WO2017185853A1 - 一种复合细胞外基质成分生物材料 - Google Patents

一种复合细胞外基质成分生物材料 Download PDF

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WO2017185853A1
WO2017185853A1 PCT/CN2017/073394 CN2017073394W WO2017185853A1 WO 2017185853 A1 WO2017185853 A1 WO 2017185853A1 CN 2017073394 W CN2017073394 W CN 2017073394W WO 2017185853 A1 WO2017185853 A1 WO 2017185853A1
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extracellular matrix
layer
ubm
matrix component
sis
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French (fr)
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张剑
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Excellence Medical Technologies (suzhou) Co Ltd
Shanghai Excellence Medical Technologies Co Ltd
Second Military Medical University SMMU
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Excellence Medical Technologies (suzhou) Co Ltd
Shanghai Excellence Medical Technologies Co Ltd
Second Military Medical University SMMU
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Application filed by Excellence Medical Technologies (suzhou) Co Ltd, Shanghai Excellence Medical Technologies Co Ltd, Second Military Medical University SMMU filed Critical Excellence Medical Technologies (suzhou) Co Ltd
Priority to EP17788509.2A priority Critical patent/EP3443990B1/en
Priority to US16/095,633 priority patent/US20190314553A1/en
Priority to JP2019503607A priority patent/JP6827522B2/ja
Publication of WO2017185853A1 publication Critical patent/WO2017185853A1/zh
Anticipated expiration legal-status Critical
Priority to US17/518,124 priority patent/US20220054707A1/en
Ceased legal-status Critical Current

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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
    • A61L27/38—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 containing added animal cells
    • A61L27/3886—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 containing added animal cells comprising two or more cell types
    • A61L27/3891—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 containing added animal cells comprising two or more cell types as distinct cell layers
    • 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
    • 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
    • A61L27/3604—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
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention belongs to the field of tissue repair materials, and particularly relates to a composite extracellular matrix component biomaterial.
  • Acellular Tissue Matrix is an important advance in the study of soft tissue repair materials in the past two decades. That is, physical or chemical methods are used to remove all cells, antigens, lipids, soluble proteins and other substances in the tissue, and the insoluble extracellular matrix (ECM) with intact appearance, histological characteristics and ultrastructure is retained.
  • ECM extracellular matrix
  • ACTM can induce "endogenous tissue regeneration”: biosignal or degradation products contained within the implant can induce macrophages around the repair area. Cells and stem cells actively and rapidly infiltrate, grow, proliferate and secrete their own extracellular matrix to replace implants. As the host tissue grows in, the ACTM gradually degrades, and the two are basically synchronized, and finally the ACTM is completely replaced by the host tissue.
  • ACTM has many advantages as a tissue repair material: 1 structure and composition close to natural tissue: the main component is structural protein such as collagen fiber, and a small amount of glycoprotein, fibronectin, glycosaminoglycan and proteoglycan, growth Factors such as factors and enzymes; 2ACTM has good degradability and controllability of degradation rate, and degradation is more in line with regeneration rules; 3ACTM has a certain porosity, which facilitates the exchange of nutrients and material air between tissues; 4ACTM has a certain mechanical strength Can support the growth of the organization. 5 has a certain tolerance to infection, can be used in tissue defects with contamination or potential infection, because it can achieve early entry of phagocytic cells, early local rapid revascularization, bacterial biofilm is difficult to form.
  • ACTM has been clinically used in the replacement of meninges, pleura, abdominal wall fascia, etc., anastomotic enhancement, pelvic floor reconstruction, bladder suspension, liver and spleen and other physical organ damage and hemostasis, as well as treatment of various complex fistula and abdominal wall defects. Contaminated abdominal wall defects, infection after implantation of synthetic patches, and secondary surgical treatment of intestinal fistula. According to statistics, the amount of ACTM in the United States has accounted for 5 to 10% of soft tissue repair materials.
  • the biological patches can be roughly divided into two categories: 1 Inert tissue (IT) source products, human corpse/pig dermis, bovine/pig/horse pericardium, bovine/porcine peritoneum, etc. representative.
  • the source tissue is a biologically inert tissue in the body.
  • the components are almost only structural proteins (collagen fibers and elastic fibers), and bioactive components such as no adhesion proteins, growth factors and proteoglycans.
