CN117205368A - Preparation method and material for improving bone formation effect of decellularized bone material - Google Patents
Preparation method and material for improving bone formation effect of decellularized bone material Download PDFInfo
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Abstract
The invention provides a preparation method and a material for improving the osteogenesis effect of a decellularized bone material, belonging to the field of medical materials, comprising the following steps: s1, soaking in-vitro bones with a certain size by adopting a surfactant, and cleaning after finishing; s2, soaking the product of the S1 by adopting lipase and a surfactant, and cleaning after finishing; s3, soaking the product in the step S2 by adopting an alkaline solution, and cleaning after finishing the soaking; s4, neutralizing, cleaning, freeze-drying and sterilizing the product of the S3 to obtain the acellular bone material; at least one soaking treatment of the steps S1-S3 is performed in a negative pressure environment, and the cleaning after the at least one soaking treatment is finished comprises: and (3) carrying out centrifugal dehydration on the corresponding product, and repeating water injection and centrifugal dehydration at least once after the centrifugal dehydration is finished. The invention reduces the intensity of enzymolysis process and alkali liquor treatment by introducing negative pressure and centrifugal dehydration, and improves the osteogenic effect of the decellularized bone material.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a preparation method and a material for improving an osteogenic effect of a decellularized bone material.
Background
An ideal bone graft substitute should have the following conditions: (1) Good biocompatibility is beneficial to adhesion, proliferation, bone conduction and bone induction activity of seed cells; (2) Biodegradable, and the degradation speed is coordinated with the generation of new bones; (3) The three-dimensional porous reticular structure can provide certain supporting strength and provide channels and spaces for bone conduction and vascularization; (4) The proper physical, chemical and biological characteristics of the surface can further regulate and control cell adhesion, proliferation and differentiation, improve the immune environment and promote bone formation.
The heterogeneous decellularized bone material is obtained from natural bone tissue, and a final decellularized product is prepared by a series of chemical reagents, wherein the components mainly comprise hydroxyapatite and collagen, and the heterogeneous decellularized bone material has the effects of biodegradation and tissue regeneration induction after being implanted into a body and is an excellent bone grafting substitute. The Chinese patent publication No. CN104174066B discloses a natural biological bone material and a preparation method thereof, and the biological bone material belongs to heterogeneous decellularized bone material.
As a decellularized product, continuous optimization of a decellularized process is one of ways to obtain improvement of biocompatibility, the decellularized process is generally realized through acid-base treatment, surfactant, enzymatic digestion and other procedures, and the procedures often damage degradation resistance of the product under the condition of obtaining improvement of the decellularized process, so that the product does not meet ideal bone grafting substitutes; therefore, the search for optimal process conditions to continuously optimize the decellularization process without damaging the degradation resistance of the product is the main direction of the research on heterogeneous decellularized bone material process.
Disclosure of Invention
In order to solve the problems, the invention provides a preparation method and a material for improving the osteogenesis effect of a decellularized bone material, which are characterized in that the intensity of enzymolysis and alkali solution treatment can be reduced and the osteogenesis effect of the decellularized bone material can be improved by adding negative pressure and centrifugal dehydration procedures in the decellularized process and combining lipase enzymolysis and alkali solution treatment.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in one aspect, the present invention provides a method for improving the osteogenic effect of a decellularized bone material, comprising: s1, soaking in-vitro bones with a certain size by adopting a surfactant, and cleaning after finishing; s2, soaking the product of the S1 by adopting lipase and a surfactant, and cleaning after finishing; s3, soaking the product in the step S2 by adopting an alkaline solution, and cleaning after finishing the soaking; s4, neutralizing, cleaning, freeze-drying and sterilizing the product of the S3 to obtain the acellular bone material; at least one soaking treatment of the steps S1-S3 is performed in a negative pressure environment, and the cleaning after the at least one soaking treatment is finished comprises: and (3) carrying out centrifugal dehydration on the corresponding product, and repeating water injection and centrifugal dehydration at least once after the centrifugal dehydration is finished.
Further, the method also comprises the step of surface modification of the product after S4 cleaning and before freeze drying, wherein the surface modification comprises the following steps: s5, treating the cleaned product by adopting an acid solution in a negative pressure environment, removing hydroxyapatite on the surface of the product, then centrifugally dehydrating, and repeatedly injecting water and centrifugally dehydrating at least once after the completion of the centrifugal dehydration; s6, performing freeze drying treatment on the product of the step S5, and then performing thermal crosslinking and low-temperature plasma treatment.
Further, the method also comprises mineralizing treatment of the product after surface modification, and after the mineralizing treatment is finished, centrifugally dewatering, and repeating the mineralizing treatment and the centrifugally dewatering at least once; washing the product after centrifugal dehydration with distilled water.
Further, the negative pressure of the negative pressure environment is controlled to be 0.1-10pa.
Further, the centrifugal dehydration process comprises the following steps: the rotating speed is 500-5000r/min, and the centrifugal time is 1-10min.
Further, in the step S1, the surfactant is one or more of triton-100, tween-80 or tween-100, the mass concentration is 0.1% -0.4%, the treatment time is 1-4h, and the treatment temperature is 10-30 ℃; in the step S1, a surfactant is adopted to soak the isolated bones with certain size, and weak base is also added, wherein the concentration is 0.1-1mol/L.
