WO2023231195A1 - 一种脱细胞基质组织纸的制备方法及应用 - Google Patents

一种脱细胞基质组织纸的制备方法及应用 Download PDF

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WO2023231195A1
WO2023231195A1 PCT/CN2022/115017 CN2022115017W WO2023231195A1 WO 2023231195 A1 WO2023231195 A1 WO 2023231195A1 CN 2022115017 W CN2022115017 W CN 2022115017W WO 2023231195 A1 WO2023231195 A1 WO 2023231195A1
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tissue paper
tissue
cross
preparation
linking
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French (fr)
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朱美峰
李雯
孔德领
张祯
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南开大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention relates to the technical field of regenerative medicine, and in particular to a preparation method and application of acellular matrix tissue paper.
  • Biomaterials can be used to replace and repair damaged and declining tissues. Biomaterials should have appropriate pore structures to provide physical structural support for cell and tissue growth, and should also have excellent biocompatibility, bioactivity and mechanical properties.
  • Decellularized extracellular matrix (dECM) material refers to an extracellular matrix material obtained by removing cells and immunogenic components from human or animal organs/tissues after decellularization. It retains a large amount of extracellular matrix components such as collagen. Protein, elastin, proteoglycans and aminoglycans, etc., as well as physical and chemical properties. Compared with commonly used natural or synthetic materials, dECM can provide a microenvironment similar to natural tissue for the regeneration and repair of damaged tissue.
  • acellular matrix materials can promote tissue regeneration and repair by regulating immune responses.
  • a variety of acellular matrix material products such as bovine pericardium, pig skin, small intestinal submucosa, etc., have been used in clinical applications, which shows that acellular matrix materials have broad application prospects.
  • Patent CN110038168A provides an anti-adhesion membrane prepared from tendon decellularized matrix.
  • the decellularized tendon tissue needs to be crushed and dissolved in an acetic acid solution, and then neutralized with a NaOH solution.
  • Patent CN113713165A provides a method for preparing acellular matrix fiber sponge.
  • the use of acetic acid destroys extracellular matrix composition and activity.
  • the purpose of the present invention is to provide a preparation method and application of acellular matrix tissue paper to solve the problems existing in the above-mentioned prior art.
  • This method does not use any acids, enzymes and organic solvents, and can retain the ECM components and activity to the greatest extent. ; And the preparation is simple and easy for industrial production.
  • the present invention provides the following solutions:
  • the invention provides a method for preparing acellular matrix tissue paper, which includes the following steps:
  • Preparation of acellular matrix material Take fresh tissue and perform decellularization treatment after disinfection and sterilization to obtain acellular matrix material;
  • tissue paper homogenize and stir the acellular matrix material, then place the homogenate on a flat-bottomed filter to filter out the water, then let the filtered homogenate sit to dry, and then freeze-dry, that is Obtain the tissue paper;
  • the cross-linked tissue paper is freeze-dried or superimposed and compacted to obtain the acellular matrix tissue paper.
  • the decellularization treatment is as follows: wash the fresh tissue and slice it into sections, disinfect it with peracetic acid and add sterile water or physiological saline to wash it, and then wash the washed tissue with a buffer containing DNase and RNase.
  • step (2) the homogenization stirring time is 1-60min, the flat-bottom filter size is 3-500 mesh, the standing drying time is 1-120h, and the freeze-drying temperature is -196°C ⁇ -20°C, time is 4-48h.
  • the cross-linking solution in step (3) is an ethanol solution containing a cross-linking agent;
  • the cross-linking agent includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N- One or more of hydroxysuccinimide, glutaraldehyde, formaldehyde and genipin.
  • cross-linking conditions are cross-linking at 4°C-27°C for 4-12 hours.
  • freeze-drying temperature in step (4) is -196°C ⁇ -20°C, and the time is 12-72h.
  • the fresh tissue includes human or animal tissue or organs.
  • the fresh tissue includes brain, heart, liver, spleen, lung, kidney, muscle, skin, fat, meninges, diaphragm, amniotic membrane, pericardium, heart valve, small intestinal submucosa, muscle, blood vessel, tendon, Ligaments, cartilage, esophagus, trachea, stomach, nerves, bladder, cornea and/or placenta.
  • the present invention also provides an acellular matrix tissue paper prepared according to the above preparation method.
  • the present invention uses traditional papermaking technology combined with freeze-drying and other technologies to prepare tissue-specific acellular matrix tissue paper.
  • No acids, enzymes or organic solvents are used in the processing of dECM.
  • the process is gentle and can retain the natural extracellular matrix components and activity to the greatest extent; and the obtained extracellular matrix tissue paper has some properties of paper and can be curled, folded and sutured. and cropping etc.
  • the mechanical properties of tissue paper can be further enhanced through cross-linking reaction or superimposed pressure of multiple layers of tissue paper.
  • the present invention also provides an application of the above-mentioned acellular matrix tissue paper in the preparation of tissue damage repair drugs.
  • decellularized materials required the use of acids, enzymes, or the addition of organic solvents, thereby damaging their composition and activity.
  • the decellularized matrix processing method adopted by the present invention does not use any acids, enzymes and organic solvents, and can retain ECM components and activity to the greatest extent; it is also simple to prepare and easy to industrialize.
  • the existing pure acellular matrix materials have a dense structure, low mechanical strength, and poor controllability, which will limit their effectiveness in promoting tissue repair.
  • the present invention can prepare acellular matrix with controllable thickness, pore size and mechanical strength by obtaining decellularized matrix materials from different tissue sources, adjusting the amount of decellularized tissue, homogenization time, drying time, freezing temperature, cross-linking time, etc. Tissue paper.
