CN113546218A - Methods and formulations for in situ construction of PRP gel tissue engineering complexes for different tissue regeneration - Google Patents
Methods and formulations for in situ construction of PRP gel tissue engineering complexes for different tissue regenerationDownload PDFInfo
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Abstract
Translated fromChinese
Description
Technical Field
The invention relates to the technical field of tissue and organ regeneration, in particular to a method and a formula for in-situ construction of an autologous PRP gel tissue engineering compound for different tissue regeneration.
Background
Regeneration and repair of large-area tissue defects is extremely difficult, bleeding, infection and new injury can be caused by adopting autologous tissue transplantation, and the source is limited. Large-area tissue defects caused by wounds, infections, tumors, burns and the like are a delicate problem for clinical treatment, and the traditional autologous tissue or allogeneic tissue transplantation and biological material filling are adopted, so that the clinical application is severely limited due to various defects and unsatisfactory treatment effect.
At present, most of the tissue repair materials are in a scaffold material loaded cell growth factor mode. The selection of scaffold materials and growth factors is an important part of tissue engineering research. Many of the scaffold materials commonly used at present are solid materials, and have remarkable effects in the preparation of tissue repair materials. However, there are also some defects such as difficulty in shaping, complicated operation for loading growth factors and seed cells, low loading rate, and cell loss. Therefore, injectable or hydrogel-like materials are the hot spot of current tissue repair material research.
The cell growth factors commonly used in the field of tissue repair materials include BMP, TGF-. beta.bFGF, VEGF, etc., which are all exogenous proteins (xenogenic or xenogenic protein factors). The preparation method analysis of these growth factors includes two kinds of allogenic tissue extraction and gene recombination, and its preparation is complex and expensive. From the immunological perspective, various growth factors commonly used at present are exogenous (non-self) proteins or polypeptides, and immune reactions exist to different degrees. The autologous growth factor which is simple to research, develop and prepare, low in price and free of immunological rejection is used for preparing the tissue repair material, and is an important problem to be solved in further clinical application of the tissue repair material.
Disclosure of Invention
The invention aims to provide a method and a formula for in-situ construction of an autologous PRP gel tissue engineering compound for different tissue regeneration, so as to provide a formula for a tissue repair material of an autologous growth factor which is simple to prepare, low in price and free of immunological rejection.
In order to achieve the purpose, the invention adopts the following technical scheme: a PRP gel tissue engineering compound in-situ construction formula for different tissue regeneration is disclosed, wherein the PRP gel comprises the following raw materials: thrombin lyophilized powder, calcium gluconate injection, and PRP solution.
A PRP gel tissue engineering compound in-situ construction formula for different tissue regeneration is disclosed, wherein the PRP gel comprises the following raw materials: thrombin lyophilized powder, calcium gluconate injection, PRP solution, and mesenchymal stem cells.
A PRP gel tissue engineering compound in-situ construction method for different tissue regeneration is disclosed, the PRP gel in-vitro construction method comprises the following steps:
s1: under the aseptic condition, 2ml of calcium gluconate injection is injected into a 2000U thrombin freeze-dried powder ampoule bottle, the mixture is shaken until the calcium gluconate injection is fully dissolved to form calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: extracting 4.5ml of PRP solution by using a 10ml syringe;
s3: respectively connecting prepared syringes of 0.5ml of calcium gluconate/thrombin mixed solution and 4.5ml of PRP solution with injection ports of internal and external trocars of a transplantation gun, simultaneously synchronously injecting the calcium gluconate/thrombin mixed solution and the PRP into a saline ampoule empty bottle, standing to obtain PRP gel, wherein the gel solution constructed in vitro comprises the components with the thrombin activity of 100U/ml and the mesenchymal stem cells of 2 multiplied by 106The concentration of the calcium gluconate injection is 10mg/ml, and the concentration of the PRP liquid is 4.5 ml;
a PRP gel tissue engineering compound in-situ construction method for different tissue regeneration is disclosed, the PRP gel in-vivo construction method comprises the following steps:
s1: under the aseptic condition, 2ml of calcium gluconate injection is injected into a 2000U thrombin freeze-dried powder ampoule bottle, the mixture is shaken until the calcium gluconate injection is fully dissolved to form calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: extracting 4.5ml of PRP solution by using a 10ml syringe;
s3: under the ultrasonic guidance of musculoskeletal tissues, 0.5ml of prepared calcium gluconate/thrombin mixed solution and 4.5ml of prepared PRP solution are respectively connected with an inner trocar injection port and an outer trocar injection port of a transplantation gun, and simultaneously, the calcium gluconate/thrombin mixed solution and the PRP are synchronously injected into a lesion part.
