WO2018157851A1 - 一种引导骨再生的骨膜修补片、制备方法和应用 - Google Patents

一种引导骨再生的骨膜修补片、制备方法和应用 Download PDF

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WO2018157851A1
WO2018157851A1 PCT/CN2018/077850 CN2018077850W WO2018157851A1 WO 2018157851 A1 WO2018157851 A1 WO 2018157851A1 CN 2018077850 W CN2018077850 W CN 2018077850W WO 2018157851 A1 WO2018157851 A1 WO 2018157851A1
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periosteal
solution
bone
small intestinal
tissue
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PCT/CN2018/077850
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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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/3641—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 site of application in the body
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    • 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
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
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    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses

Definitions

  • the invention relates to the field of medical biomaterials, in particular to a periosteal repairing piece, a preparation method and an application thereof for repairing periosteal damage or defect caused by trauma, fracture, tumor and the like, or implanting a bone graft material to prevent material from coming out.
  • periosteum is rich in microvessels, which can restore the blood supply to the bone tissue, thereby promoting the healing of the bone defect and improving the success rate of bone repair. It can also serve as an important interface between the bone tissue and the surrounding tissue to prevent muscles, tendons and tendons. Tissues such as membranes grow into bone defects, providing growth microenvironment for osteoblasts and mesenchymal stem cells, and promoting bone tissue regeneration and reconstruction. Therefore, once the periosteal defect cannot be recovered, the bone defect will be difficult to self-heal.
  • the periosteal repair materials reported at present have their insufficiency.
  • the matrix of the acellular matrix is destroyed, the DNA residue is too high, the immunogenicity is not completely removed, and the immune rejection is easy to occur.
  • the chitosan and silk fibroin are natural.
  • Biomaterials use electrospinning technology to construct three-dimensional structure, which can not achieve the porous network effect of natural extracellular matrix; artificially degradable polymer materials will cause local acidity in the degradation process, which may cause inflammatory reaction;
  • the protein membrane is insufficient in mechanical strength and degraded too fast.
  • periosteal repair material which can not only serve as a barrier, but also effectively isolate the bone injury site from the surrounding tissue, prevent the fibroblasts in the surrounding tissue from invading the bone injury site and the bone graft material, and Guide the periosteal tissue regeneration and repair, form vascularized tissue to provide nutrients and oxygen for bone regeneration, and provide growth microenvironment for osteoblasts and mesenchymal stem cells to reconstruct bone tissue.
  • the present invention is directed to the above-mentioned deficiencies of the prior art, and provides a periosteal repair patch having a multi-layer structure, low immunogenicity, controllable degradation, and retention of extracellular matrix three-dimensional structure and growth factors for guiding bone regeneration.
  • the technical solution adopted by the present invention is: a periosteal repair piece for guiding bone regeneration, the repair piece comprising a high porosity porous active layer and a low porosity dense base layer, a loose active layer and a dense base.
  • the bottom layer includes collagen, a polysaccharide substance and an active factor, and the loose active layer further includes an active factor for guiding bone regeneration, and the periosteal repair piece is implanted in the body to be degraded and absorbed.
  • the above-mentioned loose active layer and dense base layer of the present invention are described from the structural point of view, in fact, both are manufactured by the small intestinal submucosa, including collagen, polysaccharide substances and active factors are all preserved from the small intestinal submucosa itself.
  • the substance, the loose active layer also includes substances which guide bone regeneration active factors such as bone morphogenetic proteins to be added separately.
  • the high porosity porous active layer according to the present invention is a three-dimensional network porous structure having a porosity of 75 to 90%.
  • the low porosity and dense base layer according to the present invention is a three-dimensional network porous structure having a porosity of 45 to 70%.
  • the specific porosity is defined by the fact that the dense layer is used for isolation protection to prevent soft tissue from growing into the bone wound; the loose layer is suitable for vascularization, osteogenic growth, and the porosity of each of the dense and loose layers is maximized. The realization of the above effects is guaranteed.
  • the collagen of the present invention is a composition comprising collagens of type I, type III, type IV and type VI, which are essential components of a matrix material of the small intestinal submucosa.
  • the polysaccharide material of the present invention is a composition comprising chondroitin sulfate and hyaluronic acid.
  • the active factors of the present invention comprise fibronectin, laminin, integrins and growth factors.
  • the bone-inducing active factor of the present invention comprises an osteoinductive active substance, preferably a bone morphogenetic protein.
  • the periosteal repair piece of the invention can be degraded and absorbed in the body, and the specific degradation and absorption time is 3-6 months.
  • Soft tissue wound recovery usually recovers in about two weeks, while hard tissue wounds (such as bone wounds) usually recover more than three months, and six months are the time to complete bone reconstruction. Therefore, there is still a need for periosteal repair materials to achieve isolation of soft tissue at 3 months. It cannot be completely degraded for at least 3 months; and degradation can be completed after 6 months.
  • the periosteal repair piece for guiding bone regeneration according to the present invention has a length of 1-20 cm, a width of 1-10 cm, and a thickness of 1-3 mm.
  • the present invention also provides a method for preparing the above-mentioned periosteal repair piece for guiding tissue regeneration, comprising the steps of: using an animal small intestine submucosal tissue (animal small intestine submucosa) as a raw material, and then including tissue pretreatment, virus inactivation, washing , immunogen elimination, washing, slurry preparation, active layer preparation, drying, composite molding and vacuum freeze-drying steps to obtain virus-removing virus, cellular components, DNA components and ⁇ -Gal antigens, and retaining extracellular matrix components Periosteal repair tablets that guide bone regeneration.
  • the animal is a mammal, preferably a pig or a cow.
  • Virus inactivation soaking the submucosal tissue of the small intestine with peracetic acid-ethanol solution for virus inactivation;
  • washing process the small intestinal submucosal tissue obtained in the washing step (2);
  • the immunogen removal solution is used for immunogen removal, and the immunogen removal solution is a PBS solution in which trypsin and EDTA are dissolved, and the immunogen removal process is performed in a multi-frequency ultrasonic environment;
  • Cleaning process cleaning is carried out, and the cleaning process is carried out in an ultrasonic cleaning machine to obtain a submucosal matrix material of the small intestine;
  • the slurry obtained in the step (6) is freeze-dried, crushed and sieved to obtain a small intestinal submucosal matrix particle, and a bone morphogenetic protein solution (BMP) is mixed with the particle to form a paste. mixture;
  • BMP bone morphogenetic protein solution
  • the small intestinal submucosal matrix material obtained in the step (5) is fixed in a molding die and placed in an oven together with the molding die to dry;
  • the peracetic acid-ethanol solution of the step (2) of the present invention 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% (water is configured as a solution), peracetic acid
  • the volume ratio of the ethanol solution to the intestinal submucosal tissue material is (3-20): 1, the inactivation time is 2-4 hours, and the temperature range is 10-40 °C.
  • the cleaning process in the step (3) is: washing in a PBS solution having a pH of 7.2-7.4 in an ultrasonic cleaner, and then washing with water until the detection conductivity is 10 ⁇ S/cm or less, thereby obtaining a submucosal matrix material of the small intestine, wherein
  • the PBS solution has a pH in the range of 6-8, and the water used is purified purified water, preferably having an ultrasonic frequency of 40 kHz and a power of preferably 3,000 W or more.
  • the immunogen source removal solution in the step (4) comprises: a PBS solution having a pH of 6-8 in which trypsin and EDTA are dissolved; and the concentration of the trypsin in the immunogen source removal solution is 0.01-0.2%, EDTA The concentration is 0.1-1 mmol/L; further comprising: the mass percentage concentration of trypsin in the immunogen removal solution is 0.02-0.05%, the concentration of EDTA is 0.4-0.8 mmol/L, and the pH of the immunogen removal solution is 7.0-8.0.
