Preparation method of tendon gelatin and biological hot melt adhesive

Abstract

The invention discloses a preparation method of tendon gelatin and a biological hot melt adhesive. The preparation method comprises the following steps: pretreating animal tendon tissues, freezing, crushing, adding alkali liquor for soaking, cleaning and filtering, adding an acidifying agent for acidifying, cleaning and filtering, crushing and homogenizing, adding water for mixing, heating, stirring, extracting, filtering, centrifuging, dialyzing, filtering again, freeze-drying filtrate to obtain tendon gelatin, adding a buffer solution, heating, and freeze-drying to obtain the tendon gelatin. Defoaming and curing to obtain the biological hot melt adhesive. Tendon collagen hydrogen bonds are broken, a triple helix structure is unwound, then the unwinding degree of collagen is controlled, tendon gelatin containing a large number of macromolecular alpha chains and dimers (beta chains) of the alpha chains is prepared, and then the high-thermal-transition hot melt adhesive is prepared. The prepared biological hot melt adhesive shows good fluidity and tissue adhesion by adjusting the temperature, and can be applied to the fields of food, medicine, environment, biological materials and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biological glue, and in particular to a preparation method of tendon gelatin and biological hot melt adhesive. Background Art

[0002] Tissue adhesive is a medical material that can bond tissues to each other to quickly close and repair wounds, and has many advantages such as shortening surgical time and promoting tissue regeneration. As China enters an aging society, the demand for tissue adhesives has also risen sharply. At present, fibrin glue is the most commonly used tissue adhesive in clinical practice, but there are problems such as low bonding strength and risk of viral infection. Cyanoacrylate-based adhesives have high adhesion, but the degradation products are highly toxic. In addition, chemically cross-linked adhesives prepared from natural polymers show high biocompatibility, but reactive functional groups can induce inflammatory reactions, and this type of adhesive needs to dissolve the two components separately before use, which lacks the convenience of clinical operation. For the above reasons, it is necessary to develop a single-component tissue adhesive with excellent biocompatibility and tissue adhesion. If it can be successfully developed, it will have important practical significance for the development of clinical medical devices in my country.

[0003] At present, thermosensitive hydrogel materials are widely used in the biomedical field, including two major categories: synthetic and natural. Synthetic hydrogel materials mainly include pluronic, poly N-isopropylacrylamide and polyethylene glycol, while natural hydrogel materials mainly include chitosan, cellulose, collagen, gelatin and agarose. Most of these hydrogel materials are positively thermosensitive and have problems such as non-degradability, low mechanical strength and weak tissue adhesion. Agarose, collagen and gelatin have negative thermosensitivity. They are in a gel state at low temperatures and can transform from gel to sol when heated. However, considering that agarose hydrogel has a high thermal transition temperature (above 60°C) and is difficult to degrade in the body, and collagen has low solubility in phosphate buffered saline (PBS), both are difficult to use. Compared with the former two, gelatin has good solubility and tissue adhesion, and can be degraded and absorbed by biological enzymes in the body, which is more advantageous. However, the thermal transition temperature (gelation temperature) of hydrogels made of gelatin currently on the market is generally between 25-32°C, which is much lower than the body temperature of 37°C. The gelatin aqueous solution cannot cool and solidify into gel when it contacts the tissue surface, which means it cannot close and repair wounds. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a method for preparing tendon gelatin and biological hot melt adhesive. The present invention optimizes the raw materials and the preparation process, and under the action of temperature, the hydrogen bonds of tendon collagen are broken and the triple helix structure is unwound. Then, the degree of collagen unwound is controlled to prepare tendon gelatin containing a large amount of high-molecular α chains and their dimers (β chains). The tendon gelatin extracted by the present invention is subjected to heating and other processes to obtain a biological hot melt adhesive with a high thermal transition temperature above body temperature. The biological hot melt adhesive prepared by the tendon gelatin extracted by the method of the present invention exhibits good fluidity and tissue adhesion by adjusting the temperature, and can be applied to the fields of food, medicine, environment and biomaterials.

[0005] The technical solution of the present invention is as follows:

[0006] The present invention firstly provides a method for preparing tendon gelatin, comprising the following steps:

[0007] S1: pre-treating the animal tendon tissue, freezing it, and crushing it, soaking the crushed animal tendon tissue in a sodium hydroxide solution, washing and filtering it, adding an acidifier to the filtered animal tendon tissue for acidification, washing, filtering it, and then crushing and homogenizing it to obtain a tissue homogenate;

[0008] S2: Add water to the tissue homogenate, heat, stir and extract, filter and centrifuge, dialyze the supernatant, filter again, and freeze-dry the filtrate to obtain tendon gelatin.

[0009] Furthermore, in step S1, the animal tendon tissue includes at least one of pig tendon tissue, horse tendon tissue, cattle tendon tissue, sheep tendon tissue, donkey tendon tissue, camel tendon tissue, deer tendon tissue, ostrich tendon tissue, turkey tendon tissue, and alpaca tendon tissue.