  • the structure of tissue paper can be adjusted according to clinical needs, and the mechanics of tissue paper can be enhanced, thereby expanding the scope of application of tissue paper.
  • Figure 1 shows the statistics of protein types after different extracellular matrix treatment methods
  • A the Venn diagram of protein types in different treated samples
  • B the statistical results of the number of protein types in ECM samples after different treatment methods
  • a, b, c , d represents native heart tissue, decellularized heart tissue, decellularized heart tissue paper, and decellularized heart tissue digested with acetic acid and enzyme, respectively;
  • Figure 2 shows the mass spectrum peak diagram of heart tissue samples after different decellularization matrix treatment methods
  • A natural heart tissue
  • B decellularized heart tissue
  • C decellularized heart tissue paper
  • D decellularized heart tissue after acid lysis and enzymatic hydrolysis cellular heart tissue
  • Figure 3 shows the structural morphology characterization of the acellular matrix products of Examples 1-4 and Comparative Example 2; A, macro morphology and microstructure pictures; B, pore size statistical diagram of the porous structure; C, porosity statistical diagram;
  • Figure 4 is an evaluation of the mechanical properties of tissue papers from four tissue sources in Examples 1-4; A, the stress-strain curves of the four tissue papers; B, the tensile strength statistics of the four tissue papers; C, the four tissue papers Statistics on suture strength;
  • Figure 5 is the cytocompatibility evaluation of tissue paper derived from four tissues in Examples 1-4; A, cell death and survival staining diagram of bone marrow mesenchymal stem cells after culturing on tissue paper for 1 day; B, cell survival rate statistical diagram ;C, Statistical chart of CCK8 detection of stem cell proliferation in tissue paper extract.
  • the materials, instruments and reagents used in the present invention can all be obtained from commercial sources; unless otherwise specified, the experimental methods used are conventional experimental methods in this field.
  • Preparation of decellularized heart tissue Rinse the purchased fresh pig heart tissue, cut it into 1mm thick slices, put it into a glass bottle, sterilize it with 0.1% peracetic acid, add sterile water and shake it. Bed shaking and washing. After washing until clear, add 1% sodium dodecyl sulfate (SDS) solution and shake and wash for 72 hours. Change the solution every three hours and wash until the tissue turns white. Then wash with sterile water to remove residual SDS, and then use Tris-HCL buffer added with deoxyribonucleic acid (DNA) enzyme I and ribonucleic acid (RNA) enzyme to shake on a 100 rpm shaker at 37°C for 24 hours to remove the tissue. of DNA and RNA. Finally, it was washed with sterile water to obtain porcine heart extracellular matrix material.
  • SDS sodium dodecyl sulfate
  • porcine heart extracellular matrix tissue paper Weigh 10g of wet weight porcine heart decellularized tissue, add 100mL of distilled water, put it into a homogenizer and crush it, set the time to 10min, obtain the homogenate, and then pour Let it stand in a 300-mesh flat-bottomed filter with a diameter of 8cm. After filtering out the water, dry it at room temperature for 48 hours, then freeze it in a -80°C refrigerator for 4 hours, and finally transfer it to a freeze dryer to freeze-dry for 48 hours to obtain decellularized heart tissue paper.
  • Porcine heart extracellular matrix tissue paper with different thicknesses and different pore sizes can be prepared by adjusting the amount of pig heart extracellular matrix, the mesh number of the filter, and the drying time.
  • Cross-linking tissue paper First prepare 100 mL of 80% ethanol solution, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinate with a mass ratio of 4:1 imide (NHS) to a concentration of 0.3% to obtain a cross-linked solution. Then, the porcine heart extracellular matrix tissue paper was immersed in the solution and cross-linked at 4°C for 10 hours. Then it was taken out and soaked in sterile water for several times to remove residual cross-linking reagent.
  • EDC 1-ethyl-(3-dimethylaminopropyl)carbodiimide
  • NHS imide
  • Freeze-drying Freeze the cross-linked and washed tissue paper in a -20°C refrigerator for 12 hours, and then freeze-dry it in a freeze dryer for 48 hours to obtain a finished porcine heart extracellular matrix tissue paper with high mechanical strength. Multiple porcine heart tissue papers can also be stacked and compacted for later use.
  • Preparation of decellularized liver tissue rinse the purchased fresh pig liver, cut it into 1mm thick slices, put it into a glass bottle, sterilize it with 0.1% peracetic acid, add sterile water and place it on a shaker Shake and wash until clear. Add 1% SDS solution and shake for 24 hours. Change the solution every three hours and wash until the tissue turns white. Then wash with sterile water to remove residual SDS, then put it into Tris-HCL buffer containing 50U/mL DNase I and 1U/mL RNase, and shake on a 100rpm shaker at 37°C for 24h to remove the tissue. of DNA and RNA. Finally, the enzymes are removed by washing with sterile water, and the pig liver extracellular matrix material is obtained.
  • pig liver extracellular matrix tissue paper (LTP): Weigh 30g of wet weight pig liver extracellular matrix material, put it into a homogenizer and stir for 5 minutes to obtain a homogenate, then pour the homogenate into a 200-mesh container with a diameter of 10cm Let it stand in a flat-bottomed strainer to filter out the water and then dry it at room temperature for 10 hours. Then, it was placed in a -20°C refrigerator for 12 hours, and then transferred to a freeze dryer to be freeze-dried for 36 hours to obtain porcine liver extracellular matrix tissue paper.