Further, the surfactant in the step S2 is one or more of triton-100, tween-80 or tween-100, and the mass concentration is 0.01% -0.4%; the lipase is alkaline lipase, and the enzyme activity concentration is 5-20U/ml; the solvent is PBS buffer solution, the pH is 8-10, the treatment time is 0.5-24h, and the treatment temperature is 10-30 ℃.
Further, the alkaline solution in step S3 is: one or more of NaOH, ca (OH) 2 or KOH, the concentration is 0.1-1mol/L, the treatment temperature is 4-30 ℃, and the treatment time is 10-20min.
Further, the neutralization process comprises the following steps: the buffer solution is disodium hydrogen phosphate-potassium dihydrogen phosphate aqueous solution, the concentration is 0.01-0.5mol/L, the pH is 4.1-5.9, the treatment time is 10-60min, and the treatment temperature is 4-30 ℃.
In another aspect, the invention provides a bone material prepared by the preparation method.
The technical scheme provided by the embodiment of the invention has the beneficial effects that:
the process of negative pressure and centrifugal dehydration is adopted in the decellularization process, so that the intensity of enzymolysis or alkali liquor treatment is reduced, the cell extraction efficiency is improved, the damage degree of the decellularized bone material is reduced, and the bone formation effect of the decellularized bone material is improved. Specifically, in the prior art, in order to improve the cell extraction efficiency, high-concentration enzyme, alkali liquor and higher temperature are adopted to treat the bone material, but the damage degree of the high-concentration enzyme, alkali liquor and higher temperature to the acellular bone material is increased, so that the osteogenic effect of the acellular bone material is poor, and the enzyme degradation resistance is reduced. The cellular components of bone tissue are generally distributed in the bone pits, and the bone pits are mainly connected through bone tubules, so that nuclear substances are difficult to remove in the prior art, and a treatment liquid with higher solubility or higher temperature can be adopted, so that the damage degree to the decellularized bone material is increased.
Secondly, the invention adopts the following process: the process is not simple superposition, the process comprises the steps of firstly treating the isolated bone by adopting the surfactant, destroying the cell membrane to a certain extent, but still having biological macromolecular structures such as protein, fat and the like after destroying the cell membrane, and secondly decomposing the fat into substances such as fatty acid, glycerol and the like by adopting the lipase, so that the size of the molecules is reduced, and finally discharging the decomposed products under the action of the alkali liquor. The enzymolysis and alkali liquor treatment are creatively combined, so that the treatment intensity of enzymolysis or alkali liquor treatment is reduced to a certain extent, and the damage to bone materials is reduced.
Again, in the invention, at least one soaking treatment in S1-S3 is performed in a negative pressure environment, which can play the following two roles: (1) The cell components of the bone tissue are generally distributed in the bone pit, while the bone tubule has the characteristics of narrow and non-permeability, and the introduction of the negative pressure can force the solution to enter the narrow bone tubule, so that the solution can fully act on the cell components, and the cell removal efficiency is improved; (2) The breaking of the cell components is the process of decomposing the organic matters, the decomposition of the organic matters can generate a certain amount of carbon dioxide and other gas substances, and the gas in the product can be rapidly pumped out to expand the channels in the bones through the introduction of the negative pressure effect, so that the effect of cell separation can be improved, the process strength is reduced, the damage to the decellularized bone material is reduced, and the osteogenic effect is improved.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic illustration of a batch dehydration process provided by the present invention;
FIG. 2 is a graph of HE staining of sections of decellularized bone material prepared in examples and comparative examples of the invention, a-b: examples 1, c-d: examples 2, e-f: example 3, g-h: examples 4, i-j: comparative example 1, k-l: comparative example 2, m-n: comparative example 3;
FIG. 3 shows the results of the enzymolysis resistance test of the decellularized bone material prepared in the examples and comparative examples of the present invention;
FIG. 4 shows cytotoxicity test results of the decellularized bone material 24h and 48h products prepared in examples 1-2,6-7 of the present invention;
FIG. 5 shows the surface potential test results of the decellularized bone material prepared in examples 1-2 and 6-7 of the present invention;
FIG. 6 is a microstructure of examples 6-7 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and the embodiments, in order to make the objects, technical solutions and advantages of the present invention more apparent.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The overall thought is as follows:
in a first aspect, the present invention provides a method of improving the osteogenic effect of a decellularized bone material comprising:
s1, soaking the isolated bones with a certain size by adopting a surfactant, and cleaning after finishing.
Animal bones are selected as raw materials, and can be cortical bones or cancellous bones of mammals such as cattle, horses, pigs, and the like, or bones of birds such as fish, birds, and the like and marine organisms, and are cut into a specification size, preferably into a length of 0.1-3cm, by using a cutter, and cartilage and redundant muscle tissues are removed.
Preferably, fresh animal bones are adopted, pig bones which are just slaughtered are collected from slaughterhouses which are subjected to standardized management, contact with pollutants is avoided as much as possible, frozen and stored immediately after collection, frozen materials are thawed and then fully cleaned, then the bones are cut into 0.1-3cm long, cancellous bones or cortical bones are cut, and surface attachments are washed and removed.
