CN102133431A - Method for fixing bone morphogenetic protein-2 (BMP-2) with decalcified bone matrix - Google Patents
Method for fixing bone morphogenetic protein-2 (BMP-2) with decalcified bone matrix Download PDFInfo
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Abstract
The invention discloses a method for fixing a bone morphogenetic protein-2 (BMP-2) with a decalcified bone matrix. By using the decalcified bone matrix prepared by the metaphysis of pork hind leg bones as an object, the method comprises the following steps of: loading a BMP-2 solution at the concentration of 1 mu g per mu L to the decalcified bone matrix; placing into a microwave synthesizer, and processing for 30 minutes at the microwave power of 500 watts and reaction temperature 37 DEG C to fix the BMP-2 on the decalcified bone matrix; and putting the decalcified bone matrix in a 3 percent chitosan solution for 5 minutes, then taking the decalcified bone matrix out, and drying at the temperature of 37 DEG C. By adoption of the method provided by the invention, the fixation and combination of the BMP-2 and the decalcified bone matrix are stable, a fixing effect is good, the release speed of the BMP-2 is reduced, and the utilization ratio of the BMP-2 is improved; and the method has a higher practical application value in the field of medicine.
Description
Technical field
The present invention relates to the fixed method of a kind of bone morphogenesis protein-2 and decalcified bone matrix, belong to the processing and fabricating technical field of repairing the body bone tissue timbering material.
Background technology
Decalcified bone matrix is a bone tissue engineer bio-derived material commonly used, and it derives from osseous tissue, is a kind of xenogenesis or allogeneic sclerotin of the antigen deactivation from the body degraded.Nineteen sixty-five Urist (U.S. scientist Marshall R.Urist, the founder of orthopaedics basic research) confirms that the decalcification bone has the induced osteogenesis effect.Subsequently, the osteogenic induction activity of decalcification bone is confirmed that by great deal of experimental and clinical practice decalcification can make osteoinductive protein be exposed, and activates the vigor of osteoinductive protein, make allograph bone produce active bone inducibility, the absorption that helps implantable bone substitutes and the formation of bone newly.In addition, the decalcification bone also has excellent biological compatibility, biological activity and biological degradability, and is easy to merge with surrounding bone, supports the growth of new bone tissue.The decalcification bone has also kept natural reticulated cell slot sytem, and its structure and composition meet people's physiological requirement more.Yet because the inherent induced protein of decalcification bone is less, so the bone-inducting active of decalcification bone itself is limited.
Bone morphogenesis protein-2 (bone morphogenetic protein-2, BMP-2) be one of transforming growth factor member, have the ability of undifferentiated mescenchymal stem cell of inducing to chondroblast and osteoblast directed differentiation and propagation, promote the osteoblast differentiation maturation, participate in the growth promoter and the process of reconstruction thereof of bone and cartilage, and then the damaged reparation of accelerated bone.But the half-life of bone morphogenesis protein-2 is very short, has therefore limited the performance of its effect.Yet, and can control its release by the biological activity that can prolong bone morphogenesis protein-2 that combines of bone morphogenesis protein-2 with polymeric material.At present, compound bone morphogenesis protein-2 in timbering material has been considered to one of possible application process of organizational project.
But the adherence method of present bone morphogenesis protein-2 clinically and decalcified bone matrix is generally the method for physical fixation such as infusion process and negative-pressure adsorption method, though these methods are simple to operate, adhesion amount on decalcified bone matrix is few but these methods not only make bone morphogenesis protein-2, adhere to inhomogeneous, and adhere to instability, do not reach the burst size of control bone morphogenesis protein-2 and prolong the active purpose of morphogenesis protein-2, be unfavorable for the performance that bone morphogenesis protein-2 acts on clinical medicine.
Summary of the invention
The invention discloses-kind of bone morphogenesis protein-2 and the fixed method of decalcified bone matrix, present bone morphogenesis protein-2 and decalcified bone matrix are fixing inhomogeneously, fixing does not unstablely reach the burst size of control bone morphogenesis protein-2 and prolong problem such as morphogenesis protein-2 activity thereby its purpose is to solve.Adopt a kind of bone morphogenesis protein-2 of the present invention and the fixed method of decalcified bone matrix, not only can make uniform and stable being fixed on the decalcified bone matrix of bone morphogenesis protein-2, and can delay the release of bone morphogenesis protein-2, prolong the activity of bone morphogenesis protein-2 simultaneously, improved the utilization rate of bone morphogenesis protein-2.
