本发明涉及一种改良的骨修复材料，更具体地，涉及在骨修复材料上固化贻贝粘蛋白的产品。The present invention relates to an improved bone repair material and, more particularly, to a product for curing mussel mucin on a bone repair material.
长期以来，骨修复材料主要采取自体或异体骨移植物。自体骨移植在材料来源方面存在着严重的缺陷。异体骨移植因材料筛选、储存、免疫排斥反应、易感染病毒以及异体骨被取代缓慢、新生骨体积偏小等方面抑制了其发展。为了克服自体骨和异体骨移植存在的问题，人们试图通过天然或合成途径取得理想的骨修复材料。For a long time, bone repair materials have mainly used autologous or allogeneic bone grafts. Autologous bone grafts have serious drawbacks in terms of material sources. Allogeneic bone grafts inhibited the development of materials such as material screening, storage, immune rejection, vulnerability to infection, slow replacement of allogeneic bone, and small new bone volume. In order to overcome the problems of autologous bone and allogeneic bone transplantation, attempts have been made to obtain ideal bone repair materials through natural or synthetic routes.
近年来发展起来的骨修复材料，通过引入形态上与骨单位相容、且其管道可连入骨缺损组织的孔状支架结构，建立起生物材料与骨架再生之间的联系。常见的骨修复材料有天然骨材料，如牛骨烧结骨修复材料；无机非金属骨修复材料，如磷酸钙陶瓷、磷酸钙水泥；复合骨修复材料，如羟基磷灰石与金属、有机生物材料复合而成的骨修复材料。这些材料有一定的三维立体结构和良好的表面活性，如孔隙率在80％以上，高的内表面积有利于细胞的植入、贴附、营养物质的渗入及代谢废物的排出等，为细胞的生长、增殖提供良好的微环境。但是，骨科修复材料很难直接影响材料内部的细胞生长行为，使骨缺损的再生更有预见性。The bone repair material developed in recent years establishes a relationship between the biomaterial and the skeleton regeneration by introducing a pore-like scaffold structure which is morphologically compatible with the bone unit and whose tube can be connected to the bone defect tissue. Common bone repair materials include natural bone materials, such as bovine bone sintered bone repair materials; inorganic non-metallic bone repair materials such as calcium phosphate ceramics, calcium phosphate cement; composite bone repair materials such as hydroxyapatite and metals, organic biomaterials Composite bone repair material. These materials have a certain three-dimensional structure and good surface activity, such as a porosity of more than 80%, a high internal surface area is conducive to cell implantation, attachment, nutrient infiltration and metabolic waste discharge, etc. Growth and proliferation provide a good microenvironment. However, orthopedic repair materials are difficult to directly affect the cell growth behavior inside the material, making the regeneration of bone defects more predictable.
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.
目前贻贝粘蛋白产品的应用领域非常有限。商品化的贻贝粘蛋白产品有美国BD Biosciences公司的Cell-Tak，韩国Kollodis公司的MAP Trix和瑞典Biopolymer公司的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。At present, the application fields of mussel mucin products are very limited. Commercial mussel mucin products include 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 formulations and dissolved prior to use. Their primary 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.
贻贝粘蛋白由于具有促进细胞贴壁、爬行功能，可用于创面的愈合过程，但在骨科修复材料中的应用尚未见报道。Mussel mucin can be used for wound healing process because it has the function of promoting cell adhesion and crawling, but its application in orthopedic repair materials has not been reported.
发明内容Summary of the invention
本发明的目的是提供一种改良的骨修复材料产品，其不但能满足常规骨修复的要求，同时还能促进细胞生长，缩短愈合时间。It is an object of the present invention to provide an improved bone repair material product that not only meets the requirements of conventional bone repair, but also promotes cell growth and shortens healing time.
为了实现上述目的，本发明提供的一种改良的骨修复材料产品，包括：可用于骨修复的基质材料，以及可固化于基质材料上的贻贝粘蛋白。In order to achieve the above object, the present invention provides an improved bone repair material product comprising: a matrix material useful for bone repair, and mussel mucin which is curable on a matrix material.