  • 2ECM source biological material which has a complete three-dimensional ultrastructure of the extracellular matrix of living tissue and biological active components such as fibronectin, growth factor and glycosaminoglycan.
  • ECM biomaterials can be effectively integrated with the host area after implantation, actively attracting autologous stem cells to move into the lesion and promote proliferation and differentiation to achieve partial degree of specificity.
  • Functional repair such as achieving tissue regeneration such as muscle fascia and partial restoration of innervation to improve disability limb function and fingertip reconstruction.
  • 2IT source products are compact in structure and contain a large number of elastic fibers that are slowly degraded and cannot be regenerated after 25 years of age, resulting in long-term instability and loss of elasticity in the repair area.
  • 3ECM biomaterials are more resistant to infection and tissue regeneration than IT source products.
  • SIS is derived from the small intestine of mammals and is obtained by mechanically removing the muscular layer of the small intestine and the serosal layer.
  • SIS is mainly composed of type I and type III collagen, containing a small amount of IV and V collagen, glycosaminoglycan, growth factor, fibronectin, etc., which can be completely degraded after being implanted in the body, and is a good scaffold material for tissue engineering.
  • SIS has good mechanical strength, a wide range of raw materials, easy pretreatment or automated processing by machine.
  • the biological activity of SIS is relatively low, and it has a high initial bioburden due to its exposure to various antigens in food in a living environment, although it has a certain degree of decellularization, sterilization, etc.
  • the immunogenicity possibly endotoxin, Gal epitopes, etc. causes a host immune response.
  • Chinese patent CN2608014 discloses an artificial dura mater with both dural and arachnoid functions, which binds the submucosal layer of the small intestine on the human amniotic membrane layer, and has the dual functions of mechanical tensile resistance of the human dura mater and arachnoid anti-adhesion.
  • this method of preparation does not completely isolate the immunogenicity of the SIS.
  • Chinese patent CN101366979 discloses a tissue patch and a preparation method thereof, which comprises an adipose small intestinal submucosa as an inner layer and two sides covered with a decellularized amnion.
  • the decellularized amniotic membrane isolates the immunotoxicity of the submucosal layer of the acellular small intestine, while the submucosal layer of the acellular small intestine compensates for the lack of mechanical strength of the acellular amniotic membrane, has high biological activity and histocompatibility, and has no obvious immune rejection.
  • the cells are non-toxic.
  • the amnion is a human-derived material, the source is difficult to control, and there is a risk of spreading an unknown virus or disease.
  • UBM is derived from the mammalian bladder and is obtained by mechanically removing the serosa, muscle layer, submucosa, and mucosal muscle layer and then decellularizing. Compared with SIS, UBM has the following advantages: 1 very low immunogenicity, high histocompatibility: UBM does not contact biological loads such as bacteria in vivo, the level is simple, and endotoxin-free contamination can be achieved during the process. 2 high biological activity: UBM contains intact basement membrane structure and composition, which can form a supporting layer for the growth of epithelial cells such as blood vessels and skin. It can help tissue-specific cells to form continuous slices, inhibit scar tissue formation, and help strengthen tissues. Repair and wound healing and achieve tissue-specific regeneration of the defect.
  • the open wound healing time of patients using Matristem was shortened from 25.5 weeks to 9.8 weeks. Its degradation products contain more than 5,000 active ingredients, which can confirm 41 proteins or peptides related to tissue healing, including neurotrophic, angiogenic, tumor-inhibiting or other components related to tissue remodeling and wound repair (Table) 1).
  • the peeling of UBM requires a lot of labor, and the mechanical strength is insufficient, and the surface is smooth and it is difficult to produce a product having a certain thickness.
  • the technical problem to be solved by the present invention is to provide a composite extracellular matrix component biomaterial which combines the advantages of UBM and SIS: the immunogenicity of UBM isolation SIS and its direct contact with host tissues, the host after implantation -
  • the basic type of inflammatory reaction in the material junction area is the same as that of simple UBM, and the histocompatibility is high.
  • SIS can make up for the shortcomings of low mechanical strength of UBM, and the SIS is easy to prepare and has a variable thickness after splicing; it can be applied to filling and strengthening. Repair or reconstruct various soft tissue defects such as fascia, meninges, pleura, pelvic floor, dermis, and parenchymal organs, and have good clinical applicability.
