TWI690334B - 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, and is the use of a substance.

In the applicant's previous patent application, a standard preparation process has been established to effectively prepare poultry bone protein extracts containing osteoplastic 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 all beneficial to the bones, for example, osteoplastic protein 2 has the ability to induce ectopic bone or cartilage formation in vivo, transforming growth factor β It can promote osteoblasts to synthesize bone matrix, alkaline phosphatase can make bones mineralized, etc. Therefore, it is preliminary conceived that bone protein extract 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 conditions that are not harmful to living organisms. Although a standard process has 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 correct, so he has many years of experience in related technology and product design and manufacturing, upholds the excellent design concept, and researches and creates in view of the above disadvantages. The use of the poultry bone protein extract of the present invention for bone filling materials is introduced, which corrects 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 as a bone filling material 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 Used for bone filling materials.

For the purpose of the foregoing disclosure, the use of the poultry bone protein extract of the present invention as a bone filling material is to perform MTT test, lactate dehydrogenase test, endotoxin test, in vitro degradation behavior test and in vitro degradation behavior test on the prepared bone protein extract Mouse ear swelling test.

Among them, the results of the MTT test, lactate dehydrogenase test, and endotoxin test show that the bone protein extract is not toxic to cells;

The results of the in vitro degradation behavior test showed that the average pH value and weightlessness of the bone protein extract in the environment of the phantom fluid did not change significantly with time;

The results of the ear swelling test in mice show 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 risk of toxicity and sensitization to the organism, and can be maintained in a stable state in the environment of the phantom fluid, and it can be confirmed as a composition One of the ingredients is added to the use of bone filling material.

The invention relates to the use of poultry bone protein extracts for bone filling materials, wherein the poultry bone protein extracts are based on the patent application "Preparation Method of Poultry Bone Protein Extracts" filed by the applicant ”(Application No.: 1010707610), and because the case was still not disclosed at the time of the application, the preparation steps claimed in the application are described here again, including:

Crushing treatment, taking poultry bones and crushing them, and then weighing to take the predetermined weight;

Degreasing treatment, put the crushed bones into the degreasing agent for degreasing for at least 30 minutes, and then collect the bones;

For decalcification, put the degreased 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, put the washed bones in the extraction solution, perform shaking extraction in an environment with a temperature of 4-30°C for at least 4 hours, and then collect the extracted extraction solution;

Purification treatment, the extract is purified by ultrafiltration;

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

Based on the above-mentioned claimed preparation steps, the case of "Preparation Method of Poultry Bone Protein Extract" also discloses preferred examples of preparation steps:

After crushing, take the chicken bones and smash the bones with a hammer. The preferred bone fragments are about 5cm in size, and the joints should be cracked. After smashing, use paper towels to absorb blood and weigh the bones. 100g;

For degreasing, take 100% acetone as the degreasing agent, add the crushed bones to acetone, and the ratio of the volume of acetone (ml) to the weight of the bones (g) taken when weighing is 8:1. Therefore, the amount of acetone used is 800ml, and then placed in an environment of normal temperature (25 ~ 30 ℃) at a speed of 180RPM oscillator for degreasing for at least 30 minutes, then use a Buchner funnel placed in 5A filter paper to filter out the lipid-rich acetone Collect the bones. The above is the step of the degreasing process, which is repeated 3 times;

For decalcification treatment, take hydrochloric acid with an equivalent concentration of 0.6N as the decalcification agent, add the degreased bones to hydrochloric acid, and the ratio of the volume of hydrochloric acid (ml) to the weight of bones (g) taken when weighed is 8: 1. Therefore, the amount of hydrochloric acid used is 800ml, and then placed in an environment of normal temperature (25 ~ 30 ℃) in a shaker with a rotation speed of 180RPM for decalcification for at least 60 minutes, and then use a Buchner funnel placed in 5A filter paper to remove rich Mineral hydrochloric acid and bone collection, the above is the step of the decalcification treatment, the step of the decalcification treatment is repeated 4 times;

Washing process, put the decalcified bone together with 2D pure water (distilled and deionized pure water) into a sealable container, such as a serum bottle, and use the volume of pure water (ml) and scale The ratio of bone weight (g) taken at the time of weighing is 9:1, so the amount of pure water used is 900ml, then the container is capped and inverted upside down and hand shaken about 10 times, then the waste liquid is filtered with a tea bag, above It is the step of the washing process once, and the step of the washing process is repeated 3 times;

