Preparation method and application of articular cartilage regeneration membrane

Abstract

The invention discloses a preparation method and application of an articular cartilage regeneration membrane sheet, belongs to the technical field of medical materials, and specifically relates to the preparation of chondrocytes by using cartilage tissue. First, the cartilage tissue is cut into small pieces, and then treated with protease and collagenase to prepare free chondrocytes. After passage to the 2nd and 3rd generations, the chondrocytes are cultured in a culture medium containing a culture supplement to prepare the cartilage regeneration membrane sheet, thereby having the following beneficial effects: lactose amide compounds have good safety and will not cause a large number of cell apoptosis; after using a culture supplement containing at least one of fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, ITS+ reagent and lactose amide compounds, the cell growth rate can be increased, the production cycle of the cartilage regeneration membrane sheet can be reduced, and the cartilage regeneration membrane sheet with a multilayer structure can be obtained within a specific time.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and specifically relates to a preparation method and application of an articular cartilage regeneration membrane. Background Art

[0002] The surface of the connected bones in the joint is covered with a layer of articular cartilage, which is transparent. Articular cartilage has neither nerves nor blood vessels, and its nutrition is mainly supplied by synovial fluid and arterial branches around the synovial layer of the joint capsule. The surface of the articular cartilage facing the joint cavity is smooth, which is convenient for movement between bones. Articular cartilage is elastic and can buffer the vibration and impact of the connected bones during movement; the elasticity and deformation ability of articular cartilage also affect the increase of joint mobility. Usually, damage to articular cartilage will cause patients to have limited movement, inflexible joints, inability to perform strenuous activities and strong pain. Therefore, the regeneration of articular cartilage defects is particularly important. According to the current regeneration of articular cartilage defects, it is extremely difficult to achieve the regeneration of articular cartilage damage while maintaining the integrity of the original bones. In this regard, articular cartilage regeneration membrane technology is a treatment method that can improve the above situation. Compared with traditional microfracture surgery, this method can regenerate hyaline cartilage, has fewer complications, and has a lower probability of disease deterioration. Traditional microfracture surgery can only regenerate fibrocartilage and is prone to complications and serious disease deterioration.

[0003] The articular cartilage regeneration membrane is a unique culture dish that uses the cartilage regeneration membrane as the basic unit to reconstruct the tissue to form a 3D-like structure without a scaffold. CN115161268A discloses a preparation method and use of regional layered chondrocyte layers. At present, the preparation method of the cartilage regeneration membrane provided by the prior art requires the use of a cell filter to filter the cell suspension, which causes damage to the cells, affects the cell yield, and reduces the amount of cells collected, thereby making the preparation efficiency of the cartilage regeneration membrane low. At the same time, the selection of a suitable cell filter pore size will increase the production cost, and the production process is difficult to control, which is not conducive to mass production of enterprises. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing and applying an articular cartilage regeneration membrane sheet having high plasticity, good integrity, fast cell growth rate, short product production cycle and the ability to save time.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A method for preparing a cartilage regeneration membrane comprises: inoculating chondrocytes in a separation vessel, culturing them in a culture medium containing a culture supplement, and obtaining a cartilage regeneration membrane through separation treatment, wherein the culture supplement comprises at least one of fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, ITS+ reagent, and lactosylamide compound. The use of at least one of fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, ITS+ reagent, and lactosylamide compound in the present invention can increase the growth rate of chondrocytes and reduce the production cycle of the cartilage regeneration membrane, and different combinations of the above culture supplements can obtain preparation methods with different excellent effects.

[0007] Preferably, the separation vessel is a temperature-sensitive culture vessel.

[0008] Preferably, the culture medium is DMEM culture medium; or, the lactosylamide compound has a glutamine structure; or, the lactosylamide compound is obtained by the reaction of lactobionic acid and glutamine. The lactosylamide compound in the present invention has a structure formed by the combination of lactobionic acid and glutamine, and the lactosylamide compound has a certain effective concentration for its effect, and after the effective concentration, it has a good promoting effect on cell growth.

[0009] Preferably, in the culture medium containing culture supplements, the amount of fetal bovine serum used is 2-10wt%; or, the amount of ascorbic acid used is 50-200mg / L; or, the amount of sodium glycerophosphate used is 5-20mmol / L; or, the amount of dexamethasone is 5-20nmol / L; or, the amount of ITS+ reagent used is 0.5-2wt%; or, the amount of lactose amide compound used is 0.01-0.1wt%.

