WO2017101027A1 - Modified biomedical material product - Google Patents

Modified biomedical material product Download PDF

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Publication number
WO2017101027A1
WO2017101027A1 PCT/CN2015/097463 CN2015097463W WO2017101027A1 WO 2017101027 A1 WO2017101027 A1 WO 2017101027A1 CN 2015097463 W CN2015097463 W CN 2015097463W WO 2017101027 A1 WO2017101027 A1 WO 2017101027A1
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growth factor
mussel mucin
materials
biomedical material
bone
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PCT/CN2015/097463
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French (fr)
Chinese (zh)
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高敏
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江阴市本特塞缪森生命科学研究院有限公司
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Priority to PCT/CN2015/097463 priority Critical patent/WO2017101027A1/en
Publication of WO2017101027A1 publication Critical patent/WO2017101027A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances

Definitions

  • the present invention relates generally to the field of medical product technology, and more particularly to a biomedical material improved product cured with mussel mucin and growth factors.
  • Mussel adhesive protein also known as Mytilus edulis foot protein (Mefp)
  • Mefp Mytilus edulis foot protein
  • Mytilus coruscus A special protein secreted by Perna viridis. Mussels are usually attached in groups to the reefs on the coast or to the bottom of the ship, and have the ability to withstand wave impacts in the offshore. In fact, mussels can be attached extremely strongly to the substrate of any material, such as metal, wood, glass, and the like. The main reason for the above characteristics of mussels is that they can form and store this special mucin in the girth of the foot. The mussels release the mucin through the foot silk to a solid surface such as rock to form a water-resistant combination. Fix yourself.
  • Mussel mucin has two structural features: (1) containing lysine, which has a high loading of positive charge; (2) containing 3,4 dihydroxyphenylalanine (DOPA, dopa). The cells and tissues of the human body are negatively charged.
  • Mussel mucin plays a protective and therapeutic role by tightly binding cells and tissues through the electrostatic interaction between its own positive charge and the negative charge of cells and tissues.
  • dopa oxidation produces ortho-dioxins, which can be cross-linked with unoxidized dopa to form a membrane or a network scaffold, which promotes the protein to adhere more closely and firmly to the surface of the human body, thereby protecting.
  • Mussel mucin is a macromolecular protein that is completely degraded in the human body for about 3-10 days. Its ability to attach to cell tissues is excellent, so that mussel mucin can be stabilized locally and continue to function.
  • mussel mucin has the above characteristics, its current application field is very limited.
  • Commercial mussel mucin products are Cell-Tak from BD Biosciences, MAP Trix from Kollodis, Korea, and Hydrogel from Biopolymer, Sweden. These products are either used directly in the mussel mucin solution state, or are stored as lyophilized powder preparations and dissolved before use. Their main application is limited to microscopic Cell adhesion and tissue adhesives. Mussel mucin has also been reported for use in membrane repair, as a coating against seawater corrosion.
  • Biomedical materials are materials used to diagnose, treat, repair or replace diseased tissues, organs or enhance their functions. They are the basis for the study of artificial organs and medical devices and have become an important branch of contemporary materials science.
  • biomedical materials have developed rapidly, the main components that make up biomedical materials are still inorganic, organic or polymeric materials, which have poor compatibility with organisms, and often occur in tissues or cells that are difficult to use in living organisms. Growth on medical materials, etc.
  • the unique water-adhesive function of mussel mucin can effectively improve the biocompatibility of biomedical materials and the body, and at the same time, it can be combined with substances that promote the treatment such as growth factors, and is more conducive to damaged human tissues or organs. Reconstruction or rehabilitation.
  • the combination of mussel mucin and growth factors and biomedical materials effectively immobilizes growth factors and mussel mucins on biomedical materials, providing better physical and chemical stability of proteins and growth factors, and prolonging The action time of growth factors and proteins in the human body is beneficial to the action of growth factors and proteins.
  • Mussel mucin used herein refers to Mytilus edulis Linnaeus, Mytilus coruscus or Pema viridis from the Mytilidae bivalve mollusc. 11 subclasses of mussel mucin, currently known as purified from marine mussels: mefp1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, collagen pre-COL- A mixture of one or more of P, pre-COL-D, pre-COL-NG, foot silk matrix proteins PTMP and DTMP.
  • the mussel mucin used herein may have a pH of from 1.0 to 7.0 in an aqueous solution, and particularly may be in the range of from pH 3.0 to 6.5 to make the therapeutic effect better.
  • the mussel mucin used herein may also be obtained by a method of biosynthesis, comprising a mixture of one or more of the known 11 mussel mucin subclasses.
  • the artificial biosynthesized mussel mucin used herein may have a pH of 1.0 to 7.0 in aqueous solution, and particularly may be in the range of pH 3.0 to 6.5 to make the therapeutic effect better.
  • the mussel mucin used herein may also be a hydrolyzed peptide obtained by hydrolysis of mussel mucin from natural or artificial biosynthesis, or a synthetic peptide containing a functional group obtained by artificial synthesis.
  • the mussel mucin hydrolyzed peptide or synthetic peptide used herein may have a pH of 1.0 to 7.0 in an aqueous solution, and particularly may be in the range of pH 3.0 to 6.5 to make the therapeutic effect better.
  • the mussel mucin used herein can be obtained by the following preparation method, for example, a method for separating and purifying mussel mucin using mixed adsorption chromatography in Chinese Patent No. ZL200710179491.0, a kind of carboxy using Chinese Patent No. ZL200710179492.5 A method for purifying mussel mucin by methyl ion exchange chromatography, a method for separating and purifying mussel mucin using salting out and dialysis, Chinese Patent No. ZL200910087567.6.
  • the mussel mucin used herein may be in the form of a solution or a lyophilized powder, in particular, the concentration of mussel mucin in the product may be 0.1-15.0 mg/ml, and when the concentration is too low, the effect of mussel mucin Not large, when the concentration is too high, it can cause cytotoxicity, skin irritation, etc., which is not conducive to the safety of biomedical materials.
  • biomedical materials used herein include not only bone repair materials, medical scaffold materials, medical suture materials, dressings, but also artificial joints and joint prostheses, patches, waterproof tapes, artificial blood vessels and endovascular prostheses, artificial heart valves, Artificial ear implants, artificial organ prostheses, artificial soft tissue fillers, artificial amniotic membranes, intervertebral disc prostheses, spinous process implants, artificial heart valves, blood vessels, cardiovascular intubation and other cardiovascular system materials, blood purification membranes and separation Membrane, gas selective permeable membrane, contact lens and other medical membrane materials, drug release carrier materials, clinical diagnostics and biosensor materials.
  • the biomedical materials used herein can be classified into metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys according to the composition and properties of the materials, inorganic materials such as ceramics, glass, carbon, etc., polyethylene, Polypropylene, polyacrylate, aromatic polyester, polysiloxane, polyoxymethylene, collagen, linear aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polyhexyl ester, etc.
  • metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys according to the composition and properties of the materials, inorganic materials such as ceramics, glass, carbon, etc., polyethylene, Polypropylene, polyacrylate, aromatic polyester, polysiloxane, polyoxymethylene, collagen, linear aliphatic polyester, chitin, cellulose, polyamino
  • Biomedical composite materials are a composite material of two or more of the above materials, and the biological derivative material is formed by a specially treated natural biological tissue. Biomedical materials.
  • Growth factors used herein include platelet growth factor (platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor ODGF), epidermal growth factor (EGF, EGF, transforming growth factor, TGF ⁇ , and TGF ⁇ ), fibrillar Cell growth factor ( ⁇ FGF, ⁇ FGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin growth factor Sub-(IL-1, IL-1, IL-3, etc.), erythrocyte growth factor (EPO), colony stimulating factor (CSF), and the like.
  • platelet growth factor platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor ODGF
  • EGF epidermal growth factor
  • ⁇ FGF fibrillar Cell growth factor
  • IGF-I insulin-like growth factor
  • IGF-II insulin-like growth factor
  • NGF nerve growth factor
  • EPO erythrocyte growth factor
  • CSF colony stimulating factor
  • the present invention relates to a method of preparing a biomedical material cured with mussel mucin and a growth factor, comprising first combining mussel mucin with a growth factor and then curing it onto a biomedical material. .
  • the present invention also relates to a method of preparing a biomedical material cured with mussel mucin and a growth factor, comprising first curing a mussel mucin to a biomedical material, and then growing the growth factor Binds to biomedical materials that cure mussel mucin.
  • the present invention also relates to a method of preparing a product in which mussel mucin is combined with a growth factor and a biomedical material, comprising first curing a growth factor onto a biomedical material, and then placing the mussel Mucin binds to biomedical materials that cure growth factors.
  • curing refers to the physical adsorption of mussel mucin and/or growth factors onto biomedical materials. “Bonding” means that mussel mucin and growth factors are cross-linked by covalent bond or by intermolecular physic interaction.
  • the binding of mussel mucin to growth factors may be cross-linked in the form of covalent bonds or by intermolecular physical interaction.
  • the combination may be a mixture (lyophilized powder or solution). ), soaked or combined by the addition of a crosslinking agent or a modifier.
  • the crosslinking agent or modifier may be oxygen, ozone, hydrogen peroxide, iodine and its preparation, fluorine, chlorine, periodate, permanganate, nitrate, acetate, persulfuric acid. Salt, fluoride, glutaraldehyde, epichlorohydrin, 1,4-dihydroxy glycidyl ether, N-succinimidyl 3-(2-pyridyldithio)propionate or succinimide- One of or a combination of 4-(N-methylmaleimide)cyclohexane-1-carbonate.
  • mussel mucin and/or growth factors are cured to biomedical materials herein may be by spraying, soaking, and the like.
  • mussel mucin and growth factors are used in the biomedical material to uniformly cover or infiltrate the biomedical material.
  • the temperature for curing may be any, preferably 10-40 °C.
  • the period of time for curing may be any, preferably from 10 to 60 minutes.
  • the biomedical material product curable with mussel mucin and growth factor of the invention can be used for bone-to-muscle system repair materials such as bones, teeth, joints and tendons, skin, breast, esophagus, respiratory tract, bladder and the like.
  • bone-to-muscle system repair materials such as bones, teeth, joints and tendons, skin, breast, esophagus, respiratory tract, bladder and the like.
  • the biomedical material product of the present invention having mussel mucin and growth factor cured has the following advantages: the water-adhesive function unique to mussel mucin can effectively improve the biocompatibility of biomedical materials and the body, and at the same time, Combined with substances that promote treatment, such as growth factors, it is more conducive to the reconstruction or rehabilitation of damaged human tissues or organs.
  • the combination of mussel mucin and growth factors and biomedical materials effectively immobilizes growth factors and mussel mucins on biomedical materials, making proteins and growth factors better physical and It has chemical stability and prolongs the action time of growth factors and proteins in the human body, which is beneficial to the action of growth factors and proteins.
  • An improved biomedical material product comprising mussel mucin and growth factor solidified onto a biomedical material in a uniform coverage, wherein the mass ratio of mussel mucin to growth factor is from 100:1 to 1:100. .
  • biomedical material product comprising metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys, and inorganic materials such as ceramics, glass, and carbon.
  • metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys, and inorganic materials such as ceramics, glass, and carbon.
  • metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys
  • inorganic materials such as ceramics, glass, and carbon.
  • the growth factor may comprise platelet growth factor (platelet derived growth factor, PDGF; osteosarcoma derived growth factor ODGF), epidermal growth factor (epidermal growth factor, EGF, Transforming growth factor, TGF ⁇ and TGF ⁇ ), fibroblast growth factor ( ⁇ FGF, ⁇ FGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin growth factor (IL-) 1, IL-1, IL-3, etc., red fine Cytokinin (EPO), colony stimulating factor (CSF), and the like.
  • platelet growth factor platelet derived growth factor, PDGF; osteosarcoma derived growth factor ODGF
  • epidermal growth factor epidermal growth factor
  • EGF epidermal growth factor
  • TGF ⁇ and TGF ⁇ fibroblast growth factor
  • ⁇ FGF fibroblast growth factor
  • IGF-I insulin-like growth factor
  • IGF-II insulin-like growth factor
  • NGF nerve growth factor
  • the improved biomedical material product according to embodiment 1, wherein the pre-admixed mussel mucin may be in the range of pH 1.0-7.0, in particular in the range of pH 3.0-6.5.
  • the improved biomedical material product according to Embodiment 1 can be used for bone-to-muscle system repair materials such as bones, teeth, joints, tendons, soft tissue materials such as skin, breast, esophagus, respiratory tract, bladder, artificial heart valves, blood vessels, Cardiovascular system materials such as cardiovascular intubation, blood purification membrane and separation membrane, gas selective permeable membrane, contact lens and other medical membrane materials, tissue adhesive and suture material, dressing material, drug release carrier material, clinical Diagnostics and biosensor materials.
  • bone-to-muscle system repair materials such as bones, teeth, joints, tendons, soft tissue materials such as skin, breast, esophagus, respiratory tract, bladder, artificial heart valves, blood vessels, Cardiovascular system materials such as cardiovascular intubation, blood purification membrane and separation membrane, gas selective permeable membrane, contact lens and other medical membrane materials, tissue adhesive and suture material, dressing material, drug release carrier material, clinical Diagnostics and biosensor materials.
  • a method of making an improved biomedical material product comprising first combining mussel mucin with a growth factor and then curing onto a biomedical material.
  • a method of making an improved biomedical material product comprising curing a mussel mucin onto a biomedical material and then incorporating the growth factor onto the biomedical material that cures the mussel mucin.
  • a method of making an improved biomedical material product comprising: after curing a growth factor onto a biomedical material, binding the mussel mucin to the biomedical material that cures the growth factor.
  • the method according to any one of embodiments 12-14, wherein the mussel mucin and/or growth factor is spraying, soaking, and the like.
  • the method of any of embodiments 12-17, wherein the period of time for curing can be any, preferably from 10 to 60 minutes.
  • Example 1 Mussel mucin and bone morphogenetic protein were solidified into artificial bone powder for the treatment of femoral defects.
  • Preparation of mussel mucin and bone morphogenetic protein-cured bone powder Take 5ml mussel mucin solution with a concentration of 1.0mg/ml and recombinant human bone morphogenetic protein (excellent pure, Shanghai Yaochao Biological Products Co., Ltd.) 1ml, the concentration is 1.0 mg/ml, uniformly mixed to form a gel-like biomaterial. Take artificial bone powder (Shanghai Harbin Biotechnology Co., Ltd., molecular weight 4000) 10g, add the mixed gel solution of mussel mucin and bone morphogenetic protein to bone powder, mix well and cure at 40 °C for 12h, until the bone powder is completely dried. A bone powder that forms mussel mucin and bone morphogenetic protein solidification.
  • Mussel mucin blending bone powder preparation Take mussel mucin solution 5ml with concentration of 1.0mg/ml and artificial bone powder 10g, and mix it to form mussel mucin to mix bone powder.
  • Preparation of bone morphogenetic protein-adapted bone powder Take 5 ml of bone morphogenetic protein and 10 g of artificial bone powder at a concentration of 1.0 mg/ml, and mix and form bone morphogenetic protein to mix bone powder.
  • Test method 30 male New Zealand white rabbits weighing 4.0-4.5 kg were taken. After anesthesia, the hind legs were shaved. After disinfection, the skin and muscles of the legs were cut with a scalpel to expose the femur. The drill was used to cut the diameter of 1 cm in the middle of the femur. Bone, forming a bone defect. Fifteen white rabbits were taken. Each rabbit was implanted with artificial bone powder cured by mussel mucin and bone morphogenetic protein in the left hind leg, and bone powder material mixed with mussel mucin and artificial bone powder was implanted in the right hind leg.
  • Another 15 rabbits were implanted with bone powder material of bone morphogenetic protein and artificial bone powder in the left leg, and untreated bone powder was implanted into the right leg as a blank control group. After the implantation is completed, the wound is sutured and kept in a normal manner, and the healing of the bone defect is observed by X-ray every week.
  • the bone defect repaired by mussel mucin and bone morphogenetic protein-cured bone powder has been combined with the surrounding bone tissue and has angiogenesis; the bone defect repaired with mussel mucin and bone powder is combined with the surrounding bone tissue, but The degree of angiogenesis and bone formation is lower than that of mussel mucin-cured bone powder; the bone defect is repaired by bone morphogenetic protein, and the artificial bone material is not completely filled with the bone defect, and there is still a gap between the bone tissue and the normal bone tissue. Only the bone defect repaired by the artificial bone, the artificial bone material is not completely filled with the bone defect, and there is still a clear gap between the normal bone tissue and the normal bone tissue.
  • mussel mucin can promote bone regeneration and healing, especially when mussel mucin and bone morphogenetic protein are solidified onto artificial bone material, due to increased stability in body fluids and prolonged metabolic cycle. It is more conducive to the healing of bone defects.
  • Example 2 Mussel mucin and bone morphogenetic protein are solidified onto the surface of dental implant material to promote the union of implant and alveolar bone
  • Preparation of mussel mucin and bone morphogenetic protein-cured dental implants 5 ml mussel mucin solution at a concentration of 2.0 mg/ml and recombinant human bone morphogenetic protein (superior pure, Shanghai Yaochao Biological Products Co., Ltd.) 1 ml The concentration was 1.0 mg/ml, and the mixture was uniformly mixed to form a gel-like biological material.
  • the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) was used to uniformly spray the mixed gel solution of mussel mucin and bone morphogenetic protein onto the surface of the implant and solidified at 37 ° C for 4 h to form mussel mucin and bone morphology.
  • a protein-curing implant occurs.
  • Preparation of mussel mucin solidified dental implant Take 1ml of mussel mucin solution of 2.0mg/ml, soak the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) into mussel mucin solution for 5h, and remove the room temperature. Under drying, the mussel mucin-cured dental implant is obtained.
  • Preparation of bone morphogenetic protein-derived dental implant Take 1ml of 1.0mg/ml bone morphogenetic protein solution, soak the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) into bone morphogenetic protein solution for 5h, after removal
  • the bone morphogenetic protein-cured dental implant is obtained by drying at room temperature.
  • a large mongrel dog was used as a test animal. After general anesthesia, the solid teeth were removed from the left and right sides, and the upper and lower symmetrical positions were respectively removed.
  • the implants were implanted into the upper and lower alveolar bones respectively.
  • the left upper side was the untreated implant
  • the lower left side was the mussel mucin solidified implant
  • the upper right side was the bone morphogenetic protein solidified implant, bottom right.
  • the side is the implant of mussel mucin binding to bone morphogenetic protein. After implanting the implants, they were kept in the normal way. After 40 days, the implants and alveolar bone were observed.
  • Muscle mucin and bone morphogenetic protein-solidified implants are well integrated with alveolar bone, and a large number of bone cells have been grown on the implant; the number of bone cells on mussel mucin cured implants is significantly lower than that of mussel mucin and Bone morphogenetic protein-solidified implants; bone morphogenetic protein-solidified implants and blank implants have similar numbers of bone cells, which are lower than mussel mucin-cured implants. It is proved that the dental implants in which mussel mucin binding to bone morphogenetic protein is solidified are more conducive to the crawling and growth of bone cells on the implant material, and the time of binding of the implant to the bone can be significantly reduced.
  • Example 3 Effect of mussel mucin (MAP) combined with nerve growth factor (NGF) on nerve repair and regeneration after decellularized nerve scaffold transplantation.
  • MAP mussel mucin
  • NGF nerve growth factor
  • Rats 36 healthy adult Sprague-Dawley rats, male or female, weighing 180-200 g. Rats were randomly divided into 3 groups: 12 in the NS control group; 12 in the NGF treatment group; and 12 in the MAP+NGF combination treatment group. After decellularized xenografts, the animals survived for 4 weeks and 8 weeks, respectively, and 6 animals in each group at different time points.
  • the cyanotic nerve of the cyanotic blue rabbit was cut out, and the connective tissue was removed under the operating microscope.
  • the Tern X-100 solution and sodium deoxycholate solution were chemically extracted according to a certain concentration and procedure to prepare a heterogeneous nerve scaffold derived from the rabbit sacral nerve.
  • the nerve scaffolds of the NS control group and NGF (Qiyi Biotechnology Co., Ltd.) treatment group were immersed in 0.9% NS solution and NGF solution for 10 min respectively, and the nerve scaffold of the MAP+NGF combination treatment group was first immersed in 1.5 mg/ml. 10 min in MAP solution, taken out and immersed in NGF solution for 10 min.
  • the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg), the skin was cut at the lower edge of the right gluteal groove according to the principle of sterility, the muscles were separated, and the sciatic nerve was fully exposed under the operating microscope.
  • the nerve injury was caused by cutting the sciatic nerve from the lower edge of the piriformis muscle to 10 mm.
  • 10 mm long decellularized heterogeneous nerve scaffolds were sutured with a 10 mm long decellularized heterogeneous nerve scaffold and the proximal and distal ends of the rat sciatic nerve for 4-6 needles under tension without tension. Close the incision layer by layer.
  • NS control group intramuscular injection of 0.9% NS 0.1ml in the injured lateral calf of the injured rats;
  • NGF treatment group intramuscular injection of NGF (180ng/100g/d) in the injured lateral calf of the rats;
  • MAP+NGF combined treatment group NGF (180 ng/100 g/d) was injected intramuscularly into the injured leg of the injured rat. The above injections lasted for 14 days.
  • the lower limbs of the rats in each group were paralyzed, and there were paws on the ground and crawling.
  • the rats in each group had different degrees of redness and swelling, no necrosis and ulceration.
  • the swelling of the ankle and foot of the rats in each group subsided, and the affected limbs could support the ground.
  • the rats in the combined treatment group improved significantly.
  • the toes of each group were able to actively deploy, balanced and coordinated. Physical activity.
  • the nerve grafts of the NS control group had mild adhesion to the surrounding tissues, and the color and outer diameter were similar to those of the nerve trunk.
  • the proximal and distal nerve anastomosis was a small amount of connective tissue, and the anastomosis The mouth was slightly thicker; the nerve graft of the NGF treatment group had slight adhesion to the surrounding tissue, the color and outer diameter were similar to the nerve trunk, and the proximal and distal nerve anastomosis was almost flat; the MAP+NGF combined administration group was used for nerve transplantation.
