WO2014190618A1 - 一种动物源性植入性医用生物材料的制备方法 - Google Patents

一种动物源性植入性医用生物材料的制备方法 Download PDF

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WO2014190618A1
WO2014190618A1 PCT/CN2013/081050 CN2013081050W WO2014190618A1 WO 2014190618 A1 WO2014190618 A1 WO 2014190618A1 CN 2013081050 W CN2013081050 W CN 2013081050W WO 2014190618 A1 WO2014190618 A1 WO 2014190618A1
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animal
implantable medical
preparing
biomaterial according
medical biomaterial
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PCT/CN2013/081050
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English (en)
French (fr)
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赵博
王振军
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北京博辉瑞进生物科技有限公司
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Priority to US14/894,537 priority Critical patent/US9642937B2/en
Priority to DE112013007127.4T priority patent/DE112013007127B4/de
Priority to JP2016515599A priority patent/JP6189529B2/ja
Priority to GB1522042.9A priority patent/GB2530448B/en
Publication of WO2014190618A1 publication Critical patent/WO2014190618A1/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
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation

Definitions

  • the invention relates to the technical field of medical biomaterials, and in particular to a method for preparing an animal-derived implantable medical biomaterial.
  • the loss of certain tissues or organs and the loss of some or all of the functions caused by various diseases and traumas are one of the major hazards faced by human health.
  • Research and development of ideal materials for tissue repair has been an important topic in the fields of medicine, biosciences and materials science.
  • the medical biomaterials currently used in clinical repairs for tissue repair are mainly non-absorbable artificial materials, including polymer materials (such as polypropylene, polytetrafluoroethylene, polylactic acid, polyglycolic acid, silica gel, etc.) and metal materials (such as stainless steel, titanium and its alloys, inorganic materials (such as bioactive ceramics, hydroxyapatite, etc.), composite materials (carbon fiber / polymer, glass fiber / polymer, etc.).
  • the structure and composition of the above materials are far from the human tissue, and can only serve as a substitute and support in a short period of time, can not promote tissue regeneration, achieve tissue function, and can not be degraded after implantation, if not removed by surgery will be permanent In the body, its stability, tissue toxicity and carcinogenicity are difficult to control.
  • ECM extracellular matrix
  • tissue defects A serious consequence of tissue defects is the loss of “soil” and ECM, which is why the body itself cannot achieve tissue repair and regeneration.
  • Natural ECM can be used as a "soil” for tissue regeneration and is an ideal tissue repair material. Removal of cellular components from animal tissues removes most of the immunogenicity and preserves ECM components, allowing the development of ideal tissue repair materials.
  • SIS small intestinal submucosa
  • the advantages of decellularized SIS matrix materials include: 1) low immunogenicity and high histocompatibility; 2) unique structures and compositions with biological basis for active induction of various tissue regeneration; 3) wide range of applications, applicable to Repair of various soft tissues of the body; 4) Anti-microbial activity Sex.
  • Cook Biotech Incorporated's decellularized SIS matrix material has a large sample size in the fields of abdominal wall repair, burns, anal fistula, refractory wounds, plastic surgery, pelvic floor repair, tendon repair, genitourinary repair, nerve repair, etc. Clinical application.
  • the decellularization process and the virus inactivation process are the main processes and technical difficulties in the preparation of the decellularized SIS matrix material, requiring complete removal of the virus, cellular components and animal-derived DNA components of the submucosal tissue of the small intestine, while completely retaining the components and three-dimensional structure of the natural ECM.
  • growth factors such as alkaline growth factors, transforming growth factors, etc.
  • a variety of methods for decellularization and virus inactivation have been reported, but most of them cannot completely remove all animal-derived DNA components, and most of them are too long and require a variety of organic and high-strength acid-base solvents, resulting in decellularized SIS.
  • most decellularization processes do not have effective measures to control endotoxin residues.
  • the molding process is another technical difficulty in the preparation of the decellularized SIS matrix material.
  • the circumference of the small intestine is only 6 ⁇ 8cm, and the thickness of the small intestinal submucosa is less than 0.1mm. It is difficult to prepare tissue repair products of different sizes, thicknesses and different mechanical strength requirements. .
  • Cook Biotech Incorporated is formed by vacuum pressing, which compresses the spatial structure of the decellularized SIS matrix material, destroying the natural ECM three-dimensional structure and affecting the porosity of the material.
  • the technical problem to be solved by the present invention is to provide a method for preparing an animal-derived implantable medical biomaterial, and the animal-derived decellularized ECM material prepared by the method completely removes the animal source cell component and the DNA component, and is completely intact. It retains the natural ECM composition, three-dimensional structure and active growth factors that can induce tissue regeneration, without endotoxin, organic solvents and toxic solvent residues, and can form products with different sizes, thicknesses and mechanical strengths according to different indications. .
  • a method for preparing an animal-derived implantable medical biomaterial comprising the following steps:
  • the animals mentioned here theoretically include all animals, Preferred are pigs, cows, horses, and more preferably pigs.
  • the tissue includes the small intestinal submucosa, the dermis, and the pericardium.
  • the virus is inactivated by a low concentration peracetic acid-ethanol solution.
  • the step is carried out in a oscillating constant temperature ultrasonic cleaner in a washing tank, wherein the volume percentage of peracetic acid is 0.05 to 0.2% (preferably 0.1%).
  • the inactivation time is 1 to 2 h (preferably 1 h)
  • the oscillation frequency of the washing tank is 30 to 600 rpm (preferably 100 to 300 rpm, more preferably 200 rpm)
  • the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, further preferably 35 ⁇ 50KHZ, optimally 45KHZ)
  • the temperature range is 4 ⁇ 40°C
  • the phosphate buffer solution is prepared by weighing 7.9 g NaCl, 0.2 g KCK 0.24 g KH 2 P0 4 1.8 g K 2 HP0 4 , dissolved in 800 ml of distilled water, and adjusting the pH of the solution to 7.4 with HC1. Then add distilled water to a volume of 1 L.
  • This step is carried out in a constant temperature ultrasonic cleaner in which the washing tank can be oscillated.
