阅读说明：本技术 一种高强度复合引导组织骨再生材料的制备方法 (A kind of preparation method of high-strength composite guidance Tissue bone regeneration material ) 是由 陈小强 于 2019-08-01 设计创作，主要内容包括：本发明涉及一种高强度复合引导组织骨再生材料的制备方法,属于骨再生材料技术领域。本发明将聚乳酸和羟基乙酸聚合,采用静电纺丝技术制备基体纤维,将胶原涂覆于基体纤维上,并通过3D打印技术与胶原纤维束加工成骨再生材料,具有良好的机械强度,人动脉平滑肌细胞能够很好的粘附在该材料上,并沿着胶原纤维束方向生长；胶原蛋白是动物结缔组织重要的蛋白质,结缔组织除了含60～70%的水分外,胶原蛋白占了约20～30%,因为有高含量的胶原蛋白,结缔组织具有了一定的结构与机械力学性质,如张力强度、拉力、弹力等以达到支撑、保护的功能；胶原纤维束是胶原纤维在疏松结缔组织中排列成束,彼此交织吻合,纤维束常有分支,纤维具有韧性,抗牵引力强。(The present invention relates to a kind of preparation methods of high-strength composite guidance Tissue bone regeneration material, belong to bone-regeneration material technical field.Polylactic acid and hydroxyacetic acid polymerize by the present invention, matrix fiber is prepared using electrostatic spinning technique, collagen is coated on matrix fiber, and bone-regeneration material is processed by 3D printing technique and collagenous fiber bundle, with good mechanical strength, Human Arterial Smooth Muscle Cells can be good at adherency on the material, and grow along collagenous fiber bundle direction；Collagen is the important protein of animal connective tissue; connective tissue is other than containing 60~70% moisture; collagen accounts for about 20~30%; because there is the collagen of high-content; connective tissue is provided with certain structure and mechanical properties, such as tensile strength, pulling force, elastic force to reach the function of support, protection；Collagenous fiber bundle is that collagenous fibres arrange bunchy in loose connective tissue, is interlaced with one another identical, fibre bundle Chang You branch, and fiber has toughness, and anti-tractive force is strong.)

1. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material, it is characterised in that specific preparation step are as follows:

(1) lactic acid and hydroxyacetic acid are uniformly mixed, carry out vacuumize process to get mixture, mixture is stirred simultaneously Stannous chloride is added to get reactant A in cooling processing in reactant A, continues 30~40min of stirring to get mixture A, incites somebody to action Mixture A carries out vacuumizing reaction treatment, is cooled to room temperature to get presoma material；

(2) 7: 3 tetrahydrofuran and n,N-Dimethylformamide are uniformly mixed by volume to get mixed solution, in mass ratio 1 : 5 by presoma material and mixed solution after mixing, filter up to spinning solution, spinning solution be subjected to electrostatic spinning processing, i.e., Obtain matrix fiber；

(3) matrix fiber is immersed in mass fraction is to be protected from light stir process in 2% sodium periodate solution, is filtered up to precipitating, Precipitating is washed with deionized 3~5 times to get washing precipitating, it is 1% glycerin solution that washing precipitating, which is immersed in mass fraction, In after 1~2h, take out and impregnate in deionized water 20~for 24 hours, dehydration is to get pretreatment matrix fiber；Matrix will be pre-processed It is in 80% collagen solution that fiber, which is immersed in mass fraction, and immersion treatment filters up to filter residue, and filter residue, which is placed in temperature, is It dries in 80~90 DEG C of baking oven to constant weight, is cooled to room temperature up to semi-finished product, semi-finished product are placed in deionized water and impregnate 1 It, is dehydrated to get modified matrix fiber；

(4) modified matrix fiber, polyacrylamide, deionized water, collagenous fiber bundle are taken, by modified matrix fiber, polyacrylamide Amine, deionized water mixing, low whipping speed are that 10~20min is stirred under 200~300r/min to get mixed slurry, are being mixed Collagenous fiber bundle is added in slurry, continues 20~25min of stirring to get matrix slurry, matrix slurry is printed, i.e., It obtains high-strength composite and guides Tissue bone regeneration material.

2. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In: vacuumize process step described in step (1) are as follows: in mass ratio 9: 1 are uniformly mixed lactic acid and hydroxyacetic acid, are in pressure 5~10min is vacuumized under 4000~4500Pa.

3. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In stirring described in step (1) and the processing step that cools down are as follows: it by mixture in temperature is 60~65 DEG C, mixing speed is 500~ 1~2h is stirred under 600r/min to get reactant, and reactant is cooled to 25~27 DEG C, is passed through 5~10min of nitrogen.

4. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In: the mass ratio of stannous chloride described in step (1) and reactant A is 1: 5.

5. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In: step vacuumizes reaction step described in (1) are as follows: mixture A is evacuated to 400~450Pa, temperature is at 65~70 DEG C React 8~10h.

6. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In the processing of: electrostatic spinning described in step (2): in voltage being 18~20kV by spinning solution, rectangular steel plates are as reception device, needle The reception distance of head and steel plate carries out electrostatic spinning under conditions of being 20~25cm.

7. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In: step is protected from light stir process step described in (3) are as follows: in mass ratio 1: 3 matrix fiber is immersed in mass fraction is 2% high It is 40~50 DEG C in temperature in sodium iodide solution, mixing speed is to be protected from light 30~50min of stirring under 200~300r/min.

8. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In: immersion treatment step described in step (3) are as follows: it is that 80% collagen is molten that pretreatment matrix fiber, which is immersed in mass fraction, In liquid, 1~2h is impregnated in stirring at being 50~60 DEG C in temperature.

9. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist Ratio between: modified matrix fiber described in step (4), polyacrylamide, deionized water, collagenous fiber bundle is respectively as follows: According to parts by weight, 30~40 parts of modified matrix fibers, 50~60 parts of polyacrylamides, 80~100 parts of deionizations are weighed respectively Water, 20~30 parts of collagenous fiber bundles.

10. a kind of preparation method of high-strength composite guidance Tissue bone regeneration material according to claim 1, feature exist In print processing step described in step (4) are as follows: by matrix slurry in temperature be 20~25 DEG C, platform temperature is 37~39 DEG C Air pressure be 0.11~0.14MPa, speed be 8~10mm/s, printing filling spacing be 0.8~0.9mm, printing thickness be 0.28~ It is printed under 0.30mm.

Technical field

The present invention relates to a kind of preparation methods of high-strength composite guidance Tissue bone regeneration material, belong to bone-regeneration material skill Art field.

Background technique

In dentistry field, the reasons such as periodontosis, tip of a root lesion, wound cause the defect of periodontium, remain after extraction The absorption of remaining alveolar ridge, and the reasons such as Bone mineral change will cause tooth supporting tissue defect in implant prostheses.Mesh Preceding these problems, which mainly pass through, can increase bone amount in defect or improve the guided tissue regeneration (GTR) of bone mass or draw Bone tissue regeneration art (GBR) is led to be treated.In this treatment technology, GTR film and GBR timbering material are key factors, directly It connects and influences final repairing effect.In in the past few decades, the material for GTR/GBR is had changed a lot, From biological stability, permanent type substitute to Bioabsorbable, temporary substitute transition, and gradually become in clinical application Main trend.The application of absorbability GTR film and GBR timbering material avoids second operation and takes out problem, saves operation Time and cost, reduce because operation caused by complication, therefore become current oral cavity bone impairment renovation material research Emphasis.

The characteristics of physiologic theory of GTR/GBR and clinical application are for osteanagenesis process and generate and perfect.When When bone defect occurs, since the speed that different tissues cell grows into defect, migrates is different, defect will be come first It is filled, is formed soft tissue " clip pad " from the connective tissue of surrounding, and the presence of this " clip pad " will postpone the healing of bone tissue Process.Using GTR/GBR technology, above-mentioned soft tissue is mechanically hindered first and enters bone defect position, keeps bone defect portion There is a metastable regenerative environ-ment in position.New bone formation at bone defect be mainly by be located at periosteum occur the osteoblast of layer Lai It undertakes, membrane material becomes the regenerated bracket of epiphysis periosteum at this time, and guidance has osteogenic cell adherence and to defect area Migration.Timbering material at be filled in bone defect not only acts as supporting function, is able to maintain former organized shape, but also Play template action, the place of boarding of relying, growth, differentiation and proliferation be provided for osteocyte, thus realize Guided Bone Regeneration and Control the purpose of the structure of regenerating tissues.In addition, can constantly be dissolved in the degradation process of timbering material in vivo out calcium, phosphorus from Son promotes mineralising deposition process, shortens healing time.

