WO2018157848A1 - 一种宫腔内置物、制备方法及其应用 - Google Patents

一种宫腔内置物、制备方法及其应用 Download PDF

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WO2018157848A1
WO2018157848A1 PCT/CN2018/077812 CN2018077812W WO2018157848A1 WO 2018157848 A1 WO2018157848 A1 WO 2018157848A1 CN 2018077812 W CN2018077812 W CN 2018077812W WO 2018157848 A1 WO2018157848 A1 WO 2018157848A1
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uterine cavity
matrix material
intrauterine
bag
implant
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PCT/CN2018/077812
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English (en)
French (fr)
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赵博
王洪权
夏磊磊
赵延瑞
李学军
张晋辉
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北京博辉瑞进生物科技有限公司
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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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/005—Ingredients of undetermined constitution or reaction products thereof
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/046—Fibrin; Fibrinogen
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof

Definitions

  • the invention relates to the technical field of medical biomaterials, in particular to an animal source uterine cavity built-in material, a preparation method and a use thereof.
  • the intrauterine implant is an animal-derived material for the treatment and prevention of intrauterine adhesions.
  • Endometrium refers to a layer that forms the inner wall of a mammalian uterus.
  • the endometrium is divided into three layers: a dense layer, a sponge layer, and a basal layer.
  • the inner surface of the intima 2/3 is a dense layer and a sponge layer collectively called a functional layer, which is detached due to the cyclical changes caused by ovarian sex hormones.
  • the basal layer is 1/3 of the intima near the myometrium, rich in blood vessels, and is not affected by ovarian sex hormones, and does not undergo periodic changes.
  • the uterine cavity lacks endometrial coverage, and the anterior and posterior walls may undergo fibrosis, scarring and formation of intrauterine adhesions, causing endometrial or connective tissue and muscle adhesion, resulting in less menstruation, dysmenorrhea, menstrual blood reflux, amenorrhea and Habitual abortion can even lead to clinical syndromes such as infertility.
  • TCRA hysteroscopic descending intrauterine adhesions
  • the key to the prevention and treatment of uterine adhesions lies in the following three aspects: isolating wounds, repairing uterine tissue and reducing the formation of scars.
  • the methods for preventing adhesion and reducing scar after intrauterine operation in clinical use and research include: postoperative systemic administration of estrogen; mechanical barrier methods such as intrauterine device and Foley balloon catheter, hyaluronic acid membrane, fresh Amniotic membrane.
  • the researches currently promoting tissue regeneration mainly include the application of scaffold materials, growth factors, gene therapy, cell therapy (such as stem cell application), mechanical therapy, electrotherapy, and the like. Natural biomaterials such as collagen and acellular matrix have attracted more and more attention from researchers because of their good biocompatibility. Especially the acellular matrix has been proved to be a good foundation for tissue repair.
  • Ideal uterine adhesions and barriers provide barriers to prevent adhesions; provide a "template” that directs cell differentiation and growth, induce tissue regeneration, and promote uterine tissue self-healing.
  • the present invention provides an implantable material having the above functions.
  • the invention provides an implantable medical device capable of treating and preventing intrauterine adhesion, the medical device comprises a biological tissue matrix material, and the decellularization process technology of the existing biological repair material is improved to make the biological tissue matrix material of the present invention Compared with the existing products, the DNA residue is lower, the immunogenicity is lower, the anti-infective ability is higher, and the repairing ability is stronger; in addition, the implantable material provided by the present invention retains the growth factor of the extracellular matrix to promote cell growth. And differentiation, is conducive to the recovery of uterine basal layer, submucosa, mucosal layer and other tissues.
  • the invention further prepares the biological repairing material into a uterine cavity built-in structure, which is used for isolating the wound surface, repairing the uterine cavity tissue and reducing the formation of scar, thereby treating the problem of intrauterine adhesion and solving the infertility problem.
  • the invention provides a uterine cavity, characterized in that the uterine cavity has a bag-like structure, and the uterine cavity comprises a decellularized small intestinal submucosal matrix material.
  • the small intestinal submucosal matrix material comprises basic fiber growth factor (FGF-2), transforming growth factor (TGF- ⁇ 1) and vascular endothelial growth factor (VEGF).
  • FGF-2 basic fiber growth factor
  • TGF- ⁇ 1 transforming growth factor
  • VEGF vascular endothelial growth factor
  • the intrauterine inclusions include fibronectin (FN) with a mass percentage greater than 2%. Fibronectin is used to immobilize nascent cells on the extracellular matrix and contribute to the regeneration and repair of various tissues.
  • FN fibronectin
  • the pocket structure has a lumen and an opening, the lumen being surrounded by the submucosal matrix material of the small intestine.
  • the intrauterine implant further includes a support member, the lumen of the intrauterine implant being capable of receiving the support member, the support member being capable of accessing the lumen of the pocket structure at the opening.
  • the support member is used to support the uterine cavity to expand in the uterine cavity, effectively isolating the wound surface and preventing intrauterine adhesion.
  • the shape of the built-in body is substantially the following shape: a trapezoid having a base length of 0.5-6 cm and a height of 0.5-7.5 cm, the opening being located at a shorter base; preferably, at least the trapezoid a corner having an arc shape; a triangle having a base length of 0.5-6 cm and a height of 0.5-7.5 cm, the opening being located on one side; preferably, at least one corner of the triangle is an arc Linear; circular, the circular diameter is 0.5-7.5 cm, the opening is located at a circular rounded edge or a circular circular surface; or an elliptical shape, the elliptical minor axis is 0.5-6 cm, The major axis is 0.5-7.5 cm, and the opening is located at the rounded or rounded surface of the ellipse.
  • These shapes are basically adapted to the shape of the luminal cavity and are adapted to the shape of the support member, that is, can be better expanded by the support member, and is also effective for
  • the support member may be an IUD or an air bag.
  • the support member is a Foley belt airbag.
  • the lumen of the uterine cavity can accommodate the structure of the balloon, and the balloon expands after inflation. It is also the expansion of the uterine cavity, effectively isolating the wound and preventing the occurrence of intrauterine adhesions.
  • the support member is a retractable device, and the retractable device can be selected from a variety of styles such as fancy, maternal music, T-shape, meta-valve, gamma-type, and uterine cavity.
  • the small intestinal submucosa of the animal is used as a raw material, and the small intestinal submucosal matrix material is prepared by washing, virus inactivation, decellularization, drying and molding, and then the obtained small intestinal submucosal matrix material is molded into a desired bag-like structure.
  • the bag-like structure includes an open end and a closed end.
  • the open end is used to put the IUD into the bag-like structure during use, and to open it, so that the submucosal matrix material of the small intestine is in close contact with the inner wall of the uterus, which assists and promotes the repair of endometrial damage.
  • the surface of the bag-like structure in the uterine cavity may be included as needed to prevent the accumulation of tissue fluid in the bagged structure, which is advantageous for tissue repair.
  • the holes have a pitch of 0.2 to 1 cm and a hole diameter of 0.5 to 3 mm.
  • the present invention provides an implantable medical device for treating intrauterine adhesions.
  • the extracellular matrix material of the product may be derived from the intestinal submucosal tissue material of an animal, such as a small intestinal submucosal tissue material of a mammal, more preferably a small intestinal submucosal tissue material of pig or cow.
  • This product uses small intestinal submucosal tissue materials as raw materials to remove cells, DNA and other components that trigger immunogenic reactions.
  • the residual amount of animal-derived DNA (residue amount of animal-derived biological material) in the extracellular matrix of the present invention is less than 10 ng/mg, preferably less than 3 ng/mg, and the ⁇ -Gal antigen clearance rate is not less than 99%.
  • DNA and ⁇ -Gal are antigens. If the content of these substances in biological materials is too high, they will cause immunological rejection in humans, and the effective control of these contents overcomes the defects of the above-mentioned immune rejection reaction. It is achieved by the decellularization step.
  • the invention also provides a preparation method of a uterine cavity, which comprises the following steps: (1) tissue pretreatment; (2) virus inactivation: soaking small intestinal submucosal tissue material with peracetic acid-ethanol solution Virus inactivation; (3) cleaning; (4) decellularization: decellularized liquid is PBS solution in which trypsin and EDTA are dissolved, and the decellularization process is carried out in a multi-frequency ultrasonic device; (5) cleaning; (6) Vacuum freeze-drying: carried out in a vacuum freeze dryer; (7) Molding: The vacuum freeze-dried small intestinal submucosal matrix material obtained in the step (6) was formed into a bag-like structure.
  • the intestinal mucosa tissue material is taken, washed, and dried.
  • the peracetic acid-ethanol solution of the step (2) wherein the peroxoacetic acid has a volume percentage concentration of 0.1% to 5%, and the ethanol has a volume percentage concentration of 5% to 40%, and is configured to be a solution with water, peracetic acid-
  • the volume ratio of the ethanol solution to the porcine small intestine submucosal tissue material is (3-20)..1, the inactivation time is 2-4 hours, and the inactivation temperature ranges from 10 to 40 °C.
  • the porcine small intestine submucosal tissue material is cleaned by a cleaning solution
  • the cleaning solution is a PBS solution having a pH of 7.2-7.4, the temperature of the PBS solution is 20 ° C, the PBS solution and the submucosal tissue of the porcine small intestine
  • the ratio of the material (volume ratio) is (20-40)..1; then washed with purified water, the ratio of purified water to the submucosal tissue material of the small intestine of the pig is (20-40)..1, and the detection conductivity is below 10 ⁇ S/cm.
  • the cleaning process is carried out in an ultrasonic cleaner, preferably at a frequency of 40 kHz, and preferably at a power of 3000 watts or more.
  • the decellularized liquid of the step (4) of the present invention is a PBS solution containing trypsin and EDTA; the mass percentage concentration of trypsin in the decellularized liquid is 0.01-0.2%, preferably 0.02-0.05%; and the concentration of EDTA is 0.1-1 mmol/ L, preferably 0.4-0.8 mmol/L; the pH of the decellularized liquid is 7.0-8.0, preferably 7.2-7.5; the volume ratio of the decellularized liquid to the porcine small intestine submucosal tissue material is (20-40)..1,
  • the decellularization process is carried out in a dual-frequency ultrasonic device, wherein the low frequency frequency range is 20-40 KHz, and the high frequency frequency is 60-90 KHz, wherein the low frequency processing is 5-40 min, the high frequency processing is 5-40 min, and the decellularized liquid temperature range is 20 -35 ° C, ultrasonic power 5000W or more.