  • the material is placed in the washing tank, the sodium hydroxide solution is injected into the washing tank, and the washing device is turned on, and the washing time is 5 to 30 min (preferably 20 min).
  • the concentration of the sodium hydroxide solution is 5 to 100 mmol/L (preferably 5 to 20 mmol/L, further preferably 10 mmol/L), and then the washer is turned off, the sodium hydroxide solution is poured out, the phosphate buffer is injected, and the washer is turned on.
  • the cleaning time is 5 ⁇ 20min (preferably 15min)
  • the phosphate buffer cleaning is repeated 2 ⁇ 5 times
  • the pH value of the phosphate buffer after washing is detected, and when the pH reaches 6.5-7.5, the flowing water for injection cleaning is used.
  • the detection is terminated when the conductivity reaches 1.5 um/s or less.
  • the oscillating frequency of the washing tank is 100 to 300 rpm (more preferably 200 rpm)
  • the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, more preferably 35 to 50 kHz, and most preferably 45 kHz).
  • the phosphate buffer solution in this step is prepared in the same manner as in step 2.
  • This step is carried out in a constant temperature ultrasonic cleaner in which the washing tank can be oscillated.
  • the sodium chloride solution is injected into the washing tank, and the washing device is turned on.
  • the washing time is 5 to 30 min (preferably 20 min), and the concentration of the sodium chloride solution is 0.015 mol/ L or 2 mol/L (preferably 0.015 mol/L), pH not exceeding 7.8, and then the material is washed with flowing water for injection, and the conductivity is terminated when the conductivity is less than 1.5 um/s.
  • the oscillating frequency of the washing tank is 100 to 300 rpm (further preferably 200 rpm), and the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, further preferably 35 to 50 kHz, and most preferably 45 kHz).
  • This step consists of three steps of fixture fixation, freeze drying and laser micropore punching.
  • the corresponding size and shape of the tool (preferably made of stainless steel) is designed to fix the material on the tool, according to different product requirements. Overlap multiple layers, place the material that has been cleaned with water for injection and fixed in the tool in a freeze dryer, freeze-dry according to a pre-designed freeze-drying process: pre-freeze to 25 ⁇ 50 °C (preferably 25°) C), keep warm for 0.5 ⁇ 4 hours (preferably 2h), heat up to 15 °C, keep warm for 4 ⁇ 12 hours (preferably 8h), heat up 15 °C, keep warm for 0.5 ⁇ 4 hours (preferably 2h), heat up to 25 °C, keep warm for 4 hours.
  • the laser microporous punch is used for punching, and the pore diameter ranges from 0.05 to 1 mm (preferably 0.2 to 0.5 mm), and the pore spacing is 0.1 to 2 cm (preferably 0.5 to 1 cm).
  • the laser microporous punching refers to the use of laser technology to punch the material into micrometer-sized pores, and the laser microporous punching machine is used to make pores on the surface of the material to facilitate tissue repair.
  • the ultrasonic cleaning machine capable of oscillating in the cleaning tank of the present invention combines the cleaning tank of the conventional ultrasonic cleaning machine with the mechanical oscillator, so that the cleaning tank can mechanically oscillate while ultrasonic cleaning, and realizes mechanical oscillation and ultrasonic cleaning. At the same time, they work together.
  • the invention relates to a method for preparing an animal-derived implantable medical biomaterial, which can be used for preparing acellular submucosal submucosal matrix material, acellular dermal matrix material, and decellularized pericardial matrix material.
  • the invention uses an ultrasonic cleaning machine which can oscillate in the cleaning tank, realizes the simultaneous combination of mechanical oscillation and ultrasonic cleaning, and improves the animal origin.
  • the efficiency of the virus inactivation process, the decellularization process and the process of removing the animal source DNA in the preparation of the cell ECM material greatly reduces the process time and simplifies the process.
  • the entire preparation process uses only peroxyacetic acid-ethanol, sodium hydroxide and chlorine.
  • Figure 1 is a schematic view showing the cutting of the sample in the second embodiment of the present invention.
  • Figure 2 is an optical micrograph of Embodiment 2 of the present invention.
  • Fig. 3 is a view showing an ultrastructural view of an electron microscope in Embodiment 2 of the present invention.
  • Example 1 Preparation of acellular porcine small intestine submucosa matrix material 1. Pretreatment separation and initial washing of animal tissue materials
  • the small intestine tissue of freshly slaughtered pigs was cleaned, the submucosal layer of the small intestine was separated, and washed with water for injection 3 times.
  • the virus is inactivated by a low concentration peracetic acid-ethanol solution.
  • the step is carried out in a oscillating constant temperature ultrasonic cleaner in a washing tank, wherein the volume percentage of peracetic acid is 0.05 to 0.2% (preferably 0.1%).
  • the inactivation time is 1 to 2 h (preferably 1 h)
  • the oscillation frequency of the washing tank is 30 to 600 rpm (preferably 100 to 300 rpm, more preferably 200 rpm)
  • the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, further preferably 35 ⁇ 50KHZ, the most is 45KHZ)
  • the temperature range is 4 ⁇ 40°C, then wash it in phosphate buffer for 2 ⁇ 5 times, each time for 15min, check the pH of the phosphate buffer after washing, when the pH After reaching 6.5-7.5, the material is washed with running water for injection, and the conductivity is terminated when the conductivity reaches 1.5 um/s or less.
  • the phosphate buffer solution is prepared by weighing 7.9 g NaCl, 0.2 g KCK 0.24 g KH 2 P0 4 1.8 g K 2 HP0 4 , dissolved in 800 ml of distilled water, and adjusting the pH of the solution to 7.4 with HC1. Then add distilled water to a volume of 1 L.
  • This step is carried out in a constant temperature ultrasonic cleaner in which the washing tank can be oscillated.
  • the material is placed in the washing tank, the sodium hydroxide solution is injected into the washing tank, and the washing device is turned on, and the washing time is 5 to 30 min (preferably 20 min).
  • the concentration of the sodium hydroxide solution is 5 to 100 mmol/L (preferably 5 to 20 mmol/L, further preferably 10 mmol/L), and then the washer is turned off, the sodium hydroxide solution is poured out, the phosphate buffer is injected, and the washer is turned on.