The surfactant in the step S1 is one or more of triton-100, tween-80 or tween-100, the mass concentration is 0.1-0.4wt%, the treatment time is 1-4h, and the treatment temperature is 10-30 ℃; in the step S1, a surfactant is adopted to soak the isolated bones with certain size, and weak base is also added, wherein the concentration is 0.1-1mol/L. The weak base can be Na 2 CO 3 、NaHCO 3 、K 2 CO 3 、KHCO 3 The weak base can expand collagen, which is beneficial to the exchange of surfactant solution and improves the fat removal efficiency.
S2, soaking the product of the S1 by adopting lipase and a surfactant, and cleaning after the soaking is finished.
The surfactant in the step S2 is one or more of triton-100, tween-80 and tween-100, and the mass concentration is 0.01-0.4wt%; the lipase is alkaline lipase, and the enzyme activity concentration is 5-20U/ml; the solvent is PBS buffer solution, the pH is 8-10, the treatment time is 0.5-24h, and the treatment temperature is 10-30 ℃. Wherein the PBS buffer solution has pH of 8-10 by adjusting the mass ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate. It is common knowledge in the art to obtain solutions of different PH values by adjusting the content of dipotassium hydrogen phosphate and potassium dihydrogen phosphate. By this step further fat and foreign proteins in the sample are removed, wherein the lipase acts as an enzymolysis and softening agent, the surfactant helps to remove cells in the sample, and the solvent is an alkaline buffer, in order to increase the lipase activity.
Lipase was purchased from DENYPHULP PAP-6P, a biological technology Co., ltd.
And S3, soaking the product in the step S2 by adopting an alkaline solution, and cleaning after the soaking is finished.
The alkaline solution in step S3 is:NaOH、Ca(OH) 2 or one or more of KOH, the concentration is 0.1-1mol/L, the treatment temperature is 4-30 ℃, and the treatment time is 10-20min. According to the invention, non-collagen protein is destroyed and dissolved out by an alkaline solution and degreasing is carried out, and the negative pressure and centrifugal dehydration are added in the cell removing process, so that the enzymolysis process and the alkali liquor treatment intensity can be greatly reduced by combining the enzymolysis of lipase and the alkali liquor treatment, and the cell separation can be effectively carried out within 10-20min in 0.1-1mol/L, so that compared with the prior art, the damage of lipase and alkali liquor to bone materials is greatly reduced.
S4, neutralizing, cleaning, freeze-drying and sterilizing the product of the S3 to obtain the acellular bone material.
The neutralization process comprises the following steps: the buffer solution is disodium hydrogen phosphate-potassium dihydrogen phosphate aqueous solution, the concentration is 0.01-0.5mol/L, the pH is 4.1-5.9, the treatment time is 10-60min, and the treatment temperature is 4-30 ℃. The pH of the product is quickly neutralized, and the damage of alkali liquor to the product is reduced. After the treatment, the mixture was washed with a large amount of water.
The freeze drying treatment process comprises the following steps: maintained at-20℃for 8-20h, and then the ice is converted to a vapor under a higher vacuum. The sterilization adopts cobalt-60 radiation sterilization (the irradiation dose is 25 KGy).
At least one soaking treatment of the steps S1-S3 is performed in a negative pressure environment, and the cleaning after the at least one soaking treatment is finished comprises: and (3) carrying out centrifugal dehydration on the corresponding product, and repeating water injection and centrifugal dehydration at least once after the centrifugal dehydration is finished. In the invention, the repeated water injection and the centrifugal dehydration are preferably carried out for 2-4 times.
Preferably, the invention is immersed in the negative pressure environment in the steps S1-S3, so that the cells can be removed to the greatest extent, and centrifugal dehydration is adopted in the cleaning process, so that the cells in the bone pit are removed, and in order to discharge the foreign matters in the bone as much as possible, water injection is repeated, and centrifugal dehydration is carried out at least once, preferably 1-2 times, so that the treatment time is shortened, and the cell removal efficiency is improved.
Optionally, the method further comprises surface modification of the product after the step S4 is washed and before the step S is freeze-dried, wherein the surface modification comprises the following steps:
s5, treating the cleaned product by adopting an acid solution in a negative pressure environment, removing hydroxyapatite on the surface of the product, then carrying out centrifugal dehydration, and repeating water injection and centrifugal dehydration at least once after the completion of the centrifugal dehydration.
The acidic solution can be strong acid or weak acid, the strong acid is one or more of nitric acid, sulfuric acid, hydrochloric acid and the like, the weak acid is one or more of acetic acid, phosphoric acid and the like, the concentration is 0.01-2mol/L, the treatment temperature is 4-30 ℃, and the treatment time is 0.5-24h. The step is to treat the hydroxyapatite on the surface of the material and thoroughly expose the collagen scaffold tissue.
The negative pressure of the negative pressure environment is controlled to be 0.1-10pa, and the centrifugal dehydration process comprises the following steps: the rotating speed is 500-5000r/min, and the centrifugal time is 1-10min.
S6, performing freeze drying treatment on the product of the step S5, and then performing thermal crosslinking and low-temperature plasma treatment.
The freeze-drying treatment process in the step S6 is as follows: maintained at-20℃for 8-20h, and then the ice is converted to a vapor under a higher vacuum.
The heat crosslinking treatment temperature is 60-120 ℃, and the treatment time is 2-20h; the treatment process aims to improve the degradation performance of the acellular bone material and enhance the hydrophilicity of the surface of the material.
