CN111035611A - Mesenchymal stem cell biological scaffold and preparation method and application thereof - Google Patents

Mesenchymal stem cell biological scaffold and preparation method and application thereof Download PDF

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CN111035611A
CN111035611A CN201911158976.0A CN201911158976A CN111035611A CN 111035611 A CN111035611 A CN 111035611A CN 201911158976 A CN201911158976 A CN 201911158976A CN 111035611 A CN111035611 A CN 111035611A
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mesenchymal stem
stem cell
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sodium glycerophosphate
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徐斌
江良旗
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Zhongguan Sail Biotechnology Beijing Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/72—Chitin, chitosan

Abstract

The embodiment of the invention provides a temperature-sensitive chitosan hydrogel prepared by using chitosan chloride and β -sodium glycerophosphate as raw materials, namely the mesenchymal stem cell biological scaffold, which has the characteristics of stable physical property, good tissue compatibility and good biodegradation performance, particularly the characteristic of slow solidification at 37 ℃, is suitable for carrying the mesenchymal stem cells to carry out osteoarthritis joint cavity injection treatment, has a slow release effect on the release of the cells, and plays a role in long-term treatment.

Description

Mesenchymal stem cell biological scaffold and preparation method and application thereof
Technical Field
The embodiment of the invention relates to the technical field of biological products, in particular to a mesenchymal stem cell biological scaffold and a preparation method and application thereof.
Background
Osteoarthritis (OA) is a painful joint disease which is characterized by the destruction of articular cartilage, so that the incidence rate of female patients is higher than that of male patients, particularly obese women are more prone to disease, the osteoarthritis is the most common bone disease which causes disability, and the life and work of the patients are greatly influenced. The main treatment methods for arthritis include pain relieving, physical therapy, functional exercise, diet conditioning and the like, and the clinical symptoms are relieved. In the prior art, chitosan or hyaluronic acid is also used independently, but the treatment effect is poor, and a material capable of being prepared for treating arthritis is urgently needed to be found.
Disclosure of Invention
Therefore, the embodiment of the invention provides a mesenchymal stem cell biological scaffold, a preparation method and an application thereof, which aim to solve the problem that an effective material for treating arthritis is lacked in the prior art.
In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:
a preparation method of a mesenchymal stem cell biological scaffold comprises the step of dispersing mesenchymal stem cells in a mixture of chitosan chloride and β -sodium glycerophosphate to form the mesenchymal stem cell biological scaffold.
Preferably, the mixture of the chlorinated chitosan and the β -sodium glycerophosphate is formed by mixing a chlorinated chitosan solution and a β -sodium glycerophosphate solution;
wherein the mass fraction of the chlorinated chitosan solution is 2%;
the β -sodium glycerophosphate solution is 45% in mass fraction.
Preferably, the mixture of the chlorinated chitosan and the β -sodium glycerophosphate is formed by placing the chlorinated chitosan solution and the β -sodium glycerophosphate with the volume ratio of 8: 2 on an ice box and fully mixing.
Preferably, the preparation method of the mesenchymal stem cell comprises the following steps:
dividing umbilical cord tissue into 3-4cm block tissues, adding mesenchymal stem cell serum-free culture medium into the block tissues, placing the block tissues on a culture plate, placing at 37 deg.C, and adding 5% CO by volume fraction2Incubating in incubator for 30min, and adding mesenchymal stem cellsClearing the culture medium and continuing culturing;
changing the culture medium in the culture process, after the cells are confluent and grow more than 80%, using a DPBS solution for immersion washing for 1 time, adding 1ml of recombinant pancreatin for digestion to separate the cells, adding 5-6ml of soybean trypsin inhibitor diluted by the DPBS solution to stop digestion, centrifuging to remove supernatant, and suspending the cells by using complete culture medium;
the resuspended cells were seeded in T25 flasks at 37 ℃ with a volume fraction of 5% CO2Culturing in an incubator, and carrying out subculture to obtain the mesenchymal stem cells when the cultured cells are 80% fused.
Preferably, the cell density of the resuspended cells is adjusted to 5000 cells/cm when the cells are inoculated into a T25 flask2。
Preferably, the mesenchymal stem cells are resuspended in β -sodium glycerophosphate solution to form a mesenchymal stem cell resuspension, and then mixed with the chitosan chloride and β -sodium glycerophosphate mixture.
