, because it takes 2-5 minutes to load all the components into the 96-well plate. The gel formation time of thrombin with a final concentration of 1U/ml is about 15-20 minutes. -2U/ml, see specific embodiment three. the

Specific embodiment three based on the gel carrier of blood plasma and calcium chloride-thrombin:

Take medical calcium chloride (450mmol/L) and dilute it with PBS to prepare a 40mmol/L calcium ion solution;

Dilute thrombin with diluted 40mmol/L calcium ion solution, the concentrations are 0U/ml, 0.2U/ml, 0.5U/ml, 1U/ml, 2U/ml;

First add 50ul of plasma to the 96-well plate, then add 50ul of calcium ion solution containing thrombin (gently, do not generate air bubbles), at this time the calcium ion concentration is diluted to 20mmol/L, and the thrombin concentration is diluted to 0U/ml. 0.1U/ml, 0.25U/ml, 0.5U/ml, 1U/ml, 3 replicate wells for each concentration above.

Immediately after that, consecutive tests were performed by a full-wavelength microplate reader with 2-minute intervals for a total of 60 minutes. the

The blood plasma of A, B, and AB blood types was tested in the same way. There were 9 values for each concentration, and the average value of these 9 values was used to draw a graph through Origin, as shown in Figure 3. the

As shown in Figure 2-3, the concentration of calcium chloride (10mmol/L) and fibrinogen (2.48g/L) in Figure 2 is quantitative, and the concentration of thrombin (2U/ml, 31.2U/ml) is Variables, after mixing, the final concentration of calcium chloride is 5mmol/L, and the final concentration of thrombin is 1U/ml and 15.6U/ml, (the values in the accompanying drawings are all final concentrations after mixing) in Figure 3 , the concentration of calcium chloride (20mmol/L) and fibrinogen (2.48g/L) is quantitative, the concentration of thrombin (0U/ml, 0.2U/ml, 0.5U/ml, 1U/ml, 2U/ml ) is a variable; although it can be observed with the naked eye that thrombin can form gels at various concentrations after 1 hour, it can be seen from Figure 2 that thrombin has an upward curve-plateau formation only at 2U/ml. It can be seen from Figure 3 that the gel formation time is about 12-18 minutes when the thrombin concentration is between 0-2U/ml. So the following is to set the concentration of thrombin as 2U/ml, as quantitative, to observe the effect of different concentrations of fibrinogen on the gel formation time. the

Specific example four based on the influence of different concentrations of calcium ion concentration-fixed concentration of thrombin on the gelation time of fibrin/aggregate formation:

Take medical calcium chloride (450mmol/L) and dilute it with PBS to prepare 10mmol/L, 20mmol/L calcium ion solution;

Dilute thrombin with diluted 10mmol/L, 20mmol/L calcium ion solution to obtain thrombin with a concentration of 2U/ml;

In order to facilitate the gradient experiment of fibrinogen, clinical fibrinogen is selected to replace the fibrinogen contained in human plasma; since the normal range of human fibrinogen content is 2-4g/L, to purchase clinical human fibrinogen, according to Instructions for diluting into solutions with different concentrations, respectively 1mg/L, 2mg/L, 3mg/L, 5mg/L;

Take a 96-well plate, first add 50ul of fibrinogen solution of different concentrations, and then add 50ul of calcium ion solution containing thrombin (gentle action, do not generate air bubbles), 3 replicate wells for each concentration.

Immediately after that, the tests were performed continuously by a full-wavelength microplate reader, with intervals of 2 minutes, for a total of 60 minutes. the

The blood plasma of A, B, and AB blood types is tested in the same way, so there are 9 values for each concentration of the fibrinogen solution, and the average value of these 9 values is drawn by Origin; as shown in Figure 4-7 As shown, under the condition of constant thrombin concentration, gels can be formed when the concentration of calcium chloride is 5mmol/L and 10mmol/L, but the gel formation time (15-20 minutes) of 10mmol/L calcium chloride is shorter than that of 5mmol/L calcium chloride. /L gel formation time (40 minutes) was significantly shortened. Moreover, the fibrinogen content of a normal person is 2-4g/L, and the ladder test of the medical fibrinogen is consistent with the result of the human plasma test. Therefore, when the thrombin concentration is 2U/ml, it is better to choose a calcium ion concentration of 10mmol/L. the

Concrete embodiment five about the calcium ion concentration experiment of optimum seed cell growth:

When the cells are in good condition, the cells are cultured by cell plating, and the cells are replicated in 3 wells;

Take medical calcium chloride (450mmol/L) and dilute it with complete medium to prepare solutions of different concentrations of calcium ions, the concentrations are 0mmol/L, 2mmol/L, 5mmol/L, 10mmol/L, 15mmol/L, 20mmol/L 25mmol/L, 50mmol/L;

After the cells adhere to the wall, discard the supernatant, and add 200ul of the above-mentioned calcium ion solutions of different concentrations;

After 1 day, 2 days, and 3 days, CCK-8 was added to measure the OD value, so as to compare the cell proliferation.