  • the body has a neuron-like appearance, almost no adhesion to the surrounding tissue, and the proximal and distal nerve anastomosis is basically smooth and complete. There were no neuroma formation in the proximal and distal nerve grafts of each group.
  • Example 4 Curing of mussel mucin to a tricalcium phosphate artificial bone scaffold for the repair of knee osteochondral defects
  • Preparation of mussel mucin-cured tricalcium phosphate artificial bone scaffold Take 20ml of 15mg/ml mussel mucin solution and soak the tricalcium phosphate artificial bone scaffold (En ⁇ ) into mussel mucin solution 1h, after drying at 30 ° C, the mussel mucin solidified tricalcium phosphate artificial bone scaffold was obtained.
  • Bone marrow mesenchymal stem cells were isolated from the sputum and extracted from the sputum under aseptic conditions. The cells were transferred to the third passage for digestion and collection. The cell concentration was adjusted to 2 ⁇ 10 9 cells/L, and the mussel mucin was solidified.
  • the calcium artificial bone scaffold and the tricalcium phosphate artificial bone scaffold which was cured with mussel mucin were co-cultured for 24 hours, which was made into mussel mucin solidified tricalcium phosphate artificial bone scaffold-bone marrow mesenchymal stem cell complex and unused sputum.
  • the mucin-cured tricalcium phosphate artificial bone scaffold-bone marrow mesenchymal stem cell complex The osteoarticular defects of the right knee joint were prepared in both groups.
  • the experimental group was implanted with a solidified scaffold-cell complex in the defect, and the defect was implanted with the uncured scaffold-cell complex.
  • the defect of the experimental group was filled with white translucent tissue, the surface was smooth, the touch was soft, slightly higher than the surrounding cartilage surface, the distribution of chondrocytes was more uniform, and the arrangement was non-directional; at the 24th week, the filling of the experimental group was filled.
  • White translucent neonatal cartilage tissue similar in color to normal cartilage, with a tough texture and smooth surface. The boundary with normal cartilage disappears.
  • the surface cells are parallel to the articular surface.
  • the deep cells are arranged in disorder, the cells are clustered, the matrix is widely stained, and the surrounding is normal.
  • the cartilage is well connected. There was no obvious reaction in the 12th week of the control group.
  • the control group began to fill with white translucent tissue.
  • Prompt bone marrow Mesenchymal stem cells are ideal seed cells for repairing articular cartilage defects.
  • the tricalcium phosphate artificial bone scaffold is gradually degraded and absorbed. It is a suitable scaffold for tissue engineering to repair articular cartilage defects, using mussel mucin. Curing can significantly speed up the repair process.

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Abstract

A modified biomedical material product, having a mussel adhesive protein and a growth factor solidified on a biomedical material. The product not only has the functions of the original biomedical material, but also contributes to the restoration or recovery of an impaired human tissue or organ, such that the protein and the growth factor have better physical and chemical stabilities, and the action time of the growth factor and the protein in human is extended, favoring the effects of the growth factor and the protein.

Description

改良的生物医用材料产品Improved biomedical materials 技术领域Technical field
本发明大体涉及医疗产品技术领域，更具体地，涉及一种固化有贻贝粘蛋白与生长因子的生物医用材料改良产品。The present invention relates generally to the field of medical product technology, and more particularly to a biomedical material improved product cured with mussel mucin and growth factors.
背景技术Background technique
贻贝粘蛋白(Mussel adhesive protein，MAP)，也称作贻贝足丝蛋白(Mytilus edulis foot protein，Mefp)，是海洋贝类紫贻贝(Mytilus edulis Linnaeus)、厚壳贻贝(Mytilus coruscus)、翡翠贻贝(Perna viridis)等分泌的一种特殊的蛋白质。贻贝通常成群地附着在海岸边的礁石上或者轮船的底部，有在近海耐受波浪冲击的能力。实际上贻贝几乎可以极其牢固地附着在任何材料的基底上，如金属、木材、玻璃等。贻贝具有上述特性的主要原因是其足丝腺内可生成并储存这种特殊的粘蛋白，贻贝通过足丝释放粘蛋白到岩石一类的固体表面上，形成抗水的结合，从而将自己固定。Mussel adhesive protein (MAP), also known as Mytilus edulis foot protein (Mefp), is a marine shellfish, Mytilus edulis Linnaeus, and a small shell mussel (Mytilus coruscus). A special protein secreted by Perna viridis. Mussels are usually attached in groups to the reefs on the coast or to the bottom of the ship, and have the ability to withstand wave impacts in the offshore. In fact, mussels can be attached extremely strongly to the substrate of any material, such as metal, wood, glass, and the like. The main reason for the above characteristics of mussels is that they can form and store this special mucin in the girth of the foot. The mussels release the mucin through the foot silk to a solid surface such as rock to form a water-resistant combination. Fix yourself.
目前从贻贝中鉴定得到11种粘蛋白亚类，包括mefp1、mefp-2、mefp-3、mefp-4、mefp-5、mefp-6、胶原蛋白pre-COL-P、pre-COL-D、pre-COL-NG、足丝基质蛋白PTMP和DTMP(朱曜曜等，海洋科学进展，2014，32(4)：560-568)。贻贝粘蛋白具有2个结构特点：(1)含有赖氨酸，使蛋白带有高载量正电荷；(2)含3，4二羟基苯丙氨酸(DOPA，多巴)。人体的细胞和组织带有负电荷。贻贝粘蛋白通过自身正电荷与人体的细胞和组织负电荷之间的静电相互作用与细胞和组织紧密结合，发挥防护和治疗的作用。此外，多巴氧化生成邻位二醌，可以和未被氧化的多巴相互交联形成膜或是网状支架，促使蛋白质更加紧密、稳固地附着在人体表面，起到保护作用。贻贝粘蛋白是大分子蛋白质，在人体内完全降解的时间约为3-10天，其附着于细胞组织的能力优异，使贻贝粘蛋白可以稳固于局部，持续发挥作用。Currently, 11 mucin subclasses have been identified from mussels, including mefp1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, collagen pre-COL-P, pre-COL-D. , pre-COL-NG, foot silk matrix proteins PTMP and DTMP (Zhu Xi et al, Advances in Marine Science, 2014, 32(4): 560-568). Mussel mucin has two structural features: (1) containing lysine, which has a high loading of positive charge; (2) containing 3,4 dihydroxyphenylalanine (DOPA, dopa). The cells and tissues of the human body are negatively charged. Mussel mucin plays a protective and therapeutic role by tightly binding cells and tissues through the electrostatic interaction between its own positive charge and the negative charge of cells and tissues. In addition, dopa oxidation produces ortho-dioxins, which can be cross-linked with unoxidized dopa to form a membrane or a network scaffold, which promotes the protein to adhere more closely and firmly to the surface of the human body, thereby protecting. Mussel mucin is a macromolecular protein that is completely degraded in the human body for about 3-10 days. Its ability to attach to cell tissues is excellent, so that mussel mucin can be stabilized locally and continue to function.
虽然贻贝粘蛋白具有以上特点，但目前其产品应用领域非常有限。商品化的贻贝粘蛋白产品有美国BD Biosciences公司的Cell-Tak，韩国Kollodis的MAP Trix和瑞典Biopolymer的Hydrogel。这些产品或者是以贻贝粘蛋白溶液状态直接使用，或者是以冻干粉制剂保存而在使用前溶解，它们的主要应用限于微观 的细胞粘附和组织粘合剂。也有报道贻贝粘蛋白用于胎膜修复、作为抗海水腐蚀涂层等应用。Although mussel mucin has the above characteristics, its current application field is very limited. Commercial mussel mucin products are Cell-Tak from BD Biosciences, MAP Trix from Kollodis, Korea, and Hydrogel from Biopolymer, Sweden. These products are either used directly in the mussel mucin solution state, or are stored as lyophilized powder preparations and dissolved before use. Their main application is limited to microscopic Cell adhesion and tissue adhesives. Mussel mucin has also been reported for use in membrane repair, as a coating against seawater corrosion.
生物医用材料是用来对生物体进行诊断、治疗、修复或替换其病损组织、器官或增进其功能的材料，它是研究人工器官和医疗器械的基础，已成为当代材料学科的重要分支。Biomedical materials are materials used to diagnose, treat, repair or replace diseased tissues, organs or enhance their functions. They are the basis for the study of artificial organs and medical devices and have become an important branch of contemporary materials science.
近几年，虽然生物医学材料发展迅速，但由于构成生物医学材料的主体仍然是无机、有机或高分子材料，与生物体的相容性较差，在使用时经常出现组织或细胞难以在生物医学材料上生长等情况。贻贝粘蛋白特有的带水粘附功能可以有效改善生物医学材料与机体的生物相容性，同时还能与生长因子等对治疗有促进的物质相结合，更有利于受损人体组织或器官的重建或康复。贻贝粘蛋白与生长因子和生物医学材料结合形成的产品，有效地将生长因子和贻贝粘蛋白固定在生物医学材料上，使蛋白和生长因子具有更好的物理和化学稳定性，并且延长了生长因子和蛋白在人体内的作用时间，有利于生长因子和蛋白作用的发挥。In recent years, although biomedical materials have developed rapidly, the main components that make up biomedical materials are still inorganic, organic or polymeric materials, which have poor compatibility with organisms, and often occur in tissues or cells that are difficult to use in living organisms. Growth on medical materials, etc. The unique water-adhesive function of mussel mucin can effectively improve the biocompatibility of biomedical materials and the body, and at the same time, it can be combined with substances that promote the treatment such as growth factors, and is more conducive to damaged human tissues or organs. Reconstruction or rehabilitation. The combination of mussel mucin and growth factors and biomedical materials effectively immobilizes growth factors and mussel mucins on biomedical materials, providing better physical and chemical stability of proteins and growth factors, and prolonging The action time of growth factors and proteins in the human body is beneficial to the action of growth factors and proteins.
发明内容Summary of the invention
本发明的目的是提供一种改良的生物医用材料产品，包括以均匀覆盖方式固化到生物医学材料上的贻贝粘蛋白与生长因子，其中贻贝粘蛋白与生长因子的质量比为100∶1-1∶100。It is an object of the present invention to provide an improved biomedical material product comprising mussel mucin and growth factor which are cured onto a biomedical material in a uniform coverage, wherein the mass ratio of mussel mucin to growth factor is 100:1. -1:100.
在本文中使用的贻贝粘蛋白是指从贻贝科(Mytilidae)双壳类软体动物中的紫贻贝(Mytilus edulis Linnaeus)、厚壳贻贝(Mytilus coruscus)或翡翠贻贝(Pema viridis)等海洋贻贝中纯化获得的、目前已知的贻贝粘蛋白11个亚类：mefp1、mefp-2、mefp-3、mefp-4、mefp-5、mefp-6、胶原蛋白pre-COL-P、pre-COL-D、pre-COL-NG、足丝基质蛋白PTMP和DTMP中的一种或几种的混合物。在本文中使用的贻贝粘蛋白在水溶液中的酸碱度可以是pH 1.0-7.0，特别是可以在pH3.0-6.5的范围内以使其治疗效果更佳。Mussel mucin used herein refers to Mytilus edulis Linnaeus, Mytilus coruscus or Pema viridis from the Mytilidae bivalve mollusc. 11 subclasses of mussel mucin, currently known as purified from marine mussels: mefp1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, collagen pre-COL- A mixture of one or more of P, pre-COL-D, pre-COL-NG, foot silk matrix proteins PTMP and DTMP. The mussel mucin used herein may have a pH of from 1.0 to 7.0 in an aqueous solution, and particularly may be in the range of from pH 3.0 to 6.5 to make the therapeutic effect better.
