JP5767591B2 - Method for producing artificial cartilage - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a method for producing an artificial cartilage rich in elasticity using a component of living cartilage as a raw material, and more particularly, a method of thermal dehydration crosslinking treatment capable of sufficient crosslinking without causing coloring. About.

  Cartilage tissue consists of chondrocytes and a cartilage matrix (matrix). Chondrocytes are highly differentiated cells that occupy only about 10% of the cartilage tissue and rarely proliferate by cell division, but produce cartilage matrix components in the cartilage tissue. It is responsible for maintaining the cartilage matrix, which accounts for about 90% of the total.

  Attempts have been made to artificially reproduce cartilage tissue using chondrocytes and use it for the treatment of cartilage destruction / degeneration. To form cartilage-like tissue, cartilage matrix components are produced in the chondrocytes themselves. The process of making it indispensable. However, with the current technology, it is difficult to make chondrocytes efficiently produce a sufficient amount of cartilage matrix sufficient to fill the defect, and there are still many problems to be solved.

  Studies have also been made to chemically prepare tissue regeneration materials that mimic cartilage tissue. For example, Japanese Patent Application Laid-Open No. 2002-80501 (Patent Document 1) discloses a glycosaminoglycan-polycation complex for tissue regeneration matrix in which glycosaminoglycan and polycation are cross-linked by a condensation reaction. It is described as being useful as an excellent regeneration material for various tissues such as blood vessels and nerves. However, since the composite described in Patent Document 1 uses a crosslinking agent and a condensing agent in the production process, it is necessary to wash and remove these crosslinking agent, condensing agent, and these by-products. Will be asked. In addition, if these are transplanted into the body, there is a risk of chemical residue problems. Furthermore, the structure of the above complex made using a cross-linking agent or a condensing agent is nano-level and does not resemble a living tissue. Therefore, the low friction, load resistance, and biocompatibility required for cartilage functions are eliminated. There is a possibility that it cannot satisfy sex.

  International Publication No. 2007/032404 (Patent Document 2) includes (a) a step of preparing a glycosaminoglycan proteoglycan aggregate by mixing a glycosaminoglycan and a proteoglycan, and (b) the glycosaminoglycan proteoglycan. Disclosed is a method for producing a self-assembled glycosaminoglycan / proteoglycan / collagen complex including the step of mixing collagen with an aggregate, and the glycosaminoglycan / proteoglycan / collagen complex is used in cartilage regenerative medicine. It describes that it can be manufactured without using chemical substances such as a cross-linking agent because it has properties extremely suitable as a biomaterial and is manufactured by self-assembly. However, since the composite obtained by the method described in Patent Document 2 is not subjected to crosslinking treatment, the strength is greatly reduced when it is used in vivo, and improvement is desired.

  Japanese Patent Application Laid-Open No. 2011-36320 (Patent Document 3) discloses a graft material for block-like cartilage having a multilayer structure in which a porous gelatin layer having a low porosity and a porous gelatin layer having a high porosity are laminated. And a method of thermally crosslinking a freeze-dried foamed gelatin solution at normal pressure. However, when the thermal crosslinking method described in Patent Document 3 is applied to the glycosaminoglycan / proteoglycan / collagen complex disclosed in Cited Document 2, the complex is colored, and the elasticity is significantly reduced. It was.

  Haugh et al., “Novel Freeze-Drying Methods to Produce a Range of Collagen-Glycosaminoglycan Scaffolds with Tailored Mean Pore Sizes,” Tissue Engineering: Part C, vol. 16, No. 5, pp. 887-894. Document 1) describes a method of hydrothermal treatment for 24 hours under the conditions of 0.05 bar and 105 ° C. as a method for crosslinking a collagen / glycosaminoglycan scaffold. However, even when the method described in Non-Patent Document 1 is applied to the cross-linking of artificial cartilage, the occurrence of coloring of the material and the decrease in elasticity cannot be completely prevented.

JP 2002-80501 A International Publication No. 2007/032404 Pamphlet JP 2011-36320 A

Haugh et al., “Novel Freeze-Drying Methods to Produce a Range of Collagen-Glycosaminoglycan Scaffolds with Tailored Mean Pore Sizes,” Tissue Engineering: Part C, vol. 16, No. 5, pp. 887-894.

  Accordingly, an object of the present invention is to provide a method for crosslinking an artificial cartilage composed of a glycosaminoglycan / proteoglycan / collagen complex so that the artificial cartilage is not colored and does not cause a decrease in elastic modulus when used.

  As a result of diligent research in view of the above object, the inventors of the present invention performed artificial dehydration crosslinking of an artificial cartilage composed of a glycosaminoglycan / proteoglycan / collagen complex at 105 to 110 ° C. for 16 to 23 hours. The present inventors have found that it is possible to perform a crosslinking treatment with a degree of crosslinking so that coloring does not occur and the elastic modulus does not decrease during use.

  That is, the method of the present invention for producing an artificial cartilage composed of collagen, proteoglycan and hyaluronic acid is obtained by subjecting a molded body composed of collagen, proteoglycan and hyaluronic acid to thermal dehydration crosslinking at 105 to 110 ° C. and 0 Pa to atmospheric pressure for 16 to 23 hours. It has the process to perform.

