Carrier cell sheet with anti-inflammatory and osteogenic activity and preparation method thereof

Abstract

The invention discloses a cell sheet taking an acellular biological membrane as a carrier and having osteogenic activity as well as a preparation method and application of the cell sheet. The method comprises the following steps: firstly, carrying out decellularization treatment on a natural amnion, and then planting target cells on a biological membrane; and culturing the preliminarily constructed biological membrane to obtain the cell sheet taking the decellularized biological membrane as the carrier. The cell sheet prepared by the invention has excellent capability of promoting cell proliferation and osteogenic differentiation, so that tissue repair is promoted. The prepared cell sheet overcomes the defect that existing cell transplantation carriers generally lack activity, the preparation process is simple and controllable, and breakthrough of stem cell therapy in tissue engineering is facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a carrier cell sheet with anti-inflammatory and osteogenic activities and a preparation method thereof. Background Art

[0002] Bone defect refers to the destruction of the structural integrity of the bone, and its causes may be tumors, trauma, necrosis, congenital malformations, etc. Autologous bone transplantation is the gold standard for bone reconstruction because of its good compatibility and integration ability. However, when transplanting bone from a site different from the recipient area, it is very difficult to accurately match the 3D replication of the missing bone segment. In addition, the source of autologous bone is limited and it is easy to cause secondary damage and infection at the bone harvesting site, which greatly limits its clinical application.

[0003] Regenerative medicine has developed with the latest advances in stem cell research. Over the past few decades, the exciting potential of stem cells in tissue regeneration and the repair of diseased organs and systems has been demonstrated. Therefore, scaffold biomaterials infiltrated with stem cells with osteogenic differentiation ability to stimulate bone formation have good application prospects. In the selection of stem cells, adipose-derived mesenchymal stem cells (ADSCs) are stem cells with multidirectional differentiation ability and self-replication ability. They can not only differentiate into osteoblasts, chondrocytes, adipocytes, muscle cells and other cells belonging to the mesenchymal system, but also have the ability to differentiate beyond the germ layer into neurons, hepatocytes, etc. Due to its rich, easy to separate and multidirectional differentiation properties, the implantation of ADSCs scaffolds has been considered for regenerative medicine.

[0004] Current tissue engineering technology can directly implant suitable stem cells, such as ADSCs, using appropriate solutions, including culture medium or plasma as carriers in this process. However, the traditional method of injecting cell suspensions cannot ensure the effective residence of cells in the damaged area, nor can it regulate the shape and size of the repair area. In addition, since the cell suspension needs to be treated with trypsin or chemicals during the collection process before it can be peeled off and recovered from the surface of the culture container, this will cause cell damage and damage the original function. ADSCs can also be delivered in three-dimensional (3D), biocompatible and biodegradable scaffolds to replace damaged or diseased tissues. The latter can further promote the repair of bone defects through the design of the scaffold based on the activity of the stem cells themselves, so it is more widely used and more suitable for large soft tissue or hard tissue trauma.

[0005] Ideal 3D scaffold biomaterials should provide a cell microenvironment suitable for cell survival, adhesion, proliferation and differentiation to enhance the regeneration and regeneration of damaged or injured tissues. In addition, matrix biomaterials present in interstitial tissues should be favorable substrates for cell morphogenesis, migration, immune defense and wound repair, with low immunogenicity. Current cell sheets generally only serve as cell adhesion and proliferation carriers, and there is a problem of lack of active factors that promote differentiation.

[0006] Extracellular matrix materials are materials that retain three-dimensional scaffold structures, component proteins, special proteins, active factors, etc. on the basis of removing immunogenic cell components. It can provide cells cultured thereon with a microenvironment similar to that of the human body, which is conducive to cell adhesion and proliferation, and has the potential to promote cell differentiation. Amniotic membrane tissue discarded during the perinatal period is one of the important sources of extracellular matrix materials. It is widely available, easy to obtain, and has no ethical controversy, and has been a research hotspot in recent years. The amnion is the innermost layer of the fetal membrane in the placenta and is in direct contact with the fetus. It is a translucent, non-vascular and lymphatic biomaterial that is rich in a variety of collagens, non-collagenous glycoproteins, and abundant growth factors, which provides advantages for the application of amnion in tissue engineering. At the same time, the amnion has excellent properties such as promoting wound healing and anti-scar properties, antibacterial and anti-inflammatory properties, and is a good choice for preparing decellularized materials. Decellularized amnion can be prepared in a variety of ways, but there is currently no report on the application of decellularized amnion in cell implantation loads.