  • the connection between the cells and the extracellular matrix is disrupted; the cells are disrupted by low-frequency ultrasound, and high-frequency ultrasound is applied to the disrupted cells and extracellular matrix to further separate the cells from the extracellular matrix. Achieve the purpose of decellularization.
  • the various steps in the process of detaching the entire cell from the matrix are enhanced to completely detach the cells from the substrate. Reach the best immunogen removal effect.
  • the porcine small intestine submucosal tissue material is cleaned by using a cleaning solution
  • the cleaning solution is a PBS solution having a pH of 7.2-7.4, and the ratio of the PBS solution to the submucosal tissue material of the porcine small intestine (volume ratio) It is (20-40)..1; then it is washed with cooling water for injection.
  • the ratio of water for injection to the submucosal tissue of pig small intestine is (20-40)..1, the temperature of water for injection is 20-35 °C, and the water for injection is detected.
  • the difference in conductivity of the cleaning injection water is less than 1 ⁇ S/cm; the cleaning process is carried out in an ultrasonic cleaner, preferably at a frequency of 40 kHz, and preferably at a power of 3000 watts or more.
  • step (6) one or more layers of the small intestinal submucosal matrix material obtained in the step (5) are placed on a mold, and placed in a vacuum freeze dryer to freeze-dry the porcine small intestinal submucosa matrix material.
  • the mold of the above step (6) comprises a needle bottom plate and a pressure frame, and one or more layers of the porcine small intestine submucosal matrix material are laid on the needle bottom plate, and the pressure frame is placed on the porcine small intestinal submucosa matrix.
  • the needle bottom plate and the pressing frame are relatively fixed.
  • the specific structure of the mold mentioned in the present invention can be referred to the invention patent ZL201310203602.2.
  • Vacuum freeze-drying placing the mold with the submucosal matrix material of the small intestine in a vacuum freeze dryer; first pre-freezing to -45 ° C, holding for 1-2 hours; then turning on the vacuum pump, adjusting the temperature to -15 ° C, keeping 5 -7 hours, adjust the temperature to 0 ° C, keep warm for 2 hours, finally adjust the temperature to 25 ° C, keep warm for 4 hours, complete vacuum freeze-drying; the pressure in the chamber of the freeze-drying device is 1-50Pa.
  • the vacuum freeze-dried small intestinal submucosa matrix material obtained by the step (6) is used as a raw material, and is sewn or bonded into a bag-like structure;
  • the bag-like structure includes a lumen, An open end and a closed end, the open end being configured to allow a support member (eg, an IUD) to enter and exit the interior of the pocket structure at the open end, the inner cavity being capable of receiving the support member.
  • a support member eg, an IUD
  • the bag-like structure can be sewn by using a degradable thread, or can be bonded by a degradable glue to form a bag-like structure, and the degradable glue used is, for example, a protein glue.
  • the uterus is inverted flat pear shape, the front is flat, the back is slightly protruding, the wall is small, the upper end is wide and free, facing forward and upward; the lower end is narrow, cylindrical, inserted into the upper part of the vagina.
  • the uterine cavity volume of an adult woman is about 5 ml.
  • the closed end of the bag-like structure has a size greater than the size of the open end.
  • the pocket structure may be substantially trapezoidal or triangular in shape, or the obtuse or acute angle portion of the trapezoid or triangle may be cut and sewn into an arc shape.
  • the bag-like structure ensures that the IUD cannula is placed and that the IUD that has been opened therein does not fall out.
  • the bag-like structure has different size specifications and is suitable for different individual needs.
  • the suture uses a biodegradable surgical line.
  • the sewing process also requires control of sterility.
  • the pocket structure can also be round or oval to fit in the uterine cavity.
  • a circular or elliptical bag-like structure can also be applied to the corresponding shape of the device.
  • the invention also provides the application of the uterine cavity in the intrauterine medical device, the intrauterine medical device is used for repairing the endometrial or basal layer damage, for preventing and treating the intrauterine adhesion, and for preventing the anterior and posterior uterine cavity Fibrosis or scarring of the wall, or for the prevention and treatment of infertility caused by intrauterine adhesions, habitual abortion, less menstruation, dysmenorrhea, menstrual reflux or amenorrhea.
  • the invention provides a uterine cavity built-in material and a preparation method thereof, and the uterine cavity built-form formed by the preparation method is suitable for different sizes of uterus. By using it in combination with different types of IUDs, it can repair the damage of different parts of the endometrium.
  • the present invention provides a uterine cavity, the product being made of an extracellular matrix material, preferably of the type having a higher purity.
  • Preferred materials have endotoxin levels below 12 EU/g, more preferably below 5 EU/g, and most preferably below 1 EU/g.
  • the ECM material may have a bioburden of less than 1 CFU/g, more preferably less than 0.5 CFU/g.
  • the level of fungi is below 1 CFU/g, more preferably below 0.5 CFU/g.
  • the nucleic acid level is preferably less than 5 ⁇ g/mg, more preferably less than 2 ⁇ g/mg, and the virus content is preferably less than 50 PFU/g, more preferably less than 5 PFU/g.
  • the present invention provides a uterine cavity built-in material made of a small intestinal submucosal matrix material.
  • the small intestinal submucosal matrix material retains growth factors or other beneficial biologically active ingredients.
  • the small intestinal submucosal matrix material comprises basic fibroblast growth factor (FGF-2), transforming growth factor- ⁇ (TGF- ⁇ ), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and/or platelets. Derived growth factor (PDGF).
  • the extracellular matrix material further comprises heparin, heparin sulfate, hyaluronic acid and/or fibronectin.
  • the small intestinal submucosal matrix material may contain a biologically active component that directly or indirectly induces cellular responses, such as changes in morphology, division, growth, protein or gene expression.
  • the invention provides a method for preparing a uterine cavity, wherein one or more layers of small intestinal submucosal matrix material, preferably 4-6 layers, can be laid on the needle bottom plate of the mold.
  • the present invention has the following significant advantages:
  • the small intestinal submucosal matrix material is used for the isolation of the anterior and posterior wall wounds after uterine cavity operation, and does not cause secondary damage to the wound caused by mechanical isolation such as intrauterine device, and no rejection reaction occurs.
  • the small intestinal submucosal matrix material is used for the isolation of the anterior and posterior wall wounds after the uterine cavity operation, and does not produce an inflammatory reaction such as a hyaluronic acid membrane or a polylactic acid membrane for wound isolation.
  • the small intestinal submucosal matrix material is used for the isolation of the anterior and posterior wall wounds after uterine cavity operation, and does not produce a rejection reaction such as fresh amniotic membrane for wound healing.
  • the submucosal matrix material of the small intestine is used together with the IUD to effectively solve the special problem that the endometrium is not suitable for suturing, and the submucosal matrix of the repairing material is fixed in the uterine cavity by using an IUD matched with the shape of the uterine cavity. It greatly improves the level of repair function of the intestinal submucosal matrix material.
  • the small intestinal submucosal matrix material selected by the invention has less DNA residue, can reach below 10 ng/mg, and has a high removal rate of galactosidase, which can reach more than 99%; and completely retains the three-dimensional of the natural extracellular matrix.
  • Structure retaining the original beneficial components, growth factors such as basic fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF), hyaluronic acid (HA) and aminoglycan sulfate (sGAGs), etc., ensured
  • FGF2 basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • HA hyaluronic acid
  • sGAGs aminoglycan sulfate
  • the small intestinal submucosal matrix material selected by the invention is synchronized with the dehydration process of the matrix material during the preparation process, so that the multilayer structure is dense and does not delaminate, thereby greatly improving the mechanics of the acellular matrix patch. Performance and resistance to degradation. And by controlling the sterilization process, the degradation time can be effectively controlled, and products of different use periods can be prepared as needed.
  • the present invention employs a milder trypsin and EDTA complex solution and a dual-frequency sonication treatment for decellularization.
  • This step is an important step in the manufacture of biological materials, and only the amount of cells as an immunogen is lowered.
  • the material implanted in the body does not trigger an immune response, thus ensuring the safety of the material; the connection between the cell and the extracellular matrix is disrupted by trypsin and EDTA, and then low frequency ultrasound is used.
  • the cells are disrupted, and high-frequency ultrasound is applied to the broken cells and the extracellular matrix to further separate the cells from the extracellular matrix, thereby achieving the purpose of decellularization, and the corresponding methods are used for the detailed treatment of each step of decellularization.
  • the decellularization effect is better and the cell residue is lower.
  • FIG. 1 is a front side scanning electron micrograph of a decellularized and shaped small intestinal submucosal matrix material for use in a uterine cavity in accordance with an embodiment of the present invention.
  • FIG. 2 is a scanning electron micrograph of the side of a decellularized and shaped small intestinal submucosal matrix material for use in a uterine cavity in accordance with an embodiment of the present invention.
  • XRD X-ray diffraction spectrum
  • FIG. 4 is a graph showing the hydrophilic angle of a small intestinal submucosal matrix material for use in a uterine cavity in accordance with an embodiment of the present invention.
  • Figure 5 is an infrared map of the submucosal matrix material of the small intestine used in the uterine cavity.
  • Figure 6 is a graph showing the results of SDS-PAGE electrophoresis of the acellular matrix material of the intrauterine implant material and the existing matrix material.
  • Figure 7 is a photograph of Masson staining of the small intestinal submucosal matrix material after decellularization.
  • Figure 8 is a Masson stained photograph (small magnification) of the intestinal submucosal matrix material after decellularization.
  • Figure 9 is a schematic illustration of a uterine cavity in accordance with one embodiment of the present invention.
  • Figure 10 is a schematic illustration of a uterine cavity in accordance with another embodiment of the present invention.
