TW200407392A - Injectable collagen implant and methods of preparation thereof - Google Patents

Abstract

The invention discloses methods for the preparation of injectable collagen implant. The present invention provides a simplified yet more efficient method of preparing native collagen fibrils from the bovine tendons. The methodology offers advantages over the known methods in terms of increased yields, higher purity, low immunogenecity and higher degree of biocompatibility, as well as a reduction in the production costs. This pioneering purification process allows the production of collagen fibrils with increased rigidity in structure, and the resulting materials were found to exhibit a more preserved native configurations, when compared to the production utilizing the conventional known methods. The collagen fibrils generated in accordance with the above method were further processed to collagen suspension by the addition of buffering agents. The collagen suspension then underwent homogenization followed by mechanical disruption, which miniaturizes the collagen fibrils and enables it to extrude through needles as fine as 30 gauges or even needles with a finer spectrum. Appropriate amount of dispensing agents was then added to prevent recoagulation of the collagen fibrils. This injectable collagen, unlike other known collagen implants, does not need to go through a reconstitution process after it is injected into the human body in order to regain its native configuration, meanwhile it was shown to be capable of providing a structure with improved durability. The collagen fibrils could be then cross-linked to one another to generate a structure with further improved mechanical properties either by means of radiation or by the utilization of a cross-linking agent. By mixing the non-cross linked and the cross-linked collagen fibrils with different ratio will in turn achieve an optimal efficacy for different tissue augementation purposes. The above described injectable collagen material may be utilized in various human tissues augmentations, such as skin, bladder, the vocal cord, breasts, genitals and the bones.

Description

200407392 V. Description of the invention (l) TECHNICAL FIELD The present invention relates to a method for preparing collagen, particularly to a method for preparing injectable collagen, and more particularly to a method for applying to soft and hard tissues of a living body. Preparation method of filled injectable collagen. BACKGROUND OF THE INVENTION Collagen is the most abundant protein in connective tissue, and is also one of the major constituents of the extracellular matrix. Due to its special triple helix (tr ipl e-hel ix) structure, collagen not only provides a skeleton of tissue, but also provides a very good growth environment for cells. Collagen is more biocompatible, biodegradable, non-toxic, and hypoallergenic, so it has a wide range of uses in life applications. Raw materials and products have various forms, compositions, functions and uses, and are a class of products with high economic value. In terms of biomedical materials, owing to the extremely low iramunogenicity of collagen, it is being widely used by Jinda + 々cong / μ in the repair and treatment of various tissues, including heart valves, burns and scald dressings , And the use of subcutaneous implants. In particular, the application of collagen to ~ π% of soft and hard tissue repair and filling is currently one of the most popular topics that everyone cares about. The traditional method of obtaining collagen is to use acidic 1 person & ^ long white enzyme decomposition

200407392 V. Description of the invention (2) Tissue fibers, so that the collagen is completely suspended in the solution, and then extract the pure collagen from it, and then control the pressure and temperature to restore the collagen fiber tissue. For example, USP No. 3, 949, 0 73 describes the preparation of a non-crosslinked collagen composition (trade name: Zyderm II) by a conventional method; and USP No. 4, 582, 640 describes the method A cross-linked collagen composition (trade name: Z yp 1 ast) was prepared. Since the above process is mainly suitable for using animal skins such as cowhide as the main source of collagen, and the skin additionally contains a fat layer and hair, it is necessary to add steps such as removing hair and processing a high fat layer before the purification step. In addition, when various organic solvents such as acetone are used to perform degreasing and swelling steps, since the chemical treatment is used, subsequent organic solvents remain, which becomes a very difficult problem to solve. Even residues of impurities such as epitopes cause unwanted immune responses. In view of the above, today's subject is how to obtain collagen quickly, efficiently, and in large quantities, using tendons as the main source when purifying collagen. Moreover, as far as possible, the traditional chemical purification method is not used, and the collagen can maintain the triple-helix structure at all times. This point is more important for the purpose of obtaining active collagen with a triple helix structure. In addition, how to efficiently remove various impurities and various pollutants in the process is also an important effort. In addition, after the above-mentioned collagen production process, in order to further obtain injectable collagen, it is necessary to further refine collagen fibers.