[0007] The present invention mainly uses a gradient concentration of sodium dodecyl sulfate solution to shake the amniotic membrane to prepare acellular amniotic membrane (DAM), and adipose-derived mesenchymal stem cells are inoculated thereon to prepare a carrier cell sheet. Cell experiments have shown that DAM has good biocompatibility, can effectively promote the osteogenic differentiation of human adipose-derived mesenchymal stem cells, promote the expression of ALP and the formation of mineralized nodules, and is beneficial to bone repair and regeneration, which has not been reported in previous literature. Summary of the invention

[0008] In order to make up for the shortcomings of existing cell sheets for treating bone defects, the purpose of the present invention is to design and provide a cell sheet with osteogenic activity and its preparation method and application. Decellularized amniotic membrane (DAM) is prepared by subjecting amniotic membrane to a gradient concentration of sodium dodecyl sulfate oscillation treatment. As an extracellular matrix material, DAM has a microenvironment similar to that of the human body, which is conducive to the adhesion and growth of cells, thereby promoting cell proliferation. In addition, through the combined action of surface physical signals and active proteins, DAM can promote the osteogenic differentiation of stem cells, thereby promoting tissue repair.

[0009] In one aspect, the present invention provides a method for preparing an osteoblast sheet using a decellularized biofilm as a carrier, comprising:

[0010] (1) Decellularization of natural biofilm;

[0011] (2) Osteoblasts are planted on the decellularized biofilm to obtain an osteoblast sheet with the decellularized biofilm as a carrier.

[0012] In one embodiment, the aforementioned natural biological membrane is at least one of the pericardium, peritoneum, amnion, mesentery and small intestinal mucosa.

[0013] In one embodiment, the decellularization treatment in step (1) comprises the following steps: performing decellularization treatment on the biofilm carrier using at least one of the methods for preparing a decellularized matrix;

[0014] The method for preparing the decellularized matrix includes the following methods: a physical method of obtaining the decellularized matrix by physically destroying cells; a chemical method of obtaining the decellularized matrix by lysing cells with chemical reagents; and an enzymatic method of obtaining the decellularized matrix by destroying cells with enzymes.

[0015] In one embodiment, the method for preparing the osteoblast sheet comprises the following steps:

[0016] (1) Thaw the bovine placenta stored at -20°C at room temperature, peel the fetal membranes from the placenta by blunt dissection and wash them several times with sterile PBS, then physically separate the amniotic membrane (NAM) and the chorion, cut the NAM into 6×6 cm squares, and store them in freezing medium;

[0017] (2) NAM was thawed in a water bath at 37°C, washed repeatedly in sterile PBS to remove residual freezing medium, and then NAM was treated in 0.1% SDS at 37°C with shaking on a shaker for 12 hours, the SDS concentration was adjusted to 0.5%, and the treatment was repeated in the same manner for 12 hours, the SDS concentration was adjusted to 1%, and the treatment was repeated in the same manner for 6 hours. NAM was washed three times in PBS and incubated in 300 ml of 0.5% Triton X-100 for 2 hours, and terminally incubated with 200 ml of 0.1 M NaOH for 15 minutes. The resulting DAM was washed with ultrapure water for 48 hours, air-dried at 37°C for 24 hours, lyophilized, and sterilized with ethylene oxide before use;

[0018] (3) After ethylene oxide sterilization, DAM was moistened and placed in a 24-well cell culture plate at 25,000 / cm 2 Adipose-derived mesenchymal stem cells were inoculated into DAM at a density of 50000-100000 / cm2 and cultured for 48-72 hours until the cells were 100% confluent. The final cell density was about 50000-100000 / cm2. 2 .