A method for in-situ construction of PRP gel tissue engineering compound for different tissue regeneration comprises the following steps of:
s1: under the guidance of an image, a stem cell transplantation gun is used for puncturing a trocar to puncture, and the needle point is accurately positioned at a target position;
s2: synchronously injecting platelet activating agents, namely 0.2ml of calcium gluconate liquid and 2ml of PRP into the target site in vivo through two mutually isolated channels of a puncture trocar in the same time;
s3: the two liquids are fully mixed immediately before flowing out of the needle point, and a cell gel compound bracket which is matched with the lesion in shape and size is formed at the target part
Preferably, as an improvement, the mesenchymal stem cells are bone marrow mesenchymal stem cells, and the method for obtaining the bone marrow mesenchymal stem cells comprises the following steps: obtaining 200ml of bone marrow blood by using an injector containing heparin anticoagulant, and pumping the obtained bone marrow blood into a medical sterile blood collecting bag;
mixing bone marrow blood with PBS (phosphate buffer solution) with the same volume, standing at room temperature for 30min, centrifuging, removing supernatant, collecting cells, and washing the cells with PBS for 2 times; then suspending the cells by using the culture medium with the same amount of the discarded supernatant, wherein the number of nucleated cells is (1-2) multiplied by 107One per ml.
Preferably, as an improvement, the centrifugal separation mode is as follows: sucking a Percoll solution with the density position of 1.073g/ml to ensure that the ratio of the Percoll solution to the separation solution is 1: 1; centrifuging for 20-40 min under the condition of 2000r/min by using a horizontal centrifuge to divide liquid in the centrifuge tube into three layers from top to bottom, wherein the upper layer is platelets, the middle layer is separation liquid, the bottom layer is red blood cells and polymorphonuclear leukocytes, and a milky white turbid mononuclear cell layer is visible at the interface of the upper layer liquid and the middle layer liquid;
inserting a suction pipe into the mononuclear cell layer at the liquid interface of the upper layer and the middle layer, sucking nucleated cells along the edge of the test tube and transferring the nucleated cells into another centrifuge tube; suspending the cells with a culture medium more than or equal to 5 times, centrifuging for 10min at 2000r/min by using a horizontal centrifuge, and removing supernatant to obtain cells; resuspending the cells in culture medium, counting, measuring cell viability with 2% trypan blue, adjusting cell concentration to 1 × 106One per ml.
Preferably, as an improvement, the culture method of the stem cells between bone marrow is as follows: by 1.5X 105Per cm2Inoculating the bottom area to a culture dish at 37 deg.C, saturated humidity, and 5% CO2Culturing in an incubator for 48h to show a small amount of adherent cells, fusing 85% of cells after 2w, digesting, subculturing and inducing culture in a conditioned medium, and amplifying to 3 rd to 5 th generations for use. Resuspending after cell counting, adjusting to 2X 10 mesenchymal stem cells per ml nutrient solution6One per ml.
The principle and the advantages of the scheme are as follows:
(1) platelet Rich Plasma (PRP) is Plasma containing high concentration platelets separated from whole blood. In recent years, it has been found that platelet plasma (PRP) extracted from autologous venous blood by in vitro centrifugation and separation contains a large number of growth factors at high concentrations which are useful for promoting tissue regeneration. PRP is composed of platelets, leukocytes and other growth factors such as fibronectin: platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF). After the platelet is activated, a large amount of growth factors and cytokines can be released, the leucocyte has the function of resisting infection, and the fibronectin can provide a cell scaffold.
(2) In the scheme, the inner and outer pipelines of the trocar are mutually isolated, and are fully mixed and filled in a lesion part at the moment before leaving the needle point, so that the MSCs/PRP gel compound stent is ensured to be rapidly formed at one time.
(3) In the scheme, because the inner and outer pipelines of the inner and outer trocars of the used transplantation gun are mutually isolated, the special structure of the needle point promotes the solutions of the two channels to be fully mixed immediately before leaving the needle point, and the solution is quickly filled in a lesion part and then is solidified into a MSCs/PRP gel compound bracket with the shape and the size matched with the lesion, thereby being beneficial to the regeneration and repair of the form and the function of the lesion tissue and organ, and being expected to fundamentally reverse the pathological process of the tissue defect, thereby having great significance.