[0010] Preferably, in the preparation of the lactobionic acid amide compound, lactobionic acid and glutamine are reacted in a solution containing EDC·HCl and NHS to obtain the lactobionic acid amide compound.

[0011] Preferably, the amount of EDC·HCl used is 40-60wt% of lactobionic acid; or, the amount of NHS used is 20-40wt% of lactobionic acid; or, the amount of glutamine used is 40-60wt% of lactobionic acid.

[0012] Preferably, the chondrocytes are prepared by enzymatic hydrolysis of cartilage tissue; or, the chondrocytes are prepared by enzymatic hydrolysis of cartilage tissue using trypsin and / or collagenase.

[0013] Preferably, in the preparation of the lactobionic acid compound, lactobionic acid is added to water, then EDC·HCl and NHS are added and stirred, and then glutamine is added, and the reaction is carried out at 20-40° C. for 24-72 hours. After the reaction is completed, the mixture is distilled under reduced pressure, washed with methanol, and dried to obtain the lactobionic acid compound.

[0014] More preferably, in the preparation of the lactobionic acid compound, the amount of lactobionic acid used is 4-12 wt % of water.

[0015] More preferably, in the preparation of the lactobionic acid amide compound, the amount of EDC·HCl used is 40-60 wt % of lactobionic acid.

[0016] More preferably, in the preparation of the lactobionic acid amide compound, the amount of NHS used is 20-40 wt % of lactobionic acid.

[0017] More preferably, in the preparation of the lactobionic acid amide compound, the amount of glutamine used is 40-60 wt % of lactobionic acid.

[0018] Preferably, in the subculture of chondrocytes, the cartilage tissue is cut into small pieces, added to a protease solution, stirred at a temperature of 20-40°C for 30-180min, centrifuged at 300-500×g for 5-20min to remove the protease solution, and the chondrocyte precipitate is collected; the chondrocyte precipitate is added to a collagenase solution and incubated at 20-40°C until the cartilage tissue is completely digested and the chondrocytes are free in the suspension, and the incubation is completed to obtain a free chondrocyte fluid; the free chondrocyte fluid is centrifuged at 20-40×g for 5-20min, and the supernatant is taken; the supernatant is then centrifuged at 400-600×g for 5-20min to obtain a centrifuged chondrocyte precipitate; the centrifuged chondrocyte precipitate is washed, and then suspended in a basal culture medium to obtain a chondrocyte mother solution; the chondrocyte mother solution is inoculated in a basal culture medium for culture, and when the chondrocyte adhesion rate reaches more than 80%, subculture is performed, and subculture is performed to the 2nd to 3rd generation to obtain subcultured chondrocytes.

[0019] More preferably, in the subculture of chondrocytes, the cartilage tissue is cut into small pieces, the cartilage tissue is placed in a serum-free DMEM culture medium, and cut into small pieces using a surgical blade.

[0020] More preferably, in the subculture of chondrocytes, the protease solution contains 0.25-2.5wt% trypsin, and the amount of cartilage tissue used is 0.2-10wt% of the protease solution; the collagenase solution contains 0.2-2wt% collagenase, the amount of chondrocyte precipitation used is measured based on the amount of cartilage tissue, and the amount of cartilage tissue used is 0.2-10wt% of the collagenase solution.

[0021] More preferably, in the subculture of chondrocytes, the chondrocyte precipitate after centrifugation is washed first with a PBS solution and then with a DMEM medium containing 2-10% fetal bovine serum.

[0022] More preferably, in the subculture of chondrocytes, the basal culture medium is DMEM medium containing 2-10% fetal bovine serum, the amount of chondrocyte precipitate used after centrifugation is measured according to the amount of cartilage tissue, and the amount of cartilage tissue used in the chondrocyte mother solution is 0.2-10wt% of the basal culture medium.

[0023] More preferably, in the subculture of chondrocytes, the chondrocyte mother solution is inoculated in the basal culture medium at an inoculation volume of 0.1-1.0×10 4 cell / cm 2 , replace the basal culture medium every 1-4 days.

[0024] More preferably, in the subculture of chondrocytes, the cells are cultured at a temperature of 37°C and 5% CO 2 Cultivated under conditions.

