A human-derived meniscus cell autocrine biological collagen membrane and its application in promoting angiogenesis

Abstract

The present invention uses a self-made film-forming culture medium to culture autologous meniscus cells (MFCs), and finally obtains a collagen-rich extracellular matrix bio-collagen film without introducing exogenous substances throughout the process. It is found that it has the effect of promoting the formation of blood vessels by vascular endothelial cells (ECs), and the Col IV and some specific cell migration-related protein components therein can promote the migration of ECs and angiogenesis.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials, and more specifically, the present invention relates to a human-derived meniscus cell autocrine biological collagen membrane that can promote blood vessel regeneration. Background Art

[0002] The meniscus is an important structure within the knee joint, with important functions such as load transmission, shock absorption, knee joint stabilization, lubrication, and nutrition. Meniscus injury is the knee joint injury with the highest prevalence rate. According to reports in "N Engl J Med", meniscectomy is the most common surgery in the United States, with more than 700,000 cases per year. With the development of social economy and the improvement of diagnostic levels, the number of meniscus injuries in China has also been increasing year by year, becoming one of the major joint injuries that affect people's sports level and quality of life. Since only 10% - 25% of the tissue at the outer edge of the meniscus contains blood vessels, the metabolism of other parts mainly depends on the synovium and joint fluid, and the nutritional transport speed of the synovium and joint fluid is much slower than that of blood vessels. Therefore, most meniscus injuries are difficult to heal or repair spontaneously and require partial or total resection. After meniscus injury or resection, the stress on the knee joint increases significantly, and the risk of developing osteoarthritis increases by 2 - 7 times. Allogeneic meniscus transplantation can replace the meniscus defect, but its donor source is very limited. Currently, the and and other products also have insufficient blood supply and nutrition after implantation, and tissue regeneration has not been completed even after 5 years, with a failure rate as high as over 30%, resulting in severe damage to articular cartilage and seriously affecting their popularization and use. Therefore, developing early vascularization after artificial meniscus implantation is an important way to improve its long-term efficacy and promote its clinical application. There is an urgent need to develop a meniscus transplantation product that can promote rapid blood vessel regeneration. Summary of the Invention

[0003] To solve the problems existing in the prior art and accelerate meniscus vascularization to promote tissue regeneration of meniscus implants, the inventor cultured autologous meniscus cells (MFCs) using a self-made film-forming culture medium, and finally formed a collagen-rich extracellular matrix biological collagen membrane without introducing exogenous substances throughout the process, and found that it has the effect of promoting the formation of blood vessels by vascular endothelial cells (ECs). The Col IV and certain specific cell migration-related protein components therein can promote the migration and blood vessel regeneration of ECs. Specifically, the present invention provides the following technical solutions:

[0004] In the first aspect of the present invention, a collagen film-forming culture medium is provided, and the components of the culture medium include:

[0005] DMEM basal medium;

[0006] Glutmax, with an added concentration of 0.01 ml / ml;

[0007] NEAA, added at a concentration of 0.01 ml / ml;

[0008] PS, added at a concentration of 0.01 ml / ml;

[0009] Dexamethasone, added at a concentration of 100 nM;

[0010] ITS, added at a concentration of 0.01 ml / ml,;

[0011] Proline, added at a concentration of 40 μg / ml.

[0012] In a second aspect of the present invention, there is provided a biological collagen membrane, which is prepared by culturing human chondrocytes using the above-mentioned collagen film-forming medium.

[0013] In one embodiment, the human chondrocytes can be derived from human meniscus tissue or cartilage tissue.

[0014] In one embodiment, the preparation method of the biological collagen membrane is as follows: collecting the discarded meniscus tissue or cartilage tissue excised during surgery, after digestion with trypsin and collagenase, first amplifying human meniscus cells in vitro using a common medium, and after reaching a certain number, replacing the collagen film-forming medium to promote the differentiation of human meniscus cells to form a collagen biomembrane.

