( patent document 1 ) korean patent publication no . 2012 - 0125465 ( patent application no . 2012 - 7018109 , title : dry powder fibrin sealant ) has been filed . therefore , the present invention has been made in view of the above - mentioned problems , and the present invention provides a tissue sealant which collagen and fibrin are mixed and a method for preparing the same , wherein a first purpose of the present invention is to include the steps of : mixing a first material using fibrinogen and aprotinin ; mixing a second material using thrombin , calcium chloride , and collagen ; and mixing the first material and the second material with each other to prepare a third material ; according to a second purpose of the present invention , it was verified that , as a result of comparison of physical strength , a sealant containing collagen showed high strength ; according to a third purpose of the present invention , it was verified that , as a result of long - term / short - term degradability testing , a sealant containing collagen showed low degradability ; according to a fourth purpose of the present invention , it was verified that , through electron microscopic observation , collagen and fibrinogen are combined to show a stable structure ; according to a fifth purpose of the present invention , it was verified that , as a result of comparison of growth and viability using chondrocytes , osteoblasts , and adipose - derived cells , a structure containing collagen showed a good growth rate and high viability ; according to a sixth purpose of the present invention , it was verified that the inclusion of collagen maintains high strength and a stable structure and supplies a material having affinities with cells / blood , thereby greatly helping the regeneration of deleted / damaged region ; a seventh purpose of the present invention is to activate platelets included in the blood to induce the tissue regeneration ; and an eighth purpose of the present invention is to significantly improve quality and reliability of products , thereby satisfying various needs of consumers who are users , thus giving a good impression . in accordance with an aspect of the present invention , there is provided a method for preparing a tissue sealant in which collagen and fibrin are mixed , the method including : mixing fibrinogen and aprotinin to prepare a first material ; mixing thrombin , calcium chloride , and collagen to prepare a second material ; and mixing the first material and the second material with each other to prepare a third material . in accordance with another aspect of the present invention , there is provided a tissue sealant in which collagen and fibrin are mixed , the tissue sealant being prepared by steps of : mixing fibrinogen and aprotinin to prepare a first material ; mixing thrombin , calcium chloride , and collagen to prepare a second material ; and mixing the first material and the second material with each other to prepare a third material . as set forth above , the present invention includes the steps of : mixing a first material using fibrinogen and aprotinin ; mixing a second material using thrombin , calcium chloride , and collagen ; and mixing the first material and the second material with each other to prepare a third material . according to the present invention having the foregoing technical feature , it was verified that , as a result of comparison of physical strength , a sealant containing collagen showed high strength . in addition , according to the present invention , it was verified that , as a result of long - term / short - term degradability testing , a sealant containing collagen showed low degradability . in addition , according to the present invention , it was verified that , through electron microscopic observation , collagen and fibrinogen are combined to show a stable structure . in addition , according to the present invention , it was verified that , as a result of comparison of growth and viability using chondrocytes , osteoblasts , and adipose - derived cells , a structure containing collagen showed a good growth rate and high viability . in addition , according to the present invention , it was verified that the inclusion of collagen maintains high strength and a stable structure and supplies a material having affinities with cells / blood , thereby greatly helping the generation of deleted / damaged region . in addition , the present invention is to activate platelets included in the blood to induce the tissue regeneration . the present invention can significantly improve quality and reliability of products through the foregoing effects , thereby satisfying various needs of consumers as users thereof , thus giving a good impression , and thus the present invention is very useful . hereinafter , preferred embodiments of the present invention for attaining the above effects will be described in detail with reference to the accompanying drawings . fig1 is an electron micrographic image of a tissue sealant in which collage and fibrin are mixed according to the present invention ( 20 , 000 ×, critical point drying ). fig2 is a conceptual diagram showing the binding of collagen and a cell . fig3 is a conceptual diagram showing the activation of platelets in collagen . fig4 is a comparison graph showing the degradation rates of tissue sealants in which collagen and fibrin are mixed according to the present invention . fig5 is a graph showing the proliferation rates of chondrocytes in tissue sealants in which collagen and fibrin are mixed according to the present invention . fig6 illustrates images confirming the proliferation and viability of chondrocytes in tissue sealants in which collagen and fibrin are mixed according to the present invention . fig7 is a graph showing the proliferation rates of