[0025] Preferably, in the preparation of the cartilage regeneration membrane, the subcultured chondrocytes are inoculated into a temperature-sensitive culture dish, a culture medium containing a culture supplement is added, and the culture is carried out for 6-30 days. After the culture is completed, the transfer membrane is covered on the cultured membrane product, and the bubbles between the transfer membrane and the membrane product are removed. The temperature is adjusted to 20-40°C to allow the membrane product to desorb for 5-30 minutes, and the transfer membrane is removed to obtain the cartilage regeneration membrane. The subcultured chondrocytes are inoculated into the temperature-sensitive culture dish, and the inoculation amount is 0.1-1.0×10 4 cell / cm 2 , replace the culture medium containing culture supplements every 1-4 days.

[0026] More preferably, in the preparation of the cartilage regeneration membrane, in the treatment after the culture is completed, the culture medium containing the culture supplement is removed, and DMEM culture medium is added, the transfer membrane is a polyvinylidene fluoride transfer membrane, and the cartilage regeneration membrane is a single layer or a multilayer.

[0027] More preferably, in the preparation of the cartilage regeneration membrane, when adjusting the temperature to desorb the membrane product, the adjusted temperature must be lower than the culture temperature.

[0028] More preferably, in the preparation of the cartilage regeneration membrane, the cells are cultured at a temperature of 37°C and 5% CO 2 The culture medium containing the culture supplement is a mixture of the culture supplement and the DMEM medium, and the culture supplement includes at least one of the following: fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, and ITS+ reagent. The ITS+ reagent contains insulin-transferrin-sodium selenite.

[0029] More preferably, in the preparation of the cartilage regeneration membrane, the amount of fetal bovine serum used is 2-10wt%, the amount of ascorbic acid used is 50-200mg / L, the amount of sodium glycerophosphate used is 5-20mmol / L, the amount of dexamethasone used is 5-20nmol / L, and the amount of ITS+ reagent used is 0.5-2wt%.

[0030] More preferably, in the preparation of the cartilage regeneration membrane, the amount of the lactose amide compound used is 0.01-0.1 wt %.

[0031] More preferably, potassium glycol sulfate can be added to the preparation of the cartilage regeneration membrane, and the amount of potassium glycol sulfate used is 0.01-0.06wt%. The present invention finds that after using the lactose amide compound, potassium glycol sulfate can also be used. The combined use of the lactose amide compound and potassium glycol sulfate has a better effect, but it also needs to be within the effective concentration range of the lactose amide compound.

[0032] The invention discloses a cartilage regeneration membrane sheet prepared by the method.

[0033] The invention discloses the use of the cartilage regeneration membrane in preparing medical materials.

[0034] The invention discloses use of a lactose amide compound in preparing a cartilage regeneration membrane and / or culturing chondrocytes and / or a cell culture medium.

[0035] The present invention uses cartilage tissue to prepare chondrocytes. First, the cartilage tissue is cut into small pieces, and then treated with protease and collagenase to prepare free chondrocytes. After passage to 2-3 generations, the chondrocytes are cultured in a culture medium containing a culture supplement to prepare a cartilage regeneration membrane, thereby having the following beneficial effects: the lactose amide compound has good safety and will not cause a large number of cell apoptosis; after using a culture supplement containing at least one of fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, ITS+ reagent and lactose amide compounds, the cell growth rate can be increased, the production cycle of the cartilage regeneration membrane can be reduced, and a cartilage regeneration membrane with a multilayer structure can be obtained within a specific time. Therefore, the present invention is a preparation method and application of a cartilage regeneration membrane with high plasticity, good integrity, fast cell growth rate, short product production cycle and time saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is an infrared spectrum.

[0037] Figure 2 Cell apoptosis diagram.

[0038] Figure 3A growth cycle diagram.

[0039] Figure 4 This is a picture of the cartilage regeneration membrane of Example 1.

[0040] Figure 5 This is a picture of the cartilage regeneration membrane of comparative example 1.