[0015] In a third aspect of the present invention, there is provided an application of the above-mentioned collagen film-forming medium in preparing a product for promoting meniscus vascularization.

[0016] In a fourth aspect of the present invention, there is provided an application of the above-mentioned biological collagen membrane in preparing a product for treating meniscus injuries.

[0017] The present invention has achieved the following remarkable improvements compared with the prior art:

[0018] 1. This biological collagen membrane is derived from human adult somatic cells

[0019] We used a special self-made angiogenesis medium to collect the discarded meniscus tissue excised during the meniscectomy of patients. After digestion with trypsin and collagenase, we amplified human meniscus cells in vitro using a common medium. After reaching a certain number, we replaced it with a special angiogenesis medium to promote the differentiation of human meniscus cells to form a collagen biomembrane. This kind of collagen membrane is derived from autologous or allogeneic cells, which is a good reuse of the discarded tissue excised during surgery. At the same time, a small amount of the excised tissue can be passaged and amplified in vitro to meet the usage requirements of a large number of biological collagen membranes.

[0020] 2. This biological collagen membrane can promote angiogenesis of meniscus implants

[0021] Most meniscus research teams at home and abroad, including our research group, have focused their research on the construction of the meniscus tissue itself. However, there has been little research on blood supply regulation and nutrient supply after in vivo implantation. Based on the role of Col IV and specific protein components in the bio-collagen membrane in chemotaxing ECs, vascular regeneration is further promoted. According to the principle that the white area of the meniscus is not easily repaired after injury due to insufficient blood supply, this project proposes the hypothesis of "regulating the ingrowth of blood vessels and tissue regeneration in the white area of the meniscus through this bio-collagen membrane", and combines the key genes and proteins related to meniscus blood vessel ingrowth and tissue regeneration to develop a new strategy for meniscus repair and reconstruction.

[0022] 3. Promote the ingrowth of meniscus blood vessels without introducing exogenous cells or factors

[0023] In terms of cells, this project uses Col IV to initiate the chemotactic response of ECs to promote angiogenesis, thereby increasing the nutrient supply and cell source during meniscus regeneration, avoiding the implantation of exogenous cells, simplifying the operation process, and reducing the biosafety risk. In terms of factors, the Col IV used in current research related to vascular regeneration is commercially available or allogeneic, with poor biocompatibility. The inventor prepares a bio-collagen membrane using a self-developed special culture medium, which is secreted by MFCs, and no exogenous substances are introduced throughout the process.

[0024] 4. Diversified application methods

[0025] The bio-collagen membrane prepared in this project retains cell viability and can survive for a long time, which is particularly suitable for patients with severe meniscus injuries. This biofilm can be directly placed at the meniscus injury site and fixed with sutures. The live cells in the product can further proliferate in vivo and secrete and deposit extracellular matrix. At the same time, the extracellular matrix components with the ability to promote vascular regeneration can promote the ingrowth of blood vessels towards the injury site, thus providing a nutrient source for tissue regeneration; in addition, this product can also be decellularized and freeze-dried to eliminate cell surface antigens and reduce immunogenicity, and can be applied to the repair of allogeneic meniscus injuries. Brief Description of the Drawings

[0026] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0027] Figure 1 a. Gross photograph of the bio-collagen membrane (before freeze-drying); b. Gross photograph of the bio-collagen membrane (after freeze-drying); c. HE staining of the bio-collagen membrane;

[0028] Figure 2a. Gross observation of the biological collagen membrane; b. Electron microscopy; c. Col IV immunofluorescence; d. Col IV immunohistochemistry; e. qPCR analysis of the Col IV gene expression results of MFCs under two culture medium conditions;

[0029] Figure 3 a. Biological collagen membrane group; b. Col IV group; c. VEGF positive control group; d. Blank control group; e. Statistical analysis;

[0030] Figure 4 a. Biological collagen membrane group; b. Col IV group; c. VEGF positive control group; d. Blank control group; e. Statistical analysis;