  • Pig liver tissue papers with different thicknesses can be prepared by adjusting the amount of pig liver extracellular matrix.
  • Tissue paper cross-linking Take the above freeze-dried pig liver extracellular matrix tissue paper, place it in an 80% ethanol solution containing EDC/NHS with a mass ratio of 4:1, cross-link it at 4°C for 12 hours, take it out and use Soak in bacterial water and wash 5 times to remove residual cross-linking reagent.
  • Freeze-drying freeze the above-mentioned cross-linked and washed pig liver extracellular matrix tissue paper at -80°C for 4 hours, and freeze-dry it in a freeze dryer for 36 hours to obtain a mechanically enhanced finished pig liver extracellular matrix tissue paper, which can be further Stack multiple pieces of pig liver tissue paper and compact them for later use.
  • Preparation of decellularized kidney tissue Rinse the purchased fresh pig kidney, cut it into small 1mm thick slices, put it into a glass bottle, disinfect and sterilize it with 0.1% peracetic acid, add sterile water and place it in the Shake and wash until clear, then add 1% SDS solution and shake for 48 hours. Change the solution every three hours and wash until the tissue turns white. Then rinse with sterile water to remove residual SDS, then wash with Tris-HCL buffer added with DNase and RNase at 37°C overnight to remove DNA and RNA in the tissue, and finally wash with sterile water to obtain Porcine kidney extracellular matrix material.
  • KTP pig kidney extracellular matrix tissue paper
  • Cross-linking tissue paper First prepare 100 mL of 80% ethanol solution, add EDC and NHS with a mass ratio of 4:1, and prepare an EDC concentration of 0.3% to obtain a cross-linking solution. Then, the kidney extracellular matrix tissue paper was immersed in the solution for cross-linking at room temperature for 4 hours, and then taken out and soaked in sterile water for several times to remove residual cross-linking reagent.
  • Freeze-drying Freeze the cross-linked and washed tissue paper in a -80°C refrigerator for 4 hours, and then freeze-dry it in a freeze dryer for 48 hours to obtain a finished porcine kidney extracellular matrix tissue paper with high mechanical strength. Multiple porcine kidney extracellular matrix tissue papers can also be stacked and compacted for later use.
  • Achilles tendon tissue Take fresh porcine Achilles tendon tissue, remove the surrounding connective tissue and cut it into thin slices with a thickness of about 500 ⁇ m. Sterilize with 0.1% peracetic acid, wash with sterile water, and immerse in liquid Nitrogen for 2 minutes, then placed in 400 mL of physiological saline and shaken on a 100 rpm shaker at 37°C for 15 minutes, and repeated freezing and thawing 5 times.
  • the sample was placed in a reaction solution containing 50 U/mL deoxyribonuclease I and 1 U/mL ribonuclease A, and reacted on a 100 rpm shaker at 37°C for 24 hours. Subsequently, it was washed with sterile PBS to remove the enzyme solution, and decellularized Achilles tendon tissue was obtained.
  • Tissue paper cross-linking Take the above freeze-dried porcine Achilles tendon extracellular matrix tissue paper, place it in an 80% ethanol solution containing EDC/NHS with a mass ratio of 4:1, cross-link it at 4°C for 12 hours, and then cross-link it the next day. After taking it out, soak it in sterile water and wash it several times to remove the residual cross-linking reagent.
  • Freeze-drying Freeze the cross-linked and washed Achilles tendon extracellular matrix tissue paper at -80°C for 4 hours, and then quickly transfer it to a pre-cooled freeze dryer to freeze-dry for 36 hours to obtain porcine Achilles tendon extracellular matrix tissue paper.
  • the finished product can also be further stacked and compacted with multiple porcine Achilles tendon extracellular matrix tissue papers for later use.
  • Example 1 The difference from Example 1 is that the porcine heart acellular matrix material is first dissolved and digested with acetic acid and pepsin, and then tissue cross-linked.
  • Example 1 The difference from Example 1 is that there is no acellular matrix tissue paper preparation process.
  • proteomics was used to detect porcine natural heart tissue, decellularized heart tissue (Comparative Example 2), decellularized heart tissue paper (Example 1), and after dissolution and digestion with acetic acid and pepsin.
  • the matrix composition of four differently treated heart tissue samples was examined using a proteomic approach.
  • the main steps of the protein identification project process include: protein enzymatic hydrolysis, LC-MS/MS analysis, database search, data analysis, etc. This test was completed and the experimental result report was obtained by Shanghai Beipu Biotechnology Co., Ltd. The experimental results are shown in Figure 1 and Figure 2:
  • Figure 1 is a statistical diagram of protein types after different ECM processing methods.
  • A Venn diagram of protein types in different processed samples; B, statistical results of the number of protein types in ECM samples after different processing methods; where a, b, c, d represents native heart tissue, decellularized heart tissue, decellularized heart tissue paper, and acetic acid and enzyme-digested decellularized heart tissue, respectively.
  • Figure 2 shows the mass spectrum peaks of heart tissue samples after different ECM treatment methods; A, natural heart tissue; B, decellularized heart tissue; C, decellularized heart tissue paper; D, decellularized heart tissue after acid lysis. .
  • the number of protein types in the decellularized heart tissue paper is slightly lower than that of the natural heart tissue, and the difference with the decellularized heart tissue is small, indicating that the preparation process of the decellularized ECM tissue paper in the present invention can be very good.
  • the acid-lytic enzymatic treatment method reduced the number of protein types in decellularized heart tissue by about 900 types, greatly destroying the ECM material components.