  • the volume ratio of the immunogen-removing fluid to the small intestinal submucosal tissue material is (20-40):1; and the multi-frequency ultrasound in the step (4) includes at least two ultrasonic frequencies, and the low-frequency frequency range It is 20-40KHz, the high frequency frequency is 60-90KHz, wherein the low frequency processing is 5-40min, the high frequency processing is 5-40min, and the temperature range is 20-35 °C.
  • the cleaning process in the step (5) is: washing with a PBS solution in an ultrasonic cleaner, and then washing with a cooling water for injection until the conductivity difference of the water for injection before and after the detection is 1 ⁇ S/cm or less, and the submucosa of the small intestine is obtained.
  • the PBS solution has a pH in the range of 6-8, the ultrasonic frequency is preferably 40 kHz, and the power is preferably 3000 W or more.
  • the small intestinal submucosal matrix slurry in the step (6) is prepared, specifically: the small intestinal submucosal matrix material obtained by the step (5) is crushed by using a low temperature crushing device, and the acetic acid solution is added to the crushed matrix material.
  • the preparation of the active layer material of the step (7) comprises: freeze-drying the slurry obtained above, and then crushing the bulky submucosa of the small intestinal submucosa which has been lyophilized into particles by a crushing device, and screening the sieve to have a particle size of 400 ⁇ m.
  • the temperature was 20 ° C, and the mixture was allowed to stand for 24 hours after mixing to obtain a paste mixture.
  • the molding die according to the above step (8) of the present invention comprises a needle bottom plate, a cover plate and a pressing block, and one or more small intestinal submucosa matrix materials are laid on the needle bottom plate to cover the cover.
  • the plate, the cover plate is pressed with 5-10 kg of briquettes, so that the material is flat, the water overflows from the surroundings and the materials are in close contact with each other; the oven fan is turned on, preheating to 25-40 ° C, and the small intestine submucosa matrix
  • the material and the mold are placed in an oven for 8-16 hours.
  • the cover plate and the weight plate for providing pressure are slowly removed, and then placed in an oven for drying, and the drying takes place after 2-6 hours.
  • the dried small intestinal submucosa matrix is used as a base layer; the structure of the mold mentioned in the present invention can be referred to the invention patents ZL201310203588.6 and ZL201310203602.2.
  • the step (9) comprises: covering the dried small intestinal submucosa substrate obtained by the treatment in the step (8) with the cream mixture obtained in the step (7), and uniformly spreading the cream mixture on the dried
  • the upper surface of the intestinal submucosal matrix has a slurry thickness of 1-3 mm.
  • the vacuum freeze-drying according to the step (10) of the present invention specifically: placing the composite material obtained by the step (9) in a vacuum freeze dryer; first pre-freezing to -45 ° C, holding for 1-2 hours; then turning on the vacuum pump Adjust the temperature to -15 ° C, keep warm for 5-7 hours, then adjust the temperature to 0 ° C, keep warm for 2 hours, finally adjust the temperature to 25 ° C, keep warm for 4 hours, the pressure in the chamber of the freeze-drying device is 1-50Pa, complete Vacuum freeze-drying to give the final target product.
  • the method for preparing the periosteal repair piece for guiding bone regeneration according to the present invention further comprises: (11) packaging, and (12) sterilization analysis.
  • the present invention further provides the use of the above-mentioned periosteal repair piece for guiding bone regeneration.
  • it can be used to fill the extraction socket, the expansion and reconstruction of alveolar ridge, periodontal bone defect filling, alveolar iliac bone enlargement, maxillary sinus augmentation, sinus floor elevation, horizontal bone increment, vertical bone
  • Incremental periosteal defect repair, isolation of surrounding tissue, guiding bone tissue regeneration can also be used for various types of fractures, bone defects, spinal fusion, bone tumors, nonunion, joint fusion, etc. caused by periosteal defect repair of periosteal defects, isolated around Tissue, guide bone tissue regeneration.
  • the periosteal repair piece for guiding bone regeneration of the present invention wherein the basal layer provides tissue isolation, protects damaged tissue, the active layer promotes repair of periosteum, and guides bone tissue regeneration.
  • the present invention has the following significant advantages and benefits:
  • the molding process technology the periosteal repair piece for guiding bone regeneration in the present invention has a double-layer structure, and functions as a physical isolation and a micro-environment for the periosteal regeneration, and a combination of drying and freeze-drying is formed.
  • the upper layer is loose and the lower layer is dense and double-layer composite structure.
  • the control of the porosity of the two is very important.
  • the specific porosity makes the loose layer an active layer containing guided bone regeneration.
  • the porosity is large, which is beneficial to the growth of the cells and can improve the repair speed of the tissue.
  • the dense layer is the immunogen removal matrix.
  • the basal layer has an isolation function to effectively protect the repaired tissue;
  • the matrix crushing technology firstly, the submucosal tissue of the small intestine is crushed and crushed by a low temperature crushing device, and then puffed by acetic acid, and then freeze-dried to form a loose structure which can absorb a large amount of water, and the slurry is formed during the preparation process.
  • the bone morphogenetic protein technology is added: by using the strong water absorption capacity of the small intestinal micropowder, a solution containing the bone morphogenetic protein is added, and the solution can be quickly and fully entered into the micropowder particles, and then freeze-dried to make the bone morphogenetic protein exist. Released on the surface of the intestinal micropowder pores and implanted in the body;
  • the patch of the invention is used for repairing periosteal damage and defect: it can not only serve as a barrier, but also effectively isolate the bone injury site from the surrounding tissue, and prevent fibroblasts in the surrounding tissue from invading the bone injury site and bone graft material. Exfoliation can also guide the regeneration and repair of periosteal tissue, forming vascularized tissue to provide nutrients and oxygen for bone regeneration, and providing a growth microenvironment for osteoblasts and mesenchymal stem cells to reconstruct bone tissue.
  • Fig. 1 is a schematic view showing the layer structure of a periosteal repair piece for guiding bone regeneration according to the present invention.
  • a periosteal repair piece for guiding bone regeneration prepared as follows:
  • Subcutaneous submucosal tissue material such as porcine small intestine submucosal SIS, or bovine small intestine submucosa, adapted to the embodiment of the invention
  • Subcutaneous submucosal tissue material is divided into prescribed sizes, unwanted tissues (such as lymphoid tissue) are removed, washed with water, and then The cleaned small intestine submucosal tissue material is placed on a water filter such as a sieve, and allowed to stand for more than five minutes, and the water is filtered.
  • the process can be carried out in stainless steel tank, the concentration of peroxyacetic acid is 0.1% by volume, ethanol is 5% by volume, inactivation time is 2 hours, solution and small intestinal mucosa
  • the underlying tissue material has a volume ratio of 8:1 and a temperature range of 20 °C.
  • the virus was washed with an PBS solution in an ultrasonic cleaner, and then washed with water until the detection conductivity was 10 ⁇ S/cm or less.
  • the pH of the PBS solution is 7.2-7.4, and the water is purified purified water with an ultrasonic frequency of 40 kHz and an ultrasonic power of 3000 W.
  • the immunogen removal solution used was a PBS solution containing 0.1% by mass of trypsin and a concentration of 0.4 mmol/L of EDTA at pH 7.2-7.4, sonicated for 30 min, at a temperature of 20 ° C, and the submucosa of the small intestine.