Preferably, the volume ratio of the mesenchymal stem cell resuspension to the mixture of the chitosan chloride and β -sodium glycerophosphate is 1: 4.
The embodiment of the invention also provides the mesenchymal stem cell biological scaffold prepared by the method.
The embodiment of the invention also provides application of the mesenchymal stem cell biological scaffold in preparing a medicine for treating arthritis.
In the embodiment of the invention, autologous Mesenchymal Stem Cells (MSCs) can be transplanted not only by differentiating into damaged cell types through multi-differentiation potential, compensating lost components and secreting nutritional factors to promote the repair of damaged tissues of osteoarthritis, but also by exerting high-efficiency immune suppression and anti-inflammatory effects, so that the activity reaction of immune cells in damaged and/or inflammatory tissues is reduced, and the tissue damage is relieved.
Under the condition of specific induction in vivo and in vitro, the MSCs can be converted into various tissue cells such as bones, cartilages, muscles, fat, ligaments and tendons, so that a new way is opened up for the tissue engineering technology taking the MSCs as seed cells or the treatment of osteoarthritis. Meanwhile, the traditional Chinese medicine composition has the characteristics of wide sources, easy extraction and culture, low immunogenicity, multidirectional differentiation potential, strong proliferation capacity, immune regulation, anti-inflammation and the like, and can effectively treat pathological factors of osteoarthritis.
In the embodiment of the invention, chitosan is a natural renewable resource, and is a polyaminoglucose prepared by carboxymethyl substitution reaction of macromolecular compound chitin purified from shrimp shells, has positive charge, has the characteristics of good biocompatibility, no toxicity and no immunogenicity, is widely applied to the fields of medicine and pharmacology, cosmetics and tissue reconstruction, and can also improve the wound healing capacity of connective tissues. Because of its high viscoelasticity, it is similar to normal joint fluid, and after it is injected into joint cavity, it can simulate the physical action of joint synovial fluid to improve the lubrication condition of joint, relieve the pressure acting on joint cartilage surface, and can cover the cartilage surface or fill the degenerated cartilage fissure so as to prevent the harmful inflammatory cytokine in the synovia of osteoarthritis from contacting with cartilage matrix and cartilage cell. Structurally similar to mucopolysaccharides in chondrocytes, they are naturally degraded completely in vivo by enzymes. The chitosan is a good biological material for preventing tissue adhesion, has no toxicity, no irritation, no antigenicity and good biocompatibility, and is a medical biological material capable of being degraded and absorbed. The chitosan can prevent peritoneal adhesion and tendon adhesion and protect articular cartilage, has viscoelasticity and a lubricating function, can form a colloid network structure on the articular cavity cartilage surface, plays a role in biological barrier, and can also promote the secretion of synovial cells, thereby achieving the treatment effects of relieving pain and improving the joint function. The chitosan has the same effect as the mesenchymal stem cell biological scaffold, but the inhibition of the proliferation of the fiber cells, which is possessed by the chitosan, can play a positive role in the process and the progress of the osteoarthritis, and the inhibition is to be proved in clinical research of the osteoarthritis. In a word, the chitosan articular cavity injection treatment overcomes the defects of the traditional conservative therapy, not only relieves the symptoms, but also promotes the repair of articular cartilage, treats both principal and secondary aspects of diseases, and is simple and easy to implement.
On the basis of the basic characteristics of the chitosan hydrogel, the temperature-sensitive chitosan hydrogel has the characteristic of slow solidification under the condition of 37 degrees, is suitable for carrying mesenchymal stem cells to carry out osteoarthritis joint cavity injection treatment, plays a role in slow release of the cells, and plays a role in long-term treatment.
The embodiment of the invention has the following advantages:
the embodiment of the invention provides a method for preparing temperature-sensitive chitosan hydrogel by using chitosan chloride and β -sodium glycerophosphate as raw materials, namely the mesenchymal stem cell biological scaffold, which has the characteristics of stable physical properties, good histocompatibility and biodegradability, especially the characteristic of slow solidification at 37 ℃, is suitable for carrying mesenchymal stem cells to carry out osteoarthritis joint cavity injection treatment, has a slow release effect on the release of the cells, and has a long-term treatment effect.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The preparation method of the mesenchymal stem cell biological scaffold comprises the following steps:
1. preparation of chlorinated chitosan solution
0.2g of chitosan chloride is taken and stirred in 10ml of triple distilled water to respectively prepare 2 percent chitosan chloride aqueous solution. The chlorinated chitosan can not be dissolved completely instantly, is suspended into flocculent in the solution, and can be placed in a refrigerator at 4 ℃ overnight. Dissolving in triple distilled water to obtain milky clear jelly, sterilizing under high pressure, and storing in 4 deg.C refrigerator.