[0010] Furthermore, in step S1, the pretreatment is to wash the animal tendon tissue and then add an organic solvent to degrease it to obtain defatted animal tendon tissue; the organic solvent includes one or more of ethanol, ether, and acetone.

[0011] Furthermore, in step S1, the freezing temperature is -20 to -80°C and the time is 2 hours to 4 days;

[0012] The crushing refers to crushing the animal tendon into blocks;

[0013] The method also includes washing and filtering and dehydrating the pretreated animal tendon tissue before freezing;

[0014] The concentration of the sodium hydroxide solution is 2 g / L; the mass volume ratio of the crushed tendon tissue to the sodium hydroxide solution is 20 g:1 L; the immersion temperature is 4° C. and the immersion time is 24 h.

[0015] Furthermore, in step S1, the mass volume ratio of the filtered animal tendon tissue to the acidifier is 20g:1L; the acidifier is obtained by adding acetic acid to water; the content of acetic acid in the acidifier is 20-40mL / L; the acidification temperature is 0-4°C, and the time is 12-48h.

[0016] The speed of the crushing homogenate is 15000-18000 r / min, and the time is 10-30 s.

[0017] Furthermore, in step S2, the volume ratio of the tissue homogenate to water is 1:0.5 to 1:2; the extraction temperature is 50 to 70°C, and the time is 2 to 4 hours; the centrifugal speed is 5000 to 15000 r / min, and the time is 3 to 10 minutes.

[0018] Furthermore, in step S2, the freeze-drying temperature is -40 to -80°C.

[0019] The present invention also protects tendon gelatin prepared by the preparation method.

[0020] The present invention also protects a high-heat-transformation biological hot-melt adhesive prepared from the gelatin.

[0021] The present invention also protects a method for preparing the high-heat-convertible biological hot melt adhesive, which comprises: dissolving the tendon gelatin or the tendon gelatin prepared by the preparation method in a buffer solution, heating, ultrasonically defoaming, transferring to a syringe, cooling and solidifying into a gel, and obtaining the high-heat-convertible biological hot melt adhesive;

[0022] Furthermore, the buffer is PBS buffer, the PBS buffer is 1xPBS, the pH is 7.4, the mass volume ratio of the tendon gelatin to the buffer is 100-300 mg: 1 mL; the heating temperature is 60° C., and the heating time is 1 h;

[0023] Furthermore, the thermal transition temperature of the high thermal transition biological hot melt adhesive is 37.5-45°C.

[0024] The beneficial technical effects of the present invention are:

[0025] The present invention selects suitable tendon tissue and utilizes a simple unwinding rather than an enzymatic extraction process to retain a large amount of high molecular weight α chains and dimers thereof (β chains), so that a high concentration gelatin solution has a thermal transition temperature above body temperature after being heated, thereby obtaining a high thermal transition biological hot melt adhesive.

[0026] The high thermal transition temperature of the biological hot melt adhesive prepared by the present invention exceeds the normal body temperature of the human body, which solves the problem that the thermal transition temperature of the existing gelatin is far lower than the body temperature, and the gelatin aqueous solution cannot be cooled and solidified into gel when it contacts the tissue surface, thereby failing to close and repair the wound. The high thermal transition biological hot melt adhesive of the present invention can achieve a high thermal transition temperature of 37.5-45°C, exhibits good fluidity and tissue adhesion by adjusting the temperature, and because it can be used as a single-component tissue adhesive, it is easy to operate.

[0027] At the same time, the bio-hot melt adhesive of the present invention does not need to modify gelatin and add any chemical cross-linking agent, and provides a simple controllable method that greatly enhances the mechanical properties of hydrogels. In addition, since the tissue adhesive of the present invention uses gelatin as the main raw material, it has excellent biocompatibility. It can also be used as a drug delivery carrier (local delivery carrier or sustained-release delivery carrier) at the same time, for example, by mixing various drugs to deliver. The drug is not particularly limited, such as anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, biological preparations, fibroblast growth factor, vascular endothelial cell growth factor, hepatocyte growth factor, contrast agents, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of a sponge-like sample of freeze-dried gelatin prepared in an embodiment of the present invention.

[0029] Figure 2 The SDS-PAGE electrophoresis diagram of Example 8 of the present invention and Comparative Examples 1-3 is shown.

[0030] Figure 3 Circular dichroism spectra of gelatin prepared in Example 8 of the present invention and Comparative Examples 1-3.

[0031] Figure 4 The infrared spectra of the gelatin prepared in Example 8 of the present invention and Comparative Examples 1-3 are shown.

[0032] Figure 5 The UV spectra of the gelatin prepared in Example 8 of the present invention and Comparative Examples 1-3 are shown.