在本文中使用的贻贝粘蛋白也可以是采用生物合成的方法获得的，包含已知的11个贻贝粘蛋白亚类中的一种或几种的混合物。在本文中使用的人工生物合成的贻贝粘蛋白在水溶液中的酸碱度可以是pH 1.0-7.0，特别是可以在pH 3.0-6.5的范围内以使其治疗效果更佳。 The mussel mucin used herein may also be obtained by a method of biosynthesis, comprising a mixture of one or more of the known 11 mussel mucin subclasses. The artificial biosynthesized mussel mucin used herein may have a pH of 1.0 to 7.0 in aqueous solution, and particularly may be in the range of pH 3.0 to 6.5 to make the therapeutic effect better.
在本文中使用的贻贝粘蛋白也可以是天然来源或人工生物合成的贻贝粘蛋白经水解后获得的水解肽，或通过人工合成方式获得的含有功能基团的合成肽。在本文中使用的贻贝粘蛋白水解肽或合成肽在水溶液中的酸碱度可以是pH 1.0-7.0，特别是可以在pH 3.0-6.5的范围内以使其治疗效果更佳。The mussel mucin used herein may also be a hydrolyzed peptide obtained by hydrolysis of mussel mucin from natural or artificial biosynthesis, or a synthetic peptide containing a functional group obtained by artificial synthesis. The mussel mucin hydrolyzed peptide or synthetic peptide used herein may have a pH of 1.0 to 7.0 in an aqueous solution, and particularly may be in the range of pH 3.0 to 6.5 to make the therapeutic effect better.
在本文中使用的贻贝粘蛋白可以采用以下制备方法获得，例如中国专利号ZL200710179491.0的一种使用混合吸附色谱分离纯化贻贝粘蛋白的方法，中国专利号ZL200710179492.5的一种使用羧甲基离子交换色谱纯化贻贝粘蛋白的方法，中国专利号ZL200910087567.6的一种使用盐析和透析分离纯化贻贝粘蛋白的方法等。The mussel mucin used herein can be obtained by the following preparation method, for example, a method for separating and purifying mussel mucin using mixed adsorption chromatography in Chinese Patent No. ZL200710179491.0, a kind of carboxy using Chinese Patent No. ZL200710179492.5 A method for purifying mussel mucin by methyl ion exchange chromatography, a method for separating and purifying mussel mucin using salting out and dialysis, Chinese Patent No. ZL200910087567.6.
在本文中使用的贻贝粘蛋白可以是溶液或冻干粉形式，特别是贻贝粘蛋白在产品中的浓度可以是0.1-15.0mg/ml，当浓度过低时，贻贝粘蛋白的功效不大，当浓度过高时，可引起细胞毒性、皮肤刺激等作用，从而不利于保证生物医用材料的安全性。The mussel mucin used herein may be in the form of a solution or a lyophilized powder, in particular, the concentration of mussel mucin in the product may be 0.1-15.0 mg/ml, and when the concentration is too low, the effect of mussel mucin Not large, when the concentration is too high, it can cause cytotoxicity, skin irritation, etc., which is not conducive to the safety of biomedical materials.
本文中使用的生物医用材料不仅包括骨修复材料、医用支架材料、医用缝合材料、敷料，还包括人工关节及关节假体，补片，防水胶布，人工血管及血管内假体，人工心脏瓣膜，人工耳植入物，人工器官假体，人工软组织填充物，人工羊膜，椎间盘假体，棘突植入物，人工心瓣膜、血管、心血管内插管等心血管***材料，血液净化膜和分离膜、气体选择性透过膜、角膜接触镜等医用膜材料，药物释放载体材料，临床诊断及生物传感器材料等。The biomedical materials used herein include not only bone repair materials, medical scaffold materials, medical suture materials, dressings, but also artificial joints and joint prostheses, patches, waterproof tapes, artificial blood vessels and endovascular prostheses, artificial heart valves, Artificial ear implants, artificial organ prostheses, artificial soft tissue fillers, artificial amniotic membranes, intervertebral disc prostheses, spinous process implants, artificial heart valves, blood vessels, cardiovascular intubation and other cardiovascular system materials, blood purification membranes and separation Membrane, gas selective permeable membrane, contact lens and other medical membrane materials, drug release carrier materials, clinical diagnostics and biosensor materials.
在本文中使用的生物医用材料按照材料组成及性质可分为钛、钽、铌、锆、不锈钢、钴基合金和钛基合金等金属材料，陶瓷、玻璃、碳素等无机材料，聚乙烯、聚丙烯、聚丙烯酸酯、芳香聚酯、聚硅氧烷、聚甲醛、胶原、线性脂肪族聚酯、甲壳素、纤维素、聚氨基酸、聚乙烯醇、聚己丙酯等可降解或不可降解高分子材料，生物医用复合材料及生物衍生材料等，其中生物医用复合材料是由两种或两种以上的上述材料复合而成的材料，生物衍生材料是由经过特殊处理的天然生物组织形成的生物医用材料。The biomedical materials used herein can be classified into metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys according to the composition and properties of the materials, inorganic materials such as ceramics, glass, carbon, etc., polyethylene, Polypropylene, polyacrylate, aromatic polyester, polysiloxane, polyoxymethylene, collagen, linear aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polyhexyl ester, etc. Degradable or non-degradable Polymer materials, biomedical composite materials and biological derivative materials, wherein the biomedical composite material is a composite material of two or more of the above materials, and the biological derivative material is formed by a specially treated natural biological tissue. Biomedical materials.
在本文中使用的生长因子包括血小板类生长因子(血小板来源生长因子，PDGF；骨肉瘤来源生长因子ODGF)、表皮生长因子类(表皮生长因子，EGF、转化生长因子，TGFα和TGFβ)、成纤维细胞生长因子(αFGF、βFGF)、类胰岛素生长因子(IGF-I、IGF-II)、神经生长因子(NGF)、白细胞介素类生长因 子(IL-1、IL-1、IL-3等)、红细胞生长素(EPO)、集落刺激因子(CSF)等。Growth factors used herein include platelet growth factor (platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor ODGF), epidermal growth factor (EGF, EGF, transforming growth factor, TGFα, and TGFβ), fibrillar Cell growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin growth factor Sub-(IL-1, IL-1, IL-3, etc.), erythrocyte growth factor (EPO), colony stimulating factor (CSF), and the like.
根据本发明的又一个目的，本发明涉及一种制备固化有贻贝粘蛋白与生长因子的生物医用材料的方法，包括首先结合贻贝粘蛋白与生长因子，然后将其固化到生物医用材料上。According to still another object of the present invention, the present invention relates to a method of preparing a biomedical material cured with mussel mucin and a growth factor, comprising first combining mussel mucin with a growth factor and then curing it onto a biomedical material. .
根据本发明的又一个目的，本发明还涉及一种制备固化有贻贝粘蛋白与生长因子的生物医用材料的方法，包括首先将贻贝粘蛋白固化到生物医用材料上，然后再将生长因子结合到固化贻贝粘蛋白的生物医用材料上。According to still another object of the present invention, the present invention also relates to a method of preparing a biomedical material cured with mussel mucin and a growth factor, comprising first curing a mussel mucin to a biomedical material, and then growing the growth factor Binds to biomedical materials that cure mussel mucin.
根据本发明的又一个目的，本发明还涉及一种制备贻贝粘蛋白与生长因子和生物医用材料结合的产品的方法，包括首先将生长因子固化到生物医用材料上之后，然后再将贻贝粘蛋白结合到固化生长因子的生物医用材料上。According to still another object of the present invention, the present invention also relates to a method of preparing a product in which mussel mucin is combined with a growth factor and a biomedical material, comprising first curing a growth factor onto a biomedical material, and then placing the mussel Mucin binds to biomedical materials that cure growth factors.
在本发明中使用的术语“固化”是指能通过物理方法将贻贝粘蛋白和/或生长因子吸附到生物医用材料上。“结合”是指贻贝粘蛋白和生长因子通过共价键形式交联或通过分子间物理作用组合。The term "curing" as used in the present invention refers to the physical adsorption of mussel mucin and/or growth factors onto biomedical materials. "Bonding" means that mussel mucin and growth factors are cross-linked by covalent bond or by intermolecular physic interaction.
在本文中贻贝粘蛋白与生长因子的结合方式可以是以共价键的形式交联或是通过分子间物理作用结合，在本发明的实施方式中，结合可以是混合(冻干粉或溶液)、浸泡或通过加入交联剂或修饰剂等结合。In this context, the binding of mussel mucin to growth factors may be cross-linked in the form of covalent bonds or by intermolecular physical interaction. In embodiments of the invention, the combination may be a mixture (lyophilized powder or solution). ), soaked or combined by the addition of a crosslinking agent or a modifier.
在本发明的实施方式中，交联剂或修饰剂可以是氧气、臭氧、双氧水、碘及其制剂、氟、氯、高碘酸盐、高锰酸盐、硝酸盐、乙酸盐、过硫酸盐、氟化物、戊二醛、表氯醇，1，4-二羟基缩水甘油醚，N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯或琥珀酰亚胺基-4-(N-甲基马来酰亚胺)环已烷-1-碳酸酯的其中之一或其组合等。In an embodiment of the present invention, the crosslinking agent or modifier may be oxygen, ozone, hydrogen peroxide, iodine and its preparation, fluorine, chlorine, periodate, permanganate, nitrate, acetate, persulfuric acid. Salt, fluoride, glutaraldehyde, epichlorohydrin, 1,4-dihydroxy glycidyl ether, N-succinimidyl 3-(2-pyridyldithio)propionate or succinimide- One of or a combination of 4-(N-methylmaleimide)cyclohexane-1-carbonate.
在本文中贻贝粘蛋白和/或生长因子固化到生物医学材料的方式可以是喷涂、浸泡等。The manner in which mussel mucin and/or growth factors are cured to biomedical materials herein may be by spraying, soaking, and the like.
在本发明的实施方式中，贻贝粘蛋白与生长因子在生物医用材料中的用量应能均匀覆盖或浸润该生物医用材料。In an embodiment of the invention, mussel mucin and growth factors are used in the biomedical material to uniformly cover or infiltrate the biomedical material.
在本发明的实施方式中，用于固化的温度可以是任意的，优选10-40℃。In an embodiment of the invention, the temperature for curing may be any, preferably 10-40 °C.
在本发明的实施方式中，用于固化的一段时间可以是任意的，优选10-60分钟。In an embodiment of the invention, the period of time for curing may be any, preferably from 10 to 60 minutes.