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(Perna 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pH 3.0-6.5的范围内以使其效果更佳。Mussel mucin used herein refers to Mytilus edulis Linnaeus, Mytilus coruscus or Perna 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 viscosity of the mussel mucin used herein in aqueous solution may be pH 1.0-7.0,In particular, it can be in the range of pH 3.0-6.5 to make it more effective.
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 artificially 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 it more effective.
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其功能基团的合成肽。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 thereof 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 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。The mussel mucin used herein may be in the form of a solution or a lyophilized powder.
在本发明的实施方式中，骨修复材料可以是牙科植体、人工骨膜、人工骨、骨固定器、髓内装置、脊柱固定或矫形器、椎间融合器、骨蜡等。In an embodiment of the invention, the bone restorative material may be a dental implant, an artificial periosteum, an artificial bone, a bone fixator, an intramedullary device, a spinal fixation or orthosis, an interbody fusion cage, a bone wax, or the like.
本发明所述及的骨修复材料定义源于国家食品药品监督管理局颁布的《医疗器械分类目录》(2013版)。The definition of bone repair material according to the present invention is derived from the Catalogue of Medical Devices (2013 edition) promulgated by the State Food and Drug Administration.
本发明提供一种制备改良的骨修复材料的方法，包括以下步骤：The present invention provides a method of making an improved bone repair material comprising the steps of:
(1)配制贻贝粘蛋白溶液，(1) preparing a mussel mucin solution,
(2)将贻贝粘蛋白溶液固化至骨修复材料中，得到改良的骨修复材料。(2) The mussel mucin solution is solidified into a bone repair material to obtain an improved bone repair material.
在本发明中使用的术语“固化”是指通过物理手段将贻贝粘蛋白吸附到骨修复材料上。The term "curing" as used in the present invention refers to the adsorption of mussel mucin to a bone repair material by physical means.
在一个实施方式中，可以将贻贝粘蛋白溶液加入到基质材料中，一段时间后使贻贝粘蛋白固化至基质材料。In one embodiment, the mussel mucin solution can be added to the matrix material for a period of time to cure the mussel mucin to the matrix material.
在一个实施方式中，可以将贻贝粘蛋白溶液喷涂到基质材料表面，一段时间后使贻贝粘蛋白固化至基质材料。In one embodiment, the mussel mucin solution can be sprayed onto the surface of the matrix material for a period of time to cure the mussel mucin to the matrix material.
在一个实施方式中，可以将基质材料浸润于贻贝粘蛋白溶液中，一段时间后使贻贝粘蛋白固化至基质材料。In one embodiment, the matrix material can be infiltrated into the mussel mucin solution for a period of time to cure the mussel mucin to the matrix material.
在本发明的实施方式中，贻贝粘蛋白溶液可以是由酸性、中性或弱碱性溶液配制，并以酸性溶液最佳。In an embodiment of the invention, the mussel mucin solution may be formulated from an acidic, neutral or weakly alkaline solution and is most preferably an acidic solution.
在本发明的实施方式中，贻贝粘蛋白溶液可以由柠檬酸、乙酸、柠檬酸钠、草酸、碳酸、磷酸、苯甲酸、碳酸钠等配制，但不限于上述溶剂。In the embodiment of the present invention, the mussel mucin solution may be formulated from citric acid, acetic acid, sodium citrate, oxalic acid, carbonic acid, phosphoric acid, benzoic acid, sodium carbonate, or the like, but is not limited to the above solvent.
在本发明的实施方式中，贻贝粘蛋白溶液的浓度可以是0.01-150mg/ml，特别是贻贝粘蛋白溶液的浓度可以是0.1-15mg/ml。In an embodiment of the present invention, the concentration of the mussel mucin solution may be 0.01 to 150 mg/ml, and particularly the concentration of the mussel mucin solution may be 0.1 to 15 mg/ml.
在本发明的实施方式中，贻贝粘蛋白溶液的用量应能均匀覆盖或浸润骨修复材料。In an embodiment of the invention, the mussel mucin solution is used in an amount sufficient to uniformly cover or infiltrate the bone restorative 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.
