WO2019056252A1 - 一种高生物活性的细胞外基质材料及其制备方法和应用 - Google Patents

一种高生物活性的细胞外基质材料及其制备方法和应用 Download PDF

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WO2019056252A1
WO2019056252A1 PCT/CN2017/102661 CN2017102661W WO2019056252A1 WO 2019056252 A1 WO2019056252 A1 WO 2019056252A1 CN 2017102661 W CN2017102661 W CN 2017102661W WO 2019056252 A1 WO2019056252 A1 WO 2019056252A1
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extracellular matrix
matrix material
perfusion
biologically active
preparing
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PCT/CN2017/102661
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English (en)
French (fr)
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张剑
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卓阮医疗科技（苏州）有限公司
上海卓阮医疗科技有限公司
中国人民解放军第二军医大学
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Priority to PCT/CN2017/102661 priority Critical patent/WO2019056252A1/zh
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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
    • 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
    • A61L27/58—Materials at least partially resorbable by the body

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  • the invention belongs to the field of biological scaffold materials, and particularly relates to a high biological active extracellular matrix material and a preparation method and application thereof.
  • the extracellular matrix (ECM) biocomposite prepared by decellularization technology is an important development direction of soft tissue repair materials.
  • Related products are widely used in various wounds abroad, such as replacing the meninges, pleura, blood vessels, periosteum, pericardium, joint capsules, etc., liver and spleen and other solid organs are damaged and stuffed to stop bleeding, anastomotic stoma, bladder suspension, pelvic floor reconstruction And a variety of complex fistula and abdominal wall defects such as contaminated abdominal wall defects, infection after implantation of synthetic patches, secondary surgical treatment of intestinal fistula, prevention of parastomal hernia.
  • ECM is an integral part of all tissues and organs, maintaining the three-dimensional structure of various tissues and organs and storing nutrients, supporting the survival, activity and change of cells.
  • ECM has many advantages as a tissue repair and regeneration scaffold material: 1 structure and composition are close to nature, and biocompatibility is good: the main components of ECM include structural proteins (mainly type I and type III collagen fibers, containing a small amount of type IV and Type VI collagen fibers, the remainder are mainly glycoproteins, fibronectin, glycosaminoglycans (chondroitin sulfate, heparin sulfate, hyaluronic acid, proteoglycans, binding growth factors and retention water, etc.), growth factors and enzymes [Alkaline fibroblast production factor (bFGF)-2, transforming growth factor (TGF)- ⁇ , insulin-like growth factor (IGF) and vascular endothelial growth factor (VEGF), liver growth factor (HGF), etc.]; 2ECM Retaining the
  • 3ECM has good degradability and controllability of degradation rate, and degradation is more in line with regeneration law; 4ECM has A certain porosity, easy to exchange between the nutrients and material air between the tissues; 5ECM has a certain mechanical strength to support the growth of the tissue.
  • ECM biomaterials can be classified into allogeneic materials such as Acellular dermis Matrix (ADM), Anmiotic membrane, Cerebral dura mater, and heterogeneous materials such as pig small intestine according to tissue sources.
  • ADM Acellular dermis Matrix
  • Anmiotic membrane Anmiotic membrane
  • Cerebral dura mater Cerebral dura mater
  • heterogeneous materials such as pig small intestine according to tissue sources.
  • Submucosa Small intestinal submucosa, SIS
  • porcine bladder basement membrane porcine dermis
  • porcine dermis pericardium of cattle and cattle
  • Peritoneum Peritoneum
  • tissue-derived ECMs such as the small intestine submucosa, the bladder basement membrane, and the amniotic membrane contain bioactive components such as glycoprotein, fibronectin, glycosaminoglycan, and growth factors in addition to structural proteins.
  • the products such as dermis, pericardium, and peritoneum are raw materials, and their source tissues are biologically inert tissues in the body, which contain almost only structural proteins, low levels of natural adhesion proteins, growth factors, and glycosaminoglycans.
  • ECM materials are prepared from the same species, heterogeneous tissue, decellularized to remove all cells, antigens, lipids, soluble proteins and other substances in the tissue, retaining intact appearance and histology and ultrastructure Insoluble extracellular matrix.
  • the development direction of the related preparation technology is to completely remove the cells while retaining the biological active components to the utmost extent, and reducing various complications caused by the materials such as serous swelling and inelasticity of the repairing area.
  • the decellularization methods of the existing sheet-like ECM materials are generally chemical descaling agents combined with other auxiliary reagent oscillation methods, that is, the sheet materials are placed in the treatment liquid and violently oscillated. This method has many disadvantages.
  • this step can be used to promote significant expansion of the extracellular matrix material (even up to 6 times the volume), increase porosity and surface uniformity, and reduce lipid content.
  • the biologically active components in the extracellular matrix such as growth factors, glycoproteins, glycosaminoglycans, proteoglycans and the like are depleted.
  • Some manufacturers often supplement the biologically active ingredients by spraying, soaking, immersing, etc. in the subsequent steps. The operation of the oscillating process is cumbersome.
  • ECM bioremediation materials will directly affect the remodeling and repair effects in the body.
  • the decellularization protocol is very harsh, and the bioactive components will be lost (the retention of bioactive components such as fibronectin, growth factors and glycosaminoglycans is less than 30%), and only vascularized connectives can be induced after implantation.
  • the tissue fills the tissue defect and achieves anatomical repair.
  • the current clinical application of ECM materials in China including replacement of meninges, pleura, pelvic floor reconstruction, anal fistula repair, and treatment of various complex hernias and abdominal wall defects are all at this level.
  • the technical problem to be solved by the invention is to provide a high biological activity extracellular matrix material and a preparation method and application thereof, the material has good mechanical strength, complete decellularization, clean biological load such as virus, and is suitable for repairing various parts of the body.
  • Soft tissue defects including replacement of fascia, periosteum, meningeal pleura, joint capsule and other membranous tissue, filling and repairing substantial organ defects, covering various wounds such as mucosal defects, burn wounds, etc., injection of breast augmentation; and as a biological scaffold induction Functional tissue regeneration in various parts of the body.
  • the present invention provides a highly bioactive extracellular matrix material prepared by the combined use of ultra high pressure and intraluminal perfusion.
  • the invention also provides a method for preparing a highly biologically active extracellular matrix material, comprising:
  • the ultra-high pressure has a pressure value of 1-100 MPa, an operating temperature of 1-30 ° C, and a time of 1-120 minutes;
  • the raw materials in the step (1) include submucosa, serosal layer of the hollow organ of the intestine, esophagus, stomach or anterior stomach of human and mammal, skin or bladder basement membrane, dermis, pericardium, placental amnion or peritoneum .
  • the time taken should be within 4 hours of the donor's death, preferably within 30 minutes.
  • the raw material is placed in a stable buffer against infection, anti-oxidation and protease inhibition, preferably at a buffer temperature of 4 ° C or lower and maintained at a temperature not exceeding 4 ° C during transportation.