[0041] Figure 6 This is a picture of the cartilage regeneration membrane of comparative example 3. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings:

[0043] Example 1: A method for preparing an articular cartilage regeneration membrane

[0044] Subculture of chondrocytes: cut the cartilage tissue into small pieces, add it to the protease solution, stir it at 37°C for 30 minutes, centrifuge it at 400×g for 5 minutes to remove the protease solution, and collect the chondrocyte precipitate; add the chondrocyte precipitate to the collagenase solution, incubate it at 37°C until the cartilage tissue is completely digested and the chondrocytes are free in the suspension, and then complete the incubation to obtain free chondrocyte fluid; centrifuge the free chondrocyte fluid at 30×g for 5 minutes, and take the supernatant; then centrifuge the supernatant at 500×g for 10 minutes to obtain the centrifuged chondrocyte precipitate; wash the centrifuged chondrocyte precipitate, and then suspend it with the basal culture medium to obtain the chondrocyte mother liquid; inoculate the chondrocyte mother liquid in the basal culture medium for culture, and when the chondrocyte adhesion rate reaches more than 80%, subculture is carried out, and after the third generation, subcultured chondrocytes are obtained. The cartilage tissue was cut into small pieces, placed in serum-free DMEM culture medium, and cut into small pieces with a surgical blade; the protease solution contained 1wt% trypsin, and the amount of cartilage tissue used was 5wt% of the protease solution; the collagenase solution contained 1wt% collagenase, and the amount of chondrocyte precipitation used was measured based on the amount of cartilage tissue, and the amount of cartilage tissue used was 5wt% of the collagenase solution; in the washing of the chondrocyte precipitation after centrifugation, PBS solution was used first, and then DMEM culture medium containing 10% fetal bovine serum was used for washing; the basal culture medium was DMEM culture medium containing 10% fetal bovine serum, and the amount of chondrocyte precipitation used after centrifugation was measured based on the amount of cartilage tissue, and the amount of cartilage tissue used in the chondrocyte mother liquid was 5wt% of the basal culture medium; the chondrocyte mother liquid was inoculated in the basal culture medium, and the inoculation amount was 1.0×10 4 cell / cm 2 The basal medium was replaced every 2 days. During cell culture, the temperature was 37°C and 5% CO 2 Cultivated under conditions.

[0045] Preparation of cartilage regeneration membrane: subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and culture medium containing culture supplements was added for 10 days. After the culture was completed, the transfer membrane was covered on the cultured membrane product, and the bubbles between the transfer membrane and the membrane product were removed. The temperature was adjusted to 25°C to allow the membrane product to desorb for 5 minutes, and the transfer membrane was removed to obtain the cartilage regeneration membrane. Subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and the inoculation amount was 1.0×10 4 cell / cm 2 , the culture medium containing culture supplements was replaced every 2 days. In the post-culture treatment, the culture medium containing culture supplements was removed and DMEM culture medium was added. The transfer membrane was a polyvinylidene fluoride transfer membrane, and the cartilage regeneration membrane was a single layer or multiple layers. When adjusting the temperature to desorb the membrane product, the adjusted temperature must be lower than the culture temperature. In cell culture, the temperature was 37°C and 5% CO 2 The culture medium containing the culture supplement is a mixture of the culture supplement and the DMEM culture medium, and the culture supplement includes: fetal bovine serum and ascorbic acid. The amount of the fetal bovine serum used is 10wt%, and the amount of the ascorbic acid used is 100mg / L.

[0046] Example 2: A method for preparing an articular cartilage regeneration membrane

[0047] Subculture of chondrocytes: cut the cartilage tissue into small pieces, add it to the protease solution, stir it at 37°C for 120min, centrifuge it at 400×g for 5min to remove the protease solution, and collect the chondrocyte precipitate; add the chondrocyte precipitate to the collagenase solution, incubate it at 37°C until the cartilage tissue is completely digested and the chondrocytes are free in the suspension, and then complete the incubation to obtain free chondrocyte fluid; centrifuge the free chondrocyte fluid at 30×g for 5min, and take the supernatant; then centrifuge the supernatant at 500×g for 10min to obtain the centrifuged chondrocyte precipitate; wash the centrifuged chondrocyte precipitate, and then suspend it in the basal culture medium to obtain the chondrocyte mother liquid; inoculate the chondrocyte mother liquid in the basal culture medium for culture, and when the chondrocyte adhesion rate reaches more than 80%, subculture is carried out, and subculture is obtained after the third generation to obtain subcultured chondrocytes. The cartilage tissue was cut into small pieces, placed in serum-free DMEM culture medium, and cut into small pieces with a surgical blade; the protease solution contained 1wt% trypsin, and the amount of cartilage tissue used was 5wt% of the protease solution; the collagenase solution contained 1wt% collagenase, and the amount of chondrocyte precipitation used was measured based on the amount of cartilage tissue, and the amount of cartilage tissue used was 5wt% of the collagenase solution; in the washing of the chondrocyte precipitation after centrifugation, PBS solution was used first, and then DMEM culture medium containing 10% fetal bovine serum was used for washing; the basal culture medium was DMEM culture medium containing 10% fetal bovine serum, and the amount of chondrocyte precipitation used after centrifugation was measured based on the amount of cartilage tissue, and the amount of cartilage tissue used in the chondrocyte mother liquid was 5wt% of the basal culture medium; the chondrocyte mother liquid was inoculated in the basal culture medium, and the inoculation amount was 1.0×10 4 cell / cm 2 The basal medium was replaced every 4 days. During cell culture, the temperature was 37°C and 5% CO 2 Cultivated under conditions.