The low-temperature plasma treatment adopts a normal pressure plasma processor of Shenzhen Chengpeak intelligent manufacture, the gas flow is 20ml/min, the treatment time is 0.5-10min respectively, the treatment atmosphere is nitrogen, and the temperature is 40-70 ℃. The aim of the step is to change the charge of the surface collagen, so that the surface of the product is enriched with negative charge, and the adhesion and differentiation of osteoblasts are facilitated after the product is implanted into a body.
Optionally, the method further comprises mineralizing treatment of the product after surface modification, centrifuging and dehydrating after the mineralizing treatment is finished, and repeating the mineralizing treatment and centrifuging and dehydrating at least once after the mineralizing treatment is finished.
The mineralization treatment process comprises the following steps: placing the surface modified acellular bone material in 3-10mM CaCl 2 Stirring the solution to disperse the solution uniformly, standing the solution for 0.5 to 2 hours, and adding K into the solution 2 HPO 4 Controlling Ca/P to be 1.67, stirring uniformly, and regulating pH to be 1 by NaOH7 to 9, and standing for 0.5 to 2 hours.
Washing the product after centrifugal dehydration with distilled water.
In the invention, the negative pressure of the negative pressure environment is controlled to be 0.1-10pa, and the negative pressure needs to be controlled within a certain range, and if the negative pressure is too low, the channels in bones are easily damaged; if the negative pressure is too high, effective expansion of the channels in the bone cannot be ensured, so that the solution is difficult to enter the bone tubules or carbon dioxide gas is difficult to discharge.
The centrifugal dehydration process comprises the following steps: the rotational speed is 500-5000r/min, the centrifugation time is 1-10min, the nuclear material is removed from the bone pit through the centrifugation process, and the parameters of the centrifugal dehydration process need to be within the above range, so that the nuclear material can be effectively separated. If the rotating speed is too high or the centrifugal time is too long, the channels in the bones are deformed, so that the openings of the channels are closed under the action of centrifugal force, the liquid in the channels cannot be discharged, and the separation of nuclear substances is affected; if the rotational speed is too low or the centrifugation time is short, the liquid in the channel cannot be discharged against the surface tension of the liquid.
In order to further improve the removal of nuclear material by centrifugal dehydration, the centrifugal dehydration process adopts an intermittent dehydration process, and is specifically divided into at least three stages, as shown in FIG. 1, three-stage dehydration is performed, and the transverse axis T is 0 For the centrifugation time, the value range is 1-10min, wherein T 1 To T 2 Constitutes a first stage of dehydration, T 3 To T 4 Stage T constitutes the second stage of dehydration 5 To T 0 Constitutes the third stage of dehydration, the vertical axis V is the rotation speed of the centrifugal machine, V 1 To set the rotation speed, the value range is 500-5000r/min, V 0 For a preset rotation speed threshold, after the centrifugal machine is powered down, the centrifugal machine slowly decelerates under the action of inertia, and when the rotation speed is low to V 0 When the centrifugal machine is electrified and the speed is increased to the set rotating speed, wherein V 0 The value of (2) is greater than 0.5V 1 But not more than 500r/min. Through the arrangement, the scouring action on the nuclear substances in the centrifugal dehydration process is ensured, and the removal efficiency of the nuclear substances is improved.
In order to further improve the cell removal efficiency, the invention adopts a negative pressure environment in the steps S1-S3, and centrifugal dehydration is adopted after the soaking treatment is completed.
The invention also provides a bone material prepared by the preparation method.
In the invention, the determination of the properties of the decellularized bone material product comprises the following steps:
(1) And (3) cell removal detection: and (3) placing the prepared sample into a decalcification agent for treatment, fixing the decalcified sample with 14% paraformaldehyde phosphate buffer solution, embedding the sample in conventional paraffin, slicing, performing HE (high-speed) staining, and observing the cell removal effect by using a light microscope.
(2) In vitro enzymatic degradation experiment of the product: 1g of the prepared sample was taken, incubated in 1mg/ml collagenase solution for 7d, and after the end, the resulting sample was again freeze-dried and weighed.
(3) Detection of cytotoxicity of the product: the prepared samples were soaked in DMEM complete medium (0.1 g/ml) at 37 ℃ for 72 hours. L929 cells were digested with trypsin. After uniform dispersion, cells were seeded in 96-well plates at a density of 5000 cells. After 24 hours of cultivation, the old medium was replaced with the above-mentioned extraction medium, and after 24 hours or 48 hours of cultivation, the cell viability was measured by CCK-8 kit method.
(4) Solid surface potential test: the prepared samples were adhered to a test coupon and then placed in a solid surface film potentiometer for data measurement at ph=7.2.
(5) And (3) surface morphology observation: after the prepared sample surface is subjected to metal spraying treatment, SEM observation is carried out
In order to better illustrate the embodiments of the present invention, the present invention will be described in further detail by way of specific examples.