2.β preparation of sodium glycerophosphate solution
4.5g of β -sodium Glycerophosphate (GP) is dissolved in 10ml of triple distilled water to prepare 45% GP-water solution and 45% GP-DMEM solution, the salt is easy to dissolve, when the salt is dissolved in the triple distilled water, the solution is colorless clear liquid, a filter membrane with the diameter of 0.22 mu m is used for filtration and sterilization, and the solution is placed in a refrigerator at the temperature of 4 ℃ for standby.
A mixture of chitosan chloride and β -sodium glycerophosphate is prepared by mixing chitosan chloride solution and β -sodium glycerophosphate solution at a ratio of 8: 2 on ice box, standing at 37 deg.C for 20min to obtain solid gel.
3. Preparation of human umbilical cord mesenchymal stem cells
Separating and culturing human umbilical cord mesenchymal stem cells (HUC-MSCs), cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting umbilical cord tissue into 3-4cm tissue blocks, removing 3 blood vessels and blood stasis part, further separating tissue, and cutting into 2-3mm3Tissue mass of size. A small amount of the solution is dripped
Figure BDA0002285545270000051
The MSC XF Basal Medium mesenchymal stem cell serum-free culture Medium is prepared by sucking tissue blocks with 0.5-1cm distance, placing at 37 deg.C and 5% CO, and uniformly spreading in culture plate2Incubating in incubator for 30min, and slowly adding 1ml additive-containing solution into each well of 6-well culture plate
Figure BDA0002285545270000052
The MSCXF basic Medium mesenchymal stem cell serum-free culture Medium is put back into the incubator for culture.
Adding 1ml of complete culture medium into each hole on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing the cell morphology of tissue adherent growth with an inverted microscope every day, recording, observing under an inverted phase contrast microscope 5 days at the earliest, and finding that fine fusiform cells climb out from the edge. After the cells are confluent and grow more than 80%, removing the tissue mass and the culture medium in the culture plate, washing with DPBS Solution for 1 time, adding 1ml of Recombinant Trypsin EDTA Solution Recombinant pancreatin for digestion, tapping the culture plate to completely separate the cells, adding 5-6ml of diluted DPBS SolutionStopping digestion with soybean trypsin inhibitor, centrifuging to remove supernatant, suspending cells with a small amount of complete culture medium, and counting cells; the cell density was adjusted to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; the growth of the cells was observed by an inverted microscope and recorded, and when the cultured cells reached 80% confluency, subculture was performed for about 4 to 5 days.
Resuspending the mesenchymal stem cells by GP, mixing with a mixture of chitosan chloride and β -sodium glycerophosphate at a ratio of 1: 4 to obtain the mesenchymal stem cell biological scaffold, sucking by a 5ml syringe, and carrying out conventional disinfection and injection at the joint cavity part.
Example 2
The preparation method of the mesenchymal stem cell biological scaffold comprises the following steps:
1. preparation of chlorinated chitosan solution
0.2g of chitosan chloride is taken and stirred in 10ml of DMEM medium, and 2% chitosan chloride DMEM solution is taken. The chlorinated chitosan can not be dissolved completely instantly, is suspended into flocculent in the solution, and can be placed in a refrigerator at 4 ℃ overnight. Dissolving in DMEM to give pink clear viscous liquid, autoclaving, and storing in refrigerator at 4 deg.C for use.
2.β preparation of sodium glycerophosphate solution
4.5g of β -sodium Glycerophosphate (GP) is dissolved in 10ml of DMEM culture solution to prepare 45% GP-DMEM solution, the solution is clear pink solution when being dissolved in DMEM, filtration sterilization is carried out by using a filter membrane of 0.22 mu m, and the solution is placed in a refrigerator at 4 ℃ for standby.