[0033] Figure 6 This is a thermal transition temperature diagram of the hot melt adhesive prepared in Example 8 of the present invention.

[0034] Figure 7 This is a graph evaluating the stability and injectability of the hot melt adhesive prepared in Example 8 of the present invention.

[0035] In the figure: a), stability evaluation of the bio-hot melt adhesive at 37°C; b), injectability evaluation of the bio-hot melt adhesive.

[0036] Figure 8 Evaluation of the bonding strength of the hot melt adhesive prepared in Example 8 of the present invention.

[0037] In the figure: a), schematic diagram of the adhesion of biological hot melt adhesive (TG) and PG to different tissues; b), schematic diagram of the ASTM F-2258-05 standard test method; c), the adhesion strength of biological hot melt adhesive (TG) to pig blood vessels, pig lungs, and pig skin; d), the adhesion strength of pig skin gelatin (PG) to pig blood vessels, pig lungs, and pig skin. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0039] The thermal transition temperature of gelatin mainly depends on the content of proline (pro) and hydroxyproline (hyp) and the size of the molecular weight of gelatin. The higher the content of proline and hydroxyproline, the stronger the hydrogen bonding effect between molecules, and the higher the freezing point and melting point of gelatin hydrogel. The larger the molecular weight of gelatin, the stronger the interaction between molecular chains, which further improves the mechanical strength and thermal transition temperature of gelatin hydrogel.

[0040] The content of proline and hydroxyproline in gelatin mainly depends on the animal source and tissue site.

[0041] In one embodiment of the present invention, at least one of pig tendon tissue, horse tendon tissue, cattle tendon tissue, sheep tendon tissue, donkey tendon tissue, camel tendon tissue, deer tendon tissue, ostrich tendon tissue, turkey tendon tissue and alpaca tendon tissue is selected as a raw material to prepare tendon gelatin.

[0042] Furthermore, the environment in which the animal lives is also an important factor affecting the content of proline and hydroxyproline in gelatin. In one embodiment of the present invention, ostrich tendon tissue is selected, and the ostrich tendon tissue is tendon tissue from African ostrich. The raw material is commercially available from Shilichan Nan'an Shilichan Catering Service Co., Ltd. The ostrich lives in a tropical environment, and its normal body temperature can reach 40°C, so that the content of proline and hydroxyproline in ostrich gelatin is higher than that of gelatin from animals such as pigs and cattle.

[0043] The bio-hot melt adhesive prepared by the present invention can be used as a drug delivery carrier (a local delivery carrier or a sustained-release delivery carrier), for example, by mixing various drugs for delivery.

[0044] The bio-hot melt adhesive prepared by the present invention can be used as a drug delivery carrier to deliver a variety of drugs. The present invention has no particular restrictions on drugs, such as anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, biological agents, fibroblast growth factor, vascular endothelial cell growth factor, hepatocyte growth factor, contrast agents, etc.

[0045] When the biological hot melt adhesive prepared by the present invention is used, the biological hot melt adhesive is first heated to above 37.5°C, preferably 40-50°C, and after the hot melt adhesive is dissolved, it is mixed with a drug and applied to the human body. When the hot melt adhesive reaches the human body temperature of about 37°C, it solidifies, thereby avoiding the problem that the existing gelatin liquid cannot cool and solidify into glue when it contacts the tissue surface, and cannot achieve the effects of wound repair and the like.

[0046] Example 1

[0047] Pork tendon gelatin, the gelatin extraction method is as follows:

[0048] S1: The tendon tissue was removed from the pig leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water, and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then defatted with 5 times the volume of the tissue ethanol for 24 hours to obtain defatted pig tendon tissue.

[0049] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0050] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0051] Add acetic acid to water to obtain an acidifier with an acetic acid content of 20 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 48 hours. Wash the acidified foamed tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0052] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0053] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 6 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0054] The filtrate was centrifuged at 5000 rpm for 10 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0055] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0056] Take 300 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 300 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0057] Example 2

[0058] The gelatin extraction method of beef tendon gelatin is as follows:

[0059] S1: The tendon tissue was removed from the cattle leg, cleaned, and the fat, muscle, fascia, etc. on the surface were removed; the tendon tissue was repeatedly cleaned with pure water, and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then defatted with ether 5 times the volume of the tissue for 24 hours to obtain defatted cattle tendon tissue.

[0060] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent. The treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0061] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0062] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 20 mL / L. Take out 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 12 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0063] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0064] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 60°C to open the triple helix structure of collagen, heat for 4 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0065] The filtrate was centrifuged at 5000 rpm for 3 min in a high-speed centrifuge, and the supernatant filtrate was collected and impurities were finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0066] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0067] S3: Take 200 mg of freeze-dried tendon gelatin sponge solid and put it into a 5 mL ep tube. Then add 1 mL PBS and heat it at 60°C to dissolve. The gelatin concentration is 200 mg / mL. Heat it for 1 hour to fully dissolve and then perform ultrasonic defoaming. Finally, transfer it to a 2.5 mL syringe while hot and wait for it to solidify into gel at room temperature of 25°C. Seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Before use, put it in a water bath and heat it to 50°C to make it into a sol state.