  • the peptide peaks of natural heart tissue, decellularized heart tissue and decellularized heart tissue paper are basically the same, indicating that the protein components are not much different.
  • the peak position and peak intensity of the acid-lyzed sample are significantly different from natural tissue. It can be concluded that compared with the traditional processing method of acid lysis and enzymatic hydrolysis, the preparation process of the acellular matrix tissue paper of the present invention can better retain the natural extracellular matrix components.
  • Figure 3A is a picture of the macro morphology and microstructure of the acellular matrix tissue paper and conventional acellular matrix products prepared in Examples 1-4; B, the surface porous structures of four kinds of acellular matrix tissue paper and four conventional acellular matrix products Statistical diagram of pore size; C, Statistical diagram of porosity of four kinds of acellular matrix tissue paper and four kinds of conventional acellular matrix.
  • the tissue paper prepared in Examples 1-4 of the present invention is very close to "paper" in macroscopic morphology, and has a rich porous structure in microstructure, and tissue papers from different tissue sources all have large pore sizes. and higher porosity, which is conducive to cell infiltration and promotes tissue regeneration.
  • the acellular matrix material prepared according to the method of Comparative Example 2 has a dense microstructure, smaller pore size and lower porosity, which will limit cell migration and tissue regeneration.
  • Suture strength test Cut a piece of tissue paper with a length of 2cm, fix one end of it on one side of the tensile testing machine, insert a 6-0 suture line 2mm from the end of the tissue paper on the other end, knot it and fix it on the other side of the tensile testing machine. side, stretch at a speed of 10mm/min until the tissue paper breaks, and the maximum load obtained is the suture strength value of the tissue paper.
  • Figure 4 is an evaluation of the mechanical properties of the tissue papers of Examples 1-4.
  • A the stress-strain curves of the four tissue papers;
  • B the tensile strength statistics of the four tissue papers;
  • C the suture strength statistics of the four tissue papers.
  • tissue papers from four different tissue sources all have high mechanical strength, and the maximum stress is above 1MPa.
  • liver tissue paper has the highest mechanical strength, and they also have sufficient suture strength. Therefore, the prepared tissue paper can well meet the mechanical and suture strength requirements for in vivo applications.