  • the cleaning time is 5 ⁇ 20min (preferably 15min)
  • the phosphate buffer cleaning is repeated 2 ⁇ 5 times
  • the pH value of the phosphate buffer after washing is detected, and when the pH reaches 6.5-7.5, the flowing water for injection cleaning is used.
  • the detection is terminated when the conductivity reaches 1.5 um/s or less.
  • the oscillating frequency of the washing tank is 100 to 300 rpm (more preferably 200 rpm)
  • the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, more preferably 35 to 50 kHz, and most preferably 45 kHz).
  • the phosphate buffer solution in this step is prepared in the same manner as in step 2.
  • This step is carried out in a constant temperature ultrasonic cleaner in which the washing tank can be oscillated.
  • the sodium chloride solution is injected into the washing tank, and the washing device is turned on.
  • the washing time is 5 to 30 min (preferably 20 min), and the concentration of the sodium chloride solution is 0.015 mol/ L or 2 mol/L (preferably 0.015 mol/L), pH not exceeding 7.8, and then the material is washed with flowing water for injection, and the conductivity is terminated when the conductivity is less than 1.5 um/s.
  • the oscillating frequency of the washing tank is 100 to 300 rpm (further preferably 200 rpm), and the ultrasonic frequency is 20 to 80 kHz (preferably 20 to 50 kHz, further preferably 35 to 50 kHz, and most preferably 45 kHz).
  • stainless steel tools of corresponding size and shape are designed, and the materials are fixed on stainless steel tools.
  • the layers can be overlapped according to different product requirements.
  • the materials that have been cleaned with water for injection and fixed in the tool are placed in the frozen.
  • freeze-dry In the dryer, freeze-dry according to the pre-designed freeze-drying process: pre-freeze to 25 ⁇ 50 °C (preferably 25 °C), keep warm for 0.5 ⁇ 4 hours (preferably 2h), heat up to 15 °C, keep warm 4 ⁇ 12 hours (preferably 8h), heating at 15 °C, holding for 0.5 ⁇ 4 hours (preferably 2h), heating to 25 °C, holding for 4 hours, after lyophilization, punching with laser microporous punching machine,
  • the pore size ranges from 0.05 to 1 mm (preferably from 0.2 to 0.5 mm), and the pore spacing is from 0.1 to 2 cm (preferably from 0.5 to 1 cm).
  • Example 2 Physicochemical properties, histology, growth factors and biological properties of the decellularized small intestinal submucosa matrix material prepared in Example 1
  • the physical properties of the prepared 8-layer materials were tested.
  • the test items included suture retention, tensile strength, burst strength and porosity.
  • Stitch retention test Method: Use 2-0 surgical suture or stainless steel wire of the same diameter to suture 2 mm at the edge of one end of the 8-layer material, and fix the suture or stainless steel wire to the other end of the 8-layer material. Tensile force was applied 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 is greater than or equal to 5 ⁇ 0.5N.
  • Tensile strength test method Method: Using a tensile (compression) test machine, according to Figure 1, cut 8 layers of material into a sample, after cutting, the relative humidity is 40%-60%, the temperature is 22°. The test was carried out immediately after 2 h at C ⁇ 2 °C. The two ends of the sample were fixed on the chuck of the tensile tester, and were stretched outward at a speed of lOOmm/min until the sample was broken, and the longitudinal sample and the transverse sample were tested separately. Record the force at which the sample breaks in units of N. Three batches of samples were tested as described above. Result: Portrait: 15N, Landscape: 8N.
  • Blasting strength test Method: Using a tensile (compression) test machine, cut 8 layers of material into 23 X 23mm square samples for use at a relative humidity of 40%-60% and a temperature of 24 °C ⁇ 2°. The test was carried out immediately after being placed for 2 hours under the conditions of C. 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.
  • Porosity determination The porosity of the material is determined by mercury intrusion method. Result: The porosity is not less than 85%.
  • test solution Take the thickness of the sample, cut into lcm 2 pieces, wash with water, dry, and then add to the glass container, according to the total surface area (cm 2 ) and water inside and outside the sample ( The ratio of mL) is 5:1. Add water, cover and place in a pressure steam sterilizer, heat at 121 °C ⁇ rC for 30 min. After heating, separate the sample from the liquid and cool to room temperature as 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 Method: Select pseudorabies virus as indicator virus, use real-time quantitative PCR to detect viral DNA copy number, and test 3 batches of samples. Result: Virus DNA copy number 0.
  • Endotoxin Prepare test solution according to the ratio of 6cm 2 sample plus lml extraction medium, 37 ⁇ C, 72 ⁇ 2hr, extraction medium: normal saline. According to the method specified in GB/T 14233.2 -2005 ("Test methods for medical infusion, blood transfusion, injecting instruments, part 2: biological test methods"), three batches of samples were tested. Results: The endotoxin content was less than 5 EU/g.
  • DNA residue detection DNA residue of the sample provided in Example 1 was detected by fluorescence staining according to the method for detecting residual DNA of biological agents (Chinese Pharmacopoeia 2010, Appendix IX-B Exogenous DNA Residue Determination) the amount. Result: The DNA residue of the material did not exceed 150 pg/g.
  • the test solution was prepared according to the ratio of 6 cm 2 sample plus 1 ml of the extraction medium, 37 ⁇ C, 72 ⁇ 2 hr, and the extraction medium: physiological saline.
  • the alkaline growth factor (bFGF) and vascular endothelial growth factor (VEGF) levels in the extract were determined by ELLISA method. Results: The bFGF content was 121.8 ⁇ 2.683 ng/L, and the VEGF content was 93.8 ⁇ 3.033 ng/L.
  • Bio performance testing, testing items including cytotoxicity, delayed type hypersensitivity, intradermal reaction.
  • Cytotoxicity Method: Prepare test solution according to the ratio of 6cm 2 sample plus lml extraction medium, 37 ⁇ 1 °C, 24 ⁇ 2 hr, extraction medium: serum-containing MEM medium. The test solution was tested in accordance with the test method specified in GB/T 16886.5-2003 ("Biological evaluation of medical devices Part 5: In vitro cytotoxicity test"). Results: The cytotoxic response was less than or equal to grade 1.