[0048] Preparation of cartilage regeneration membrane: subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and a culture medium containing culture supplements was added for 24 days. After the culture was completed, the transfer membrane was covered on the cultured membrane product, and the bubbles between the transfer membrane and the membrane product were removed. The temperature was adjusted to 25°C to allow the membrane product to desorb for 30 minutes, and the transfer membrane was removed to obtain the cartilage regeneration membrane. Subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and the inoculation amount was 1.0×10 4 cell / cm 2, the culture medium containing culture supplements was replaced every 4 days. In the post-culture treatment, the culture medium containing culture supplements was removed and DMEM culture medium was added. The transfer membrane was a polyvinylidene fluoride transfer membrane, and the cartilage regeneration membrane was a single layer or multiple layers. When adjusting the temperature to desorb the membrane product, the adjusted temperature must be lower than the culture temperature. In cell culture, the temperature was 37°C and 5% CO 2 The culture medium containing culture supplements is a mixture of culture supplements and DMEM culture medium, and the culture supplements include: fetal bovine serum, ascorbic acid, sodium glycerophosphate, and dexamethasone. ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of fetal bovine serum used is 10wt%, the amount of ascorbic acid used is 100mg / L, the amount of sodium glycerophosphate used is 10mmol / L, and the amount of dexamethasone is 10nmol / L.

[0049] Example 3: A method for preparing an articular cartilage regeneration membrane

[0050] The subculture of chondrocytes is the same as the subculture steps in Example 1. Preparation of cartilage regeneration membrane: subcultured chondrocytes are inoculated into a temperature-sensitive culture dish, and a culture medium containing a culture supplement is added and cultured for 10 days. After the culture is completed, the transfer membrane is covered on the cultured membrane product, and the bubbles between the transfer membrane and the membrane product are removed. The temperature is adjusted to 25°C to allow the membrane product to desorb for 5 minutes, and the transfer membrane is removed to obtain a cartilage regeneration membrane. Subcultured chondrocytes are inoculated into a temperature-sensitive culture dish, and the inoculation amount is 1.0×10 4 cell / cm 2 , the culture medium containing culture supplements was replaced every 2 days. In the post-culture treatment, the culture medium containing culture supplements was removed and DMEM culture medium was added. The transfer membrane was a polyvinylidene fluoride transfer membrane, and the cartilage regeneration membrane was a single layer or multiple layers. When adjusting the temperature to desorb the membrane product, the adjusted temperature must be lower than the culture temperature. In cell culture, the temperature was 37°C and 5% CO 2 The culture medium containing the culture supplement is prepared by mixing the culture supplement and the DMEM culture medium, and the culture supplement includes ascorbic acid and ITS+ reagent. The ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, and the amount of ITS+ reagent used is 1 wt%.

[0051] Example 4: A method for preparing an articular cartilage regeneration membrane

[0052] The subculture of chondrocytes is the same as the subculture steps in Example 2.

[0053] Preparation of cartilage regeneration membrane: subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and a culture medium containing culture supplements was added for 24 days. After the culture was completed, the transfer membrane was covered on the cultured membrane product, and the bubbles between the transfer membrane and the membrane product were removed. The temperature was adjusted to 25°C to allow the membrane product to desorb for 30 minutes, and the transfer membrane was removed to obtain the cartilage regeneration membrane. Subcultured chondrocytes were inoculated into a temperature-sensitive culture dish, and the inoculation amount was 1.0×10 4 cell / cm 2 , the culture medium containing culture supplements was replaced every 4 days. In the post-culture treatment, the culture medium containing culture supplements was removed and DMEM culture medium was added. The transfer membrane was a polyvinylidene fluoride transfer membrane, and the cartilage regeneration membrane was a single layer or multiple layers. When adjusting the temperature to desorb the membrane product, the adjusted temperature must be lower than the culture temperature. In cell culture, the temperature was 37°C and 5% CO 2 The culture medium containing the culture supplement is a mixture of the culture supplement and the DMEM culture medium, and the culture supplement includes ascorbic acid, sodium glycerophosphate, dexamethasone, and ITS+ reagent. The ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of sodium glycerophosphate used is 10 mmol / L, the amount of dexamethasone is 10 nmol / L, and the amount of ITS+ reagent used is 1 wt%.