Meanwhile, the scheme includes the following advantages:
1. the method has the advantages that the autologous PRP is collected, so that immune rejection, ethical limitation and risk of infection diseases are avoided, the operation feasibility is high, and the method is suitable for clinical application;
2. the in situ pathological microenvironment can be greatly improved: PRP contains various anti-inflammatory factors, such as soluble tumor necrosis factor receptor (sTNF-R), interleukin-receptor antagonist (IL-RA), interleukin 4(IL-4), IL-10, IL-13, and the like;
3. can provide nutrition and a large amount of growth factors required by the survival of the stem cells, rebuilds the regeneration microenvironment of damaged tissues and organs: PRP contains far supraphysiological concentrations of platelets, which upon activation release large amounts of growth factors, mediating tissue repair, such as transforming growth factor- β 1(TGF- β 1), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), EGF, etc.;
4. adhesion, chemotactic factors and the like in the PRP can induce endogenous stem cells to migrate to the damaged part to participate in the regeneration and repair of the damaged tissue and organ;
5. the self-PRP gel is high-permeability fibrin gel, so that the self-PRP gel can become an excellent and suitable cell scaffold, can provide an attachment place for MSCs, can promote the diffusion of nutrients and metabolites, is easy to establish the contact between cells and promotes the formation of functionalized tissues and organs;
6. injecting the gel forming liquid S1 and the gel forming liquid S2 into the damaged tissue organ through a trocar inside and outside the transplantation gun, filling the damaged tissue organ with the gel forming liquid S1 and the gel forming liquid S2 before curing, and instantly forming a cell gel compound bracket which is matched with the shape and the size of the damaged tissue organ to participate in the perfect remodeling of the shape and the function.
Drawings
FIG. 1 is a graph showing the correlation between PRP aggregation at 1h in A and B in example 1 of the present invention.
FIG. 2 is a graph comparing the aggregation of three sets of PRPs A, B and C during stabilization in an embodiment of the present invention.
FIG. 3 is a schematic view of the inner and outer trocars in an embodiment of the present invention.
FIG. 4 is an enlarged view of a portion of an inner and outer trocar in accordance with an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail below by way of specific embodiments:
reference numerals in the drawings of the specification include: anouter trocar 11, a liquid inlet hole A12, aninner trocar 13, a liquid inlet hole B13, agap 15 and anoutlet position 16.
The first embodiment is as follows:
a formula for in-situ construction of PRP gel tissue engineering compound for different tissue regeneration comprises the following raw materials: thrombin lyophilized powder, calcium gluconate injection, and PRP solution.
A method for in-situ construction of PRP gel tissue engineering compound for different tissue regeneration comprises the following steps:
the PRP gel in-vitro construction comprises the following steps:
s1: under the aseptic condition, 2ml of calcium gluconate injection (1 g/10ml of calcium gluconate) is injected into a 2000U ampoule bottle of thrombin freeze-dried powder, the ampoule bottle is shaken until the calcium gluconate injection is fully dissolved to form calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: 4.5ml of PRP solution was extracted with a 10ml syringe and PRP was obtained by either mechanical or manual collection as is currently done.
S3: connecting prepared 0.5ml of mixed solution of calcium gluconate and thrombin and 4.5ml of PRP solution respectively with the injection ports of the inner and outer trocars of the transplantation gunAnd synchronously injecting the calcium gluconate/thrombin mixed solution and the PRP into a saline ampoule empty bottle, and standing to obtain PRP gel. The gel liquid constructed in vitro comprises thrombin with activity of 100U/ml and mesenchymal stem cells of 2 × 106The concentration of the calcium gluconate injection is 10mg/ml, and the concentration of the PRP liquid is 4.5 ml.