  • Delayed type hypersensitivity reaction Method: The ratio of 6cm 2 sample plus lml extraction medium, 37 ⁇ 1 °C, 72 ⁇ The test solution was prepared in 2 hr, and the leaching medium was physiological saline and cottonseed oil. The test was carried out in accordance with the test method specified in GB/T 16886.10-2005 ("Medical Device Biological Evaluation Part 10: Stimulating and Delayed Hypersensitivity Test"). Results: There was no delayed hypersensitivity reaction.
  • Intradermal reaction The test solution was prepared according to the ratio of 6 cm 2 sample plus 1 ml of the extraction medium, 37 ° C, 72 ⁇ 2 hr, and the extraction medium was physiological saline and cottonseed oil. The test was carried out in accordance with the test method specified in GB/T 16886.10-2005 ("Medical Device Biological Evaluation Part 10: Stimulating and Delayed Hypersensitivity Test"). Results: The difference between the average score of the test sample and the solvent control was less than 1.0.
  • Example 3 Preparation of acellular porcine dermal matrix material
  • the dermal tissue of freshly slaughtered pigs was taken as a raw material, and the preparation method was the same as in Example 1.
  • Example 4 Physicochemical properties, histology, growth factor and biological properties of the acellular porcine dermal matrix material prepared in Example 3
  • the acellular dermal matrix material prepared in Example 3 had a suture tensile strength greater than 5 N, a transverse and longitudinal tensile strength greater than 20 N, a burst strength greater than 120 N, a porosity greater than 80%, and a viral DNA copy number of 0.
  • the endotoxin content is less than 5 EU/g, the DNA residue is not more than 150 pg/g, there is no delayed type hypersensitivity reaction, and no intradermal reaction.
  • Example 5 Preparation of acellular pig pericardium matrix material
  • the pericardial tissue of freshly slaughtered pigs was taken as a raw material, and the preparation method was the same as in Example 1.
  • Example 6 Physicochemical properties, histology, growth factor and biological properties of the decellularized pig pericardium matrix material prepared in Example 5
  • the acellular dermal matrix material prepared in Example 5 had a suture tensile strength greater than 5 N, a transverse and longitudinal tensile strength greater than 20 N, a burst strength greater than 120 N, a porosity greater than 85%, and a viral DNA copy number of 0.
  • the endotoxin content is less than 5 EU/g, the DNA residue is not more than 150 pg/g, there is no delayed type hypersensitivity reaction, and no intradermal reaction.

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Abstract

一种动物源性植入性医用生物材料的制备方法，它包括以下操作步骤：动物组织材料的前置处理与初洗；病毒灭活；脱细胞；氯化钠处理；成型；包装灭菌。用该方法制备的动物源性脱细胞ECM材料完全去除了动物源细胞成分和DNA成分，同时完整的保留了天然ECM的成分、三维结构和能够诱导促进组织再生的活性生长因子，无内毒素、有机溶剂和有毒溶剂残留，并可根据不同的适应症形成具有不同尺寸、厚度和力学强度的产品。

Description

一种动物源性植入性医用生 料的制备方法
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性植入性医用生物材料的 制备方法。