  • Figure 11 is a schematic illustration of a uterine cavity in accordance with another embodiment of the present invention.
  • Figure 12 is a schematic view of an IUD
  • Figure 13 is a schematic illustration of an IUD placement device.
  • FIG. 1 and 2 are scanning electron micrographs of the front and side surfaces of the decellularized and shaped small intestinal submucosal matrix material used in the uterine cavity of the present invention.
  • the matrix material of the invention adopts a non-crosslinking process, and the multi-layered acellular matrix is combined to form a collagen microfiber network, and the excellent mechanical properties are sufficient for the use requirement, so that no chemical crosslinking agent is needed in the whole production process, and the chemical is avoided.
  • Adverse reactions such as fiber wrapping, erosion, calcification and inflammation caused by cross-linking. It can be seen from Fig.
  • the matrix material of the small intestine submucosa has a porous network structure, and the porosity can reach more than 90%, providing a suitable microenvironment for guiding cell growth.
  • the small intestinal submucosal matrix material is processed to form a multi-layered structure in which the collagen fibers are layered, and the degradation performance and mechanical properties of the material can be adjusted to meet the needs of different tissue repair.
  • the submucosal submucosal matrix material has a high bursting strength and suturing strength, and can be applied to surgical conditions requiring suturing and bearing.
  • the round peak indicates that the matrix material has changed the crystal structure of the original collagen after the decellularization process and the molding process, forming an amorphous structure, thereby widening and narrowing the X-ray diffraction peak.
  • Such a microstructure can greatly eliminate the anisotropy of the material in the microstructure.
  • the anisotropy in the microstructure of the material leads to the anisotropy of the cell growth distribution, forming an advantageous growth in some directions, thereby forming a scar.
  • the uterine cavity of the present invention has no anisotropy in cell growth distribution, thereby avoiding scarring and preventing intrauterine adhesions.
  • Figure 4 is a graph showing the hydrophilic angle of the small intestinal submucosal matrix material used in the uterine cavity.
  • the hydrophilicity of the surface of the biomaterial has an important influence on the conformation of the surface-adsorbed protein.
  • the submucosal matrix material used in the uterine cavity has a hydrophilic angle of 45°, which is very favorable for cell adhesion and spreading.
  • Figure 5 is an infrared map of the submucosal matrix material of the small intestine used in the uterine cavity.
  • the vicinity of cm -1 is the NH bending vibration of the amide II band; the absorption peak near 1066.41-1548.46 cm -1 indicates the integrity of the collagen triple helix structure.
  • the NH stretching vibration of collagen at 3308.26 cm -1 illustrates the presence of hydrogen bonds between peptide bonds.
  • FIG. 6 shows the results of SDS-PAGE electrophoresis of the acellular matrix material of the intrauterine implant material and the existing matrix material.
  • the leftmost stripe is the stripe corresponding to the different molecular weight markers
  • the middle stripe is the electrophoretic stripe of the existing matrix material
  • the rightmost stripe is the extracellular matrix material of the intrauterine implant material prepared by the method of the present invention.
  • Electrophoretic stripes, in which the stripes arranged from top to bottom represent the type I collagen fibers, the ⁇ chain, the ⁇ chain, the ⁇ 1 chain, and the ⁇ 2 chain.
  • the present invention contains type I collagen.
  • the matrix material component using the non-crosslinking process has a small amount of elastin and type III, type IV and type VI collagen in addition to type I collagen.
  • the matrix material of the non-crosslinking process contains trace amounts of active biomolecules, such as fibronectin and laminin, which facilitate cell adhesion and growth; glycosaminoglycans, proteoglycans and the like act as growth factor binding sites, which is beneficial to Preservation of various growth factors, and inhibition of collagenase degradation, increase cell migration ability.
  • the non-crosslinking process also allows the matrix material to retain basic fiber growth factor (FGF-2), transforming growth factor (TGF- ⁇ 1) and vascular endothelial growth factor (VEGF) in the original animal tissues, and the retention rate is higher than 45%, these growth factors play a key role in tissue vascularization and functional reconstruction.
  • FGF-2 basic fiber growth factor
  • TGF- ⁇ 1 transforming growth factor
  • VEGF vascular endothelial growth factor
  • the small intestinal submucosal matrix material is rich in biologically active factors, which enable it to actively induce cells to enter the material, form vascularized tissue, and regulate the regeneration and repair process of the host tissue.
  • Fibronectin is a large glycoprotein present in all vertebrates with a molecular weight of 4.5-9.5%.
  • the structure of the sugar chain varies depending on the tissue source and differentiation state.
  • FN can connect cells to the extracellular matrix.
  • Some of the short peptide sequences in the FN peptide chain are the smallest structural units for the recognition and binding of various FN receptors on the cell surface.
  • the mass percentage of FN in the small intestinal submucosal matrix material used in uterine implant materials was >5%; the FN mass percentage was above 2.6% by ELISA.
  • the existing extracellular matrix material uses a tissue having a large elasticity such as skin, arteries, and pericardium, and contains a large amount of elastin in addition to type I collagen.
  • Type I collagen is more than 100 times stronger than elastin, providing mechanical strength to the extracellular matrix, while elastin imparts excellent elasticity to the matrix, allowing the extracellular matrix to elongate and contract. Due to the presence of elastin, the collagen of the acellular matrix is degraded continuously, the matrix is stretched by the force, and shrinks and deforms when the force is not applied, so that the tissue cannot be reshaped into a normal shape, and the surgery is repaired due to the deformation of the material. failure.
  • Fig. 7 is a photograph of Masson staining of the intestinal submucosal matrix material after decellularization, in which inelastic protein remains.
  • the small intestinal submucosal matrix material used in the intrauterine implant material is an animal-derived acellular material
  • the relevant immunogen derived from the heterologous material should be removed as much as possible.
  • a photograph of HE stained sections of the product prepared by the decellularization process is shown in Fig. 8, and no residual nuclei were found.
  • the DNA residue reached 3.8 ng/mg or less, which was much lower than that of similar acellular materials, and the ⁇ -Gal antigen clearance rate reached 99.4%.
  • This data indicates that the matrix material used in uterine implant materials is completely decellularized and has a high immunogenic removal rate, minimizing the risk of causing immune rejection.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • Figure 9 is a schematic illustration of a uterine cavity in accordance with an embodiment of the present invention having a pocket-like structure having a generally trapezoidal shape with at least a portion of the corner being curved.
  • the opening 2 is located on the shorter side of the trapezoid.
  • a piece of vacuum-freeze-dried small intestinal submucosal matrix material can be used to fold it, or two or more pieces of matrix material can be used.
  • the uterine cavity can be placed in the uterine cavity and deployed to isolate and protect the wound, promote the recovery of the basal membrane and mucosa of the uterus, and prevent adhesion.
  • the uterine cavity of the above embodiment may further comprise a support structure to expand the interior within the bag-like structure to further prevent the occurrence of intrauterine adhesions.
  • the support structure can be an air bag, preferably a Foley belt air bag.
  • the support structure can also be an IUD.
  • Figure 12 is a schematic view of an IUD
  • Figure 13 is a schematic view of an IUD placement device.
  • the intrauterine implant 1 can be placed in the uterine cavity, and the IUD device 4 with the IUD 3 can be passed through the uterine ostium and the uterine cavity.
  • the opening 2 of 1 is then disengaged from the IUD device 4 into the lumen of the uterine cavity 1 and the IUD 3 is automatically expanded in the lumen of the member 1 and left in the palace together with the uterine cavity 1 Inside the cavity.
  • the IUD 3 pre-assemble the IUD 3 into the lumen of the intrauterine implant 1 and place it together in the IUD placement device 4. Then, the IUD placement device 4 is passed through the uterine ostium, and then the uterine cavity 1 is disengaged from the IUD device to enter the uterine cavity. The IUD 3 automatically expands in the lumen of the member and expands the uterine cavity 1 The uterine cavity formed by the uterine cavity 1 and the IUD 3 is left in the uterine cavity.
  • Embodiment 2 is a diagrammatic representation of Embodiment 1:
  • FIG 10 is a schematic illustration of a uterine cavity in accordance with another aspect of the present invention.
  • the uterine cavity built-in material 1 has a bag-like structure and has a substantially triangular shape, and at least a part of the corner is curved.
  • the opening 2 is located on one side of the triangle, and the length of the opening 2 is less than the length of the side so that the respective ends of the IUD 3 can be caught at the end of the substantially triangular bag-like structure 1.
  • the contraceptive device 3 can be placed in advance in the lumen of the intrauterine implant 1 and placed together in the device placement device 4.
  • the IUD placement device 4 is passed through the uterine ostium, and then the uterine cavity is placed out of the IUD device to enter the uterine cavity.
  • the IUD 3 automatically expands in the lumen of the member and expands the uterine cavity 1 so that The uterine cavity built into the uterine cavity 1 and the IUD 3 is left in the uterine cavity.
  • Embodiment 3 is a diagrammatic representation of Embodiment 3
  • FIG 11 is a schematic illustration of a uterine cavity in accordance with another aspect of the present invention.
  • the uterine cavity built-in material 1 has a bag-like structure and has a substantially circular or elliptical shape.
  • the opening 2 is located on a circular or elliptical circular or rounded edge.
  • the IUD 3 and the uterine cavity 1 can be placed in the uterine cavity by performing the 1 or 2 placement.
  • Embodiment 4 Method for preparing uterine cavity with bag-like structure
  • Raw material selection and initial treatment freshly slaughtered pig small intestine tissue cleaned, split the small intestine submucosa, divide the small intestine submucosal tissue material, remove lymphoid tissue, rinse with tap water for 1-3 times, then rinse with purified water The surface is free of stains, and then the cleaned porcine small intestine submucosal tissue material is placed on a water filter such as a sieve, and allowed to stand for more than five minutes to filter the water.
  • a water filter such as a sieve
  • the cleaning solution is PBS solution with pH value of 7.2-7.4, the temperature of PBS solution is 20 °C, the ratio of PBS solution to submucosal tissue material of pig small intestine (volume ratio) ) is 30..1, preferably washed 3 times for 20 minutes each time; then washed with purified water, the ratio of purified water to porcine small intestine submucosal tissue material is 30..1, until the detection conductivity is below 10 ⁇ S/cm; cleaning process It is carried out in an ultrasonic cleaner with a frequency of 40 kHz and a power of 3000 W.