Example two:
the difference between the second embodiment and the first embodiment is that the method for in-situ construction of the PRP gel tissue engineering compound for different tissue regeneration,
the PRP gel in vivo construction comprises the following steps:
s1: under aseptic conditions, 2ml of calcium gluconate injection (1 g/10ml of calcium gluconate) is injected into a 2000U ampoule of thrombin freeze-dried powder, the mixture is shaken up to be fully dissolved into calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: the PRP solution (4.5 ml) was extracted with a 10ml syringe
S3: under the ultrasonic guidance of musculoskeletal bones, 0.5ml of prepared calcium gluconate/thrombin mixed solution and 4.5ml of PRP solution are respectively connected with an inner trocar injection port and an outer trocar injection port of a transplantation gun, simultaneously, the calcium gluconate/thrombin mixed solution and the PRP are synchronously injected into a lesion part, PRP gel is formed at the lesion part, the ratio of each component of the gel solution constructed in vivo is that the thrombin activity is 100U/ml, and the mesenchymal stem cells are 2 multiplied by 106The concentration of the calcium gluconate injection is 10mg/ml, and the concentration of the PRP liquid is 4.5 ml;
s4: after the operation, the trocar is pulled out, the aseptic dressing is pasted for 20 minutes after the operation (after the temporary operation for 20 minutes, the aseptic dressing can be adjusted according to the in-vitro PRP/calcium gluconate gel forming time), the gel forming condition of the PRP/calcium gluconate implanted target position in the body is observed by using ultrasonic wave, and the observation is compared with the PRP/NS.
EXAMPLE III
The difference between the third embodiment and the second embodiment is that a method for in-situ construction of a PRP gel tissue engineering composite for different tissue regeneration, in this embodiment, PRP gel shaping is performed in situ on lumbar disc degeneration, which includes the following steps:
s1: under the guidance of an image, a stem cell transplantation gun is used for puncturing a trocar to puncture, and the needle point is accurately positioned at a target position;
s2: synchronously injecting platelet activating agents, namely 0.2ml of calcium gluconate liquid and 2ml of PRP into the target site in vivo through two mutually isolated channels of a puncture trocar in the same time;
s3: the two liquids are fully mixed immediately before flowing out of the needle point, and a cell gel compound scaffold which is matched with the lesion in shape and size is formed at a target part.
The experiments were as follows:
a, simultaneously and respectively connecting a prepared 0.5ml NS syringe and a prepared 5ml PRP syringe with aninner sleeve opening 14 and anouter sleeve opening 12 for injection of an inner outer sleeve needle of a self-made transplantation gun, synchronously and simultaneously injecting the liquid in the two syringes into a1 st 10ml saline ampoule empty bottle, and standing and observing;
b, operating the same as A, injecting 0.5ml of calcium gluconate and 5ml of PRP into a 2 nd 10ml saline ampoule empty bottle, and standing and observing;
c, the operation is the same as A, 0.5ml of calcium gluconate/thrombin mixed solution and 5ml of PRP are injected into a 3 rd 10ml saline ampoule empty bottle, and the mixture is kept stand and observed. The observation results are shown in FIG. 1, FIG. 2 and FIG. 3.
Example four:
example four differs from examples one to three in that a method for the in situ construction of PRP gel tissue engineering complex for regeneration of different tissues, in this example, for in situ reconstruction of MSCs/PRP gel complex scaffolds in vivo, comprises the steps of,
s1: under aseptic condition, 2ml of calcium gluconate injection (1 g/10ml of calcium gluconate) is injected into 2000U of thrombin lyophilized powder ampoule bottle, and bone marrow mesenchymal stem cells are added to obtain 2 × 106Shaking until the calcium gluconate/thrombin mixed solution is fully dissolved, and extracting 0.5ml of the mixed solution by using a 1ml syringe; in the present embodiment, the mesenchymal stem cells are not limited to bone marrow mesenchymal stem cells, and umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, and other mesenchymal stem cells that can be used may be used.
S2: the PRP solution (4.5 ml) was extracted with a 10ml syringe
S3: and (3) respectively connecting the prepared injectors of 0.5ml of calcium gluconate/thrombin mixed solution and 4.5ml of PRP solution with the injection ports of the inner trocar and the outer trocar of the transplantation gun, simultaneously synchronously injecting the calcium gluconate/thrombin mixed solution and the PRP into an empty saline ampoule bottle, and standing to obtain PRP gel. The gel liquid constructed in vitro comprises thrombin with activity of 100U/ml and mesenchymal stem cells of 2 × 106The concentration of the calcium gluconate injection is 10mg/ml, and the concentration of the PRP liquid is 4.5 ml.
