The content of the invention
The technical problems to be solved by the invention：For natural macromolecular material mechanical property is not good enough, degradation rate and performanceThe problem of being not sufficiently stable provides a kind of nano bone repair material and preparation method thereof.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
The bone renovating material includes following weight parts raw material：1~2 part of pearl protein, 4~8 parts of air bladder collagens, 50~100 parts of biphasic calcium phosphate powder.
The pearl protein be the pearl powder being ground up, sieved extract at 40~50 DEG C 1 through edta solution~It is freeze-dried and is made after 2h.
The mass fraction of the edta solution is 1%, and dosage is 50~100 times of pearl powder quality.
The air bladder collagen is
The freeze-drying after the deionized water of 5~10 times of its quality extract 2~3h at 50~60 DEG C of pretreatment air bladder is obtained.
The pretreatment air bladder drains to be washed after fresh catfish fish glue removal blood vessel and mucous membrane, and is immersed in mass fraction and is8~10h in 5% sodium carbonate liquor, washing pulverize and sieve obtained after air-drying.
The biphasic calcium phosphate powder is crossed 200 mesh sieves for ball milling after bovine bone powder calcining and is made.
The calcination process is warming up to 500~600 DEG C to be fitted into Muffle furnace, with 5~10 DEG C/min rates, calcine 1~After 2h, 1350~1400 DEG C of 6~8h of calcining are continuously heating to, then stands and is cooled to 1000~1100 DEG C of 1~2h of calcining.
The bovine bone powder is fresh ox bone, is cut into bulk after removing the soft tissue and marrow adhered on ox bone, and is immersed inMass fraction is that 6~8h is impregnated in 4% sodium hydroxide solution, then is immersed in mass fraction as 10~12h in 20% hydrogen peroxide, uses phosphorusDrying and crushing is crossed 20 mesh sieves and is made after sour sodium buffer solution for cleaning.
It concretely comprises the following steps：
（1）By ground 400 mesh sieve after pearl laundry air-dry, pearl powder is obtained；
（2）Pearl powder is added in edta solution, 1~2h is extracted at 40~50 DEG C, is freezed after centrifugationIt is dry, obtain pearl protein；
（3）Fresh catfish fish glue is removed wash after blood vessel and mucous membrane and is drained, and be immersed in mass fraction be 8 in 5% sodium carbonate liquor~10h, washing pulverize and sieve after air-drying, and obtain pretreatment air bladder；
（4）Pretreatment air bladder is added in deionized water, 2~3h is extracted at 50~60 DEG C, is freeze-dried, obtains after centrifugationAir bladder collagen；
（5）By fresh ox bone, bulk is cut into after removing the soft tissue and marrow adhered on ox bone, and is immersed in mass fraction as 4%6~8h is impregnated in sodium hydroxide solution, then is immersed in mass fraction as 10~12h in 20% hydrogen peroxide, it is clear with sodium phosphate bufferIt washes rear drying and crushing and crosses 20 mesh sieves, obtain bovine bone powder；
（6）Ball milling crosses 200 mesh sieves after bovine bone powder is calcined, and obtains biphasic calcium phosphate powder；
（7）Pearl protein, air bladder collagen are taken, adds in deionized water and is uniformly mixed, add biphasic calcium phosphate powder, microwaveIt is heated to 90~100 DEG C and is evaporated to paste, then paste is fitted into mold, cure 20~30h at 110~120 DEG C, take offNano bone repair material is obtained after mould.