  • the decellularized solution was prepared by using a trypsin solution containing 0.1% by mass of a concentration and a PBS solution of EDTA at a concentration of 0.5 mmol/L.
  • the washing solution is a PBS solution having a pH of 7.2-7.4, the temperature of the PBS solution is 20 ° C, and the ratio (volume ratio) of the PBS solution to the submucosal tissue material of the porcine small intestine is 30..1, preferably 3 times for 20 minutes each time;
  • the water is washed with water at a temperature of 20 ° C.
  • the ratio of the water content of the water for injection to the submucosal tissue of the small intestine is 30..1, and the difference between the conductivity of the cleaned and injected water is less than 1 ⁇ S/cm. 40 kHz, the power is preferably 3000 W or more.
  • Vacuum freeze-drying one or more layers of the porcine intestinal submucosa matrix material obtained in the step (5) are placed on a mold, and placed in a vacuum freeze dryer to freeze-dry the porcine small intestinal submucosal matrix material.
  • the mold of the above step (6) comprises a needle bottom plate and a pressure frame, and one or more layers of the porcine small intestine submucosal matrix material are laid on the needle bottom plate, and the pressure frame is placed on the porcine small intestinal submucosa matrix. In the material, the needle bottom plate and the pressing frame are relatively fixed.
  • the specific structure of the mold mentioned in the present invention can be referred to the invention patent ZL201310203602.2.
  • Vacuum freeze-drying placing a mold with a small intestinal submucosal matrix material in a vacuum freeze dryer; first pre-freezing to -45 ° C, holding for 1 hour; then turning on the vacuum pump, adjusting the temperature to -15 ° C, holding for 6 hours, The temperature was adjusted to 0 ° C, the temperature was kept for 2 hours, and finally the temperature was adjusted to 25 ° C, and the temperature was kept for 4 hours to complete the vacuum freeze drying; the pressure in the chamber of the freeze-drying device was 25-30 Pa. A lyophilized small intestinal submucosal matrix material is obtained.
  • the punching and packaging step (8), the step (8) is perforated packaging, specifically: the dry material is cut into a fixed shape (including a square, a circle or other shape) on the mold, and then placed in a mechanical punching machine. Perforated at a pitch of 0.9 cm, with a hole diameter of 1.5 mm, and then packaged in a Tyvek bag.
  • Sterilization analysis step (9), the step (9) sterilization analysis step is specifically: using ethylene oxide for sterilization, the sterilization conditions are: first temperature 20-40 ° C holding time 2-4 hours, humidity 30 -70% (preferably 60%), then pass through a concentration of 300-1000 mg / L (preferably 800 mg / L) of ethylene oxide, sterilized for 4-8 hours; the analytical process is carried out in a ventilated analytical chamber, temperature control at 10 Between -30 ° C and 14-28 days.
  • the above-mentioned step of punching can prevent the liquid from accumulating in the bag-shaped inner body and cause infection.
  • the punching step facilitates tissue repair.
  • Porosity measurement The porosity of the material was measured by a porosity meter, and the porosity of the sample provided in Embodiment 4 was 90%.
  • Stitch retention test Method: suture 2 mm of the end edge of the biomaterial with 2-0 surgical suture or stainless steel wire of the same diameter, and fix the suture or stainless steel wire to the other end of the biomaterial on the tension meter. The stretching was performed at a speed of 20 mm/min until the stitching point was torn, and the pulling force when the stitching point was torn was recorded. Three batches of samples were tested as described above. Result: The suture tensile strength was greater than or equal to 6N.
  • Tensile strength test method Method: Using a tensile (compression) test machine, the patch is cut into strip samples, and after cutting, the relative humidity is 40%-60%, and the temperature is 22 °C ⁇ 2 °C. The test was carried out immediately after standing for 2 hours. The two ends of the sample were fixed on the chuck of the tensile tester, and were sequentially stretched outward at a speed of 100 mm/min until the sample was broken, and the force at which the sample was broken was recorded in units of N. Three batches of samples were tested as described above. The result is greater than 200N.
  • Blasting strength test The method uses a tensile (compression) test machine to cut the material into a 23 ⁇ 23mm square pattern for use, and is placed at a relative humidity of 40%-60% and a temperature of 22°C ⁇ 2°C. The test was carried out immediately after 2 hours. The specimen was fixed on the table of the tensile tester with a ring clamp, and the spherical probe was passed through the sample at a speed of 750 mm/min, and the force of the probe to pierce the sample was recorded. Three batches of samples were tested as described above. Result: The bursting strength is greater than 120N.
  • test solution Take a uniform portion of the thickness of the sample, cut into pieces of 1 cm 2 , wash with water, dry it, and then add it to a glass container. The ratio of total surface area (cm 2 ) to water (mL) is 5 Add water to the ratio of 1:1, put it in a pressure steam sterilizer, heat it at 121 °C for 30 min, and separate the sample from the liquid after heating, and cool to room temperature as a test solution. The same volume of water was placed in a glass container, and a blank control solution was prepared in the same manner.
  • Virus detection The pseudorabies virus was selected as the indicator virus, and the DNA copy number of the virus was detected by real-time quantitative PCR, and three batches of samples were detected. Result: The viral DNA copy number is zero.
  • Bacterial endotoxin According to GB/T 14233.2-2005 "medical infusion, blood transfusion, injector test method Part 2: biological test method" for testing, a total of 3 batches of samples, the results: bacterial endotoxicity is 20EU / set.
  • Ethylene oxide residue According to the method specified in GB/T14233.1-2008 "Test methods for medical infusion, blood transfusion, and injecting tools - Part 1: Chemical analysis", the results are as follows: The amount does not exceed 10 ⁇ g / set.
  • the animal model was selected from 12-week-old adult female New Zealand white rabbits weighing about 2500 grams, which was normal. Endometrial growth was synchronized by injecting 50 IU of chorionic gonadotropin into each rabbit's ear margin 24 h before the experiment. The longitudinal incision was taken about 2 cm long and the longitudinal incision was made into the abdomen. The bilateral uterus was found, and the abdominal incision was sutured according to the group treatment.
  • Group B Modeling group: Mechanical damage modeling method: Right uterus, 1.5 cm longitudinal incision in the lower third of the uterus, resection of the semi-intimal endometrium and submucosa; left uterus, resection of the half-sided palace Endoluminal and submucosal, saline flushes the uterine cavity.
  • the size of the uterine repair material placed through the incision is 3*0.5 cm.
  • Group C Treatment group: right uterus, 1.5 cm longitudinal incision in the lower third of the uterus, resection of the endometrium and submucosa of the whole uterus; left uterine hysterectomy to remove the endometrium and submucosa. Wash the uterine cavity with saline. The size of the uterine repair material placed through the incision is 3*0.5 cm. Antibiotics were applied during the perioperative period for 3 days. The animals were sacrificed at 7 days, 14 days, and 28 days after surgery. The uterus specimens were taken and HE staining and Masson staining were performed to determine the adhesion and the number of endometrial glands and the degree of fibrosis.