In this embodiment, the method for obtaining mesenchymal stem cells is as follows: obtaining 200ml of bone marrow blood by using an injector containing heparin anticoagulant, and pumping the obtained bone marrow blood into a medical sterile blood collecting bag;
mixing bone marrow blood with PBS (phosphate buffer solution) with the same volume, standing for 30min at room temperature, and performing centrifugal separation in the following manner: taking a Percoll solution with the density position of 1.073g/ml, and leading the ratio of the Percoll solution to a separation solution to be 1: 1; centrifuging for 20-40 min under the condition of 2000r/min by using a horizontal centrifuge to ensure that liquid in the centrifuge tube is divided into three layers from top to bottom, wherein the upper layer is platelets, the middle layer is separation liquid, the bottom layer is red blood cells and polymorphonuclear leukocytes, and a milky white turbid mononuclear cell layer is visible at the interface of the upper layer liquid and the middle layer liquid;
inserting a suction pipe into the mononuclear cell layer at the liquid interface of the upper layer and the middle layer, sucking nucleated cells along the edge of the test tube and transferring the nucleated cells into another centrifuge tube; suspending cells with culture medium more than or equal to 5 times, centrifuging for 10min at 2000r/min with horizontal centrifuge, removing supernatant, collecting cells, and washing cells with PBS for 2 times; then suspending the cells in the culture medium with the same amount of the discarded supernatant, wherein the number of the nucleated cells is (1-2) multiplied by 107Per ml; resuspending the cells in culture medium, counting, measuring cell viability with 2% trypan blue, adjusting cell concentration to 1 × 106One per ml.
The bone marrow mesenchymal stem cells are cultured in the following way: by 1.5X 105Per cm2The bottom area was inoculated into a petri dish and cultured in a 5% CO2 incubator at 37 ℃ with saturated humidity. After 48 hours of culture, a small amount of cells can adhere to the wall, and after 2w, 85 percent of cells are fused, and digestion and passage can be carried outInoculating, culturing in the condition culture medium, and amplifying to 3-5 generation. Resuspending after cell counting, adjusting to 2X 10 mesenchymal stem cells per ml nutrient solution6One per ml.
In the first to fourth embodiments of the present invention, as shown in fig. 4, the inner and outer cannula needles of the transplantation gun are provided with anouter cannula needle 11 and aninner cannula needle 13, theinner cannula needle 13 is detachably connected to theouter cannula needle 11, a needle tube portion of theinner cannula needle 13 is located in a needle tube portion of theouter cannula needle 11, and agap 15 is formed between the needle tube portion of theinner cannula needle 13 and the needle tube portion of theouter cannula needle 11.
Theouter trocar 11 is provided with a liquid inlet A12 communicated with the needle tube, theinner trocar 13 is provided with a liquid inlet B14 communicated with the needle tube, and theouter trocar 11 is communicated with theinner trocar 13.
The use mode in this example is as follows: communicating a prepared injector with 0.5ml of calcium gluconate/thrombin mixed solution with a liquid inlet B14, communicating an injector with 4.5ml of PRP solution with a liquid inlet A12, simultaneously synchronously injecting the calcium gluconate/thrombin mixed solution and the PRP solution into aninner trocar 13 and anouter trocar 11 respectively, enabling 0.5ml of the calcium gluconate/thrombin mixed solution in theinner trocar 13 to flow to anoutlet 16, enabling 4.5ml of the PRP solution in theouter trocar 11 to flow to theoutlet 16 in agap 16, enabling the two solutions to meet and mix, and then enabling the two solutions to enter a defect position. (in this embodiment, the solid arrows indicate the direction of fluid flow in the inner trocar, the hollow arrows indicate the direction of fluid flow in the outer trocar, and the solid arrows are staggered with the hollow arrows indicate the mixing of fluid in the inner and outer cannulae).
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (8)
1. An autologous PRP gel tissue engineering compound in-situ construction formula for different tissue regeneration is characterized in that: the PRP gel comprises the following raw materials: thrombin lyophilized powder, calcium gluconate injection, and PRP solution.
2. The formulation for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 1, wherein: the PRP gel comprises the following raw materials: thrombin lyophilized powder, calcium gluconate injection, PRP solution, and mesenchymal stem cells.