本发明提供一种改良的骨修复材料，该伤骨修复材料是在基质材料上包被贻贝粘蛋白后形成的，其能在骨修复的同时，促进细胞生长，缩短愈合时间。The invention provides an improved bone repairing material which is formed by coating mussel mucin on a matrix material, which can promote cell growth and shorten healing time while repairing bone.
本发明还提供一种改良的牙科植体，其中将贻贝粘蛋白固化到现有的牙科植体上。牙科植体可以包括：骨内种植体、骨膜下种植体、根管内种植体、穿骨种植体等。The present invention also provides an improved dental implant in which mussel mucin is cured onto an existing dental implant. Dental implants can include: intraosseous implants, subperiosteal implants, root canal implants, bone implants, and the like.
本发明还提供一种改良的人工骨膜，其中将贻贝粘蛋白固化到现有的人工骨膜上。人工骨膜可以包括：牙骨膜、关节骨膜、骨骨膜等。The present invention also provides an improved artificial periosteum in which mussel mucin is cured onto an existing artificial periosteum. The artificial periosteum may include: a periosteum, a joint periosteum, a periosteum, and the like.
本发明还提供一种改良的人工骨，其中将贻贝粘蛋白固化到现有的人工骨上。人工骨可以包括：无机材料人工骨、有机材料人工骨及复合材料人工骨。The present invention also provides an improved artificial bone in which mussel mucin is cured onto existing artificial bone. The artificial bone may include: an inorganic material artificial bone, an organic material artificial bone, and a composite artificial bone.
本发明还提供一种改良的骨固定器，其中将贻贝粘蛋白固化到现有的骨固定器上。骨固定器可以包括：不锈钢、钴铬钼合金、钛和钛合金骨固定器等。The present invention also provides an improved bone anchor in which mussel mucin is cured onto an existing bone anchor. The bone anchors may include: stainless steel, cobalt chromium molybdenum alloy, titanium and titanium alloy bone anchors, and the like.
本发明还提供一种改良的髓内装置，其中将贻贝粘蛋白固化到现有的髓内装置上。The present invention also provides an improved intramedullary device in which mussel mucin is cured onto an existing intramedullary device.
本发明还提供一种改良的脊柱固定或矫形器，其中将贻贝粘蛋白固化到现有的脊柱固定或矫形器上。The present invention also provides an improved spinal fixation or orthosis wherein the mussel mucin is cured onto an existing spinal fixation or orthosis.
本发明还提供一种改良的椎间融合器，其中将贻贝粘蛋白固化到现有的椎间融合器上。The present invention also provides an improved interbody fusion cage in which mussel mucin is cured to an existing one.On the interbody cage.
本发明还提供一种改良的骨蜡，其中将贻贝粘蛋白固化到现有的骨蜡上。The present invention also provides an improved bone wax in which mussel mucin is cured onto an existing bone wax.
本发明的所有产品均可通过上述制备改良的骨修复材料的方法进行制备。All of the products of the present invention can be prepared by the above-described method of preparing a modified bone repair material.
本发明人出人意料地发现，将贻贝粘蛋白固化到骨修复材料上，可以在骨修复材料上发挥贻贝粘蛋白的活性，赋予骨修复材料生物结构，如加速愈合，更有利于重建或康复受损的人体组织或器官。并且特别是，贻贝粘蛋白固化骨修复材料后，提高了蛋白的物理、化学稳定性，延长了蛋白的降解周期，更有利于贻贝粘蛋白生物活性的发挥。The present inventors have surprisingly found that the mussel mucin can be cured on the bone repair material to exert the activity of mussel mucin on the bone repair material, and to impart the biological structure of the bone repair material, such as accelerated healing, which is more conducive to reconstruction or rehabilitation. Damaged human tissue or organ. In particular, after mussel mucin solidifies bone repair material, it improves the physical and chemical stability of the protein, prolongs the degradation cycle of the protein, and is more conducive to the biological activity of mussel mucin.