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Abstract

一种宫腔内置物、其制备方法及用途。宫腔内置物具有袋状结构，包括经脱细胞处理的动物小肠粘膜下层基质材料，经组织前置处理、病毒灭活、清洗、多频超声装置中进行脱细胞、清洗、真空冷冻干燥和成型步骤制成。用于宫腔内置医疗器械，能够治疗和预防宫腔粘连，对生物修补材料的脱细胞工艺进行改进，使得其DNA残留量更低，免疫原性更低、抗感染能力更高、修复能力更强，有利于宫腔基底层、粘膜下层、粘膜层等组织的恢复。作为宫腔内置结构，用于隔离创面、宫腔组织修复和减少瘢痕的形成，从而处理宫腔粘连问题，并解决由此产生的不孕问题。

Description

一种宫腔内置物、制备方法及其应用 技术领域
本发明涉及医用生物材料技术领域，具体涉及一种动物源宫腔内置物、制备方法和用途。该宫腔内置物为动物源性材料，用于治疗和预防宫腔粘连。
背景技术
子宫内膜(endometrium)是指构成哺乳类子宫内壁的一层。子宫内膜分为致密层、海绵层和基底层三层。内膜表面2/3为致密层和海绵层统称功能层，受卵巢性激素影响发生周期变化而脱落。基底层为靠近子宫肌层的1/3内膜，富有血管，不受卵巢性激素影响，不发生周期性的变化。
临床上，当子宫内壁出现创伤时，如吸宫或刮宫、子宫肌瘤剔除术、子宫内膜结核等，造成内膜过度损伤，进而损伤子宫内膜基底层，结果会导致上皮、间质细胞再生障碍，新生血管形成受损，内膜难以实现自我修复。此时，宫腔缺乏内膜覆盖，前后壁可发生纤维化、瘢痕化及形成宫腔粘连，引起子宫内膜或结缔组织、肌肉粘着，导致出现月经过少、痛经、经血反流、闭经和习惯性流产甚至导致不孕等临床综合征。
20世纪70年代以来，随着宫腔镜等技术的发展，宫腔镜下行宫腔粘连分离术(TCRA)目前已成为治疗宫腔粘连以及由此治疗不孕的标准方法。但是行TCRA术后宫腔再次粘连仍有可能发生，且一旦再发宫腔粘连，子宫内膜修复将无法顺利完成，临床表现为月经量无明显增加或仍然闭经，故TCRA术后仍需要采取一定防粘连措施。
子宫粘连预防和治疗的关键在于以下三个方面：隔离创面、宫腔组织修复和减少瘢痕的形成。目前临床使用及研究中的宫腔操作后防止粘连和减少瘢痕的方法有：术后全身给予雌激素治疗；机械屏障法如宫内节育器和Foley带气囊导尿管、透明质酸膜，新鲜羊膜。目前研究的促进组织再生的研究主要包括支架材料的应用、生长因子、基因疗法、细胞疗法(如干细胞的应用)、机械疗法、电疗等。天然生物材料如胶原、脱细胞基质因其良好的生物相容性而越来越受到科研人员的重视，尤其是脱细胞基质已有研究证明可作为组织修复的良好基础。
理想的子宫粘连和是提供屏障隔离创面以防止粘连的发生；提供引导细胞分化、生长的“模板”，诱导组织再生，促进宫腔组织自我修复。本发明提供具有上述功能的植入性材料。
发明内容
技术问题
本发明提供一种能够治疗和预防宫腔粘连的植入性医疗器械，该医疗器械包括生物组织基质材料，对现有生物修补材料的脱细胞工艺技术进行改进，使本发明生物组织基质材料与现有产品相比，DNA残留量更低，免疫原性更低、抗感染能力更高、修复能力更强；此外本发明提供的植入性材料保留了细胞外基质的生长因子促进细胞的生长和分化，有利于宫腔基底层、粘膜下层、粘膜层等组织的恢复。此外本发明更将生物修补材料制作为宫腔内置结构，用于隔离创面、宫腔组织修复和减少瘢痕的形成，从而处理宫腔粘连问题，并解决由此产生的不孕问题。
解决方案
本发明提供一种宫腔内置物，其特征在于，所述宫腔内置物具有袋状结构，所述宫腔内置物包括经脱细胞处理的小肠粘膜下层基质材料。
小肠粘膜下层基质材料包含碱性纤维生长因子(FGF-2)、转化生长因子(TGF-β1)和血管内皮生长因子(VEGF)。
宫腔内置物包括纤维粘连蛋白(fibronectin，FN)，其质量百分比含量大于2％。纤维粘连蛋白用于新生细胞在细胞外基质上的固定，有助于各组织的再生和修复。
袋状结构具有内腔和开口，所述内腔由所述小肠粘膜下层基质材料围成。
所述宫腔内置物还包括支撑构件，所述宫腔内置物的内腔能够容纳所述支撑构件，所述支撑构件能够在所述开口处进出所述袋状结构的所述内腔。支撑构件用于支撑宫腔内置物，使其在宫腔内扩张，有效隔离创面，防止发生宫腔粘连。
所述内置物的形状基本上呈以下形状：梯形，所述梯形底边长为0.5-6cm，高度为0.5-7.5cm，所述开口位于较短底边处；优选地，所述梯形 的至少一个角部为弧线形；三角形，所述三角形底边长为0.5-6cm，高度为0.5-7.5cm，所述开口位于一个侧边上；优选地，所述三角形的至少一个角部为弧线形；圆形，所述圆形直径为0.5-7.5cm，所述开口位于圆形的圆边处或圆形的圆面上；或者椭圆形，所述椭圆形短轴为0.5-6cm，长轴为0.5-7.5cm，所述开口位于椭圆形的圆边处或圆面上。这些形状与宫腔内腔形状基本上适配，并与支撑构件形状适配，即能被支撑构件较好地扩张，也有利于有效隔离创面，防止发生宫腔粘连。
其中支撑构件可以为节育器或气囊。
其中支撑构件为Foley带气囊。宫腔内置物的内腔可以容纳气囊结构，气囊充气后扩张，也是宫腔内置物扩张，有效隔离创面，防止发生宫腔粘连。
其中的支撑构件为可收缩节育器，该可收缩节育器可选用花式、母体乐、T形、元宫形、γ型、宫腔形等各种样式。
其中用动物的小肠粘膜下层为原料，经过清洗、病毒灭活、脱细胞、干燥和成型处理制备小肠粘膜下层基质材料，然后将得到的小肠粘膜下层基质材料成型为所需要的袋状结构。所述袋状结构，包括开口端和封闭端。
开口端用于在使用时将节育器放入袋状结构中，将其撑开，使小肠粘膜下层基质材料与子宫内壁紧密的接触，辅助并促进子宫内膜损伤的修复。
根据需要在宫腔内置物的袋状结构表面可包括通孔，免于组织液在袋装结构内积累，利于组织修复。优选地，所述孔间距0.2-1厘米，孔直径0.5-3毫米。
本发明提供一种治疗宫腔粘连的植入性医疗器械。该产品的细胞外基质材料可以来自于动物的小肠粘膜下层组织材料，例如哺乳动物的小肠粘膜下层组织材料，更优选猪或牛的的小肠粘膜下层组织材料。本产品以小肠粘膜下层组织材料为原料，去除细胞、DNA及其它引发免疫原反应的成分。本发明所述的细胞外基质中动物源DNA残留量(动物源性生物材料DNA残留量)小于10ng/mg，优选小于3ng/mg，α-Gal抗原清除率不低于99％。DNA和α-Gal是抗原，如果生物材料中这些物质含量过高，放入人体后会使人体发生免疫排斥反应，而上述含量的有效控制克服了上述免疫排斥反应的缺陷，上述这些物质的去除是通过脱细胞步骤实现的。
本发明还提供一种宫腔内置物的制备方法，其特征在于，包括以下步 骤：(1)组织前置处理；(2)病毒灭活：采用过氧乙酸-乙醇溶液浸泡小肠粘膜下层组织材料进行病毒灭活；(3)清洗；(4)脱细胞：脱细胞液液为溶有胰蛋白酶和EDTA的PBS溶液，脱细胞过程在多频超声波装置中进行；(5)清洗；(6)真空冷冻干燥：在真空冷冻干燥机中进行；(7)成型：将由步骤(6)得到的经真空冷冻干燥的小肠粘膜下层基质材料制成袋状结构。
所述步骤(1)中，取小肠粘膜下层组织材料，清洗，滤干。
所述步骤(2)的过氧乙酸-乙醇溶液，其中过氧乙酸的体积百分比浓度为0.1％-5％、乙醇的体积百分比浓度为5％-40％，用水配置成溶液，过氧乙酸-乙醇溶液与猪小肠粘膜下层组织材料的体积比为(3-20)︰1，灭活时间2-4小时，灭活的温度范围为10-40℃。
本发明步骤(3)的清洗过程中，采用清洗液清洗猪小肠粘膜下层组织材料，清洗液为pH值为7.2-7.4的PBS溶液，PBS溶液温度为20℃，PBS溶液与猪小肠粘膜下层组织材料的比例(体积比)为(20-40)︰1；然后采用纯化水清洗，纯化水与猪小肠粘膜下层组织材料比例为(20-40)︰1，至检测电导率为10μS/cm以下终止；清洗过程在超声波清洗机中进行，频率优选40kHz，功率优选3000W以上。
本发明步骤(4)的脱细胞液为含有胰蛋白酶和EDTA的PBS溶液；脱细胞液中胰蛋白酶的质量百分比浓度为0.01-0.2％，优选0.02-0.05％；EDTA的浓度为0.1-1mmol/L，优选0.4-0.8mmol/L；脱细胞液的pH值为7.0-8.0，优选为7.2-7.5；所述脱细胞液与猪小肠粘膜下层组织材料体积比为(20-40)︰1，脱细胞过程在双频超声波装置中进行，其中低频频率范围为20-40KHz，高频频率为60-90KHz，其中低频处理5-40min，高频处理5-40min，脱细胞液的温度范围为20-35℃，超声功率5000W以上。采用胰蛋白酶和EDTA，使细胞与细胞外基质之间的连接被破坏；采用低频超声对细胞进行破碎，同时使用高频超声作用于破碎的细胞及细胞外基质，进一步使细胞脱离细胞外基质，达到脱细胞目的。采用上述方式，对整个细胞脱离基质过程中的各个步骤进行强化，使细胞被从基质上完全脱离。到达最佳的免疫原去除效果。
本发明步骤(5)的清洗过程中，采用清洗液清洗猪小肠粘膜下层组织材料，清洗液为pH值为7.2-7.4的PBS溶液，PBS溶液与猪小肠粘膜下层组织材料的比例(体积比)为(20-40)︰1；然后采用降温的注射用水清洗，注射用水与 猪小肠粘膜下层组织材料比例为(20-40)︰1，注射用水温度20-35℃，检测清洗注射用水与未清洗注射用水电导率之差小于1μS/cm终止；清洗过程在超声波清洗机中进行，频率优选40kHz，功率优选3000W以上。