  • the raw materials can be repeatedly frozen and rewarmed.
  • the combined intraluminal and external perfusion in the step (1) is as follows: the liquid flow inside and outside the cavity is independent, and the type or pressure of the flowing liquid inside and outside the cavity can be different; the volume of the liquid outside the cavity exceeds the volume in the lumen and the extracellular matrix material is fully immersed. In the fluid outside the lumen.
  • hydrostatic pressure or no pressure is maintained outside the lumen.
  • the perfusion time in the step (1) is not more than 1 hour, preferably not more than 30 minutes, more preferably not more than 15 minutes.
  • the perfusion mode is cyclic or acyclic perfusion. After disinfection, PBS and deionized water were alternately perfused. The perfusion rate is updated every 15 minutes for the fluid inside and outside the lumen.
  • the pre-sterilization in the step (1) reduces the bioburden of the raw material to not more than 2 CFU/g, the endotoxin amount to not more than 20 EU/g, and the indicated virus amount to at least 6 logs.
  • the bioactive ingredient content is lost no more than 10%, more preferably no more than 5%, in this step.
  • the pressure value is 1-100 MPa, the preferred pressure value is 40-100 MPa; the high pressure working temperature is 1-30 ° C, and the working temperature is adjusted according to the pressure value, preferably 4 ° C; the high pressure treatment time 1-120 minutes, preferably 10-30 minutes. It may be before, during or after the steps of degreasing (optional step), washing out cell contents, removing antigen, and the like.
  • the perfusion liquid for washing out the cell contents in the step (3) is one or more of a phosphate buffer salt, a basic aqueous buffer salt, a DNA solubilizing buffer salt, purified water, and the like, and the washing time is 0.1- 10 hours. Preferably, it is about 0.5 hour, and a strong alkaline liquid such as NaOH is not used.
  • the perfusion liquid for removing the antigen in the step (3) is an ionic or nonionic surfactant; the removal time is not more than 0.5 hours.
  • the concentration is 0.005-0.1% sodium lauryl sulfate or 0.01-1% Triton-X, the surfactant treatment can assist degreasing and further reduce the DNA content.
  • Washing out the cell contents perfusion liquid, reducing the immunogenicity and blocking the antigen liquid can simultaneously inject and maintain the osmotic pressure inside and outside the cavity, and the preferred solution is to wash out the cell contents, perfusion liquid, high osmotic pressure, immunogenicity and low antigen-encapsulating liquid.
  • Osmotic pressure, the osmotic pressure inside and outside the lumen is 2-10X, preferably 2-4X.
  • the type of liquid can be exchanged inside and outside the lumen.
  • Degreasing is also included prior to sterilization in the step (3).
  • the defatting treatment can be carried out before, during or after the step of washing out the cell contents and removing the antigen.
  • the degreased perfusion liquid includes a mixture of one or more of methanol, ethanol, isopropanol and an organic solvent such as butanol, acetone, chloroform or the like. Ultrasonic oscillations can be assisted during processing.
  • the degreasing step determines if it is needed based on the end use of the product.
  • the perfusion treatment includes intraluminal perfusion and extraluminal perfusion.
  • the type and pressure of the flowing liquid inside and outside the lumen may be different (such as DNA solubilizing buffer salts, ionic or nonionic surfactants, etc.), and the pressure difference may be alternately used internally and externally.
  • the perfusion mode is cyclic or non-circulating perfusion, and the intraluminal and extraluminal perfusion systems are independent.
  • the volume ratio of the mass of the extraluminal fluid to the extracellular matrix is ⁇ 2:1 and the material is required to be completely immersed in the fluid outside the lumen.
  • the flow of liquid inside and outside the lumen is required to be updated every 15 minutes.
  • the full working temperature is 1-30 ° C, preferably 4 ° C.
  • the sterilization in the step (3) includes pre-sterilization using a full reactor system, full-sterilization liquid perfusion, perfusion of a disinfectant (alcohol, peroxide or another oxidizing or non-oxidizing disinfectant), One or more of ethylene oxide, supercritical CO 2 , plasma treatment, gamma ray radiation sterilization, and the like.
  • a disinfectant alcohol, peroxide or another oxidizing or non-oxidizing disinfectant
  • Sterilization can assist in decellularization, immunogenicity and blocking of antigens, but sterilization can result in the loss of bioactive components, with losses ranging from about 20% to 100%. Different sterilization methods are selected for different target uses.
  • the perfusion reactor is filled with degreasing, washing out the contents of the cells, removing the antigen, disinfecting and sterilizing, and instilling and cleaning during the operation steps or during the exchange of liquid inside and outside the lumen to remove the introduced chemical residues remaining in the material or on the material. .
  • Including repeated alternating perfusion of deionized water, 1X phosphate buffer salt or saline, can assist in ultrasonic oscillation.
  • the preservation in the step (3) includes preservation in a freeze-drying preservation or preservation solution.
  • the bioactive component such as the basement membrane component, the fibronectin, the growth factor, the glycoprotein, the glycosaminoglycan or the proteoglycan in the extracellular matrix material obtained in the step (3) accounts for nearly 5% of the dry weight of the extracellular matrix.
  • the content of the above components in the dry weight of the natural extracellular matrix is close.
  • the biosafety of the material is equivalent or superior, and the biological activity is significantly higher than that obtained by mechanical oscillating, chemical reagent or biological reagent decellularization.
  • the invention also provides for the use of a highly biologically active extracellular matrix material for the preparation of microparticles, fluidized compositions, gels or active peptides.
  • extracellular matrix material and the synthetic high molecular polymer fiber network constitute a "sandwich” structure "bio-polymer” composite biological patch
  • non-crosslinked and cross-linked extracellular matrix material constitutes a "sandwich” structure repair area. Long-term stable, no-elastic biological patch.
  • perfusion is achieved with one-tenth of the chemical concentration and one-half of the processing time.
  • the mechanical oscillating method removes DNA and blocks the antigen while retaining a higher bioactive content.
  • all the processes of the perfusion method can be completed by computer control.
  • other steps can be programmed, and it is not necessary to manually unwind the membrane-like ECM as in the mechanical oscillation method. Material entanglement, liquid change, suitable for industrial production.
  • ultra high pressure The full name of biological high-pressure treatment technology is high hydrostatic pressure, referred to as ultra high pressure (UHP), which is the pressure transmitted by fluid medium.
  • UHP ultra high pressure
  • the non-common bonds such as hydrogen bonding, hydrophobic bonding, and ionic bonding in the stereostructure of biopolymers have changed, and studies have confirmed that the pressure is higher than 40 MPa (the Von-Mises stress at the cell surface will reach 6.5 MPa). It will cause cell membrane rupture and nuclear structure destruction, including intracellular components including nuclear composition; from the histological observation of ECM materials treated with ultra-high pressure, ultra-high pressure treatment can significantly deform and break cells in ECM materials.