Example 1
The embodiment provides a preparation method for improving the osteogenic effect of a decellularized bone material, which comprises the following steps:
s1, cutting an in-vitro bone into 1cm long, and adopting 0.1mol/L Na at 10 DEG C 2 CO 3 Soaking the cut in-vitro bones in 0.1wt% Tween-80 solution for 1h, and placing the soaked bones in a negative pressure environment, wherein the negative pressure is controlled at 0.1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 3000r/min, the centrifugation time is 2min, and the process is repeated once again after the completion.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 0.5h, wherein the lipase activity concentration in the solution is 5U/ml, the triton-100 concentration is 0.01wt%, the pH of the solution is 8.0, the treatment temperature is 10 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled to be 0.1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 3000r/min, the centrifugation time is 2min, and the process is repeated once again after the completion.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 0.1mol/L, the treatment time is 10min, the treatment temperature is 4 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 0.1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 3000r/min, the centrifugation time is 2min, and the process is repeated once again after the completion.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH value of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 5.0, the concentration is 0.4mol/L, the temperature is 20 ℃, and the treatment time is 60min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
Example 2
The embodiment provides a preparation method for improving the osteogenic effect of a decellularized bone material, which comprises the following steps:
s1, cutting an in-vitro skeleton into 1cm long, and adopting 0.5mol/L Na at 20 DEG C 2 CO 3 Soaking the cut in-vitro bones in 0.2wt% Tween-80 solution for 2h, and placing the soaked bones in a negative pressure environment, wherein the negative pressure is controlled at 1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 5000r/min, and the centrifugal time is 1min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 5000r/min, the centrifugation time is 1min, and the process is repeated three times after the completion.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 3 hours, wherein the lipase activity concentration in the solution is 10U/ml, the triton-100 concentration is 0.1wt%, the solution pH is 9.0, the treatment temperature is 20 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled to be 1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 5000r/min, and the centrifugal time is 1min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 5000r/min, the centrifugation time is 1min, and the process is repeated three times after the completion.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 0.3mol/L, the treatment time is 12min, the treatment temperature is 15 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 1pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 5000r/min, and the centrifugal time is 1min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 5000r/min, the centrifugation time is 1min, and the process is repeated three times after the completion.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH value of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 4.1, the concentration is 0.5mol/L, the temperature is 4 ℃, and the treatment time is 10min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
Example 3
The embodiment provides a preparation method for improving the osteogenic effect of a decellularized bone material, which comprises the following steps:
s1, cutting an in-vitro bone into a length of 0.5cm, and adopting 0.8mol/L Na at 30 DEG C 2 CO 3 Soaking the cut in-vitro bones in 0.3wt% Tween-80 solution for 4 hours, and placing the soaked bones in a negative pressure environment, wherein the negative pressure is controlled at 5pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated once again after the completion.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 10 hours, wherein the lipase activity concentration in the solution is 10U/ml, the triton-100 concentration is 0.3wt%, the solution pH is 9.0, the treatment temperature is 30 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 5pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated once again after the completion.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 0.5mol/L, the treatment time is 18min, the treatment temperature is 30 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 5pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated three times after the process is finished.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 5.9, the concentration is 0.01mol/L, the temperature is 30 ℃, and the treatment time is 60min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
Example 4
The embodiment provides a preparation method for improving the osteogenic effect of a decellularized bone material, which comprises the following steps:
S1, cutting an in-vitro bone into 3cm long, and adopting 1mol/L Na at 30 DEG C 2 CO 3 Soaking the cut in-vitro bones in 0.4wt% Tween-80 solution for 4 hours, and placing the soaked bones in a negative pressure environment, wherein the negative pressure is controlled at 10pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated three times after the process is finished.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 24 hours, wherein the lipase activity concentration in the solution is 20U/ml, the triton-100 concentration is 0.4wt%, the solution pH is 10, the treatment temperature is 30 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 10pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated three times after the process is finished.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 1mol/L, the treatment time is 20min, the treatment temperature is 30 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 10pa.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated four times: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated three times after the process is finished.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH value of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 5.0, the concentration is 0.4mol/L, the temperature is 20 ℃, and the treatment time is 60min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
Example 5
The embodiment provides a preparation method for improving the osteogenic effect of a decellularized bone material, which comprises the following steps:
s1, cutting an in-vitro bone into a length of 0.1cm, and adopting 1mol/L Na at 30 DEG C 2 CO 3 Soaking the cut in-vitro bones in 0.4wt% Tween-80 solution for 4 hours, and placing the soaked bones in a negative pressure environment, wherein the negative pressure is controlled at 10pa.
After the soaking treatment is finished, centrifugally dewatering, placing the product in a centrifugal machine, removing excessive water in the product in a high-speed centrifugal mode, wherein the rotation speed of the centrifugal machine is 500r/min, the centrifugal time is 1min, and the centrifugal dewatering process adopts an intermittent dewatering process, and the embodiment adopts three stages, wherein V is as follows 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated once: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, and distilled water is injected into the centrifugeAfter the water is fully absorbed, opening a centrifugal machine, performing high-speed centrifugation at 500r/min for 1min, and adopting three stages, wherein V 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 24 hours, wherein the lipase activity concentration in the solution is 20U/ml, the triton-100 concentration is 0.4wt%, the solution pH is 10, the treatment temperature is 30 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 0.1pa.
After the soaking treatment is finished, centrifugally dewatering, placing the product in a centrifugal machine, removing excessive water in the product in a high-speed centrifugal mode, wherein the rotation speed of the centrifugal machine is 500r/min, the centrifugal time is 1min, and the centrifugal dewatering process adopts an intermittent dewatering process, and the embodiment adopts three stages, wherein V is as follows 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated once: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 1min, and three stages are adopted, wherein V is as follows 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 1mol/L, the treatment time is 20min, the treatment temperature is 20 ℃, the soaking treatment is carried out in a negative pressure environment, and the negative pressure is controlled at 0.5pa.