  • the volume ratio of the tissue material is 20:1; the multi-frequency ultrasound includes at least two ultrasonic frequencies, the low frequency range is 25KHz, and the high frequency frequency is 65KHz, wherein the low frequency processing is 10min, the high frequency processing is 15min, and the temperature range is 20-35°C.
  • the power is above 5000W.
  • low temperature crushing device crushes the small intestinal submucosal matrix material obtained by step (5); low temperature crushing device can use liquid nitrogen freezing and crushing device to feed liquid nitrogen and small intestinal submucosal material into liquid nitrogen
  • the cooling and crushing device the small intestinal submucosal material is frozen by liquid nitrogen, and is broken by a high-speed rotating cutter head of the crushing device.
  • the acetic acid solution is added to the crushed matrix material to form a small intestinal submucosal matrix material slurry; wherein the acetic acid solution has a mass percentage concentration of 0.3%, and the mass ratio of the intestinal submucosal matrix material to the acetic acid solution is 1:60.
  • a small intestinal submucosa slurry is formed.
  • the slurry obtained in the step (6) is freeze-dried, crushed and sieved to obtain a small intestinal submucosal matrix particle; and a bone morphogenetic protein (BMP) aqueous solution having a mass percentage concentration of 0.02% is The particles were mixed, and the mass ratio of the particles to the bone morphogenetic protein solution was 1:1, and the temperature was 20 ° C. After mixing, the mixture was allowed to stand for 24 hours, and a paste mixture was obtained.
  • BMP bone morphogenetic protein
  • the one or more layers of small intestinal submucosa matrix material obtained by the step (5) are laid on the needle bottom plate of the molding die, covering the cover plate, and the cover plate is pressed with a pressure block of 5-10 kg to make the material flat.
  • the water overflows from the surroundings and the materials are in close contact with each other.
  • the small intestinal submucosal matrix material dried in the step (8) is covered with the creamy mixture obtained in the step (7), and the creamy mixture is evenly spread on the upper surface of the dried small intestinal submucosal matrix material using a flattening tool.
  • the thickness is 1 to 3 mm.
  • the composite material obtained in the step (9) is placed in a vacuum freeze dryer, the door of the freeze-drying chamber is closed, the circulation pump is turned on for about 1 minute, the compressor is turned on to cool the freeze-drying box, and the product is pre-frozen to -45 ° C.
  • the vacuum pump was turned on to adjust the product temperature to about -15 ° C.
  • the product temperature was adjusted to 0 ° C, the temperature was kept for 2 hours, the product temperature was adjusted to 25 ° C, and the temperature was kept for 4 hours, and vacuum freeze-drying was completed.
  • the lyophilization chamber pressure during vacuum drying is 1-50 Pa.
  • the periosteal repair piece for guiding bone regeneration can be obtained by the above method.
  • the patch comprises a dense layer formed by a small intestinal submucosal matrix material obtained by drying and then freezing, and a loose layer formed by exposing the lyophilized small intestinal submucosa matrix material.
  • the preparation method of the present invention may further comprise the following steps:
  • Sterilization analysis The product is sterilized by ethylene oxide.
  • the sterilization conditions are: temperature 40 ° C, holding time 4 h, humidity 70%, concentration 500 mg / L, sterilization time 6 h; analytical process: ventilation analysis room The temperature is controlled between 20 ° C and the time is about 14 days.
  • the periosteal repair sheet for guiding bone regeneration comprises two parts: a lyophilized loose portion 1 and a dried dense portion 2.
  • the lyophilized loose portion 1 comprises small intestinal submucosa lyophilized particles and comprises bone morphogenetic proteins; and the dense portion 2 comprises one or more layers of small intestinal submucosa material.
  • Collagen proteins of type I, III, IV and VI were detected by immunohistochemical staining, serial sections of 3 ⁇ m thick, dewaxed by xylene, and dehydrated by gradient ethanol.
  • the slice was transferred into a rice cooker water bath (containing 0.01 mol/L of trisodium citrate buffer at pH 6.0), the temperature was kept at 95-100 ° C, and boiled for 20 min to carry out antigen retrieval, and after taking out, it was naturally cooled at room temperature. Wash in phosphate buffer (PBS) for 5 min x 3 times.
  • PBS phosphate buffer
  • I, III, IV and VI collagen monoclonal antibody primary antibody were added dropwise at a concentration of 1:100, incubated at room temperature for 60 min at room temperature overnight, and washed three times with PBS.
  • the Envision reaction solution was added dropwise and incubated at room temperature for 30 min. Wash 3 times with PBS.
  • 0.05% of 3,3 diaminobenzidine + 0.03% H 2 O 2 developed color for 5-10 min.
  • Incremental gradient ethanol dehydration, xylene transparent, conventional resin sealing The results showed that all the four stained specimens under the microscope were stained with brownish yellow, which was positive, indicating that type I, III, IV and VI collagen could be detected in the sample.
  • bFGF alkaline growth factor
  • VEGF vascular endothelial growth factor
  • the pseudorabies virus was selected as the indicator virus, and the DNA copy number of the virus was detected by real-time quantitative PCR method, and three batches of samples were detected. Result: The viral DNA copy number is zero.
  • DNA residue According to the method for detecting residual DNA of biological agents, the fourth part of the Chinese Pharmacopoeia 2015 edition, the sample DNA residue provided by the examples was detected by fluorescence staining. Results: The average DNA residue of the samples provided in the examples was average. It is 3.15 ⁇ 0.42 ng / mg.
  • Galactosidase ( ⁇ -Gal) clearance rate Take Gal-positive reference material of animal-derived biological material, 2mg of Gal antigen-negative reference substance, add 1ml of lysate, lyse 30-90min, make 20, 10, 5 , 2.5, 1.25, 0.625 ⁇ g Gal standard curve sample, test the immunogen before and after the test article taken 50mg, add lysate 2ml, lysis 30-90min; take the lysate and M86 antibody reaction supernatant, add 96 Orifice plate, plus secondary antibody, add coloring agent, absorb the absorbance value by 450nm by ELISA method, calculate the Gal value of the sample according to the standard curve, and the Gal value of the material before the immunogen removal treatment is (22.61 ⁇ 2.40) ⁇ 10 14 /mg In the examples, the Gal value of the sample was (0.13 ⁇ 0.01) ⁇ 10 14 /mg, and the galactosidase ( ⁇ -Gal) clearance rate was 99.43%.
  • Bacterial endotoxin According to GB/T14233.2-2005 "medical infusion, blood transfusion, injector test method part 2: biological test method" for testing, a total of 3 batches of samples, the results: bacterial endotoxin is less than 20EU / packaging .
  • Porosity According to the Archimedes principle, using ethanol as the extraction medium, the porosity of the dense base layer of the sample is calculated to be 48.09 ⁇ 5.12%, and the porosity of the loose active layer is 85.23 ⁇ 6.89%.
  • Stitching tensile strength Samples were prepared according to the examples, and 3-0 non-absorbable sutures were used to fix the suture and the other end of the patch on the tensile force at 2 mm at the edge of one end of the patch, at 20 mm/min. The speed is stretched until the stitching point is torn, and the maximum force value is recorded. The result shows that the maximum value can reach 13N.
  • Tensile strength Samples were prepared according to the examples, and the samples were cut into 2 x 5 cm sizes, and tested immediately after being placed at a relative humidity of 40% - 60% and a temperature of 22 ° C ⁇ 2 ° C for 2 hours. The two ends of the sample were fixed on the chuck of the tensile testing machine, and were sequentially stretched outward at a speed of 100 mm/min until the sample was broken, and the longitudinal sample and the transverse sample were respectively tested. The final measurement results show that the longitudinal tensile strength can reach 78N and the transverse tensile strength can reach 65N.
