TW552295B - 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

552295

V. Description of the invention (1) ^ S Technical field _ The present invention relates to a method for preparing collagen, especially to the preparation method of injectable collagen, especially to the preparation method of injectable collagen. _ A method for preparing injectable collagen for filling soft and hard tissues of living bodies. BACKGROUND OF THE INVENTION Collagen is the most abundant protein in connective tissue, and it is also the main component of extracellular matrix ~. Due to its special double helix (t r p 1 e _ h e 1 i X) 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 is widely used in daily life applications, covering biomedical materials, medical drugs, cosmetics, food industry, chemical materials Products have various forms, compositions, functions and uses, and are a class of products with high economic value. In terms of biomedical materials, collagen is widely used in the repair of various tissues due to its low immunogenicity, including the use of heart valves, burns and scald dressings, and the use of subcutaneous implants. . In particular, the application of collagen to the repair and filling of soft and hard tissues is one of the most popular topics of great interest. The traditional method of obtaining collagen is to use acidic protein enzymes to break down

Page 10 552295 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, US patent IL SP No. 3, 949, 0 73 describes a conventional method for preparing a non-crosslinked collagen composition (trade name: Zyderm II); and US Patent No. 4, 582, 640 describes that A cross-linked collagen composition (trade name: Zyp last) was prepared by a method. Because the above process is mainly suitable to use animal skin 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 the presence of impurities such as antigenic determinants can cause unnecessary immune responses. In view of the above, today's problem 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, do not use traditional chemical purification methods, and keep the collagen three-helix structure. This 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 manufacturing 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.

552295 V. Description of the invention (3) The above-mentioned two types of collagen compositions (that is, U.S. patent U. s · ρ · Ν〇 · 3,94 9,0 73 and U.S. patent USP · ν〇 · 4,5 82, 64 0 instructors) in the 'cross-linked vicinal protein composition will be stronger than the non-cross-linked, but because they are obtained by traditional methods, that is, after the decomposition of recombinant collagen', the common problem is the structure It is relatively loose, easily degraded and cannot resist resistance migration, resulting in collagen implanted in the affected area or in the main shot or after injection, the phenomenon of syneres is, resulting in uneven implant viscosity, Easily decomposed and absorbed and cannot be persisted. One of the methods to improve the durability and reduce the phenomenon of desalting is to increase the collagen concentration in the soft and hard tissue filling composition. The difficulty of the affected area is quite high, and many people have worked hard to solve this problem. For example, USP No. 4,8 0 3,0 7 5 proposes to combine cross-linked injectable collagen with a liquid lubricant, but obviously it cannot effectively solve the problem of the phenomenon of slurry. Patent U_S.P. No. 4, 424, 20 8 describes a collagen composition comprising cross-linked injectable collagen and reconstituted collagen fibers, the durability of which has been improved, but ingress and egress The injectability of syringes has not been resolved. US patent U.S.P. No. 5, 428, 024 teaches homogenization to reduce the particle size of collagen fibers. After homogenization by a suitable homogenizer, the average fiber of non-crosslinked collagen can be averaged. Reduced size by at least 25%, and reduced average fiber size of crosslinked collagen by at least 50%.

Page 12 552295 V. Description of the invention (4) However, because the purification process still uses the aforementioned traditional chemical method, it still has problems such as complicated steps and residual organic solvents, and the collagen fiber particles will still reappear after a period of time The agglomeration and formation of fibrous granules of different sizes still destroy the uniformity and stability of injectable collagen. None of the above patents can successfully solve this problem, and therefore it has become a suitable direction to be solved today. In summary, the present inventor proposes a novel method for preparing injectable collagen, which provides a high concentration, durability, and injectability through simple and harmless collagen purification steps and homogenization process' And injectable collagen with good preservability, and completed the present invention. In the present invention, the original collagen fibers are purified by using beef tendon in a novel enzyme / acid washing / salt washing step. Prepare a collagen suspension with a buffer solution, and then homogenize the collagen suspension with a homogenizer to obtain a preset collagen fiber size of 150 to 250 # m. This homogenized collagen suspension 'is then miniaturized to a collagen fiber of 50 to 100 / m by a crusher. This crushing and miniaturization step can be performed by, for example, a Micrf luidics M110EH or M110Y homogenizer, a Brinkmann homogenizer, or a Virtis homogenizer.