  • a composite extracellular matrix component biomaterial of the present invention wherein the biomaterial is an adipose small intestinal submucosal SIS as an intermediate layer, and a decellularized bladder mucosa base film UBM is an upper and lower layer; the upper and lower layers are completely covered with an intermediate layer.
  • Sandwich structure wherein the biomaterial is an adipose small intestinal submucosal SIS as an intermediate layer, and a decellularized bladder mucosa base film UBM is an upper and lower layer; the upper and lower layers are completely covered with an intermediate layer.
  • the SIS is obtained by mechanically removing the serosa layer and the muscular layer from the small intestine of the mammal and then decellularizing the membrane to obtain a membrane-like material.
  • the UBM is obtained by mechanically removing the serosa, muscle layer, submucosa, and mucosal muscle layer from a mammalian bladder, and then decellularizing the membrane to obtain a membrane-like material.
  • the number of layers of the intermediate layer is 1 to 20 layers.
  • the number of layers in the upper and lower layers is 1 to 10 layers each.
  • the intermediate layer and the upper and lower layers, the intermediate layer and the upper and lower layers are fixed by one or several methods of medical adhesive, suture binding, and vacuum lamination.
  • the upper and lower layers have high biological activity and effectively isolate the immunogenicity of the intermediate layer without altering the host-material interface inflammatory response type of the outer UBM.
  • the intermediate layer can significantly increase the mechanical strength and material thickness of the material.
  • the medical adhesive is one or more of components such as chitosan, collagen, fibrin glue, hyaluronic acid, chondroitin sulfate, hydrogel, bone glue, gelatin or pectin. Medical adhesives and sutures are preferably absorbable components.
  • the process parameters of the vacuum lamination are: a vacuum pressure of -50 to -760 mmHg, and an action time of 0.5 to 72 h.
  • the biomaterial also includes holes through the material.
  • the holes have a diameter of 1 to 5 mm and a hole pitch of 0.5 to 5 cm.
  • the surface layer of UBM basement membrane tissue can release a large number of active factors supporting and regulating life behaviors such as cell growth and differentiation, such as basic fibroblast growth factor, epidermal growth factor, liver Cell growth factor, keratinocyte growth factor and other components promote cell adhesion and migration, induce differentiation, reduce apoptosis; regulate epithelial cells to preferentially fibroblast invasion and inhibit excessive fibrinogen exudation, predominating in fibroblasts
  • the epithelial tissue is formed before, and the basement membrane structure with smooth surface of the joint material reduces adhesion and inhibits scar tissue formation.
  • the intermediate layer can improve the mechanical strength of the material as a whole, and the number of layers of the SIS can be appropriately increased or decreased to adapt to the tissue repair of different mechanical requirements.
  • the combination of medical adhesive and vacuum lamination enhances the peel strength of the material, ensuring the integrity of the multilayer material after hydration or tailoring, and is less prone to loosening.
  • the invention can greatly reduce the amount of production and production cycle limiting factors - UBM. Reduce material costs, shorten production cycles, and reduce manpower input.
  • 1 is a schematic view showing the structure of the present invention; wherein 1 is a decellularized small intestinal submucosal SIS, and 2 is a decellularized bladder mucosal basement membrane UBM.
  • a porcine-derived acellular bladder mucosal basement membrane (UBM) and a decellularized small intestinal submucosa (SIS) were prepared by the Abraham method.
  • UBM human body mucosal basement membrane
  • SIS small intestinal submucosa
  • One layer of UBM is flattened (smooth face down), and a single film-shaped SIS is spliced into independent layers in a manner of 50% interlayer dislocation, which is laid on the surface of UBM, and four layers are laid at intervals of 90° between layers.
  • a layer of UBM (smooth face up) is placed on top of it. Air bubbles were removed and the layers were bonded using a medical chitosan adhesive and pressed together at a pressure of -250 mm Hg for 24 h.
  • the material is perforated in a full layer with a hole spacing of 5 mm and a diameter of 1 mm.
  • a porcine-derived acellular bladder mucosal basement membrane (UBM) and a decellularized small intestinal submucosa (SIS) were prepared by the Abraham method.
  • the two-layer UBM is flattened (smooth face down), and the single-film SIS is spliced into independent layers in a manner of 50% interlayer dislocation, which is laid on the surface of the UBM, and the layers are offset by 90° to lay 6 layers. It is then laid with two layers of UBM (smooth facing up). Air bubbles were removed and the layers were bonded using a medical collagen adhesive and pressed together at a pressure of -300 mm Hg for 36 h. The material is perforated in a full layer with a hole spacing of 8 mm and a diameter of 2 mm.