[0068] Example 3

[0069] Camel tendon gelatin, the gelatin extraction method is as follows:

[0070] S1 The tendon tissue was removed from the camel leg, cleaned, and the fat, muscle, fascia, etc. on the surface were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then defatted with acetone 5 times the volume of the tissue for 24 hours to obtain the defatted camel tendon tissue.

[0071] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0072] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0073] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 30 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0074] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0075] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 70°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0076] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0077] Take 300 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 100 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set aside, put it in a water bath and heat to 50 ° C before use, and use it in a sol state.

[0078] Example 4

[0079] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0080] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0081] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0082] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0083] Add acetic acid to water to obtain an acidifying solution with an acetic acid content of 30 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifying solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0084] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0085] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0086] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0087] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0088] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0089] Example 5

[0090] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0091] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0092] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0093] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0094] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0095] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0096] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 70°C to open the triple helix structure of collagen, heat for 3 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0097] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0098] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0099] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0100] Example 6

[0101] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0102] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0103] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0104] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0105] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0106] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0107] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 60°C to open the triple helix structure of collagen, heat for 4 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0108] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0109] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0110] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0111] Example 7

[0112] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0113] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0114] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0115] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0116] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0117] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0118] S2: Add 1 volume of distilled water to the homogenate, stir and heat at 70°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0119] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0120] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0121] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0122] Example 8

[0123] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0124] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0125] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0126] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0127] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0128] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0129] S2: Add 1 volume of distilled water to the homogenate, stir and heat at 60°C to open the triple helix structure of collagen, heat for 3 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0130] The filtrate was centrifuged at 10000 rpm for 5 min using a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens. The filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent. The dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge solid (such as Figure 1 ).

[0131] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0132] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0133] Example 9

[0134] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0135] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0136] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0137] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0138] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0139] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0140] S2: Add 1 volume of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 4 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0141] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0142] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0143] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0144] Example 10

[0145] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0146] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0147] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0148] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0149] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0150] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0151] S2: Add 2 volumes of distilled water to the homogenate, stir and heat at 60°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0152] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0153] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0154] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0155] Embodiment 11

[0156] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0157] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0158] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0159] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0160] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0161] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0162] S2: Add 2 volumes of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 3 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0163] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0164] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0165] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0166] Example 12

[0167] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0168] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then degreased with acetone 5 times the volume of the tissue for 24 hours.

[0169] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -20°C for 2 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0170] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0171] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 40 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 4°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0172] The washed tendon tissue was broken and homogenized using a biological homogenizer (load speed 18000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0173] S2: Add 2 volumes of distilled water to the homogenate, stir and heat at 70°C to open the triple helix structure of collagen, heat for 4 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0174] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -80°C to obtain a tendon gelatin sponge-like solid.

[0175] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0176] Take 100 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 100 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0177] Embodiment 13

[0178] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0179] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then defatted with ether 5 times the volume of the tissue for 24 hours.

[0180] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -60°C for 24 hours; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0181] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0182] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 20 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 0°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0183] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 15000 rpm / min, homogenization time 10 s) to obtain a homogenate.

[0184] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0185] The filtrate was centrifuged at 15000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -40°C to obtain a tendon gelatin sponge-like solid.

[0186] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0187] Take 200 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 200 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0188] Embodiment 14

[0189] Ostrich tendon gelatin, the gelatin extraction method is as follows:

[0190] S1 The tendon tissue was removed from the ostrich leg, cleaned, and the surface fat, muscle, fascia, etc. were removed; the tendon tissue was repeatedly cleaned with pure water and immersed in physiological saline at 4°C for 24 hours; the tendon tissue was cleaned with pure water again, and then defatted with 5 times the volume of the tissue in ethanol for 24 hours.

[0191] The defatted tendon tissue was rinsed with pure water and immersed in pure water at 4°C for 24 hours to remove the defatted organic solvent; the treated tendon tissue was filtered and dehydrated, and then frozen at -80°C for 4 days; the frozen tendon tissue was taken out and sliced ​​into 5mm thick slices with a meat slicer, and then crushed to a volume less than 125mm 3 of fragments.

[0192] The mass volume ratio of the crushed tendon tissue fragments to the sodium hydroxide solution is 20g:1L; add 2g / L sodium hydroxide solution to the crushed animal tendon tissue fragments, soak at 4°C for 24 hours, and then wash and filter.

[0193] Add acetic acid to water to obtain an acidifier solution with an acetic acid content of 20 mL / L. Take 20 g of the filtered tendon tissue and soak it in 1 L of the acidifier solution. Acidify at 2°C for 24 hours. Wash the acidified soaked tissue with pure water until the washing solution becomes neutral, and filter it using a 100-mesh nylon filter.