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，本发明的固化有贻贝粘蛋白的骨修复材料产品，均可由上述方法制备，并且在本发明范围内均已测试并验证了效果，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 should be noted that the bone repair material product of the present invention having mussel mucin cured can be prepared by the above method, and the effects have been tested and verified within the scope of the present invention. Hereinafter, only for explanation, only in the A few of the examples are described in the examples, which 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：贻贝粘蛋白骨粉用于肱骨骨缺损治疗Example 1: Mussel mucin bone powder for the treatment of tibia bone defect
试验材料：雄性新西兰白兔15只、戊巴比妥钠、电子称、刀片、手术剪刀、止血钳、5％聚维酮碘、酒精、脱脂棉、纱布、手术缝合针、手术缝合线、注射器、电钻、口罩、手套、X-光机、灭菌锅等。Test materials: 15 male New Zealand white rabbits, sodium pentobarbital, electronic scale, blade, surgical scissors, hemostat, 5% povidone iodine, alcohol, absorbent cotton, gauze, surgical suture needle, surgical suture, syringe, Electric drills, masks, gloves, X-ray machines, sterilizers, etc.
贻贝粘蛋白固化骨粉制备：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，将贻贝粘蛋白溶液加入骨粉中，均匀混合后于40℃固化12h，至骨粉完全干燥后形成贻贝粘蛋白固化骨粉。Mussel mucin solidified bone powder preparation: Take mussel mucin solution 5ml with concentration of 1.0mg/ml and artificial bone powder (Shanghai Harbin Biotechnology Co., Ltd., molecular weight 4000) 10g, add mussel mucin solution to bone powder, evenly After mixing, it was cured at 40 ° C for 12 h until the bone powder was completely dried to form mussel mucin cured bone powder.
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，混合均匀后立即使用。Preparation of mussel mucin blended bone powder: Take 5 ml of mussel mucin solution and 10 g of artificial bone powder at a concentration of 1.0 mg/ml, and mix them immediately before use.
试验方法：取10只体重4.0-4.5kg的雄性新西兰白兔，麻醉后剃去前腿毛，消毒后用手术刀切开前腿皮肤和肌肉露出肱骨，用电钻在肱骨中间位置截取直径1cm的骨，形成骨缺损。每只白兔左前腿中植入经贻贝粘蛋白固化的人工骨粉，右前腿植入贻贝粘蛋白与人工骨粉调合的骨粉材料。另取5只体重4.0-4.5kg的雄性新西兰白兔，按上述相同方法制造骨缺损，仅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。至术后8周，用贻贝粘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15只新西兰白兔处死，取出骨缺损部位的肱骨，观察愈合情况。Test method: 10 male New Zealand white rabbits weighing 4.0-4.5 kg were taken before shaving before anesthesiaLeg hair, after disinfection, use a scalpel to cut the skin and muscles of the front leg to expose the humerus, and use an electric drill to intercept the bone of 1 cm in diameter at the middle of the humerus to form a bone defect. Each white rabbit was implanted with artificial bone powder solidified by mussel mucin in the left front leg, and bone powder material blended with mussel mucin and artificial bone powder was implanted in the right front leg. Five male New Zealand white rabbits weighing 4.0-4.5 kg were used to make bone defects in the same manner as above, and only untreated bone powder was implanted 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. By the 8th week after surgery, the bone defect of the bone defect repaired with mussel mucin as a repair material was blurred and 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 surgery, 15 New Zealand white rabbits were sacrificed, and the humerus at the defect site was removed to observe the healing.
贻贝粘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；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。The bone defect repaired by mussel mucin solidified 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 to grow, but the blood vessel level and bone are generated. The amount is lower than that of mussel mucin solidified bone powder; only the bone defect is repaired by artificial bone, the artificial bone material is not completely filled with the bone defect, and the gap can be seen 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 is cured onto artificial bone material, which is more conducive to promotion due to increased stability in body fluids and prolonged metabolic cycle. The healing of bone defects.