The soaking treatment is finishedAfter centrifugal dehydration, placing the product in a centrifugal machine, removing excessive water in the product by a high-speed centrifugal mode, wherein the rotation speed of the centrifugal machine is 500r/min, the centrifugal time is 1min, the centrifugal dehydration process adopts an intermittent dehydration process, and the embodiment adopts three stages, wherein V 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated once: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 1min, and three stages are adopted, wherein V is as follows 0 The value is 260r/min, T 1 To T 2 Constitutes a first stage of dehydration, for a period of 15s, T 3 To T 4 The stage constitutes a second stage of dehydration, for a period of 15s, T 5 To T 0 A third stage of dewatering is constituted.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH value of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 5.0, the concentration is 0.4mol/L, the temperature is 20 ℃, and the treatment time is 60min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
Example 6
In comparison with example 1, this example also includes surface modification of the product after S4 washing and before freeze-drying, the surface modification includes:
s5, in a negative pressure environment of 0.5Pa, placing the cleaned product into 0.1mol/L HCl solution for treatment, removing hydroxyapatite on the surface of the product, wherein the treatment temperature is 4 ℃, the treatment time is 2 hours, then, carrying out centrifugal dehydration, placing the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal way, wherein the rotation speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, the centrifugation time is 10min, and the process is repeated once again after the completion.
S6, performing freeze drying treatment on the product of the step S5, and then performing thermal crosslinking and low-temperature plasma treatment.
The freeze-drying treatment process in the step S6 is as follows: hold at-20 ℃ for 8 hours, then convert the ice to vapor under higher vacuum. The heat crosslinking treatment temperature is 120 ℃, and the treatment time is 20 hours. Low temperature plasma process gas. The flow rate is 20ml/min, the treatment time is 2min, the treatment atmosphere is nitrogen, and the temperature is 40 ℃.
Example 7
In comparison with example 6, this example also includes mineralization and spin-drying of the surface-modified product, which are repeated at least once after the completion of the mineralization and spin-drying. This example includes four mineralization treatments and spin-drying.
The first mineralization treatment process comprises the following steps: the product of step S6 was placed in 5mM CaCl 2 Stirring the solution to uniformly disperse the solution, and standing for 1h; after which K is added 2 HPO 4 The Ca/P is controlled to be 1.67, the mixture is stirred uniformly, the pH is regulated to be 7.4 by NaOH, and the mixture is kept stand for 1h. And (3) after the completion of the centrifugal dehydration, placing the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 500r/min, and the centrifugal time is 10min.
The second mineralization treatment process comprises the following steps: the product of the first mineralization treatment was placed in 10mM CaCl 2 Stirring the solution to uniformly disperse the solution, and standing for 0.5h; after which K is added 2 HPO 4 The Ca/P is controlled to be 1.67, the mixture is stirred uniformly, the pH is regulated to 9 by NaOH, and the mixture is kept stand for 0.5h. And (3) placing the product in a centrifugal machine, removing excessive water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
The third mineralization treatment process comprises the following steps: placing the product of the second mineralization treatment into 3mM CaCl2 solution, stirring to disperse uniformly, standing2h; after that, K2H is added into the mixture P O 4 The Ca/P is controlled to be 1.67, the mixture is stirred uniformly, the pH is regulated to 7 by NaOH, and the mixture is kept stand for 2 hours. And (3) placing the product in a centrifugal machine, removing excessive water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
The fourth mineralization treatment process comprises the following steps: placing the product of the third mineralization treatment into a 5mM CaCl2 solution, stirring to uniformly disperse the product, and standing for 1h; after which K is added 2 HPO 4 The Ca/P is controlled to be 1.67, the mixture is stirred uniformly, the pH is regulated to be 7.2 by NaOH, and the mixture is kept stand for 1h. And (3) placing the product in a centrifugal machine, removing excessive water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
After the end, the product was rinsed with a large amount of distilled water. The product is put into distilled water, and is cleaned by ultrasonic waves.
Comparative example 1
The present comparative example provides a preparation method for improving the osteogenic effect of a decellularized bone material, comprising:
s1, cutting an in-vitro bone into 1cm long, and adopting 0.1mol/L Na at 10 DEG C 2 CO 3 And treating the excised bones for 1h by using a 0.1wt% Tween-80 solution, and cleaning the products by using distilled water after finishing.
S2, soaking the product obtained in the step S1 in a lipase and triton-100 compound solution for 0.5h, wherein the concentration of lipase activity in the solution is 5U/ml, the concentration of triton-100 is 0.01wt%, the pH of the solution is 8.0, the treatment temperature is 10 ℃, and distilled water is adopted to clean the product after the completion of the treatment.
S3, putting the product obtained in the step S2 into alkali liquor for treatment, wherein NaOH is selected, the concentration of the solution is 0.1mol/L, the treatment time is 10min, the treatment temperature is 4 ℃, and distilled water is adopted to clean the product after the treatment is finished.
S4, carrying out neutralization treatment on the product in the step S3 by using a disodium hydrogen phosphate-monopotassium phosphate aqueous solution, wherein the pH value of the disodium hydrogen phosphate-monopotassium phosphate aqueous solution is 5.0, the concentration is 0.4mol/L, the temperature is 20 ℃, and the treatment time is 60min. And after the treatment is finished, cleaning, freeze-drying and sterilizing to obtain the acellular bone material.