所述步骤(6)中，将一层或多层由步骤(5)得到的小肠粘膜下层基质材料放置在模具上，放入真空冷冻干燥机中进行猪小肠粘膜下层基质材料的冷冻干燥。上述步骤(6)所述的模具包括带针底板与压框，将一或多层猪小肠粘膜下层基质材料平铺于所述带针底板上，将所述压框放置于猪小肠粘膜下层基质材料上，将所述带针底板和所述压框相对固定。本发明提到的模具，具体的结构可以参考发明专利ZL201310203602.2。真空冷冻干燥为：将带有小肠粘膜下层基质材料的模具放置于真空冷冻干燥机中；先预冻至-45℃，保温1-2小时；然后开启真空泵，调节温度至-15℃，保温5-7小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4小时，完成真空冷冻干燥；冷冻干燥装置的腔室内的压强为1-50Pa。
所述步骤(7)中，以由步骤(6)得到的经真空冷冻干燥的小肠粘膜下层基质材料为原料，将其缝制或粘接成袋状结构；所述袋状结构包括内腔、开口端和封闭端，所述开口端能够允许支撑构件(例如节育器)在所述开口端处进出所述袋状结构的内腔，所述内腔能够容纳所述支撑构件。
其中袋状结构可以使用的可降解线缝制，也可以通过可降解胶粘接而成袋状结构，所用的可降解胶例如是蛋白胶。
子宫呈倒置扁梨形，前面扁平，后面稍突出，壁宽腔小，上端宽而游离，朝前上方；下端较窄，呈圆柱状，插入阴道的上部。成年女性的子宫宫腔容量约5ml。所述袋状结构的封闭端的尺寸大于所述开口端的尺寸。袋状结构可以基本上呈梯形或三角形形状，或者将梯形或三角形的钝角或锐角部分裁切并缝制成弧线形。所述袋状结构能够保证节育器套管放入，且保证已经在其内打开的节育器不会掉出来。袋状结构具有不同的大小规格，适合不同个体需要。缝线使用可生物降解的外科手术线。缝制过程亦需控制无菌。袋状结构还可以呈圆形或椭圆，以安放于宫腔中。圆形或椭圆形的袋状结构也可以适用于相应形状的节育器。
本发明还提供宫腔内置物在宫腔内置医疗器械中的应用，宫腔内置医疗器械用于修复宫腔内膜或基底层损伤，用于预防和治疗宫腔粘连，用于防 止宫腔前后壁发生纤维化或瘢痕化，或者用于预防和治疗由宫腔粘连所导致的不孕、习惯性流产、月经过少、痛经、经血反流或闭经。
本发明提供一种宫腔内置物及其制备方法，经该制备方法成型的宫腔内置物，适用于不同大小的子宫。通过与不同类型的节育器等配合使用，针对性的修复子宫内膜不同部位的损伤。
本发明提供一种宫腔内置物，该产品由细胞外基质材料制成，所用细胞外基质材料优选具有较高的纯度的类型。优选材料的内毒素水平低于12EU/g，更优选低于5EU/g，最优选的是低于1EU/g。作为额外的偏好，ECM材料可的生物负载低于1CFU/g，更优选的是低于0.5CFU/g。真菌的水平低于1CFU/g，更优选的是低于0.5CFU/g。核酸水平最好小于5μg/mg，更优选的是小于2μg/mg，病毒含量优选小于50PFU/g，更优选的是小于5PFU/g。
本发明提供一种宫腔内置物，由小肠粘膜下层基质材料制成。小肠粘膜下层基质材料保留生长因子或其他的有益的生物活性成分。小肠粘膜下层基质材料包含碱性成纤维细胞生长因子(FGF-2)，转化生长因子-β(TGF-β)、表皮生长因子(EGF)，血管内皮细胞生长因子(VEGF)，和/或血小板衍生生长因子(PDGF)。细胞外基质材料还包含肝素、硫酸肝素、透明质酸和/或纤维粘连蛋白。通常来说，小肠粘膜下层基质材料可能包含一个生物活性成分，能够直接或间接的诱导细胞反应，如细胞在形态、分裂、生长、蛋白质或基因表达上的变化。
本发明提供一种宫腔内置物的制备方法中，可以在模具的带针底板上铺设一层或多层小肠粘膜下层基质材料，优选4-6层。
有益效果
相比现有技术，本发明具有以下显著优点：
小肠粘膜下层基质材料用于宫腔操作后前后壁创面的隔离，不会造成如宫内节育器等机械隔离对创面造成的二次伤害，也不会发生排异反应。
可选的，小肠粘膜下层基质材料用于宫腔操作后前后壁创面的隔离，不会产生如透明质酸膜、聚乳酸膜用于创面隔离的炎症反应。
可选的，小肠粘膜下层基质材料用于宫腔操作后前后壁创面的隔离，不会产生如新鲜羊膜用于创面隔离修复的排异反应。
可选的，小肠粘膜下层基质材料与节育器配合使用，有效解决了子宫 内膜不适合缝合的特殊问题，使用与宫腔形状契合的节育器将修复材料小肠粘膜下层基质固定于宫腔内，极大地提高了小肠粘膜下层基质材料发挥修复功能的水平。
可选的，本发明选用的小肠粘膜下层基质材料，DNA残留少，能够达到10ng/mg以下，半乳糖苷酶去除率较高，能够达到99％以上；且完整保留了天然细胞外基质的三维结构，保留了本源性有益成分，生长因子如碱性成纤维细胞生长因子(FGF2)、血管内皮细胞生长因子(VEGF)，透明质酸(HA)和硫酸氨基聚糖(sGAGs)等，保证了产品具有低的免疫原性，高的抗感染能力，并具有诱导细胞分化、促进细胞生长的修复功能。
可选的，本发明选用的小肠粘膜下层基质材料，因其在制备过程中将基质材料定型与脱水过程同步化，使多层结构致密，不分层，极大地提高脱细胞基质补片的力学性能与抗降解能力。并且通过控制灭菌工艺，能够有效的控制降解时间，可以根据需要制备不同使用周期的产品。
可选的，本发明采用更温和的胰蛋白酶和EDTA复合溶液以及双频超声处理相配合进行脱细胞处理，这一步骤是制造生物材料很重要的一个环节，只有将作为免疫原的细胞含量降至极低或完全去除，植入体内的材料才不会引发免疫反应，从而保证了材料的安全性；采用胰蛋白酶和EDTA使细胞与细胞外基质之间的连接被破坏，然后采用低频超声对细胞进行破碎，同时使用高频超声作用于破碎的细胞及细胞外基质，进一步使细胞脱离细胞外基质，从而达到脱细胞目的，针对脱细胞的各个环节采用对应的方法进行细节上的处理，使脱细胞效果更好，细胞残留更低。
根据下面参考附图对示例性实施例的详细说明，本发明的其它特征及方面将变得清楚。
附图说明
包含在说明书中并且构成说明书的一部分的附图与说明书一起示出了本发明的示例性实施例、特征和方面，并且用于解释本发明的原理。
图1是根据本发明一个实施方式的宫腔内置物所用的经脱细胞及成型处理的小肠粘膜下层基质材料的正面扫描电镜图。
图2是根据本发明一个实施方式的宫腔内置物所用的经脱细胞及成型 处理的小肠粘膜下层基质材料的侧面的扫描电镜图。
图3为根据本发明一个实施方式的宫腔内置物所用的小肠粘膜下层基质材料的X射线衍射谱(XRD)。
图4是根据本发明一个实施方式的宫腔内置物所用的小肠粘膜下层基质材料的亲水角测试图。
图5为宫腔内置物所用的小肠粘膜下层基质材料的红外图谱。
图6示出宫腔植入材料的脱细胞基质材料与现有基质材料的SDS-PAGE电泳结果图。
图7为经脱细胞处理后的小肠粘膜下层基质材料经Masson染色照片。
图8为经脱细胞处理后的小肠粘膜下层基质材料经Masson染色照片(小倍率)。
图9根据本发明的一个实施方式的宫腔内置物的示意图；
图10是根据本发明的另一个实施方式的宫腔内置物的示意图；
图11是根据本发明的另一个实施方式的宫腔内置物的示意图；
图12是一种节育器的示意图；
图13为一种节育器安放装置的示意图。
具体实施方式
以下将参考附图详细说明本发明的各种示例性实施例、特征和方面。附图中相同的附图标记表示功能相同或相似的元件。尽管在附图中示出了实施例的各种方面，但是除非特别指出，不必按比例绘制附图。
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。
图1和图2是本发明中宫腔内置物所用的经脱细胞及成型处理的小肠粘膜下层基质材料的正面和侧面的扫描电镜图。本发明的基质材料采用非交联工艺，将多层脱细胞基质复合后形成胶原蛋白微纤维网络，优秀的力学性能足以满足使用需求，因此整个生产过程中无需使用化学交联剂，避免了化学 交联产生的纤维包裹、糜烂、钙化及炎症等不良反应。从图1中可以看到小肠粘膜下层基质材料呈多孔网络结构，孔隙率可以达到90％以上，为引导细胞长入提供适宜的微环境。从图2中可以看到，小肠粘膜下层基质材料经过处理使胶原蛋白纤维形成层层叠加的多层结构，可以调整材料的降解性能与力学性能，以适应不同组织修复的需要。另外，小肠粘膜下层基质材料的顶破强度与缝合强度较高，可以应用于需要缝合、承力的手术条件。
图3为宫腔内置物所用的小肠粘膜下层基质材料的X射线衍射谱(XRD)结果，其中在2θ＝8°范围存在一个较尖锐的峰，而2θ＝15-25°存在一个较为平缓的圆峰，表明基质材料在经过脱细胞工艺与成型工艺处理后，已改变了原来胶原蛋白的晶体结构，形成无定形结构，从而使X射线的衍射峰变宽变圆。这样的微结构，能够极大消除材料在微结构中的各向异性。而材料微结构中的各向异性导致细胞生长分布的各向异性，形成某些方向上的优势生长，从而形成瘢痕。而本发明的宫腔内置物使细胞生长分布无各向异性，从而避免瘢痕产生，防止宫腔粘连。
图4为宫腔内置物所用的小肠粘膜下层基质材料的亲水角测试图。生物材料表面的亲疏水性对表面吸附蛋白质的构象具有重要的影响，宫腔内置物所用的小肠粘膜下层基质材料的亲水角为45°，非常有利于细胞的黏附和铺展。