[0054] Example 5: A method for preparing an articular cartilage regeneration membrane

[0055] The difference between this embodiment and embodiment 3 lies in the preparation of the cartilage regeneration membrane.

[0056] In this embodiment, in the preparation of the cartilage regeneration membrane, the culture supplement includes: ascorbic acid, ITS+ reagent and lactose amide compound. The ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of ITS+ reagent used is 1wt%, and the amount of lactose amide compound used is 0.03wt%. Others are the same.

[0057] Lactobionic acid is added to water, and then EDC·HCl and NHS are added and stirred and mixed, and then glutamine is added, and the reaction is carried out at 30°C for 48 hours. After the reaction is completed, the mixture is distilled under reduced pressure, washed with methanol, and dried to obtain a lactobionic acid compound. The amount of lactobionic acid used is 8wt% of water, the amount of EDC·HCl used is 50wt% of lactobionic acid, the amount of NHS used is 30wt% of lactobionic acid, and the amount of glutamine used is 50wt% of lactobionic acid.

[0058] Example 6: A method for preparing an articular cartilage regeneration membrane

[0059] The difference between this embodiment and embodiment 5 lies in the preparation of the cartilage regeneration membrane.

[0060] In this embodiment, in the preparation of the cartilage regeneration membrane, the culture supplement includes: ascorbic acid, ITS+ reagent and lactose amide compound. The ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of ITS+ reagent used is 1wt%, and the amount of lactose amide compound used is 0.08wt%. Others are the same.

[0061] Example 7: A method for preparing an articular cartilage regeneration membrane

[0062] The difference between this embodiment and embodiment 5 lies in the preparation of the cartilage regeneration membrane.

[0063] In this embodiment, in the preparation of the cartilage regeneration membrane, the culture supplement includes: ascorbic acid, ITS+ reagent, potassium glycol sulfate and lactose amide compound. ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of ITS+ reagent used is 1wt%, the amount of potassium glycol sulfate used is 0.02wt%, and the amount of lactose amide compound used is 0.03wt%. Others are the same.

[0064] Example 8: A method for preparing an articular cartilage regeneration membrane

[0065] The difference between this embodiment and embodiment 7 lies in the preparation of the cartilage regeneration membrane.

[0066] In this embodiment, in the preparation of the cartilage regeneration membrane, the culture supplement includes: ascorbic acid, ITS+ reagent, potassium glycol sulfate and lactose amide compound. ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of ITS+ reagent used is 1wt%, the amount of potassium glycol sulfate used is 0.04wt%, and the amount of lactose amide compound used is 0.03wt%. Others are the same.

[0067] Comparative Example 1: A method for preparing a membrane for articular cartilage regeneration

[0068] The difference between this comparative example and Example 1 lies in the preparation of the cartilage regeneration membrane.

[0069] In this comparative example, in the preparation of the cartilage regeneration membrane, the temperature-sensitive culture dish was replaced with a common culture dish. All other conditions were the same. After using the common culture dish, a complete cartilage regeneration membrane could not be separated.

[0070] Comparative Example 2: A method for preparing an articular cartilage regeneration membrane

[0071] The difference between this comparative example and Example 1 lies in the preparation of the cartilage regeneration membrane.

[0072] In this comparative example, in the preparation of the cartilage regeneration membrane, the culture supplement includes: fetal bovine serum. The amount of fetal bovine serum used is 10wt%. Others are the same.

[0073] Comparative Example 3: A method for preparing an articular cartilage regeneration membrane

[0074] The difference between this comparative example and Example 1 lies in the preparation of the cartilage regeneration membrane.

[0075] In this comparative example, in the preparation of the cartilage regeneration membrane, the transfer membrane was replaced by PVC from polyvinylidene fluoride. The regeneration membrane could not be adsorbed from the temperature-sensitive culture dish by using PVC.