  • the nucleus of the residual cells is convergent and chromatin staining. Under this pressure, bioactive components such as glycosaminoglycans and growth factors do not change, and various short peptide components that are important for maintaining stem cell structure and function in ECM are not lost. Ultra-high pressure treatment will also enhance the biomechanics of the extracellular matrix.
  • the present invention combines the use of ultra-high pressure and endoluminal perfusion to prepare high bioactive ECM, because the structure and components are closer to natural and the degradation is more in line with the regeneration law, retaining the "tissue specific" microenvironment, so after implantation Effective integration with the residential area, actively attracting semi-specialized autologous stem cells to migrate from other parts of the body and promote proliferation and differentiation to form the original specific tissue of the lesion, achieving partial degree of functional recovery;
  • the extracellular matrix obtained by the invention by mechanical demagnetization, chemical reagent or biological reagent method significantly increases the adhesion of fibronectin, growth factors, proteoglycans, glycoproteins, and glucosamine groups.
  • biological active ingredients such as sugars; at the same time, the mechanical strength is retained, the cells are completely removed, and the biological load such as viruses is cleaned; industrial production is convenient; Diversified, including membranous products, particles of various diameters, gels and active peptides; the products are suitable for repairing soft tissue defects in various parts of the body, including membranous tissues such as fascia, periosteum, meninges, and joint capsules. Filling and repairing substantial organ defects, covering various types of wounds such as mucosal defects, burn wounds, etc., injection of breast augmentation; and as a biological scaffold to induce functional tissue regeneration in various parts of the body.
  • Figure 1 is a side view of the "perfusion-high pressure-perfusion" automatic decellularization reactor used in the present invention
  • Figure 2 is a derivative product of the present invention, wherein the left image is a microparticle; the right image is a gel;
  • Figure 3 is a flow chart of preparing an active peptide of the present invention.
  • Figure 4 is a staining diagram of the stromal tissue of the present invention; wherein A is Movat's staining (100 times); B is picric acid-Sirius red staining (40 times); C is Verhoeff-Van Gieson staining (100 times); D is Herovici staining (40 times);
  • Figure 5 is a test for chemotaxis of cells.
  • Raw material acquisition Large pigs kept closed by plant source feed, weighing more than 150 kg, and taking out the jejunum half an hour after death. After rinsing in the cavity, it was placed in the long-term No.-1 organ preservation solution at 2 °C, and the temperature was maintained at 4 °C during transportation.
  • Pre-sterilization pig jejunal access to perfusion reaction rod, intracavitary circulation of 0.1% peroxyacetic acid / 4% ethanol mixed disinfectant, perfusion rate of 100ml / min / m, while external cavity soaked in 0.1% peracetic acid / 4% Ethanol mixed disinfectant, treatment time 5 minutes. It has been confirmed that the porcine capsular virus can be completely inactivated after 5 minutes of treatment, the bioburden is reduced to no more than 2 CFU/g, the endotoxin amount is not higher than 20 EU/g, and the indicated virus amount is reduced to 100 PFU/g. bFGF is here. The loss in one step does not exceed 10%.
  • the pig jejunum was prepared using a general-purpose pig casing machine (3-U-400 Stridhs type, AB Stridhs Maskiner, Sweden). Wash PBS and deionized water alternately.
  • Ultra-high pressure treatment The obtained pig jejunum SIS is placed in a soft plastic bag, sealed, placed in a closed ultra-high pressure container, and then subjected to a pressure of 60 MPa through a fluid medium (deionized water) at an operating temperature of 4 ° C.
  • the high pressure treatment time is 15 minutes.
  • the specific scheme is to increase the pressure to 40 MPa, stay for 5 minutes, and then increase the pressure to 60 MPa for 15 minutes.
  • the perfusion reactor was connected, and PBS and deionized water were alternately perfused for 15 minutes each.
  • Degreasing treatment reversed intraluminal and external perfusion 50%, 70%, 90%, 100%, ethanol for 15 minutes, circulating perfusion, intracavity pressure is higher than the cavity, intraluminal perfusion rate 100ml / min / m, auxiliary 170kHz Ultrasonic oscillations. After the end, deionized water was perfused for 15 minutes to proceed to the next step.
  • the cell contents were washed out: the DNA solubilized buffer salt was reversely perfused inside and outside the lumen for 30 minutes, non-circulating perfusion, the intracavity pressure was higher than the extraluminal cavity, and the intraluminal perfusion rate was 100 ml/min/m, which assisted the ultrasonic oscillation of 170 kHz. After the end, deionized water was perfused for 15 minutes to proceed to the next step.
  • Antigen blocking 0.005% sodium lauryl sulfate was reversely perfused inside and outside the lumen for 30 minutes, non-circulating perfusion, the intracavity pressure was higher than the extraluminal cavity, and the intraluminal perfusion rate was 100 ml/min/m. After the end, deionized water and 1X PBS were alternately perfused for 2 rounds, each time for 15 minutes, and the next step was taken.
  • the porcine jejunal SIS matrix prepared in this example can be derived from medical products such as microparticles, fluidized compositions, gels and active peptides, as shown in FIG.
  • the microparticle product is obtained by vacuum-drying the dried body of the porcine jejunal SIS matrix by high-speed rotation, and includes a plurality of product grades ( ⁇ 38 ⁇ m, 38-100 ⁇ m, 100-250 ⁇ m), and can be used as a wound covering, a defect filling material and the like.
  • the fluidized composition is obtained by pepsin digestion of porcine jejunal SIS matrix microparticles in an acidic environment.
  • the gel product is obtained by re-concentrating and neutralizing the uniform fluidized composition, including different concentration levels, and can be used for skeletal muscle wound covering, filling skeletal muscle defect for injection, and treating muscular dystrophy. Techniques for preparing the above derivatives are well known to those skilled in the art.
  • the preparation of the porcine jejunal SIS matrix active peptide according to the embodiment as shown in FIG. 3, after ultracentrifugation ( ⁇ 13000 rpm) of the porcine jejunal SIS matrix fluidized composition, the supernatant is vacuum freeze-dried, and the obtained dried body is obtained.
  • an active peptide of the porcine jejunal SIS matrix in vitro tests demonstrated that its ability to promote chemokinegic melanocyte chemotaxis, proliferation and differentiation is much more efficient than the porcine jejunal SIS matrix fluidization composition.
  • Biosafety measurement endotoxin content was 0.25 EU/g, bioburden, fungus, virus detection were 0, no SDS residue was detected, IgA content was 0.1 ⁇ g/g, and lipid content was 0.5%.
  • Pig jejunal SIS stromal tissue staining As shown in Figure 4, HE staining, Movat's staining, Verhoeff-Van Gieson staining, VVG staining, Herrovici staining showed no nuclear structure, structural integrity, mainly type I and type III collagen fibers, with a small amount Elastin, type IV and type VI collagen fibers containing glycosaminoglycan/proteoglycan components.