There are many type of the GTR/GBR film of studies and clinical application at present, by biodegradability be divided into Absorbable membrane and Nonabsorable film two major classes.Nonabsorable film is primarily used for guide tissue regeneration, and this kind of material has very strong biologically inert, Property is stablized, and does not react, but because it cannot be absorbed by tissue with tissue, needs to carry out two after 4 ~ 6 weeks after being implanted into human body Secondary operation is taken out, and the chance of wound is increased, thus is difficult to be accepted by patients, and clinical therapeutic efficacy is also undesirable, therefore gradually Replaced Absorbable membrane.Absorbability membrane material has good biocompatibility, nonantigenic interference, takes without second of operation Out, degradation time can regulate and control, can directly participate in the features such as tissue repair and bootable cell growth of intrinsic reticular structure, As ideal Guided bone regeneration membrane material.Ideal GTR/GBR film should have following characteristics:

(1) good biocompatibility and with the matched degradation rate of regeneration；

(2) good permeability also ensures the supply of nutriment and organizes metabolism while playing barrier action It carries out；(3) suitable mechanical strength and clinical operability；

It (4) being capable of slow release active material promotion new bone formation.From materialogy angle, single material and single knot Structure can not meet above-mentioned requirements simultaneously, therefore clinical existing guide tissue regeneration film material has been all made of multilayer and has answered Close structure.

At present mainly using collagen or artificial macromolecule as the Absorbable membrane of main material used in clinic.Collagem membrane has Following advantages:

(1) it is the main component of connective tissue, participates in tissue metabolism；

(2) there is higher inductivity to fibroblast；

(3) there is the characteristic for inhibiting epithelial cell mobile；

(4) poor antigen；

(5) there is anastalsis.What application was more at present is pigskin collagen film, due to the how purified processing of collagen, is generally not easy to draw Play rejection.In addition, collagem membrane is generally gradually absorbed by organisms after implanting 8 weeks, remove the hardship of second operation from.Experiment It confirms, collagem membrane is used on mouse nod the Guided Bone Regeneration reparation of bone defect, can not only play the role of obstructing fibr tissue, It can also promote the regeneration of bone and participate in induced osteogenesis differentiation.

Bone tissue has very strong power of regeneration, when fracture or defect occur for bone tissue, forms hemotoncus in damaged zone first, And then it is changed into granulation, and fibr tissue machine forms fibrocartilage, with the progress of mineral deposit and growing into for blood vessel, at Osteocyte is also grown into therewith, and synthesis collagenous fibres are laid equal stress on the raw bone tissue of renewing, is finally completed entire regenerative process.It can be complete It heals related with the gap size of damaged zone, if gap is excessive, osteoblast is difficult to get over gap and cannot occur normal Agglutination is only filled by fibr tissue, in this case fracture or bone defect will be difficult to heal, need using timbering material into Row fills the completion to assist knitting.

According to the difference of material source, osteanagenesis timbering material can be divided into autologous bone, homogeneous allogenic bone, bone- xenograft, Decalcified bone matrix and artificial synthesis bone etc..Allograph bone is possible to cause rejection in vivo；Autologous bone meets bone tissue reparation Requirement, but being limited source makes it be not widely deployed for clinic；And artificial synthesis bone, such as tricalcium phosphate, due to material The characteristic for expecting itself, is difficult structure required for meeting carrying cell, these materials are difficult to meet the needs of clinical repair.With The fast development of organizational engineering carries out Bone Defect Repari using organizational engineering principle, it has also become the hot spot studied at present.

Summary of the invention

The technical problems to be solved by the invention: aiming at the problem that existing bone-regeneration material bad mechanical strength, one is provided The preparation method of kind high-strength composite guidance Tissue bone regeneration material.

In order to solve the above technical problems, the technical solution adopted by the present invention is that:

(1) lactic acid and hydroxyacetic acid are uniformly mixed, carry out vacuumize process to get mixture, mixture is stirred simultaneously Stannous chloride is added to get reactant A in cooling processing in reactant A, continues 30~40min of stirring to get mixture A, incites somebody to action Mixture A carries out vacuumizing reaction treatment, is cooled to room temperature to get presoma material；

(2) 7: 3 tetrahydrofuran and n,N-Dimethylformamide are uniformly mixed by volume to get mixed solution, in mass ratio 1 : 5 by presoma material and mixed solution after mixing, filter up to spinning solution, spinning solution be subjected to electrostatic spinning processing, i.e., Obtain matrix fiber；