  • Three samples were prepared to be 2 cm ⁇ 5 cm, and both ends were fixed on the upper and lower grippers of the tensile machine, and continuously peeled off at a stable speed of 10 mm/min until the overlapping portions of the samples were layered. Measure the load force during delamination.
  • the peel strength between the SIS-SIS and the UBM-SIS was 6 ⁇ 2 N/cm, and the peeling maintenance force was 1.5 ⁇ 0.5 N/cm.
  • the cytotoxicity of the material was evaluated according to the method specified in GB/T 16886.5. NIH3T3 and L929 were used as model cells. The cell culture medium was used as the extraction medium, and the cells were cultured with a gradient concentration of the extract medium, and the cell survival rate was determined by MTT assay. The cytotoxicity of the material is from 0 to I.
  • the material was pulverized at a low temperature, and the protease was digested, and the concentration of the enzymatic hydrolyzed product was 50 ⁇ g/mL. After the cells were cultured for 24 hours, the Boyden chamber method was used to determine the cell migration for 6 hours. The cells containing 10% fetal bovine serum and serum-free medium were positive and negative controls, respectively. The cell migration of the repair material was 2056 ⁇ 72, the positive control was 2105 ⁇ 35, and the negative control was 1328 ⁇ 65. There was no significant difference between the material group and the positive control group (P>0.05).
  • the endotoxin content of the material was tested according to the method specified in GB/T14233.2. Endotoxin test water as extraction medium, 37 ° C Dip for 24h. The dynamic turbidity method was used to determine the endotoxin content of the material, and the endotoxin content of the diluted extract was retested to eliminate interference.
  • the material has an endotoxin content ⁇ 5 EU / device.
  • the blood compatibility of the material was determined according to the method specified in GB/T14233.2.
  • Contact group the back of the rat was depilated, and the enzymatic hydrolysate was applied at a concentration of 50 ⁇ g/mL, applied once a day for 20 days; the oral ingestion group: 1 mL of the extract was taken orally every other day for 7 days;
  • the intramuscular group and the intravenous group were injected with a concentration of 0.15 mL of the extract every other day for 7 days, and each injection was performed 4 times.
  • the animals were sacrificed in two batches on the 30th and 90th days of exposure, and venous blood was taken for testing.
  • the intradermal irritancy of the material was evaluated according to the method specified in GB/T 16886.10.
  • the white rabbits were intradermally injected with 0.2 mL of the extract and the control solution (PBS).
  • the skin reaction of the experimental area was observed at 15 min, 1 h, 2 d, and 3 d after the injection, and scored according to the erythema and edema.
  • the material is non-irritating.
  • the material sensitization was evaluated according to the maximum dose method specified in GB/T 16886.10.
  • the pure starch solution was used as a negative control group, and it was orally administered for 1 week. After stopping the drug, the observation was continued for 1 week.
  • the body weight of the rats was recorded daily, and the clinical signs of toxicity were observed and the toxicity level was recorded. At the end of the experiment, the routine histopathological sections of the rats were sacrificed. The material has no delayed hypersensitivity reaction.
  • An animal model of canine rectus abdominis anterior sheath and rectus abdominis defect was constructed.
  • the defect area was 10 ⁇ 5 cm 2 .
  • the composite soft tissue repair material was cut to a certain size for repair.
  • the simple SIS and simple UBM were used as controls.
  • the incidence of seroma in the repair area of the simple SIS control group was 33%, and no seroma occurred in the simple UBM and composite soft tissue repair materials.
  • the repaired area was removed at 2 weeks, 1 month, 2 months, and 4 months after operation.
  • Tissue sections were stained with CD68, CCR7, and CD163 to observe the infiltrating cell type and density, and the ratio of M1/M2 macrophages confirmed that the material did not change UBM.
  • the host-material inflammatory response is of a basic type and the tissue repair effect is similar to that of pure UBM.