A mixture of chitosan chloride and β -sodium glycerophosphate is prepared by mixing chitosan chloride solution and β -sodium glycerophosphate solution at a ratio of 8: 2 on ice box, standing at 37 deg.C for 20min to obtain solid gel.
3. Preparation of human umbilical cord mesenchymal stem cells
Separating and culturing human umbilical cord mesenchymal stem cells (HUC-MSCs), cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting umbilical cord tissue into 3-4cm tissue blocks, removing 3 blood vessels and blood stasis parts, further separating tissue,cutting into 2-3mm3Tissue mass of size. A small amount of the solution is dripped
Figure BDA0002285545270000061
The MSC XF Basal Medium mesenchymal stem cell serum-free culture Medium is prepared by sucking tissue blocks with 0.5-1cm distance, placing at 37 deg.C and 5% CO, and uniformly spreading in culture plate2Incubating in incubator for 30min, and slowly adding 1ml additive-containing solution into each well of 6-well culture plate
Figure BDA0002285545270000062
The MSCXF basic Medium mesenchymal stem cell serum-free culture Medium is put back into the incubator for culture.
Adding 1ml of complete culture medium into each hole on day 1, changing the culture medium in half on day 3, changing the culture medium in full on day 6, changing the culture medium 2 times per week, observing the cell morphology of tissue adherent growth with an inverted microscope every day, recording, observing under an inverted phase contrast microscope 5 days at the earliest, and finding that fine fusiform cells climb out from the edge. After the cells are confluent and grow by more than 80 percent, absorbing and removing tissue blocks and culture media in the culture plate, washing for 1 time by using a DPBS Solution, adding 1ml of recombined Trypsin EDTA Solution for digestion, tapping the culture plate to completely separate the cells, adding 5-6ml of soybean Trypsin inhibitor diluted by the DPBS Solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells; the cell density was adjusted to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; the growth of the cells was observed by an inverted microscope and recorded, and when the cultured cells reached 80% confluency, subculture was performed for about 4 to 5 days.
Resuspending the mesenchymal stem cells by GP, mixing with a mixture of chitosan chloride and β -sodium glycerophosphate at a ratio of 1: 4 to obtain the mesenchymal stem cell biological scaffold, sucking by a 5ml syringe, and carrying out conventional disinfection and injection at the joint cavity part.
Example 3
The preparation method of the mesenchymal stem cell biological scaffold comprises the following steps:
1. preparation of chlorinated chitosan solution
0.2g of chitosan chloride is taken and stirred in 10ml of triple distilled water to prepare 2 percent chitosan chloride aqueous solution. The chlorinated chitosan can not be dissolved completely instantly, is suspended into flocculent in the solution, and can be placed in a refrigerator at 4 ℃ overnight. The product is milky clear jelly-like liquid with uniform properties dissolved in triple distilled water.
2.β preparation of sodium glycerophosphate solution
4.5g of β -sodium Glycerophosphate (GP) is dissolved in 10ml of DMEM culture solution to prepare 45% GP-DMEM solution which is clear pink solution when dissolved in DMEM, filtration sterilization is carried out by using 0.22 mu m filter membranes, and the solution is placed in a refrigerator at 4 ℃ for standby.
The mixture of chitosan chloride and β -sodium glycerophosphate is prepared by mixing chitosan chloride solution and β -sodium glycerophosphate solution at a ratio of 8: 2 on ice box, standing at 37 deg.C for 20min to obtain solid gel.
3. Preparation of human umbilical cord mesenchymal stem cells
Separating and culturing human umbilical cord mesenchymal stem cells (HUC-MSCs), cleaning umbilical cord blood stain with DPBS under aseptic condition, cutting umbilical cord tissue into 3-4cm tissue blocks, removing 3 blood vessels and blood stasis part, further separating tissue, and cutting into 2-3mm3Tissue mass of size. A small amount of the solution is dripped
Figure BDA0002285545270000071
The MSC XF Basal Medium mesenchymal stem cell serum-free culture Medium is prepared by sucking tissue blocks with 0.5-1cm distance, placing at 37 deg.C and 5% CO, and uniformly spreading in culture plate2Incubating in incubator for 30min, and slowly adding 1ml additive-containing solution into each well of 6-well culture plate
Figure BDA0002285545270000081
The MSCXF basic Medium mesenchymal stem cell serum-free culture Medium is put back into the incubator for culture.