osteoblasts in tissue sealants in which collagen and fibrin are mixed according to the present invention . fig8 illustrates images confirming the proliferation and viability of osteoblasts in tissue sealants in which collagen and fibrin are mixed according to the present invention . fig9 illustrates images confirming the proliferation and viability of adipose - derived cells in tissue sealants in which collagen and fibrin are mixed according to the present invention . fig1 is a diagram of a state in which a tissue sealant in which collagen and fibrin are mixed according to the present invention is loaded in a two - way syringe . a tissue sealant in which collagen and fibrin are mixed and a method for preparing the same according to the present invention are as shown in fig1 to 10 . in the following descriptions , when it is determined that detailed descriptions of known functions or constitutions associated with the present invention obscure the gist of the present invention , detailed descriptions thereof will be omitted . in addition , the terms to be later described are defined in consideration of functions in the present invention , and thus the definitions of the terms are to be interpreted throughout the present specification since the terms may be interpreted by the intention of the producer or custom . first , the present invention includes a step of mixing a first material using fibrinogen and aprotinin . in addition , the present invention includes a step of mixing a second material using thrombin , calcium chloride , and collagen . in addition , the present invention includes a step of mixing the first material and the second material with each other to prepare a third material , and thus a tissue sealant in which collagen and fibrinogen are mixed is prepared . especially , according to the present invention , preferably , the fibrinogen has a concentration of 65 - 130 mg / ml and the aprotinin has a concentration of 1 , 000 - 3 , 000 kiu / ml . in addition , preferably , the thrombin has a concentration of 40 - 600 u / ml , the calcium chloride has a concentration of 4 - 140 mmol / ml , and the collagen has a concentration of no more than 60 mg / ml . here , the concentration of the fibrinogen is preferably 65 - 130 mg / ml . less than 65 mg / ml of fibrinogen weakens physical strength , and more than 130 mg / ml of fibrinogen leads to the densification of the physical structure , resulting in reducing the pore sizes , thereby inhibiting cellular activity , and thus the concentration of fibrinogen is preferably 65 - 130 mg / ml . in addition , the concentration of the aprotinin is preferably 1 , 000 - 3 , 000 kiu / ml . less than 1 , 000 kiu / ml of aprotinin accelerates the degradation of a composition , and more than 3 , 000 kiu / ml of aprotinin increases the risk of causing anaphylaxis , and thus the concentration of aprotinin is preferably 1 , 000 - 3 , 000 kiu / ml . in addition , the concentration of the thrombin is preferably 40 - 600 kiu / ml . less than 40 u / ml of thrombin weakens the physical strength of a composition , and more than 60 u / ml of thrombin leads to the densification of the structure of the composition , which therefore has no affinity with cells and rapidly increases the gelation rate , failing to serve as a sealant in an applied region , and thus , the concentration of the thrombin is preferably 40 - 60 u / ml . in addition , the concentration of calcium chloride is preferably 4 - 140 mmol / ml . less than 4 mmol / ml of calcium chloride too slows down the gelation rate , and more than 140 mmol / ml of calcium chloride may have a bad influence on cells due to a high osmotic pressure , and thus the concentration of calcium chloride is preferably 4 - 140 mmol / ml . last , the concentration of the collagen is preferably no more than 60 mg / ml . especially , the preferable concentration of the collagen is 10 - 30 mg / ml . that is , less than 10 mg / ml of collagen weakens physical strength , and more than 30 mg / ml has a bad influence on degradability and a stable structure and has no affinity with cells and blood , and thus the concentration of the collagen is preferably 10 - 30 mg / ml . meanwhile , the method for preparing a tissue sealant in which collagen and fibrin are mixed according to the present invention will be specifically described as follows . first , a step of preparing a first material including fibrinogen and aprotinin is conducted . after that , a step of preparing a second material including thrombin , calcium chloride , and collagen is conducted . then , a step of putting the first material in one side of a two - way syringe and the second material in the other side of the two - way syringe and then mixing the first and second materials with each other is conducted , and thus , a tissue sealant in which collagen and fibrin are mixed is prepared . according to the present invention , aprotinin and calcium solutions are injected into the fibrinogen and the thrombin , respectively , and the thrombin is mixed with a collagen solution , and then the resultant solutions are loaded in the two - way syringe , thereby preparing a tissue sealant in which collagen and fibrin are mixed . according to the present invention , a tissue sealant in which collagen and fibrin are mixed can be prepared by going through the respective steps for preparing a tissue sealant in which collagen and fibrin are mixed . the tissue sealant in which collagen and fibrin are mixed and the method for preparing the same according to the present invention will be described by giving examples . in order to verify physical properties of the present invention , the maximum stress , the gel strength , and the tensile strength were checked using a physical property meter . 