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Abstract

一种复合细胞外基质成分生物材料，以脱细胞小肠粘膜下层SIS（1）为中间层，脱细胞膀胱粘膜层基底层UBM（2）为上下表层。该上下表层完全包覆中间层形成三明治结构。通过这种三明治结构，UBM（2）隔绝SIS（1）的免疫原性，并且与宿主组织直接接触，组织相容性高，SIS（1）能弥补UBM（2）力学强度低的缺点。该复合细胞外基质成分生物材料可用于填充、加强、修复或重建筋膜、脑膜、胸膜、盆底、真皮、实质脏器等各种软组织缺损。

Description

一种复合细胞外基质成分生物材料 技术领域
本发明属于组织修复材料领域，特别涉及一种复合细胞外基质成分生物材料。
背景技术
脱细胞基质生物材料(Acellular Tissue Matrix，ACTM)是近二十年来软组织修复材料研究的重要进展。即运用物理或化学方法脱去组织中的所有细胞、抗原、脂质、可溶性蛋白质等物质、保留下的具有完整外观形态、组织学特性及超微结构的不溶性细胞外基质(Extracellular Matrix，ECM)作为生物支架。ECM是生物进化过程中高度保守的部分，不同种属间相同组织内ECM差异小，经脱细胞技术去除引起免疫排斥反应的细胞成分和抗原后的ACTM是能够安全的异种异体移植的。与合成材料的引发慢性炎症刺激，导致纤维细胞包裹瘢痕修复的机理不同，ACTM可以诱导“内源性组织再生”：植入后其内所含的生物信号或降解产物可以诱导修复区周围巨噬细胞和干细胞主动、快速浸润，生长、增殖并分泌自身细胞外基质替代植入物。随着宿主组织的长入，ACTM逐步降解，两者基本同步，最终ACTM完全被宿主组织代替。因此，ACTM作为组织修复材料具有较多优势：①结构和组分接近自然组织：主要成分是胶原纤维等结构蛋白，和少量糖蛋白、纤粘蛋白、糖胺聚糖和蛋白多糖类、生长因子与酶类等；②ACTM有良好的可降解性和降解速率可控性，降解更符合再生规律；③ACTM具有一定的孔隙率，便于组织间的营养和物质空气的交换；④ACTM具有一定的机械强度能够对组织的生长起到支持作用。⑤具备一定的耐受感染能力，可以用在伴有污染或潜在感染的组织缺损，因为其可以实现吞噬细胞早期进入、早期局部快速再血管化，细菌生物膜难以形成。
ACTM目前已临床应用于替代脑膜、胸膜、腹壁筋膜等，吻合口加强、盆底重建、膀胱悬吊、肝脾等实质脏器破损填塞止血以及各种复杂疝和腹壁缺损的治疗如伴有污染的腹壁缺损、合成补片植入后感染、肠瘘二次手术治疗等。据统计，在美国ACTM的用量已占到软组织修复材料的5～10％。根据组织来源的不同生物补片可大体分为两类：①惰性组织(Inert tissue，IT)源产品，以人尸/猪的真皮、牛/猪/马的心包、牛/猪的腹膜等为代表。来源组织属机体内生物惰性组织，成分几乎仅为结构蛋白(胶原纤维和弹力纤维)，无粘连蛋白、生长因子和蛋白聚糖类等生物活性成分。②ECM源生物材料，其拥有完整的活体组织细胞外基质的三维超微结构和粘连蛋白类、生长因子类、糖胺聚糖类等生物活性成分。这类产品以小肠粘膜下层(Small Intestinal Submucosa,SIS)、人羊膜、膀胱粘膜层基底膜(Urinary Bladder Matrix,UBM)等为代表。ECM生物材料的制备技术(包括脱细胞处理和成型工艺)较IT源产品更复杂但ECM生物材料较IT源产品拥有更多修复组织缺损的优势：①IT源产品仅能诱导血管 化的结缔组织再生填充组织缺损、实现解剖层面的修复，而ECM生物材料植入后可与宿区有效整合，主动吸引自体干细胞迁入损伤处并促进增殖和分化而实现部分程度的特异性、功能性修复，如实现肌肉筋膜等组织再生和神经支配部分恢复而改善残疾肢体功能和指尖再造。②IT源产品结构致密，含有大量降解缓慢且人体25岁后不能再生的弹性纤维，导致修复区远期不稳定，易失弹性。③ECM生物材料较IT源产品更耐受感染、组织再生速度更快。