Compared with other methods, advantageous effects are the present invention：
（1）The present invention generates the α ﹣ tricalcium phosphates crystal forms and high degradability of high intensity using high-temperature calcination bovine bone powder, and high biology is livingProperty, the biphasic calcium phosphate of the β ﹣ tricalcium phosphate crystal forms of outstanding osteoinductive, and using it as skeleton, pass through pearl protein and air bladderCollagen cross-linking is filled into the porous structure of biphasic calcium phosphate, improves the brittleness and mechanical property of nano bone repair material；
（2）During guide tissue regeneration, the degradation rate of nano bone repair material prepared by the present invention reaches gives birth to tissueIt is long to be adapted, both maintained spacial ability at bone defect, and prevented fibr tissue from entering bone defect area, and prevent catabolite quickly intoEnter body, influence regeneration at bone defect, while regulate and control CaCO using pearl protein3Deposition velocity and configuration of surface are adjustedWith calcium binding situation, accelerate the nucleation of calcium carbonate crystal, also promote the activity of alkaline phosphatase, induced osteogenesis cell pointChange, Mineral nodules is promoted to be formed, inhibit osteoclast differentiation and bone tissue absorbs, effectively facilitate Bone Defect Repari；
（3）The present invention has preferable biological degradability, and catabolite is without any toxicity and side effects, can be with the natural physiology of bodyIt is metabolized and excretes, so as to complete the reparation of bone tissue and reconstruction, bone formation performance is good, has preferable osteoconductiveAnd osteoinductive, while compression strength and elasticity modulus are higher, are firmly combined with bone defect position, can be rapidly completed bone injury portionThe reparation of position.
Specific embodiment
Take 300~500g pearls, natural wind dry doubling, which is fitted into grinder, after washes clean grinds, and crosses 400 mesh sieves, takes 100It is in 1% edta solution, at 40~50 DEG C that pearl powder after~200g sievings, which adds in 10~20kg mass fractions,1~2h is stirred with 300~400r/min, is fitted into after being cooled to room temperature in centrifuge, 10 are centrifuged with 6000~8000r/min~15min, removal precipitation are placed in vacuum freezing drying oven, dry 20 at -30~-20 DEG C~for 24 hours, and pearl protein is obtained,The fresh catfish fish glues of 300~500g are taken, remove air bladder medium vessels and mucous membrane, and air bladder are washed with deionized 3~5 times, will be cleanedAir bladder afterwards drains, and is immersed in 3~5kg mass fractions as in 5% sodium carbonate liquor, with 300~400r/min stirrings 8~10h takes out air bladder and being washed with deionized to cleaning solution and is in neutrality, is fitted into pulverizer and crushes after natural air drying, 60 mesh of mistakeSieve, obtains pretreatment air bladder, and 100~200g pretreatment air bladders is taken to add in 1~2L deionized waters, at 50~60 DEG C with 300~400r/min stirring extraction 2~3h, is fitted into after being cooled to room temperature in centrifuge, with 6000~8000r/min centrifugation 10~15min, removal precipitation are placed in vacuum freezing drying oven, dry 20 at -30~-20 DEG C~for 24 hours, and obtain air bladder collagen eggIn vain, the fresh ox bones of 1~2kg are taken, bulk is cut into after removing the soft tissue and marrow adhered on ox bone, and is immersed in 1~2kg massFraction is in 4% sodium hydroxide solution, and 6~8h is impregnated at 60~70 DEG C, ox bone is further taken out and is transferred to 1~2L mass fractions and be10~12h is impregnated in 20% hydrogen peroxide, take out ox bone and is cleaned 2~3 times for 1% sodium phosphate buffer with mass fraction, then is transferred toIt in air dry oven, is fitted into pulverizer and crushes after being dried at 105~110 DEG C to constant weight, cross 20 mesh sieves, obtain bovine bone powder, it willBovine bone powder is fitted into Muffle furnace, is warming up to 500~600 DEG C with 5~10 DEG C/min rates, after calcining 1~2h, is continuously heating to1350~1400 DEG C of 6~8h of calcining, then stand and be cooled to 1000~1100 DEG C of 1~2h of calcining, after being cooled to room temperature, calcining is producedObject is transferred in ball mill, with 300~400r/min rotating speed 30~40min of ball milling, is crossed 200 mesh sieves, is obtained biphasic calcium phosphate powder, take 1~2g pearl proteins, 4~8g air bladder collagens are added in 500~1000mL deionized waters, are stirred with 300~400r/min20~30min adds 50~100g biphasic calcium phosphate powder, and microwave heating is to 90~100 DEG C after mixing, and is evaporated to creamShape, then paste is fitted into mold, cure 20~30h at 110~120 DEG C, nano bone repair material is obtained after the demoulding.