[0031] Figure 5 Results of the angiogenesis-promoting experiment of the biological collagen membrane (a) and vaseline gauze (b) in repairing the subcutaneous tissue of rats. Specific implementation manners

[0032] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0033] Example 1 Preparation and Characterization of Autocrine Bio-Collagen Membrane of MFCs

[0034] 1. Culture of human-derived MFCs (autologous meniscus cells):

[0035] First, collect the discarded meniscus tissues removed during the operation. After rinsing the tissues three times with PBS, cut them into 1*1*1 mm particles, and treat them with 0.25% trypsin at 37°C. After half an hour, discard the trypsin and replace it with collagenase (0.2% type I collagenase: 0.2% type II collagenase = 1:1) and incubate overnight at 37°C. After all the tissue particles are digested, add fetal bovine serum to terminate the digestion. Gently pipette and resuspend the cells with DMEM containing 10% fetal bovine serum, and ensure that the cell concentration is 6*10^5 / ml. Replace the culture medium every three days.

[0036] 2. Preparation method of ECM (extracellular matrix membrane) with a certain thickness:

[0037] After the above cells are cultured to P2, digest them with 0.25% trypsin at 37°C and passage them into six-well plates, with 1*10^5 cells in each well. First, culture them with ordinary DMEM medium until the cell density reaches 90%, and then replace it with a specially formulated membrane-forming medium. After three days, an extracellular matrix membrane (biological collagen membrane) with a certain thickness can be observed.

[0038] The specially formulated membrane-forming medium is prepared from the following components (final concentration after addition, manufacturer):

[0039] DMEM basal medium (low glucose, Sigma Adrich, USA), Glutmax (0.01 ml / ml, Sigma Adrich, USA), NEAA (0.01 ml / ml, Sigma Adrich, USA), PS (0.01 ml / ml, Sigma Adrich, USA), Dexamethasone (100 nM, 1 μl / ml, Sigma Adrich, USA), ITS (0.01 ml / ml, Sigma Adrich, USA), Proline (40 μg / ml, Sigma Adrich, USA).

[0040] 3. Scanning electron microscopy detection:

[0041] Prepare collagen membrane tissue with a volume of 3 mm 3 according to the above steps, fix it with 2.5% glutaraldehyde for 30 minutes, dehydrate it with gradient alcohol, freeze-dry it overnight in a vacuum freeze dryer, send it to the electron microscopy room for gold spraying, and observe it under the microscope.

[0042] 4. COL IV immunofluorescence:

[0043] First, fix the collagen membrane with 4% paraformaldehyde at room temperature, permeabilize it with PBS containing 0.2 - 0.5% Triton-100 at room temperature, block it with 5% bovine serum albumin (BSA) at room temperature or 37 °C, incubate it with anti-COL IV primary antibody overnight at 4 °C, wash it with phosphate buffer (PBS), incubate it with a fluorescein-labeled secondary antibody and DAPI at an appropriate dilution concentration, wash it with PBS, add an appropriate amount of anti-fluorescence quencher, mount the slides, and detect it with a fluorescence confocal microscope.

[0044] 5. COL IV immunohistochemistry:

[0045] First, fix the collagen membrane with 4% paraformaldehyde at room temperature, permeabilize it with PBS containing 0.2 - 0.5% Triton-100 at room temperature, block it with 5% bovine serum albumin (BSA) at room temperature or 37 °C, incubate it with anti-COL IV primary antibody overnight at 4 °C, wash it with phosphate buffer (PBS), incubate it with an immunohistochemical secondary antibody and DAPI at an appropriate dilution concentration, wash it with PBS, add an appropriate amount of anti-fluorescence quencher, mount the slides, and detect it with a fluorescence confocal microscope.