(3) matrix fiber is immersed in mass fraction is to be protected from light stir process in 2% sodium periodate solution, is filtered up to precipitating, Precipitating is washed with deionized 3~5 times to get washing precipitating, it is 1% glycerin solution that washing precipitating, which is immersed in mass fraction, In after 1~2h, take out and impregnate in deionized water 20~for 24 hours, dehydration is to get pretreatment matrix fiber；Matrix will be pre-processed It is in 80% collagen solution that fiber, which is immersed in mass fraction, and immersion treatment filters up to filter residue, and filter residue, which is placed in temperature, is It dries in 80~90 DEG C of baking oven to constant weight, is cooled to room temperature up to semi-finished product, semi-finished product are placed in deionized water and impregnate 1 It, is dehydrated to get modified matrix fiber；

(4) modified matrix fiber, polyacrylamide, deionized water, collagenous fiber bundle are taken, by modified matrix fiber, polyacrylamide Amine, deionized water mixing, low whipping speed are that 10~20min is stirred under 200~300r/min to get mixed slurry, are being mixed Collagenous fiber bundle is added in slurry, continues 20~25min of stirring to get matrix slurry, matrix slurry is printed, i.e., It obtains high-strength composite and guides Tissue bone regeneration material.

Vacuumize process step described in step (1) are as follows: in mass ratio 9: 1 are uniformly mixed lactic acid and hydroxyacetic acid, In Pressure is to vacuumize 5~10min under 4000~4500Pa.

Stirring described in step (1) and the processing step that cools down are as follows: by mixture in temperature be 60~65 DEG C, mixing speed is 1~2h is stirred under 500~600r/min to get reactant, and reactant is cooled to 25~27 DEG C, is passed through 5~10min of nitrogen.

The mass ratio of stannous chloride described in step (1) and reactant A is 1: 5.

Step vacuumizes reaction step described in (1) are as follows: mixture A is evacuated to 400~450Pa, temperature is 65~ 8~10h is reacted at 70 DEG C.

Electrostatic spinning described in step (2) processing: by spinning solution voltage be 18~20kV, rectangular steel plates as receive dress It sets, the reception distance of syringe needle and steel plate carries out electrostatic spinning under conditions of being 20~25cm.

Step is protected from light stir process step described in (3) are as follows: matrix fiber is immersed in mass fraction by mass ratio 1: 3 is It is 40~50 DEG C in temperature in 2% sodium periodate solution, mixing speed is to be protected from light 30~50min of stirring under 200~300r/min.

Immersion treatment step described in step (3) are as follows: it is 80% collagen egg that pretreatment matrix fiber, which is immersed in mass fraction, In white solution, 1~2h is impregnated in stirring at being 50~60 DEG C in temperature.

Modified matrix fiber described in step (4), polyacrylamide, deionized water, the ratio point between collagenous fiber bundle Not are as follows: according to parts by weight, weigh respectively 30~40 parts of modified matrix fibers, 50~60 parts of polyacrylamides, 80~100 parts go Ionized water, 20~30 parts of collagenous fiber bundles.

Print processing step described in step (4) are as follows: by matrix slurry temperature be 20~25 DEG C, platform temperature be 37~ 39 DEG C of air pressures are 0.11~0.14MPa, and speed is 8~10mm/s, and printing filling spacing is 0.8~0.9mm, and printing thickness is It is printed under 0.28~0.30mm.

The present invention is compared with other methods, and advantageous effects are:

(1) polylactic acid and hydroxyacetic acid polymerize by the present invention, prepare matrix fiber using electrostatic spinning technique, collagen is coated on On matrix fiber, and bone-regeneration material is processed by 3D printing technique and collagenous fiber bundle, there is good mechanical strength, people Arterial smooth muscle cell can be good at adherency on the material, and grow along collagenous fiber bundle direction；Collagen is The important protein of object connective tissue, for connective tissue other than containing 60~70% moisture, collagen accounts for about 20~30%, because To there is the collagen of high-content, connective tissue is provided with certain structure and mechanical properties, as tensile strength, pulling force, Elastic force etc. is to reach the function of support, protection；Collagenous fiber bundle is that collagenous fibres arrange bunchy in loose connective tissue, each other Interweave and coincide, fibre bundle Chang You branch, fiber has toughness, and anti-tractive force is strong；