  • the content of GAGs in the dry weight of SIS of pig jejunum was 2745 ⁇ 540 ⁇ g/gg
  • the content of VEGF was 36 ⁇ 22ng/g
  • the content of bFGF was 393 ⁇ 230ng/g
  • the content of TGF- ⁇ 1 was 17 ⁇ . 8ng/mg.
  • the prepared porcine jejunal SIS/UBM matrix fluidized composition was neutralized at different concentrations (1000 ⁇ g/ml, 500 ⁇ g/ml, 250 ⁇ g/ml, 100 ⁇ g/ml). , 50 ⁇ g/ml, 25 ⁇ g/ml, 10 ⁇ g/ml, 5 ⁇ g/ml) Comparing the chemotaxis of fetal bovine serum to skeletal muscle myoblasts and MJK/PJK (Boyden chamber method), it was found that the jejunal SIS matrix had obvious stem cell chemotaxis and reached the highest concentration at 25 ⁇ g/ml. Good effect.
  • the total weight of the extracellular matrix component in the dry weight of natural pig SIS was 780.67 mg, and it became 622.13 mg after decellularization.
  • ECM collagen and proteoglycan form a fibrous network complex on the cell surface, which is directly linked to cell surface receptors by fibronectin or laminin and other connecting molecules; or attached To the receptor. Since the receptor is mostly a membrane integrin and is linked to the intracellular matrix protein, the extracellular matrix connects the extracellular and intracellular cells through a membrane integrin.
  • Collagen I/III The main source of ECM biomechanics. Collagen I/III consists of ⁇ 1, ⁇ 2, ⁇ 3 peptide chains, which can bind to specific receptors on the cell surface, activate signal transduction pathways, promote cell adhesion, proliferation and differentiation, and a large number of hydroxyl groups. Can promote cell adhesion. Collagen is involved in cell differentiation, controls cell adhesion, regulates cell growth, and helps maintain the cytoplasmic structure of the cell matrix.
  • Elastin An important structural protein that constitutes an elastic fiber network within the ECM to provide elasticity, texture, durability, and retraction after elongation. Extensive cross-linking of soluble elastinogen monomers forms a broad covalent array that confers protein elasticity. Due to its extensive lysine cross-linking, elastin is particularly insoluble and therefore difficult to quantify in biological samples.
  • Collagen IV An important component in the basement membrane that promotes cell adhesion and proliferation.
  • Collagen VII the main structural components of the basement membrane and connective tissue
  • GAGs including chondroitin sulfate / dermatan sulfate / keratan sulfate / heparin / heparan sulfate / hyaluronic acid. GAGs bind to growth factors and cytokines and control the moisture retention and gel properties within the ECM. The heparan-binding properties of many cell surface receptors and many growth factors (eg, FGF family, VEGF) make heparan-rich GAG an ideal component for tissue repair scaffolds.
  • Proteoglycan is a glycoprotein that is heavily glycosylated: the basic proteoglycan unit consists of a "core protein" with one or more covalently bound glycosaminoglycan chains. .
  • Fibroblast growth factor-2 bFGF stimulates angiogenesis and neurite outgrowth; increases endothelial cell viability, promotes proliferation, increases plasminogen activator (PA) and matrix metalloproteinase 1 (MMP1) Secretion; increase the chemotaxis of endothelial cells.
  • bFGF stimulates most cell proliferation associated with wound healing, including vascular endothelial cells, fibroblasts, smooth muscle cells, osteoblasts, and chondrocytes, which stimulate the proliferation and migration of risky vascular endothelial cells and create other vascular regenerative growth. conditions of.
  • Submucosal tissue is isolated from the small intestine and minimally invasive treatment, for example, only by rinsing, the FGF-2 content can exceed 100, or even potentially higher levels, such as 200 or 400 ng/g dry weight.
  • VEGF Vascular Endothelial Cell Growth
  • HGF Liver growth factor
  • Epidermal growth factor promotes mitosis of fibroblasts, smooth muscle cells, and keratinocytes, which is important for re-epithelialization.
  • TGF- ⁇ 1 Transforming growth factor (TGF- ⁇ 1): regulates the ECM component produced by fibroblasts and inhibits the proliferation of certain cells. TGF ⁇ 1 can promote the differentiation and proliferation of mesenchymal cells, increase the synthesis of type I collagen, osteonectin and osteopontin, and regulate the transcription of various matrix proteins, including collagen, fibronectin, glucosamine, matrix-degrading protease and Its grafts, etc., ultimately increase the accumulation of matrix proteins.
  • SDF-1 Stromal Cell Derived Factor
  • Fibronectin involved in tissue repair, embryogenesis, blood coagulation and cell migration/adhesion, promoting and directing the attachment of cells and extracellular matrices
  • Connexin A key role in regulating cell development, differentiation, and migration, promoting and directing the attachment of cells and extracellular matrices.