图5为宫腔内置物所用的小肠粘膜下层基质材料的红外图谱。材料的红外图谱中主要峰均体现为I型胶原蛋白的官能团振动峰，其中3308.26cm -1峰值附近，是N-H伸缩振动；1640.33cm -1附近，是酰胺I带的C＝O伸缩振动，1548.46cm -1附近是酰胺Ⅱ带的N-H弯曲振动；1066.41-1548.46cm -1附近的吸收峰表明了胶原蛋白三股螺旋结构的完整性。另外，胶原的N-H伸缩振动位于3308.26cm -1说明了肽键间氢键的存在。1240cm -1与1450cm -1峰强度的比值为0.99，基本上与I型胶原蛋白特征值1.0相同。因此，基质材料中，I型胶原蛋白三股螺旋结构保持较完整。为进一步明确基质材料中含有I型胶原，图6示出宫腔植入材料的脱细胞基质材料与现有基质材料的SDS-PAGE电泳结果图。图6中，最左侧条纹为对应于不同分子量标记物条纹，中间条纹为现有基质材料的电泳条纹，最右侧条纹为采用本发明方法制备的宫腔植入材料的细胞外基质材料的电泳条纹，其中表示从上至下排列的条纹依次表示I型胶 原纤维、Γ链、β链、α 1链和α 2链。表面本发明中含有I型胶原蛋白。
此外，采用非交联工艺的基质材料成分除了I型胶原蛋白，还有少量的弹性蛋白与III型、IV型和VI型胶原蛋白。非交联工艺的基质材料含有有微量的活性生物分子，如纤维粘连蛋白、层粘连蛋白有利于细胞的粘附、生长；糖胺聚糖、蛋白多糖等分子作为生长因子结合位点，有利于保存各种生长因子，并阻碍胶原蛋白酶的降解，增加细胞迁移能力。此外，非交联工艺还使基质材料保留了原动物组织中的碱性纤维生长因子(FGF-2)、转化生长因子(TGF-β1)和血管内皮生长因子(VEGF)等，保留率高于45％，这些生长因子在组织血管化、功能重建过程起到关键性作用。与其他脱细胞材料所不同的是，小肠粘膜下层基质材料含有丰富的生物活性因子，这些活性因子使其能够积极诱导细胞进入材料，形成血管化组织，调控宿主组织的再生修复过程。
纤维粘连蛋白(fibronectin，FN)是一种大型的糖蛋白，存在于所有脊椎动物，分子含糖4.5-9.5％，糖链结构依组织细胞来源及分化状态而异。FN可将细胞连接到细胞外基质上。FN肽链中的一些短肽序列为细胞表面的各种FN受体识别与结合的最小结构单位。经质谱法测试，FN在宫腔植入材料所用的小肠粘膜下层基质材料的中的质量百分比含量>5％；经ELISA法检测，FN质量百分比含量在2.6％以上。
现有的细胞外基质材料采用皮肤、动脉、心包等弹性较大的组织，除I型胶原蛋白外，还含有较大含量的弹性蛋白。I型胶原蛋白强度比弹性蛋白强100倍以上，为细胞外基质提供力学强度，而弹性蛋白赋予基质优良的弹性，使细胞外基质可以伸长与收缩。由于弹性蛋白的存在，脱细胞基质的胶原蛋白不断降解，基质会因受力而伸长，不受力时则收缩变形，从而使组织无法重塑为正常形状，并因材料变形而使手术修复失败。另外，由于弹性蛋白在体内很难降解，而使脱细胞基质降解不完全，形成纤维包裹。而本发明所采用的粘膜下层材料中弹性蛋白含量极低，且经处理后，基本上被去除。图7为经脱细胞处理后的小肠粘膜下层基质材料经Masson染色照片，其中无弹性蛋白存留。
由于宫腔植入材料所用的小肠粘膜下层基质材料为动物源性脱细胞材料，来源于异种材料的相关免疫原应尽量去除。通过脱细胞工艺处理制备的产品HE染色切片照片如图8所示，未发现残留细胞核。且DNA残留达到 3.8ng/mg以下，远低于同类脱细胞材料水平，而α-Gal抗原清除率可达到99.4％。该项数据表明宫腔植入材料所用的基质材料脱细胞过程彻底，免疫原性去除率高，最大程度上降低了引起免疫排斥反应的风险。
实施方式1：
图9是根据本发明的一个实施方式的宫腔内置物的示意图，该宫腔内置物为袋状结构，外形大致为梯形，至少部分角部为弧形。开口2位于梯形较短的底边一侧。可以使用一片经真空冷冻干燥的小肠粘膜下层基质材料对折后制作，或者使用两片或多片基质材料制作。该宫腔内置物可以被放置于宫腔内并展开，从而达到隔离和保护创面，促进宫腔基底膜和粘膜等组织的恢复，防止粘连的发生。
进一步地，以上实施方式的宫腔内置物还可以包含支撑结构，从而在袋状结构内部撑开所述内置物，以进一步地防止发生宫腔粘连。支撑结构可以是气囊，优选Foley带气囊。通过预先将宫腔内置物包覆于未充气气囊表面，并置于宫腔内，后充气使气囊膨胀以支撑宫腔内置物，以进一步地防止发生宫腔粘连。
支撑结构还可以是节育器。图12是一种节育器的示意图；图13为一种节育器安放装置的示意图。在安放本实施方式的宫腔内置物1的过程中，可以将宫腔内置物1放置于宫腔内，通过将带有节育器3的节育器安放装置4穿过子宫口和宫腔内置物1的开口2，然后使节育器3脱离节育器安放装置4，进入宫腔内置物1的内腔，节育器3在构件1的内腔内自动扩张，与宫腔内置物1一起留置于宫腔内。
也可以将节育器3预先组装至宫腔内置物1的内腔中，并一起放置在节育器安放装置4中。然后使节育器安放装置4穿过子宫口，再使宫腔内置物1脱离节育器安放装置，进入宫腔，节育器3在构件的内腔内自动扩张并使宫腔内置物1扩张，从而使宫腔内置物1和节育器3一起形成的宫腔内置物留置于宫腔内。
实施方式2：
图10是根据本发明的另一种方式的宫腔内置物的示意图。该宫腔内置物1为袋状结构，外形大致为三角形，至少部分角部为弧形。开口2位于三角形的一个侧边上，开口2长度小于侧边长度，使得节育器3的各个端部可以卡 在大致呈三角形的袋状结构1的端部。在安放本实施方式的宫腔内置物的过程中，可以将节育器3预先放置于宫腔内置物1的内腔中，并共同放置于节育器安放装置4中。然后使节育器安放装置4穿过子宫口，再使宫腔内置物脱离节育器安放装置，进入宫腔，节育器3在构件的内腔内自动扩张并使宫腔内置物1扩张，从而使宫腔内置物1和节育器3一起形成的宫腔内置物留置于宫腔内。
实施方式3：
图11是根据本发明的另一种方式的宫腔内置物的示意图。该宫腔内置物1为袋状结构，外形大致呈圆形或椭圆形。开口2位于圆形或椭圆形的圆面上或圆边处。可以通过实施1或2的安放方式将节育器3和宫腔内置物1放置于宫腔内。
实施方式4：一种具有袋状结构的宫腔内置物的制备方法
(1)原料选择与初处理：取新鲜屠宰的猪小肠组织清洗洁净，分裂出小肠粘膜下层，将小肠粘膜下层组织材料分割，剔除淋巴组织，用自来水冲洗1-3次，再用纯化水冲洗至表面无污渍，然后将清洗后的猪小肠粘膜下层组织材料置于筛网等滤水装置上，静置五分钟以上，以将水滤干。
(2)病毒灭活：采用过氧乙酸-乙醇溶液浸泡猪小肠粘膜下层组织材料，该过程可在不锈钢桶中进行，过氧乙酸体积百分比浓度采用0.1％，乙醇体积百分比浓度采用5％，灭活时间2小时，溶液与猪小肠粘膜下层组织材料比例为5：1，温度为20℃；完成后在超声波清洗机中清洗数次至清洗液检测电导率为10以下终止。
(3)清洗：采用清洗液清洗猪小肠粘膜下层组织材料，清洗液为pH值为7.2-7.4的PBS溶液，PBS溶液温度为20℃，PBS溶液与猪小肠粘膜下层组织材料的比例(体积比)为30︰1，优选清洗3次，每次20分钟；然后采用纯化水清洗，纯化水与猪小肠粘膜下层组织材料比例为30︰1，至检测电导率为10μS/cm以下终止；清洗过程在超声波清洗机中进行，频率40kHz，功率3000W。
(4)脱细胞：脱细胞液采用含有质量百分比浓度0.1％的胰蛋白酶溶液和浓度为0.5mmol/L的EDTA的PBS溶液，脱细胞液pH＝7.2-7.5，超声振荡清洗30分钟，温度20℃，溶液与猪小肠粘膜下层组织材料比例为20：1，脱细胞 过程在双频超声波装置中进行，包含低频和高频两个频率，其中低频频率为20KHz，高频频率为80KHz，超声功率为5KW，其中低频处理10min，高频处理10min，温度为30℃；功率5000W。
(5)清洗：采用清洗液进行清洗，清洗过程在超声波清洗机中进行，超声波的功率为3000W以上。清洗液为pH7.2-7.4的PBS溶液，PBS溶液温度为20℃，PBS溶液与猪小肠粘膜下层组织材料的比例(体积比)为30︰1，优选清洗3次，每次20分钟；然后采用20℃的降温的注射用水清洗，注射用水与猪小肠粘膜下层组织材料比例(体积比)为30︰1，检测清洗注射用水与未清洗注射用水电导率之差小于1μS/cm终止，频率优选40kHz，功率优选3000W以上。
(6)真空冷冻干燥：将一层或多层由步骤(5)得到的猪小肠粘膜下层基质材料放置在模具上，放入真空冷冻干燥机中进行猪小肠粘膜下层基质材料的冷冻干燥。上述步骤(6)所述的模具包括带针底板与压框，将一或多层猪小肠粘膜下层基质材料平铺于所述带针底板上，将所述压框放置于猪小肠粘膜下层基质材料上，将所述带针底板和所述压框相对固定。本发明提到的模具，具体的结构可以参考发明专利ZL201310203602.2。真空冷冻干燥为：将带有小肠粘膜下层基质材料的模具放置于真空冷冻干燥机中；先预冻至-45℃，保温1小时；然后开启真空泵，调节温度至-15℃，保温6小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4小时，完成真空冷冻干燥；冷冻干燥装置的腔室内的压强为25-30Pa。得到冷冻干燥后的小肠粘膜下层基质材料。
(7)成型：取由步骤(6)得到的冷冻干燥后的小肠粘膜下层基质材料，以折叠处为长底边缝制梯形的袋子。缝制成的袋子，短底边处开口，短底边约为长底边的1/3左右，能够保证节育器套管放入，且保证已经在其内打开的节育器不会掉出来。设计为不同的大小规格，适合不同个体需要。缝线使用可生物降解的外科手术线。缝制过程亦需控制无菌。
本实施方式还可以进一步包括以下步骤：
打孔包装步骤(8)，所述步骤(8)打孔包装，具体为：干燥材料在模具上切割成固定的形状(包括方形、圆形或其他形状)，然后放入机械打孔机中，以间距0.9cm进行打孔，孔直径1.5毫米，然后采用特卫强包装袋包装。