200407392 V. Description of the invention (3) The above-mentioned two types of collagen compositions (that is, U.S. patent U. s. Ρ · NO. 3,949,07 3 and U.S. patent usp. No. 4,582,64 0 Tutor) The 'cross-linked collagen composition will be stronger than non-cross-linked ones, but because they are obtained by traditional methods, that is, collagen that is recombined after decomposition', the common problem is that the structure is looser, Easily degradable and unable to resist migration, causing the collagen implanted in the affected area to undergo syneresis in the main shot or after injection, resulting in uneven implant viscosity and easy to be absorbed by decomposition And it cannot persist. One of the methods to improve the durability and reduce the phenomenon of desalting is to increase the concentration of collagen in the composition applied to the soft and hard tissue filling, but the difficulty of injecting the high concentration composition into the affected area. Quite high, many people have worked hard to solve this problem. For example, U.S. ρ. No. 4,8 0,3 0,7 U.S. patent suggests combining cross-linked injectable collagen with a liquid lubricant, but obviously cannot effectively solve the problem. The problem of pulp phenomenon; US Patent No. 4, 424, 208 describes a collagen composition containing parental injectable collagen and reconstituted collagen fibers, the durability of which has been improved , But the injectability of the injection syringe is not resolved. U.S. Patent No. 5, 42 8, 0 24 teaches homogenization to reduce the particle size of collagen fibers. After homogenization with an appropriate homogenizer, the average fiber size of non-crosslinked collagen can be reduced. Reduced by at least 25%, and the average fiber size of cross-linked collagen was reduced by at least 50 ° / 〇.

Page 12 200407392 V. Description of the invention (4) ~~ However, since the purification process still uses the traditional chemical method mentioned above, it still has the problems of complicated steps and residual organic solvents, and the collagen fiber particles still remain after a period of time. Will be re-assembled to form fiber particles of different sizes, or will cause the destruction of the homogeneity and stability of injectable collagen 'The above-mentioned several patents cannot successfully solve this problem, so it has become a direction to be urgently addressed today. In summary, the present inventors have proposed a novel method for preparing injectable collagen, which provides a high concentration, long-lasting, injectable collagen through a simple and harmless collagen purification and homogenization process. And injectable collagen with good preservability, and completed the present invention. In the present invention, the original collagen fibers are purified using beef tendon in a novel enzyme / acid washing / salt washing step. A collagen suspension suspension solution is prepared with a buffer solution, and this collagen suspension is homogenized with a homogenizer to obtain a preset collagen fiber size of 150 to 250 # m. This homogenized victorious protein suspension is then micronized to 50 ~ 100 0 // m with a crusher. This crushing and miniaturization step can be performed by, for example, μ crQ f u u d i c s M110EH or M110Y homogenizer, Brinkmann homogenizer, or virtis homogenizer. In addition, in order to prevent the collagen fiber particles from re-aggregating to change the consistency of the collagen fiber size, a dispersant can be further added to the collagen suspension to improve the uniformity and stability of the injectable primogenin. The dispersant includes, for example, gelatin, alginate, pectin, and the like. The gelatinous substance can help fix the position of the collagen fiber structure.

200407392 V. Description of the invention (5), it can successfully prevent the collagen fibers from agglomerating again, and destroy the uniformity and stability of the injectable collagen. Examples Hereinafter, the present invention will be further described by examples, but the present invention is not limited to these examples, and anyone who works based on the same spirit disclosed by the present invention should be included in the scope of the present invention. Example 1 Process for Purifying Collagen Raw materials are obtained from mammals, and include skin, tendons, blood vessels, dura mater, ligament, heart valve and heart valve. In this embodiment, we use beef tendon as the raw material for purifying collagen. Before initial cutting, excess tissue and fat from beef tendon must be removed. After the initial cut is completed, the beef tendon is washed with a first buffer solution such as an alcoholic aqueous solution at a low temperature, and then washed with a second buffer solution such as a neutral aqueous sodium acid / sodium chloride mixed aqueous solution. The washed beef tendons are placed in a freezer and frozen at -20 ° C. Then, the frozen beef tendon is sliced to obtain a slice of beef tendon having a predetermined thickness or weight. Taking this implementation example as an example, the weight of the beef tendon slice is about 100 grams. Then, 2 liters of 0.5 N acidic solution containing 0.5 g / L enzyme and 1 M salt (such as sodium chloride), including acetic acid, lactic acid, etc., were added to a stainless steel container containing beef tendon slices. Among them, the enzyme such as pepsin, papain, but because of pepsin