Adding 1ml of complete culture medium into each well on day 1, changing medium half on day 3, changing medium full on day 6, and changing medium 2 times per weekThe morphology of the cells growing adherent to the tissue is observed and recorded by an inverted microscope, and the cells are observed under an inverted phase contrast microscope at the earliest 5 days, and the cells with fine fusiform shapes climb out from the edge. After the cells are confluent and grow by more than 80 percent, absorbing and removing tissue blocks and culture media in the culture plate, washing for 1 time by using a DPBS Solution, adding 1ml of recombined Trypsin EDTA Solution for digestion, tapping the culture plate to completely separate the cells, adding 5-6ml of soybean Trypsin inhibitor diluted by the DPBS Solution to stop digestion, centrifuging to remove supernatant, suspending the cells by using a small amount of complete culture medium, and counting the cells; the cell density was adjusted to 5000 cells/cm2Inoculated in a T25 flask at 37 ℃ with 5% CO2Culturing in an incubator; the growth of the cells was observed by an inverted microscope and recorded, and when the cultured cells reached 80% confluency, subculture was performed for about 4 to 5 days.
Resuspending the mesenchymal stem cells by GP, mixing with a mixture of chitosan chloride and β -sodium glycerophosphate at a ratio of 1: 4 to obtain the mesenchymal stem cell biological scaffold, sucking by a 5ml syringe, and carrying out conventional disinfection and injection at the joint cavity part.
The mesenchymal stem cell biological scaffold prepared in the embodiments 1 to 3 of the invention utilizes the characteristic of slow solidification under the condition of 37 degrees, is suitable for carrying mesenchymal stem cells to carry out joint cavity injection treatment of osteoarthritis, has the slow release effect on the release of the cells, and has the long-term treatment effect.
Test examples
1. Data:
42 cases of knee osteoarthritis in orthopedic hospitalization at the hospital in 2011, 9-2015, 10 are selected, and are randomly divided into 15 cases (23 knees) of chitosan group, 22 cases (25 knees) of hyaluronic acid group, 19 cases (23 knees) of simple mesenchymal stem cell group and 16 cases (20 knees) of mesenchymal stem cell biological scaffold group implemented by the invention. Chitosan group 8 men (13 knees), 7 women (10 knees); age 44-69 years, mean (56.3 ± 12.5 years), course 6 months-30 years; hyaluronic acid group 8 men (10 knees), 14 women (18 knees); the age is 37-67 years, the average (50.5 + -16.4) years, and the course is 8 months-27 years. The mesenchymal stem cell group alone was 10 men (12 knees) and 9 women (11 knees); the age is 40-70 years, the average (45.5 +/-14.4) years, and the course is 8 months-30 years. Mesenchymal stem cell bioscaffold group male 7 (9 knees), female 9 (11 knees); the age is 34-63 years, the average (47.5 +/-15.4) years, and the course is 8 months-28 years. All four groups of patients have proven knee osteoarthritis, have osteophytes formed in different degrees, have partial joint gaps changed and meet diagnosis standards recommended by the American college of rheumatology.
2. Method of treatment
The patient lies down, and the affected knee is in the extended position or in the 90-degree flexed position. After the knee joint area is disinfected conventionally, the infrapatellar puncture of the knee joint of the affected limb is carried out according to the aseptic requirement, the effusion in the joint cavity is firstly extracted, the effusion is stored and detected at the temperature of minus 20 ℃, and 2ml of 1 percent chitosan solution, 2ml of 1 percent mesenchymal stem cell biological scaffold, hyaluronic acid and 1.5 multiplied by 10 are respectively injected into the joint cavity according to the components7The knee joint is moved for 10-15min after injection, so that the injectant is uniformly distributed on the joint surface, the contraction and relaxation exercise of the quadriceps femoris muscle is started 24h after operation, the flexion and extension exercise is exercised on the 3 rd-4 th day, the knee joint is moved to the lower part after 1 week, the injection is carried out for 3 times within 1 month as 1 treatment course, 4 treatment courses within 1 year, joint synovial fluid is taken and stored for testing respectively at the last 1 time of each treatment course, namely joint fluid testing is kept at the 1 st, 4 th, 7 th and 12 th month of the treatment, and the L-1, IL-6 and TNF- α levels are tested.