A kind of bone morphogenetic protein-2 and the fixed method of decalcified bone matrix, carry out according to following steps:
(1) decalcified bone matrix with the metaphyseal spongy bone preparation of pig back bone is an object;
(2) the BMP-2 lyophilized powder is mixed with the BMP-2 solution that concentration is 1 μ g/ μ L with sterilized water;
(3) the BMP-2 solution that configures in (2) is loaded into (1) decalcified bone matrix (on 1cm * 1cm * 3mm); The amount of the BMP-2 of institute solution is 20 μ L.
(4) in the microwave synthesizer, with load in (3) decalcified bone matrix of BMP-2 handle 30min;
(5) decalcified bone matrix of having fixed BMP-2 in (4) being placed concentration is to take out behind 3% the chitosan solution 5min;
(6) with the decalcified bone matrix drying that obtains in (5), baking temperature is 37 ℃.
Described microwave treatment conditions is: microwave power is 500W, and reaction temperature is 37 ℃.
Advantage of the present invention and good effect are: adopt the present invention's a kind of bone morphogenetic protein-2 and the fixed new method of decalcified bone matrix, not only can make bone morphogenesis protein-2 be fixed on the decalcified bone matrix uniformly and stably, and can delay the release of bone morphogenesis protein-2, prolong the activity of bone morphogenesis protein-2 simultaneously, improved the utilization rate of bone morphogenesis protein-2.
Description of drawings
Figure 1B radford Protein Detection method standard curve;
Fig. 2 infusion process is to the fixed effect of BMP-2 and decalcified bone matrix;
Fig. 3 negative-pressure adsorption method is to the fixed effect of BMP-2 and decalcified bone matrix;
Fig. 4 microwave power is to the fixed effect of BMP-2 and decalcified bone matrix;
Fig. 5 microwave fixation (500W) is to the fixed effect of BMP-2 and decalcified bone matrix;
Fig. 6 microwave fixes-and the chitosan coating method is to the fixed effect of BMP-2 and decalcified bone matrix.
The specific embodiment
Below infusion process, negative-pressure adsorption method, microwave fixation and microwave are fixed-four kinds of methods of chitosan coating method; the decalcification bone supporting stand of separate sources and the fixed effect of BMP-2 are made embodiment; and in conjunction with the accompanying drawings the present invention is described in further detail; but present embodiment is not limited to the present invention; every employing similarity method of the present invention and similar variation thereof all should be listed protection scope of the present invention in.
Embodiment 1: the dipping fixation
One, the metaphyseal spongy bone of pig back bone prepares decalcified bone matrix, and concrete preparation process comprises the steps:
(1) cartilage, cortical bone and the adhering tissue on removal spongy bone surface;
(2) with the spongy bone section, the flushing that obtain in (1);
(3) spongy bone that obtains in (2) is cut into the bone piece of 1cm * 1cm * 3mm;
(4) spongy bone that obtains in (3) is handled 48 hours to remove the cell on the spongy bone with Triton X-100;
(5) spongy bone that obtains in (4) is handled 48 hours to remove the fatty oils and fats on the spongy bone with absolute ether;
(6) spongy bone that obtains in (5) is placed 0.6mol/L hydrochloric acid handled 12 hours.
(7) with the decalcification spongy bone that obtains in (6) distilled water flushing 30min, obtain decalcification bone supporting stand;
(8) decalcification bone supporting stand that obtains in (7) is placed dehydrated alcohol preserve.
Two, the dipping fixing means of bone morphogenetic protein-2 and decalcified bone matrix, concrete implementation step is as follows:
(1) decalcified bone matrix that utilizes the metaphyseal spongy bone of pig back bone to prepare;
(2) be the BMP-2 solution of 1 μ g/ μ L with the BMP-2 lyophilized powder with the concentration that sterilized water is mixed with solution;
(3) the BMP-2 solution that configures in (2) is loaded on the decalcified bone matrix of (1) preparation; (amount of the BMP-2 solution on 1cm * 1cm * 3mm) is 20 μ L to be loaded into decalcified bone matrix;
(4) with load in (3) decalcified bone matrix of BMP-2 dry down at 37 ℃.
Three, the mensuration of bone morphogenetic protein-2 and decalcified bone matrix fixed effect:
A) bone morphogenetic protein-2 is as follows with the detailed process of decalcified bone matrix fixed effect mensuration:
The decalcified bone matrix of having fixed bone morphogenetic protein-2 is put into centrifuge tube, in centrifuge tube, add the 2ml distilled water.Centrifuge tube placed to vibrate on the shaking table sway, vibration velocity is 150r/min, time of vibration is 1-5 days, detect the content that discharges bone morphogenetic protein-2 in the centrifuge tube every day, the content that dipping fixation, negative-pressure adsorption fixation and fixing means of the present invention are discharged bone morphogenetic protein-2 every day carries out determination and analysis respectively, to contrast fixed effect.