实施例2：贻贝粘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 is cured to the surface of dental implant material to promote the bonding of the implant to the alveolar bone
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，取出后室温干燥，得到贻贝粘蛋白固化的牙科植体。Take 1ml of 2.0mg/ml mussel mucin solution, soak the dental implant (Foshan Gaoan Tooth Biotechnology Co., Ltd.) into mussel mucin solution for 5h, remove it and dry at room temperature to obtain the mussel mucin-cured dental implant. body.
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。左侧为对照组，将在使用前喷涂贻贝粘蛋白溶液的植体分别植入至上、下牙槽骨；右侧为试验组，将贻贝粘蛋白固化的植体分别植入至上、下牙槽骨。植入植体后继续按照正常方式饲养，2个月后观察植体与牙槽骨结合情况。试验组植体与牙槽骨结合良好，植体上已有大量骨细胞生长；对照组植体上骨细胞的数量明显低于试验组。证明贻贝粘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 left side is the control group, and the implants that spray the mussel mucin solution before use are implanted into the upper and lower alveolar bones respectively; the right side is the test group, and the mussel mucin-solidified implants are implanted into the upper and lower parts respectively. Alveolar bone. After implantation, the implants were continued in the normal manner, and the binding of the implants to the alveolar bone was observed 2 months later. The implants in the experimental group were well integrated with the alveolar bone, and a large number of bone cells were grown on the implants; the number of bone cells on the implants in the control group was significantly lower than that in the experimental group. It is proved that the dental implants cured by mussel mucin are more conducive to the crawling and growth of bone cells on the implant material, and can significantly reduce the time of the implant and the bone.
实施例3：贻贝粘蛋白固化到人工骨膜上促进骨细胞生长Example 3: Mussel mucin solidified to artificial periosteum to promote bone cell growth
贻贝粘蛋白固化人工骨膜的制备：取1.5mg/ml的贻贝粘蛋白溶液10ml，将人工骨膜(贝朗，德国，1.5cm×3.0cm)浸泡到贻贝粘蛋白溶液中，2小时后取出，室温晾干后备用。Preparation of mussel mucin solidified artificial periosteum: Take 10ml of mussel mucin solution of 1.5mg/ml, and soak the artificial periosteum (Berlang, Germany, 1.5cm×3.0cm) into mussel mucin solution for 2 hours. Take out, dry at room temperature and set aside.
收集2例人工骨膜移植手术患者，分别采用普通骨膜和用贻贝粘白固化的骨膜治疗。术后分别于1、4、5、6及9个月拍摄x射线片，观察骨膜愈合情况。至术后第4个月，采用贻贝粘蛋白固化的人工骨膜已完全愈合，至术后第6个月，采用普通骨膜治疗的部位完全愈合。可以看出，固化贻贝粘蛋白人工骨膜患者康复明显更快。Two patients undergoing artificial periosteal transplantation were treated with common periosteum and periosteum cured with mussels. X-ray films were taken at 1, 4, 5, 6 and 9 months after surgery to observe the healing of periosteum. By the fourth month after surgery, the artificial periosteum cured with mussel mucin had completely healed, and by the sixth month after surgery, the site treated with common periosteum completely healed. It can be seen that the rehabilitation of mussel mucin artificial periosteum patients is significantly faster.
实施例4：贻贝粘蛋白固化到羟基磷灰石人工骨上用于颌骨骨缺损的治疗Example 4: Curing of mussel mucin to hydroxyapatite artificial bone for the treatment of jaw bone defects
贻贝粘蛋白固化羟基磷灰石人工骨的制备：取150mg/ml的贻贝粘蛋白溶液2ml，均匀喷涂到羟基磷灰石人工骨(12um，含量96％，上海浦陈生物技术有限公司)表面，40℃恒温保持30分钟，取出后备用。Preparation of mussel mucin cured hydroxyapatite artificial bone: Take 2ml of mussel mucin solution of 150mg/ml and spray evenly onto hydroxyapatite artificial bone (12um, content 96%, Shanghai Puchen Biotechnology Co., Ltd.) The surface was kept at a constant temperature of 40 ° C for 30 minutes, and taken out after use.