3. Index detection
Evaluating knee joint function (joint function index), observing and recording the rest pain, activity pain, tenderness, swelling and joint function self-evaluation of the knee joint, and summing the evaluation results to obtain the joint function index. The joint function index is divided into 15 grades, 0 grade, 1-5 grades, 6-10 grades and 11-15 grades. The efficacy evaluation was effective in achieving "0" after treatment and increasing by 1 grade, as shown in table 1.
TABLE 1 evaluation criteria for Knee Joint function
Figure BDA0002285545270000091
Figure BDA0002285545270000101
4. Results
According to the knee joint function evaluation results and the curative effect evaluation, the joint function indexes after 4, 7 and 12 months of treatment of the pure mesenchymal stem cell group, the chitosan group, the hyaluronic acid group and the mesenchymal stem cell biological scaffold group are obviously lower than those before treatment (P is less than 0.05, and P is less than 0.01), and the effective rate groups at 1, 4, 7 and 12 months are obviously different from each other, as shown in table 2.
TABLE 2 articular function index change, i.e. curative effect assessment results
Figure BDA0002285545270000102
Figure BDA0002285545270000111
Compared with the pretreatment, ＊ P is less than 0.05, ＊＊ P is less than 0.01
As shown in tables 2 and 3, the results show that the mesenchymal stem cell bioscaffold treatment of the present invention can significantly improve the symptoms and dysfunctional conditions of joint pain of knee osteoarthritis patients and has a long-lasting effective therapeutic effect.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (9)

1. A preparation method of a mesenchymal stem cell biological scaffold is characterized in that mesenchymal stem cells are dispersed in a mixture of chitosan chloride and β -sodium glycerophosphate to form the mesenchymal stem cell biological scaffold.
2. The method of preparing a mesenchymal stem cell bioscaffold of claim 1,
the mixture of the chlorinated chitosan and the β -sodium glycerophosphate is formed by mixing a chlorinated chitosan solution and a β -sodium glycerophosphate solution;
wherein the mass fraction of the chlorinated chitosan solution is 2%;
the β -sodium glycerophosphate solution is 45% in mass fraction.
3. The method of preparing a mesenchymal stem cell bioscaffold of claim 1,
the mixture of the chlorinated chitosan and the β -sodium glycerophosphate is formed by placing the chlorinated chitosan solution and the β -sodium glycerophosphate with the volume ratio of 8: 2 on an ice box and fully mixing.
4. The method of preparing a mesenchymal stem cell bioscaffold of claim 1,
the preparation method of the mesenchymal stem cells comprises the following steps:
dividing umbilical cord tissue into 3-4cm block tissues, adding mesenchymal stem cell serum-free culture medium into the block tissues, placing the block tissues on a culture plate, placing at 37 deg.C, and adding 5% CO by volume fraction2After the incubator is incubated for 30min, then, a mesenchymal stem cell serum-free culture medium is added for continuous culture;
changing the culture medium in the culture process, after the cells are confluent and grow more than 80%, using a DPBS solution for immersion washing for 1 time, adding 1ml of recombinant pancreatin for digestion to separate the cells, adding 5-6ml of soybean trypsin inhibitor diluted by the DPBS solution to stop digestion, centrifuging to remove supernatant, and suspending the cells by using complete culture medium;
the resuspended cells were seeded in T25 flasks at 37 ℃ with a volume fraction of 5% CO2Culturing in an incubator, and carrying out subculture to obtain the mesenchymal stem cells when the cultured cells are 80% fused.
5. The method of preparing a mesenchymal stem cell bioscaffold of claim 4,
the weightWhen the suspension cells were inoculated into a T25 flask, the cell density was adjusted to 5000 cells/cm2。
6. The method of preparing a mesenchymal stem cell bioscaffold of claim 4,
the mesenchymal stem cells are resuspended with β -sodium glycerophosphate solution to form a mesenchymal stem cell resuspension, and then mixed with the chitosan chloride and β -sodium glycerophosphate mixture.
7. The method of preparing a mesenchymal stem cell bioscaffold of claim 6,
the volume ratio of the mesenchymal stem cell resuspension to the mixture of the chitosan chloride and β -sodium glycerophosphate is 1: 4.
8. A mesenchymal stem cell bioscaffold prepared by the method of any one of claims 1-7.
9. Use of a mesenchymal stem cell bioscaffold of claim 8 in the preparation of a medicament for the treatment of arthritis.
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