  • Plant 4 ⁇ 10 4 stem cells on a tissue paper membrane in a 48-well plate After culturing for 24 hours, aspirate the culture medium, add Calcein AM and PI live and alive staining solution, incubate at room temperature for 20 minutes in the dark, and observe and take pictures with a confocal microscope. , and count the number of dead and living cells and their survival rate.
  • tissue paper extract we first soaked 0.1g of tissue paper in 5 ml of culture medium and incubated it for 24 hours to prepare the tissue paper extract. Then inoculate 3 ⁇ 10 3 stem cells/well into a 96-well plate, add 100 ⁇ l of extract solution for culture, take out the well plate at the time points of day 1, day 3 and day 5, and add 10 ⁇ l of CCK8 working solution. Incubate at 37°C for 2 hours, and detect the absorbance with a microplate reader.
  • Figure 5 shows the cytocompatibility of four types of acellular matrix tissue paper.
  • A cell death and survival staining of bone marrow mesenchymal stem cells after culturing on tissue paper for 1 day;
  • B statistical chart of cell survival rate;
  • C CCK8 detection of stem cells in tissue Proliferation in paper extracts.
  • the extracted bone marrow mesenchymal stem cells can adhere and spread well on all four tissue papers, and the cell survival rate is above 90%, indicating that the tissue papers have good cell compatibility.
  • tissue paper extract culture significantly promoted the proliferation of stem cells, indicating that tissue paper has good biological activity.

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Abstract

本发明公开了一种脱细胞基质组织纸的制备方法及应用，属于再生医学技术领域。该制备方法包括以下步骤：(1)制备脱细胞基质材料；(2)制备组织纸：将制备的脱细胞基质材料进行匀浆搅拌，之后置于平底滤网滤出水分，再将过滤后的匀浆液静置晾干，进行冷冻干燥，即得所述组织纸；(3)组织纸交联：将所述组织纸置于交联溶液中进行交联；(4)将交联后的组织纸进行冷冻干燥或进行叠加压实。本发明采用传统造纸技术结合冷冻干燥技术制备具有组织特异性的脱细胞基质组织纸，在加工过程中未使用酸、酶或有机溶剂，工艺温和，可最大程度保留天然细胞外基质组分及活性；同时该组织纸具有丰富的多孔结构，更利于细胞浸润及促进组织再生。

Description

一种脱细胞基质组织纸的制备方法及应用 技术领域
本发明涉及再生医学技术领域，特别是涉及一种脱细胞基质组织纸的制备方法及应用。
背景技术
生物材料可用于替代、修复损伤及衰退的组织。生物材料应具备适宜的孔结构为细胞和组织生长提供物理结构支撑，同时应具有优异的生物相容性、生物活性及力学性能。脱细胞基质(decellularized extracellular matrix，dECM)材料是指将人体或动物器官/组织经过脱细胞处理后去除细胞及免疫原性成分而得到的细胞外基质材料，它保留有大量细胞外基质成分如胶原蛋白、弹性蛋白、蛋白聚糖和氨基聚糖等，以及理化性质。与常用的天然或人工合成材料相比，dECM可为损伤组织再生修复提供近似于天然组织的微环境。大量研究表明脱细胞基质材料可通过调控免疫应答反应来促进组织再生修复。目前，已经有多种脱细胞基质材料的产品如牛心包、猪皮、小肠粘膜下层等应用于临床，这表明脱细胞基质材料具有广阔的应用前景。
然而，这些临床常用产品通常是将获取的组织进行脱细胞处理后直接交联而得到，缺乏材料宏观和微观结构的可控性。同时，这些产品结构致密，会限制细胞迁移和组织再生。为解决该问题，大量研究通过引入多种工艺如静电纺丝、3D打印、水凝胶、微囊化、微载体等来改善dECM材料的性能。然而，这些工艺中都需要利用酸、酶溶液或有机溶剂将dECM溶解或消化，从而破坏或损失细胞外基质组分，降低其生物活性。