本发明的固化有贻贝粘蛋白与生长因子的生物医用材料产品可用于骨、牙、关节、肌腱等骨骼-肌肉***修复材料，皮肤、***、食道、呼吸道、膀胱等软 组织材料，人工心瓣膜、血管、心血管内插管等心血管***材料，血液净化膜和分离膜、气体选择性透过膜、角膜接触镜等医用膜材料，组织粘合剂和缝线材料，敷料材料，药物释放载体材料，临床诊断及生物传感器材料等。The biomedical material product curable with mussel mucin and growth factor of the invention can be used for bone-to-muscle system repair materials such as bones, teeth, joints and tendons, skin, breast, esophagus, respiratory tract, bladder and the like. Tissue materials, artificial heart valves, blood vessels, cardiovascular intubation and other cardiovascular system materials, blood purification membranes and separation membranes, gas selective permeable membranes, contact lens and other medical membrane materials, tissue adhesives and suture materials, Dressing materials, drug release carrier materials, clinical diagnostics and biosensor materials.
本发明的固化有贻贝粘蛋白与生长因子的生物医用材料产品具有以下优点：贻贝粘蛋白特有的带水粘附功能可以有效改善生物医学材料与机体的生物相容性，同时，还能与生长因子等对治疗有促进的物质相结合，更有利于受损人体组织或器官的重建或康复。此外，出人意料地发现，贻贝粘蛋白与生长因子和生物医学材料结合形成的产品，有效地将生长因子和贻贝粘蛋白固定在生物医学材料上，使蛋白和生长因子具有更好的物理和化学稳定性，并且延长了生长因子和蛋白在人体内的作用时间，有利于生长因子和蛋白作用的发挥。The biomedical material product of the present invention having mussel mucin and growth factor cured has the following advantages: the water-adhesive function unique to mussel mucin can effectively improve the biocompatibility of biomedical materials and the body, and at the same time, Combined with substances that promote treatment, such as growth factors, it is more conducive to the reconstruction or rehabilitation of damaged human tissues or organs. In addition, it has been surprisingly discovered that the combination of mussel mucin and growth factors and biomedical materials effectively immobilizes growth factors and mussel mucins on biomedical materials, making proteins and growth factors better physical and It has chemical stability and prolongs the action time of growth factors and proteins in the human body, which is beneficial to the action of growth factors and proteins.
具体实施方式detailed description
本发明的实施方式包括：Embodiments of the invention include:
1、一种改良的生物医学材料产品，包括以均匀覆盖方式固化到生物医学材料上的贻贝粘蛋白与生长因子，其中贻贝粘蛋白与生长因子的质量比为100∶1-1∶100。1. An improved biomedical material product comprising mussel mucin and growth factor solidified onto a biomedical material in a uniform coverage, wherein the mass ratio of mussel mucin to growth factor is from 100:1 to 1:100. .
2、根据实施方式1的改良的生物医学材料产品，其中所述贻贝粘蛋白是来自亚类：mefp1、mefp-2、mefp-3、mefp-4、mefp-5、mefp-6、胶原蛋白pre-COL-P、pre-COL-D、pre-COL-NG、足丝基质蛋白PTMP和DTMP中的一种或几种的混合物。2. The improved biomedical material product according to embodiment 1, wherein the mussel mucin is from a subclass: mefp1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, collagen A mixture of one or more of pre-COL-P, pre-COL-D, pre-COL-NG, foot silk matrix proteins PTMP and DTMP.
3、根据实施方式1的改良的生物医学材料产品，其中生物医用材料可以包括钛、钽、铌、锆、不锈钢、钴基合金和钛基合金等金属材料，陶瓷、玻璃、碳素等无机材料，聚乙烯、聚丙烯、聚丙烯酸酯、芳香聚酯、聚硅氧烷、聚甲醛、胶原、线性脂肪族聚酯、甲壳素、纤维素、聚氨基酸、聚乙烯醇、聚己丙酯等可降解或不可降解高分子材料，生物医用复合材料及生物衍生材料等。3. The improved biomedical material product according to embodiment 1, wherein the biomedical material comprises metal materials such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloys and titanium-based alloys, and inorganic materials such as ceramics, glass, and carbon. , polyethylene, polypropylene, polyacrylate, aromatic polyester, polysiloxane, polyoxymethylene, collagen, linear aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polyhexyl ester, etc. Degradable or non-degradable polymer materials, biomedical composite materials and biologically derived materials.
4、根据实施方式1的改良的生物医学材料产品，其中生长因子可以包括血小板类生长因子(血小板来源生长因子，PDGF；骨肉瘤来源生长因子ODGF)、表皮生长因子类(表皮生长因子，EGF、转化生长因子，TGFα和TGFβ)、成纤维细胞生长因子(αFGF、βFGF)、类胰岛素生长因子(IGF-I、IGF-II)、神经生长因子(NGF)、白细胞介素类生长因子(IL-1、IL-1、IL-3等)、红细 胞生长素(EPO)、集落刺激因子(CSF)等。4. The improved biomedical material product according to embodiment 1, wherein the growth factor may comprise platelet growth factor (platelet derived growth factor, PDGF; osteosarcoma derived growth factor ODGF), epidermal growth factor (epidermal growth factor, EGF, Transforming growth factor, TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin growth factor (IL-) 1, IL-1, IL-3, etc., red fine Cytokinin (EPO), colony stimulating factor (CSF), and the like.
5、根据实施方式1的改良的生物医学材料产品，其中所述贻贝粘蛋白可以是采用从贻贝中提取获得的，包含已知的11个贻贝粘蛋白亚类中的一种或几种的混合物。5. The improved biomedical material product according to embodiment 1, wherein the mussel mucin is obtained by extracting from mussels, comprising one or more of the known 11 mussel mucin subclasses. Kind of mixture.
6、根据实施方式1的改良的生物医学材料产品，其中所述贻贝粘蛋白可以是生物合成的方法获得的，包含已知的11个贻贝粘蛋白亚类中的一种或几种的混合物。6. The improved biomedical material product according to embodiment 1, wherein the mussel mucin is obtained by a method of biosynthesis, comprising one or more of the known 11 mussel mucin subclasses. mixture.
7、根据实施方式1的改良的生物医学材料产品，其中所述的贻贝粘蛋白也可以是天然来源或人工生物合成贻贝粘蛋白经水解后获得的水解肽，或通过人工合成方式获得的含有功能基团的合成肽。7. The improved biomedical material product according to embodiment 1, wherein the mussel mucin is also a hydrolyzed peptide obtained by hydrolysis of natural or artificial biosynthesis mussel mucin, or obtained by artificial synthesis. A synthetic peptide containing a functional group.
8、根据实施方式1的改良的生物医学材料产品，其中结合前贻贝粘蛋白浓度可以是0.1-15.0mg/ml。8. The improved biomedical material product according to embodiment 1, wherein the pre-bonded mussel mucin concentration may be from 0.1 to 15.0 mg/ml.
9、根据实施方式1的改良的生物医学材料产品，其中结合前贻贝粘蛋白可以是在pH 1.0-7.0的范围内，特别是在pH 3.0-6.5的范围内。9. The improved biomedical material product according to embodiment 1, wherein the pre-admixed mussel mucin may be in the range of pH 1.0-7.0, in particular in the range of pH 3.0-6.5.
10、根据实施方式1的改良的生物医学材料产品，其中贻贝粘蛋白与生长因子的结合方式可以是以共价键的形式交联或是通过分子间物理作用结合。10. The improved biomedical material product according to embodiment 1, wherein the mussel mucin binds to the growth factor in a manner of cross-linking in the form of a covalent bond or by intermolecular physical interaction.
11、根据实施方式1的改良的生物医学材料产品，可用于骨、牙、关节、肌腱等骨骼-肌肉***修复材料，皮肤、***、食道、呼吸道、膀胱等软组织材料，人工心瓣膜、血管、心血管内插管等心血管***材料，血液净化膜和分离膜、气体选择性透过膜、角膜接触镜等医用膜材料，组织粘合剂和缝线材料，敷料材料，药物释放载体材料，临床诊断及生物传感器材料等。11. The improved biomedical material product according to Embodiment 1 can be used for bone-to-muscle system repair materials such as bones, teeth, joints, tendons, soft tissue materials such as skin, breast, esophagus, respiratory tract, bladder, artificial heart valves, blood vessels, Cardiovascular system materials such as cardiovascular intubation, blood purification membrane and separation membrane, gas selective permeable membrane, contact lens and other medical membrane materials, tissue adhesive and suture material, dressing material, drug release carrier material, clinical Diagnostics and biosensor materials.
12、一种制备改良的生物医学材料产品的方法，包括首先将贻贝粘蛋白与生长因子结合，然后固化到生物医用材料上。12. A method of making an improved biomedical material product comprising first combining mussel mucin with a growth factor and then curing onto a biomedical material.
13、一种制备改良的生物医学材料产品的方法，包括将贻贝粘蛋白固化到生物医用材料上之后，再将生长因子结合到固化贻贝粘蛋白的生物医用材料上。13. A method of making an improved biomedical material product comprising curing a mussel mucin onto a biomedical material and then incorporating the growth factor onto the biomedical material that cures the mussel mucin.
14、一种制备改良的生物医学材料产品的方法，包括将生长因子固化到生物医用材料上之后，再将贻贝粘蛋白结合到固化生长因子的生物医用材料上。14. A method of making an improved biomedical material product comprising: after curing a growth factor onto a biomedical material, binding the mussel mucin to the biomedical material that cures the growth factor.
15、根据实施方式12-14中任一项的方法，其中贻贝粘蛋白与生长因子结合的方式为混合(冻干粉或溶液)、浸泡等。The method according to any one of embodiments 12-14, wherein the mussel mucin is combined with the growth factor by mixing (lyophilized powder or solution), soaking or the like.
16、根据实施方式12-14中任一项的方法，其中贻贝粘蛋白和/或生长因子 与生物医学材料的固化方式为喷涂、浸泡等。The method according to any one of embodiments 12-14, wherein the mussel mucin and/or growth factor The curing method with biomedical materials is spraying, soaking, and the like.
17、根据实施方式12-16中任一项的方法，其中用于固化的温度可以是任意的，优选10-40℃。The method of any of embodiments 12-16, wherein the temperature for curing can be any, preferably 10-40 °C.
18、根据实施方式12-17中任一项的方法，其中用于固化的一段时间可以是任意的，优选10-60分钟。The method of any of embodiments 12-17, wherein the period of time for curing can be any, preferably from 10 to 60 minutes.
下面将结合具体实施例对本发明作进一步说明。需要指出的是，由本发明中的贻贝粘蛋白与生长因子和生物医用材料形成的产品，在本发明范围内均已测试，下文中，仅仅是为说明，只在实施例中描述了其中一少部分，然而不应将其理解为对本发明的限制。以下非限制性的实施例中进一步说明了本发明产品的性质和优点。The invention will now be further described in conjunction with specific embodiments. It is to be noted that the products formed from the mussel mucin of the present invention and growth factors and biomedical materials have been tested within the scope of the present invention. Hereinafter, for the sake of explanation, only one of them is described in the examples. It is a few, but should not be construed as limiting the invention. The nature and advantages of the products of the present invention are further illustrated in the following non-limiting examples.
实施例1：贻贝粘蛋白与骨形态发生蛋白固化到人工骨粉用于股骨缺损治疗。Example 1: Mussel mucin and bone morphogenetic protein were solidified into artificial bone powder for the treatment of femoral defects.