[0019] In one embodiment, the aforementioned freezing medium formula is 60% DMEM, 20% FBS, 10% DMSO (v / v), in which NAM is immersed and stored at -80°C.

[0020] On the other hand, the present invention provides an osteoblast sheet using a decellularized biofilm as a carrier obtained by the above preparation method.

[0021] In another aspect, the present invention provides use of the osteoblast sheet.

[0022] In one embodiment, the osteoblast sheet using the decellularized amniotic membrane as a carrier is used to prepare medical devices.

[0023] Beneficial Effects

[0024] The carrier cell sheet provided by the present invention has good biocompatibility and a natural matrix microenvironment, which can provide cells with a 3D growth space similar to that of the human body. At the same time, the collagen fibers, proteoglycans, glycosaminoglycans and certain potential growth factors rich in natural amniotic membrane are retained, which is more conducive to the proliferation and adhesion of cells. In addition, the carrier cell sheet provided by the present invention has the ability to promote the differentiation of stem cells into osteoblasts, is rich in a variety of growth factors, can significantly promote the differentiation of adipose mesenchymal stem cells into osteoblasts, and can more effectively promote the repair of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 DAPI staining of decellularized amnion. Scale bar: 150 μm. NAM, native amnion; DAM, decellularized amnion.

[0026] Figure 2 The DNA quantification of decellularized amniotic membrane (**P<0.01).

[0027] Figure 3 HE staining of decellularized amniotic membrane. Scale bar: 50 μm. NAM, natural amniotic membrane; DAM, decellularized amniotic membrane.

[0028] Figure 4 Masson staining of decellularized amnion. Scale bar: 25 μm. NAM, native amnion; DAM, decellularized amnion.

[0029] Figure 5 This is the infrared spectrum of decellularized amniotic membrane. NAM, natural amniotic membrane; DAM, decellularized amniotic membrane.

[0030] Figure 6 Scanning electron micrographs of decellularized amniotic membrane. NAM, native amniotic membrane; DAM, decellularized amniotic membrane. Scale bar: 15 μm.

[0031] Figure 7Water contact angle test of decellularized amniotic membrane. NAM, natural amniotic membrane; DAM, decellularized amniotic membrane (**P<0.01).

[0032] Figure 8 The proliferation of human adipose-derived mesenchymal stem cells after inoculation on the cell sheet.

[0033] Fig. 9 ALP staining of human adipose-derived mesenchymal stem cells (ADMSC) seeded on a cell sheet and cultured for a certain period of time.

[0034] Fig.10 Human adipose-derived mesenchymal stem cells (ADMSC) were seeded on the cell sheet and cultured for a certain period of time, and the ALP quantitative results were obtained. Different letters represent significant differences (P < 0.05).

[0035] Fig.11 ARS staining of human adipose-derived mesenchymal stem cells (ADMSC) seeded on a cell sheet and cultured for a certain period of differentiation.

[0036] Fig.12 Human adipose-derived mesenchymal stem cells (ADMSCs) were seeded on cell sheets and cultured for 21 days before transcriptomic analysis of differentially expressed genes.

[0037] Fig.13 Human adipose-derived mesenchymal stem cells (ADMSCs) were seeded on cell sheets and cultured for 21 days before transcriptomic analysis of differentially expressed genes.

[0038] Fig.14 Human adipose-derived mesenchymal stem cells (ADMSCs) were seeded on cell sheets and cultured for 21 days before transcriptomic analysis and KEGG analysis of differentially expressed genes. DETAILED DESCRIPTION

[0039] In this specification, the term "cell sheet" refers to a sheet-like structure in which a plurality of cells are adhered to each other.