Abstract

一种高生物活性的细胞外基质材料及其制备方法和应用，将原材料通过联合使用超高压和腔内外灌注制备而得。制备方法主要包含：原材料的获取和准备、前置处理；细胞外基质的机械分离；超高压破碎和脱细胞；洗出细胞内容物、清除抗原；最终消毒灭菌和保存。较以机械振荡法、化学试剂或生物试剂法脱细胞所获得的细胞外基质明显提高了粘连蛋白类、生长因子类、蛋白聚糖类、糖蛋白类、葡糖胺基聚糖类等生物活性成分的保留量；同时力学强度保留好、脱细胞彻底，病毒等生物负载清除干净；工业化生产方便。

Description

一种高生物活性的细胞外基质材料及其制备方法和应用 技术领域
本发明属于生物支架材料领域，特别涉及一种高生物活性的细胞外基质材料及其制备方法和应用。
背景技术
脱细胞技术制备的细胞外基质(Extracellular matrix，ECM)成分生物补片是软组织修复材料的重要发展方向。相关产品在国外被广泛的用于各种创面，如代替脑膜、胸膜、血管、骨膜、心包、关节囊等，肝脾等实质脏器破损填塞止血、吻合口加强、膀胱悬吊、盆底重建以及各种复杂疝和腹壁缺损的治疗如伴有污染的腹壁缺损、合成补片植入后感染、肠瘘二次手术治疗、造口旁疝预防等。
ECM是所有组织器官的组成部分，维持着各种组织器官的三维结构和储存营养物质，支撑着细胞赖生存、活动和变化。ECM作为组织修复和再生支架材料具有较多优势：①结构和组分接近自然，生物相容性佳：ECM的主要成分包括结构蛋白(主要为I型和Ⅲ型胶原纤维，含少量Ⅳ型和Ⅵ型胶原纤维，其余部分主要为糖蛋白、纤粘蛋白、糖胺聚糖类(硫酸软骨素、硫酸肝素、透明质酸、蛋白多糖、结合生长因子和储留水等)、生长因子与酶类[碱性成纤维细胞生成因子(bFGF)-2、转化生长因子(TGF)-β、胰岛素样生长因子(IGF)和血管内皮生长因子(VEGF)、肝脏生长因子(HGF)等]；②ECM保留“组织特异性”微环境，能够维持生长其上的细胞形态和表型、增进细胞的粘附、增殖和分化：以骨骼肌ECM为例，其ECM高IV型胶原含量(骨骼肌基底膜的主要成分)，具有复杂的、具有启动功能的三维超微结构，包括平行排列的基底膜(肌内膜管)结构、残存的神经和血管通路，利于成肌细胞粘附、迁移、激活和融合[21][22][23]，且其降解产物对维持成肌细胞和卫星细胞的活力非常重要。③ECM有良好的可降解性和降解速率可控性，降解更符合再生规律；④ECM具有一定的孔隙率，便于组织间的营养和物质空气的交换；⑤ECM具有一定的机械强度能够对组织的生长起到支持作用。
目前临床常用的ECM生物材料根据组织来源可分为同种异体材料如人尸真皮(Acellular dermis Matrix,ADM)、羊膜(Anmiotic membrane)、硬脑膜等(Cerebral dura mater)，异种异体材料如猪小肠粘膜下层(Small intestinal submucosa,SIS)、猪膀胱基底膜、猪真皮、牛马的心包(Pericardium)、猪牛腹膜(Peritoneum)等。国内外当前已有数十种的商品化的生物补片。需要指出的是，小肠粘膜下层、膀胱基底膜、羊膜等组织来源的ECM，其组织成分中除了结构蛋白外，还含有糖蛋白、纤粘蛋白、糖胺聚糖、生长因子等生物活性成分，而真皮、心包、腹膜等为原材料的产品，因其来源组织属机体内生物惰性组织，几乎仅含结构蛋白，天然粘连蛋白、生长因子和糖胺聚糖类等含量低。
ECM材料的制备均是将取自同种、异种的组织，经脱细胞处理去除组织中的所有细胞、抗原、脂质、可溶性蛋白质等物质、保留下具有完整外观形态和组织学及超微结构的不溶性细胞外基质。
相关制备技术发展的方向即是彻底脱细胞的同时最大限度保留生物活性成分、并减少因材料引起的各种并发症如浆液肿、修复区失弹性等。已有的片状ECM材料的脱细胞方法一般都是化学除垢剂联合其他辅助试剂振荡法，即将片状材料置于处理液中剧烈震荡摇摆。这一方法存在较多缺点，振荡过程中极易导致片状的细胞外基质材料纠结而致使不能全部得到彻底的脱细胞处理，部分细胞外基质材料片段上的内毒素、核酸含量以及生物负载如真菌、病毒等灭活不充分，植入引起较高水平的炎症反应、降低材料重塑效果；此外，振荡洗出DNA、抗原等物质需要较长的处理时间、生物活性成分丢失多，如细胞外基质制备过程中都经历了碱处理，这一步骤虽然可用于促进细胞外基质材料的显著扩张(体积增加甚至达到6倍)、提高了孔隙率和表面的均匀性、减少脂质含量，但同时也几乎全部耗尽了细胞外基质中生物活性成分如生长因子、糖蛋白、葡萄糖胺聚糖、蛋白聚糖等。部分厂家常在之后步骤中，再次通过喷雾、浸泡、沉浸等方法补充生物活性成分。振荡工艺过程操作繁琐。
ECM生物修补材料携带生物活性成分的多少将直接影响其体内重塑和修复效果。脱细胞方案十分严酷，生物活性成分将丢失殆尽(粘连蛋白、生长因子和糖胺聚糖类等生物活性成分含量保留量低于30％)，植入后仅可诱导再生出血管化的结缔组织填充组织缺损、实现解剖层面的修复。中国目前临床上已有的关于ECM材料的应用，包括替代脑膜、胸膜，盆底重建、肛瘘修补以及各种复杂疝和腹壁缺损的治疗等均属于这一水平。
发明内容
本发明所要解决的技术问题是提供一种高生物活性的细胞外基质材料及其制备方法和应用，该材料力学强度保留好、脱细胞彻底，病毒等生物负载清除干净；适用于修复机体各个部位的软组织缺损，包括替代筋膜、骨膜、脑膜胸膜、关节囊等膜状组织，填充修复实质性脏器缺损、覆盖各类创面如粘膜缺损、烧伤创面等，注射隆胸；以及做为生物支架诱导体内各部位功能性组织再生等。
本发明提供了一种高生物活性的细胞外基质材料，将原材料通过联合使用超高压和腔内外灌注制备而得。
本发明还提供了一种高生物活性的细胞外基质材料的制备方法，包括：
(1)将原材料腔内灌注或腔内外灌注消毒液同时联合超声振荡预灭菌，随后进行机械分离；
(2)使用超高压破碎和脱细胞；其中，超高压的压力值为1-100MPa，工作温度1-30℃，时间为1-120分钟；
(3)灌注洗出细胞内容物、清除抗原，最后消毒灭菌、保存，得到高生物活性的细胞外基质材料。
所述步骤(1)中的原材料包括人和哺乳动物的肠、食管、胃或前胃等空腔脏器的粘膜下层、浆膜层，皮肤或膀胱基底膜、真皮、心包、胎盘羊膜或腹膜。
根据目标产品的不同用途选择不同年龄和体重的供体。年轻供体的细胞外基质内粘连蛋白、生长因子、蛋白聚糖、糖蛋白、葡糖胺基聚糖等生物活性成分含量显著高于高龄供体。大体重的供体能获得较大面积的细胞外基质，同一供体不同部位的细胞外基质用途有区别。
取材时间应于供体死亡后4小时内，优选30分钟内。取材后原材料置于抗感染、抗氧化、抑制蛋白酶的稳定缓冲液中，优选缓冲液温度4℃以下且运输过程中维持温度不超过4℃。原材料可反复冷冻、复温。