Considering the limited use of thrombin in clinical practice, thrombin was not added in this group of experiments for optimal cell growth. As shown in Figure 6, the most suitable cell growth is 5mmol/L, 10mmol/L, because considering the time problem of forming the gel scaffold as shown in Figure 4-5, so finally choose 10mmol/L as the most suitable cell growth Calcium chloride concentration, which is consistent with the observation of cell growth under a microscope. When the concentration of calcium chloride is too high (25mmol/L, 50mmol/L), the cell growth will be poor, and there will be precipitates in the medium, which will affect the growth of the cells. . the

Concrete embodiment six about the plasma ratio experiment of optimum seed cell growth:

Take A, B, and AB blood types and dilute them with complete medium to prepare medium containing 0, 5%, 10%, 20%, 30%, 40%, 50%, and 100% plasma;

When the cells are in good condition, the cells are cultured by cell plating, and the cells are replicated in 3 wells;

After the cells adhere to the wall, discard the supernatant and add 200ul of the above-mentioned medium with different plasma concentrations;

After 1 day, 2 days, and 3 days, CCK-8 was added to measure the OD value, so as to compare the cell proliferation.

As shown in Figure 7, during the cell culture process, the difference between plasma and serum is that serum does not contain fibrinogen, while plasma contains coagulation factors and fibrinogen, and other nutritional components are similar. So the proportion gradient of plasma is set to 5%, 10%, 20%, 30%, 40%, 50%, 100%. Observed under a microscope, the cells in 100% of the plasma cannot grow; the growth state of the cells in 50% of the plasma is smaller than that of the plasma, the first reason may be that it is not suitable for cell growth; the second reason may be that the nutrients in the plasma are too high and the cells grow Super fast, so that the cells grow excessively and age prematurely; while the cells in 5% plasma grow slowly; the cells in 10%-40% plasma grow better. As shown in Figure 7, the best plasma ratio for cell growth was 20-40%. Therefore, in this group of experiments, 30% was selected as the suitable plasma ratio for cell growth based on the microscopic observation of cell growth and the ability to form a gel. the

About rat stroke experimental model (permanent cerebral ischemia model of middle cerebral artery occlusion (MCAO) established by nylon monofilament suture to occlude blood vessels):

Male rats, weighing 300-350 g, were anesthetized with 2.0% isoflurane, 30% oxygen, and 70% nitrous oxide spray. Rectal temperature was maintained at 37 ± 0.5 °C with a thermostatically controlled heating blanket (Harvard Instruments). The National Bureau of Statistics portable clinical analyzer and blood gas biochemical multiple rapid test cassettes (EC8+, Heska) were used to analyze blood; the anterior midline of the neck was cut to expose the left external carotid artery, ligated with 4-0 silk suture, and the distal end was cut Open, its branches electrocoagulated; separate the left internal carotid artery and vagus nerve. A 4-0 surgical monofilament nylon suture (Devis and GECK) was cut flat at the base and passed through the external carotid artery to the left internal carotid artery, 9 to 10 mm anterior to the carotid bifurcation. The skin was sutured within 10 minutes of the incision. After recovering from anesthesia, mice were kept in an air-conditioned room at 20 °C. Rats in the control group underwent the same surgery except that the suturing thread was not inserted.

Table S1 shows the results of arterial blood studies before and after ischemia. the

Table S1 time mean arterial pressure arterial partial pressure of oxygen difference Arterial CO ₂ partial pressure difference pH glucose Before ischemia 105 ±5 142 ±13 40 ±2 7.40±0.01 114 ±12 108 ±5 147 ±3 38 ±2 7.42±0.00 111 ±13 111 ±8 141 ±3 38 ±3 7.41±0.01 105 ±15 107 ±8 152 ±8 37 ±2 7.41±0.01 119 ±19 after ischemia 121±9 151 ±17 38 ±1 7.42±0.01 122 ±5 116 ±11 143 ±8 41±2 7.41±0.00 121 ±8 124 ±6 143±7 38 ±2 7.40±0.02 119 ±23 113 ±11 140 ±17 38 ±1 7.41±0.01 129 ±15

repair experiment

(1) Preparation of neural stem cells and scaffolds

(1) Preparation of seed cells: extract autologous bone marrow cells, culture and proliferate in vitro by differential culture method, the medium used is complete medium, and obtain bone marrow mesenchymal stem cells with a cell density of the order of 10 ⁶ -10 ⁸ ; Suspended in artificial cerebrospinal fluid (ACSF), the concentrations were 1x10 ³ cells/μl, 1x10 ⁴ cells/μl, 1x10 ⁵ cells/μl or 1x10 ⁶ cells/μl.

(2) Preparation of plasma: Obtain autologous venous blood with anticoagulant-containing blood collection tubes, centrifuge at 3000 rpm for 5-10 min, extract supernatant, and obtain plasma containing fibrinogen and cell growth factors;

(3) Preparation of calcium chloride-thrombin mixed solution: (3a) Take medical calcium chloride with a concentration of 450mmol/L and dilute it with PBS diluent to prepare a calcium ion solution with a concentration of 30mmol/L; (3b) Take (3a ) in the calcium ion solution dilutes the thrombin with a concentration of 250U/ml to 0.1U/ml to obtain a calcium chloride-thrombin solution;

(4) adding the calcium chloride-thrombin mixture obtained in step (3) to the plasma obtained in step (2), and mixing evenly to obtain a fibrin/aggregate carrier;

(5) Take the bone marrow mesenchymal stem cells obtained in step (1) and add them to the fibrin/condensate carrier obtained in step (4) to obtain bone marrow mesenchymal stem cells-fibrin carrier, transfer to 37°C after 1-2 hours at room temperature. ℃ thermostat storage.