  • Residual ethylene oxide Tested 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 residual amount does not exceed 10 ⁇ g/package.
  • Heavy metal inspection lead and chromium shall be tested according to the method specified in 5.9.1 “Test methods for medical infusion, blood transfusion and injecting instruments, Part 1: Chemical analysis method” in GB/T14233.1-2008. Mercury and arsenic shall be in accordance with GB/T14233. In the method of 5.9.1, “Test methods for medical infusion, blood transfusion, and injecting instruments, Part 1: Chemical analysis method", the total heavy metal content of lead, chromium, mercury, and arsenic in the product test solution is less than 1 ⁇ g/g. .
  • the samples in the examples were subjected to biocompatibility experiments including: pyrogen, cytotoxicity, delayed type hypersensitivity reaction, intradermal reaction, acute systemic toxicity, Ames test, mouse lymphoma cell mutation test, chromosome aberration, Implantation, subchronic toxicity.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline. According to the method specified in GB/T14233.2-2005, the product has no pyrogen reaction.
  • the test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 24 ⁇ 2 hr, and the leaching medium: serum-containing MEM medium.
  • the test solution was tested according to the test method specified in GB/T16886.5-2003, and the cytotoxic reaction of the product was not more than grade 1.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and cottonseed oil. According to the provisions of GB/T16886.10-2005 Part 10: Stimulation and delayed type hypersensitivity test method, the product has no delayed type hypersensitivity reaction.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and cottonseed oil. According to the test method specified in GB/T16886.10-2005 Part 10: Stimulation and delayed-type hypersensitivity test, the results show that the difference between the average score of the test sample and the solvent control is less than 1.0.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and cottonseed oil.
  • the test solution was tested according to the test method specified in GB/T16886.11-2011. The results showed that the product had no acute systemic toxicity.
  • test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and DMSO. According to the method specified in GB/T16886.3-2008, the result is that the Ames test of the product is negative.
  • the test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and DMSO. According to the method specified in GB/T16886.3-2008, the results: the mouse lymphoma cell mutation test of the product is a negative result
  • muscle implantation for 1 week neutrophils, lymphocytes and macrophage infiltration around the sample, no cyst formation
  • muscle implantation for 4 weeks A small amount of macrophages and lymphocytes were seen around the sample, collagen fibers and fibroblasts proliferated, and fibrous cysts were formed.
  • the samples in the examples were subjected to animal experiments to observe the effects of periosteal growth, material degradation, and guiding bone formation.
  • the Beagle dog was used to make the periosteal defect of the tibia in the forelimb, and the cortical bone was taken as the bone defect model, covering the patch, and the contralateral leg was a blank control.
  • the animal After the anesthesia is successful, the animal is placed on its back, and the legs and mouth are tied and fixed with a bandage, and the hair is removed (be careful to avoid damage to the skin), iodophor disinfection, and sterile towel.
  • An arc-shaped incision was made on the volar side of the middle arm of the forearm to cut the skin and subcutaneous tissue, expose the leg bone, and make a periosteum and bone defect model.
  • the bone defect was inserted into the sample in the example.
  • the control group was not implanted with any material, and was not fixed during the operation. After washing with sterile saline, it was sutured layer by layer. No external fixation was performed after operation.
  • each dog was given 800,000 units of penicillin intramuscularly, once a day for 3 consecutive days, and caged for feeding.
  • the experimental results are shown in Table 2 below.