[0040] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0041] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0042] Example 1: Preparation and application of osteoblast sheets

[0043] 1. Preparation of myocardial patch with bone marrow mesenchymal stem cells using decellularized amniotic membrane as carrier

[0044] (1) Decellularization of natural amniotic membrane

[0045] Bovine placenta was stored at -20°C until transported to the laboratory for processing. After thawing at room temperature, the fetal membranes were stripped from the placenta by blunt dissection and washed 3-5 times with sterile PBS until there was no obvious blood on the surface of the amnion, and then the amnion (NAM) and chorion were physically separated. The NAM was cut into 6×6 cm squares and stored in freezing medium. The freezing medium formula was 60% DMEM, 20% FBS, 10% DMSO (v / v), and the NAM was immersed in it and stored at -80°C.

[0046] The NAM was thawed in a water bath at 37°C and repeatedly washed in sterile PBS to remove residual freezing medium. Then, the cellular components of the NAM were removed using a gradient concentration of sodium dodecyl sulfate (SDS) solution. First, the NAM was treated with 0.1% SDS at 37°C for 12 hours on a shaker. The SDS concentration was adjusted to 0.5% and the treatment was repeated in the same manner for 12 hours. Then, the NAM was treated with 1% SDS for about 6 hours. Next, the NAM was washed three times in PBS and incubated in 300ml of 0.5% Triton X-100 for 2 hours to remove residual DNA. To ensure complete cell lysis, a terminal incubation was performed with 200ml of 0.1M NaOH for 15 minutes. The treated decellularized amniotic membrane (DAM) was washed with ultrapure water for 48 hours, air-dried at 37°C for 24 hours, and lyophilized. The DAM was sterilized with ethylene oxide before use and stored aseptically at room temperature.

[0047] (2) Planting target cells on decellularized amniotic membrane

[0048] After ethylene oxide sterilization, DAM was moistened and placed in a 24-well cell culture plate at 25,000 / cm 2 Adipose-derived mesenchymal stem cells were seeded in DAM at a density of 100% and cultured for 48-72 hours until the cells were 100% confluent. The final cell density was about 50,000-100,000 / cm 2 , and obtain a cell sheet with decellularized amniotic membrane as carrier.

[0049] 2. Basic Characterization of Cell Sheet Carriers

[0050] To characterize the decellularization effect of the amniotic membrane, the amniotic membrane before and after decellularization was added to a 4% paraformaldehyde solution for 30 minutes. Next, the cells were counterstained with DAPI nuclear dye to observe whether there were any cell remnants in the DAM. The fixed NAM and DAM were dehydrated with gradient ethanol, embedded in paraffin, and cut into 4 μm thick slices. The sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome, respectively. In the decellularized samples, there was no DAPI staining, indicating that there were no nuclear remnants ( Figure 1 ). Quantification of DNA residues also confirmed this result. After decellularization, the amount of DNA dropped below 50 ng / mg tissue dry weight (3.51 ± 0.85 ng / mg), which is required for successful decellularization, while the residual amount of DNA in NAM was quantified as 1579.24 ± 112.14 ng / mg tissue dry weight ( Figure 2 ). Similarly, H&E staining of DAM did not reveal any cellular components compared to NAM ( Figure 3 ). According to the standard of decellularized biomaterials, NAM was successfully decellularized, showing the immunogenicity safety of DAM. Masson's trichrome staining showed collagen fibers (blue). Regardless of whether it was decellularized or not, the amniotic mesenchymal layer showed abundant collagen ( Figure 4 ), the decellularization process did not cause significant damage to the collagen component of the amniotic membrane.

[0051] The amniotic membrane was tested by Fourier transform infrared spectrometer (FT-IR) at 400-4000cm -1 Infrared absorption in the wavelength range. FTIR spectra show the structure of DAM and NAM ( Figure 5 In the spectrum of the NAM sample, we can see the amino group NH (3289 cm -1 )、CO(1650cm -1 ) and CN(1538cm -1 and nearly 1000cm -1 ) characteristic absorption peak. In addition, at 1227cm -1 A characteristic absorption peak appears at , which is related to the triple helix structure of collagen. The above peaks are typical peaks of collagen, which constitutes the main body of NAM. The DAM spectrum shows similar results.