B) bone morphogenetic protein-2 Determination on content method:
Adopt Bradford Protein Detection method to measure the content of bone morphogenetic protein-2.The measuring principle of this method is based on Coomassie brilliant blue, under 95% ethanol and acid condition, can be made into light blue solution, when with protein bound after, produce blue chemical compound, this is swift in response and stablizes.Compound of reaction has maximum absorbance value at 465～595nm place, the depth of chemical compound color and the height of protein concentration are proportional, and the size of absorbance value that therefore can be by detection 595nm is calculated proteic content.Concrete grammar is as follows:
(1) preparation of Bradford working solution (seeing Table 1.1)
The preparation of the Bradford working solution of table 1.1 500ml
In proper order: after G250 is dissolved in ethanol fully, add phosphoric acid, leave standstill 30min after, add distilled water, it is standby to keep in Dark Place.
(2) preparation standard protein solution: accurate weighing 100mg bovine serum albumin, put into the volumetric flask of 100ml, adds an amount of distilled water and dissolve, accurate quantification is to 100ml again, and obtaining concentration is the standard bovine serum albumen solution of 1mg/ml.
(3) protein standard curve plotting:
Measure the bovine serum albumen solution of the 1mg/ml of 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, 60 μ l respectively with accurate liquid-transfering gun, add distilled water and Bradford working solution (accurately liquid-transfering gun is measured), measure it in the OD at 595nm place value with ultra-violet and visible spectrophotometer, each experimental point is done 3 times, get the meansigma methods of 3 OD values, concrete operations see Table 1.2.
The OD value of table 1.2 standard protein detects
With the standard protein addition is X-axis, and pairing OD average (595nm) is made curve chart for Y-axis, get final product Bradford Protein Detection method standard curve (see figure 1).
Through linear regression analysis, draw straight line, its equation is:
Y=0.0101x+0.0021 formula 1-(1)
Like this, rely on the Coomassie brilliant blue standard curve, we just can calculate the protein content of rhBMP-2 to be measured, suppose to measure solution V to be measured, record the OD value and are Y, can try to achieve the standard protein addition X on the corresponding standard curve:
X=(Y-0.0021)/0.0101 formula 1-(2)
Then can be in the hope of the concentration C of unknown rhBMP-2:
C=(X/V) * 1mg/ml formula 1-(3)
(4) according to the absorbance of institute's test sample product, on standard curve, find the corresponding proteins quality, thereby calculate the content of bone morphogenetic protein-2
As shown in Figure 2, prolong with duration of oscillation, the burst size of the BMP-2 on the timbering material of each preparation method all increases gradually, and preparation method is to the burst size of BMP-2 on the material do not make significant difference (P＞0.05).The theoretical release concentration of BMP-2 is 10 μ g/ml on the timbering material, test data shows, vibrate the 1st day the time, after the application infusion process is fixing, the average burst size of BMP-2 on three kinds of timbering materials is just up to (4.75 ± 0.21) μ g/ml, the BMP-2 that existing (47.5 ± 2.1) % is described discharges from decalcified bone matrix, and vibrating, the rate of release of BMP-2 slows down relatively in the 2nd to 5 day.Use Excel2003 BMP-2 burst size on three kinds of timbering materials carried out match with the data that the vibration natural law changes, show that the burst size of BMP-2 meets following non-linear formula with vibration natural law Changing Pattern:
m＝-0.1509t
2+1.7204t+3.1139，R
2＝0.9924，
M:BMP-2 burst size (μ g/ml); T: duration of oscillation (d).
Use and show after this formula calculates: vibrate the 6th day the time, the burst size of BMP-2 is near balance, and the content of BMP-2 is 8.0 μ g/ml in this moment solution, illustrates that 80% BMP-2 discharges.This be because, after infusion process is fixing, just fixed between BMP-2 and the decalcified bone matrix by the hydrophobic interaction power on decalcified bone matrix and BMP-2 surface, and this adhesion relatively a little less than, make BMP-2 under the effect of vibration, can discharge in a large number from decalcified bone matrix fast in the short time, thus relatively poor to the fixed effect of BMP-2 and follow-up slow releasing function.
Embodiment 2: the negative-pressure adsorption method
One, the metaphyseal spongy bone of pig back bone prepares decalcified bone matrix, and concrete preparation process comprises the steps:
(1) cartilage, cortical bone and the adhering tissue on removal spongy bone surface;
(2) with the spongy bone section, the flushing that obtain in (1);
(3) spongy bone that obtains in (2) is cut into the bone piece of 1cm * 1cm * 3mm;
(4) spongy bone that obtains in (3) is handled 48 hours to remove the cell on the spongy bone with Triton X-100;
(5) spongy bone that obtains in (4) is handled 48 hours to remove the fatty oils and fats on the spongy bone with absolute ether;
(6) spongy bone that obtains in (5) is placed 0.6mol/L hydrochloric acid handled 12 hours.