In the extraction process, the extract is taken, and the washed bones are placed in the extract. The extract contains trimethylolmethylamine with a molar concentration of 0.6M, 0.5M ethylenediaminetetraacetic acid, 4M guanidine hydrochloride, and The volume concentration of pepsin is 1%, and the ratio of the volume of extract (ml) used to the bone weight (g) taken during weighing is 6:1, so the amount of extract used is 600ml, followed by normal temperature (25 ~30℃) placed in a shaker at 180 RPM for 12 hours, then filter out the bones with a double-layer tea bag and collect the extract containing bone protein;

For purification, the collected extract is put into a hollow fiber column with a molecular weight cutoff of 3 kDa for ultrafiltration purification;

In the drying process, the purified material is freeze-dried in an environment with 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 embodiment, the "preparation method of poultry bone protein extract" also adopts different condition parameters for the extraction processing step and the purification processing step to obtain multiple groups of bone protein extract samples, The condition parameters are the extraction time of 4 or 12 hours in the extraction treatment step, the vibration extraction or ultrasonic extraction in the extraction method, the extraction solution containing pepsin or not, and the purification treatment The hollow fiber column of the step can use a cut-off molecular weight of 3kDa or 10kDa. The preparation conditions and extraction rates of each group of samples are listed in the following Table 1. (The crude extract with the number 0 is used without pepsin during the extraction process step. The extraction liquid is allowed to stand for 4 hours without shaking extraction or ultrasonic extraction, and the crude extract is directly subjected to the drying treatment step without purification treatment step): Table 1:

In addition, in the "Preparation of Poultry Bone Protein Extract", related tests were conducted on the prepared bone protein extract to confirm 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 sample No. 5 in Table 1 (extraction 4hr, oscillating extraction, pepsin addition, molecular weight 3 kDa or less) as an example The BMP-2 content is 242.671 pg/mg, the TGF-β1 content is 56 ng/100 mg, the ALP content is 6.172 ng/g, and the OCN content is 0.489 ng/g.

In this case, a biocompatibility test was performed on each group of prepared 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, and The biocompatibility test includes MTT test, lactate dehydrogenase test, endotoxin test, in vitro degradation behavior test, and mouse ear swelling test. The following describes the test methods and test results of each test.

1. MTT test: MTT is a yellow water-soluble solid, which can be metabolized and reduced by the dehydrogenase in live cells to produce a purple precipitate formazan. Only active cells have active enzymes in the line body, so it can be Evaluate the survival of the cells by measuring the production of the sediment 量來.

The MTT test procedure in this case is as follows:

(1) Human chondrosarcoma cells (SW-1353) were seeded in a cell culture dish at a growth density of 1×10 ⁴ and cultured for 24 hours, and then a sample of bone protein extract with a predetermined concentration was given and cultured for another 24 hours.

(2) After absorbing the culture solution from the cell sample to be tested for activity, rinse with PBS 數 times. Add 1mL of fresh culture medium containing MTT and place in a 37°C incubator for 3 hours.

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

(4) Take 200 μL of formazan-dissolved DMSO under an enzyme immunoassay analyzer (ELISA), and measure its absorbance at a wavelength of 570 nm, and the test wavelength is 650 nm.

[Please refer to the first picture and the second picture] And in this case, the results of the MTT test for some of the samples in Table 1 are shown in the first picture and the second picture. 0.1 mg/ml (the value in the first figure is expressed as mean±SE, the number of samples n=3, *P<0.05, compared with the control group), and the concentration of the samples in each group in the second figure is 1 mg /ml (the value in the second figure is also expressed as mean±SE, the number of samples n=3, **P<0.01, *P<0.05, compared with the control group), and from the test results, we can see the extraction The resulting sample is not toxic to the cells at a concentration of 0.1 mg/ml (P-value> 0.1), and the cell survival 率 is above 100%. Only 6-7 are left after the high concentration of 1 mg/ml is given The survival rate shows that the sample will not cause damage to the cells at the normal processing concentration (0.1 mg/ml). If viewed from the perspective of hyperplasia, the samples No. 1 and 2 (ultrasonic extraction, pepsin, The samples extracted for 4 hours) and number 13 and 14 (shaking extraction, pepsin addition, 12 hours extraction) were significantly different from the control group, indicating that the bone protein extract extracted under these two conditions can proliferate the cells.