Homogeneity and stability of collagen In addition, in order to prevent the collagen fiber particles from re-aggregating to change the consistency of the collagen fiber size, a dispersing agent can be added to the collagen suspension to improve the injectability The dispersant includes, for example, gelatin, alginate (al # 1 1 nate), and pectin (pec11 η). The dispersing agent can help the coffee with the gelatinous substance, and belongs to the position of the collagen fiber structure that helps U 疋. special

552295 V. Description of the invention (5), it can successfully prevent the collagen fibers from agglomerating again, and the uniformity and stability of the injectable collagen can be damaged. EXAMPLES Hereinafter, the present invention will be further described with reference to examples, but the present invention is not limited to these examples. 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 example, we use beef tendon as the raw material for purifying collagen. Before initial cutting, the excess tissue and fat from beef tendon must be removed. After the initial cut is completed, the tendon is first washed at a low temperature with a first buffer solution such as an aqueous alcohol solution, and then washed with a second buffer solution such as a neutral aqueous solution of sodium phosphate / sodium chloride. The cleaned beef tendons are placed in a freezer and frozen at -20 ° C. Next, the frozen beef tendon is sliced to obtain a slice of beef tendon of a predetermined thickness or weight. Taking this embodiment as an example, the weight of the beef tendon slice is about 1000 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 and lactic acid, 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 652295 V. Description of the invention (6) One element is more easily deactivated and Rongxian white shed is required to be removed. Therefore, in this embodiment, gastric protease is used to mix and enzyme in the collagen production process. Digest and repeat the above steps: for a wide range, in a suitable refrigerator for at least 48 hours. After the step, the square protein solution in which the solution is stored is transitioned with a cloth and the remaining solution in the filter cake is squeezed out in a squeeze manner. The filling weight of the collagen falls within a predetermined range: then *, :: Dough (d0ugh) -like collagen containing 0.5 ΛΛ: containing liquid, such as acetic acid, lactic acid, etc., thoroughly mixed with Λ, washing out the unreacted: protein protein enzymes. Then, #filter it by squeeze filtration and repeat the above steps several times. The acid-washed collagen is then washed with a 4 M salt buffer solution, such as a mixed aqueous solution of sodium phosphate / sodium gasification, and filtered and dried. Repeat the above steps several times. Finally, the collagen is returned to neutral (ρΗ value is about 6.8-7.2), and the weight is controlled within a predetermined range (for example, 600-64 g). After the salt-washed collagen is washed with 4 liters of an alcohol solution, such as an isopropanol aqueous 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 600-680 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 is under a special condition, and the fibrous collagen based protein is decomposed by proteinases to remove non-colloidal protein materials, and a relatively simple process can be used to obtain a high-purity, inter-product cover

552295 V. Description of the invention (7) Low-cost ’does not produce immune system rejection and has 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 (i) 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 / ml of 2 liters of collagen suspension. Then, homogenize the collagen suspension at a temperature of about 1 or lower with a homogenizer (for example, poly tron) for several minutes to obtain a preset collagen fiber size, such as 15〇—25〇 # m. Then 'pulverize this slurry collagen suspension high-pressure crusher, such as Microfluidizer M-110Y (Microfluidics Internationa Corporat) for several minutes to achieve uniformity and apply to about 30 gauge. (Gauge) or its / s needle's collagen fiber size. Taking this embodiment as an example, at this time, the size of the glue H white fibers is about 50-100 // m. It should be specifically stated here that, if the collagen suspension is not homogenized to 150 ~ 25 ## / mu (D, the above crusher treatment may block the tube groove of the crusher and be connected, Therefore, the appropriate homogenization is to continue to break the collagen fibers in two necessary pre-treatment steps · 'and (2) the small size of collagen fiber particles will affect the durability of its application to soft and hard tissue filling, 1 Shuyuan