贻贝粘蛋白与骨形态发生蛋白固化骨粉制备：取浓度为1.0mg/ml的贻贝粘蛋白溶液5ml及重组人骨形态发生蛋白(优级纯，上海药巢生物制品有限公司)1ml，浓度为1.0mg/ml，混合均匀形成凝胶状的生物材料。取人工骨粉(上海哈研生物科技有限公司，分子量4000)10g，将贻贝粘蛋白与骨形态发生蛋白的混合凝胶溶液加入骨粉中，混合均匀后于40℃固化12h，至骨粉完全干燥后形成贻贝粘蛋白与骨形态发生蛋白固化的骨粉。Preparation of mussel mucin and bone morphogenetic protein-cured bone powder: Take 5ml mussel mucin solution with a concentration of 1.0mg/ml and recombinant human bone morphogenetic protein (excellent pure, Shanghai Yaochao Biological Products Co., Ltd.) 1ml, the concentration is 1.0 mg/ml, uniformly mixed to form a gel-like biomaterial. Take artificial bone powder (Shanghai Harbin Biotechnology Co., Ltd., molecular weight 4000) 10g, add the mixed gel solution of mussel mucin and bone morphogenetic protein to bone powder, mix well and cure at 40 °C for 12h, until the bone powder is completely dried. A bone powder that forms mussel mucin and bone morphogenetic protein solidification.
贻贝粘蛋白调合骨粉制备：取浓度为1.0mg/ml的贻贝粘蛋白溶液5ml及人工骨粉10g，混合均匀后形成贻贝粘蛋白调合骨粉。Mussel mucin blending bone powder preparation: Take mussel mucin solution 5ml with concentration of 1.0mg/ml and artificial bone powder 10g, and mix it to form mussel mucin to mix bone powder.
骨形态发生蛋白调合骨粉制备：取浓度为1.0mg/ml的骨形态发生蛋白5ml及人工骨粉10g，混合均匀后形成骨形态发生蛋白调合骨粉。Preparation of bone morphogenetic protein-adapted bone powder: Take 5 ml of bone morphogenetic protein and 10 g of artificial bone powder at a concentration of 1.0 mg/ml, and mix and form bone morphogenetic protein to mix bone powder.
试验方法：取30只体重4.0-4.5kg的雄性新西兰白兔，麻醉后剃去后腿毛，消毒后用手术刀切开腿部皮肤和肌肉露出股骨，用电钻在股骨中间位置截取直径1cm的骨，形成骨缺损。取15只白兔，每只白兔左后腿中植入经贻贝粘蛋白与骨形态发生蛋白固化的人工骨粉，右后腿植入贻贝粘蛋白与人工骨粉调合的骨粉材料。另取15只白兔左腿植入骨形态发生蛋白与人工骨粉调合的骨粉材料，右腿植入未经处理的骨粉，作为空白对照组。植入完成后，将伤口缝合，按正常方法饲养，每周采用X射线观察骨缺损愈合情况。 Test method: 30 male New Zealand white rabbits weighing 4.0-4.5 kg were taken. After anesthesia, the hind legs were shaved. After disinfection, the skin and muscles of the legs were cut with a scalpel to expose the femur. The drill was used to cut the diameter of 1 cm in the middle of the femur. Bone, forming a bone defect. Fifteen white rabbits were taken. Each rabbit was implanted with artificial bone powder cured by mussel mucin and bone morphogenetic protein in the left hind leg, and bone powder material mixed with mussel mucin and artificial bone powder was implanted in the right hind leg. Another 15 rabbits were implanted with bone powder material of bone morphogenetic protein and artificial bone powder in the left leg, and untreated bone powder was implanted into the right leg as a blank control group. After the implantation is completed, the wound is sutured and kept in a normal manner, and the healing of the bone defect is observed by X-ray every week.
至术后7周，用贻贝粘蛋白和骨形态发生蛋白固化骨粉作修复材料的骨缺损处缺损边缘模糊，已完全愈合。至术后10周，用贻贝粘蛋白调合骨粉作修复材料的骨缺损完全愈合。术后10周，将30只新西兰白兔处死，取出骨缺损部位的股骨，观察愈合情况。贻贝粘蛋白与骨形态发生蛋白固化骨粉修复的骨缺损处已与周围骨组织结合生长，并且有血管生成；用贻贝粘蛋白调合骨粉修复的骨缺损处与周围骨组织结合生长，但生成血管程度及骨的量低于贻贝粘蛋白固化骨粉；有用骨形态发生蛋白修复的骨缺损处，人工骨材料未完全充填骨缺损处，与正常骨组织间仍有缝隙。只采用人工骨修复的骨缺处，人工骨材料未完全充填骨缺损处，与正常骨组织间仍能看能明显缝隙。以上结果可以看出，贻贝粘蛋白可以促进骨的再生和愈合，尤其是当贻贝粘蛋白与骨形态发生蛋白固化到人工骨材料上时，由于在体液中的稳定性增加、代谢周期延长，更有利于促进骨缺损的愈合。By the 7th week after surgery, the defect of the defect in the bone defect with mussel mucin and bone morphogenetic protein-cured bone powder was completely healed. By 10 weeks after surgery, the bone defect with the mussel mucin blended bone powder as a repair material completely healed. Ten weeks after the operation, 30 New Zealand white rabbits were sacrificed, and the femur of the bone defect site was taken out to observe the healing condition. The bone defect repaired by mussel mucin and bone morphogenetic protein-cured bone powder has been combined with the surrounding bone tissue and has angiogenesis; the bone defect repaired with mussel mucin and bone powder is combined with the surrounding bone tissue, but The degree of angiogenesis and bone formation is lower than that of mussel mucin-cured bone powder; the bone defect is repaired by bone morphogenetic protein, and the artificial bone material is not completely filled with the bone defect, and there is still a gap between the bone tissue and the normal bone tissue. Only the bone defect repaired by the artificial bone, the artificial bone material is not completely filled with the bone defect, and there is still a clear gap between the normal bone tissue and the normal bone tissue. The above results show that mussel mucin can promote bone regeneration and healing, especially when mussel mucin and bone morphogenetic protein are solidified onto artificial bone material, due to increased stability in body fluids and prolonged metabolic cycle. It is more conducive to the healing of bone defects.
实施例2：贻贝粘蛋白与骨形态发生蛋白固化到牙科植体材料表面促进植体与牙槽骨的结合Example 2: Mussel mucin and bone morphogenetic protein are solidified onto the surface of dental implant material to promote the union of implant and alveolar bone
贻贝粘蛋白与骨形态发生蛋白固化牙科植体的制备：取浓度为2.0mg/ml的贻贝粘蛋白溶液5ml及重组人骨形态发生蛋白(优级纯，上海药巢生物制品有限公司)1ml，浓度为1.0mg/ml，混合均匀形成凝胶状的生物材料。取牙科植体(佛山高安齿生物科技有限公司)，将贻贝粘蛋白与骨形态发生蛋白的混合凝胶溶液均匀喷涂到种植体表面，于37℃固化4h，形成贻贝粘蛋白与骨形态发生蛋白固化的种植体。Preparation of mussel mucin and bone morphogenetic protein-cured dental implants: 5 ml mussel mucin solution at a concentration of 2.0 mg/ml and recombinant human bone morphogenetic protein (superior pure, Shanghai Yaochao Biological Products Co., Ltd.) 1 ml The concentration was 1.0 mg/ml, and the mixture was uniformly mixed to form a gel-like biological material. The dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) was used to uniformly spray the mixed gel solution of mussel mucin and bone morphogenetic protein onto the surface of the implant and solidified at 37 ° C for 4 h to form mussel mucin and bone morphology. A protein-curing implant occurs.
贻贝粘蛋白固化牙植体的制备：取2.0mg/ml的贻贝粘蛋白溶液1ml，将牙科植体(佛山高安齿生物科技有限公司)浸泡至贻贝粘蛋白溶液中5h，取出后室温下干燥得到贻贝粘蛋白固化的牙科植体。Preparation of mussel mucin solidified dental implant: Take 1ml of mussel mucin solution of 2.0mg/ml, soak the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) into mussel mucin solution for 5h, and remove the room temperature. Under drying, the mussel mucin-cured dental implant is obtained.
骨形态发生蛋白因化牙植体的制备：取1.0mg/ml的骨形态发生蛋白溶液1ml，将牙科植体(佛山高安齿生物科技有限公司)浸泡至骨形态发生蛋白溶液中5h，取出后室温下干燥得到骨形态发生蛋白固化的牙科植体。Preparation of bone morphogenetic protein-derived dental implant: Take 1ml of 1.0mg/ml bone morphogenetic protein solution, soak the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) into bone morphogenetic protein solution for 5h, after removal The bone morphogenetic protein-cured dental implant is obtained by drying at room temperature.
采用大型杂种犬作为试验动物，全身麻醉后，分别从左、右侧，上下对称位置拔掉实牙。将植体分别植入至上、下牙槽骨，左上侧为未作处理的植体，左下侧为贻贝粘蛋白固化的植体，右上侧为骨形态发生蛋白固化的植体，右下 侧为贻贝粘蛋白结合骨形态发生蛋白固化的植体。植入植体后继续按照正常方式饲养，40天后观察植体与牙槽骨结合情况。贻贝粘蛋白与骨形态发生蛋白固化的植体与牙槽骨结合良好，植体上已有大量骨细胞生长；贻贝粘蛋白固化植体上骨细胞的数量明显低于贻贝粘蛋白与骨形态发生蛋白固化植体；骨形态发生蛋白固化植体与空白植体上骨细胞的数量相近，均低于贻贝粘蛋白固化植体。证明贻贝粘蛋白结合骨形态发生蛋白固化的牙科植体更有利于骨细胞在植体材料上的爬行和生长，可以明显降低植体与牙骨结合的时间。A large mongrel dog was used as a test animal. After general anesthesia, the solid teeth were removed from the left and right sides, and the upper and lower symmetrical positions were respectively removed. The implants were implanted into the upper and lower alveolar bones respectively. The left upper side was the untreated implant, the lower left side was the mussel mucin solidified implant, and the upper right side was the bone morphogenetic protein solidified implant, bottom right. The side is the implant of mussel mucin binding to bone morphogenetic protein. After implanting the implants, they were kept in the normal way. After 40 days, the implants and alveolar bone were observed. Muscle mucin and bone morphogenetic protein-solidified implants are well integrated with alveolar bone, and a large number of bone cells have been grown on the implant; the number of bone cells on mussel mucin cured implants is significantly lower than that of mussel mucin and Bone morphogenetic protein-solidified implants; bone morphogenetic protein-solidified implants and blank implants have similar numbers of bone cells, which are lower than mussel mucin-cured implants. It is proved that the dental implants in which mussel mucin binding to bone morphogenetic protein is solidified are more conducive to the crawling and growth of bone cells on the implant material, and the time of binding of the implant to the bone can be significantly reduced.
实施例3：贻贝粘蛋白(MAP)与神经生长因子(NGF)联合应用对去细胞神经支架移植后神经修复和再生的影响。Example 3: Effect of mussel mucin (MAP) combined with nerve growth factor (NGF) on nerve repair and regeneration after decellularized nerve scaffold transplantation.
1、实验动物及分组1. Experimental animals and grouping
(1)健康成年青紫蓝兔10只，雌雄不限，体重2000-2500g。(1) 10 healthy adult blue-purple rabbits, male or female, weighing 2000-2500g.