Example 1
Take 300g pearls, natural wind dry doubling, which is fitted into grinder, after washes clean grinds, and crosses 400 mesh sieves, after 100g is taken to sievePearl powder adds in 10kg mass fractions as in 1% edta solution, 1h, cooling are stirred with 300r/min at 40 DEG CIt being fitted into after to room temperature in centrifuge, 10min is centrifuged with 6000r/min, removal precipitation is placed in vacuum freezing drying oven,Dry 20h, obtains pearl protein at -30 DEG C, takes the fresh catfish fish glues of 300g, removes air bladder medium vessels and mucous membrane, and uses deionizationWater washing air bladder 3 times drains the air bladder after cleaning, and it is in 5% sodium carbonate liquor, with 300r/ to be immersed in 3kg mass fractionsMin stirs 8h, takes out air bladder and being washed with deionized to cleaning solution and is in neutrality, is fitted into pulverizer and crushes after natural air drying,60 mesh sieves are crossed, obtain pretreatment air bladder, 100g pretreatment air bladders is taken to add in 1L deionized waters, are stirred at 50 DEG C with 300r/min2h is extracted, is fitted into after being cooled to room temperature in centrifuge, 10min is centrifuged with 6000r/min, it is cold that removal precipitation is placed on vacuumFreeze in drying box, dry 20h, obtains air bladder collagen at -30 DEG C, takes the fresh ox bones of 1kg, removes soft group adhered on ox boneIt knits and bulk is cut into after marrow, and be immersed in 1kg mass fractions in 4% sodium hydroxide solution, 6h to be impregnated at 60 DEG C, then is takenGo out ox bone and be transferred to 1L mass fractions to impregnate 10h in 20% hydrogen peroxide, take out ox bone and be 1% sodium phosphate buffer with mass fractionLiquid cleans 2 times, then is transferred in air dry oven, is fitted into pulverizer and crushes after being dried at 105 DEG C to constant weight, crosses 20 mesh sieves,Bovine bone powder is obtained, bovine bone powder is fitted into Muffle furnace, 500 DEG C is warming up to 5 DEG C/min rates, after calcining 1h, is continuously heating to1350 DEG C of calcining 6h, then stand and be cooled to 1000 DEG C of calcining 1h, after being cooled to room temperature, calcined product is transferred in ball mill, with300r/min rotating speed ball milling 30min cross 200 mesh sieves, obtain biphasic calcium phosphate powder, take 1g pearl proteins, 4g air bladder collagens addEnter in 500mL deionized waters, 20min is stirred with 300r/min, adds 50g biphasic calcium phosphate powder, microwave adds after mixingHeat is evaporated to paste to 90 DEG C, then paste is fitted into mold, cures 20h at 110 DEG C, and nano bone is obtained after the demoulding and is repaiiedMultiple material.
Example 2
Take 400g pearls, natural wind dry doubling, which is fitted into grinder, after washes clean grinds, and crosses 400 mesh sieves, after 150g is taken to sievePearl powder adds in 15kg mass fractions as in 1% edta solution, 1h, cooling are stirred with 350r/min at 45 DEG CIt being fitted into after to room temperature in centrifuge, 12min is centrifuged with 7000r/min, removal precipitation is placed in vacuum freezing drying oven,Dry 22h, obtains pearl protein at -25 DEG C, takes the fresh catfish fish glues of 400g, removes air bladder medium vessels and mucous membrane, and uses deionizationWater washing air bladder 4 times drains the air bladder after cleaning, and it is in 5% sodium carbonate liquor, with 350r/ to be immersed in 4kg mass fractionsMin stirs 9h, takes out air bladder and being washed with deionized to cleaning solution and is in neutrality, is fitted into pulverizer and crushes after natural air drying,60 mesh sieves are crossed, obtain pretreatment air bladder, 150g pretreatment air bladders is taken to add in 1L deionized waters, are stirred at 55 DEG C with 350r/min2h is extracted, is fitted into after being cooled to room temperature in centrifuge, 12min is centrifuged with 7000r/min, it is cold that removal precipitation is placed on vacuumFreeze in drying box, dry 22h, obtains air bladder collagen at -25 DEG C, takes the fresh ox bones of 2kg, removes soft group adhered on ox boneIt knits and bulk is cut into after marrow, and be immersed in 2kg mass fractions in 4% sodium hydroxide solution, 7h to be impregnated at 65 DEG C, then is takenGo out ox bone and be transferred to 2L mass fractions to impregnate 11h in 20% hydrogen peroxide, take out ox bone and be 1% sodium phosphate buffer with mass fractionLiquid cleans 2 times, then is transferred in air dry oven, is fitted into pulverizer and crushes after being dried at 107 DEG C to constant weight, crosses 20 mesh sieves,Bovine bone powder is obtained, bovine bone powder is fitted into Muffle furnace, 550 DEG C is warming up to 7 DEG C/min rates, after calcining 1h, is continuously heating to1370 DEG C of calcining 7h, then stand and be cooled to 1050 DEG C of calcining 1h, after being cooled to room temperature, calcined product is transferred in ball mill, with350r/min rotating speed ball milling 35min cross 200 mesh sieves, obtain biphasic calcium phosphate powder, take 1g pearl proteins, 6g air bladder collagens addEnter in 750mL deionized waters, 25min is stirred with 350r/min, adds 75g biphasic calcium phosphate powder, microwave adds after mixingHeat is evaporated to paste to 95 DEG C, then paste is fitted into mold, cures 25h at 115 DEG C, and nano bone is obtained after the demoulding and is repaiiedMultiple material.