背景技术
各种疾病、创伤所造成的机体某些组织或器官的缺损以及功能的部分或全部丧失是 人类健康所面临的主要危害之一。研究和开发理想的材料用于组织修复一直是医学、 生 物科学以及材料科学等领域的重要课题。 目前广泛应用于临床的用于组织修复的医学生 物材料主要是不可吸收的人工材料， 包括高分子材料（如聚丙烯、聚四氟乙烯、聚乳酸、 聚乙醇酸、 硅胶等）、 金属材料 （如不锈钢、 钛金属及其合金等）、 无机材料 （如生物活 性陶瓷， 羟基磷灰石等）、 复合材料 （碳纤维 /聚合物、 玻璃纤维 /聚合物等）。 上述材 料的结构及组成均与人体组织相差甚远， 只能在短期内起到替代和支撑作用， 不能促进 组织再生， 实现组织功能， 并且， 植入后不能降解， 如不通过手术取出会永久存留体内， 其稳定性、 组织毒性反应及致癌性等均难以控制。
在欧美发达国家， 植入性医用生物材料正进入重大产业革命时期， 即从传统的不可 吸收材料向可降解的、 能够主动诱导组织再生的新型生物材料变革。 而基于组织工程学 原理的以动物组织为原料的细胞外基质（extracellular matrix, ECM)材料是主要发展方 向。 ECM 是由多种大分子物质如胶原、 非胶原糖蛋白、 氨基聚糖、 蛋白聚糖、 弹性蛋 白等成分， 按一定比例和结构建成的复杂的有机的三维整体， 为各种细胞的生存及活动 提供适宜的场所和微环境， 能够调节各种细胞的生长、形状、代谢、迁移、增殖和分化， 进而调控组织和器官功能。 组织缺损的一个严重后果是 "土壤" 一 ECM 的丧失， 这也 是机体自身无法实现组织修复和再生的原因。天然的 ECM可以作为组织再生的 "土壤"， 是理想的组织修复材料。 去除动物组织的细胞成分可以去除大部分的免疫原性， 并保留 ECM成分， 可以开发出理想的组织修复材料。
国外已有来源于猪、 马、 牛等动物的真皮、 心包、 小肠等组织的动物源性脱细胞 ECM材料产品进入临床应用。其中脱细胞小肠粘膜下层（small intestinal submucosa, SIS ) 基质材料是学术界公认的最理想的软组织修复材料。 脱细胞 SIS基质材料的优势包括： 1 ) 免疫原性低， 组织相容性高； 2)特有的结构和组成具备主动诱导各种组织再生的生 物学基础； 3 )应用领域广泛， 可以适用于机体各种软组织的修复； 4) 具有抗微生物活 性。 美国 Cook Biotech Incorporated 的脱细胞 SIS基质材料在腹壁修复、 烧伤、 肛痿、 难治性伤口、 整形手术、 盆底修复、 肌腱修复、 泌尿生殖道修复、 神经修复等领域已有 较大样本量的临床应用。
国内外专利和文献中关于脱细胞 SIS基质材料制备方法的报道很多， 但到目前为止 仍只有 Cook Biotech Incorporated公司一家脱细胞 SIS基质材料产品进入临床应用。
脱细胞工艺和病毒灭活工艺是脱细胞 SIS基质材料制备的主要工艺和技术难点， 要 求完全去除小肠粘膜下层组织的病毒、 细胞成分和动物源 DNA成分， 同时完整保留天 然 ECM的成分和三维结构， 特别是促进组织再生的生长因子 （如碱性生长因子、 转化 生长因子等）。 已报道的脱细胞和病毒灭活方法种类繁多， 但大多不能完全去除所有的 动物源性 DNA成分， 并且大多耗时过长， 需要使用多种有机和高强度酸碱溶剂， 导致 了脱细胞 SIS基质材料中活性细胞外基质成分的破坏， 有害溶剂残留会导致细胞毒性， 从而影响组织修复效果。 另外， 大部分脱细胞工艺没有控制内毒素残留的有效措施。 成 型工艺是脱细胞 SIS基质材料制备的另一技术难点， 小肠的周径只有 6〜8cm， 小肠粘膜 下层的厚度少于 0.1mm， 难以制备不同尺寸、 厚度和适应不同力学强度要求的组织修复 产品。 Cook Biotech Incorporated采用真空压制法成型， 这种方法会使脱细胞 SIS基质材 料的空间结构压缩， 破坏了天然的 ECM三维结构， 影响材料的孔隙率。
临床研究显示， Cook Biotech Incorporated 的脱细胞 SIS 基质材料产品 （surgisis BIODESIGN®等）在临床应用中出现了浆液肿、 感染、 免疫排斥反应、 组织愈合不良等 并发症， 其中浆液肿发生率最高。 并发症可能导致疾病的复发， 甚至需要二次手术去除 已植入的脱细胞 SIS基质材料。 研究已经证实， 浆液肿是由 Th2 炎性细胞因子反应引 起的， 而该反应与产品的动物源 DNA残留密切相关。
发明内容
本发明所要解决的技术问题是提供一种动物源性植入性医用生物材料的制备方法， 用该方法制备的动物源性脱细胞 ECM材料完全去除了动物源细胞成分和 DNA成分， 同时完整地保留了天然 ECM的成分、 三维结构和能够诱导促进组织再生的活性生长因 子，无内毒素、有机溶剂和有毒溶剂残留，并可根据不同的适应症形成具有不同的尺寸、 厚度和力学强度的产品。
本发明所采用的技术方案为：
一种动物源性植入性医用生物材料的制备方法， 包括以下操作步骤：
1.动物组织材料的前置处理分离与初洗
取新鲜动物的组织，使用注射用水冲洗 3遍。这里所说的动物理论上包括所有动物， 优选为猪、 牛、 马， 更优选是猪。 所述的组织包括小肠粘膜下层、 真皮、 心包。
2.病毒灭活
采用低浓度过氧乙酸-乙醇溶液法灭活病毒，该步骤在清洗槽可振荡的恒温超声波清 洗器中进行， 其中过氧乙酸的体积百分含量为 0.05~0.2% (优选为 0.1%)， 灭活时间为 l~2h (优选为 lh)， 清洗槽振荡频率为 30~600rpm (优选为 100~300rpm， 进一步优选为 200rpm)， 超声波频率为 20~80KHZ (优选为 20~50KHZ， 进一步优选为 35~50KHZ， 最 优为 45KHZ), 温度范围为 4~40°C， 然后在磷酸盐缓冲液中清洗 2~5次， 每次 15min， 检测清洗后的磷酸盐缓冲液的 pH值， 当 pH达到 6.5-7.5后， 以流动的注射用水清洗材 料， 检测电导率达到 1.5um/s以下时终止。 该步骤中磷酸盐缓冲液的配制方法为称 7.9g NaCl、 0.2g KCK 0.24g KH2P04 1.8g K2HP04， 溶于 800 ml蒸熘水中， 用 HC1调节溶 液的 pH值至 7.4， 然后加蒸熘水定容至 1 L。
3.脱细胞
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 首先将材料置入清洗槽中， 在 清洗槽中注入氢氧化钠溶液， 开启清洗器， 清洗时间为 5~30min (优选为 20min)， 氢氧 化钠溶液浓度为 5~100mmol/L (优选为 5~20mmol/L， 进一步优选为 10mmol/L)， 然后 关闭清洗器，将氢氧化钠溶液倾出，注入磷酸盐缓冲液，开启清洗器，清洗时间为 5~20min (优选为 15min)，磷酸盐缓冲液清洗重复 2~5次，检测清洗后的磷酸盐缓冲液的 pH值， 当 pH达到 6.5-7.5后，流动的注射用水清洗材料，检测电导率达到 1.5um/s以下时终止。 该步骤中清洗槽振荡频率为 100~300rpm (进一步优选为 200rpm )， 超声波频率为 20~80KHZ (优选为 20~50KHZ， 进一步优选为 35~50KHZ， 最优为 45KHZ)。 该步骤中 磷酸盐缓冲液的配制方法同步骤 2。