[0046] 6. COL IV q-PCR detection:

[0047] The relative expression levels of COL IV gene were evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR). TRIzol (Thermo Corporation, USA) was used for lysis to extract RNA, and reverse transcription was performed using a reverse transcription kit, with GAPDH as the internal reference gene. The relative expression levels were calculated using the ΔΔCt method.

[0048] The results of electron microscopy showed that the biofilm had a loose and porous structure ( Figure 2 b); by Col IV immunofluorescence ( Figure 2 c), immunohistochemistry ( Figure 2 d), it had abundant Col IV and other protein components (such as PRELP, NID1, etc.); MFCs were cultured using special medium and ordinary medium respectively, and qPCR analysis indicated that the special medium group had a higher Col IV gene expression level ( Figure 2 e).

[0049] Example 2 Results of in Vitro Vascular Endothelial Cell Migration and in Vitro Tube Formation Assays

[0050] I. Transwell cell co-culture system migration experiment (to verify the migration effect of the biological collagen membrane on ECs):

[0051] 1. Preparation of cell suspension and Col IV-RECM decellularized solution

[0052] The cells were digested, and after terminating the digestion, the culture medium was discarded by centrifugation (washed 1-2 times with PBS), and resuspended in serum-free medium containing BSA. The cell density was adjusted to 5*10^5 / ml; the Col IV-RECM biomaterial was first subjected to decellularization treatment using the repeated freeze-thaw method to obtain freeze-dried Col IV-RECM material, dissolved in acetic acid, and after completely dissolving at a temperature below 10°C, NaOH solution was added dropwise to adjust the pH to neutral, and then placed at a low temperature of 4-15°C to obtain the Col IV-RECM decellularized solution. In the experiment using Col IV instead of ColIV-RECM, commercial Col IV was dissolved in the lower chamber medium at a certain ratio.

[0053] 2. Seeding cells

[0054] 100 μl of the ECs cell suspension was placed into the Transwell insert, and 600 μl of the culture medium was added to the lower chamber of the 24-well plate. Cells or the Col IV-RECM decellularized solution were added according to the grouping.

[0055] 3. Culturing cells

[0056] Cultivate routinely for 12 - 48 h.

[0057] 4. Result statistics

[0058] Take out the Transwell chamber, discard the culture medium in the well, wash twice with calcium - free PBS, fix with methanol for 30 minutes, and air - dry the chamber appropriately. Stain with 0.1% crystal violet for 20 min, gently wipe off the non - migrated ECs on the upper layer with a cotton swab, and wash three times with PBS. Observe and count the cells in 5 random fields under a 400 - fold microscope.

[0059] For the biological collagen membrane group ( Figure 3 a), simple Col IV group ( Figure 3 b), VEGF positive control group ( Figure 3 c) and blank control group ( Figure 3 d), the result statistical analysis shows that: there is a significant difference between the biological collagen membrane group and the blank control group, indicating that the biological collagen membrane can promote the migration of ECs; there is a significant difference between the biological collagen membrane group and the Col IV group. Combining with the mass spectrometry analysis results of the biological collagen membrane, it is suggested that there are other related proteins in the biological collagen membrane that can synergistically promote the migration of ECs in addition to Col IV ( Figure 3 e).

[0060] II. In vitro tube - formation experiment (to verify the promoting effect of biological collagen membrane on the in vitro angiogenesis of ECs):

[0061] Inoculate the ECs suspension in a 96 - well culture plate pre - coated with Matrigel matrix gel (Matrigel matrix gel, Corning, USA). Measure the tube - formation rate of each group after 8 h. Evaluate in vitro by measuring migration and duct formation.

[0062] For the biological collagen membrane group ( Figure 4 a), Col IV group ( Figure 4 b), positive control group ( Figure 4 c), blank control group ( Figure 4 d), perform statistical analysis on the tube - formation rate. The results show that: there is a significant difference between the biological collagen membrane group and the blank control group, indicating that the biological collagen membrane can promote the in vitro angiogenesis of ECs; there is a significant difference between the biological collagen membrane group and the Col IV group. Combining with the mass spectrometry analysis results of the biological collagen membrane, it is suggested that there are other related protein components in the biological collagen membrane that can synergistically promote the in vitro angiogenesis of ECs in addition to Col IV ( Figure 4 e).