2. Lactate dehydrogenase (LDH) test: LDH will be released when the tissue is necrotic, so LDH can be measured as a substitute for tissue decomposition, such as hemolysis, etc. By measuring the LDH value compared with the normal range also helps medical For the diagnosis, the LDH test in this case measures the concentration of LDH contained 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 (Automatic System Calibration Solution) for instrument calibration.

(2) Centrifuge the sample at 1500rcf 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 place it in the instrument for testing.

(5) Calculate the activity of lactate dehydrogenase in each sample by analyzing the automatic instrument.

The test results are shown in Table 2 below: Table 2:

The extracellular toxicity is measured by lactate dehydrogenase. When the cell membrane is damaged or ruptured, the lactate dehydrogenase in the cell will flow into the cell culture fluid. It is known that the amount of lactate dehydrogenase released 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 mortality of the cells, and the results of this test showed that the bone was administered in human chondrosarcoma cells (SW-1353) After the crude protein extract and the bone protein extract under different preparation conditions, the concentration of lactate dehydrogenase will not be higher than that of the control group, so it can be preliminarily determined that the bone protein extract is not toxic to the cells.

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

(1) Take 100μl of the appropriately diluted bone protein extract sample.

(2) Add the sample to a test tube that has been filled with 100 μl of LAL reagent in advance.

(3) Place the test tube in a 37±0.5℃ non-circulating water bath.

(4) Judgment result: the content of the test tube is agglomerated, which means that the endotoxin is positive, and if the content is gelatinous but slips or is thick, and the liquid state is maintained, it does not agglomerate, which means that the endotoxin is present. Negative and positive means that the endotoxin concentration is greater than the sensitivity concentration set by this product, and negative is the opposite.

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 results of the endotoxin test in this case are shown in Table 3 below: Table 3:

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 agglomerate, which means that the endotoxin content is less than 0.25 EU/mL. The test results show that the crude protein extract of human chondrosarcoma cells (SW-1353) and the extraction of bone protein with different preparation conditions After the substance, the endotoxins are negative, and it can be judged that the extraction process avoids the possible endotoxin effects on the cells.

4. In vitro degradation behavior test: The purpose is to observe the changes in the state of bone protein extracts in the phantom fluid environment to simulate whether the degradation behavior after implantation in vivo conforms to the actual needs of clinical bone defect healing (refer to Fang Xuwei (2012)-行政The Atomic Energy Commission of the Chinese Academy of Sciences entrusted the research report of the research project "Medical 療 Bone Materials 臨 Pre-bed Biological Test Applied Research"). The in vitro degradation behavior test in this case is divided into two types:

a. After lyophilizing the bone protein extract sample, measure its weight 量 (Wo), and then put it into the 離 heart tube containing PBS, and then put it into an incubator with a temperature 度 of 37°C and 5% CO ₂裡, after 4, After 7, 14, 28 and 42 days, take out the samples of each group, and then go through the 冷 freeze drying place 理 after the same steps to measure 量 weight 量 (Wt).

b. After lyophilizing the bone protein extract samples, put them into the 離 heart tube containing PBS (pH value 7.4, 25℃), and then put them into the incubator with temperature 度 of 37℃ and 5% CO ₂裡4. After 7, 7, 14, 28, and 42 days 量 measure the pH value of the extracts of each group of samples, and record the change in pH value during the solution of the sample.

[Please refer to the third figure] The results of the in vitro degradation behavior test 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 by the incubation time, which means that the bone protein extract will not be degraded in vitro Significant changes.