[0052] In order to detect the changes in the surface morphology of the amniotic membrane before and after decellularization, NAM and DAM were fixed with 2.5% glutaraldehyde for 4 hours, washed with PBS, and dehydrated with gradient ethanol. Then, they were air-dried overnight at room temperature and freeze-dried for 24 hours. After gold spraying, the obtained samples were detected by scanning electron microscopy (voltage 10kV). Scanning electron microscopy showed that the basement membrane was smooth. There was no significant difference in the surface morphology of DAM and NAM ( Figure 6 ).

[0053] NAM and DAM were stuck to the edge of a glass slide, and then a drop of water was dropped on it. The water contact angle was measured after 5 seconds. When measuring the contact angle, 5 points were randomly selected from each sample surface and the average value was taken. Figure 7 As shown in the figure, the water contact angle of NAM is 70.20°±1.82°, and the water contact angle of DAM is 58.68°±2.88°. Compared with NAM, DAM has a significant improvement in hydrophilicity, which is beneficial to cell adhesion (P<0.01).

[0054] The above experiments show that the decellularization method used in the present invention achieves a certain balance between removing cell components and retaining the components and functions of the extracellular matrix.

[0055] 3. Biocompatibility of decellularized amniotic membrane (DAM)

[0056] When ADMSCs are seeded on the surface of the amniotic membrane, Figure 8 As shown in the figure, there was no significant difference in the growth of ADMSCs in the DAM group compared with the control group, while the proliferation of ADMSCs was significantly inhibited in the NAM group. The decellularization method used in this experiment significantly improved the cell adhesion and proliferation on the amniotic membrane, which was equivalent to the effect on the collagen-coated culture plate.

[0057] 4. Characterization of the osteogenic differentiation properties of decellularized amniotic membrane (DAM)

[0058] The osteogenic differentiation-promoting property of the present invention is verified by inoculating ADMSCs in NAM and DAM and using differentiation medium for differentiation culture, characterizing the expression of alkaline phosphatase and mineralized nodules after a certain period of time, and performing transcriptomic analysis.

[0059] The specific operation is as follows: NAM and DAM were cut into squares (1.4 cm × 1.4 cm) and placed in a 24-well plate. ADMSCs were inoculated on the sample surface at a density of 40,000 cells / well and cultured until 100% confluence, and then the differentiation medium was replaced. The control group (CON) was cells cultured on a normal culture plate. The preparation method of the differentiation medium was as follows: 10 nmol / L dexamethasone, 10 mmol / L sodium β-glycerophosphate and 5 μg / ml ascorbic acid were added to high-glucose DMEM supplemented with 10% fetal bovine serum. The differentiation medium was replaced every 3 days, and alkaline phosphatase was specifically stained using the BCIP / NBT colorimetric kit at 3, 5, 7, 14 and 21 days of differentiation. At the same time, the cells were lysed, the ALP expression was detected using an alkaline phosphatase detection kit, and the total protein content of the cells was normalized by testing the BCA protein quantification kit. In addition, the mineralized nodules after differentiation culture were stained with Alizarin Red S staining solution. Total RNA was extracted from ADMSCs cultured for 21 days for transcriptomic analysis.

[0060] The results of ALP staining showed that at different stages of osteogenic differentiation, the ALP expression in the DAM group was better than that in the control group, with more uniform ALP staining and more significant deposition ( Fig. 9 ). The relative quantitative results of ALP showed that in the early stage of osteogenic differentiation, the ALP activity of the DAM group was significantly higher than that of the control group, while in the late stage of osteogenic differentiation, there was no significant difference between the DAM group and the control group, which indicates that DAM mainly promotes the expression of ALP in the early stage of differentiation ( Fig.10 This is consistent with the expression characteristics of ALP during osteogenic differentiation, and cooperates with the early DAM to inhibit excessive inflammation, which helps DAM to promote bone tissue repair. The results of Alizarin Red S (ARS) staining showed that at all time points, the ARS staining results of the DAM group were better than those of the control group, and DAM significantly promoted the formation of mineralized nodules during osteogenic differentiation ( Fig.11 ).