2 ) for components of the present invention , dried fibrinogen and thrombin of greenplast were dissolved in an aprotinin solution and a calcium solution added thereto , respectively . here , the thrombin solution was mixed with a 3 % collagen solution . the resultant solutions were loaded in a two - way syringe . 3 ) for the measurement of physical properties , each sample was put in a cylindrical - shaped mold ( φ12 × 15 mm ) to manufacture a form . 2 ) test items : maximum stress ( n ), gel strength ( g - cm ), tensile strength ( g / cm2 ) 3 ) test conditions : entry distance ( 7 . 5 mm ), table speed ( 50 mm / min ), maximum stress ( 10 kg ), adapter ( no . 1 φ20 mm ) in order to verify degradability of the composition of the present invention , the degradability of the fibrin glue product and the material for a predetermined period was checked . in the prior art , the greenplast product was used . for components of the present invention , dried fibrinogen and thrombin of greenplast were dissolved in an aprotinin solution and a calcium solution added thereto , respectively . here , the thrombin solution was mixed with a 3 % collagen solution . the resultant solutions were loaded in a two - way syringe . for the measurement of physical properties , each sample was put in a cylindrical - shaped mold ( φ8 × 5 mm ) to manufacture a form . two conditions were confirmed for the solvent . a condition of using only a deme medium and a condition of using a dmem medium containing liberase tm were confirmed ( the concentration of liberase tm was set to 10 ug / ml ). the sample was put in a 12 - well plate , and the degradation aspect was checked as a residue weight of the composition in the 2 - hour unit for 12 hours . it was verified that , in the condition of degradation through the enzyme treatment , over 90 % of the formulation of the prior art was degraded within 12 hours , and about 80 % of the composition of the present invention remained for 12 hours . in the dmed condition , the degradation was not verified for 12 hours . in the prior art , the greenplast product was used . for components of the present invention , dried fibrinogen and thrombin of greenplast were dissolved in an aprotinin solution and a calcium solution added thereto , respectively . here , the thrombin solution was mixed with a 3 % collagen solution . the resultant solutions were loaded in a two - way syringe . for the measurement of physical properties , each sample was put in a cylindrical - shaped mold ( φ12 × 15 mm ) to manufacture a form . dmem was used for the solvent , and the naked eye observation was conducted at 37 for one month . the composition of the present invention was observed for one month or longer , but the formulation of the prior art was degraded within three weeks . it was verified that the degradation period of the composition of the present invention was longer than that of the formulation of the prior art . the structure of the composition of the present invention was observed by an electron microscope . for the preparation of the composition , a fibrinogen solution of greenplast and a collagen - containing thrombin solution / calcium solution were prepared . the concentration of the collagen solution was 3 % ( w / v ). each of the prepared solutions was applied to a two - way syringe to be dispensed on trays for electron microscopic observation , and then gelated . the composition of the present invention was dried at the critical point , and then observed by an electron microscope . the critical point drying of the composition was conducted through alcohol treatment in a critical point drier ( hitachi , hcp - 2 ). the sample for electron microscopic observation was cut and gold - coated , and then observed using sem ( hitachi , s3500 ). the electron microscopic analysis was conducted at the magnification of 20 , 000 . as a result of observing in the electron microscope at the magnification of 20 , 000 , it was verified that collagen and fibrin cross - linked with each other . the fibrous structure of collagen was also observed . it can be anticipated that such a material cross - linkage enhances physical properties . in order to verify proliferation and viability of chondrocytes in the composition of the present invention , cck - 8 assay and calcein - am & amp ; ethd - 1 staining were used . chondrocytes of animals excluding human were used . the number of cells was 12 , 000 , 000 in the composition mixture liquid . for the composition , greenplast , which is the fibrin glue product , was mixed with 3 % and 6 % of collagen , respectively . the composition was prepared by dissolving dried fibrinogen of greenplast in 1 ml of a solution containing chondrocytes and mixing the thrombin / calcium solution with 1 ml of a collagen solution with each concentration . the fibrinogen solution containing chondrocytes and the thrombin solution containing collagen were used to prepare a total of 2 ml of collagen - fibrin solution . for culture , the prepared collagen - fibrin component was dispensed in a 24 - well plate at 0 . 2 ml for each well , and observation was conducted for 20 days while the dmem medium was exchanged every 2 - 3 days . the medium was removed from each well ( 24 - well plate ) containing a cultured material . 