[0063] Example 3 In Vivo Experimental Verification of the Promotion of Vascular Regeneration by Autocrine Bio-Collagen Membrane of Meniscus

[0064] Pass the biological collagen membrane and oil - soaked gauze through Co 60Sterilization was carried out, and then the mice were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital according to body weight. The back skin was prepared, disinfected with iodophor and alcohol. The back skin of the mice was longitudinally incised with a scalpel for 2-3 cm. The sterilized biological collagen membrane and oil gauze were sutured layer by layer to close the incision. After 14 days, the mice were euthanized, and the samples were fixed in 4% paraformaldehyde for 48 h after sampling; an automatic dehydrator was used for integrated dehydration: 75% ethanol for 40 min, 80% ethanol I and II for 40 min each, 95% ethanol I and II for 75 min each, 100% ethanol I and II for 75 min each; similarly, the subsequent clearing and infiltration operations were completed using an automatic dehydrator: xylene I, II, III, and IV for 20 minutes each for clearing, benzene wax for 1 hour, and paraffin I, II, and III for 20 minutes each for infiltration; after infiltration was completed, embedding was carried out; after cooling, the wax blocks containing the tissues were trimmed neatly and fixed on a microtome for sectioning. First, the section thickness was adjusted to 30 μm for rough cutting, and then changed to 5 μm for fine cutting when the tissue layer we wanted to observe was reached; after sectioning was completed, the sections were first placed in distilled water containing alcohol for 1 minute, and then placed in warm water at 42 °C for spreading. After spreading was completed, the sections were picked up using an adhesive glass slide; after ensuring that the tissues on the sections were well extended, baking was carried out, overnight at 60 °C and then for 2 hours at 37 °C to firmly adhere the tissues to the glass slide to prevent detachment during subsequent experiments.

[0065] The sections were stained with hematoxylin and eosin (HE): First, the prepared tissue sections were dewaxed to water: xylene I, II, and III for 10 minutes each, absolute ethanol I, II, and III for 5 minutes each, 95% alcohol, 80% alcohol, and 75% alcohol for 5 minutes each, distilled water I and II for 2 minutes each; after dewaxing and hydration, hematoxylin staining was carried out for 20 minutes; rinsed with distilled water; differentiated with hematoxylin differentiating solution (75% hydrochloric acid ethanol) for 10 seconds; rinsed with tap water; rinsed with distilled water; blued with hematoxylin bluing solution (ammonia water) for 3 seconds; rinsed with distilled water; stained with 1% eosin ethanol staining solution for 2 minutes; rinsed with distilled water to remove floating color; after staining was completed, dehydration, clearing, and mounting were carried out: placed in 95% ethanol I and II for 1 minute each, 100% ethanol I and II for 1 minute each for dehydration, placed in xylene I and II for 2 minutes each for clearing, and mounted with neutral gum mounting agent; observed under an optical microscope.

[0066] The results showed that after sampling 14 days after the implantation of the biological collagen membrane, histology indicated good biocompatibility, and at the same time, it had good ability to promote angiogenesis compared with the oil gauze group ( Figure 5 ).

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A collagen film-forming culture medium, characterized in that Its components include: DMEM basal medium; L-alanyl-L-glutamine (GlutMAX), with an added concentration of 0.01 ml / ml; Non-essential amino acids (NEAA), with an added concentration of 0.01 ml / ml; Penicillin-streptomycin antibiotic mixture (PS), with an added concentration of 0.01 ml / ml; Dexamethasone, with an added concentration of 100 nM; Insulin-transferrin-selenite additive (ITS), with an added concentration of 0.01 ml / ml; and Proline, with an added concentration of 40 μg / ml.

Images