Page 14 200407392 V. Description of the invention (6) The element is more easily deactivated and can be easily removed from the original protein process. Therefore, in this embodiment, the gastric protein enzyme is the best. After proper mixing and enzyme digestion, and repeating the above steps, store the solution in a refrigerator at 0 ~ 4 ° C for at least 48 hours. After the enzyme reaction, the collagen solution is filtered with a filter cloth and the excess solution in the filter cake is squeezed out to squeeze the collagen sufficiently, so that the weight of the collagen falls within a predetermined range. Next, the dough-shaped collagen formed is thoroughly mixed and mixed with a 0.5 M acidic solution containing 1 M salts (such as sodium chloride), such as acetic acid, lactic acid, etc. Wash out the enzyme. Then, it was filtered by squeeze filtration, and the above steps were repeated several times. The acid-washed collagen is washed with a 4 M salt buffer solution, such as a mixed aqueous solution of sodium phosphate / sodium chloride, and filtered and dried. Repeat the above steps several times. Finally, the collagen is returned to neutral (pH is about 6 · 8-7 · 2), and the weight is controlled within a predetermined range (for example, 6 0-6 40 g). After the salt-washed collagen is washed with 4 liters of an alcohol solution, such as an aqueous isopropanol solution (isopropanol: water = 1: 4), filtered and dried. Repeat the above steps several times so that the weight of the collagen falls within a preset weight range (for example, about 6 0-6 8 g). Finally, store this dough-like collagen in a refrigerator at -20 ° C for easy access. In the above collagen purification process, the collagen tissue under a special condition decomposes the fibrous collagen matrix through protease to remove the non-colloidal protein material, and can obtain high purity and high yield in a simpler process.

Page 15 200407392 V. Description of the invention (7) Low cost, no immune system rejection, and highly biocompatible native collagen fibers. Compared with the traditional collagen protein purification technology, the purification process adopted by us can obtain the original collagen with complete and strong structure, low degradability and fiber size of about 1000 times that of recombinant collagen. Example 2 Preparation of injectable collagen (1) A fixed weight of the above-mentioned collagen is prepared in a low concentration buffer solution (for example, a 10% (v / v) alcohol / 10% (v / v) glycerol aqueous solution) to prepare a concentration of approximately It is 5 ~ 15 mg / m 1 of 2 liters of collagen suspension. Then, homogenize the collagen suspension at a temperature of about 15 ° C or lower with a homogenizer (such as a polytron) for several minutes to obtain a preset collagen fiber size. , For example 1 5 0-2 5 0 // m.

Then ’this slurry-like collagen suspension is crushed into small pieces with a high-pressure crusher such as Microfluidizer M-110Y (Microfluidics International Corporation)

Take several minutes to achieve a collagen fiber size that is uniform and suitable for about 30 gauge or even λ lambda needles. Taking this embodiment as an example, at this time, the glue = the size of the white fiber is about 5 0-1 0 0 " m. It should be specifically explained here that if the egg collagen suspension is not homogenized to 1525 m, the above-mentioned breaks: the treatment will cause the condition of the tube of the crusher to be blocked; The homogenization is to continue to break the collagen fibers into tiny pieces: J step; and (2) due to the size of the collagen fiber particles, g / a, and the durability of the soft and hard tissue filling, that is, collagen