552295 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. In the process, it is very important to really control the size of collagen fibers. Then, 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 / 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 paying special attention 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 it is injected 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 552295 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, cross-linking technology can be used, such as the use of carbodiimide cross-linking agents, to form a denser cross-linked collagen that is less resistant to degradation. . Among them, the use of mixed cross-linked and non-cross-linked collagen in combination with each other can improve 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 steps of Example 1 were repeated to obtain a fixed weight of collagen. Prepare a 2 liter collagen suspension with a low concentration buffer solution (eg 10% (v / v) alcohols / 10% (v / v) glycerol in water) . The steps of Example 2 are repeated until the collagen cake is centrifuged and filtered with a low-concentration buffer solution (for example, 10% (v / v) alcohols / 1 10% (v / v) glycerol aqueous solution). Injectable collagen with a concentration of about 3 8-4 5 mg / m 1. Next, another gelatinous substance, such as gelatin, alginate, and pectin, is added to the injectable collagen. The source of the gelatin, for example, the aforementioned dough-like collagen can be dissolved in water for injection, and adjusted to a uniform suspension having a concentration of about 1% and a pH of about 2.5-4. Then warm up to a temperature of about 100-180 ° C for about 4 5-7 5 minutes, and then cool at room temperature. Finally, filter it with a filter cloth with a specific pore size, and adjust the filtrate to a pH 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 §52295 V. Description of the invention (ίο) Injectable collagen mg / m 1 ° Adjust the concentration of the injectable collagen to about 3 5 Finally, send the injection syringe prepared above to sterilization. Sterilized at low temperature, it can form a jelly state, which does not cause aggregation, or causes the product to dehydrate and maintain a homogeneous and small homogeneous property. It can be filled with a complete collagen solution after a predetermined injection collagen is stored in the fruit state. Fiber shrinkage or pulping. That is, the preservation of fibers. The above are only one embodiment of the present invention, for example, any biocompatible and

Injectable biomedical materials that meet the medical requirements can be incorporated into the present invention for use. Comparative examples are U.S. Patent No. 3,949,073, U.S. Patent No. 4,582,640, and U.S. Patent 11.8 ·? .— · The collagen purification process (traditional technique) described in 5, 4 2 8, 0 2 4 is compared with the one described in Example 1 of the present invention, and the following differences can be found:

Page 19 552295 Simple illustration

Item Traditional technology The present invention Purification method: The white enzyme decomposes the tissue fiber, so that the original protein is completely suspended in the solution. There are many production steps and low product recovery. Without damaging the fibrous tissue of collagen, the impurities are directly washed from the tendon to retain collagen. There are fewer steps in the production process. The injectable collagen of the product passes the 20 gauge protein fiber size. Stable fibers of the injectable collagen fiber dosage form are suspended in physiological saline. Difficult 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. The yield is high. The addition of alginate, such as a 30 gauge or fine needle, can effectively solve the phenomenon of detachable pulp, and improve the stability of fiber homogeneity and ease of injection. ———_

Claims (1)

f552295 ί y, minus _ —_ ί. A method for preparing injectable collagen, the method includes: (ί) automatic process to obtain a collagen by a process that does not damage the fibrous tissue of collagen; that is, the non-destructive The process of collagen fibrous tissue includes: I. Add animal tissue containing collagen and suspend it in proteolytic enzyme solution, and decompose the tissue without destroying the collagen's native structure and triple helix structure; Π. Continue the suspension until the non-collagenous substances contained in the animal tissues are decomposed; and ii. Isolate the remaining enzymes and non-collagenous substances. (2) suspending the collagen in a physiologically acceptable matrix to make it a suspension containing 20 to 120 mg / ml of insoluble primary collagen fibers; and (3) using a mechanical device to collagen The fiber suspension is homogenized 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 according to item 1 of the scope of patent application, wherein the animal tissue includes beef tendon. 3. The method for preparing injectable collagen according to item 1 of the patent application, wherein the matrix comprises an alcohol / glycerin solution. 4. The alcohol as described in item 3 of the scope of patent application, wherein the alcohol includes alcohols having 2 to 3 carbon atoms. 5. Preparation of injectable collagen as described in item 1 of the scope of patent application Page 21 552295 6. Scope of patent application The preparation method, wherein the mechanical device includes a homogenizer or a high-pressure crusher. 6. The method for preparing injectable collagen according to item 5 of the scope of the patent application, wherein the homogenizer includes ρ ο 1 y t r ο η. 7. The method for preparing injectable collagen as described in item 5 of the scope of patent application, wherein the high-pressure crusher further comprises a Microfluidizer (Microfluidics International Corporation) series, J 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 may 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 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 a dispersant for preventing re-aggregation of collagen protein fiber particles can be added after the homogenization step. 1 1. 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 patent application, wherein the dispersant further comprises gelatin at a temperature of 0 to 1 ° C. 1 3. The injectable collagen protein as described in claims 1 to 12 of the scope of patent application, which is used for soft and hard tissue filling. 14. The injectable collagen according to item 13 of the scope of patent application, wherein the soft and hard tissues include human soft and hard tissues. * 552295 Page 23