(2)健康成年Sprague-Dawley大鼠36只，雌雄不限，体重180-200g。大鼠随机分为3组：NS对照组12只；NGF治疗组12只；MAP+NGF联合治疗组12只。去细胞异种神经支架移植术后，动物分别存活4周和8周，不同时段各组动物为6只。(2) 36 healthy adult Sprague-Dawley rats, male or female, weighing 180-200 g. Rats were randomly divided into 3 groups: 12 in the NS control group; 12 in the NGF treatment group; and 12 in the MAP+NGF combination treatment group. After decellularized xenografts, the animals survived for 4 weeks and 8 weeks, respectively, and 6 animals in each group at different time points.
2、去细胞神经基膜管支架的制备2, preparation of decellularized nerve basement tube stent
切取青紫蓝兔胫神经，在手术显微镜下去除外覆***，以TritionX-100溶液和脱氧胆酸钠溶液按一定浓度和程序进行化学萃取，制备兔胫神经来源的异种神经支架，放入内含100μg/ml青霉素和100μg/ml链霉素的无菌PBS缓冲液(pH 7.4)中，4℃冰箱保存，2周内使用。The cyanotic nerve of the cyanotic blue rabbit was cut out, and the connective tissue was removed under the operating microscope. The Tern X-100 solution and sodium deoxycholate solution were chemically extracted according to a certain concentration and procedure to prepare a heterogeneous nerve scaffold derived from the rabbit sacral nerve. 100 μg/ml penicillin and 100 μg/ml streptomycin in sterile PBS buffer (pH 7.4), stored in a refrigerator at 4 ° C, and used within 2 weeks.
3、去细胞异种神经支架移植前处理3, decellularized heterogeneous nerve stent before transplantation
移植前，将NS对照组、NGF(齐一生物科技有限公司)治疗组的神经支架分别浸入0.9％NS液、NGF液中10min，将MAP+NGF联合治疗组的神经支架先浸入1.5mg/ml MAP溶液中10min，取出后浸入NGF溶液中10min。Before transplantation, the nerve scaffolds of the NS control group and NGF (Qiyi Biotechnology Co., Ltd.) treatment group were immersed in 0.9% NS solution and NGF solution for 10 min respectively, and the nerve scaffold of the MAP+NGF combination treatment group was first immersed in 1.5 mg/ml. 10 min in MAP solution, taken out and immersed in NGF solution for 10 min.
4、手术移植4, surgical transplantation
将大鼠用2％异戊巴比妥钠(50mg/kg)腹腔内注射麻醉，按无菌原则于右侧臀沟下缘处切开皮肤，分离肌肉，于手术显微镜下充分暴露并游离坐骨神经，在距梨状肌下缘切断坐骨神经造成神经缺损达10mm。在16倍手术显微镜下，用10-0无损伤医用缝合线在无张力下将10mm长的去细胞异种神经支架与大鼠坐骨神经的近、远断端行神经外膜缝合4-6针，然后逐层关闭切口。 The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (50 mg/kg), the skin was cut at the lower edge of the right gluteal groove according to the principle of sterility, the muscles were separated, and the sciatic nerve was fully exposed under the operating microscope. The nerve injury was caused by cutting the sciatic nerve from the lower edge of the piriformis muscle to 10 mm. Under a 16-fold surgical microscope, 10 mm long decellularized heterogeneous nerve scaffolds were sutured with a 10 mm long decellularized heterogeneous nerve scaffold and the proximal and distal ends of the rat sciatic nerve for 4-6 needles under tension without tension. Close the incision layer by layer.
5、术后给药途径和剂量5, postoperative route and dose
NS对照组：术后大鼠损伤侧小腿肌内注射0.9％NS 0.1ml；NGF治疗组：术后大鼠损伤侧小腿肌内注射NGF(180ng/100g/d)；MAP+NGF联合治疗组：术后大鼠损伤侧小腿肌内注射NGF(180ng/100g/d)；上述注射均持续14天。NS control group: intramuscular injection of 0.9% NS 0.1ml in the injured lateral calf of the injured rats; NGF treatment group: intramuscular injection of NGF (180ng/100g/d) in the injured lateral calf of the rats; MAP+NGF combined treatment group: NGF (180 ng/100 g/d) was injected intramuscularly into the injured leg of the injured rat. The above injections lasted for 14 days.
6、检测结果6, test results
(1)大体及手术显微镜下观察(1) Observing under general and operating microscope
术后各组大鼠手术侧下肢立即瘫痪，出现爪背着地，托步爬行现象；术后2周，各组大鼠术侧足踝部有不同程度红肿，未发生坏死及溃疡；术后4周，各组大鼠术侧足踝部肿胀消退，患肢可支撑着地，联合治疗组大鼠跛行改善明显；术后8周，各组大鼠足趾已能主动展开，平衡、协调地配合身体活动。Immediately after operation, the lower limbs of the rats in each group were paralyzed, and there were paws on the ground and crawling. After 2 weeks, the rats in each group had different degrees of redness and swelling, no necrosis and ulceration. Week, the swelling of the ankle and foot of the rats in each group subsided, and the affected limbs could support the ground. The rats in the combined treatment group improved significantly. After 8 weeks, the toes of each group were able to actively deploy, balanced and coordinated. Physical activity.
取材时观察所见：术后4周，NS对照组大鼠神经移植体与周围组织有轻度粘连，色泽、外径与神经干相似，近、远端神经吻合口为少量***包裹，吻合口处稍粗大；NGF治疗组大鼠神经移植体与周围组织有轻微粘连，色泽、外径与神经干相似，近、远端神经吻合口基本平整；MAP+NGF联合给药组大鼠神经移植体呈神经样外观，与周围组织几乎无粘连，近、远端神经吻合口基本光滑完整。各组大鼠神经移植体近、远端无神经瘤形成。Observations at the time of observation: At 4 weeks after operation, the nerve grafts of the NS control group had mild adhesion to the surrounding tissues, and the color and outer diameter were similar to those of the nerve trunk. The proximal and distal nerve anastomosis was a small amount of connective tissue, and the anastomosis The mouth was slightly thicker; the nerve graft of the NGF treatment group had slight adhesion to the surrounding tissue, the color and outer diameter were similar to the nerve trunk, and the proximal and distal nerve anastomosis was almost flat; the MAP+NGF combined administration group was used for nerve transplantation. The body has a neuron-like appearance, almost no adhesion to the surrounding tissue, and the proximal and distal nerve anastomosis is basically smooth and complete. There were no neuroma formation in the proximal and distal nerve grafts of each group.
(2)神经电生理检测结果(2) Results of neurophysiological examination
术后4周及8周，各处理组测量结果见下表1和表2。同一时间点联合用药组比单独用药组神经传导速度快，波幅高，差异有显著性。At 4 weeks and 8 weeks after surgery, the results of each treatment group are shown in Table 1 and Table 2 below. At the same time point, the combined drug group had faster nerve conduction velocity and higher amplitude than the drug alone group, and the difference was significant.
表1：坐骨神经运动传导速度恢复率的比较(％，-x±s)：Table 1: Comparison of recovery rates of sciatic nerve motor conduction velocity (%, -x ± s):
组别Group 术后4周4 weeks after surgery 术后8周8 weeks after surgery
MAP+NGFMAP+NGF 60.53±2.8060.53±2.80 71.52±4.6571.52±4.65
NGFNGF 51.19±2.9151.19±2.91 56.34±5.5256.34±5.52
NSNS 28.66±4.6528.66±4.65 47.85±1.0547.85±1.05
表2：小腿腓肠肌复合动作电位波幅恢复率的比较(％，-x±s)Table 2: Comparison of recovery rate of combined action potential amplitude of calf gastrocnemius muscle (%, -x±s)
组别Group 术后4周4 weeks after surgery 术后8周8 weeks after surgery
MAP+NGFMAP+NGF 36.15±1.8236.15±1.82 76.80±1.1876.80±1.18
NGFNGF 24.09±0.9024.09±0.90 55.24±1.6155.24±1.61
NSNS 17.65±0.6617.65±0.66 34.88±0.8634.88±0.86
(3)小腿三头肌湿重恢复率检测结果 (3) Test results of wet weight recovery rate of triceps
术后第4及第8周，各处理组测量结果见下表3。同一时间点联合用药组比单独用药组小腿三头肌湿重恢复率高，差异显著。At the 4th and 8th week after surgery, the measurement results of each treatment group are shown in Table 3 below. At the same time point, the recovery rate of the wet weight of the triceps was higher than that of the combination group, and the difference was significant.
表3：小腿三头肌湿重恢复率的比较(％，-x±s)Table 3: Comparison of wet weight recovery rates of calf triceps (%, -x ± s)
组别Group 术后4周4 weeks after surgery 术后8周8 weeks after surgery
MAP+NGFMAP+NGF 48.65±1.1248.65±1.12 58.80±1.8858.80±1.88
NGFNGF 36.39±0.9036.39±0.90 44.94±2.6144.94±2.61
NSNS 27.85±1.2227.85±1.22 37.48±1.7237.48±1.72
经以上试验可以看出，单独使用或不使用NGF的神经支架对损伤神经的修复不够理想，贻贝粘蛋白(MAP)联合了贻贝粘蛋白的神经生长因子能更好的与神经支架结合，促进神经细胞的再生，增强神经生长因子修复效果。It can be seen from the above experiments that the nerve scaffold with or without NGF is not ideal for repairing injured nerves. Mussel mucin (MAP) combined with mussel mucin's nerve growth factor can better bind to nerve scaffold. Promote the regeneration of nerve cells and enhance the repair effect of nerve growth factor.
实施例4：贻贝粘蛋白固化到磷酸三钙人工骨支架上用于膝关节骨软骨缺损修复治疗Example 4: Curing of mussel mucin to a tricalcium phosphate artificial bone scaffold for the repair of knee osteochondral defects
贻贝粘蛋白固化磷酸三钙人工骨支架的制备：取15mg/ml的贻贝粘蛋白溶液20ml，将磷酸三钙人工骨支架(恩赛华垦科技有限公司)浸泡至贻贝粘蛋白溶液中1h，取出后30℃下干燥得到贻贝粘蛋白固化的磷酸三钙人工骨支架。Preparation of mussel mucin-cured tricalcium phosphate artificial bone scaffold: Take 20ml of 15mg/ml mussel mucin solution and soak the tricalcium phosphate artificial bone scaffold (En赛华垦科技有限公司) into mussel mucin solution 1h, after drying at 30 ° C, the mussel mucin solidified tricalcium phosphate artificial bone scaffold was obtained.