SIS来源于哺乳动物小肠，是机械除去小肠肌层和浆膜层后经脱细胞处理而得。SIS主要由Ⅰ、Ⅲ型胶原组成，含有少量的Ⅳ、Ⅴ型胶原，糖胺聚糖、生长因子、纤连蛋白等，植入体内后可完全降解，是组织工程良好的支架材料。SIS具有良好的力学强度，原材料来源广泛，预处理较为简便或借助机器实现自动化处理。但SIS的生物活性相对较低，且由于其在活体环境中可能接触到食物中各种抗原而致其带有较高的初始生物负载，虽然经过严格的脱细胞、灭菌等处理仍具有一定的免疫原性(可能的内毒素、Gal抗原表位等残留)，引起宿主免疫反应。
中国专利CN2608014公开了一种兼有硬膜和蛛网膜功能的人造硬脑膜，在人羊膜层上粘合小肠粘膜下层，具有人体硬脑膜的机械抗张能力和蛛网膜防粘连的双重功能。但这一制备方法没有完全隔绝SIS的免疫原性。
中国专利CN101366979公开了一种组织补片及其制备方法，以脱细胞小肠粘膜下层为内层，两面包裹覆有脱细胞羊膜所构成。以脱细胞羊膜隔绝了脱细胞小肠粘膜下层的免疫毒性，而脱细胞小肠粘膜下层补偿了脱细胞羊膜的机械强度不足，具有较高的生物活性和组织相容性，无明显免疫排斥反应，对细胞无毒性。但羊膜为人源性材料，来源难以控制，存在传播未知病毒或疾病的风险。
UBM来源于哺乳动物膀胱，经机械方法除去浆膜、肌层、粘膜下层、粘膜肌层后脱细胞处理所制得。与SIS相比，UBM具有如下优势：①极低免疫原性，高组织相容性：UBM在活体内不接触细菌等生物负载，层次简单，且取材过程中可以做到无内毒素污染。②生物活性高：UBM含完整基底膜结构和成分，可构成血管、皮肤等上皮组织细胞生长的支撑层，可以帮助组织特异性细胞形成连续的片层、抑制疤痕组织生成，有助于增强组织修复和伤口愈合并实现缺损组织特异性的再生。应用Matristem(Acell公司UBM产品)的患者开放性伤口愈合时间由25.5周缩短至9.8周。其降解产物含有超过5000种活性成分，其中可以确证与组织愈合相关的蛋白或多肽有41种，包括神经营养、促血管新生、抑制肿瘤活性或其他与组织重塑、伤口修复相关的成分(表1)。但在实际生产过程中，UBM的剥离需耗费大量人工，且机械强度不足，表面光滑难以制成具有一定厚度的产品。
表1UBM降解产物活性成分
Figure PCTCN2017073394-appb-000001
Figure PCTCN2017073394-appb-000002
发明内容
本发明所要解决的技术问题是提供一种复合细胞外基质成分生物材料，该材料综合了UBM和SIS的优点：UBM隔绝SIS的免疫原性及其与宿主组织的直接接触，植入后的宿主-材料交界区炎症反应基本类型与单纯UBM相同，组织相容性高；另一方面，SIS能弥补UBM力学强度低的缺点，且SIS制备方便、拼接后厚度可变；可应用于填充、加强、修复或重建筋膜、脑膜、胸膜、盆底、真皮、实质脏器等各种软组织缺损，具备良好的临床实用性。
本发明的一种复合细胞外基质成分生物材料，所述生物材料以脱细胞小肠粘膜下层SIS为中间层，脱细胞膀胱粘膜层基底膜UBM为上下表层；所述上下表层完全包覆中间层形成三明治结构。
所述SIS由哺乳动物小肠经机械方法除去浆膜层和肌层后脱细胞处理制得，为膜片状材料。
所述UBM由哺乳动物膀胱经机械方法除去浆膜、肌层、粘膜下层、粘膜肌层后脱细胞处理制得，为膜片状材料。
所述中间层的层数为1～20层。
所述上下表层的层数各为1～10层。
所述中间层与上下表层间、中间层与上下表层内(即多层UBM间、多层SIS间)以医用粘合剂、缝合捆扎、真空层压中的一种或几种方式固定。
所述上下表层具有高生物活性，且有效隔绝中间层的免疫原性，不改变外层UBM的宿主-材料交界区炎症反应类型。所述中间层可显著提高材料的力学强度与材料厚度。
所述医用粘合剂为壳聚糖、胶原蛋白、纤维蛋白胶、透明质酸、硫酸软骨素、水凝胶、骨胶、明胶或果胶等成分中一种或几种。医用粘合剂、缝合线优选可吸收成分。
所述真空层压的工艺参数为：真空压力为-50～-760mmHg，作用时间为0.5～72h。
所述生物材料还包括贯穿材料的孔洞。
