TW202033229A - Use of bone protein extracts of poultry for bone graft - Google Patents

Abstract

The present invention related to an use of bone protein extracts of poultry for bone graft. It main content is to conduct several biocompatibility experiments to confirm the non-toxic and non-allergenic properties of the bone protein extracts which produced by following predetermined steps. So the bone protein extracts can be added into bone graft as the component of it.

Description

Use of poultry bone protein extract for bone filling material

The present invention relates to the use of a poultry bone protein extract for bone filling materials, which is a kind of use.

In the applicant’s previous patent application, a standard preparation process has been established, which can effectively prepare poultry bone protein extract containing bone shaping protein 2, transforming growth factor β, alkaline phosphatase, and osteocalcin .

After the establishment of the standard preparation process, it is considered that the relevant ingredients contained in the extract are beneficial to bones. For example, bone shaping protein 2 has the ability to induce ectopic bone or cartilage in the body, and transforming growth factor β It can promote osteoblasts to synthesize bone matrix, alkaline phosphatase can mineralize bones, and so on. Therefore, it is initially conceived that bone protein extracts should be added as one of the components in bone filling materials.

However, bone filling materials are materials used in living organisms, so they should meet the basic requirements of being harmless to living organisms. Although standard manufacturing processes have been established for bone protein extracts, their biocompatibility is still unknown.

In view of the above shortcomings, the inventor believes that it is necessary to make corrections, so with years of experience in related technology and product design and manufacturing, upholding the excellent design concept, researching and creating the above shortcomings, after continuous efforts, finally The use of poultry bone protein extract of the present invention for bone filling materials is introduced to correct the product structure to enhance the excellent efficacy of the product.

The main purpose of the use of the poultry bone protein extract of the present invention for bone filling materials is to conduct a biocompatibility test on the poultry bone protein extract prepared according to a predetermined process to confirm that it can be added as one of the components For bone filling materials.

In order to achieve the purpose of the foregoing disclosure, the use of the poultry bone protein extract of the present invention for bone filling materials is to perform MTT test, lactate dehydrogenase test, endotoxin test, in vitro degradation behavior test on the prepared bone protein extract and Mouse ear swelling test.

Among them, the results of MTT test, lactate dehydrogenase test, and endotoxin test show that bone protein extract is non-toxic to cells;

The results of the in vitro degradation behavior test show that the average pH and weight loss of the bone protein extract will not change significantly due to the prolonged time when the bone protein extract is in the environment of the body fluid;

The results of the mouse ear swelling test showed that the bone protein extract is a non-irritating substance that does not cause allergic reactions.

From the integration of the results of each test, the poultry bone protein extract prepared according to the predetermined steps has no toxicity and sensitization risk to the organism, and can maintain a stable state in the simulated body fluid environment, and it is confirmed that it can be used as a composition One of the ingredients is added to the use of bone filling materials.

The present invention relates to the use of a poultry bone protein extract for bone filling materials. The poultry bone protein extract is based on the applicant’s prior patent application "Preparation method of poultry bone protein extract" "(Application case number: 108107610). Since the case has not yet been published at the time of the application of this case, the preparation steps claimed in the application of this case are described again here, including:

Shredding, take poultry bones and break them, and then weigh them to get the predetermined weight;

For degreasing treatment, put the broken bones into a degreasing agent for degreasing for at least 30 minutes, and then collect the bones;

For decalcification, put the defatted bones in a decalcifying agent for decalcification for at least 60 minutes, and then collect the bones;

Washing treatment: Put the decalcified bones into pure water for washing, and then collect the bones;

In the extraction process, the cleaned bones are put into the extraction liquid, and the extraction is carried out in an environment with a temperature of 4~30℃ for at least 4 hours, and then the extracted liquid is collected;

Purification treatment, the extract is purified by ultrafiltration;

In the drying process, the purified material is freeze-dried to obtain a cotton-like bone protein extract.