(7) with the decalcification spongy bone that obtains in (6) distilled water flushing 30min, obtain decalcification bone supporting stand;
(8) decalcification bone supporting stand that obtains in (7) is placed dehydrated alcohol preserve.
Two, the negative-pressure adsorption fixing means of bone morphogenetic protein-2 and decalcified bone matrix, concrete implementation step is as follows:
(1) decalcified bone matrix that utilizes the metaphyseal spongy bone of pig back bone to prepare;
(2) be the BMP-2 solution of 1 μ g/ μ L with the BMP-2 lyophilized powder with the concentration that sterilized water is mixed with solution;
(3) the BMP-2 solution that configures in (2) is loaded on the decalcified bone matrix of (1) microwave preparation; (amount of the BMP-2 solution on 1cm * 1cm * 3mm) is 20 μ L to be loaded into decalcified bone matrix.
(4) with load in (3) decalcified bone matrix of BMP-2 put in the vacuum drying oven, its vacuum is-0.1MPa, reaction temperature is 37 ℃, the response time is 30min;
(5) decalcified bone matrix drying under 37 ℃ of BMP-2 will have been fixed in (4) by the negative-pressure adsorption method.
Three, the mensuration of bone morphogenetic protein-2 and decalcified bone matrix fixed effect:
A) bone morphogenetic protein-2 is as follows with the detailed process of decalcified bone matrix fixed effect mensuration:
The decalcified bone matrix of having fixed bone morphogenetic protein-2 is put into centrifuge tube, in centrifuge tube, add the 2ml distilled water.Centrifuge tube placed to vibrate on the shaking table sway, vibration velocity is 150r/min, time of vibration is 1-5 days, detect the content that discharges bone morphogenetic protein-2 in the centrifuge tube every day, the content that dipping fixation, negative-pressure adsorption fixation and fixing means of the present invention are discharged bone morphogenetic protein-2 every day carries out determination and analysis respectively, to contrast fixed effect.
B) bone morphogenetic protein-2 Determination on content method:
Adopt Bradford Protein Detection method to measure the content of bone morphogenetic protein-2.The measuring principle of this method is based on Coomassie brilliant blue, under 95% ethanol and acid condition, can be made into light blue solution, when with protein bound after, produce blue chemical compound, this is swift in response and stablizes.Compound of reaction has maximum absorbance value at 465～595nm place, the depth of chemical compound color and the height of protein concentration are proportional, and the size of absorbance value that therefore can be by detection 595nm is calculated proteic content.Concrete grammar is as follows:
(2) preparation of Bradford working solution (seeing Table 1.1)
The preparation of the Bradford working solution of table 1.1 500ml
In proper order: after G250 is dissolved in ethanol fully, add phosphoric acid, leave standstill 30min after, add distilled water, it is standby to keep in Dark Place.
(2) preparation standard protein solution: accurate weighing 100mg bovine serum albumin, put into the volumetric flask of 100ml, adds an amount of distilled water and dissolve, accurate quantification is to 100ml again, and obtaining concentration is the standard bovine serum albumen solution of 1mg/ml.
(3) protein standard curve plotting:
Measure the bovine serum albumen solution of the 1mg/ml of 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, 60 μ l respectively with accurate liquid-transfering gun, add distilled water and Bradford working solution (accurately liquid-transfering gun is measured), measure it in the OD at 595nm place value with ultra-violet and visible spectrophotometer, each experimental point is done 3 times, get the meansigma methods of 3 OD values, concrete operations see Table 1.2.
The OD value of table 1.2 standard protein detects
With the standard protein addition is X-axis, and pairing OD average (595nm) is made curve chart for Y-axis, get final product Bradford Protein Detection method standard curve (see figure 1).