[0194] The washed and filtered tendon tissue was broken and homogenized using a biological homogenizer (load speed 16000 rpm / min, homogenization time 30 s) to obtain a homogenate.

[0195] S2: Add 0.5 times the volume of distilled water to the homogenate, stir and heat at 50°C to open the triple helix structure of collagen, heat for 2 hours to obtain a gelatin-rich extract, cool to room temperature 25°C for use; use a 100-mesh nylon filter for preliminary filtration, and then use a 250-mesh nylon filter for further filtration.

[0196] The filtrate was centrifuged at 10000 rpm for 5 min in a high-speed centrifuge, and the supernatant filtrate was collected and finely filtered using two 250-mesh nylon filter screens; the filtrate was placed in a dialysis bag (molecular weight cutoff 3500) and dialyzed for 3 days to filter out trace amounts of degreasing solvent and acidifying agent; the dialyzed solution was freeze-dried at -60°C to obtain a tendon gelatin sponge-like solid.

[0197] The biological hot melt adhesive is prepared using the tendon gelatin prepared above, and the specific preparation method is as follows:

[0198] Take 100 mg of S3 freeze-dried to obtain tendon gelatin sponge solid, put it into a 5 mL ep tube, then add 1 mL PBS, heat at 60 ° C to dissolve, the gelatin concentration is 100 mg / mL, heat for 1 hour to fully dissolve, then ultrasonically defoam, finally transfer to a 2.5 mL syringe while hot, and wait for solidification into gel at room temperature 25 ° C; seal the syringe (containing 1 mL hot melt adhesive) and set it aside. Put it in a water bath and heat it to 50 ° C before use to make it into a sol state.

[0199] Comparative Example 1

[0200] Commercially available porcine skin gelatin, specifically porcine skin gelatin (PG) purchased from Sigma-Aldrich.

[0201] Comparative Example 2

[0202] Commercially available bovine bone gelatin, specifically bovine bone gelatin (BG) purchased from Admas.

[0203] Comparative Example 3

[0204] Commercially available fish skin gelatin, specifically fish skin gelatin (FG) purchased from Admas.

[0205] Comparative Example 4

[0206] The same as Example 8, except that the volume of distilled water added in step S2 is changed, specifically, one-third of the volume of distilled water is added to the homogenate.

[0207] Comparative Example 5

[0208] The same as Example 8, except that the acidification time is changed to 6 hours.

[0209] Comparative Example 6

[0210] The same as Example 8, except that in step S2, after adding distilled water, the temperature of stirring and heating to hydrolyze the collagen component is changed. In this comparative example, the heating and hydrolysis temperature is 80°C.

[0211] Comparative Example 7

[0212] The same as Example 8, except that the time for stirring and heating to hydrolyze the collagen components after adding distilled water in step S2, that is, the extraction time, is changed. The extraction time in this comparative example is 6 hours.

[0213] Test Case

[0214] (1) Gelatin performance test

[0215] The tendon gelatin (TG) prepared in Example 8 of the present invention and the commercially available gelatin of Comparative Examples 1-3 were subjected to characterization and analysis such as polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), gel permeation chromatography (GPC), automatic amino acid analyzer, infrared spectroscopy (FT-IR), and ultraviolet spectroscopy (UV).

[0216] (1-1) SDS-PAGE spectrum analysis

[0217] 8% separation gel and 5% concentrating gel were used for SDS-PAGE electrophoresis, Coomassie Brilliant Blue R-250 staining, sample concentration was 5 mg / mL, and the sample volume was 20 μL. Figure 2 As shown. As can be seen from the figure, the peptide chains of the tendon gelatin extracted using the present invention are more concentrated in the high molecular weight area, containing at least two α chains (α1 and α2) and dimers (β chains), which are similar to type I collagen. Compared with PG (pigskin gelatin of Comparative Example 1), FG (fish skin gelatin of Comparative Example 3) and BG (bovine bone gelatin of Comparative Example 2) sold on the market, TG (Example 8 of the present invention) has an extremely high content of dimer β chains, which shows that the use of the present invention to extract gelatin can control the degree of hydrolysis and inhibit the reduction of molecular weight. The molecular weight distribution of gelatin determines the gel strength and thermal transition temperature of gelatin. The higher the content of β and α subunits, the better the gel properties of gelatin.