On the basis of the above-mentioned preparation steps, the case of "Preparation Method of Poultry Bone Protein Extract" also discloses examples of better preparation steps:

For shredding, take the chicken bones from the ground and break them with a hammer. The best bone fragment size is about 5cm. Note that the joints should be cracked. After breaking, use a paper towel to absorb the blood and weigh the bones. 100g;

For degreasing treatment, take 100% acetone as a degreasing agent, add the crushed bones to the acetone, and the ratio of the volume of acetone (ml) used to the weight of the bones (g) taken during weighing is 8:1, Therefore, the amount of acetone used is 800ml, and then placed in a 180RPM shaker at room temperature (25~30℃) for degreasing for at least 30 minutes, and then use a Buchner funnel with 5A filter paper to filter out the lipid-rich acetone And collect the bones, the above is the step of the degreasing treatment once, and the step of the degreasing treatment is repeated 3 times;

For decalcification, take hydrochloric acid with an equivalent concentration of 0.6N as a decalcification agent, add the defatted bones to hydrochloric acid, and the ratio of the volume of hydrochloric acid (ml) used to the weight of the bones (g) taken during weighing is 8: 1. So the amount of hydrochloric acid used is 800ml, and then placed in a 180RPM shaker at room temperature (25~30℃) for decalcification for at least 60 minutes, and then use a Buchner funnel with 5A filter paper to filter out the rich Mineral hydrochloric acid and collecting bones, the above is the step of decalcification treatment once, and the step of decalcification treatment is repeated 4 times;

After washing, put the decalcified bones and 2D pure water (distilled and deionized pure water) into a closable container, such as a serum bottle, and use the pure water volume (ml) and scale The ratio of the bone weight (g) taken when the weight is heavy is 9:1, so the amount of pure water used is 900ml, then the container is closed and the container is turned upside down and hand shaked about 10 times, and the waste liquid is filtered out with a tea bag. This is one step of the washing treatment, and the steps of the washing treatment are repeated 3 times;

Extraction treatment, take the extract, put the washed bones into the extract, the extract contains 0.6M trimethylolmethylamine, 0.5M ethylenediaminetetraacetic acid, 4M guanidine hydrochloride and Pepsin with a volume concentration of 1%, and the ratio of the volume of extract used (ml) to the weight of the bone (g) taken during weighing is 6:1, so the amount of extract used is 600ml, and then at room temperature (25 ~30℃) placed in an oscillator with a rotation speed of 180RPM for 12 hours of shaking extraction, then use a double-layer tea bag to filter out the bones and collect the bone protein-containing extract;

Purification treatment: Put the collected extract into a hollow fiber column with a molecular weight cutoff of 3kDa for ultrafiltration purification;

In the drying process, the purified material is freeze-dried in an environment at a temperature of -40°C or lower and a vacuum pressure of 200 psi or lower to obtain a cotton-like bone protein extract.

Further, based on the above-mentioned embodiment, the "Preparation Method of Poultry Bone Protein Extract" also uses different condition parameters for the extraction processing step and the purification processing step to obtain multiple sets of bone protein extract samples. The condition parameters are that the extraction time in the extraction treatment step is 4 or 12 hours, the extraction method is shaking extraction or ultrasonic extraction, the extraction solution is pepsin-containing or not, and the purification treatment The hollow fiber column of the step can use 3kDa or 10kDa molecular weight cut-off. The preparation conditions and extraction rate of each group of samples are listed in the following table (the crude extract with number 0 is used in the extraction processing step without pepsin The extract is allowed to stand for 4 hours without shaking extraction or ultrasonic extraction, and the crude extract is directly subjected to drying treatment step without purification treatment step): Table 1: Numbering Extraction time (hr) Extraction method 1% pepsin Molecular weight (kDa) Extraction rate (mg/g) 0 Crude extract 202.08 1 4 Ultrasound Have 3↓ 191.10 2 4 Ultrasound Have 10↓ 164.66 3 4 Ultrasound no 3↓ 141.47 4 4 Ultrasound no 10↓ 153.50 5 4 oscillation Have 3↓ 136.95 6 4 oscillation Have 10↓ 179.13 7 4 oscillation no 3↓ 159.83 8 4 oscillation no 10↓ 150.62 9 12 Ultrasound Have 3↓ 157.64 10 12 Ultrasound Have 10↓ 143.38 11 12 Ultrasound no 3↓ 140.36 12 12 Ultrasound no 10↓ 177.39 13 12 oscillation Have 3↓ 157.64 14 12 oscillation Have 10↓ 157.26 15 12 oscillation no 3↓ 141.54 16 12 oscillation no 10↓ 136.10