The cleaning method of this comparative example adopts a conventional cleaning method such as ultrasonic cleaning, distilled water rinsing, etc., and the comparative example adopts an ultrasonic cleaning method.
Comparative example 2
Unlike comparative example 1, the present example centrifugally dewaters the corresponding product after the soaking process of steps S1, S2, S3 in comparative example 1 is completed, and water injection is repeated twice after the completion.
And after the soaking treatment is finished, centrifugally dewatering, putting the product into a centrifugal machine, removing redundant water in the product in a high-speed centrifugal mode, wherein the rotating speed of the centrifugal machine is 3000r/min, and the centrifugal time is 2min.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated twice: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 3000r/min, the centrifugation time is 2min, and the process is repeated once again after the completion.
Comparative example 3
Unlike comparative example 1, the soaking treatment of steps S1, S2, S3 in comparative example 1 was performed in a negative pressure atmosphere.
Wherein the soaking process of steps S1, S2 and S3 is under a negative pressure of 0.1 pa.
Comparative example 4
Unlike example 5, the spin-drying process in comparative example 4 is: the rotation speed of the centrifugal machine is 500r/min, the centrifugal time is 1min, and an intermittent dehydration process is not adopted.
After the centrifugal dehydration is finished, the centrifugal dehydration is repeated once: specifically, after the centrifuge is stopped, a water outlet valve of the centrifuge is closed, distilled water is injected into the centrifuge, after the centrifuge is fully full of water, the centrifuge is opened for high-speed centrifugation, the rotational speed of the centrifuge is 500r/min, and the centrifugation time is 1min.
Measurement of the properties of decellularized bone material products.
(1) Decellularization assay, table 1 shows the results of the decellularization of each example.
Table 1 results of the cell removal in the examples
As shown in fig. 2, comparative example 1, as shown in fig. 2i and 2j, does not use centrifugation and negative pressure treatment, and the product is distributed with a large amount of nuclear material residues; when the centrifugal treatment is introduced, the nuclear material components in the product are greatly reduced (comparative example 2, fig. 2k and 2 l), and the analysis of the change is caused because the cell components of the bone tissue are generally distributed in the bone pits, and the bone pits are connected through the bone tubules, so that the nuclear material can be quickly removed from the bone pits under the action of high-speed centrifugation, and the cell removal efficiency is improved. In the process of the decellularization treatment of bone tissues, the improvement of the cell removal efficiency and the reduction of the product damage degree are the balance points of process pursuit, and the cell removal efficiency can be greatly improved through the introduction of a centrifugal process.
As shown in fig. 2m and 2n, the negative pressure treatment condition is introduced in comparative example 3 based on comparative example 1, so that the cell removal efficiency is greatly improved, and the analysis reasons are mainly as follows: (1) the cell components of the bone tissue are generally distributed in the bone pit, while the bone tubule has the characteristics of narrow and non-permeability, and the introduction of the negative pressure can force the solution to enter the narrow bone tubule, so that the solution can fully act on the cell components, and the cell removal efficiency is improved; (2) the breaking of the cell components is the process of decomposing the organic matters, the decomposition of the organic matters can generate a certain amount of carbon dioxide and other gas substances, the gas substances in the product can be rapidly extracted through the introduction of the negative pressure effect, the solution exchange efficiency is improved, the decomposition process of the organic matters is accelerated, and the cell removal efficiency is improved. But small amounts of nuclear material remain.
As shown in fig. 2a-2h, the cell removal efficiency of the product is greatly improved by introducing the centrifugal action and the negative pressure action, so that the combination of the centrifugal action and the negative pressure action can prove that the decomposition of the cell tissues can be rapidly promoted, and the high-speed centrifugal action can ensure the rapid discharge of the decomposed nuclear substances, so that the cell removal effect is achieved.
As shown in fig. 2c-2h, it can be found that the material prepared by the process has good decellularization after the concentration of each solution is reduced or the treatment time is shortened.
Based on the decellularized results of example 5 and comparative example 4, decellularized bone material can be obtained in a short time using a batch dehydration process at the same dehydration time and rotation speed, but cells cannot be removed efficiently using a one-time dehydration process. Therefore, the intermittent dehydration process can greatly improve the decellularization efficiency.
(2) In vitro enzymatic degradation experiment of product
The degradation stability between the various groups of materials was further evaluated by measuring resistance to enzymatic degradation. As can be seen from fig. 3, compared with example 1, example 2 shows a similar weight loss rate, so that it can be proved that even if centrifugation is introduced and a negative pressure treatment device obtains a good cell removal effect, the material can still maintain high degradation resistance, and the mechanical superiority of the product is ensured; while examples 1 to 5 show good resistance to enzymatic degradation compared with comparative examples 1 to 2, the main reason is that the improvement of the degradation resistance of the product can be obtained by the reduction of the concentration of the solution and the shortening of the treatment period under the premise of ensuring the decellularization effect under the introduction of centrifugation and negative pressure treatment. Examples 6 to 7 show good resistance to enzymatic degradation compared with comparative examples 1 to 2, because on the one hand, improvement of degradation resistance of the product can be obtained by reduction of the concentration of the solution and shortening of the treatment time period under the premise of ensuring the decellularizing effect by introducing centrifugation and negative pressure treatment; on the other hand, the thermal crosslinking of the product brings the complexation of hydrogen bond and amino group, thereby enhancing the degradation resistance of the product.