[0218] (1-2) Circular dichroism spectrum

[0219] The secondary structure of gelatin was evaluated using a circular dichroism spectrometer. The tendon gelatin prepared in Example 8 was heated after adding water to obtain a 0.1 mg / mL tendon gelatin solution. Similarly, the gelatin of Comparative Examples 1-3 was heated after adding water to obtain a 0.1 mg / mL gelatin solution. 400 μL of the gelatin solutions of the examples and comparative examples 1-3 were respectively taken and placed in a cuvette for testing, and the absorbance at wavelengths from 190 nm to 250 nm was measured. The α-helix structure has a double negative peak at 222 nm and 208 nm, and a positive peak near 190 nm; the β-fold has a negative peak at 217-218 nm, and a strong positive peak at 195-198 nm; the random coil conformation has a negative peak near 198 nm, and a small and wide positive peak near 220 nm. Figure 3 As shown, all gelatins have no α-helical structure, which indicates that the triple helical structure of collagen is destroyed and all the products obtained are unwinding products. The extracted tendon gelatin has a smaller positive peak at 193nm than the gelatin sold on the market, but a larger negative peak at 198nm, which indicates that the extracted tendon gelatin has a larger molecular weight, such as the α chain and dimer (β chain) form a random coil conformation.

[0220] (1-3) GPC molecular weight determination

[0221] The gelatin of Example 8 and Comparative Examples 1-3 was dissolved in sodium chloride solution, and the sample concentration was 5 mg / mL. After filtration with a 0.22 μm filter membrane, ultrasonic degassing was performed, and room temperature must be restored after ultrasonication for use. The calibration curve has a total of 8 molecular weights, and 2 standard samples with large molecular weight differences are matched together, for a total of 4 standard samples. The molecular weight was determined using a gel permeation chromatograph (model: HLC-8320GPCEcoSEC), and the chromatographic column was first rinsed with a mobile phase (flow rate of 0.1 mL / min) to ensure that there were no impurities in the chromatographic column to interfere with the experimental results. The molecular weights of TG and commercially available gelatin are shown in Table 1. As can be seen from the table, the molecular weight of commercially available gelatin is 60,000-80,000, while the molecular weight of TG reaches 180,000, which is almost three times the molecular weight of commercially available gelatin. This also proves that the reduction in the molecular weight of gelatin can be controlled by using the extraction method of the present invention.

[0222] Table 1. Molecular weight of TG extracted by the present invention and gelatin sold on the market

[0223] Abbreviation M Mw / Mn PG <![CDATA[6.1×10 4 (±3.7%)]]> 2.1 FG <![CDATA[7.8×10 4 (±3.4%)]]> 1.5 BG <![CDATA[7.1×10 4 (±4.2%)]]> 1.9 TG <![CDATA[1.8×10 5 (±2.6%)]]> 2.2

[0224] In the table: Mw is the weight average molecular weight; Mn is the number average molecular weight.

[0225] (1-4) Determination of amino acid composition

[0226] The amino acid composition of each gelatin was analyzed using a high performance liquid chromatograph for hydrolysis amino acid analysis. As can be seen from Table 2, the content of imino acids (proline and hydroxyproline) in TG was 27.32%, which is higher than that of gelatin sold on the market.

[0227] Table 2. Amino acid composition of gelatin (content g / 100g)

[0228]

[0229]

[0230] (1-5) FTIR spectrum

[0231] Use ultrapure water to dissolve gelatin, the sample concentration is 10mg / mL. Pour the gelatin solution into a centrifuge tube and freeze-dry to obtain a gelatin sponge film. During the test, place the sample on the BRUKER Tensor II test bench, the scanning area is 500~4500nm, and the number of scans is 64. Figure 4 As shown in Figure 2, the infrared spectrum of commercially available gelatin is similar to that of TG amide A, which appears at a lower frequency (3306 cm -1 ), indicating that more NH groups are involved in the formation of hydrogen bonds; the amide B peak occurs at a lower wave number (2932cm -1), indicating that there are more CH2 groups cross-linking between peptide chains; the frequencies of amide I peak and II peak are higher (1645cm -1 and 1550cm -1 ), indicating a higher degree of cross-linking between molecules; the amide III peak is related to the triple helix structure, and its higher amplitude indicates a more ordered molecular structure.

[0232] (1-6) UV spectrum

[0233] The content of phenylalanine (phe) and tyrosine (tyr) containing benzene amino acids was analyzed using a UV-visible spectrophotometer. 400 μL of 0.1 mg / mL tendon gelatin, pig skin gelatin, bovine bone gelatin and fish skin gelatin were taken into a cuvette for testing, and the absorbance value near the wavelength of 280 nm was measured. Figure 5 As shown in the figure, all gelatins have strong UV absorption peaks at 220-230nm, indicating that these gelatin molecules contain amino acids such as phe and tyr. The extracted tendon gelatin has a larger absorption peak than the gelatin sold on the market (PG, FG and BG), indicating that it contains more phenyl amino acids, which is consistent with the results of the amino acid composition determination.