在犬下颌骨体上设计两个10mm×10mm×5mm的箱状缺损，在其中一个缺损植入相应大小的贻贝粘蛋白固化羟基磷灰石人工骨预制块，另一缺损植入相应大小的直接喷涂贻贝粘蛋白的羟基磷灰石人工骨预制块做空白对照。于术后2、4、8、12、16周切取组织标本，进行X线摄片和组织学切片检查，观察骨缺损修复情况。Two 10mm×10mm×5mm box-shaped defects were designed on the canine mandibular body, and one of the defects was implanted with the corresponding size mussel mucin solidified hydroxyapatite artificial bone preform, and the other defect was implanted into the corresponding size. A hydroxyapatite artificial bone preform directly sprayed with mussel mucin as a blank control. Tissue samples were taken at 2, 4, 8, 12, and 16 weeks after surgery, and X-ray and histological sections were performed to observe the repair of bone defects.
结果显示：植入贻贝粘蛋白固化的羟基磷灰石人工骨的术区未见明显的排斥反应。第2～8周人工骨周围被纤维结缔组织包裹，12周时人工骨组织界面上出现膜性成骨迹象，第16周贻贝粘蛋白固化羟基磷灰石人工骨被新生骨组织完全包裹，受植床与人工骨形成骨性结合；植入直接喷涂贻贝粘蛋白的羟基磷灰石人工骨的术区亦未见明显的排斥反应。第6～12周观察到人工骨周围开始被纤维结缔组织包裹，16周时显现人工骨组织界面上出现膜性成骨迹象。可以看出，贻贝粘蛋白固化的羟基磷灰石人工骨具有更好的生物相容性，更强的骨引导和潜在的骨诱导作用。The results showed that there was no obvious rejection in the hydroxyapatite artificial bone implanted with mussel mucin. At the 2nd to 8th week, the artificial bone was surrounded by fibrous connective tissue. At 12 weeks, the membranous osteogenesis showed on the artificial bone tissue interface. At the 16th week, the mussel mucin solidified hydroxyapatite artificial bone was completely wrapped by the new bone tissue. The bed formed a bony bond with the artificial bone; there was no obvious rejection in the operation area of the hydroxyapatite artificial bone implanted with mussel mucin directly. From the 6th to the 12th week, it was observed that the artificial bone was surrounded by fibrous connective tissue, and at 16 weeks, the membranous osteogenesis showed on the artificial bone tissue interface. It can be seen that mussel mucin-cured hydroxyapatite artificial bone has better biocompatibility, stronger bone guidance and potential osteoinduction.
实施例5：贻贝粘蛋固化磷酸钙骨水泥用于松质骨骨缺损治疗。Example 5: Mussel sticky egg solidified calcium phosphate cement for the treatment of cancellous bone defect.
贻贝粘蛋白固化磷酸钙骨水泥制备：取浓度为0.01mg/ml的贻贝粘蛋白溶液10ml及人工骨粉10g，将贻贝粘蛋白溶液加入磷酸钙骨水泥(含量≥98％，苏州鼎安科技有限公司)中，将该骨水泥调制成可用注射器和16号穿刺针操作的注射物，混合均匀后于10℃固化1h保存备用。对照组采用生理盐水，将该骨水泥调制成可用注射器和16号穿刺针操作的注射物。Mussel mucin solidified calcium phosphate bone cement preparation: mussel mucin with concentration of 0.01mg/ml10ml of solution and 10g of artificial bone powder, the mussel mucin solution was added to calcium phosphate cement (content ≥98%, Suzhou Ding'an Technology Co., Ltd.), and the bone cement was prepared into an injection that can be operated with a syringe and a 16-gauge needle. After mixing, it was cured at 10 ° C for 1 h and stored for use. The control group was prepared with physiological saline, and the bone cement was prepared into an injection product which can be operated with a syringe and a 16-gauge needle.