(2) copolymer that polylactic acid and hydroxyacetic acid polymerize in the present invention is that one kind can be with biodegradable macromolecule material Material, has excellent mechanical performance, biocompatibility and biodegradability and degradation speed is controllable, copolymer quilt in human body It is degraded to lactic acid and light acetic acid, carbon dioxide and water are then generated by metabolism, to excrete, pair nontoxic to the human body Effect；

(3) collagen has good biocompatibility and biodegradable in the present invention, and biocompatibility refers to collagen and place There is good interaction between chief cell and tissue or between the matrix of cell peripheral, there is interactional coordination Property, and collagen can become a part of cell and tissue, the physiological function brought into normal play jointly；Collagen is beaten using 3D Print technology is coated to matrix fiber, and matrix fiber is made to have good biocompatibility；

(4) 3D printing technique is total with Clinical Computer tomoscan, magnetic as a kind of novel digital forming technique in the present invention Vibration imaging etc. combines, and using CAD personalization implantation material, realization is precisely controlled pore structure, design with The substantially identical bone-regeneration material of patient bone defect area is conducive to the biocompatibility for improving implantation material；By glue The matrix resin of original coating has good mechanical strength, and matrix resin and collagenous fiber bundle mixing, collagenous fibres element are distributed in On matrix fiber, under load effect, the collagenous fibres network across crackle may prevent crackle from expanding and propagate, and make forced section Position crack, which glances off, generates a large amount of matrix crackles, and the matrix debris and the plane of disruption of the more voluminous life of crackle are also more, can absorb More energy provide some strength for bone-regeneration material.

Specific embodiment

In mass ratio 9: 1 are uniformly mixed lactic acid and hydroxyacetic acid, 5 are vacuumized in the case where pressure is 4000~4500Pa~ Mixture in temperature is 60~65 DEG C to get mixture by 10min, and mixing speed is that 1~2h is stirred under 500~600r/min, Up to reactant, reactant is cooled to 25~27 DEG C, is passed through 5~10min of nitrogen to get reactant A, in mass ratio 1: 5 Stannous chloride is added in reactant A, continues 30~40min of stirring to get mixture A, mixture A is evacuated to 400~ 450Pa, temperature are that 8~10h is reacted at 65~70 DEG C, are cooled to room temperature to get presoma material；7: 3 by tetrahydro by volume Furans and n,N-Dimethylformamide are uniformly mixed to get mixed solution, and in mass ratio 1: 5 by presoma material and mixed solution After mixing, it filters up to spinning solution, in voltage is 18~20kV by spinning solution, rectangular steel plates are as reception device, syringe needle Reception distance with steel plate carries out electrostatic spinning under conditions of being 20~25cm to get matrix fiber；In mass ratio 1: 3 by matrix It is in 2% sodium periodate solution that fiber, which is immersed in mass fraction, is 40~50 DEG C in temperature, mixing speed is 200~300r/min Under be protected from light 30~50min of stirring, filter up to precipitate, precipitating is washed with deionized 3~5 times to get washing precipitating, will wash Precipitating be immersed in mass fraction and be in 1% glycerin solution after 1~2h take out and impregnate in deionized water 20~for 24 hours, dehydration, Up to pretreatment matrix fiber；It is to be in temperature in 80% collagen solution that pretreatment matrix fiber, which is immersed in mass fraction, 1~2h is impregnated in stirring at 50~60 DEG C, filters up to filter residue, filter residue is placed in the baking oven that temperature is 80~90 DEG C and is dried to perseverance Weight is cooled to room temperature up to semi-finished product, semi-finished product is placed in deionized water and are impregnated 1 day, is dehydrated to get modified matrix fiber； According to parts by weight, 30~40 parts of modified matrix fibers, 50~60 parts of polyacrylamides, 80~100 parts of deionizations are weighed respectively Water, 20~30 parts of collagenous fiber bundles mix modified matrix fiber, polyacrylamide, deionized water, low whipping speed 200 10~20min is stirred under~300r/min to get mixed slurry, and collagenous fiber bundle is added in mixed slurry, continues stirring 20 Matrix slurry in temperature is 20~25 DEG C, platform temperature is that 37~39 DEG C of air pressures are 0.11 to get matrix slurry by~25min ~0.14MPa, speed are 8~10mm/s, and printing filling spacing is 0.8~0.9mm, and printing thickness, which is that 0.28~0.30mm is lower, beats Print guides Tissue bone regeneration material to get high-strength composite.