[0234] (2) Performance evaluation of bio-hot melt adhesive

[0235] (2-1) Thermal transition temperature measurement

[0236] Rheological measurements were performed using a rheometer (DHR3, TA Instruments, USA). The bio-hot melt adhesive (Example 8) and the gelatin of Comparative Examples 1-3 (all with a concentration of 20 w / v%) dissolved in phosphate buffered saline were heated to 60°C, and the solution was placed on the stage of the rheometer using a pipette, and a fixture with a diameter of 40 mm was set at a gap of 500 μm. After removing the excess solution, the edges were sealed with silicone oil, and a temperature scan measurement from 25°C to 45°C was started at a rate of 1°C / min. The measurement was carried out at a frequency of 10 rad / s and a strain of 1%. The intersection of the storage modulus (G′) and the loss modulus (G″) curves was taken as the gel-sol transition temperature. Figure 6 As shown, the bio-hot melt adhesive has extremely high thermal stability, with a thermal transition temperature of 42°C, far exceeding the body temperature of 37°C, and more than 10°C higher than the thermal transition temperature of gelatin sold on the market. This ensures that the bio-hot melt adhesive can be turned into a liquid with good fluidity when heated to 45-50°C, and can be quickly solidified into glue when cooled to 37°C body temperature.

[0237] The thermal transition temperature test results of Examples 4-12 are shown in Table 3.

[0238] Table 3 Comparison of Example Parameters and Thermal Transition Temperatures

[0239] serial number Material-liquid ratio Extraction temperature ℃ Extraction time Thermal transition temperature℃ Example 4 2:1 50 2 44 Example 5 2:1 70 3 38 Example 6 2:1 60 4 41 Example 7 1:1 70 2 39 Example 8 1:1 60 3 42 Example 9 1:1 50 4 42.5 Example 10 1:2 60 2 43 Embodiment 11 1:2 50 3 43 Example 12 1:2 70 4 37.5 Comparative Example 4 3:1 60 3 Insoluble in PBS Comparative Example 5 1:1 60 3 Insoluble in PBS Comparative Example 6 1:1 80 3 32 Comparative Example 7 1:1 60 6 35

[0240] As can be seen from Table 3, the thermal transition temperatures of the hot melt adhesives prepared in Examples 4-12 are all greater than normal human body temperature and can be heated to a liquid state. At the same time, they solidify when the temperature drops to normal human body temperature, avoiding the problem that traditional gelatin liquid cannot solidify at normal human body temperature. At the same time, it can be seen from Comparative Examples 6-7 that when the extraction time or temperature changes, the thermal transition temperature of the prepared hot melt adhesive is low, and it can be seen from Comparative Examples 4-5 that when the volume of distilled water or the acidification time is changed, the prepared hydrogel is insoluble in PBS and a hot melt adhesive cannot be obtained.

[0241] (2-2) Evaluation of the stability of bio-hot melt adhesive at 37°C

[0242] After the bio-hot melt adhesive prepared in Example 8 of the present invention was solidified, it was placed in a 37°C water bath and heated for 12 hours, and then taken out and observed. Figure 7 As shown in the figure (a), the bio-hot melt adhesive does not melt after being heated at body temperature (37°C) for a long time, indicating that it has excellent stability below the thermal transition temperature (42°C). This characteristic is mainly due to the strong hydrogen bonding of the high molecular weight α chain and the dimer β chain below the thermal transition temperature. Only when the temperature is higher than 42°C can the strong hydrogen bonding be broken and the adhesive has fluidity.

[0243] (2-3) Investigation of injectability of bio-hot melt adhesive

[0244] The bio-hot melt adhesive prepared in Example 8 of the present invention was put into a syringe and solidified at room temperature of 25°C. Then, the front end of the syringe was sealed and placed in a 50°C water bath for 30 seconds to heat, and then quickly taken out for injection. Figure 7 As shown in Figure b), when the bio-hot melt adhesive is heated above the thermal transition temperature, the strong hydrogen bonds between the gelatin molecules are destroyed, showing excellent injectability and can be injected with a 27G needle.

[0245] (2-4) Evaluation of bonding strength

[0246] The adhesion of bio-hot melt adhesive (TG) to various tissues was evaluated according to ASTM F-2258-05 (tissue adhesion strength performance test) standard.

[0247] Cut the animal tissues (pig blood vessels, pig lungs, pig skin) into 2.5cm*2.5cm squares, and fix two pieces of animal tissues on a clamp (the size is the same as the tissue size) and a heating plate (the temperature is controlled to be constant at 37°C) respectively. Preheat the biological hot melt adhesive (TG) prepared in Example 8 to 50°C, squeeze out 0.2mL of molten hot melt adhesive and evenly apply it on the tissue surface on the heating plate, quickly press down the clamp, and press with a force of 2N for 3min. After the time is up, pull up the clamp at a speed of 10mm / min to measure the bonding strength of the hot melt adhesive. As a control, the bonding strength of commercially available pig skin gelatin (PG) solution was also evaluated. Figure 8As shown in Figure a), when the temperature of biological hot melt glue (TG) drops from 50℃ to 37℃, it changes from a fluid to a solid, and its viscosity also increases significantly, which can firmly bond tissues together. In contrast, commercially available PG cannot solidify at 37℃ and remains in liquid form, which cannot bond tissues. Figure 8 The adhesion strength was evaluated by the standard of Figure b), and it was found that the adhesion strength of biological hot melt adhesive (TG) to pig blood vessels, pig lungs, and pig skin reached 5.6KPa, 6.2KPa, and 3.6KPa, respectively. The adhesion of biological hot melt adhesive (TG) to internal tissues is stronger than that to skin, which indicates that it has a stronger affinity for hydrophilic tissue surfaces. Commercially available PG cannot be cured at 37°C, and the tissue adhesion strength is at a lower level of ~0.4KPa.