(3) Detection of cytotoxicity of products
The samples of examples 1-2, 6-7 were subjected to product cytotoxicity assays. As shown in fig. 4, after 24 hours or 48 hours, the cell viability of the four groups of samples was high, indicating that the surface modification and in vitro mineralization treatment of the decellularized bone material did not affect the compatibility of the product, demonstrating that the four groups of materials all had good cell compatibility.
(4) Solid surface potential test
After the decellularized bone material samples prepared in examples 1-2 and 6-7 were sterilized under the same process conditions, each group of samples was adhered to a test sample stage, and then placed in a solid surface film potentiometer at ph=7.2 for data measurement. The comparison of the potential conditions between groups was further confirmed by testing the surface potential conditions. As can be seen from fig. 5, example 2 shows a similar surface potential compared to example 1, thereby proving that the introduction of centrifugation and negative pressure treatment means did not change the surface functional groups of the material; whereas examples 6-7 have lower negative charge characteristics than examples 1-2, because plasma treatment can alter the collagen surface profile.
(5) Surface topography observation
The samples of examples 6 to 7 were subjected to surface metal spraying treatment and then SEM observation was performed. By SEM observation, the surface morphology change of the mineralized product can be observed. According to fig. 6, compared with example 6, example 7 shows a rough surface and has a concave-convex structure with different sizes, so that it can be obtained that in vitro mineralization can improve the surface roughness of the product, facilitate the deposition and differentiation of cells, and effectively improve the biocompatibility of the product.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A method of preparing a bone effect improving decellularized bone material comprising:
s1, soaking in-vitro bones with a certain size by adopting a surfactant, and cleaning after finishing;
s2, soaking the product of the S1 by adopting lipase and a surfactant, and cleaning after finishing;
s3, soaking the product in the step S2 by adopting an alkaline solution, and cleaning after finishing the soaking;
s4, neutralizing, cleaning, freeze-drying and sterilizing the product of the S3 to obtain the acellular bone material;
at least one soaking treatment of the steps S1-S3 is performed in a negative pressure environment, and the cleaning after the at least one soaking treatment is finished comprises: and (3) carrying out centrifugal dehydration on the corresponding product, and repeating water injection and centrifugal dehydration at least once after the centrifugal dehydration is finished.
2. The method of claim 1, further comprising surface modification of the product after S4 washing and before freeze drying, the surface modification comprising:
S5, treating the cleaned product by adopting an acid solution in a negative pressure environment, removing hydroxyapatite on the surface of the product, then centrifugally dehydrating, and repeatedly injecting water and centrifugally dehydrating at least once after the completion of the centrifugal dehydration;
s6, performing freeze drying treatment on the product of the step S5, and then performing thermal crosslinking and low-temperature plasma treatment.
3. The method according to claim 2, further comprising mineralizing the surface-modified product, centrifuging the product after the mineralizing, and repeating the mineralizing and centrifuging at least once after the mineralizing;
washing the product after centrifugal dehydration with distilled water.
4. A method of producing according to any one of claims 1 to 3, wherein the negative pressure in the negative pressure atmosphere is controlled to be 0.1 to 10pa.
5. The method according to claim 4, wherein the centrifugal dehydration process is: the rotating speed is 500-5000r/min, and the centrifugal time is 1-10min.
6. The preparation method according to claim 5, wherein the surfactant in the step S1 is one or more of triton-100, tween-80 and tween-100, the mass concentration is 0.1-0.4wt%, the treatment time is 1-4h, and the treatment temperature is 10-30 ℃;
In the step S1, a surfactant is adopted to soak the isolated bones with certain size, and weak base is also added, wherein the concentration is 0.1-1mol/L.
7. The preparation method according to claim 4, wherein the surfactant in the step S2 is one or more of triton-100, tween-80 and tween-100, and the mass concentration is 0.01-0.4wt%;
the lipase is alkaline lipase, and the enzyme activity concentration is 5-20U/ml;
the solvent is PBS buffer solution, the pH is 8-10, the treatment time is 0.5-24h, and the treatment temperature is 10-30 ℃.
8. The method according to claim 4, wherein the alkaline solution in step S3 is: naOH, ca (OH) 2 Or one or more of KOH, the concentration is 0.1-1mol/L, the treatment temperature is 4-30 ℃, and the treatment time is 10-20min.
9. The method according to claim 4, wherein the neutralization process is as follows: the buffer solution is disodium hydrogen phosphate-potassium dihydrogen phosphate aqueous solution, the concentration is 0.01-0.5mol/L, the pH is 4.1-5.9, the treatment time is 10-60min, and the treatment temperature is 4-30 ℃.
10. A bone material prepared by the method of any one of claims 1-9.
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| US20030135284A1 (en) * | 1998-06-30 | 2003-07-17 | Katrina Crouch | Plasticized bone grafts and methods of making and using same |
| US20030087428A1 (en) * | 1999-06-07 | 2003-05-08 | Lloyd Wolfinbarger | Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants produced |
| US20100003301A1 (en) * | 2006-11-11 | 2010-01-07 | The University Of Tokyo | Bone Defect Filler, Release-Controlled Carrier, And Their Production Methods |
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