试验方法：取10只体重4.0-4.5kg的雄性新西兰白兔，麻醉后剃去前腿毛，消毒后用手术刀切开前腿皮肤和肌肉露出股骨髁部，用电钻在股骨髁部中间位置钻取直径2mm骨洞，形成骨缺损。每只白兔左前腿中注入经贻贝粘蛋白固化的磷酸钙骨水泥，右前腿植入贻贝粘蛋白未固化的磷酸钙骨水泥和贻贝粘蛋白混合物。另取10只体重4.0-4.5kg的雄性新西兰白兔，按上述相同方法制造骨缺损，仅植入未经处理的磷酸钙骨水泥，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。至术后8周，用贻贝粘蛋白固化磷酸钙骨水泥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，用贻贝粘蛋白调合磷酸钙骨水泥作修复材料的骨缺损完全愈合。术后10周，将20只新西兰白兔处死，取出骨缺损部位的股骨髁部，观察愈合情况。贻贝粘蛋白固化磷酸钙骨水泥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；用贻贝粘蛋白调合磷酸钙骨水泥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磷酸钙骨水泥；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。Test method: 10 male New Zealand white rabbits weighing 4.0-4.5 kg were taken. After anesthesia, the front leg hair was shaved. After disinfection, the skin of the forelegs and muscles were excised with a scalpel to expose the femoral condyle. The electric drill was placed in the middle of the femoral condyle. A 2 mm diameter bone hole was drilled to form a bone defect. Each rabbit was injected with mussel mucin-cured calcium phosphate cement in the left front leg, and a mussel mucin uncured calcium phosphate cement and a mussel mucin mixture were implanted in the right front leg. Another 10 male New Zealand white rabbits weighing 4.0-4.5 kg were used to make bone defects in the same manner as above, and only untreated calcium phosphate cement was implanted 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. At 8 weeks after surgery, the defect of the defect in the bone defect with mussel mucin solidified calcium phosphate cement as a repair material was blurred and completely healed. By 10 weeks after surgery, the bone defect with mussel mucin and calcium phosphate cement as a repair material completely healed. Ten weeks after the operation, 20 New Zealand white rabbits were sacrificed, and the femoral condyle of the bone defect site was taken out to observe the healing condition. The bone defect repaired by mussel mucin solidified calcium phosphate cement has been combined with the surrounding bone tissue and has angiogenesis; the bone defect repaired with mussel mucin and calcium phosphate cement is combined with the surrounding bone tissue. However, the degree of blood vessel formation and bone volume is lower than that of mussel mucin solidified calcium phosphate cement; only the bone defect is repaired by artificial bone, and the artificial bone material is not completely filled with the bone defect, and it can still be seen between normal bone tissue. Gap.
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。The above results show that mussel mucin can promote bone regeneration and healing, especially when mussel mucin is cured onto artificial bone material, which is more conducive to promotion due to increased stability in body fluids and prolonged metabolic cycle. The healing of bone defects.
实施例6：贻贝粘蛋白固化到羟基磷灰石涂层金属螺钉和外支架上用于治疗中老年开放性胫骨、肱骨骨折Example 6: Mussel mucin is cured onto hydroxyapatite coated metal screws and external stents for the treatment of open tibia and fibula fractures in middle-aged and elderly patients
贻贝粘蛋白固化羟基磷灰石涂层金属螺钉和外支架的制备：取0.1mg/ml的贻贝粘蛋白溶液100ml，将羟基磷灰石涂层金属螺钉和外支架浸泡至贻贝粘蛋白溶液中45分钟，取出后室温下干燥得到贻贝粘蛋白固化的羟基磷灰石涂层金属螺钉和外支架。Preparation of mussel mucin cured hydroxyapatite coated metal screw and external scaffold: Take 100ml of 0.1mg/ml mussel mucin solution, soak hydroxyapatite coated metal screw and outer scaffold to mussel mucin The solution was dried for 45 minutes, and then dried at room temperature to obtain a mussel-mucin-cured hydroxyapatite-coated metal screw and an outer stent.