1 ml of a new medium was put in each well . a cck - 8 ( dojindo , ck04 - 11 ) reagent was added in 100 ul for each well , which corresponds to 10 % of the volume of the medium , followed by a reaction in 5 % co2 incubator at 37 for 3 hours . upon completion of the reaction , the absorbance ( 450 nm ) of the reaction liquid was read using a microplate reader . the cell proliferation for each culture period was verified using the measured od value . a working solution was prepared by mixing a buffer solution with 2 μm and 4 μm of calcein am and ethd - 1 of the live / deadviability / cytotoxicity assay kit ( invitrogen , l3224 ), respectively . the cultured material was transferred to a new 24 - well plate , and then 1 ml of the working solution was put therein , followed by a reaction in the condition where the light is blocked for 20 minutes . after that , live cells and dead cells were observed using a fluorescent microscope . the live cells were observed as green and the dead cells were observed as red . in order to verify proliferation and viability of osteoblasts in the composition of the present invention , cck - 8 assay and calcein - am & amp ; ethd - 1 staining were used . osteoblasts of animals excluding human were used . the number of cells was 12 , 000 , 000 in the composition mixture liquid . the composition was configured by mixing greenplast , which is the fibrin glue product , with 3 % and 6 % of collagen , respectively . the composition was prepared by dissolving dried fibrinogen of greenplast in 1 ml of a solution containing chondrocytes and mixing the thrombin / calcium solution with 1 ml of a collagen solution with each concentration . the fibrinogen solution containing osteoblasts and the thrombin solution containing collagen were use to prepare a total of 2 ml of collagen - fibrin solution . for culture , the prepared collagen - fibrin component was dispensed in a 24 - well plate at 0 . 2 ml for each well , and observation was conducted for 20 days while the α - dmem medium was exchanged every 2 - 3 days . the medium containing cultured material was removed from each well ( 24 - well plate ). 1 ml of a new medium was put in each well . a cck - 8 ( dojindo , ck04 - 11 ) reagent was added in 100 ul for each well , which corresponds to 10 % of the volume of the medium , followed by a reaction in 5 % co2 incubator at 37 for 3 hours . upon completion of the reaction , the absorbance ( 450 nm ) of the reaction liquid was read using a microplate reader . the cell proliferation for each culture period was verified using the measured od value . a working solution was prepared by mixing a buffer solution with 2 μm and 4 μm of calcein am and ethd - 1 of the live / deadviability / cytotoxicity assay kit ( invitrogen , l3224 ), respectively . the cultured material was transferred to a new 24 - well plate , and then 1 ml of the working solution was put therein , followed by a reaction in the condition where the light is blocked for 20 minutes . after that , live cells and dead cells were observed using a fluorescent microscope . in order to verify proliferation and viability of adipose - derived cells in the composition of the present invention , cck - 8 assay and calcein - am & amp ; ethd - 1 staining were used . adipose - derived cells were used . the number of cells was 12 , 000 , 000 in the composition mixture liquid . the composition was configured by mixing greenplast , which is the fibrin glue product , with 3 % of collagen , respectively . the composition was prepared by dissolving dried fibrinogen of greenplast in 1 ml of a solution containing adipose - derived cells and mixing the thrombin / calcium solution with 1 ml of a collagen solution with each concentration . the fibrinogen solution containing adipose - derived cells and the thrombin solution containing collagen were used to prepare a total of 2 ml of collagen - fibrin solution . for culture , the prepared collagen - fibrin component was dispensed in a 24 - well plate at 0 . 2 ml for each well , and observation was conducted for 20 days while the dmem medium was exchanged every 2 - 3 days . a working solution was prepared by mixing pbs with 2 μm and 4 μm of calcein am and ethd - 1 of the live / deadviability / cytotoxicity assay kit ( invitrogen , l3224 ), respectively . the cultured material was transferred to a new 24 - well plate , and then 1 ml of the working solution was put therein , followed by a reaction in the condition where the light is blocked for 20 minutes . after that , live cells and dead cells were observed using a fluorescent microscope . meanwhile , the present invention can be modified in various ways and embodied in many different forms for the application of the foregoing feature . however , it shall be noted that it is not intended to limit the present invention to specific embodiments described in the detailed description , but intended to cover all the modifications , equivalents or substitutions belonging to the technical idea and technical scope of the present invention , which are defined by the accompanying claims . technical spirits of the tissue sealant in which collagen and fibrin are mixed and the method for preparing the same of the present invention are sufficiently worthy of protection since the same results are actually replicable , and , especially , the implementation of the present invention can promote the technical development and contribute to the industrial development .
the present invention relates to a tissue sealant in which collagen and fibrin are mixed and to a method for preparing same . to this end , the method of the present invention comprises the steps of : mixing a first material using fibrinogen and aprotinin ; mixing a second material using thrombin , calcium chloride , and collagen ; and preparing a third material by mixing the first material and the second material . the tissue sealant prepared by the method may supplement weaknesses , i . e . strength and degradability , of the fibrin sealant which is currently available in the commercial marketplace . further , the tissue sealant of the present invention is cytophilic and activates blood platelets contained in blood so as to induce tissue regeneration . thus , quality and reliability of products can be significantly improved so as to satisfy various needs of consumers who are users , thereby presenting good image .