Through linear regression analysis, draw straight line, its equation is:
Y=0.0101x+0.0021 formula 1-(1)
Like this, rely on the Coomassie brilliant blue standard curve, we just can calculate the protein content of rhBMP-2 to be measured, suppose to measure solution V to be measured, record the OD value and are Y, can try to achieve the standard protein addition X on the corresponding standard curve:
X=(Y-0.0021)/0.0101 formula 1-(2)
Then can be in the hope of the concentration C of unknown rhBMP-2:
C=(X/V) * 1mg/ml formula 1-(3)
(4) according to the absorbance of institute's test sample product, on standard curve, find the corresponding proteins quality, thereby calculate the content of bone morphogenetic protein-2
As shown in Figure 3, after negative-pressure adsorption is fixing, prolong with duration of oscillation, the burst size of the BMP-2 on the timbering material of each preparation method all increases gradually, and preparation method is to the burst size of BMP-2 on the material do not make significant difference (P＞0.05).The theoretical release concentration of BMP-2 is 10 μ g/ml on the timbering material, vibrate after 1 day, in the solution on three kinds of timbering materials the average content of BMP-2 be (3.88 ± 0.19) μ g/ml, the BMP-2 that (38.8 ± 1.9) % is described discharges from decalcified bone matrix, and these data are significantly less than the burst size (P＜0.05) of the fixed BMP-2 of contemporaneity infusion process.Vibrated the 2nd day to the 5th day, the rate of release of BMP-2 slows down.Use Excel2003 the burst size of BMP-2 on three kinds of timbering materials is carried out match with the delta data of duration of oscillation, the burst size that shows BMP-2 meets following non-linear formula with the Changing Pattern of duration of oscillation:
m＝-0.1725t
2+1.778t+2.236，R
2＝0.998，
M:BMP-2 burst size (μ g/ml); T: duration of oscillation (d).
Use and show after this formula calculates: during the 5th day of vibration, the burst size of BMP-2 is near balance, the content of BMP-2 is 6.78 μ g/ml in the solution at this moment, illustrate that 67.8% BMP-2 discharges, fixed effect than infusion process significantly improves (P＜0.05), but since between BMP-2 and the decalcified bone matrix also just by hydrophobic interaction power and fixed, suction function is the gap structure that enters into decalcified bone matrix that makes BMP-2 more deep, so fixed effect is better than infusion process.But this adhesion still relatively a little less than, make BMP-2 under the effect of vibration, can discharge in a large number from decalcified bone matrix fast, thereby still can not play slow releasing function effectively BMP-2 in the short time.
Embodiment 3: the microwave fixation
One, the metaphyseal spongy bone of pig back bone prepares decalcified bone matrix, and concrete preparation process comprises the steps:
(1) cartilage, cortical bone and the adhering tissue on removal spongy bone surface;
(2) with the spongy bone section, the flushing that obtain in (1);
(3) spongy bone that obtains in (2) is cut into the bone piece of 1cm * 1cm * 3mm;
(4) spongy bone that obtains in (3) is handled 48 hours to remove the cell on the spongy bone with Triton X-100;
(5) spongy bone that obtains in (4) is handled 48 hours to remove the fatty oils and fats on the spongy bone with absolute ether;
(6) spongy bone that obtains in (5) is placed 0.6mol/L hydrochloric acid handled 12 hours.
(7) with the decalcification spongy bone that obtains in (6) distilled water flushing 30min, obtain decalcification bone supporting stand;
(8) decalcification bone supporting stand that obtains in (7) is placed dehydrated alcohol preserve.
Two, the microwave fixing means of bone morphogenetic protein-2 and decalcified bone matrix, concrete implementation step is as follows:
(1) decalcified bone matrix that utilizes the metaphyseal spongy bone of pig back bone to prepare;
(2) be the BMP-2 solution of 1 μ g/ μ L with the BMP-2 lyophilized powder with the concentration that sterilized water is mixed with solution;
(3) the BMP-2 solution that configures in (2) is loaded on the decalcified bone matrix of (1) microwave preparation; (amount of the BMP-2 solution on 1cm * 1cm * 3mm) is 20 μ L to be loaded into decalcified bone matrix.
(4) with load in (3) decalcified bone matrix of BMP-2 put in the microwave synthesizer, its power is 100-900W, reaction temperature is 37 ℃, the response time is 30min; By microwave effect BMP-2 is fixed on the decalcified bone matrix;
(5) decalcified bone matrix drying under 37 ℃ of BMP-2 will have been fixed in (4) by microwave method.
Three, the mensuration of bone morphogenetic protein-2 and decalcified bone matrix fixed effect:
A) bone morphogenetic protein-2 is as follows with the detailed process of decalcified bone matrix fixed effect mensuration:
The decalcified bone matrix of having fixed bone morphogenetic protein-2 is put into centrifuge tube, in centrifuge tube, add the 2ml distilled water.Centrifuge tube placed to vibrate on the shaking table sway, vibration velocity is 150r/min, time of vibration is 1-5 days, detect the content that discharges bone morphogenetic protein-2 in the centrifuge tube every day, the content that dipping fixation, negative-pressure adsorption fixation and fixing means of the present invention are discharged bone morphogenetic protein-2 every day carries out determination and analysis respectively, to contrast fixed effect.