5. Mouse ear swelling test: The purpose is to determine whether the extracted bone protein extract can cause allergies, so this test is carried out. In the case of epidermal allergies, the ears will swell and react when the mouse is stimulated by the sample. In the next two days, the late-onset hypersensitivity was quantified by measuring the increase in ear thickness compared to before stimulation. Refer to Johansen Pal [Note 1] and Shayne Cox Gad [Note 2] to test the samples for allergic irritation through the epidermis To stimulate the ear swelling test to measure its allergic reaction, the mouse ear swelling test in this case is as follows:

Take female BCA mice 6-10 weeks old, randomly group 3 mice into each group, and sensitize the mice by applying 10 mM bone protein extract on the skin surface. The bone protein extract is diluted in 0.9% saline, as referenced in Shayne Cox Gad As described in [Note 2], 20 μl of the extract dilution was injected into the left and right ears, and 20 μl of saline was injected into the right ear as a negative control. The swelling thickness was recorded 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 allergic reaction was quantified by measuring the increase in the ear thickness value compared to before the stimulation, and measured using a precision digital thickness meter.

[Please refer to the fourth figure] The mouse ear swelling test in this case was tested with sample No. 5 in Table 1 (extraction time 4 hours, oscillating extraction, pepsin addition, molecular weight 3 kDa or less). The test results are shown in the fourth figure As shown, if the ear swelling percentage of more than 120% is represented as a stimulant, the results show that the mouse ear thickness has no allergic reactions (not reaching the stimulus index value), indicating that the bone protein extract is a non-irritating substance.

Conclusion: After conducting MTT test, lactate dehydrogenase test and endotoxin test on bone protein extract, it shows that there is no risk of toxicity at normal concentration, and allergy test results show that it has no obvious sensitization and degradation in vitro The test results also showed that the average pH value and average weight loss did not change significantly due to the prolonged incubation 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.

The above is only one of the preferred embodiments of the present invention, and it should not be used to limit the scope of the present invention. That is to say, all equal changes and modifications made according to the scope of the patent application should still fall within the scope of this creative patent.

In summary, when the invention is known to be novel and progressive, and the invention has not been seen in any publication, it must comply with the provisions of 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 graph is the result graph of the MTT test performed in the use of the poultry bone protein extract of the present invention as a bone filling material. The second graph is the result graph of another test condition MTT test performed in the use of the poultry bone protein extract of the present invention as a bone filling material. The third figure is a graph showing the results of an in vitro degradation behavior test conducted in the use of the poultry bone protein extract of the present invention as a bone filling material. The fourth figure is a graph showing the results of a mouse ear swelling test performed in the use of the poultry bone protein extract of the present invention as a bone filling material.

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Claims (9)

A poultry bone protein extract is used as a bone filling material. The bone protein extract is added to the bone filling material as one of the components. The bone protein extract is made according to the following steps: crushed, taken Poultry bones are crushed, and then weighed to take a predetermined weight; degreasing treatment, put the crushed bones into a degreasing agent for degreasing for at least 30 minutes, and then collect the bones; decalcification treatment, put the degreased bones into the degreasing Carry out decalcification in calcium for at least 60 minutes, and then collect the bones; wash treatment, put the decalcified bones in pure water for washing, then collect the bones; extract treatment, put the washed bones in the extraction solution In the extract, the extract contains 0.6M trimethylolmethylamine, 0.5M ethylenediaminetetraacetic acid and 4M guanidine hydrochloride. Vibration extraction is carried out in an environment at a temperature of 4~30℃ for at least 4 hours, after which the extraction is completed The extraction solution; purification treatment, the extraction solution is purified by ultrafiltration; drying treatment, the purified material is freeze-dried to obtain cotton-like bone protein extract. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the degreasing agent used in the degreasing step uses acetone, and the volume of acetone (ml) used when weighed The ratio of bone weight (g) taken was 8:1. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the decalcifying agent used in the decalcification process uses hydrochloric acid, and the volume of hydrochloric acid (ml) used and the scale The ratio of bone weight (g) taken when weighing is 8:1. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the steps of degreasing and decalcification are carried out in an environment with a temperature of 25 to 30°C. The use of the poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the volume of the extract (ml) used in the extraction process step and the weight of the bone taken during weighing ( g) The ratio is 6:1. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling material, wherein the extract also contains pepsin at a volume concentration of 1%. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the extraction process step is changed to ultrasonic extraction. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein a hollow fiber column with a molecular weight cutoff of 3 kDa or more is used in the purification process. The use of poultry bone protein extract as described in item 1 of the patent application scope for bone filling materials, wherein the freeze-drying conditions in the step of the drying process are those with a temperature of at least -40°C and a vacuum pressure of at least 200 psi.

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