所述孔洞的直径为1～5mm，孔间距为0.5～5cm。
有益效果
(1)高生物活性，组织粘连轻，无过量瘢痕：表层UBM基底膜组织成分可以释放大量支持和调控细胞生长分化等生命行为的活性因子，如碱性成纤维生长因子、表皮生长因子、肝细胞生长因子、角蛋白细胞生长因子等成分，促进细胞的黏附和迁移，诱导分化，减少凋亡；调控上皮细胞优先于成纤维细胞侵入并抑制过量纤维蛋白原渗出，在成纤维细胞占优势前形成上皮组织，联合材料表面光滑的基底膜结构，减少粘连，抑制瘢痕组织生成。
(2)高组织相容性、低免疫原性、不改变UBM的宿主-材料交界区炎症反应基本类型：SIS被包裹在极低免疫原性的UBM中，保证植入初期无法直接接触到周围组织，延迟可能引起免疫反应的成分释放，免疫反应类型和程度与单纯UBM植入相同。伴随UBM的降解，组织同步长入，植入后期SIS的逐渐暴露将不再改变组织修复结果。贯穿整个材料的孔洞，加速周围组织细胞长入，利于组织液流动，故血清肿等局部并发症发生率低。
(3)良好的力学强度，厚度可变，适应不同创面修复的需要。中间层可为材料整体提升力学强度，且SIS的层数可以适当增减以适应不同力学需求的组织修复。医用粘合剂和真空层压的联合使用增强了材料的剥离强度，保证多层材料水化或剪裁后的完整性，不易松散。
(4)降低原料预处理难度，价格低廉：在兼顾UBM和SIS优势的同时，本发明可以大幅度降低产量和生产周期限制因素-UBM的用量。降低材料成本，缩短生产周期，减少人力投入。
附图说明
图1为本发明的结构示意图；其中，1为脱细胞小肠粘膜下层SIS，2为脱细胞膀胱粘膜层基底膜UBM。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实施例1
以Abraham法制备猪源性脱细胞膀胱粘膜层基底膜(UBM)和脱细胞小肠粘膜下层(SIS)。一层UBM铺平(光滑面向下)，将单片膜状SIS按照层间错位50％的方式拼接为独立片层，平铺于UBM表面，层间错位90°铺设4层。其上再铺设一层UBM(光滑面向上)。排除气泡，各层间使用医用壳聚糖粘合剂粘合，再以-250mm Hg的压力经过24h将其压为一体。材料全层打孔，孔间距为5mm，直径为1mm。
实施例2
以Abraham法制备猪源性脱细胞膀胱粘膜层基底膜(UBM)和脱细胞小肠粘膜下层(SIS)。两层UBM铺平(光滑面向下)，将单片膜状SIS按照层间错位50％的方式拼接为独立片层，平铺于UBM表面，层间错位90°铺设6层。其上再铺设两层UBM(光滑面向上)。排除气泡，各层间使用医用胶原蛋白粘合剂粘合，再以-300mm Hg的压力经过36h将其压为一体。材料全层打孔，孔间距为8mm，直径为2mm。
实施例3
根据GB/T528-2009，取3个样品制作成4cm×1cm的哑铃状，水化后用材料力学试验机固定样品两端，以10mm/min速度拉伸，测算材料抗张强度为34±3N/cm。
取3个样品制作成2cm×5cm，两端固定在拉力机上下夹持器上，以10mm/min稳定的速度连续剥离，直到样品重叠部分分层。测量分层时负载力。SIS-SIS间、UBM-SIS间的剥离强度为6±2N/cm，剥离维持力为1.5±0.5N/cm。
根据GB/T 16886.5规定的方法评价材料细胞毒性。以NIH3T3和L929为模式细胞。以细胞培养基为浸提介质，以梯度浓度的浸提液替代培养基培养细胞，MTT法测定细胞存活率。材料的细胞毒性为0～Ⅰ级。
细胞迁移实验：将材料低温粉碎，蛋白酶酶解，酶解产物浓度为50μg/mL。细胞经饥饿培养24h后，Boyden小室法测定6h细胞迁移量，以含10％胎牛血清培养基和不含血清培养基分别为阳性和阴性对照。修复材料细胞迁移量为2056±72，阳性对照为2105±35，阴性对照为1328±65。材料组与阳性对照组无显著性差异(P>0.05)。
根据GB/T14233.2规定的方法检测材料内毒素含量。以内毒素检测用水为浸提介质，37℃ 浸提24h。动态浊度法测定材料内毒素含量，复测稀释浸提液内毒素含量以排除干扰。材料内毒素含量≤5EU/装置。