B) bone morphogenetic protein-2 Determination on content method:
Adopt Bradford Protein Detection method to measure the content of bone morphogenetic protein-2.The measuring principle of this method is based on Coomassie brilliant blue, under 95% ethanol and acid condition, can be made into light blue solution, when with protein bound after, produce blue chemical compound, this is swift in response and stablizes.Compound of reaction has maximum absorbance value at 465～595nm place, the depth of chemical compound color and the height of protein concentration are proportional, and the size of absorbance value that therefore can be by detection 595nm is calculated proteic content.Concrete grammar is as follows:
(3) preparation of Bradford working solution (seeing Table 1.1)
The preparation of the Bradford working solution of table 1.1 500ml
In proper order: after G250 is dissolved in ethanol fully, add phosphoric acid, leave standstill 30min after, add distilled water, it is standby to keep in Dark Place.
(2) preparation standard protein solution: accurate weighing 100mg bovine serum albumin, put into the volumetric flask of 100ml, adds an amount of distilled water and dissolve, accurate quantification is to 100ml again, and obtaining concentration is the standard bovine serum albumen solution of 1mg/ml.
(3) protein standard curve plotting:
Measure the bovine serum albumen solution of the 1mg/ml of 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, 60 μ l respectively with accurate liquid-transfering gun, add distilled water and Bradford working solution (accurately liquid-transfering gun is measured), measure it in the OD at 595nm place value with ultra-violet and visible spectrophotometer, each experimental point is done 3 times, get the meansigma methods of 3 OD values, concrete operations see Table 1.2.
The OD value of table 1.2 standard protein detects
With the standard protein addition is X-axis, and pairing OD average (595nm) is made curve chart for Y-axis, get final product Bradford Protein Detection method standard curve (see figure 1).
Through linear regression analysis, draw straight line, its equation is:
Y=0.0101x+0.0021 formula 1-(1)
Like this, rely on the Coomassie brilliant blue standard curve, we just can calculate the protein content of rhBMP-2 to be measured, suppose to measure solution V to be measured, record the OD value and are Y, can try to achieve the standard protein addition X on the corresponding standard curve:
X=(Y-0.0021)/0.0101 formula 1-(2)
Then can be in the hope of the concentration C of unknown rhBMP-2:
C=(X/V) * 1mg/ml formula 1-(3)
(4) according to the absorbance of institute's test sample product, on standard curve, find the corresponding proteins quality, thereby calculate the content of bone morphogenetic protein-2
As shown in Figure 4, though under each constant power, the burst size of BMP-2 all increases gradually along with the prolongation of duration of oscillation.But there is appreciable impact (P＜0.05) in microwave power to the burst size of BMP-2.Suitable microwave power is the effectively fixing prerequisite of BMP-2, and when microwave frequency was too high, the intensity of microwave was excessive, and the electromagnetic wave that microwave produces just can influence the activation energy and the pre-exponential factor of reaction, thereby suppresses the process of chemical reaction.And microwave power is crossed when hanging down, and the electromagnetic energy that microwave produced is too small, and the reaction activity that provides is just less, thereby not obvious to the facilitation of chemical reaction.Result of the test shows, when microwave power was 500W, the burst size of BMP-2 every day was relatively low, illustrates that BMP-2 is relative with the combination of decalcified bone matrix firm under this condition.
As shown in Figure 5, after microwave is fixing, prolong with duration of oscillation, the burst size of the BMP-2 on the timbering material of each preparation method all increases gradually, and preparation method is to the burst size of BMP-2 on the material do not make significant difference (P＞0.05).The theoretical release concentration of BMP-2 is 10 μ g/ml on the timbering material, vibrate after 1 day, BMP-2 average content in the solution on three kinds of timbering materials is (1.79 ± 0.14) μ g/ml, the BMP-2 that (17.9 ± 1.4) % is described discharges from decalcified bone matrix, significantly floods the result (P＜0.05) of fixation, negative-pressure adsorption method or glutaraldehyde cross-linking method less than contemporaneity.Vibrated the 2nd to 5 day, the rate of release of BMP-2 eases up gradually.Use Excel2003 the burst size of BMP-2 on three kinds of timbering materials carried out match with the data that duration of oscillation changes, show that the BMP-2 burst size meets following non-linear formula with the Changing Pattern of duration of oscillation:
m＝-0.1104t
2+1.4132t+0.5913，R
2＝0.9895，
M:BMP-2 burst size (μ g/ml); T: duration of oscillation (d).