专利CN110038168A提供了一种由肌腱脱细胞基质制备的防粘连膜，该 技术方案中，需要将脱细胞肌腱组织粉碎后溶于乙酸溶液中，随后用NaOH溶液进行中和。专利CN113713165A提供了一种脱细胞基质纤维海绵的制备方法，在该技术方案中也需要将脱细胞基质与乙酸和水混合。然而，乙酸的使用会破坏细胞外基质成分和活性。
因此，现有的脱细胞基质材料制备的技术方案均存在明显的缺陷，开发一种新型的可有效保留细胞外基质成分和活性且绿色温和的制备工艺十分必要。
发明内容
本发明的目的是提供一种脱细胞基质组织纸的制备方法及应用，以解决上述现有技术存在的问题，该方法没有使用任何酸、酶以及有机溶剂，可最大程度地保留ECM成分和活性；且制备简单，易工业化生产。
为实现上述目的，本发明提供了如下方案：
本发明提供一种脱细胞基质组织纸的制备方法，包括以下步骤：
(1)制备脱细胞基质材料：取新鲜组织消毒灭菌后进行脱细胞处理，获得脱细胞基质材料；
(2)制备组织纸：将所述脱细胞基质材料进行匀浆搅拌，之后将匀浆液置于平底滤网滤出水分，再将过滤后的匀浆液静置晾干，然后进行冷冻干燥，即得所述组织纸；
(3)组织纸交联：将步骤(2)获得的所述组织纸置于交联溶液中进行交联；
(4)将交联后的组织纸进行冷冻干燥或进行叠加压实，即得所述脱细胞基质组织纸。
进一步地，所述脱细胞处理为：将新鲜组织洗净后切片，用过氧乙酸消毒后加入无菌水或生理盐水洗涤，之后用含有DNA酶和RNA酶的缓冲液清洗洗涤后的组织。
进一步地，步骤(2)中，所述匀浆搅拌时间为1-60min，所述平底滤网尺寸为3-500目，所述静置晾干时间为1-120h，所述冷冻干燥温度为-196℃～-20℃，时间为4-48h。
进一步地，步骤(3)所述交联溶液为含有交联剂的乙醇溶液；所述交联剂包括1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺、戊二醛、甲醛和京尼平中的一种或多种。
进一步地，所述交联条件为4℃-27℃下交联4-12h。
进一步地，步骤(4)所述冷冻干燥温度为-196℃～-20℃，时间为12-72h。
进一步地，所述新鲜组织包括人体或动物的组织或器官。
进一步地，所述新鲜组织包括脑、心脏、肝脏、脾脏、肺、肾、肌肉、皮肤、脂肪、脑膜、膈膜、羊膜、心包膜、心脏瓣膜、小肠粘膜下层、肌肉、血管、肌腱、韧带、软骨、食道、气管、胃、神经、膀胱、角膜和/或胎盘。
本发明还提供一种根据上述的制备方法制得的脱细胞基质组织纸。
进一步地，本发明采用传统造纸技术结合冷冻干燥等技术制备具有组织特异性的脱细胞基质组织纸。在加工dECM过程中未使用酸、酶或有机溶剂，工艺温和，可最大程度保留天然细胞外基质组分及活性；且获得的细胞外基质组织纸具有纸张的一些性质，可以卷曲、折叠、缝合和裁剪等。此外，可通过交联反应或者多层组织纸的叠加加压来进一步增强组织纸力学性能。
本发明还提供一种上述的脱细胞基质组织纸在制备组织损伤修复药物中 的应用。
本发明公开了以下技术效果：
1、以往脱细胞材料加工过程中需要使用酸、酶溶解或添加有机溶剂，从而损害其成分和活性。本发明采用的脱细胞基质加工方法没有使用任何酸、酶以及有机溶剂，可最大程度地保留ECM成分和活性；且制备简单，易工业化生产。
2、现有纯的脱细胞基质材料结构致密，力学强度较低，可控性差，因而会限制其促进组织修复的效果。本发明可通过获得不同组织来源的脱细胞基质材料、调节脱细胞组织的用量、匀浆时间、晾干时间、冷冻温度、交联时间等来制备厚度、孔径及力学强度可控的脱细胞基质组织纸。同时可根据临床需求调整组织纸结构，增强组织纸力学，进而扩大组织纸的适用范围。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为不同细胞外基质处理方式后的蛋白质种类统计；A，不同处理样品的蛋白质种类韦恩图；B，不同处理方式后的ECM样品中蛋白质种类数的统计结果；其中a，b，c，d分别表示天然心脏组织、脱细胞心脏组织、脱细胞心脏组织纸以及乙酸和酶消化的脱细胞心脏组织；
图2为不同脱细胞基质处理方式后的心脏组织样品的质谱峰图；A，天然心脏组织；B，脱细胞心脏组织；C，脱细胞心脏组织纸；D，酸溶酶解后 的的脱细胞心脏组织；
图3为实施例1-4和对比例2的脱细胞基质产品的结构形貌表征；A，宏观形貌及微观结构图片；B，多孔结构的孔径统计图；C，孔隙率统计图；
图4为实施例1-4中四种组织来源的组织纸的力学性能评价；A，四种组织纸的应力应变曲线；B，四种组织纸的拉伸强度统计；C，四种组织纸的缝合强度统计；
图5为实施例1-4中四种组织来源的组织纸的细胞相容性评价；A，骨髓间充质干细胞在组织纸上培养1天后的细胞死活染色图；B，细胞存活率统计图；C，CCK8检测干细胞在组织纸浸提液中的增殖情况统计图。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述 与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
本发明所使用的材料、仪器及试剂如无特殊说明，均可由商业途径获得；所使用的实验方法如无特殊说明，均为本领域常规实验方法。
实施例1猪心脏组织纸的制备
脱细胞心脏组织的制备：将购买的新鲜猪心脏组织冲洗干净后，切成1mm厚的薄片，装入玻璃瓶中，用0.1％的过氧乙酸消毒灭菌后，加入无菌水置于摇床震荡洗涤，洗至澄清后，加入1％的十二烷基硫酸钠(SDS)溶液震荡洗涤72h，每三小时换一次液，洗至组织发白。随后再用无菌水洗涤以去除残留的SDS，接着用添加脱氧核糖核酸(DNA)酶I和核糖核酸(RNA)酶的的Tris-HCL缓冲液在37℃，100rpm摇床上震荡24h去除组织中的DNA和RNA。最后用无菌水洗涤，获得猪心脏细胞外基质材料。
猪心脏细胞外基质组织纸(HTP)的制备：称取10g湿重的猪心脏脱细胞组织，加入100mL蒸馏水，放入匀浆机器中打碎，设置时间为10min，得到匀浆液，然后倒入直径8cm的300目的平底滤网中静置，滤出水分后室温晾干48h，随后放入-80℃冰箱冷冻4h，最后转移到冷冻干燥机中冻干48h， 得到脱细胞心脏组织纸。可通过调节猪心脏细胞外基质的用量、滤网目数和晾干时间等制备具有不同厚度、不同孔径的猪心脏细胞外基质组织纸。