[0076] Comparative Example 4: A method for preparing an articular cartilage regeneration membrane

[0077] The difference between this comparative example and Example 1 lies in the preparation of the cartilage regeneration membrane.

[0078] In this comparative example, in the preparation of the cartilage regeneration membrane, the transfer membrane was replaced by PET from polyvinylidene fluoride. The regeneration membrane could not be adsorbed from the temperature-sensitive culture dish by using PET.

[0079] Comparative Example 5: A method for preparing an articular cartilage regeneration membrane

[0080] The difference between this comparative example and Example 5 lies in the preparation of the cartilage regeneration membrane.

[0081] In this comparative example, in the preparation of the cartilage regeneration membrane, the culture supplement includes: ascorbic acid, ITS+ reagent and lactose amide compound. The ITS+ reagent contains insulin-transferrin-sodium selenite. The amount of ascorbic acid used is 100 mg / L, the amount of ITS+ reagent used is 1wt%, and the amount of lactose amide compound used is 0.005wt%. Others are the same.

[0082] Comparative Example 6: A method for preparing an articular cartilage regeneration membrane

[0083] The difference between this comparative example and Example 7 lies in the preparation of the cartilage regeneration membrane.

[0084] In this comparative example, no lactose amide compound was used in the culture supplement in the preparation of the cartilage regeneration membrane, and all other conditions were the same.

[0085] Comparative Example 7: A method for preparing an articular cartilage regeneration membrane

[0086] The difference between this comparative example and Example 8 lies in the preparation of the cartilage regeneration membrane.

[0087] In this comparative example, no lactose amide compound was used in the culture supplement in the preparation of the cartilage regeneration membrane, and all other conditions were the same.

[0088] Test example:

[0089] Infrared characterization

[0090] The present invention performs infrared testing on the lactose amide compound prepared in Example 5, and the results are as follows: Figure 1 As shown, 3000-3600cm -1 The absorption peak of hydroxyl is 2800-3000cm -1 The absorption peak of alkyl is 1608cm -1 The absorption peak of carbonyl is 1408cm -1 The absorption peaks of carbon and nitrogen in amide are 1148 and 1057 cm -1 The absorption peak at is carbon oxygen, indicating that the lactobionic acid and glutamine react to obtain the lactobionic acid amide compound.

[0091] Apoptosis test

[0092] L929 cells were tested for apoptosis using flow cytometry. Lactose amide compounds were added as a sample group. L929 cells were inoculated in a 6-well cell culture plate at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator, and when the cell density reached 60%, they were treated with 0, 0.01, 0.05 and 0.1 wt% lactose amide compounds for 24 h. The cells in the blank-treated wells were used as single-stained wells and blank wells for flow cytometry gate setting.

[0093] Cell apoptosis test results Figure 2 As shown, when the lactose amide compound is used at a concentration of 0.01-0.1wt%, the cell apoptosis rate is not significantly different from that when the concentration is 0wt%, indicating that the lactose amide compound prepared by the present invention has good biosafety.

[0094] Cell adhesion test

[0095] The time required for the cell adhesion rate to reach 100% in each embodiment of the present invention and the comparative example was observed and recorded. It should be noted that after the cell adhesion rate reached 100%, continued culture could obtain a multilayer cartilage regeneration membrane.