Data analysis shows: vibrate the 7th day the time, the burst size of BMP-2 is near balance, and the content of BMP-2 is 5.07 μ g/ml in this moment solution, illustrates that 50.7% BMP-2 is released.Test data also shows the final balance burst size of BMP-2 significantly less than infusion process, negative-pressure adsorption method (P＜0.05), and the time that discharges is also long than infusion process and negative-pressure adsorption method.This be because, radiation effects by microwave, make BMP-2 go up amino residue and the generation cross-linking reaction of the functional group on the decalcified bone matrix, part BMP-2 is by the effect and firm the combining of decalcified bone matrix of chemical bond, so, even still have nearly 49.3% BMP-2 to be fixed in the decalcified bone matrix after 7 days in vibration, these BMP-2 can be along with the degraded of decalcified bone matrix discharging gradually, thereby prolonged the activity of BMP-2, improved the utilization rate of BMP-2, illustrate that the microwave fixation has fixed effect preferably, and pair cell etc. has no side effect, be applicable to the fixedly research of BMP-2 and decalcified bone matrix.
Embodiment 4: microwave is fixed-the chitosan coating method
One, the metaphyseal spongy bone of pig back bone prepares decalcified bone matrix, and concrete preparation process comprises the steps:
(1) cartilage, cortical bone and the adhering tissue on removal spongy bone surface;
(2) with the spongy bone section, the flushing that obtain in (1);
(3) spongy bone that obtains in (2) is cut into the bone piece of 1cm * 1cm * 3mm;
(4) spongy bone that obtains in (3) is handled 48 hours to remove the cell on the spongy bone with Triton X-100;
(5) spongy bone that obtains in (4) is handled 48 hours to remove the fatty oils and fats on the spongy bone with absolute ether;
(6) spongy bone that obtains in (5) is placed 0.6mol/L hydrochloric acid handled 12 hours.
(7) with the decalcification spongy bone that obtains in (6) distilled water flushing 30min, obtain decalcification bone supporting stand;
(8) decalcification bone supporting stand that obtains in (7) is placed dehydrated alcohol preserve.
Two, bone morphogenetic protein-2 is fixed-the chitosan coating method with the microwave of decalcified bone matrix, and concrete implementation step is as follows:
(1) decalcified bone matrix that utilizes the metaphyseal spongy bone of pig back bone to prepare;
(2) be the BMP-2 solution of 1 μ g/ μ L with the BMP-2 lyophilized powder with the concentration that sterilized water is mixed with solution;
(3) the BMP-2 solution that configures in (2) is loaded on the decalcified bone matrix of (1) microwave preparation; (amount of the BMP-2 solution on 1cm * 1cm * 3mm) is 20 μ L to be loaded into decalcified bone matrix.
(4) with load in (3) decalcified bone matrix of BMP-2 put in the microwave synthesizer, its power is 500W, reaction temperature is 37 ℃, the response time is 30min; By microwave effect BMP-2 is fixed on the decalcified bone matrix;
(5) decalcified bone matrix of having fixed BMP-2 in (4) being placed concentration is to take out behind 3% the chitosan solution 5min;
(6) the decalcification bone that has applied chitosan in (5) is dry down at 37 ℃.
Three, the mensuration of bone morphogenetic protein-2 and decalcified bone matrix fixed effect:
A) bone morphogenetic protein-2 is as follows with the detailed process of decalcified bone matrix fixed effect mensuration:
The decalcified bone matrix of having fixed bone morphogenetic protein-2 is put into centrifuge tube, in centrifuge tube, add the 2ml distilled water.Centrifuge tube placed to vibrate on the shaking table sway, vibration velocity is 150r/min, time of vibration is 1-5 days, detect the content that discharges bone morphogenetic protein-2 in the centrifuge tube every day, the content that dipping fixation, negative-pressure adsorption fixation and fixing means of the present invention are discharged bone morphogenetic protein-2 every day carries out determination and analysis respectively, to contrast fixed effect.
B) bone morphogenetic protein-2 Determination on content method:
Adopt Bradford Protein Detection method to measure the content of bone morphogenetic protein-2.The measuring principle of this method is based on Coomassie brilliant blue, under 95% ethanol and acid condition, can be made into light blue solution, when with protein bound after, produce blue chemical compound, this is swift in response and stablizes.Compound of reaction has maximum absorbance value at 465～595nm place, the depth of chemical compound color and the height of protein concentration are proportional, and the size of absorbance value that therefore can be by detection 595nm is calculated proteic content.Concrete grammar is as follows:
(4) preparation of Bradford working solution (seeing Table 1.1)
The preparation of the Bradford working solution of table 1.1 500ml
In proper order: after G250 is dissolved in ethanol fully, add phosphoric acid, leave standstill 30min after, add distilled water, it is standby to keep in Dark Place.
(2) preparation standard protein solution: accurate weighing 100mg bovine serum albumin, put into the volumetric flask of 100ml, adds an amount of distilled water and dissolve, accurate quantification is to 100ml again, and obtaining concentration is the standard bovine serum albumen solution of 1mg/ml.