收集55岁以上肱骨和胫骨开放性骨折患者18例，随机分配至试验组或对照组，采用贻贝粘蛋白固化的羟基磷灰石涂层金属螺钉和外支架(Orthofix，Italy)和直接喷涂贻贝粘蛋白的羟基磷灰石涂层金属螺钉和外支架(Orthofix，Italy)治疗。试验组9例患者在术后平均18周内达到临床愈合；对照组8例患者在术后平均24周内达到临床愈合。试验组无支架、螺钉松动发生，对照组1例发生螺钉松动。可以看出，贻贝粘蛋白固化的羟基磷灰石涂层金属螺钉和外支架治疗中老年开放性胫骨、肱骨骨折具有稳定性好、康复快。Eighteen patients with open fractures of the tibia and fibula over the age of 55 were randomly assigned to the test group or the control group. The hydroxyapatite-coated metal screws and outer stents (Orthofix, Italy) and direct spray 固化 were cured with mussel mucin. Shellfish mucin hydroxyapatite coated metal screws and external stents (Orthofix, Italy) treatment. Nine patients in the experimental group achieved clinical healing within an average of 18 weeks after surgery; 8 patients in the control group achieved clinical healing within an average of 24 weeks after surgery. There was no stent and screw loosening in the test group, and screw loosening occurred in 1 case of the control group. It can be seen that mussel mucin-cured hydroxyapatite-coated metal screws and external stents have good stability and rapid recovery in the treatment of open tibia and fibula fractures in middle-aged and elderly patients.
实施例7：贻贝粘蛋白固化到牛骨烧结骨上用于颌骨缺损的修复Example 7: Curing of mussel mucin to bovine bone sintered bone for repair of jaw defect
贻贝粘蛋白固化牛骨烧结骨的制备：取10mg/ml的贻贝粘蛋白溶液5ml，喷涂到牛骨烧结骨(北大口腔医院自制)至表面完全湿润，室温下保持30分钟后干燥，得到贻贝粘蛋白固化的牛骨烧结骨。Mussel mucin solidified bovine bone sintered bone preparation: Take 10ml of mussel mucin solution of 10mg/ml, spray it onto bovine bone sintered bone (made by Peking University Stomatological Hospital) until the surface is completely wet, keep it at room temperature for 30 minutes and then dry it. Mussel bone mucin-cured bovine bone sintered bone.
收集颌骨缺损患者20例，由骨科医生确认后入组，随机分配至试验组或对照组，采用贻贝粘蛋白固化的牛骨烧结骨或直接喷涂贻贝粘蛋白的牛骨烧结骨对缺损进行修复。试验组和对照组均进行X线及免疫学、r骨显像等观察。经6～12个月追踪观察，试验组10例患者创口愈合正常，未见免疫排斥反应，术后3周左右植骨成活，术后2个月有新骨形成。对照组10例患者创口愈合正常，未见免疫排斥反应，术后4周左右植骨成活，术后3个月有新骨形成。可以看出，贻贝粘蛋白固化的牛骨烧结骨具有良好的组织相容性和骨传导能力。Twenty patients with jaw defects were enrolled and confirmed by orthopedic surgeons. They were randomly assigned to the experimental group or the control group, and the bovine bone sintered bones with mussel mucin solidified or the bovine bone sintered bone directly sprayed with mussel mucin Make a repair. X-ray, immunology, and r-bone imaging were observed in both the experimental group and the control group. After 6 to 12 months of follow-up observation, 10 patients in the experimental group had normal wound healing, no immune rejection, bone graft survival around 3 weeks after surgery, and new bone formation 2 months after surgery. In the control group, 10 patients had normal wound healing, no immune rejection, bone graft survival around 4 weeks after surgery, and new bone formation 3 months after surgery. It can be seen that mussel-mucin-cured bovine bone sintered bone has good tissue compatibility and bone conduction ability.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2015/097450WO2017101021A1 (en) | 2015-12-15 | 2015-12-15 | Modified bone repairing material |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2015/097450WO2017101021A1 (en) | 2015-12-15 | 2015-12-15 | Modified bone repairing material |
| Publication Number | Publication Date |
|---|---|
| WO2017101021A1true WO2017101021A1 (en) | 2017-06-22 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2015/097450CeasedWO2017101021A1 (en) | 2015-12-15 | 2015-12-15 | Modified bone repairing material |
| Country | Link |
|---|---|
| WO (1) | WO2017101021A1 (en) |
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