灭菌解析步骤(9)，所述步骤(9)灭菌解析步骤具体为：采用环氧乙烷进 行灭菌，灭菌条件为：先温度20-40℃保温时间2-4小时，湿度30-70％(优选60％)，然后通入浓度300-1000mg/L(优选800mg/L)环氧乙烷，灭菌4-8小时；解析过程在通风的解析室中进行，温度控制在10-30℃之间，时间14-28天。
还可以根据实际需要，在真空冷冻干燥步骤(6)与成型步骤(7)之间完成打孔，相应地，包装步骤中，则无需再次打孔。
上述打孔的步骤可以避免袋状内置物中积存液体，而引发感染。打孔步骤利于组织修复。
对上述实施例所制备的生物材料进行理化性质、生物学检测。
1.对制备的生物材料进行物理性能检测
1)孔隙率测定：采用孔隙率测定仪测定材料的孔隙率，实施方式4提供的样品的孔隙率为90％。
2)缝合保持力检测：方法：用2-0号外科缝线或相同直径的不锈钢丝缝合在生物材料一端边缘2毫米处，将缝线或不锈钢丝与生物材料的另一端固定在拉力仪上，以20mm/min的速度进行拉伸，直到缝合点被撕裂，记录下缝合点被撕裂时的拉力。按上述方法对3批样品进行检测。结果：缝合抗拉强度大于或等于6N。
3)抗张强度检测方法：方法：使用拉伸(压缩)试验机，将补片裁剪成条状试样，裁剪后在相对湿度40％-60％，温度为22℃±2℃的条件下放置2小时后立即进行试验。将试样两端固定在拉伸试验机的夹头上，以100mm/min的速度依次向外拉伸直到试样断裂，以N为单位记录下试样断裂时的力。按照上述方法对3批样品进行检测。结果大于200N。
4)爆破强度检测：方法，使用拉伸(压缩)试验机，将材料裁剪成23×23mm的正方形式样备用，在相对湿度为40％-60％，温度为22℃±2℃的条件下放置2小时后立即进行试验。用环形夹具将试样固定在拉伸试验机的工作台上，使球形探头以750mm/min的速度穿过试样，记录下探头穿破试样的力。按上述方法对3批样品进行检测。结果：爆破强度大于120N。
2.化学性能检测
1)检验液制备：取样品的厚度均匀部分，切成1cm 2的碎片，用水洗净后晾干，然后加入玻璃容器中，按样品总表面积(cm 2)与水(mL)的比为5:1的比例加水，加盖后置于压力蒸汽灭菌器中，在121℃加热30min，加热结束后 将样品与液体分离，冷至室温作为检验液。取同体积水置于玻璃容器中，同法制备空白对照液。
2)病毒检测：选择伪狂犬病毒为指示病毒，采用实时定量PCR法检测病毒的DNA拷贝数，检测3批样品。结果：病毒DNA拷贝数为0。
3)酸碱度：按GB/T 14233.2-2005中5.4.1《医用输液、输血、注射器具检验方法第1部分：化学分析法》中规定的方法试验，结果：检验液与空白对照液的PH值之差不超过1.5。
4)DNA残留检测：依据生物制剂残留DNA检测方法《中国药典》2015年版第四部，采用荧光染色法检测实施例所提供的样品DNA残留量。结果：实施例所提供的样品的DNA残留量小于10ng/mg。
5)细菌内毒素：按照GB/T 14233.2-2005《医用输液、输血、注射器具检验方法第2部分：生物学试验方法》进行检测，共3批样品，结果：细菌内毒为20EU/套。
6)环氧乙烷残留：按GB/T14233.1-2008《医用输液、输血、注射器具检验方法第1部分：化学分析法》中9的规定的方法试验，结果：产品环氧乙烷残留量不超过10μg/套。
3.动物实验
动物模型选取12周龄成年雌性新西兰大白兔，体重2500克左右，普通级。实验前24h每只兔耳缘静脉注射绒毛膜促性腺激素50IU，使内膜生长同步。取下腹正中长约2cm纵切口进腹，查见双侧子宫，根据分组给予相应处理后缝合腹部切口。实验分组：A组：对照组：右侧子宫，于子宫中下1/3处作1.5cm纵切口，经切口放置袋状子宫修复材料大小3*0.5cm。缝合，作为补片对照。左侧子宫仅做子宫切口后缝合作为空白对照。B组：建模组：机械损伤建模法：右侧子宫，于子宫中下1/3处作1.5cm纵切口，切除半侧宫腔内膜及粘膜下层；左侧子宫，切除半侧宫腔内膜及粘膜下层，生理盐水冲洗宫腔。经切口放置子宫修复材料大小3*0.5cm。C组：治疗组：右侧子宫，于子宫中下1/3处作1.5cm纵切口，切除全宫腔内膜及粘膜下层；左侧子宫宫腔切除全宫腔内膜及粘膜下层。生理盐水冲洗宫腔。经切口放置子宫修复材料大小3*0.5cm。围手术期应用抗生素3天。分别于手术后7d，14d，28d处死动物，留取子宫标本，行HE染色和Masson染色，明确粘连情况及子宫内膜腺 体数量、创面纤维化程度。结果表明，以A组：对照组中样品空白对照组与空白对照组为基线，观察B组：建模组、C组：治疗组中，在造模后创面无子宫修复材料的宫腔修复时纤维化面积比例增加，腺体数量减少，发生明显粘连，妊娠能力明显下降，有显著差异(P＜0.05)。而在造模后创面加子宫修复材料的宫腔修复时纤维化面积比例、腺体数量减少两项指标与对照组相比无明显差异，未发现粘连发生，相比于无修复材料组明显改善子宫内膜修复过程，有显著差异(P＜0.05)。
Figure PCTCN2018077812-appb-000001
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (13)

  1. 一种宫腔内置物，其特征在于，所述宫腔内置物具有袋状结构，所述宫腔内置物包括经脱细胞处理的动物小肠粘膜下层基质材料。
  2. 根据权利要求1所述的宫腔内置物，其特征在于，所述小肠粘膜下层基质材料包含碱性纤维生长因子、转化生长因子和血管内皮生长因子。
  3. 根据权利要求1或2所述的宫腔内置物，其特征在于，所述小肠粘膜下层基质材料中纤粘连蛋白的质量百分比含量在2％以上。
  4. 根据权利要求1所述的宫腔内置物，其特征在于，所述袋状结构具有内腔和开口，所述内腔由所述小肠粘膜下层基质材料围成。
  5. 根据权利要求4所述的宫腔内置物，其特征在于，所述宫腔内置物还包括支撑构件，所述宫腔内置物的内腔能够容纳所述支撑构件，所述支撑构件能够在所述开口处进出所述袋状结构的所述内腔。
  6. 根据权利要求5所述的宫腔内置物，其特征在于，所述支撑构件为节育器或气囊。
  7. 根据权利要求1-6中的任意一项所述的宫腔内置物，其特征在于，所述内置物的形状基本上呈以下形状：梯形，所述梯形底边长为0.5-6cm，高度为0.5-7.5cm，所述开口位于较短底边处；优选地，所述梯形的至少一个角部为弧线形；三角形，所述三角形底边长为0.5-6cm，高度为0.5-7.5cm，所述开口位于一个侧边上；优选地，所述三角形的至少一个角部为弧线形；圆形，所述圆形直径为0.5-7.5cm，所述开口位于圆形的圆边处或圆形的圆面上；或者椭圆形，所述椭圆形短轴为0.5-6cm，长轴为0.5-7.5cm，所述开口位于椭圆形的圆边处或圆面上。
  8. 根据权利要求7中的任意一项所述的宫腔内置物，其特征在于，所述袋状结构表面打有孔；优选地，所述孔间距0.2-1厘米，孔直径0.5-3毫米。
  9. 一种宫腔内置物的制备方法，其特征在于，包括以下步骤：(1)组织前置处理；(2)病毒灭活：采用过氧乙酸-乙醇溶液浸泡小肠粘膜下层组织材料进行病毒灭活；(3)清洗；(4)脱细胞：脱细胞液为溶有胰蛋白酶和EDTA的PBS溶液，脱细胞过程在多频超声波装置中进行；(5)清洗，得到小肠粘膜下层基质材料；(6)真空冷冻干燥：在真空冷冻干燥机中进行；(7)成型：将由步骤(6)得到的经真空冷冻干燥的小肠粘膜下层基质材料制成袋状结构。
  10. 根据权利要求9所述的制备方法，其特征在于，所述步骤(4)中，脱细 胞液中胰蛋白酶的质量百分比浓度为0.01-0.2％，优选0.02-0.05％；EDTA的浓度为0.1-1mmol/L，优选0.4-0.8mmol/L；脱细胞液的pH值为7.0-8.0，优选为7.2-7.5；所述脱细胞液与小肠粘膜下层组织材料体积比为(20-40)︰1，脱细胞过程在双频超声波装置中进行，其中低频频率范围为20-40KHz，高频频率为60-90KHz，其中低频处理5-40min，高频处理5-40min，脱细胞液的温度范围为20-35℃；超声功率5000W以上。
  11. 根据权利要求9所述的制备方法，其特征在于，所述步骤(6)中，将一层或多层由步骤(5)得到的小肠粘膜下层基质材料放置在模具上，放入真空冷冻干燥机中进行小肠粘膜下层基质材料的冷冻干燥；其中将带有小肠粘膜下层基质材料的模具放置于真空冷冻干燥机中；先预冻至-45℃，保温1-2小时；然后开启真空泵，调节温度至-15℃，保温5-7小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4小时，完成真空冷冻干燥；冷冻干燥装置的腔室内的压强为1-50Pa。
  12. 根据权利要求9中任意一项所述的制备方法，其特征在于，所述步骤(7)中，所述袋状结构包括内腔和开口，所述开口能够允许支撑构件在所述开口处进出所述袋状结构的内腔，所述内腔能够容纳所述支撑构件。
  13. 一种宫腔内置物在宫腔内置医疗器械中的应用，其特征在于，所述宫腔内置医疗器械用于修复宫腔内膜或基底层损伤，用于预防和治疗宫腔粘连，用于防止宫腔前后壁发生纤维化或瘢痕化，或者用于预防和治疗由宫腔粘连所导致的不孕、习惯性流产、月经过少、痛经、经血反流或闭经。
PCT/CN2018/077812 2017-03-03 2018-03-02 一种宫腔内置物、制备方法及其应用 WO2018157848A1 (zh)

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