4.氯化钠处理
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 将氯化钠溶液注入清洗槽中， 开启清洗器， 清洗时间为 5~30min (优选为 20min)， 氯化钠溶液浓度为 0.015mol/L或 2mol/L (优选为 0.015mol/L)、 pH值不超过 7.8， 然后以流动的注射用水清洗材料， 检 测电导率达到 1.5um/s以下时终止。该步骤中清洗槽振荡频率为 100~300rpm (进一步优 选为 200rpm)，超声波频率为 20~80KHZ (优选为 20~50KHZ，进一步优选为 35~50KHZ， 最优为 45KHZ)。
5.成型
该步骤包括制具固定、 冷冻干燥和激光微孔打孔 3步。 按照不同产品要求设计相应 尺寸和形状的制具 （优选为不锈钢制具）， 将材料固定于制具上， 可根据不同产品要求 重叠多层， 将已用注射用水清洗洁净、 已固定于制具的材料放于冷冻干燥机中， 按照预 先设计的冻干流程进行冷冻干燥： 预冻至 25~50°C (优选为 25°C )， 保温 0.5~4小时（优 选为 2h)， 升温至 15°C， 保温 4~12小时（优选为 8h)， 升温 15°C， 保温 0.5~4小时（优 选为 2h)， 升温至 25°C， 保温 4小时。 冻干完成后， 使用激光微孔打孔机打孔， 孔径范 围为 0.05~lmm (优选为 0.2~0.5mm)， 孔间隔为 0.1~2cm (优选为 0.5~lcm)。 所述的激 光微孔打孔是指利用激光技术将材料打出微米级的小孔，使用激光微孔打孔机打孔是为 了使材料表面形成孔隙， 以利于组织修复。
6.包装灭菌
在无菌的状态下进行包装， 一层采用特卫强纸、 另一层采用聚乙烯塑料， 包装完成 后采用环氧乙烷进行灭菌。
本发明中注射用水的使用标准依照国家药典中规定。
本发明中所述的清洗槽可振荡的超声波清洗机是将传统超声波清洗机的清洗槽与 机械震荡器相结合， 使清洗槽能够在超声波清洗的同时发生机械震荡， 实现了机械振荡 与超声波清洗同时联合发挥作用。
本发明涉及的一种动物源性植入性医用生物材料的制备方法，可用于制备脱细胞小 肠粘膜下层基质材料、 脱细胞真皮基质材料、 脱细胞心包基质材料。
与现有技术相比， 本发明具有以下显著优点和有益效果： 本发明使用一种清洗槽可 振荡的超声波清洗机， 实现了机械振荡与超声波清洗同时性联合发挥作用， 提高了动物 源性脱细胞 ECM材料制备中病毒灭活工艺、 脱细胞工艺和清除动物源 DNA工艺的效 率， 大大降低了工艺耗时， 简化了工艺流程， 整个制备过程仅使用过氧乙酸-乙醇、 氢 氧化钠和氯化钠 3种溶液， 并且浓度均远远低于现有的制备技术， 使制备的材料完全去 除了免疫原性， 完整保留了天然 ECM的结构和生长因子等有效成分； 并且， 在成型工 艺中创新性地将冷冻干燥技术和激光微孔技术有效结合， 在不破坏天然 ECM三维结构 的前提下， 根据不同适应症形成具有不同的形状、 尺寸、 厚度和力学强度的产品。 附图说明
图 1所示的是本发明实施例 2中的试样剪裁示意图；
图 2所示的是本发明实施例 2中的光学显微镜图；
图 3所示的是本发明实施例 2中的电镜超微结构图。
具体实 式
以下结合实施例对本发明作进一步具体描述， 但不局限于此。
实施例 1 : 脱细胞猪小肠粘膜下层基质材料的制备 1.动物组织材料的前置处理分离与初洗
取新鲜屠宰的猪的小肠组织清洗洁净， 分离出小肠粘膜下层， 使用注射用水冲洗 3 遍。
2.病毒灭活
采用低浓度过氧乙酸-乙醇溶液法灭活病毒，该步骤在清洗槽可振荡的恒温超声波清 洗器中进行， 其中过氧乙酸的体积百分含量为 0.05~0.2% (优选为 0.1%)， 灭活时间为 l~2h (优选为 lh)， 清洗槽振荡频率为 30~600rpm (优选为 100~300rpm， 进一步优选为 200rpm)， 超声波频率为 20~80KHZ (优选为 20~50KHZ， 进一步优选为 35~50KHZ， 最 有为 45KHZ), 温度范围为 4~40°C， 然后在磷酸盐缓冲液中清洗 2~5次， 每次 15min， 检测清洗后的磷酸盐缓冲液的 pH值， 当 pH达到 6.5-7.5后， 以流动的注射用水清洗材 料， 检测电导率达到 1.5um/s以下时终止。 该步骤中磷酸盐缓冲液的配制方法为称 7.9g NaCl、 0.2g KCK 0.24g KH2P04 1.8g K2HP04, 溶于 800 ml蒸熘水中， 用 HC1调节溶 液的 pH值至 7.4， 然后加蒸熘水定容至 1 L。
3.脱细胞
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 首先将材料置入清洗槽中， 在 清洗槽中注入氢氧化钠溶液， 开启清洗器， 清洗时间为 5~30min (优选为 20min)， 氢氧 化钠溶液浓度为 5~100mmol/L (优选为 5~20mmol/L， 进一步优选为 10mmol/L)， 然后 关闭清洗器，将氢氧化钠溶液倾出，注入磷酸盐缓冲液，开启清洗器，清洗时间为 5~20min (优选为 15min)，磷酸盐缓冲液清洗重复 2~5次，检测清洗后的磷酸盐缓冲液的 pH值， 当 pH达到 6.5-7.5后，流动的注射用水清洗材料，检测电导率达到 1.5um/s以下时终止。 该步骤中清洗槽振荡频率为 100~300rpm (进一步优选为 200rpm )， 超声波频率为 20~80KHZ (优选为 20~50KHZ， 进一步优选为 35~50KHZ， 最优为 45KHZ)。 该步骤中 磷酸盐缓冲液的配制方法同步骤 2。
4.氯化钠处理
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 将氯化钠溶液注入清洗槽中， 开启清洗器， 清洗时间为 5~30min (优选为 20min)， 氯化钠溶液浓度为 0.015mol/L或 2mol/L (优选为 0.015mol/L)、 pH值不超过 7.8， 然后以流动的注射用水清洗材料， 检 测电导率达到 1.5um/s以下时终止。该步骤中清洗槽振荡频率为 100~300rpm (进一步优 选为 200rpm)，超声波频率为 20~80KHZ (优选为 20~50KHZ，进一步优选为 35~50KHZ， 最优为 45KHZ)。