In addition, in the "Preparation Method of Poultry Bone Protein Extract", related tests were conducted on the obtained bone protein extract, and it was confirmed that it contained Bone morphogenetic protein 2, BMP-2 and transforming growth factor β ( TGF-β), alkaline phosphatase (Alkaline phosphatase, ALP), osteocalcin (OCN), for example, take the sample No. 5 in Table 1 (extraction for 4 hours, extraction with shaking, addition of pepsin, molecular weight below 3kDa) as an example The content of BMP-2 is 242.671pg/mg, the content of TGF-β1 is 56ng/100mg, the content of ALP is 6.172ng/g, and the content of OCN is 0.489ng/g.

In this case, a biocompatibility test was performed on each set of bone protein extract samples to confirm whether the bone protein extract can be added to the bone filling material used by humans or animals as one of the components. The biocompatibility test includes MTT test, lactate dehydrogenase test, endotoxin test, in vitro degradation behavior test and mouse ear swelling test. The methods and results of each test are described below.

1. MTT test: MTT is a yellow water-soluble solid, which can be metabolized and reduced by dehydrogenase in the mitochondria of living cells to produce purple precipitate formazan. Only living cells have active mitochondrial enzymes, so it can The survival rate of the cells was evaluated by measuring the yield of the sediment.

The MTT test steps in this case are as follows:

(1) human chondrosarcoma cells (SW-1353) to grow the inoculation density of 1x10 ⁴ cells to culture dishes and cultured for 24 hours, to give a predetermined concentration of the bone protein extract samples were further incubated for 24 hours.

(2) After aspirating the culture solution from the cell sample to be tested for viability, rinse with PBS several times. Add 1 mL of fresh culture medium containing MTT and place it in an incubator at 37°C for 3 hours.

(3) After removing the culture medium containing MTT, add 500 μL of DMSO and shake with vortex until the purple formazan particles are completely dissolved.

(4) Take 200μL of DMSO with formazan dissolved in an enzyme immunoassay (ELISA) and measure its absorbance at a wavelength of 570nm. The reference wavelength is 650nm.

[Please refer to the first and second graphs] In this case, the results of the MTT test of several samples in Table 1 are shown in the first and second graphs. The concentration of each group of samples in the first graph is all 0.1 mg/ml (the value in the first figure is represented by mean±SE, the number of samples is n=3, *P＜0.05, compared with the control group), the concentration of each group of samples in the second figure is 1 mg /ml (the value in the second figure is also represented by mean±SE, the number of samples is n=3, **P＜0.01, *P＜0.05, compared with the control group), and from the test results, it can be seen that the extraction The sample is not toxic to cells at a concentration of 0.1 mg/ml (P-value ＞ 0.1), and the cell survival rate is above 100%. After a high concentration of 1 mg/ml is given, only 6-7 remain. The survival rate of success shows that the sample will not cause damage to the cells under the normal treatment concentration (0.1 mg/ml). If you look at it from the proliferation point of view, the samples numbered 1 and 2 (ultrasonic extraction, pepsin addition, Compared with the control group, samples with numbers 13 and 14 (vibration extraction, pepsin addition, and 12 hours extraction) were significantly different from the control group, indicating that the bone protein extract extracted under these two conditions can proliferate cells.