所述步骤(1)中的腔内外联合灌注具体为：腔内外液体流动各自独立，腔内外流动液体的种类或压力可不同；腔外液体的体积超过管腔内体积且细胞外基质材料全部浸于管腔外液体中。单纯腔内灌注时，腔外维持静水压或无压力。
所述步骤(1)中的灌注时间不超过1小时，优选不超过30分钟，更优不超过15分钟。
所述灌注方式为循环或非循环灌注。消毒后PBS和去离子水交替灌注冲洗。灌注速度为管腔内外液体每15分钟更新一遍。
所述步骤(1)中的预灭菌将原材料的生物负载量降至不高于2CFU/g、内毒素量降至不高于20EU/g、指示病毒量至少降低6logs。优选方案是生物活性成分含量在这一步骤中丢失不超过10％，更优的是不超过5％。
所述步骤(1)中的超高压处理，其压力值1-100MPa，优选的压力值40-100MPa；高压工作温度1-30℃，根据压力值调整工作温度，优选为4℃；高压处理时间1-120分钟，优选10-30分钟。可在脱脂(可选步骤)、洗出细胞内容物、清除抗原等步骤之前、之中或之后。
所述步骤(3)中的洗出细胞内容物的灌注液体为磷酸缓冲盐、碱性含水缓冲盐、DNA增溶性缓冲盐、纯化水等中的一种或多种，洗出时间为0.1-10小时。优选约0.5小时，不使用NaOH等强碱性液体。
所述步骤(3)中的清除抗原的灌注液体为离子型或非离子型表面活性剂；清除时间不超过0.5小时。如浓度0.005-0.1％的十二烷基硫酸钠或0.01-1％的Triton-X，表面活性剂处理同时可协助脱脂、进一步降低DNA含量。
洗出细胞内容物灌注液体、降免疫原性和封闭抗原液体可腔内外同时灌注但保持不同渗透压，优选方案是洗出细胞内容物灌注液体高渗透压、降免疫原性和封闭抗原液体低渗透压，管腔内外渗透压差别2-10X，优选2-4X。管腔内外可交换液体种类。
所述步骤(3)中消毒灭菌之前还包括脱脂。脱脂处理，可在洗出细胞内容物、清除抗原步骤的之前、之中或之后进行。
所述脱脂灌注的液体包括甲醇、乙醇、异丙醇和丁醇、丙酮、氯仿等有机溶剂中一个或多个的混合物。处理过程中可辅助超声振荡。脱脂步骤根据产品最终用途决定是否需要。
脱脂(可选步骤)、洗出细胞内容物、清除抗原处理，在灌注反应器进行。灌注处理包括管腔内灌注和管腔外灌注，管腔内外流动液体的种类和压力可不同(如DNA增溶性缓冲盐、离子或非离子型表面活性剂等)，可内外反复交替利用压力差产生透壁流动。灌注方式为循环或非循环灌注，管腔内外灌注系统各自独立。管腔外液体的质量与细胞外基质的体积比≥2:1且要求材料全部浸于管腔外液体中。管腔内外液体流量要求每15分钟全部更新一遍。全程工作温度1-30℃，优选为4℃。
所述步骤(3)中的消毒灭菌包括使用全反应器系统预灭菌、全程灭菌液体灌注、灌注消毒液(醇、过氧化物或另一种氧化性或非氧化性消毒剂)、环氧乙烷、超临界CO2、等离子体处理、γ射线辐射灭菌等方法中一种或多种。
消毒灭菌处理可辅助脱细胞、降免疫原性和封闭抗原，但灭菌会带来生物活性成分的丢失，丢失量范围约20％-100％。针对不同目标用途选择不同灭菌方式。
灌注反应器中灌注脱脂、洗出细胞内容物、清除抗原、消毒灭菌，各操作步骤间或管腔内外液体交换时均予以灌注清洗，以便除去保留在材料内或材料上的引进的化学残留物。包括反复交替灌注去离子水、1X的磷酸缓冲盐或生理盐水，可辅助超声振荡。
所述步骤(3)中的保存包括冷冻干燥保存或保存液中保存。
所述步骤(3)得到的细胞外基质材料中基底膜成分、粘连蛋白、生长因子、糖蛋白、葡萄糖胺聚糖或蛋白聚糖等生物活性成分占细胞外基质干重的比例接近5％，接近天然细胞外基质干重中上述成分的含量。该材料的生物安全性等同或优于、生物活性显著高于机械振荡法、化学试剂或生物试剂法脱细胞所获得的细胞外基质。
制备方法中全部操作步骤，除最初接入反应器需要人手操作外，全程处理可由生产线自动完成。
本发明还提供了一种高生物活性的细胞外基质材料的应用，所述材料应用于制备微粒、流体化组合物、凝胶或活性肽。此外还包括该细胞外基质材料与合成高分子聚合物纤维网构成“三明治”结构“生物-高分子”复合生物补片、非交联和交联细胞外基质材料构成“三明治”结构的修复区远期稳定无失弹性生物补片。
灌注法理论优势：相对于振荡法的不足①振荡过程中极易导致片状的细胞外基质材料纠结而致使不能全部得到彻底的脱细胞处理，②振荡洗出DNA、抗原等物质需要较长的处理时 间、生物活性成分丢失多，③工艺过程操作繁琐；腔内外联合灌注制备法处理膜状或管状细胞外基质材料，通过压力形成内腔、外腔等壁两侧可反复交替的透壁液体流动，以温和的方式持续不断的洗去DNA、抗原等物质。使用灌注可使用最少的液体流量、最短的处理时间即可脱脂、洗走细胞成分、清除抗原和消毒，如灌注法以十分之一的化学试剂浓度、二分之一的处理时间即获得与机械振荡法一样的DNA去除和封闭抗原效果，同时保留有更高的生物活性成分含量。此外，灌注法全部流程都可以使用电脑控制完成，除了最初将膜状ECM材料接入反应器需要人工操作，其他步骤均可程控完成，不需要像机械振荡法那样反复需人工解开膜状ECM材料纠结、换液，适合工业化生产。
超高压理论优势：生物高压处理技术的全称是超高冷等静压(high hydrostatic pressure)，简称为超高压(ultra high pressure,UHP)，是用流体介质传递压力。在超高压条件下，生物体高分子立体结构中的氢键结合、疏水结合、离子结合等非共有结合发生变化，研究证实高于40MPa(细胞表面的Von-Mises应力则会达到6.5MPa)的压力将致细胞膜破裂和细胞核结构破坏，包括细胞核组成在内的胞内成分外漏；从单纯超高压处理的ECM材料的组织学观察可以看到，超高压处理可使ECM材料中细胞明显变形及破碎，残留细胞的细胞核呈现同缩以及染色质浓染。而在这一压力下，糖胺聚糖、生长因子等生物活性成分不发生变化，ECM中各种对维持干细胞结构和功能十分重要的短肽成分等也没有损失。超高压处理还将提升细胞外基质的生物力学。
ECM经超高压处理后形态学的变化证实了通过超高压处理可以降低化学脱细胞试剂浓度或处理时间的理论基础。联合使用超高压和腔内外灌注，超高压破碎细胞膜、致细胞内容物漏出，腔内外灌注将细胞内容物洗出，透壁流动确保了洗出彻底。整个脱细胞过程彻底且温和，并较既往方法大幅减少了所用化学试剂的浓度和作用时间，更好的保留生物活性成分含量。
有益效果