  • the pathological results of HE showed that the blood supply of periosteum was good and no obvious inflammatory reaction was found.
  • the periosteal repair piece for guiding bone regeneration of the present invention has the following advantages:
  • Double-layer structure which plays the dual role of physical isolation and microenvironment for periosteal regeneration.
  • the combination of drying and lyophilization forms a double-layer composite structure with loose upper layer and dense lower layer.
  • the loose layer is an active layer containing guided bone regeneration, which has a large porosity, is favorable for cell ingrowth, and can improve the repair speed of the tissue, and the dense layer has an isolation function for removing the basal layer of the immunogen to effectively protect the repaired tissue;
  • the DNA residue can reach 10 ng/mg or less, 30-50 ng/mg lower than similar products, and the galactosidase removal rate is higher, which can reach more than 99%;
  • BMP bone morphogenetic protein

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Abstract

一种引导骨再生的骨膜修补片、制备方法和应用。骨膜修补片包括高孔隙率的疏松活性层与低孔隙率的致密基底层，包括胶原蛋白、多糖物质和活性因子，疏松活性层还包括引导骨再生活性因子，骨膜修补片植入体内可被降解吸收，其制备方法和应用也被提及。该修补片形成了上层疏松、下层致密的双层复合结构，有利于细胞的长入，能提高组织的修复速度，致密层为免疫原去除基质基底层有隔离作用，有效保护被修复的组织；添加了骨形态发生蛋白用于骨膜损伤与缺损修复。

Description

一种引导骨再生的骨膜修补片、制备方法和应用 技术领域
本发明涉及医用生物材料领域，具体为一种用于修复因创伤、骨折、肿瘤等原因引起的骨膜损伤、缺损，或配合骨移植材料植入防止材料脱出的骨膜修补片、制备方法和应用。
背景技术
由于老龄化、创伤、肿瘤等原因造成骨折、骨不连、骨延迟愈合、骨缺损等是临床上常见的骨科疾病，其治疗研究关注于骨移植材料，而忽视骨膜重修复的重要性。而事实上，骨膜具有丰富的微血管能让骨组织重新恢复血供，从而促进骨缺损部位愈合，提高骨修复成功率高；并且可作为骨组织与周围组织的重要界面，防止肌肉、肌腱、筋膜等组织长入骨缺损处，为成骨细胞、间充质干细胞提供生长微环境，促进骨组织再生与重建。因此一旦骨膜缺失无法恢复，骨缺损将难以自愈合。
目前报道的骨膜修复材料都存在不足之处，采用脱细胞基质材料基质破坏较大，DNA残留过高，免疫原性去除不彻底，易引起免疫排斥反应；采用壳聚糖、丝素蛋白等天然生物材料多采用电纺丝技术构建三维结构，无法达到天然细胞外基质的多孔网络效果；采用人工合成可降解高分子材料则会在降解过程中造成局部酸性过强，易引起炎症反应；而胶原蛋白膜则力学强度不足，降解过快，使用化学交联剂会造成组织感染、炎症与纤维包裹；或者基质中需要添加BMP、VEGF、bFGF等生长因子提高材料对组织的诱导再生能力。因此还没有一种较好的骨膜修复材料，既能起到屏障作用，可将骨损伤部位与周围组织有效隔离，防止周围组织中的成纤维细胞侵入骨损伤部位和骨移植材料脱出，又能引导骨膜组织再生修复，形成血管化组织为骨再生提供营养物质与氧气，为成骨细胞、间充质干细胞重建骨组织提供生长微环境。
发明内容
本发明针对现有技术的上述不足，提供一种具有多层结构、低免疫原性、降解可控、保留细胞外基质三维结构与生长因子的引导骨再生的骨膜修补片。
为了解决上述技术问题，本发明采用的技术方案为：一种引导骨再生的骨膜修补片，该修补片包括高孔隙率的疏松活性层与低孔隙率的致密基底层，疏松活性层和致密基底层均包括胶原蛋白、多糖物质和活性因子，疏松活性层还包括引导骨再生活性因子，所述骨膜修补片植入体内能被降解吸收。
本发明上述的疏松活性层和致密基底层是从结构角度的描述，实际上二者都是由小肠粘膜下层制造，包括的胶原蛋白、多糖物质和活性因子均是制造过程从小肠粘膜下层本身保留的物质，疏松活性层还包括引导骨再生活性因子如骨形态发生蛋白是另行加入的物质。
本发明所述的高孔隙率的疏松活性层为孔隙率75～90％的三维网状多孔结构。
本发明所述的低孔隙率的致密基底层为孔隙率45～70％的三维网状多孔结构。
上述特定的孔隙率的限定是因为：致密层用于隔离保护，防止软组织长入骨创面；而疏松层适于血管化、成骨细胞长入，致密层与疏松层各自的孔隙率范围最大程度上保证了上述效果的实现。
本发明所述的胶原蛋白为包含I型、III型、IV型和VI型胶原蛋白的组合物，这些组合物是小肠粘膜下层基质材料的基本组分。
本发明所述的多糖物质为包含硫酸软骨素和透明质酸的组合物。
本发明所述的活性因子包含纤维粘连蛋白、层粘连蛋白、整合素及生长因子。
本发明所述的引导骨再生活性因子包括诱导骨形成活性物质，优选骨形态发生蛋白。
本发明所述的骨膜修补片植入体内能被降解吸收，具体的能降解吸收的时间为3-6个月。软组织创面恢复通常在两周左右恢复，而硬组织创面(例如骨创面)恢复通常在三个月以上，六个月是骨重建完成的时间。因此，在 3个月时仍需要骨膜修复材料存在以实现软组织的隔离。至少在3个月内不能完全降解；且在6个月以后完成降解即可。
作为一些实施方式，本发明所述引导骨再生的骨膜修补片，长1-20cm，宽1-10cm，厚度1-3mm。
本发明还提供一种上述引导组织再生的骨膜修补片的制备方法，包括以下步骤：采用动物小肠粘膜下层组织(动物小肠粘膜下层)作为原料，然后经过包括组织前置处理、病毒灭活、清洗、免疫原消除、清洗、浆料制备、活性层制备、干燥、复合成型和真空冷冻干燥的步骤，获得清除动物源病毒风险、细胞成分、DNA成分和α-Gal抗原，保留细胞外基质成分的引导骨再生的骨膜修补片。
所述动物为哺乳动物，优选猪或牛。
本发明上述具体的引导骨再生的骨膜修补片的制备方法，其特征在于：步骤包括：
(1)原料的前置处理：取小肠粘膜下层组织材料进行前置处理；
(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进行病毒灭活；
(3)清洗过程：清洗步骤(2)获得的小肠粘膜下层组织；
(4)免疫原去除：采用的免疫原去除液进行免疫原去除，免疫原去除液为溶有胰蛋白酶和EDTA的PBS溶液，免疫原去除过程在多频超声环境下进行；
(5)清洗过程：进行清洗，清洗过程在超声波清洗机中进行，得到小肠粘膜下层基质材料；
(6)小肠粘膜下层基质浆料制备：使用低温破碎装置破碎由步骤(5)得到的小肠粘膜下层基质材料，向破碎后的所述基质材料加入醋酸溶液，形成小肠粘膜下层基质材料浆料；
(7)活性层材料制备：将由步骤(6)得到的浆料冷冻干燥后破碎并过筛，得到小肠粘膜下层基质颗粒，将骨形态发生蛋白溶液(BMP)与所述颗粒混合，形成膏状混合物；
(8)干燥：将步骤(5)中得到的小肠粘膜下层基质材料固定于成型模具并与成型模具一同放入烘箱中干燥；
(9)复合成型：在由步骤(8)得到的小肠粘膜下层材料上覆盖由步骤(7)中得到的已经加入骨形态发生蛋白的小肠粘膜下层基质浆料；
(10)真空冷冻干燥：将步骤(9)中得到的复合材料置于真空冷冻干燥机中进行真空冷冻干燥。
本发明步骤(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，温度范围为10-40℃。
所述步骤(3)中清洗过程为：在超声波清洗机中用pH值为7.2-7.4的PBS溶液清洗，然后用水清洗至检测电导率为10μS/cm以下终止，得到小肠粘膜下层基质材料，其中PBS溶液pH值范围为6-8，所使用的水为经过纯化处理的纯化水，超声频率优选40kHz，功率优选3000W以上。
所述步骤(4)中的免疫源去除液包括：溶解有胰蛋白酶和EDTA的pH值为6-8的PBS溶液；所述免疫源去除液中胰蛋白酶质量百分比浓度为0.01-0.2％，EDTA的浓度0.1-1mmol/L；进一步包括：免疫源去除液中胰蛋白酶的质量百分比浓度为0.02-0.05％，EDTA的浓度为0.4-0.8mmol/L，免疫源去除液的pH值为7.0-8.0，优选为7.2-7.5；所述免疫源去除液与小肠粘膜下层组织材料体积比为(20-40)：1；所述步骤(4)中多频超声至少包括两个超声频率，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，温度范围为20-35℃。