[0061] Transcriptomics revealed that compared with the control group, 189 genes were upregulated and 337 genes were downregulated in ADMSC cultured on DAM after 21 days of osteogenic differentiation ( Fig.12 ). GO functional annotation was performed on the differentially expressed genes to find genes related to osteogenic differentiation and immune regulation. GO analysis found that the differentially expressed genes were involved in biological processes such as cell adhesion, extracellular matrix formation, and the production of vascular endothelial growth factor ( Fig.13 ). KEGG pathway annotation found that NF-κB, Wnt signaling pathways, etc. were disturbed inflammation-related signaling pathways. Compared with the control group, the levels of related pathways in the DAM group were significantly downregulated; osteoclast differentiation and other pathways were pathways related to osteogenic differentiation ( Fig.14 ). These are all closely related to bone tissue repair.

[0062] The above results indicate that decellularized amniotic membrane significantly promotes the osteogenic differentiation of ADMSCs and has great potential and good prospects in playing an osteogenic repair role as a guided bone regeneration (GBR) membrane.

[0063] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an osteoblast sheet using a decellularized biofilm as a carrier, comprising: (1) Decellularization of natural biofilm; (2) Osteoblasts are planted on the decellularized biofilm to obtain an osteoblast sheet with the decellularized biofilm as a carrier.

2. The preparation method according to claim 1, wherein the natural biological membrane is at least one of pericardium, peritoneum, amnion, mesentery and small intestinal mucosa.

3. The preparation method according to claim 1 or 2, wherein the decellularization treatment in step (1) comprises the following steps: performing decellularization treatment on the biofilm carrier using at least one of the methods for preparing a decellularized matrix; The method for preparing the decellularized matrix includes the following methods: a physical method of obtaining the decellularized matrix by physically destroying cells; a chemical method of obtaining the decellularized matrix by lysing cells with chemical reagents; and an enzymatic method of obtaining the decellularized matrix by destroying cells with enzymes.

4. The preparation method according to any one of claims 1 to 3, wherein the preparation method of the osteoblast sheet comprises the following steps: (1) Thaw the bovine placenta stored at -20°C at room temperature, peel the fetal membranes from the placenta by blunt dissection and wash them several times with sterile PBS, then physically separate the amniotic membrane (NAM) and the chorion, cut the NAM into 6×6 cm squares, and store them in freezing medium; (2) NAM was thawed in a water bath at 37°C, washed repeatedly in sterile PBS to remove residual freezing medium, and then NAM was treated in 0.1% SDS at 37°C with shaking on a shaker for 12 hours, the SDS concentration was adjusted to 0.5%, and the treatment was repeated in the same manner for 12 hours, the SDS concentration was adjusted to 1%, and the treatment was repeated in the same manner for 6 hours. NAM was washed three times in PBS and incubated in 300 ml of 0.5% Triton X-100 for 2 hours, and terminally incubated with 200 ml of 0.1 M NaOH for 15 minutes. The resulting DAM was washed with ultrapure water for 48 hours, air-dried at 37°C for 24 hours, lyophilized, and sterilized with ethylene oxide before use; (3) After ethylene oxide sterilization, DAM was moistened and placed in a 24-well cell culture plate at 25,000 / cm 2 Adipose-derived mesenchymal stem cells were inoculated into DAM at a density of 50000-100000 / cm2 and cultured for 48-72 hours until the cells were 100% confluent. The final cell density was about 50000-100000 / cm2. 2 .

5. The preparation method according to claim 4, wherein the freezing medium formula is 60% DMEM, 20% FBS, 10% DMSO (v / v), NAM is immersed therein and stored at -80°C.

6. An osteoblast sheet with acellular biofilm as a carrier obtained according to the preparation method according to any one of claims 1 to 5.

7. Use of the osteoblast sheet according to claim 6.

8. The use according to claim 7, wherein the use is for preparing medical devices.

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