200407392 V. Description of the invention (8) The smaller the size of the protein fiber, the easier it is to migrate from the application site to other places without forming clumps, and the product preservation will prolong the time and cause the phenomenon of pulp separation. Therefore, In the process, it is very important to control the size of collagen fibers. Next, the collagen suspension is centrifuged in a centrifuge, or vacuum-filtered with a special filter having a pore size of about 5 // m to collect a collagen deposit or a filter cake. Control the concentration of collagen to a predetermined range (for example, about 40-50 m g / m 1). Finally, the collagen deposit or filter cake is diluted with a low-concentration buffer solution, such as a 10% (v / v) alcohol / 1/10% (v / v) glycerol solution to prepare a concentration of about 35 mg / ml Of injectable collagen. The concentration of this collagen protein can be adjusted by repeating the filtration / centrifugation times as needed. Here, it is worth noting that the main purpose of adding glycerin is to act as a dispersant to prevent the aggregation of collagen fiber particles, and even to form fiber particles of different sizes, which destroys the injection type collagen Uniformity and stability. Finally, the prepared injectable collagen solution is filled into a predetermined injection syringe, tightly packed, and then sent to sterilization. Since the injectable collagen of the present invention has its fixed structure determined before injection into the target tissue, its efficacy can be seen immediately after injection. This point is very different from other conventional inventions, which need to be warmed up after being injected into the human body for a period of time and then recombined to form the required stronger structure. In addition, the injectable collagen of the present invention is a native collagen, and its structure is more compact and dense than the conventional recombinant collagen, so it has high mechanical strength and filling

Page 17 200407392 V. Description of the invention (9) The durability of the affected part is greatly improved. In addition, in order to further strengthen the compactness of collagen fibers, it can be combined with cross-linking technology. For example, a carb οn diimide cross-linking agent is used to form a cross-linked collagen with a denser structure and less degradation. . Among them, the use of mixed cross-linked and non-cross-linked collagen in combination with each other can enhance the durability of the filled in the affected part, and achieve the ideal soft and hard tissue filling effect. Example 3 Preparation of injectable collagen (2)

The procedure of Example 1 was repeated to obtain a fixed weight of collagen. This collagen is prepared in a low-concentration buffer solution (for example, 10% (v / v) alcohols / 10% (v / v) glycerol aqueous solution) to prepare a 2 liter collagen suspension with a concentration of about 5 mg / m 1 liquid. The steps of Example 2 are repeated until the collagen cake is centrifuged and filtered with a low-concentration buffer solution (for example, a 10% (v / v) alcohol / 10% (v / v) glycerol aqueous solution). Injectable collagen at a concentration of about 38-45 mg / m 1.

Next, another gelatinous substance, such as gelatin, alginate, and pectin, is added to the injectable collagen. The origin of the gelatin may be, for example, dissolving the aforementioned dough-like collagen in water for injection to prepare a uniform suspension having a concentration of about 1% and a pH value of about 2.5 to 4. Then warm to a temperature of about 100-180 ° C for 4 5-7 5 minutes, and then cool at room temperature. Finally, it is filtered with a filter cloth of a specific pore size, and the filtrate is adjusted to a pH value of about 6. 8-7. 2 to obtain the gelatin required for this experiment. Thereafter, the above-produced gelatin having a concentration of about 1% was added to the above-prepared

Page 18, 200,407,392 5. Description of the invention (一) --- Injectable collagen, adjust the concentration of the injectable collagen to about mg / m 1 0. Finally, fill the injectable collagen solution prepared above. Put it into the predetermined syringe and send it to sterilization. The sterilized injectable collagen is stored at low temperature to form a jelly state. The jelly state will prevent the collagen fibers from agglomerating, or cause the product to dehydrate and condense or get rid of pulp. That is, the homogeneous and minute characteristics of the dimension can be completely preserved. The above is only one example of the present invention. For example, any injectable biomedical material that is biocompatible and meets medical requirements can be incorporated into the present invention. The comparative example is based on US Patent No. 3,949,073, US Patent Comparing the collagen purification process (traditional technique) described in USP No. 4,582,640 and the US patent uSP No. 5, 42 8, 0 24 with those described in Example 1 of the present invention, the following differences can be found:

Page 19 200407392 Schematic description

Item ~ ^^ _ Traditional technology Purification method The white enzyme decomposes tissue fibers, so that the collagen is completely suspended in the solution, and is repeatedly killed by adding salt, re-dissolved, and re-fibrous structure. There are many steps in the production level and the product recovery rate is low. Without damaging the fibrous tissue of collagen, the impurities are directly washed from the tendon to retain collagen. The production process has fewer steps and high product recovery. The size of injectable collagen fibers passes through "ϋ gauge. Pass 30 gauge or smaller needles. Injectable collagen fiber dosage form. Stable fibers are suspended in physiological saline. The fibers tend to aggregate and cause the product to dislodge. The particles are not evenly distributed and the injection is not easy. Problem. Or it can be improved by increasing the content of collagen, but it will cause the calcification of the implant in the application site and increase the difficulty of application. With the addition of dispersant glycerin, gelatin, alginate, etc., it can effectively solve the possible pulping phenomenon, and improve the fiber homogeneity and stability and injection ease. ----- Page 20

Claims (1)

200407392 VI. Application Patent Scope 1. A method for preparing injectable collagen, the method includes: (1) using a process that does not damage the fibrous tissue of collagen, automatically obtain a collagen; (2) suspending the collagen In a physiologically acceptable matrix, make it a suspension containing insoluble primary collagen fibers at a concentration of about 20 to 120 mg / ml; and (3) homogenize the collagen fiber suspension with a mechanical device to Reduce its average particle size so that it can pass through 30 gauge or even smaller needles. 2. The method for preparing injectable collagen as described in item 1 of the scope of patent application, wherein the process of not damaging the fibrous tissue of collagen includes: (1) adding and suspending animal tissue containing collagen in proteolytic enzymes In solution, the tissue is decomposed without destroying the native structure and triple helix structure of collagen; (2) Continue the suspension until the non-collagenous substances contained in the animal tissue are decomposed; and (3) Isolate Residual enzymes and non-collagenous substances. 3. The method for preparing injectable collagen according to item 2 of the patent application, wherein the animal tissue includes beef tendon. 4. The method for preparing injectable collagen according to item 1 of the patent application scope, wherein the matrix comprises an alcohol / glycerin solution. 5. The method for preparing injectable collagen according to item 1 of the patent application scope, wherein the mechanical device comprises a homogenizer or a high-pressure crusher. 6. Preparation of injectable collagen as described in item 5 of the scope of patent application Page 21 200407392 6. Scope of patent application The preparation method, wherein the homogenizer includes a polytron. 7. The method for preparing injectable collagen according to item 5 of the scope of patent application, wherein the high-pressure crusher further includes a Microfluidizer (Microfluidics International Corporation) series 歹 1J homogenizer. 8. The method for preparing injectable collagen as described in item 1 of the patent application scope, wherein after the homogenization step, a cross-linking agent or a physical cross-linking method can be used to form a mixture of cross-linked collagen and non- A collagen suspension of cross-linked collagen. 9. The method for preparing injectable collagen according to item 8 of the scope of the patent application, wherein the cross-linking agent comprises carbodiimide or glutaric acid. 10. The method for preparing injectable collagen as described in item 1 of the scope of the patent application, wherein after the homogenizing step, a dispersant for preventing the collagen protein fiber particles from agglomerating again can be added. 11. The method for preparing injectable collagen according to item 10 of the patent application scope, wherein the dispersant comprises gelatin, alginate or pectin. 12. The method for preparing injectable collagen according to item 11 of the scope of the patent application, wherein the dispersant further comprises gelatin at a temperature of 0 to 10 ° C. 1 3. A type of injectable collagen, which is prepared according to any one of the methods in claims 1 to 12. 14. The injectable collagen according to item 13 of the scope of patent application, which is used for filling soft and hard tissues. 15. The injectable collagen according to item 14 of the scope of patent application, wherein the soft and hard tissues include human soft and hard tissues. Page 22 ^ 200407392 Page 23