取12月龄杂种犬10只，随机分为实验组、缺损对照组。无菌条件下自髂后嵴处抽取骨髓分离培养骨髓间充质干细胞，传至第3代消化、收集，调整细胞浓度为2×109个细胞/L，与贻贝粘蛋白固化的磷酸三钙人工骨支架和为使用贻贝粘蛋白固化的磷酸三钙人工骨支架共培养24h，即制成贻贝粘蛋白固化的磷酸三钙人工骨支架-骨髓间充质干细胞复合物和未使用贻贝粘蛋白固化的磷酸三钙人工骨支架-骨髓间充质干细胞复合物。两组犬均制备右膝关节骨软骨缺损，实验组于缺损处植入固化支架-细胞复合物，缺损植入未固化支架-细胞复合物。Ten 12-month-old mongrel dogs were randomly divided into experimental group and defect control group. Bone marrow mesenchymal stem cells were isolated from the sputum and extracted from the sputum under aseptic conditions. The cells were transferred to the third passage for digestion and collection. The cell concentration was adjusted to 2×10 9 cells/L, and the mussel mucin was solidified. The calcium artificial bone scaffold and the tricalcium phosphate artificial bone scaffold which was cured with mussel mucin were co-cultured for 24 hours, which was made into mussel mucin solidified tricalcium phosphate artificial bone scaffold-bone marrow mesenchymal stem cell complex and unused sputum. The mucin-cured tricalcium phosphate artificial bone scaffold-bone marrow mesenchymal stem cell complex. The osteoarticular defects of the right knee joint were prepared in both groups. The experimental group was implanted with a solidified scaffold-cell complex in the defect, and the defect was implanted with the uncured scaffold-cell complex.
第12周，实验组缺损内充填以白色半透明组织，表面光滑，触之较软，稍高出周围软骨面，软骨细胞分布较均一，排列无方向性；第24周，实验组缺损内充填白色半透明新生软骨组织，色泽与正常软骨相似，质韧，表面平整，与正常软骨界限消失，表面细胞平行于关节面，深层细胞排列紊乱，细胞呈团状，基质异染广泛，与周围正常软骨连接良好。对照组第12周未见明显反应，第24周开始，对照组缺损内开始充填以白色半透明组织，表面光滑，触之较软，稍高出周围软骨面，软骨细胞分布较均一，排列无方向性。(这个有图？)提示骨髓 间充质干细胞是修复关节软骨缺损较理想的种子细胞，在新生软骨形成的同时，磷酸三钙人工骨支架逐渐降解吸收，是组织工程修复关节软骨缺损适宜的支架材料，使用贻贝粘蛋白进行固化能明显加速修复过程。 At the 12th week, the defect of the experimental group was filled with white translucent tissue, the surface was smooth, the touch was soft, slightly higher than the surrounding cartilage surface, the distribution of chondrocytes was more uniform, and the arrangement was non-directional; at the 24th week, the filling of the experimental group was filled. White translucent neonatal cartilage tissue, similar in color to normal cartilage, with a tough texture and smooth surface. The boundary with normal cartilage disappears. The surface cells are parallel to the articular surface. The deep cells are arranged in disorder, the cells are clustered, the matrix is widely stained, and the surrounding is normal. The cartilage is well connected. There was no obvious reaction in the 12th week of the control group. At the 24th week, the control group began to fill with white translucent tissue. The surface was smooth, the touch was soft, slightly higher than the surrounding cartilage surface, and the chondrocytes were more evenly distributed. Directionality. (This picture?) Prompt bone marrow Mesenchymal stem cells are ideal seed cells for repairing articular cartilage defects. At the same time of neonatal cartilage formation, the tricalcium phosphate artificial bone scaffold is gradually degraded and absorbed. It is a suitable scaffold for tissue engineering to repair articular cartilage defects, using mussel mucin. Curing can significantly speed up the repair process.

Claims (10)

  1. 一种改良的生物医学材料，包括以均匀覆盖方式固化到生物医学材料上的贻贝粘蛋白与生长因子，其中贻贝粘蛋白与生长因子的质量比为100∶1-1∶100。An improved biomedical material comprising mussel mucin and growth factor solidified onto a biomedical material in a uniform coverage, wherein the mass ratio of mussel mucin to growth factor is from 100:1 to 1:100.
  2. 根据权利要求1的改良的生物医学材料，其中所述贻贝粘蛋白是来自亚类：mefp1、mefp-2、mefp-3、mefp-4、mefp-5、mefp-6、胶原蛋白pre-COL-P、pre-COL-D、pre-COL-NG、足丝基质蛋白PTMP和DTMP中的一种或几种的混合物。The improved biomedical material according to claim 1 wherein said mussel mucin is from a subclass: mefp1, mefp-2, mefp-3, mefp-4, mefp-5, mefp-6, collagen pre-COL a mixture of one or more of -P, pre-COL-D, pre-COL-NG, foot silk matrix proteins PTMP and DTMP.
  3. 根据权利要求1的改良的生物医学材料，其中生物医用材料为钛、钽、铌、锆、不锈钢、钴基合金和钛基合金等金属材料，陶瓷、玻璃、碳素等无机材料，聚乙烯、聚丙烯、聚丙烯酸酯、芳香聚酯、聚硅氧烷、聚甲醛、胶原、线性脂肪族聚酯、甲壳素、纤维素、聚氨基酸、聚乙烯醇、聚己丙酯等可降解或不可降解高分子材料，生物医用复合材料及生物衍生材料。The improved biomedical material according to claim 1, wherein the biomedical material is a metal material such as titanium, tantalum, niobium, zirconium, stainless steel, cobalt-based alloy or titanium-based alloy, inorganic materials such as ceramics, glass, carbon, etc., polyethylene, Polypropylene, polyacrylate, aromatic polyester, polysiloxane, polyoxymethylene, collagen, linear aliphatic polyester, chitin, cellulose, polyamino acid, polyvinyl alcohol, polyhexyl ester, etc. Degradable or non-degradable Polymer materials, biomedical composites and biologically derived materials.
  4. 根据权利要求1的改良的生物医学材料，其中生长因子包括血小板类生长因子(血小板来源生长因子，PDGF；骨肉瘤来源生长因子ODGF)、表皮生长因子类(表皮生长因子，EGF、转化生长因子，TGFα和TGFβ)、成纤维细胞生长因子(αFGF、βFGF)、类胰岛素生长因子(IGF-I、IGF-II)、神经生长因子(NGF)、白细胞介素类生长因子(IL-1、IL-1、IL-3等)、红细胞生长素(EPO)、集落刺激因子(CSF)。The improved biomedical material according to claim 1, wherein the growth factor comprises platelet-like growth factor (platelet-derived growth factor, PDGF; osteosarcoma-derived growth factor ODGF), epidermal growth factor (epidermal growth factor, EGF, transforming growth factor, TGFα and TGFβ), fibroblast growth factor (αFGF, βFGF), insulin-like growth factor (IGF-I, IGF-II), nerve growth factor (NGF), interleukin growth factor (IL-1, IL-) 1. IL-3, etc., erythrocyte growth factor (EPO), colony stimulating factor (CSF).
  5. 根据权利要求1的改良的生物医学材料，其中贻贝粘蛋白与生长因子是以共价键的形式交联或是通过分子间物理作用结合的。The improved biomedical material according to claim 1 wherein the mussel mucin and the growth factor are crosslinked in the form of covalent bonds or by intermolecular physical interaction.
  6. 根据权利要求1的改良的生物医学材料，可用于骨、牙、关节、肌腱等骨骼-肌肉***修复材料，皮肤、***、食道、呼吸道、膀胱等软组织材料，人工心瓣膜、血管、心血管内插管等心血管***材料，血液净化膜和分离膜、气体选择性透过膜、角膜接触镜等医用膜材料，组织粘合剂和缝线材料，敷料材料，药物释放载体材料，临床诊断及生物传感器材料。The improved biomedical material according to claim 1 can be used for bone-to-muscle system repair materials such as bones, teeth, joints, tendons, soft tissue materials such as skin, breast, esophagus, respiratory tract, bladder, artificial heart valves, blood vessels, and cardiovascular interpolation. Cardiovascular system materials, blood purification membranes and separation membranes, gas selective permeable membranes, contact lens and other medical membrane materials, tissue adhesives and suture materials, dressing materials, drug release carrier materials, clinical diagnosis and biology Sensor material.
  7. 一种制备改良的生物医学材料的方法，包括：A method of making an improved biomedical material comprising:
    将贻贝粘蛋白与生长因子结合后，固化到生物医用材料上；或Combining mussel mucin with growth factors and curing onto biomedical materials; or
    将贻贝粘蛋白固化到生物医用材料上之后，再将生长因子结合到固化贻贝粘蛋白的生物医用材料上；或 After the mussel mucin is cured onto the biomedical material, the growth factor is then bound to the biomedical material that cures the mussel mucin; or
    将生长因子固化到生物医用材料上之后，再将贻贝粘蛋白结合到固化生长因子的生物医用材料上，After the growth factor is cured onto the biomedical material, the mussel mucin is bound to the biomedical material that cures the growth factor,
    其中贻贝粘蛋白与生长因子的质量比为100∶1-1∶100。The mass ratio of mussel mucin to growth factor is from 100:1 to 1:100.
  8. 根据权利要求7的方法，其中固化温度为10-40℃。The method of claim 7 wherein the curing temperature is from 10 to 40 °C.
  9. 根据权利要求7的方法，其中固化时间为10-60分钟。The method of claim 7 wherein the curing time is from 10 to 60 minutes.
  10. 根据权利要求7的方法，其中结合是以共价键的形式交联或是通过分子间物理作用结合。 A method according to claim 7 wherein the binding is crosslinked in the form of a covalent bond or by intermolecular physical interaction.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109813911A (en) * 2017-11-22 2019-05-28 江阴贝瑞森生化技术有限公司 A kind of method quantifying traceable measurement sea-mussel mucin degradation
CN110665060A (en) * 2019-10-18 2020-01-10 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof
WO2022041401A1 (en) * 2020-08-27 2022-03-03 振德医疗用品股份有限公司 Wound covering and preparation method therefor
CN114668889A (en) * 2022-03-30 2022-06-28 绽妍生物科技有限公司 Regeneration and repair dressing containing mussel-like mucin and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130021951A (en) * 2011-08-24 2013-03-06 포항공과대학교 산학협력단 Surface immobilization of various functional biomolecules using mussel adhesive protein
CN103638554A (en) * 2013-11-13 2014-03-19 浙江大学 Preparation method of composite material of silk fibroin and marine mussel adhesion protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20130021951A (en) * 2011-08-24 2013-03-06 포항공과대학교 산학협력단 Surface immobilization of various functional biomolecules using mussel adhesive protein
CN103638554A (en) * 2013-11-13 2014-03-19 浙江大学 Preparation method of composite material of silk fibroin and marine mussel adhesion protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAO, MIN ET AL.: "Overview of Mussel Adhesive Protein", JOURNAL OF ANHUI AGRICULTURAL SCIENCES, vol. 39, no. 32, 20 October 2015 (2015-10-20), pages 19860, ISSN: 0517-6611 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109813911A (en) * 2017-11-22 2019-05-28 江阴贝瑞森生化技术有限公司 A kind of method quantifying traceable measurement sea-mussel mucin degradation
CN110665060A (en) * 2019-10-18 2020-01-10 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof
CN110665060B (en) * 2019-10-18 2021-09-21 浙江瑞谷生物科技有限公司 Bone repair material and preparation method and application thereof
WO2022041401A1 (en) * 2020-08-27 2022-03-03 振德医疗用品股份有限公司 Wound covering and preparation method therefor
CN114668889A (en) * 2022-03-30 2022-06-28 绽妍生物科技有限公司 Regeneration and repair dressing containing mussel-like mucin and preparation method thereof

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