(1)本发明联合使用超高压和腔内外灌注法制备高生物活性ECM，因为结构和组分更接近天然并且降解更符合再生规律、保留了“组织特异性”微环境，故植入后可与宿区有效整合，主动吸引半特化的自体干细胞从身体其他部位迁入并促进增殖和分化而形成损伤处原有的特异性组织，实现部分程度的功能恢复；
(2)本发明较以机械振荡法、化学试剂或生物试剂法脱细胞所获得的细胞外基质明显提高了粘连蛋白类、生长因子类、蛋白聚糖类、糖蛋白类、葡糖胺基聚糖类等生物活性成分的保留量；同时力学强度保留好、脱细胞彻底，病毒等生物负载清除干净；工业化生产方便；产品 多样，包括膜状产品、各种直径的微粒、凝胶和活性肽；所制备的产品适用于修复机体各个部位的软组织缺损，包括替代筋膜、骨膜、脑膜胸膜、关节囊等膜状组织，填充修复实质性脏器缺损、覆盖各类创面如粘膜缺损、烧伤创面等，注射隆胸；以及做为生物支架诱导体内各部位功能性组织再生等。
附图说明
图1为本发明采用的“灌注-高压-灌注”自动脱细胞反应器的侧视图；
图2为本发明的衍生产品，其中，左图为微粒；右图为凝胶；
图3为本发明制备活性肽的流程图；
图4为本发明的基质组织染色图；其中，A为Movat's染色(100倍)；B为苦味酸-天狼猩红染色(40倍)；C为Verhoeff-Van Gieson染色(100倍)；D为Herovici染色(40倍)；
图5为对细胞趋化作用的测试。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
原材料获取：植物源饲料封闭饲养的大猪，体重150公斤以上，死亡后半小时取出空肠。腔内冲洗后置于2℃的长征-1号器官保存液中，运输过程中维持温度不超过4℃。
预灭菌：猪空肠接入灌注反应杆，腔内循环灌注0.1％过氧乙酸/4％乙醇混合消毒液，灌注速度100ml/min/m，同时腔外浸泡于0.1％过氧乙酸/4％乙醇混合消毒液，处理时间5分钟。已证实处理5分钟可以完全灭活猪荚膜病毒，生物负载量降至不高于2CFU/g、内毒素量降至不高于20EU/g、指示病毒量降至100PFU/g，bFGF在这一步骤中损失不超过10％。
分离猪小肠粘膜下层：猪空肠使用通用机猪肠衣机(3-U-400Stridhs型，瑞典AB Stridhs Maskiner公司)制备。PBS和去离子水交替灌注冲洗。
超高压处理：获取的猪空肠SIS放入柔软的的塑料袋里，密封后，将其置于密闭的超高压容器中，然后通过流体介质(去离子水)施加60Mpa的压力，工作温度4℃，高压处理时间15分钟，具体方案是升压至40MPa，停留5分钟，再升压至60MPa，维持15分钟。处理后再接入灌注反应器，PBS和去离子水交替灌注冲洗，各15分钟。
脱脂处理：依次管腔内外反向灌注50％、70％、90％、100％、乙醇各15分钟，循环灌注，腔内压力高于腔外，腔内灌注速度100ml/min/m，辅助170kHz的超声振荡。结束后去离子水灌注15分钟进入下一步骤。
洗出细胞内容物：管腔内外反向灌注DNA增溶性缓冲盐30分钟，非循环灌注，腔内压力高于腔外，腔内灌注速度100ml/min/m，辅助170kHz的超声振荡。结束后去离子水灌注15分钟进入下一步骤。
抗原封闭：管腔内外反向灌注0.005％的十二烷基硫酸钠30分钟，非循环灌注，腔内压力高于腔外，腔内灌注速度100ml/min/m。结束后去离子水、1XPBS液交替灌注2轮，每次各灌注15分钟，进入下一步骤
消毒和保存：自0.1％PAA/4％ETOH灌注后，所有灌注的液体，包括去离子水，均为已灭菌液体。最终获得猪空肠SIS基质真空冷冻干燥后，γ射线辐射灭菌。
本实施例所制备的猪空肠SIS基质可衍生出微粒、流体化组合物、凝胶和活性肽等医疗产品，如图2所示。微粒产品为将猪空肠SIS基质真空冷冻干燥后的干燥体经高速旋转粉碎所得，包括多个产品等级(≤38μm,38-100μm,100-250μm)，可用作创面覆盖、缺损充填材料等。流体化组合物为将猪空肠SIS基质微粒在酸性环境中使用胃蛋白酶消化所得。凝胶产品为均匀流体化组合物再浓缩、中性化处理所得，包括不同浓度等级，可用作骨骼肌创面覆盖、注射用充填骨骼肌缺损、治疗肌营养不良等。制备上述衍生产品的技术均是本领域技术人员所熟知的。
本实施例涉及的猪空肠SIS基质活性肽的制备：如图3所示，将猪空肠SIS基质流体化组合物经超高速离心(≥13000rpm)后，上清液真空冷冻干燥，所得干燥体即为猪空肠SIS基质的活性肽，体外测试证实其促骨骼肌成肌细胞趋化、增殖和分化的能力远较猪空肠SIS基质流体化组合物更高效。
脱细胞彻底性测定：组织学染色未见细胞核结构，DAPI染色阴性，使用Picogreen法测定猪空肠SIS基质干重中残留DNA的含量低于500ng/mg。残留DNA链的长度小于300bp。
生物安全性测定：内毒素含量0.25EU/g，生物负载、真菌、病毒检测均为0，未检测到SDS残留，IgA含量0.1μg/g，脂质含量0.5％。
猪空肠SIS基质组织染色：如图4所示，HE染色、Movat's染色、Verhoeff-Van Gieson染色、VVG染色、Herovici染色未见细胞核结构，结构完整，主要为I型和Ⅲ型胶原纤维，含少量弹性蛋白、Ⅳ型和Ⅵ型胶原纤维，含有糖胺聚糖/蛋白聚糖成分。
生物活性测定：经ELISA法测定，猪空肠SIS基质干重中GAGs含量是2745±540μg/gg、VEGF含量是36±22ng/g，bFGF含量是393±230ng/g，TGF-β1含量是17±8ng/mg。
对细胞趋化作用：如图5所示，所制备的猪空肠SIS/UBM基质流体化组合物经中性化后，以不同浓度(1000μg/ml、500μg/ml、250μg/ml、100μg/ml、50μg/ml、25μg/ml、10μg/ml、5μg/ml) 比较与胎牛血清对骨骼肌成肌细胞、MJK/PJK的趋化作用(Boyden chamber法)，结果发现猪空肠SIS基质具有明显的促干细胞趋化作用，且在浓度25μg/ml时即达到最佳作用。
联合使用超高压和腔内外灌注法，制备猪SIS、UBM来源的脱细胞基质，获得的产品生物安全性标准如下：
内毒素 <5EU/g
生物负载 <0.5CFU/g
真菌 <0.5CFU/g
DNA/RNA含量 <500ng/mg
病毒 <5PFU/g
病毒灭活标准(log TCID50) ≥6.0
残留化合物 <0.1ppm/g
IgA <1μg/g
脂质 <2.5％
联合使用超高压和腔内外灌注法，制备猪SIS来源的脱细胞基质，获得的产品成分含量如下：
天然猪SIS干重1g中总体细胞外基质成分质量780.67mg，经脱细胞后变为622.13mg。
Figure PCTCN2017102661-appb-000001
各种生物成分的生理效应：