所述步骤(5)中清洗过程为：在超声波清洗机中用PBS溶液清洗，然后用降温的注射用水清洗至检测清洗前后的注射用水电导率差为1μS/cm以下终止，得到小肠粘膜下层基质材料。其中PBS溶液pH值范围为6-8，超声频率优选40kHz，功率优选3000W以上。
所述步骤(6)中的小肠粘膜下层基质浆料制备，具体的为：使用低温破碎装置破碎由步骤(5)处理得到的小肠粘膜下层基质材料，向破碎后的所述基质材料加入醋酸溶液形成小肠粘膜下层基质材料浆料；其中所述醋酸溶液的质量百分比浓度0.1％-0.8％，优选0.2％-0.3％，小肠粘膜下层基质材料与醋酸溶液质量比为1:25-1:500，优选1:50-1:100。
所述步骤(7)的活性层材料制备包括：将上述获得的浆料进行冷冻干 燥，然后用破碎装置将已经冻干的小肠粘膜下层基质蓬松块体破碎成颗粒，过筛筛选出粒径400μm以下颗粒；将质量百分比为0.01％-0.1％的骨形态发生蛋白(bone morpHogenetic protein，BMP)溶液加入到所述颗粒中，颗粒质量与骨形态发生蛋白溶液的质量比为1:1-5:1，温度20℃，混合后静置24小时备用，得到膏状混合物。
本发明上述步骤(8)所述的成型模具由带针底板、盖板与压块三部分组成，将一或多层小肠粘膜下层基质材料平铺于所述带针底板上，覆盖所述盖板，所述盖板上压5-10公斤的压块，使得材料平整、水分从四周溢出并且材料上下层之间紧密接触；开启烘箱风机，预热至25-40℃，将小肠粘膜下层基质材料和模具放于烘箱中，时间8-16小时，将用于提供压力的盖板和重物板缓缓取下，再次放于烘箱中干燥，时间需2-6小时后干燥完成。烘干后的小肠粘膜下层基质作为基底层；本发明提到的模具的结构可以参考发明专利ZL201310203588.6和ZL201310203602.2。
所述步骤(9)包括：在步骤(8)处理获得的烘干型的小肠粘膜下层基质上覆盖步骤(7)中得到的膏状混合物，使所述膏状混合物均匀平铺在烘干的小肠粘膜下层基质的上表面，浆料厚度为1-3mm。
本发明步骤(10)所述的真空冷冻干燥，具体为：将由步骤(9)得到的复合材料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，冷冻干燥装置的腔室内的压强为1-50Pa，完成真空冷冻干燥，得到最终目标产物。
本发明上述的引导骨再生的骨膜修补片的制备方法，步骤还包括：(11)包装，(12)灭菌解析。
本发明进一步提供上述引导骨再生的骨膜修补片的应用。具体为：可用于填充拔牙窝、牙槽嵴的扩展与重建、牙周骨缺损充填、牙槽嵴骨增量术、上颌窦提升术、窦底抬高、水平向骨增量、垂直向骨增量的骨膜缺损修复，隔离周围组织，引导骨组织再生；也可用于各类骨折、骨缺损、脊柱融合、骨肿瘤、骨不连、关节融合等造成骨膜缺损时的骨膜缺损修复，隔离周围组织，引导骨组织再生。
本发明的引导骨再生的骨膜修补片，其中的基底层提供组织隔离作用、 保护受损组织，活性层促进骨膜修复，引导骨组织再生。
与现有技术相比，本发明具有以下显著优点和有益效果：
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。
可选的，成型工艺技术：本发明引导骨再生的骨膜修补片为双层结构，起到物理隔离与为骨膜再生提供微环境的双重作用，采用烘干与冻干相结合的方式，形成了上层疏松、下层致密的双层复合结构。二者孔隙率的控制非常关键，特定的孔隙率使得疏松层为含有引导骨再生的活性层，孔隙率大，有利于细胞的长入，能提高组织的修复速度，致密层为免疫原去除基质基底层有隔离作用，有效保护被修复的组织；
可选的，基质破碎技术：首先采用低温破碎装置将小肠粘膜下层组织粉碎碎，然后通过醋酸膨化处理，再经过冻干形成蓬松状、可大量吸水的疏松结构，制备过程中形成浆料便于涂布成型；
可选的，添加骨形态发生蛋白技术：利用小肠微粉的强力吸水能力，加入含有骨形态发生蛋白的溶液，可以使溶液迅速充分的进入微粉颗粒内部，再经过冻干，使骨形态发生蛋白存在于小肠微粉孔径表面并在植入体内后释放；
可选的，本发明补片用于骨膜损伤与缺损修复：既能起到屏障作用，可将骨损伤部位与周围组织有效隔离，防止周围组织中的成纤维细胞侵入骨损伤部位和骨移植材料脱出，又能引导骨膜组织再生修复，形成血管化组织为骨再生提供营养物质与氧气，为成骨细胞、间充质干细胞重建骨组织提供生长微环境。
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所示的是本发明一种引导骨再生的骨膜修补片的层结构示意图。
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。
实施例1：
一种引导骨再生的骨膜修补片，按如下述方法制备：
(1)组织前置处理：
取小肠粘膜下层组织材料(如可以是猪小肠粘膜下层SIS，或者牛小肠粘膜下层，均适应于本发明的实施例)分割成规定尺寸，剔除无用组织(例如淋巴组织)，用水清洗，然后将清洗后的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，将水滤干。
(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，该过程可在不锈钢桶中进行，浓度过氧乙酸采用体积百分比0.1％，乙醇采用体积百分比5％，灭活时间2小时，溶液与小肠粘膜下层组织材料体积比为8:1，温度范围为20℃。
(3)清洗过程：
完成病毒灭活后在超声波清洗机中用PBS溶液清洗，然后用水清洗至检测电导率为10μS/cm以下终止。其中PBS溶液pH值为7.2-7.4，水为经过纯化处理的纯化水，超声频率40kHz，超声功率3000W。
(4)免疫原去除：
采用的免疫原去除液为含有质量百分比浓度0.1％的胰蛋白酶和浓度为0.4mmol/L的EDTA的pH值为7.2-7.4的PBS溶液，超声振荡处理30min，温度20℃，溶液与小肠粘膜下层组织材料体积比为20:1；多频超声包括至少两个超声频率，低频频率范围为25KHz，高频频率为65KHz，其中低频处理10min，高频处理15min，温度范围为20-35℃，超声波功率在5000W以上。
(5)清洗过程：完成免疫原去除后在超声波清洗机中用PBS溶液(磷酸盐缓冲溶液)清洗，然后用22℃的注射用水清洗至清洗前后检测注射用水电导率差值为1μS/cm以下终止，得到小肠粘膜下层基质材料。其中PBS溶液pH值为7.2-7.4。
(6)小肠粘膜下层基质浆料制备：低温破碎装置破碎由步骤(5)得到的小肠粘膜下层基质材料；低温破碎装置可用液氮冷冻破碎装置，将液氮与小肠粘膜下层材料送入液氮冷却破碎装置，小肠粘膜下层材料被液氮冷冻，利用破碎装置的高速旋转刀头进行破碎。向破碎后的所述基质材料加入醋酸溶液形成小肠粘膜下层基质材料浆料；其中所述醋酸溶液质量百分比浓度为0.3％，小肠粘膜下层基质材料与醋酸溶液质量比为1:60。形成小肠粘膜下层浆料。
(7)活性层材料制备：将由步骤(6)得到的浆料冷冻干燥后破碎并过筛，得到小肠粘膜下层基质颗粒；将质量百分比浓度为0.02％的骨形态发生蛋白(BMP)水溶液与所述颗粒混合，颗粒与骨形态发生蛋白溶液的质量比为1:1，温度20℃，混合后静置24小时备用，得到膏状混合物。
(8)干燥
将由步骤(5)处理得到的一层或多层小肠粘膜下层基质材料平铺于成型模具的带针底板上，覆盖盖板，所述盖板上压5-10公斤的压块，使得材料平整、水分从四周溢出并且材料上下层之间紧密接触。开启烘箱风机，预热至25-40℃，将小肠粘膜下层基质材料和模具放于烘箱中，时间10小时，将用于提供压力的盖板和重物板缓缓取下，再次放于烘箱中干燥，3小时后干 燥完成。
(9)复合成型
在(8)步骤中烘干的小肠粘膜下层基质材料上覆盖步骤(7)中得到的膏状混合物，使用刮平工具将膏状混合物均匀平铺在烘干的小肠粘膜下层基质材料的上表面，厚度为1～3mm。
(10)真空冷冻干燥
将步骤(9)中得到的复合材料放置于真空冷冻干燥机中，关闭冻干室的门，打开循环泵约1min，开启压缩机对冻干箱致冷，将产品预冻至-45℃，保温2小时，开启真空泵，调节产品温度约-15℃升华，6小时后，调节产品温度0℃，保温2小时，调节产品温度25℃，保温4小时，真空冷冻干燥完成。真空干燥过程中冻干室气压为1-50Pa。
通过以上方法可以得到引导骨再生的骨膜修补片。修补片中包括经历先烘干再冷冻得到的小肠粘膜下层基质材料形成的致密层以及经历先破碎后冻干的小肠粘膜下层基质材料形成的疏松层。
实施例2
本发明制备方法还可以进一步包括以下步骤：
(11)包装
烘干的产品取出后，在模具上切割，采用双层特卫强包装袋包装，该过程需要无菌转运与操作。
(12)灭菌解析：产品采用环氧乙烷灭菌，灭菌条件：温度40℃，保温时间4h，湿度70％，浓度为500mg/L，灭菌时间6h；解析过程：通风的解析室中，温度控制在20℃之间，时间约14天。
由图1可以看到，引导骨再生的骨膜修补片包括两个部分：冻干的疏松部分1和烘干的致密部分2。冻干的疏松部分1包括小肠粘膜下层冻干颗粒，并包括骨形态发生蛋白；而致密部分2包括一层或多层小肠粘膜下层材料。
对本实施例所得材料进行化学成分检测，结果如下表1所示：
蛋白(％) 碳水化合物(％) 脂类(％) 水分％ 灰分％ 生长因子％
75-85 15-25 <1 <5 <1 <2
对实施例中样品进行性能检测，检测项目与结果如下：
1)胶原蛋白亚型鉴别：采用免疫组化染色法检测I、III、IV型和VI型胶原蛋白，3μm厚连续切片，二甲苯脱蜡，梯度乙醇脱水。将切片移入电饭煲水浴中(内含0.01mol/L，pH6.0的枸橼酸三钠缓冲液)，温度保持在95-100℃，煮20min，进行抗原修复，取出后在室温下自然冷却。磷酸盐缓冲液(PBS)洗涤，5min×3次。二步法免疫组化：分别滴加I、III、IV型和VI型胶原蛋白单克隆抗体一抗，浓度1:100，4℃冰箱过夜室温下孵育60min，PBS洗涤3次。滴加Envision反应液，室温下孵育30min。PBS洗涤3次。0.05％的3,3一二氨基联苯胺+0.03％的H 2O 2显色5-10min。流水洗，苏木精衬染。递增梯度乙醇脱水，二甲苯透明，常规树脂封固。结果表明，显微镜下观察四种染色标本皆可见棕黄染色，为阳性，表明样品中可检测到I、III、IV型和VI型胶原蛋白。