组织纸交联：首先配置80％的乙醇溶液100mL，加入质量比为4：1的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)，配成0.3％的浓度，得到交联溶液。然后将猪心脏细胞外基质组织纸浸入溶液中，4℃交联10h，随后取出用无菌水浸泡清洗数次，去除残留的交联试剂。
冷冻干燥：将交联后洗净的组织纸于-20℃冰箱冷冻12小时，而后于冷冻干燥机中冻干48小时，得到力学强度较高的猪心脏细胞外基质组织纸成品。也可进一步将多张猪心脏组织纸叠加并压实后备用。
实施例2猪肝脏组织纸的制备
脱细胞肝脏组织的制备：将购买的新鲜猪肝冲洗干净后，切成1mm厚的薄片，装入玻璃瓶中，用0.1％的过氧乙酸消毒灭菌后，加入无菌水置于摇床震荡洗涤，洗至澄清后加入1％的SDS溶液震荡洗涤24h，每三小时换一次液，洗至组织变白。随后再用无菌水洗净去除残留的SDS，接着放入含有50U/mL的DNA酶I和1U/mL的RNA酶的Tris-HCL缓冲液中，在37℃，100rpm摇床上震荡24h去除组织中的DNA和RNA。最后用无菌水洗涤除去酶，得到猪肝脏细胞外基质材料。
猪肝脏细胞外基质组织纸(LTP)的制备：称取30g湿重的猪肝脏细胞外基质材料，放入匀浆机器中搅拌5min得到匀浆液，然后将匀浆液倒入直径为10cm的200目的平底滤网中静置，滤出水分后室温晾干10h。随后放入-20℃冰箱冷冻12h后，转移到冷冻干燥机中冻干36h，得到猪肝脏细胞外基质组织纸。可通过调节猪肝脏细胞外基质的用量制备具有不同厚度的猪肝脏组织 纸。
组织纸交联：取上述冻干的猪肝脏细胞外基质组织纸，置于含有质量比为4：1的EDC/NHS的80％的乙醇溶液中，于4℃交联12h，取出后用无菌水浸泡洗涤5次，去除残留的交联试剂。
冷冻干燥：将上述交联洗净的猪肝脏细胞外基质组织纸置于-80℃冷冻4小时，于冷冻干燥机中冻干36h，得到力学增强的猪肝脏细胞外基质组织纸成品，可进一步将多张猪肝脏组织纸叠加并压实后备用。
实施例3猪肾脏组织纸的制备
脱细胞肾脏组织的制备：将购买的新鲜猪肾冲洗干净后，切成小的1mm厚的薄片，装入玻璃瓶中，用0.1％的过氧乙酸消毒灭菌后，加入无菌水置于摇床震荡洗涤至澄清后加入1％的SDS溶液震荡洗涤48h，每三小时换一次液，洗至组织发白。随后再用无菌水冲洗去除残留的SDS，接着用添加DNA酶和RNA酶的Tris-HCL缓冲液在37℃条件下清洗过夜，去除组织中的DNA和RNA，最后用无菌水洗净得到猪肾脏细胞外基质材料。
猪肾脏细胞外基质组织纸(KTP)的制备：称取20g湿重的猪肾脏脱细胞组织，加入200mL蒸馏水放入匀浆机器中搅碎10min得到匀浆液，然后倒入10cm直径的200目的平底滤网中静置，滤出水分后室温晾干72h，随后放入-20℃冰箱冷冻12h，最后转移到冷冻干燥机中冻干48h，得到猪肾脏细胞外基质组织纸。
组织纸交联：首先配置80％的乙醇溶液100mL，加入质量比为4：1的EDC和NHS，配成0.3％的EDC浓度，得到交联溶液。然后将肾脏细胞外基质组织纸浸入溶液中室温交联4h，随后取出用无菌水浸泡清洗数次，去除残 留的交联试剂。
冷冻干燥：将交联后洗净的组织纸于-80℃冰箱冷冻4小时，而后在冷冻干燥机中冻干48小时，得到力学强度较高的猪肾脏细胞外基质组织纸成品。也可进一步将多张猪肾脏细胞外基质组织纸叠加并压实后备用。
实施例4跟腱组织纸的制备
脱细胞跟腱组织的制备：取新鲜猪跟腱组织，剔除周围的结缔组织后切割成约500μm厚度的薄片，用0.1％的过氧乙酸消毒灭菌后，无菌水洗净，浸没在液氮中2min，然后放入400mL生理盐水中于37℃在100rpm摇床震荡15min，如此反复冻融5次。为去除残余细胞核，最后一次洗涤后，将样品放入含有50U/mL的脱氧核糖核酸酶I和1U/mL的核糖核酸酶A的反应液中，于37℃，100rpm摇床上反应24h。随后用无菌PBS洗涤以去除酶液，得到脱细胞跟腱组织。
猪跟腱细胞外基质组织纸(TTP)的制备：称取50g湿重的猪肌腱脱细胞组织，放入匀浆机中搅碎30min得到匀浆液，然后倒入直径为10cm的200目的平底滤网中静置，滤出水分后室温晾干36h，随后液氮速冻后于冷冻干燥机中冻干24h，得到跟腱细胞外基质组织纸。
组织纸交联：取上述冻干的猪跟腱细胞外基质组织纸，置于含有质量比为4：1的EDC/NHS的80％的乙醇溶液中，于4℃交联12h，第二天取出后用无菌水浸泡洗涤数次，去除残留的交联试剂。
冷冻干燥：将上述交联洗净的跟腱细胞外基质组织纸于-80℃冷冻4小时，而后快速转移到预冷的冷冻干燥机中冻干36小时，得到猪跟腱细胞外基质组织纸成品，也可进一步将多张猪跟腱细胞外基质组织纸叠加并压实后备用。
对比例1
与实施例1的区别在于，将猪心脏脱细胞基质材料先进行乙酸和胃蛋白酶溶解消化处理后，再进行组织交联。
对比例2
与实施例1的区别在于，缺少脱细胞基质组织纸制备过程。
效果例1
以猪心脏组织为例，通过蛋白组学分别检测了猪源天然心脏组织、脱细胞心脏组织(对比例2)、脱细胞心脏组织纸(实施例1)以及用乙酸和胃蛋白酶溶解消化处理后的脱细胞心脏组织(对比例1)样品中的蛋白质种类数及肽段丰度。
检测方法：
四种不同处理的心脏组织样品的基质成分组成我们使用蛋白质组学方法进行了检测。蛋白质鉴定项目流程的主要步骤包括：蛋白质酶解、LC-MS/MS分析、数据库检索、数据分析等。该项测试依托上海拜谱生物科技有限公司完成并取得实验结果报告。实验结果如图1和图2：
图1为不同ECM处理方式后的蛋白质种类统计图，A，不同处理样品的蛋白质种类韦恩图；B，不同处理方式后的ECM样品中蛋白质种类数的统计结果；其中a,b,c,d分别表示天然心脏组织、脱细胞心脏组织、脱细胞心脏组织纸以及乙酸和酶消化的脱细胞心脏组织。图2为不同ECM处理方式后的心脏组织样品的质谱峰图；A，天然心脏组织；B，脱细胞心脏组织；C，脱细胞心脏组织纸；D，酸溶酶解后的脱细胞心脏组织。