Example 3
Take 500g pearls, natural wind dry doubling, which is fitted into grinder, after washes clean grinds, and crosses 400 mesh sieves, after 200g is taken to sievePearl powder adds in 20kg mass fractions as in 1% edta solution, 2h, cooling are stirred with 400r/min at 50 DEG CIt being fitted into after to room temperature in centrifuge, 15min is centrifuged with 8000r/min, removal precipitation is placed in vacuum freezing drying oven,It is dry at -20 DEG C to obtain pearl protein for 24 hours, the fresh catfish fish glues of 500g are taken, air bladder medium vessels and mucous membrane is removed, and uses deionizationWater washing air bladder 5 times drains the air bladder after cleaning, and it is in 5% sodium carbonate liquor, with 400r/ to be immersed in 5kg mass fractionsMin stirs 10h, takes out air bladder and being washed with deionized to cleaning solution and is in neutrality, is fitted into pulverizer and crushes after natural air drying,60 mesh sieves are crossed, obtain pretreatment air bladder, 200g pretreatment air bladders is taken to add in 2L deionized waters, are stirred at 60 DEG C with 400r/min3h is extracted, is fitted into after being cooled to room temperature in centrifuge, 15min is centrifuged with 8000r/min, it is cold that removal precipitation is placed on vacuumFreeze in drying box, it is dry at -20 DEG C to obtain air bladder collagen for 24 hours, the fresh ox bones of 2kg are taken, remove soft group adhered on ox boneIt knits and bulk is cut into after marrow, and be immersed in 2kg mass fractions in 4% sodium hydroxide solution, 8h to be impregnated at 70 DEG C, then is takenGo out ox bone and be transferred to 2L mass fractions to impregnate 12h in 20% hydrogen peroxide, take out ox bone and be 1% sodium phosphate buffer with mass fractionLiquid cleans 3 times, then is transferred in air dry oven, is fitted into pulverizer and crushes after being dried at 110 DEG C to constant weight, crosses 20 mesh sieves,Bovine bone powder is obtained, bovine bone powder is fitted into Muffle furnace, 600 DEG C is warming up to 10 DEG C/min rates, after calcining 2h, is continuously heating to1400 DEG C of calcining 8h, then stand and be cooled to 1100 DEG C of calcining 2h, after being cooled to room temperature, calcined product is transferred in ball mill, with400r/min rotating speed ball milling 40min cross 200 mesh sieves, obtain biphasic calcium phosphate powder, take 2g pearl proteins, 8g air bladder collagens addEnter in 1000mL deionized waters, 30min is stirred with 400r/min, adds 100g biphasic calcium phosphate powder, after mixing microwave100 DEG C are heated to, and is evaporated to paste, then paste is fitted into mold, cures 30h at 120 DEG C, nano bone is obtained after the demouldingRepair materials.
Nano bone repair material prepared by the present invention and the nano bone repair material of Beijing company production are detected,Specific testing result such as following table table 1：
1 nano bone repair material performance characterization of table
The nano bone repair material that as shown in Table 1 prepared by the present invention, compression strength and elasticity modulus are high, possess excellent degradabilityCan, nontoxic to body, wound healing time is short.