2. Lactate dehydrogenase (LDH) test: LDH is released when tissue is necrotic, so LDH can be measured as a substitute for tissue decomposition, such as hemolysis, etc. It is also helpful for medicine to compare the LDH value with the normal range. For the diagnosis, the LDH test in this case determines the LDH concentration in the cell culture medium in the aforementioned MTT test. The test instrument uses Hitachi MODULAR P/D analyzers: ACN 080. The test steps are as follows:

(1) First use S1 0.9% NaCl, S2 C.F.A.S (automated system calibration fluid) for instrument calibration.

(2) Centrifuge the sample at 1500 rcf for 10 minutes.

(3) Take the supernatant, place the liquid in a microcentrifuge tube, and add 100ul of 0.9% saline.

(4) After mixing, transfer the supernatant to a clean centrifuge tube and put it in the instrument for testing.

(5) Calculate the activity of lactate dehydrogenase in each sample through an automatic analyzer.

The test results are shown in the following table two: Table two: Numbering Extraction time (hr) Extraction method 1% pepsin Molecular weight (kDa) LDH content (U/L) no Control group 44 0 Crude extract 39 1 4 Ultrasound Have 3↓ 36 2 4 Ultrasound Have 10↓ 39 5 4 oscillation Have 3↓ 39 6 4 oscillation Have 10↓ 37 7 4 oscillation no 3↓ 39 8 4 oscillation no 10↓ 39 9 12 Ultrasound Have 3↓ 38 13 12 oscillation Have 3↓ 38 14 12 oscillation Have 10↓ 39

Extracellular toxicity is based on lactate dehydrogenase as an indicator. When the cell membrane is damaged or ruptured, the intracellular lactate dehydrogenase will flow into the cell culture medium. It is known that the release of lactate dehydrogenase is positively correlated with the degree of cell death Changes (Koh et al., 1987), so by measuring the activity of lactate dehydrogenase in the cell culture fluid can represent the cell death rate, and the results of this test show that in human chondrosarcoma cells (SW-1353) given bone The crude protein extract and the bone protein extract with different preparation conditions did not make the concentration of lactate dehydrogenase higher than the control group. Therefore, it can be initially determined that the bone protein extract is not toxic to cells.

3. Endotoxin test: Endotoxin is a compound that exists in pathogens (such as bacteria). It has potential toxicity and may cause abnormal immune response in the human body. Therefore, it can be used as a test target for the risk of contamination by pathogens. , And the endotoxin test procedure in this case is as follows:

(1) Take 100μl of a properly diluted bone protein extract sample.

(2) Add the sample to the test tube which has been pre-filled with 100μl LAL reagent.

(3) Put the test tube into the 37±0.5℃ non-circulating water bath.

(4) Judgment result: if the content of the test tube is gelatinous, it is agglutination, which means endotoxin is positive, and if the content is gelatinous but slips down or thick and remains liquid, it is non-agglutination, which means endotoxin is Negative and positive means that the endotoxin concentration is greater than the sensitivity concentration specified for this product, and negative is the opposite.

The results of the endotoxin test in this case are shown in Table 3 below: Table 3: Numbering Time(hr) Extraction method 1% pepsin Molecular weight (kDa) Endotoxin(EU/mL) 0 Crude extract ＜ 0.25 1 4 Ultrasound Have 3↓ ＜ 0.25 2 4 Ultrasound Have 10↓ ＜ 0.25 5 4 oscillation Have 3↓ ＜ 0.25 6 4 oscillation Have 10↓ ＜ 0.25 7 4 oscillation no 3↓ ＜ 0.25 8 4 oscillation no 10↓ ＜ 0.25 9 12 Ultrasound Have 3↓ ＜ 0.25 13 12 oscillation Have 3↓ ＜ 0.25 14 12 oscillation Have 10↓ ＜ 0.25 1.Acceptable range for dialysate as recommended by the AAMI guidelines: acceptable range: ＜ 2.0 EU/Ml. 2.Action level: ＞1.0 EU/Ml.