[0096] The time required for the cell adhesion rate to reach 100% in the present invention is as follows: Figure 3As shown, wherein S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7, S8 is Example 8, D1 is Comparative Example 1, D2 is Comparative Example 2, D3 is Comparative Example 3, D4 is Comparative Example 4, D5 is Comparative Example 5, D6 is Comparative Example 6, and D7 is Comparative Example 7. In the present invention, cartilage tissue is first processed to prepare subcultured chondrocytes. Subcultured chondrocytes can be prepared by a known method, subcultured chondrocytes that meet the conditions are cultured, and then the chondrocytes are cultured using a culture medium containing a culture supplement to prepare. A cartilage regeneration membrane is obtained. The culture supplement in the present invention comprises at least one of fetal bovine serum, ascorbic acid, sodium glycerophosphate, dexamethasone, ITS+ reagent and lactose amide compound. When only fetal bovine serum is used, the time required for cell adhesion to reach 100% is longer; after using fetal bovine serum, further using ascorbic acid can reduce the time required for cell adhesion to reach 100%; after using fetal bovine serum and ascorbic acid, further using sodium glycerophosphate and dexamethasone can further reduce the time required for cell adhesion to reach 100%; in the present invention, ascorbic acid and ITS+ reagent can also be used as culture supplements, compared with fetal bovine serum and ascorbic acid, , has a better effect, indicating that ascorbic acid and ITS+ reagents are not only better than the use of fetal bovine serum, but also better than the combined use of fetal bovine serum and ascorbic acid; further, after using ascorbic acid and ITS+ reagents, further using sodium glycerophosphate and dexamethasone, the time required for cell adhesion to reach 100% is shorter; after using ascorbic acid and ITS+ reagents, lactose amide compounds can also be added. Lactose amide compounds are made by the reaction of lactobionic acid and glutamine. The use of lactose amide compounds can also reduce the time required for cell adhesion to reach 100%, indicating that lactose amide compounds can increase cell growth rate; further, After using ascorbic acid, ITS+ reagent and lactosamide compounds, potassium glycol sulfate can also be added. The use of potassium glycol sulfate requires the presence of lactosamide compounds, indicating that the use of potassium glycol sulfate and lactosamide compounds together can reduce the time required for cell adhesion to reach 100%; the lactosamide compound needs to reach a certain content in order to take effect. When the content is low, it has no effect on reducing the time required for cell adhesion to reach 100%, and the increase in the amount of lactosamide compounds used has little effect on the growth cycle, indicating that after the lactosamide compounds reach the effective concentration, the change in amount has basically no effect on the growth cycle of chondrocytes.

[0097] The present invention selects some products prepared in the embodiments or comparative examples for photographic comparison. Figure 4 The cartilage regeneration membrane prepared in Example 1 can be successfully separated; Figure 5The cartilage regeneration membrane prepared in Comparative Example 1 cannot be separated. Figure 6 The cartilage regeneration membrane prepared in Example 3 was unable to be adsorbed by PVC from the temperature-sensitive culture dish.

[0098] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0099] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a cartilage regeneration membrane, comprising: The chondrocytes are inoculated in a separation vessel, cultured in a culture medium containing a culture supplement, and separated to obtain a cartilage regeneration membrane, wherein the culture supplement at least includes ascorbic acid, ITS+ reagent and lactosylamide compound; the separation vessel is a temperature-sensitive culture vessel; the amount of ascorbic acid used is 50-200 mg / L; the amount of ITS+ reagent used is 0.5-2wt%; the amount of lactosylamide compound used is 0.01-0.1wt%; and the culture medium is DMEM culture medium; The lactobionic acid amide compound is obtained by reacting lactobionic acid with glutamine. In the preparation of the lactobionic acid amide compound, lactobionic acid and glutamine react in a solution containing EDC·HCl and NHS to obtain the lactobionic acid amide compound; the amount of EDC·HCl used is 40-60wt% of the lactobionic acid; the amount of NHS used is 20-40wt% of the lactobionic acid; and the amount of glutamine used is 40-60wt% of the lactobionic acid.

2. The method for preparing a cartilage regeneration membrane according to claim 1, characterized in that: The culture supplement also includes at least one of fetal bovine serum, sodium glycerophosphate and dexamethasone.

3. The method for preparing a cartilage regeneration membrane according to claim 2, characterized in that: In the culture medium containing the culture supplement, the amount of fetal bovine serum used is 2-10wt%; or the amount of sodium glycerophosphate used is 5-20mmol / L; or the amount of dexamethasone used is 5-20nmol / L.

4. The method for preparing a cartilage regeneration membrane according to claim 1, characterized in that: The chondrocytes are prepared from cartilage tissue by enzymatic hydrolysis with trypsin and / or collagenase.

5. The cartilage regeneration membrane prepared by the method according to any one of claims 1 to 4.

6. Use of the cartilage regeneration membrane according to claim 5 in the preparation of medical materials.

7. Use of a lactobionic acid amide compound in preparing a cartilage regeneration membrane and / or culturing chondrocytes; the lactobionic acid amide compound is obtained by reacting lactobionic acid with glutamine. In the preparation of the lactobionic acid amide compound, lactobionic acid and glutamine are reacted in a solution containing EDC·HCl and NHS to obtain the lactobionic acid amide compound; the amount of EDC·HCl used is 40-60wt% of the lactobionic acid; the amount of NHS used is 20-40wt% of the lactobionic acid; the amount of glutamine used is 40-60wt% of the lactobionic acid.

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