[1].ECM胶原和蛋白聚糖为基本骨架在细胞表面形成纤维网状复合物,这种复合物通过纤粘连蛋白或层粘连蛋白以及其他的连接分子直接与细胞表面受体连接；或附着到受体上。由于受体多数是膜整合蛋白,并与细胞内的骨架蛋白相连,所以细胞外基质通过膜整合蛋白将细胞外与细胞内连成了一个整体
[2].胶原I/III：ECM生物力学的主要来源。胶原I/III均由α1、α2、α3肽链组成，其中α1肽链可与细胞表面的特异受体结合，激活信号传导通路，促进细胞的粘附、增殖和分化、大量的羟基基团也能促进细胞粘附。胶原蛋白参与细胞分化、控制细胞粘附，调节细胞生长，有助于维持细胞基质的网状结构。
[3].弹性蛋白：构成ECM内弹性纤维网络以提供弹性、质地、耐久性和伸长后缩回能力的重要结构蛋白。可溶性弹性蛋白原单体的广泛交联形成广泛的共价阵列赋予了蛋白质弹性。由于其广泛的赖氨酸交联，弹性蛋白尤其不可溶，因此难以在生物学样品中定量测定。
[4].胶原IV：基底膜中促进细胞粘着和增殖的重要组分。
[5].胶原蛋白VII：基底膜和结缔组织的主要结构成分
[6].糖胺聚糖(Glycosaminoglycan,GAGs)：包括硫酸软骨素/硫酸皮肤素/硫酸角质素/肝素/硫酸乙酰肝素/透明质酸。GAGs与生长因子和细胞因子结合，并控制ECM内的水分保持和凝胶特性。许多细胞表面受体和许多生长因子(例如，FGF家族、VEGF)的乙酰肝素结合特性使得富含乙酰肝素的GAG成为组织修复支架的极其理想组分。
[7].蛋白聚糖(proteoglycan)是被大量糖基化了的糖蛋白：基本的蛋白聚糖单位由一个“核心蛋白质”与一个或多个共价结合着的糖胺聚糖链所组成。
[8].联合使用超高压和腔内外灌注法，制备猪SIS、UBM来源的脱细胞基质，获得的产品生物活性标准如下：
Figure PCTCN2017102661-appb-000002
Figure PCTCN2017102661-appb-000003
各种生物活性成分作用：
[1].成纤维细胞生长因子-2(bFGF):bFGF刺激血管生成、神经突生长；提高内皮细胞的活力、促进增殖，增加纤溶酶原激活物(PA)和基质金属蛋白酶1(MMP1)的分泌；提高内皮细胞的趋化性。bFGF能够刺激大多数与创面愈合有关的细胞增殖，包括血管内皮细胞、成纤维细胞、平滑肌细胞、成骨细胞和软骨细胞等，能够刺激冒险血管内皮细胞的增殖和迁移并创造其他血管再生长入的条件。粘膜下层组织分离自小肠和微创治疗，例如只有通过漂洗，FGF-2含量可以超过100，甚至潜在的更高的水平，如200或400ng/g干重。
[2].血管内皮细胞生长(VEGF)：诱导血管内皮细胞增殖和迁移、新生血管生成和保存，并增加血管内皮细胞的通透性。
[3].肝脏生长因子(HGF)：刺激生长和增强上皮细胞和内皮细胞的运动性，诱导上皮小管形成
[4].表皮生长因子(EGF)：促进成纤维细胞，平滑肌细胞，角质形成细胞的有丝分裂，对再上皮化非常重要
[5].转化生长因子(TGF-β1)：调控成纤维细胞产生的ECM成分、抑制某些细胞的增殖。TGFβ1可以促进间充质细胞分化增殖，增加I型胶原、骨连接蛋白和骨桥蛋白的合成，同时能够调节多种基质蛋白的转录，包括胶原、纤维结合素、葡糖胺、基质降解蛋白酶及其移植物等，最终是基质蛋白积聚的增加。
[6].基质细胞衍生因子(Stromal Cell Derived Factor,SDF-1):刺激祖细胞的增殖、趋化吸引祖细胞
[7].纤连蛋白：参与组织修复、胚胎发生、血液凝固和细胞迁移/粘附，促进和指导细胞和细胞外基质的附着
[8].层连蛋白：调节细胞的发育、分化和迁移的关键角色，促进和指导细胞和细胞外基质的附着。
联合使用超高压和腔内外灌注法，制备猪SIS(体重100公斤猪的空肠)来源的脱细胞基质，获得的产品生物力学标准如下：
单层力学指标
Figure PCTCN2017102661-appb-000004
多层力学指标
Figure PCTCN2017102661-appb-000005

Claims (10)

  1. 一种高生物活性的细胞外基质材料，其特征在于：将原材料通过联合使用超高压和腔内外灌注制备而得。
  2. 一种高生物活性的细胞外基质材料的制备方法，包括：
    (1)将原材料腔内灌注或腔内外灌注消毒液同时联合超声振荡预灭菌，随后进行机械分离；
    (2)使用超高压破碎和脱细胞；其中，超高压的压力值为1-100MPa，工作温度0-30℃，时间为1-120分钟；
    (3)灌注洗出细胞内容物、清除抗原，最后消毒灭菌、保存，得到高生物活性的细胞外基质材料。
  3. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(1)中的原材料包括人和哺乳动物的肠、食管、胃或前胃空腔脏器的粘膜下层、浆膜层，皮肤或膀胱基底膜、真皮、心包、胎盘羊膜或腹膜。
  4. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(1)中的腔内外联合灌注具体为：腔内外液体流动各自独立；腔外液体的体积超过管腔内体积且细胞外基质材料全部浸于管腔外液体中。
  5. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(1)中的灌注时间不超过1小时。
  6. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(1)中的预灭菌将原材料的生物负载量降至不高于2CFU/g、内毒素量降至不高于20EU/g、指示病毒量至少降低6logs。
  7. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(3)中的洗出细胞内容物的灌注液体为磷酸缓冲盐、碱性含水缓冲盐、DNA增溶性缓冲盐、纯化水中的一种或多种，洗出时间为0.1-10小时。
  8. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(3)中的清除抗原的灌注液体为离子型或非离子型表面活性剂；清除时间不超过0.5小时。
  9. 根据权利要求1所述的一种高生物活性的细胞外基质材料的制备方法，其特征在于：所述步骤(3)中消毒灭菌之前还包括脱脂。
  10. 一种如权利要求1所述的高生物活性的细胞外基质材料的应用，其特征在于：所述材料应用于制备微粒、流体化组合物、凝胶或活性肽。
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