The source of the data is that the content of the test tube does not agglutinate, which means that the endotoxin content is less than 0.25 EU/mL. The test results show that the crude bone protein extract and bone protein extraction under different preparation conditions were given in human chondrosarcoma cells (SW-1353) After the treatment, the endotoxins were all negative. It can be judged that the extraction process has avoided the impact of possible endotoxins on the cells.

4. In vitro degradation behavior test: The purpose is to observe the state change of bone protein extract in a simulated body fluid environment to simulate whether the degradation behavior after implantation in a living body meets the actual needs of clinical bone defect healing (refer to Fang Xuwei (2012)-Administration The “Application Research of Preclinical Biological Test of Medical Bone Material” in the research report commissioned by the Atomic Energy Committee of the Institute), the in vitro degradation behavior test in this case is divided into two types:

a. After freeze-drying the bone protein extract sample, measure its weight (Wo), then put it in a centrifuge tube containing PBS, and then put it in a 37°C, 5% CO ₂ incubator, and go through 4, After 7, 14, 28 and 42 days, samples of each group were taken out, and the weight (Wt) was measured after freeze-drying in the same step.

b. After freeze-drying the bone protein extract sample, it is then placed in a centrifuge tube containing PBS (pH 7.4, 25°C), and then placed in a 37°C, 5% CO ₂ incubator, and passed through After 4, 7, 14, 28 and 42 days, measure the pH value of the extracts of each group of samples, and record the pH changes during the degradation of the samples.

[Please refer to the third figure] The in vitro degradation behavior test results of this case are shown in the third figure. The results show that the average pH and average weight loss of the sample will not be prolonged due to the incubation time, which means that the bone protein extract will not be degraded in vitro Make a significant change.

5. Mouse ear swelling test: The purpose is to determine whether the extracted bone protein extract can cause allergies. Therefore, this test is carried out. In the case of epidermal allergies, mice will experience redness and swelling reaction when stimulated by the sample. In the next two days, the delayed-type allergic reaction was quantified by measuring the increase in ear thickness compared to the value before the stimulation. Refer to the literature of Johansen Pal[Note 1] and Shayne Cox Gad[Note 2] to test the allergic irritation of the sample through the epidermis. The irritation-induced ear swelling test is used to measure the allergic reaction. The mouse ear swelling test method in this case is as follows:

Take female BCA mice 6-10 weeks old and randomly divide them into groups of 3. The mice were sensitized by applying 10mM bone protein extract on the skin surface. The bone protein extract was diluted in 0.9% saline, as in the reference Shayne Cox Gad [Note 2] As described above, 20 μl of extract diluent was injected into the left ear for both positive and negative tests, and 20 μl of saline was injected into the right ear as a negative control. Record the thickness of swelling before and 24/48 hours after application. In the case of epidermal hypersensitivity, the mice were also stimulated by the sample. Two days after the stimulation, the delayed type hypersensitivity reaction was quantified by measuring the increase in ear thickness compared with that before the stimulation, and the measurement was performed with a precision digital thickness meter.

[Please refer to the fourth figure] The mouse ear swelling test in this case takes the sample No. 5 in Table 1 (extraction time 4 hours, shaking extraction, with added pepsin, molecular weight below 3kDa) for the test, the test results are shown in the fourth figure As shown, if the ear swelling percentage exceeds 120%, it represents an irritating substance. The results show that the mouse ear thickness has no allergic reaction (not reaching the irritant index value), indicating that the bone protein extract is a non-irritating substance.

Conclusion: After MTT test, lactate dehydrogenase test and endotoxin test on bone protein extract, it is shown that there is no risk of toxicity under normal concentration, and the results of allergy test show that it has no obvious sensitization and degradation in vitro. The test results also show that the average pH value and average weight loss will not change significantly due to the lengthening of the culture time, indicating that the bone protein extract prepared according to the predetermined steps can indeed be added to the bone filling material as one of the components.