(2) Transplantation of neural stem cells and scaffolds

Three weeks after focal cerebral ischemia, the rats were anesthetized.

Different ratios of MSC/fibrin-aggregate vehicle, MSCs alone, fibrin/aggregate alone, or artificial cerebrospinal fluid (ACSF) alone were injected using a 10-μl Hamilton syringe driven by a syringe pump. Inject directly into the infarct cavity. The injection volume was determined by estimating the volume of the cerebral infarct cavity three weeks after focal cerebral ischemia in rats. In this experiment, the injection volume of infarct lumen transplantation was 15 μl. Inject into the center of the cavity (0.7 mm anterior to bregma, 4.5 mm lateral to the midline, and 2.5 mm below the deep dura). The injection speed is 3 μL for 10 minutes, and the needle will remain for 5 minutes after injection, and then slowly withdraw from the injection site. After injection, the bone wound was sealed with bone wax. the

In addition, transplantation was also performed in control rats. BMSCs, fibrin/aggregate carrier, BMSC/fibrin-aggregate carrier or artificial cerebrospinal fluid (ACSF) alone were infused. the

The above two groups of experiments will analyze focal cerebral ischemia rats receiving and not receiving chamber transplantation at 1, 2 and 6 months after transplantation. the

Detection experiment:

Cresyl violet staining of posterior coronal rat brain sections of the above two groups of rats, as shown in Figure 8 (A)-(B); the volume of the infarct area of the rats injected with bone marrow mesenchymal stem cells/fibrin carrier was significantly reduced At the same time, as shown in Figure 9, the limb function also recovered obviously.

The present invention uses autologous plasma to construct a bone marrow mesenchymal stem cell/fibrin gel carrier as a central nervous repair material for the treatment of brain damage caused by stroke. The nerve repair material overcomes the problem of insufficient mechanical strength of natural materials. And solve the problem of stent graft versus host. the

Claims (6)

1. the tissue engineering nerve repair material built by autologous plasma, it is characterized in that: comprise carrier and seed cell, described carrier is built by the autologous plasma containing Fibrinogen and cell growth factor and condensation product and forms, and described seed cell is autologous bone marrow mesenchymal stem cells.

2. the tissue engineering nerve repair material built by autologous plasma according to claim 1, is characterized in that: described containing fibrinogenic blood plasma and condensation product with volume ratio 1:1 ~ 3:7 mixing.

3. the tissue engineering nerve repair material built by autologous plasma according to claim 1 and 2, is characterized in that: described condensation product is the one in ionic calcium soln or calcium chloride-thrombin mixed solution.

4. the tissue engineering nerve repair material built by autologous plasma according to claim 3, is characterized in that: described calcium ion concentration is 10 ~ 50mmol/L; Described concentration of thrombin is 0 ~ 2U/ml.

5. prepare a preparation method for the tissue engineering nerve repair material built by autologous plasma according to claim 1, it is characterized in that comprising the following steps:

(1) preparation of seed cell: obtain autologous vein blood, the centrifugal 5-10min of 3000rpm with the blood taking tube containing anticoagulant, extract the supernatant, obtain the blood plasma containing Fibrinogen and cell growth factor;

(2) preparation of blood plasma: obtain autologous vein blood, leave standstill layering, extracts the supernatant, obtains the blood plasma containing Fibrinogen and cell growth factor;

(3) preparation of calcium chloride-thrombin mixed solution: (3a) compound concentration is the ionic calcium soln of 10 ~ 50mmol/L; (3b) get ionic calcium soln dilution thrombin to the 0 ~ 1U/ml in step (3a), obtain calcium chloride-thrombin solution;

(4) added by calcium chloride-thrombin mixed liquor that step (3) obtains in the blood plasma that step (2) obtains, mix homogeneously, obtains fibrin/condensation product carrier;

(5) get mesenchymal stem cells MSCs that step (1) obtains to add in fibrin/condensation product carrier that step (4) obtains, obtain mesenchymal stem cells MSCs/fibrin-condensation product nerve repair material.

6. the preparation method of the tissue engineering nerve repair material built by autologous plasma according to claim 5, is characterized in that:

The preparation of seed cell in described step (1): with differential culture method In vitro culture autologous bone marrow cells, cultivate propagation by complete medium;

The preparation method of the tissue engineering nerve repair material built by autologous plasma according to claim 5 or 6, is characterized in that:

The preparation of calcium chloride-thrombin mixed solution in described step (3): it be the medical calcium chloride PBS diluted compound concentration of 450mmol/L is the ionic calcium soln of 10 ~ 50mmol/L that step (3a) gets concentration.