(3) protein standard curve plotting:
Measure the bovine serum albumen solution of the 1mg/ml of 10 μ l, 20 μ l, 30 μ l, 40 μ l, 50 μ l, 60 μ l respectively with accurate liquid-transfering gun, add distilled water and Bradford working solution (accurately liquid-transfering gun is measured), measure it in the OD at 595nm place value with ultra-violet and visible spectrophotometer, each experimental point is done 3 times, get the meansigma methods of 3 OD values, concrete operations see Table 1.2.
The OD value of table 1.2 standard protein detects
With the standard protein addition is X-axis, and pairing OD average (595nm) is made curve chart for Y-axis, get final product Bradford Protein Detection method standard curve (see figure 1).
Through linear regression analysis, draw straight line, its equation is:
Y=0.0101x+0.0021 formula 1-(1)
Like this, rely on the Coomassie brilliant blue standard curve, we just can calculate the protein content of rhBMP-2 to be measured, suppose to measure solution V to be measured, record the OD value and are Y, can try to achieve the standard protein addition X on the corresponding standard curve:
X=(Y-0.0021)/0.0101 formula 1-(2)
Then can be in the hope of the concentration C of unknown rhBMP-2:
C=(X/V) * 1mg/ml formula 1-(3)
(4) according to the absorbance of institute's test sample product, on standard curve, find the corresponding proteins quality, thereby calculate the content of bone morphogenetic protein-2
As shown in Figure 6, fix-after the chitosan coating method is fixing, prolong with duration of oscillation through microwave, the burst size of the BMP-2 on the timbering material of each preparation method all increases gradually, and preparation method is to the burst size of material B MP-2 do not make significant difference (P＞0.05).The theoretical release concentration of BMP-2 is 10 μ g/ml on the timbering material, vibrate after 1 day, the average content of BMP-2 in the solution on three kinds of timbering materials is (1.11 ± 0.13) μ g/ml, the BMP-2 that (11.1 ± 1.3) % is described discharges from decalcified bone matrix, and these data are significantly flooded the result (P＜0.05) of fixation, negative-pressure adsorption method, glutaraldehyde cross-linking method or microwave fixation less than contemporaneity.Vibrated the 2nd to 5 day, the rate of release of BMP-2 eases up gradually.Use Excel2003 the burst size of BMP-2 on three kinds of timbering materials carried out match with the data that duration of oscillation changes, show that the BMP-2 burst size meets following non-linear formula with the Changing Pattern of duration of oscillation:
m＝-0.0259t
2+0.9191t+0.1084，R
2＝0.9804，
M:BMP-2 burst size (μ g/ml); T: duration of oscillation (d).
Data analysis shows: vibrated the 18th day, the burst size of BMP-2 can be near balance, and the content of the BMP-2 in the solution is 8.26 μ g/ml, and explanation can have 82.6% BMP-2 to be released.Test data shows that the rate of release of BMP-2 is significantly less than infusion process, negative-pressure adsorption method and microwave fixation.This is because on the fixed basis of microwave, chitosan coating has played the effect of strengthening fixed effect; Simultaneously, chitosan coat can also play the effect that the gentle slow release of protection is put to BMP-2, helps prolonging the raising of BMP-2 activity and utilization rate.Therefore, microwave fixes-and the chitosan coating method is the best approach of a kind of fixedly BMP-2 and decalcified bone matrix, and the research of repairing medical science for clinical bone has great importance.
The present invention investigated infusion process, negative-pressure adsorption method,, microwave fixation and microwave fix-the chitosan coating method is to the decalcification bone supporting stand of separate sources and the fixed effect of BMP-2.By relatively discovery to fixed effect and slow release effect, vibrate after 1 day, microwave fixes-the chitosan coating method the burst size of fixed BMP-2 only be (1.11 ± 0.13) μ g/ml, release rate is (11.1 ± 1.3) %, and just arrive the release balance after 18 days in vibration, fixing preferably and slow release effect are arranged, and are a kind of ideal BMP-2 fixing meanss.This research is significant to the raising of the raising of bone defect repair speed and BMP-2 utilization rate.
Claims (1)
1. bone morphogenetic protein-2 and the fixed method of decalcified bone matrix are object with the decalcified bone matrix of the metaphyseal spongy bone preparation of pig back bone, and the BMP-2 solution that configures is loaded on the decalcified bone matrix, it is characterized in that carrying out according to following steps:
(1) with load the decalcified bone matrix of BMP-2 put into the microwave synthesizer, microwave power is 500W, reaction temperature is 37 ℃, processing time 30min;
(2) decalcified bone matrix of having fixed BMP-2 in (1) being placed concentration is to take out behind 3% the chitosan solution 5min;
(3) with the decalcified bone matrix drying that obtains in (2), baking temperature is 37 ℃.
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