3. The method for construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 1, wherein:
s1: under the aseptic condition, 2ml of calcium gluconate injection is injected into a 2000U thrombin freeze-dried powder ampoule bottle, the mixture is shaken until the calcium gluconate injection is fully dissolved to form calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: extracting 4.5ml of PRP solution by using a 10ml syringe;
s3: and (3) respectively connecting the prepared 0.5ml of calcium gluconate/thrombin mixed solution and 4.5ml of autologous PRP solution injectors with the injection ports of the inner trocar and the outer trocar of the transplantation gun, simultaneously synchronously injecting the calcium gluconate/thrombin mixed solution and the PRP into a saline ampoule empty bottle, and standing to obtain PRP gel, wherein the gel solution constructed in vitro comprises the components with the thrombin activity of 100U/ml, the calcium gluconate injection concentration of 10mg/ml and the PRP solution of 4.5 ml.
4. The method for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 2, wherein:
s1: under the aseptic condition, 2ml of calcium gluconate injection is injected into a 2000U thrombin freeze-dried powder ampoule bottle, the mixture is shaken until the calcium gluconate injection is fully dissolved to form calcium gluconate/thrombin mixed solution, and a 1ml syringe is used for extracting 0.5ml of the solution;
s2: extracting 4.5ml of PRP solution by using a 10ml syringe;
s3: under the ultrasonic guidance of musculoskeletal tissues, 0.5ml of prepared calcium gluconate/thrombin mixed solution and 4.5ml of prepared PRP solution are respectively connected with an inner trocar injection port and an outer trocar injection port of a transplantation gun, and simultaneously, the calcium gluconate/thrombin mixed solution and the PRP are synchronously injected into a lesion part.
5. The method for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 2, comprising the steps of:
s1: under the guidance of an image, a stem cell transplantation gun is used for puncturing a trocar to puncture, and the needle point is accurately positioned at a target position;
s2: synchronously injecting platelet activating agents, namely 0.2ml of calcium gluconate liquid and 2ml of PRP into the target site in vivo through two mutually isolated channels of a puncture trocar in the same time;
s3: the two liquids are fully mixed immediately before flowing out of the needle point, and a cell gel compound scaffold which is matched with the lesion in shape and size is formed at a target part.
6. The method for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 2, wherein:
the mesenchymal stem cells are bone marrow mesenchymal stem cells, and the method for obtaining the bone marrow mesenchymal stem cells comprises the following steps: obtaining 200ml of bone marrow blood by using an injector containing heparin anticoagulant, and pumping the obtained bone marrow blood into a medical sterile blood collecting bag;
mixing bone marrow blood with PBS (phosphate buffer solution) with the same volume, standing at room temperature for 30min, centrifuging, removing supernatant, collecting cells, and washing the cells with PBS for 2 times; then suspending the cells by using the culture medium with the same amount of the discarded supernatant, wherein the number of nucleated cells is (1-2) multiplied by 107One per ml.
7. The method for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 5, wherein:
the centrifugation method is as follows: sucking a density position 1.073g/ml Percoll solution to ensure that the ratio of the density position to the separation solution is 1: 1; centrifuging for 20-40 min under the condition of 2000r/min by using a horizontal centrifuge to ensure that liquid in the centrifuge tube is divided into three layers from top to bottom, wherein the upper layer is platelets, the middle layer is separation liquid, the bottom layer is red blood cells and polymorphonuclear leukocytes, and a milky white turbid mononuclear cell layer is visible at the interface of the upper layer liquid and the middle layer liquid;
inserting a suction pipe into the mononuclear cell layer at the liquid interface of the upper layer and the middle layer, sucking nucleated cells along the edge of the test tube and transferring the nucleated cells into another centrifuge tube; suspending the cells with a culture medium more than or equal to 5 times, centrifuging for 10min at 2000r/min by using a horizontal centrifuge, and removing supernatant to obtain cells; resuspending the cells in culture medium, counting, measuring cell viability with 2% trypan blue, adjusting cell concentration to 1 × 106One per ml.
8. The method for the in situ construction of PRP gel tissue engineering complex for differential tissue regeneration according to claim 6, wherein:
the marrow stem cell culture mode is as follows: by 1.5X 105Per cm2Inoculating the bottom area to a culture dish at 37 deg.C, saturated humidity, and 5% CO2Culturing in an incubator for 48h to show a small amount of adherent cells, fusing 85% of cells after 2w, digesting, subculturing and inducing culture in a conditioned medium, and amplifying to 3 rd to 5 th generations for use. Resuspending after cell counting, adjusting to 2X 10 mesenchymal stem cells per ml nutrient solution6One per ml.
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