5.成型 按照不同产品要求设计相应尺寸和形状的不锈钢制具， 将材料固定于不锈钢制具 上， 可根据不同产品要求重叠多层， 将已用注射用水清洗洁净、 已固定于制具的材料放 于冷冻干燥机中， 按照预先设计的冻干流程进行冷冻干燥： 预冻至 25〜50°C (优选为 25 °C )， 保温 0.5~4小时 （优选为 2h)， 升温至 15 °C， 保温 4~12小时 （优选为 8h)， 升 温 15 °C， 保温 0.5~4小时 （优选为 2h)， 升温至 25 °C， 保温 4小时， 冻干完成后， 使用 激光微孔打孔机打孔， 孔径范围为 0.05~lmm (优选为 0.2~0.5mm)， 孔间隔为 0.1~2cm (优选为 0.5~lcm)。
6.包装灭菌
在无菌的状态下进行包装， 一层采用特卫强纸、 另一层采用聚乙烯塑料， 包装完成 后采用环氧乙烷进行灭菌。
实施例 2: 实施例 1所制备的脱细胞小肠粘膜下层基质材料的理化性质、 组织学、 生长因子和生物学性能检测
1.对制备的 8层材料进行物理性能检测， 检测项目包括缝合保持力、 抗张强度、 爆 破强度和孔隙率。
1 ) 缝合保持力检测： 方法： 用 2-0的外科缝线或相同直径的不锈钢丝缝合在 8层 材料一端边缘内 2毫米处， 将缝线或不锈钢丝与 8层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 5 ± 0.5N。
2) 抗张强度检测方法： 方法： 使用拉伸 （压缩） 试验机， 按照图 1所示， 将 8层 材料裁剪成试样， 裁剪后在相对湿度为 40%-60%， 温度为 22°C ±2°C的条件下放置 2h 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 lOOmm/min 的速度依次 向外拉伸直到试样断裂，纵向试样和横向试样分别进行试验。 以 N为单位记录下试样断 裂时的力。 按上述方法对 3批样品进行检测。 结果： 纵向： 15N， 横向： 8N。
3 )爆破强度检测： 方法： 使用拉伸 （压缩）试验机， 将 8层材料裁剪成 23 X 23mm 的正方形试样备用， 在相对湿度为 40%-60%， 温度为 24°C ±2°C的条件下放置 2h后立 即进行试验。 用环形夹具将试样固定在拉伸仪的工作台上， 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。
4) 孔隙率测定： 采用压汞法测定材料的孔隙率。 结果： 孔隙率不低于 85%。
2.对制备的 8层材料进行化学性能检测，检测项目包括病毒、酸碱度、内毒素和 DNA 残留量。 1 )检验液制备： 检验液制备:取样品的厚度均勾部分， 切成 lcm2的碎片， 用水洗净 后晾干， 然后加入玻璃容器中， 按样品内外总表面积 （cm2) 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 °C ± rC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： 方法： 选择伪狂犬病毒为指示病毒， 采用实时定量 PCR法检测病毒 DNA拷贝数， 检测 3批样品。 结果： 病毒 DNA拷贝数 0。
3 )酸碱度检测： 按 GB/T14233.1 (《医用输液、 输血、 注射器具检验方法第 1部分： 化学分析方法》） 中 5.4.1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) 内毒素： 按 6cm2样品加 lml浸提介质的比例， 37士 C， 72±2hr制备试验液， 浸提介质： 生理盐水。 按 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》)规定的方法进行，检测 3批样品。结果：内毒素含量小于 5EU/g。
5 ) DNA残留量检测： 依据生物制剂残留 DNA检测方法（《中国药典》 2010， 附录 IX-B 外源性 DNA残留量测定法），采用荧光染色法检测实施例 1所提供的样品的 DNA 残留量。 结果： 材料的 DNA残留量不超过 150pg/g。
3. 组织学检测
1 ) 光学显微镜观察： 方法： 石蜡包膜后的材料行苏木精一伊红染色， 倒置相差显 微镜观察。 结果： 无细胞和细胞碎片残留， 胶原显微连续无断裂， 如图 2所示。
2) 超微结构观察。 结果： 材料呈多孔结构， 纤维无断裂， 孔径均勾， 平均孔径大 小为 200um， 孔隙率大于 85%， 如图 3所示。
4.生长因子检测
按 6cm2样品加 lml浸提介质的比例， 37士 C， 72±2hr制备试验液， 浸提介质： 生理盐水。 采用 ELLISA法检测浸提液中碱性生长因子 （bFGF) 和血管内皮生长因子 (VEGF) 含量。 结果： bFGF含量为 121.8±2.683ng/L， VEGF含量为 93.8±3.033ng/L。
5. 生物学性能检测， 检测项目包括细胞毒性、 迟发型超敏反应、 皮内反应。
1 )细胞毒性： 方法： 按 6cm2样品加 lml浸提介质的比例， 37士 1 °C， 24±2hr制备 试验液， 浸提介质： 含血清的 MEM培养基。 取试验液按照 GB/T 16886.5-2003 (《医疗 器械生物学评价第 5部分： 体外细胞毒性试验》） 中规定的试验方法进行试验。 结果： 细胞毒性反应小于或等于 1级。
2) 迟发型超敏反应： 方法： 按 6cm2样品加 lml浸提介质的比例， 37士 1 °C， 72士 2hr制备试验液， 浸提介质为生理盐水和棉籽油。 按照 GB/T 16886.10-2005 (《医疗器械 生物学评价第 10部分： 剌激与迟发型超敏反应试验》） 试验方法规定进行试验。 结果： 无迟发型超敏反应。
3 )皮内反应： 按 6cm2样品加 lml浸提介质的比例， 37士 C， 72±2hr制备试验液， 浸提介质为生理盐水和棉籽油。 按照 GB/T 16886.10-2005 (《医疗器械生物学评价第 10 部分： 剌激与迟发型超敏反应试验》） 试验方法规定进行试验。 结果： 试验样品与溶剂 对照平均记分之差小于 1.0。
实施例 3: 脱细胞猪真皮基质材料的制备
取新鲜屠宰的猪的真皮组织作为原材料， 制备方法同实施例 1。
实施例 4: 实施例 3所制备的脱细胞猪真皮基质材料的理化性质、 组织学、 生长因 子和生物学性能检测
按照实施例 2所述的方法进行检测。
结果显示： 实施例 3所制备的脱细胞真皮基质材料的缝合抗拉强度大于 5N， 横向 和纵向抗张强度均大于 20N， 爆破强度大于 120N， 孔隙率大于 80%， 病毒 DNA拷贝数 为 0， 内毒素含量小于 5EU/g， DNA残留量不超过 150pg/g， 无迟发型超敏反应， 无皮 内反应。
实施例 5: 脱细胞猪心包基质材料的制备
取新鲜屠宰的猪的心包组织作为原材料， 制备方法同实施例 1。
实施例 6: 实施例 5所制备的脱细胞猪心包基质材料的理化性质、 组织学、 生长因 子和生物学性能检测
按照实施例 2所述的方法进行检测。