根据GB/T14233.2规定的方法测定材料血液相容性。接触组：大鼠背部脱毛，涂抹浓度为50μg/mL的酶解产物，每天涂抹1次，连续涂抹20d；经口摄入组：7d内隔天口服1mL浸提液，共摄入4次；肌肉注射组和静脉注射组：7d内隔天注射浓度为0.15mL浸提液，各注射4次。分别于染毒第30天和90天两批处死动物，取静脉血用于测试。溶血率按下列公式计算：溶血率(％)＝(样品吸光度－阴性吸光度)/(阳性吸光度－阴性吸光度)×100％。材料溶血率≤5％。
根据GB/T 16886.10规定的方法评价材料皮内刺激性。于白兔皮内注射浸提液及对照液(PBS)各0.2mL，于注射后15min，1h，2d，3d观察实验区皮肤反应，根据红斑和水肿情况打分。材料无刺激性。
根据GB/T 16886.10规定的最大剂量法评价材料致敏性。纯淀粉溶液为阴性对照组，经口灌胃，持续1周，停药后继续观察1周。每天记录大鼠体重，观察动物临床毒性体征，记录毒性级别。实验结束后处死大鼠常规组织病理切片。材料无迟发型超敏反应。
构建犬腹直肌前鞘和腹直肌缺损动物模型，缺损面积为10×5cm2，将复合软组织修复材料裁剪至一定大小进行修复，以单纯SIS、单纯UBM为对照。单纯SIS对照组术后修复区血清肿发生率为33％，单纯UBM和复合软组织修复材料组未发生血清肿。术后2周、1个月、2个月、4个月取出修复区，组织切片经CD68、CCR7、CD163染色观察浸润细胞类型和密度，M1/M2巨噬细胞比例，证实材料不改变UBM的宿主-材料炎症反应基本类型，且组织修复效果与纯UBM相近。

Claims (10)

  1. 一种复合细胞外基质成分生物材料，其特征在于：所述生物材料以脱细胞小肠粘膜下层SIS为中间层，脱细胞膀胱粘膜层基底膜UBM为上下表层；所述上下表层完全包覆中间层形成三明治结构。
  2. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述SIS由哺乳动物小肠经机械方法除去浆膜层和肌层后脱细胞处理制得。
  3. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述UBM由哺乳动物膀胱经机械方法除去浆膜、肌层、粘膜下层、粘膜肌层后脱细胞处理制得。
  4. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述中间层的层数为1～20层。
  5. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述上下表层的层数各为1～10层。
  6. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述中间层与上下表层间、中间层与上下表层内以医用粘合剂、缝合捆扎、真空层压中的一种或几种方式固定。
  7. 根据权利要求6所述的一种复合细胞外基质成分生物材料，其特征在于：所述医用粘合剂为壳聚糖、胶原蛋白、纤维蛋白胶、透明质酸、硫酸软骨素、水凝胶、骨胶、明胶或果胶等成分中一种或几种。
  8. 根据权利要求6所述的一种复合细胞外基质成分生物材料，其特征在于：所述真空层压的工艺参数为：真空压力为-50～-760mmHg，作用时间为0.5～72h。
  9. 根据权利要求1所述的一种复合细胞外基质成分生物材料，其特征在于：所述生物材料还包括贯穿材料的孔洞。
  10. 根据权利要求9所述的一种复合细胞外基质成分生物材料，其特征在于：所述孔洞的直径为1～5mm，孔间距为0.5～5cm。
PCT/CN2017/073394 2016-04-25 2017-02-13 一种复合细胞外基质成分生物材料 Ceased WO2017185853A1 (zh)

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