[0248] The gelatin extracted from tendon tissue has a high content of proline and hydroxyproline. The present invention limits the source of tendon tissue and controls the degree of unwinding. The ostrich tendon tissue is used to extract gelatin, which can retain a large amount of high-molecular α-chain dimers (β-chains). The tendon gelatin extracted using a mild process has excellent solubility. The high-concentration tendon gelatin has a super-body temperature thermal transition temperature (up to 42°C) and can be injected by heating to 40-50°C. The molten sol has excellent tissue viscosity. When the surface of the contacting tissue is cooled to 37 degrees, it will gradually solidify into a hydrogel and adhere firmly. These properties of the prepared ostrich tendon gelatin are very suitable for use as a single-component biological hot melt adhesive.

[0249] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A method for preparing tendon gelatin, characterized in that: The preparation method is as follows: S1: pre-treating the animal tendon tissue, freezing it, and crushing it, soaking the crushed animal tendon tissue in a sodium hydroxide solution, washing and filtering it, adding an acidifier to the filtered animal tendon tissue for acidification, washing, filtering it, and then crushing and homogenizing it to obtain a tissue homogenate; S2: Add water to the tissue homogenate, heat, stir and extract, filter and centrifuge, dialyze the supernatant, filter again, and freeze-dry the filtrate to obtain tendon gelatin.

2. The preparation method according to claim 1, characterized in that: In step S1, the animal tendon tissue includes at least one of pig tendon tissue, horse tendon tissue, cattle tendon tissue, sheep tendon tissue, donkey tendon tissue, camel tendon tissue, deer tendon tissue, ostrich tendon tissue, turkey tendon tissue, and alpaca tendon tissue.

3. The preparation method according to claim 1, characterized in that: In step S1, the pretreatment is to wash the animal tendon tissue and then add an organic solvent to degrease it to obtain defatted animal tendon tissue; the organic solvent includes one or more of ethanol, ether, and acetone.

4. The preparation method according to claim 1, characterized in that: In step S1, the freezing temperature is -20 to -80°C and the time is 2 hours to 4 days; The crushing refers to crushing the animal tendon into blocks; The method also includes washing and filtering and dehydrating the pretreated animal tendon tissue before freezing; The concentration of the sodium hydroxide solution is 2 g / L; the mass volume ratio of the crushed tendon tissue to the sodium hydroxide solution is 20 g:1 L; the immersion temperature is 4° C. and the immersion time is 24 h.

5. The preparation method according to claim 1, characterized in that: In step S1, the mass volume ratio of the filtered animal tendon tissue to the acidifier is 20g:1L; the acidifier is obtained by adding acetic acid to water; the content of acetic acid in the acidifier is 20-40mL / L; the acidification temperature is 0-4°C, and the time is 12-48h; The speed of the crushing homogenate is 15000-18000 r / min, and the time is 10-30 s.

6. The preparation method according to claim 1, characterized in that: In step S2, the volume ratio of the tissue homogenate to water is 1:0.5 to 1:2; the extraction temperature is 50 to 70°C, and the time is 2 to 4 hours; the centrifugal speed is 5000 to 15000 r / min, and the time is 3 to 10 minutes.

7. The preparation method according to claim 1, characterized in that: In step S2, the freeze-drying temperature is -40 to -80°C.

8. Tendon gelatin prepared by the preparation method according to any one of claims 1 to 7.

9. A high thermal transition bio-hot melt adhesive prepared from the gelatin according to claim 8.

10. A method for preparing the high thermal conversion bio-hot melt adhesive according to claim 9, characterized in that: The preparation method comprises: dissolving the tendon gelatin described in claim 8 or the tendon gelatin prepared by the preparation method of any one of claims 1 to 7 in a buffer solution, heating, ultrasonically defoaming, transferring to a syringe, cooling and solidifying into a gel, and obtaining a high-heat-converted biological hot melt adhesive; The buffer solution is PBS buffer solution, the mass volume ratio of the tendon gelatin to the buffer solution is 100-300 mg:1 mL; the heating temperature is 60° C., and the heating time is 1 hour; the thermal transition temperature of the high thermal transition biological hot melt adhesive is 37.5-45° C.

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