2)多糖物质含量检测：取10个样品，取样，浸提，用Biocolor硫酸软骨素检测试剂盒测试硫酸软骨素含量，样品中硫酸软骨素含量平均值为5627±243μg/g；用透明质酸检测试剂盒测试透明质酸(HA)含量，结果显示，样品的透明质酸(HA)保留量平均值为302±73μg/g。
3)活性因子种类鉴别：将样品裁剪成20×20mm尺寸的3个样品，PBS浸泡24h后,固定于4％多聚甲醛5-10min，用0.1mol/LPBS洗3次，每次5min，然后用玻璃细管转至涂有多聚赖氨酸的玻片上,进行免疫组织化学染色。LN抗体、FN抗体和整合素效价均为1:100，0.5％胰酶消化3-5min暴露抗原，0.1％Triton X100作用10min增加抗体的穿透性。免疫组织化学染色显阳性，表面样品中包含纤维粘连蛋白、层粘连蛋白、整合素及其配体的等物质。
4)生长因子残留量：对实施例中样品采用ELISA法检测样品中碱性生长因子(bFGF)和血管内皮生长因子(VEGF)含量，并对免疫原去除前动 物组织作为对照。结果发现碱性生长因子(bFGF)免疫原去除前后含量分别为2312±202ng/L、1142±124ng/L，保留生长因子45％以上；血管内皮生长因子(VEGF)含量免疫原去除前后含量分别为810±66ng/L、341±23ng/L，保留生长因子30％以上。
5)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。
6)DNA残留：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残留量平均为3.15±0.42ng/mg。
7)半乳糖苷酶(α-Gal)清除率：取动物源性生物材料Gal阳性参考品，Gal抗原阴性参考品各2mg，加裂解液1ml，裂解30-90min，配制成20、10、5、2.5、1.25、0.625μg的Gal标准曲线样品，测试免疫原去除前后的测试品各取50mg，加裂解液2ml，裂解30-90min；取裂解液与M86抗体反应后的上清液，加入96孔板，加二抗，加显色剂，采用ELISA方法450nm检测吸光度值，按标准曲线计算出样品的Gal值，免疫原去除处理前材料的Gal值为(22.61±2.40)×10 14/mg，实施例中样品的Gal值为(0.13±0.01)×10 14/mg，半乳糖苷酶(α-Gal)清除率在99.43％。
8)细菌内毒：按照GB/T14233.2-2005《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：细菌内毒小于20EU/包装。
9)孔隙率：按照阿基米德原理，以乙醇作为浸提介质，计算样品致密基底层孔隙率为48.09±5.12％，疏松活性层孔隙率为85.23±6.89％。
10)缝合抗拉强度：按照实施例制备样品，用3-0非吸收缝合线在修补片一端边缘2毫米处，将缝合线与修补片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，记录最大力值，结果显示，最大值可达13N。
11)抗张强度：按照实施例制备样品，将样品裁剪成2×5cm尺寸，在相 对湿度为40％-60％，温度为22℃±2℃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100mm/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。最后的测定结果显示纵向抗张强度可达78N，横向抗张强度可达65N。
12)环氧乙烷残留量：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/包装。
13)重金属检查：铅、铬按GB/T14233.1-2008中5.9.1《医用输液、输血、注射器具检验方法第1部分：化学分析法》规定的方法试验，汞、砷按GB/T14233.1-2008中5.9.3《医用输液、输血、注射器具检验方法第1部分：化学分析法》规定的方法试验，产品检验液中铅、铬、汞、砷总重金属含量少于1μg/g。
对实施例中样品进行生物相容性实验，检测项目包括：热原、细胞毒性、迟发型超敏反应、皮内反应、急性全身毒性、Ames试验、小鼠淋巴瘤细胞突变试验、染色体畸变、植入、亚慢性毒性。
1)热原
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水。按GB/T14233.2-2005规定的方法进行，产品无热原反应。
2)细胞毒性
按质量比1:5浸提介质的比例，37±1℃，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/T16886.5-2003中规定的试验方法进行试验，结果产品的细胞毒性反应不大于1级。
3)迟发型超敏反应
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和棉籽油。按照GB/T16886.10-2005第10部分：刺激与迟发型超敏反应试验方法规定进行试验，结果产品无迟发型超敏反应。
4)皮内反应
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和棉籽油。按照GB/T16886.10-2005第10部分：刺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。
5)急性全身毒性
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和棉籽油。取试验液按照GB/T16886.11-2011规定的试验方法进行试验，结果：产品无急性全身毒性反应。
6)Ames试验
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO。按GB/T16886.3-2008规定的方法进行，结果：产品的Ames试验为阴性。
7)小鼠淋巴瘤细胞突变试验
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO。按GB/T16886.3-2008规定的方法进行，结果：产品的小鼠淋巴瘤细胞突变试验为阴性结果
8)染色体畸变试验
按质量比1:5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO，按GB/T16886.3-2008规定的方法进行，结果：产品的染色体畸变试验为阴性
9)植入
按GB/T16886.6-1997规定的方法进行，结果：肌肉植入1周：样品周围可见嗜中性粒细胞、淋巴细胞和巨噬细胞浸润，应无囊腔形成；肌肉植入4周：样品周围可见少量巨噬细胞和淋巴细胞，胶原纤维和纤维母细胞增生，有纤维囊腔形成；肌肉植入12周：样品周围可见少量淋巴细胞、胶原纤维、纤维囊腔较致密规整。
10)亚慢性毒性
按GB/T16886.11规定的方法进行，结果：无亚慢性毒性反应。
对实施例中样品进行动物实验，观察骨膜生长、材料降解、引导骨生成效果。选择Beagle犬在前肢造桡骨骨膜缺损，并取皮质骨作骨缺损模型，覆盖补片，对侧腿为空白对照。动物实验过程：用速眠新注射液1.5ml+盐酸氯胺酮注射液0.19+阿托品0.5mg混合液按0.4ml/kg，行肌肉注射麻醉。麻醉成功后，将动物仰卧，用绷带将其双后腿及嘴巴系住固定，褪毛(注意避免损破皮肤)、碘伏消毒、铺无菌巾。于前臂中段掌侧取弧形切口，切开皮肤及皮下组织，暴露腿骨，造骨膜、骨缺损模型。实验组骨缺损处嵌插植入实施例中样品，对照组不植入任何材料，术中不予固定，无菌生理盐水冲洗后，逐层缝合。术后未予外固定，为预防感染，每只犬给予青霉素80万单位肌肉注射，1次/天，连续3天，分笼喂养。实验结果如下表2所示。HE病理切片结果表明骨膜形成血供良好，未发现明显的炎症反应。
表2本发明实施例样品使用效果
Figure PCTCN2018077850-appb-000001
综上，本发明的引导骨再生的骨膜修补片，具有如下的优势：
(1)保留细胞外基质中胶原蛋白纤维的三维空间结构；
(2)双层结构，起到物理隔离与为骨膜再生提供微环境的双重作用，采用烘干与冻干相结合的方式，形成了上层疏松、下层致密的双层复合结构。疏松层为含有引导骨再生的活性层，孔隙率大，有利于细胞的长入，能提高组织的修复速度，致密层为免疫原去除基质基底层有隔离作用，有效保护被 修复的组织；
(3)力学强度高，可控降解，与骨组织再生周期同步；
(4)促进组织血管化，降低引起感染、炎症或纤维包裹的风险；
(5)DNA残留能够达到10ng/mg以下，较同类产品低30-50ng/mg、半乳糖苷酶去除率较高，能够达到99％以上；
(6)保留细胞外基质中活性生长因子，添加骨形态蛋白(BMP)。
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 (10)

  1. 一种引导骨再生的骨膜修补片，其特征在于：该修补片包括高孔隙率的疏松活性层与低孔隙率的致密基底层，疏松活性层和致密基底层均包括胶原蛋白、多糖物质和活性因子，疏松活性层还包括引导骨再生活性因子，所述骨膜修补片植入体内能被降解吸收。
  2. 根据权利要求1所述的骨膜修补片，其特征在于，疏松活性层和致密基底层由哺乳动物的小肠粘膜下层组织材料制成，优选猪或牛的小肠粘膜下层组织材料。
  3. 根据权利要求1所述的引导骨再生的骨膜修补片，其特征在于：所述的疏松活性层为三维网状多孔结构，其孔隙率为75～90％；所述的致密基底层为三维网状多孔结构，其孔隙率为45～70％.