Only the above is only a preferred embodiment of the present invention, and should not be used to limit the scope of the present invention. That is to say, all equal changes and modifications made in accordance with the scope of the patent application should still fall within the scope of this creation patent.

In summary, when it is known that the present invention is novel and progressive, and that the present invention has not been seen in any publications, it should comply with Article 22 of the Patent Law.

[Note 1]: Johansen P, Wäckerle-Men Y, Senti G, Kündig TM. Nickel sensitisation in mice: a critical appraisal. J Dermatol Sci. 2010 Jun;58(3):186-92. doi: 10.1016/j. jdermsci.2010.03.011. Epub 2010 Mar 23.

[Note 2]: Shayne Cox Gad, Mouse Ear Swelling Test, Springer-Verlag Berlin Heidelberg 2014.

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The first figure is the result figure of the MTT test carried out in the use of the poultry bone protein extract of the present invention for bone filling materials. The second figure is the result of the MTT test under another test condition in the use of the poultry bone protein extract of the present invention for bone filling materials. The third figure is the result figure of the in vitro degradation behavior test conducted in the use of the poultry bone protein extract of the present invention for bone filling materials. The fourth figure is the result of the mouse ear swelling test in the use of the poultry bone protein extract of the present invention for bone filling materials.

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Claims (10)

A use of poultry bone protein extract for bone filling material is to add the bone protein extract as one of the components to the bone filling material. The use of the poultry bone protein extract described in item 1 of the scope of patent application for bone filling materials, wherein the bone protein extract is prepared according to the following steps: Shredding, take poultry bones and break them, and then weigh them to get the predetermined weight; For degreasing treatment, put the broken bones into a degreasing agent for degreasing for at least 30 minutes, and then collect the bones; For decalcification, put the defatted bones in a decalcifying agent for decalcification for at least 60 minutes, and then collect the bones; Washing treatment: Put the decalcified bones into pure water for washing, and then collect the bones; In the extraction process, the cleaned bones are put into the extraction liquid, and the extraction is carried out in an environment with a temperature of 4~30℃ for at least 4 hours, and then the extracted liquid is collected; Purification treatment, the extract is purified by ultrafiltration; Drying treatment, freeze-drying the purified material to obtain a cotton-like bone protein extract. The use of the poultry bone protein extract described in item 2 of the scope of patent application for bone filling materials, wherein the degreasing agent in the degreasing process uses acetone, and the volume (ml) of acetone used is compared with the time when weighing The ratio of bone weight (g) taken is 8:1. The use of the poultry bone protein extract described in item 2 of the scope of patent application for bone filling materials, wherein the decalcification agent in the decalcification step uses hydrochloric acid, and the volume (ml) of hydrochloric acid used is proportional to the scale The ratio of bone weight (g) taken when the weight is 8:1. The poultry bone protein extract described in item 2 of the scope of patent application is used for bone filling materials, wherein the steps of the degreasing treatment and the decalcification treatment are both performed in an environment with a temperature of 25-30°C. The poultry bone protein extract described in item 2 of the scope of patent application is used for bone filling materials, wherein the extract used in the extraction process contains 0.6M trimethylolamine and 0.5M The ratio of ethylenediaminetetraacetic acid to 4M guanidine hydrochloride, and the volume of the extract used (ml) to the weight of the bone (g) taken when weighing is 6:1. The poultry bone protein extract described in item 6 of the scope of patent application is used for bone filling materials, wherein the extract also contains pepsin at a volume concentration of 1%. The use of the poultry bone protein extract described in item 2 of the scope of patent application for bone filling materials, wherein the ultrasonic extraction method is used in the extraction process. The poultry bone protein extract described in item 2 of the scope of patent application is used for bone filling materials, wherein, in the purification process, a hollow fiber column with a molecular weight cut-off of 3kDa or above is used. The use of the poultry bone protein extract described in item 2 of the scope of patent application for bone filling materials, wherein the freeze-drying conditions in the drying process are at least -40°C and vacuum pressure at least 200 psi.

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