结果显示： 实施例 5所制备的脱细胞真皮基质材料的缝合抗拉强度大于 5N， 横向 和纵向抗张强度均大于 20N， 爆破强度大于 120N， 孔隙率大于 85%， 病毒 DNA拷贝数 为 0， 内毒素含量小于 5EU/g， DNA残留量不超过 150pg/g， 无迟发型超敏反应， 无皮 内反应。
本发明的上述实施例是对本发明的说明而不能用于限制本发明，与本发明的权利要 求书相当的含义和范围内的任何改变， 都应认为是包括在权利要求书的范围内。

Claims

权利要求书
1.一种动物源性植入性医用生物材料的制备方法， 其特征在于包括以下操作步骤：
( 1 ) 动物组织材料的前置处理分离与初洗
取新鲜动物的组织， 使用注射用水冲洗 3遍；
(2) 病毒灭活
采用低浓度过氧乙酸-乙醇溶液法灭活病毒，该步骤在清洗槽可振荡的恒温超声波清 洗器中进行， 其中过氧乙酸的体积百分含量为 0.05~0.2%， 灭活时间为 l~2h， 温度范围 为 4~40°C ; 然后在磷酸盐缓冲液中清洗 2~5次， 每次 15min， 检测清洗后的磷酸盐缓冲 液的 pH值，当 pH达到 6.5-7.5后，以流动的注射用水清洗材料，检测电导率达到 1.5um/s 以下时终止；
( 3 ) 脱细胞
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 首先将材料置入清洗槽中， 在 清洗槽中注入氢氧化钠溶液， 开启清洗器， 清洗时间为 5~30min， 氢氧化钠溶液浓度为 5~100mmol/L, 然后关闭清洗器， 将氢氧化钠溶液倾出， 注入磷酸盐缓冲液， 开启清洗 器， 清洗时间为 5~20min， 磷酸盐缓冲液清洗重复 2~5次， 检测清洗后的磷酸盐缓冲液 的 pH值， 当 pH达到 6.5-7.5后， 流动的注射用水清洗材料， 检测电导率达到 1.5um/s 以下时终止；
(4) 氯化钠处理
该步骤在清洗槽可振荡的恒温超声波清洗器中进行， 将氯化钠溶液注入清洗槽中， 开启清洗器， 清洗时间为 5~30min， 氯化钠溶液浓度为 0.015mol/L或 2mol/L、 pH值不 超过 7.8， 然后以流动的注射用水清洗材料， 检测电导率达到 1.5um/s以下时终止；
( 5 ) 成型
该步骤包括制具固定、 冷冻干燥和激光微孔打孔 3步， 按照不同产品要求设计相应 尺寸和形状的制具， 将材料固定于制具上， 将已用注射用水清洗洁净、 已固定于制具的 材料放于冷冻干燥机中， 按照预先设计的冻干流程进行冷冻干燥： 预冻至 25〜50°C， 保 温 0.5~4小时，升温至 15 °C，保温 4~12小时，升温 15 °C，保温 0.5~4小时，升温至 25 °C， 保温 4小时， 冻干完成后， 使用激光微孔打孔机打孔， 孔径范围为 0.05〜lmm， 孔间隔 为 0.1~2cm;
( 6) 包装灭菌
在无菌的状态下进行包装， 一层采用特卫强纸、 另一层采用聚乙烯塑料， 包装完成 后采用环氧乙烷进行灭菌。
2.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （1 ) 中的动物包括猪、 牛、 马。
3.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （1 ) 中的组织包括小肠粘膜下层、 真皮、 心包。
4.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2) 中过氧乙酸的体积百分含量为 0.1%。
5.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2) 中灭活时间为 lh。
6.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2)、 （3 )、 （4) 中清洗槽振荡频率为 100~300rpm。
7.根据权利要求 6所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2)、 （3 )、 （4) 中清洗槽振荡频率为 200rpm。
8.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2)、 （3 )、 （4) 中超声波频率为 20~80KHZ。
9.根据权利要求 8所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （2)、 （3 )、 （4) 中超声波频率为 45KHZ。
10.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （3 ) 中用氢氧化钠溶液对材料进行清洗的清洗时间为 20min。
11.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法， 其特征在 于： 所述步骤 （3 ) 中氢氧化钠溶液浓度为 5~20mmol/L。
12.根据权利要求 11所述的一种动物源性植入性医用生物材料的制备方法， 其特征 在于： 所述步骤 （3 ) 中氢氧化钠溶液浓度为 10mmol/L。
13.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （3 ) 中用磷酸盐缓冲液对材料进行清洗的清洗时间为 15min。
14.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （4) 中氯化钠溶液清洗时间为 20min。
15.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （4) 中氯化钠溶液浓度为 0.015mol/L。
16.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （5 ) 中的制具为不锈钢制具。
17.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤（5 ) 中预先设计的冻干流程为： 预冻至 25°C， 保温 2h， 升温至 15°C， 保 温 8h， 升温 15°C， 保温 2h， 升温至 25°C， 保温 4小时。
18.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （5 ) 中激光微孔打孔机打孔的孔径范围为 0.2~0.5mm。
19.根据权利要求 1所述的一种动物源性植入性医用生物材料的制备方法，其特征在 于： 所述步骤 （5 ) 中激光微孔打孔机打孔的孔间隔为 0.5~lcm。
20.权利要求 1所述的一种动物源性植入性医用生物材料的制备方法的应用，其特征 在于： 包括用于制备脱细胞小肠粘膜下层基质材料、 脱细胞真皮基质材料、 脱细胞心包 基质材料。
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