根据图1可以看出，脱细胞心脏组织纸中的蛋白种类的数量略低于天然 心脏组织，与脱细胞心脏组织差异较小，说明本发明中脱细胞ECM组织纸的制备工艺能够很好地保留原始ECM组织组分。而酸溶酶解的处理方法使脱细胞心脏组织中的蛋白质种类数减少了约900种，极大地破坏了ECM材料组分。根据图2可知，天然心脏组织、脱细胞心脏组织和脱细胞心脏组织纸的肽段峰基本一致，说明蛋白质组分相差不大，酸溶酶解的样品中峰位置及峰强度均显著区别于天然组织。由此可得，与传统的酸溶酶解的加工方法相比，本发明脱细胞基质组织纸的制备工艺能够更好地保留天然细胞外基质组分。
效果例2
检测本发明实施例1-4及按照对比例2常规脱细胞基质制备方法制得的脱细胞基质产品(心脏基质、肝脏基质、肾脏基质和跟腱基质)的结构形貌表征。
检测过程：
我们对制备的四种组织纸和四种常规脱细胞基质的宏观形貌进行了拍照观察，并在表面喷金后，在15kV的加速电压下，使用扫描电子显微镜(SEM)对组织纸表面及脱细胞基质产品的微观结构进行了细致的拍照记录。最后使用Image J软件对组织纸的孔径进行了测量，利用液体浸入法测定组织纸材料的孔隙率。
图3A为实施例1-4制得的脱细胞基质组织纸和常规脱细胞基质产品的宏观形貌及微观结构图片；B，四种脱细胞基质组织纸和四种常规脱细胞基质表面多孔结构的孔径统计图；C，四种脱细胞基质组织纸和四种常规脱细胞基质的孔隙率统计图。
由图3可见本发明实施例1-4制备出的组织纸在宏观形貌上与“纸”十 分接近，而在微观结构上具有丰富的多孔结构，且不同组织来源的组织纸均具有大孔径及较高的孔隙率，有利于细胞浸润及促进组织再生。而按照对比例2方法制备的脱细胞基质材料微观结构上较为致密，且孔径较小及孔隙率比较低，会限制细胞迁移和组织再生。
效果例3
本发明制得的脱细胞基质组织纸的性能评价
1.力学性能评价
检测方法：
将组织纸裁剪成3cm×1cm大小的矩形形状，用单轴力学拉伸测量仪对组织纸的力学性能进行测试。设置拉伸参数为10mm/min，对组织纸进行纵向拉伸，得到拉伸应力及应力应变曲线。缝合强度测试：裁剪长度为2cm的组织纸，将其一端于拉力测试机一侧固定，另一端在距离组织纸末端2mm处插入6-0缝合线，将其打结后固定在拉力机另一侧，以10mm/min的速度拉伸直至组织纸断裂，得到的最大载荷即为组织纸的缝合强度值。
图4为实施例1-4组织纸的力学性能评价，A，四种组织纸的应力应变曲线；B，四种组织纸的拉伸强度统计；C，四种组织纸的缝合强度统计。
从图4可以看出，四种不同组织来源的组织纸均具有较高的力学强度，最大应力均在1MPa以上，其中肝脏组织纸力学强度最高，另外也都具有足够的缝合强度。因此，制备得到的组织纸能够很好地满足体内应用时的力学和缝合强度要求。
2.细胞相容性评价
检测方法：
将4×10 4个干细胞种植于48孔板中的组织纸膜片上，培养24h后，吸去培养液，加入Calcein AM和PI死活染色液，室温避光孵育20min，用共聚焦显微镜观察拍照，并统计死活细胞数量及存活率。
细胞增殖实验中，我们首先将0.1g组织纸浸泡于5ml的培养基中孵育24h制备得到组织纸浸提液。随后将干细胞接种3×10 3个/孔到96孔板中，加入100μl的浸提液培养，分别在第1天、3天和第5天时间点取出孔板，加入10μl的CCK8工作液，37℃孵育2h，用酶标仪检测吸光度。
图5为四种脱细胞基质组织纸的细胞相容性，A，骨髓间充质干细胞在组织纸上培养1天后的细胞死活染色；B，细胞存活率统计图；C，CCK8检测干细胞在组织纸浸提液中的增殖情况。
由图5可见，提取的骨髓间充质干细胞在四种组织纸上均能良好地黏附铺展，细胞存活率在90％以上，表明组织纸具有很好的细胞相容性。另外，与空白培养基对照相比，组织纸浸提液培养显著促进了干细胞的增殖，说明组织纸具有很好的生物活性。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (10)

  1. 一种脱细胞基质组织纸的制备方法，其特征在于，包括以下步骤：
    (1)制备脱细胞基质材料：取新鲜组织清洗消毒后进行脱细胞处理，获得脱细胞基质材料；
    (2)制备组织纸：将所述脱细胞基质材料进行匀浆搅拌，之后将匀浆液置于平底滤网滤出水分，再将过滤后的匀浆液静置晾干，然后进行冷冻干燥，即得所述组织纸；
    (3)组织纸交联：将步骤(2)获得的所述组织纸置于交联溶液中进行交联；
    (4)将交联后的组织纸进行冷冻干燥或进行叠加压实，即得所述脱细胞基质组织纸。
  2. 根据权利要求1所述的制备方法，其特征在于，所述脱细胞处理为：将新鲜组织洗净后切片，用过氧乙酸消毒后加入无菌水或生理盐水洗涤，之后用含有DNA酶和RNA酶的缓冲液清洗洗涤后的组织。
  3. 根据权利要求1所述的制备方法，其特征在于，步骤(2)中，所述匀浆搅拌时间为1-60min，所述平底滤网尺寸为3-500目，所述静置晾干时间为1-120h，所述冷冻干燥温度为-196℃～-20℃，时间为4-48h。
  4. 根据权利要求1所述的制备方法，其特征在于，步骤(3)所述交联溶液为含有交联剂的乙醇溶液；所述交联剂包括1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、N-羟基琥珀酰亚胺、戊二醛、甲醛和京尼平中的一种或多种。
  5. 根据权利要求1所述的制备方法，其特征在于，所述交联条件为4℃-27℃下交联4-12h。
  6. 根据权利要求1所述的制备方法，其特征在于，步骤(4)所述冷冻干燥温度为-196℃～-20℃，时间为12-72h。
  7. 根据权利要求1所述的制备方法，其特征在于，所述新鲜组织包括人体或动物的组织或器官。
  8. 根据权利要求7所述的制备方法，其特征在于，所述新鲜组织包括脑、心脏、肝脏、脾脏、肺、肾、肌肉、皮肤、脂肪、脑膜、膈膜、羊膜、心包膜、心脏瓣膜、小肠粘膜下层、肌肉、血管、肌腱、韧带、软骨、食道、气管、胃、神经、膀胱、角膜和/或胎盘。
  9. 一种根据权利要求1-8任一项所述的制备方法制得的脱细胞基质组织纸。
  10. 一种权利要求9所述的脱细胞基质组织纸在制备组织损伤修复药物中的应用。
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