  4. 根据权利要求1所述的引导骨再生的骨膜修补片，其特征在于：所述的胶原蛋白为包含I型、III型、IV型和VI型胶原蛋白的组合物；所述的多糖物质为包含硫酸软骨素和透明质酸的组合物；所述的活性因子包含纤维粘连蛋白、层粘连蛋白、整合素及生长因子；所述的引导骨再生活性因子包括诱导骨形成活性物质。
  5. 根据权利要求1所述的引导骨再生的骨膜修补片，其特征在于：所述的植入体内能被降解吸收时间为3-6个月。
  6. 根据权利要求1所述的引导骨再生的骨膜修补片，其特征在于：所述该修补片的长1-20cm、宽1-10cm、厚度1-3mm。
  7. 一种引导骨再生的骨膜修补片的制备方法，其特征在于：采用动物小肠粘膜下层组织作为原料，步骤包括：
    (1)原料的前置处理：取动物小肠粘膜下层组织材料进行前置处理；
    (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进行病毒灭活；
    (3)清洗过程：清洗小肠粘膜下层组织；
    (4)免疫原去除：采用的免疫原去除液为溶有胰蛋白酶和EDTA的PBS溶液，免疫原去除过程在多频超声环境下进行；
    (5)清洗过程：进行清洗，清洗过程在超声波清洗机中进行，得到小肠粘膜下层基质材料；
    (6)小肠粘膜下层基质浆料制备：使用低温破碎装置破碎由步骤(5) 得到的小肠粘膜下层基质材料，向破碎后的所述基质材料加入醋酸溶液，形成小肠粘膜下层基质材料浆料；
    (7)活性层材料制备：将由步骤(6)得到的浆料冷冻干燥后破碎并过筛，得到小肠粘膜下层基质颗粒，将骨形态发生蛋白溶液(BMP)与所述颗粒混合，形成膏状混合物；
    (8)干燥：将步骤(5)中得到的小肠粘膜下层基质材料固定于成型模具上、并与成型模具一同放入烘箱中进行干燥；
    (9)复合成型：在(8)步骤中烘干型的产品上覆盖步骤(7)得到的膏状混合物；
    (10)真空冷冻干燥：将步骤(9)中得到的材料与模具置于真空冷冻干燥机中进行真空冷冻干燥。
  8. 根据权利要求7所述的引导骨再生的骨膜修补片的制备方法，其特征在于：
    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％，过氧乙酸-乙醇溶液与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，温度范围为10-40℃；
    所述步骤(3)中清洗过程为：在超声波清洗机中用PBS溶液清洗，然后用水清洗至检测电导率为10μS/cm以下终止，得到小肠粘膜下层基质材料，其中PBS溶液pH值范围为6-8，所使用的水为经过纯化处理的纯化水；
    所述步骤(4)中的免疫源去除液包括：溶解有胰蛋白酶和EDTA的PBS溶液；所述免疫源去除液中胰蛋白酶质量百分比浓度为0.01-0.2％，EDTA的浓度0.1-1mmol/L，免疫源去除液的pH值为7.0-8.0，所述免疫源去除液与小肠粘膜下层组织材料体积比为(20-40)：1；所述步骤(4)中多频超声至少包括两个超声频率，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，温度范围为20-35℃，超声功率5000W以上；
    步骤(5)中清洗过程为：在超声波清洗机中用PBS溶液清洗，然后用降温的注射用水清洗至检测清洗前后的注射用水电导率差为1μS/cm以下终止，得到小肠粘膜下层基质材料，其中PBS溶液pH值范围为6-8；
    所述的步骤(6)小肠粘膜下层基质浆料制备为：使用低温破碎装置破碎由步骤(5)处理得到的小肠粘膜下层基质材料，向破碎后的所述基质材料加入醋酸溶液形成小肠粘膜下层基质材料浆料；其中所述醋酸溶液的质量百分比浓度为0.1％-0.8％，浆料中的小肠粘膜下层基质材料与醋酸溶液质量比为1:25-1:500；
    所述的步骤(7)活性层制备为：向小肠粘膜下层基质颗粒中加骨形态发生蛋白溶液，骨形态发生蛋白溶液的质量百分比浓度为0.01-0.1％，小肠粘膜下层基质颗粒与骨形态发生蛋白溶液的质量比为(1-10)：2；
    所述的步骤(10)真空冷冻干燥为：将由步骤(9)得到的复合材料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，冷冻干燥装置的腔室内的压强为1-50Pa，完成真空冷冻干燥。
  9. 根据权利要求8所述的引导骨再生的骨膜修补片的制备方法，其特征在于：所述的步骤(4)免疫源去除液中胰蛋白酶的质量百分比浓度为0.02-0.05％，EDTA的浓度为0.4-0.8mmol/L，免疫源去除液的pH值为7.2-7.5；所述的步骤(8)干燥温度25-40℃后，时间为8-16小时；所述的步骤(6)醋酸溶液的质量百分比浓度为0.2％-0.3％，浆料中的小肠粘膜下层基质材料与醋酸溶液质量比为1:50-1:100。
  10. 一种引导骨再生的骨膜修补片在骨膜修补材料中的用途，其特征在于：所述骨膜修补材料用于填充拔牙窝、牙槽嵴的扩展与重建、牙周骨缺损充填、牙槽嵴骨增量术、上颌窦提升术、窦底抬高、水平向骨增量、垂直向骨增量的骨膜缺损修复，隔离周围组织，引导骨组织再生；或用于各类骨折、骨缺损、脊柱融合、骨肿瘤、骨不连、关节融合等造成骨膜缺损时的骨膜缺损修复，隔离周围组织，引导骨组织再生。
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