  The molded body is preferably obtained by freeze-drying a composition comprising collagen, proteoglycan and hyaluronic acid.

  The composition preferably comprises 15 to 95% by weight collagen, 4.9 to 70% by weight proteoglycan and 0.1 to 20% by weight hyaluronic acid.

  It is preferable to subject the artificial cartilage after crosslinking to gamma irradiation treatment.

[1] Artificial cartilage The artificial cartilage is composed of collagen, proteoglycan and hyaluronic acid, and preferably contains 15 to 95% by mass of collagen, 4.9 to 70% by mass of proteoglycan and 0.1 to 20% by mass of hyaluronic acid. By adding hyaluronic acid and / or proteoglycan to collagen fibers, an artificial cartilage having excellent flexibility can be obtained. More preferably, the amounts of collagen, proteoglycan and hyaluronic acid in the artificial cartilage are 45 to 65% by mass, 20 to 40% by mass and 2 to 5% by mass, respectively.

  When the collagen content is less than 15% by mass, the expansion rate when inserted into a living body increases, making it difficult to adapt to a cartilage defect, and the porosity tends to decrease due to expansion. When the collagen content exceeds 95% by mass, coloring increases. When the proteoglycan content is less than 4.9% by mass, the elastic modulus is lowered and the performance as cartilage is lowered. When the proteoglycan content is more than 70% by mass, the change in size due to expansion becomes large, and the porosity tends to decrease. When the hyaluronic acid content is less than 0.1% by mass, the elastic modulus is lowered, the performance as cartilage is lowered, and the lubricity (low friction property) of the artificial cartilage surface is lowered. If the hyaluronic acid content exceeds 20% by mass, it will greatly exceed the proportion contained in living cartilage, and it will be a material with components different from living cartilage, so depending on the application site, the desired content of collagen and proteoglycan It becomes difficult to secure the ratio.

  It does not specifically limit as collagen, What was extracted from the animal etc. can be used. Also, the species, tissue site, age, etc. of the animal from which it is derived are not particularly limited. In general, collagen obtained from the skin, bone, cartilage, tendon, organ, etc. of mammals (eg, cows, pigs, horses, rabbits, mice, etc.) and birds (eg, chickens, etc.) can be used. Collagen-like proteins obtained from the skin, bones, cartilage, fins, scales, organs, etc. of fish (eg cod, flounder, flounder, salmon, trout, tuna, mackerel, Thai, sardine, shark etc.) may also be used. . In addition, the extraction method of collagen is not specifically limited, A general extraction method can be used. Further, instead of extraction from animal tissue, synthetic collagen or collagen obtained by gene recombination technology may be used.

  Glycosaminoglycan is an acidic polysaccharide consisting of a repeating structure of disaccharides in which an amino sugar and uronic acid or galactose are bound. The glycosaminoglycan used in the present invention may be any of chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, heparin, and hyaluronic acid, but it is preferable to use hyaluronic acid.

  A proteoglycan is a protein in which one or many glycosaminoglycan chains are bound to one core protein. The proteoglycan is not particularly limited, and examples include aggrecan, versican, neurocan, brevican, decorin, biglycan, serglycine, perlecan, syndecan, glypican, lumican, keratocan, etc., but it is preferable to use aggrecan.

  There are no particular restrictions on the origin of proteoglycans, depending on the intended use of the complex, mammals (human, cow, pig, etc.), birds (chicken, etc.), fish (sharks, sharks, etc.), crustaceans (crabs, shrimp, etc.) It can be appropriately selected from various animal origins such as In particular, if the artificial cartilage of the present invention is used for the treatment of human cartilage defect or degeneration, it is desirable to select from those having low immunogenicity in humans.

  Collagen in the artificial cartilage can be quantified by measuring UV absorption, HPLC or the like. Hyaluronic acid can be quantified by a carbazole sulfate method, an inhibition method using a hyaluronic acid binding protein, HPLC, or the like. Proteoglycan can be quantified by a colorimetric method using the dye DMMB, HPLC or the like.

  Artificial cartilage is subjected to thermal dehydration crosslinking treatment in order to increase mechanical strength and to hold the artificial cartilage inserted into the body for a long period of time. The artificial cartilage is preferably sterilized by a method such as gamma ray treatment.

[2] Manufacturing method The method of the present invention for manufacturing artificial cartilage includes a step of thermally dehydrating and crosslinking a molded body comprising collagen, proteoglycan and hyaluronic acid at 105 to 110 ° C. and 0 Pa to atmospheric pressure for 16 to 23 hours. The molded body composed of collagen, proteoglycan and hyaluronic acid preferably comprises a step of obtaining a mixture of collagen, proteoglycan and hyaluronic acid, and a step of freeze-drying the mixture. The lyophilized product may be pulverized and dispersed in water, and then the dispersion may be freeze-dried again.

(a) Preparation and mixing The step of obtaining a mixture of collagen, proteoglycan and hyaluronic acid includes the steps of obtaining a first composition comprising hyaluronic acid and collagen, and obtaining a second composition comprising proteoglycan and collagen. And the step of mixing the first and second compositions.

  In the step of obtaining the first composition, the mixing ratio of hyaluronic acid and collagen is preferably 10000: 1 to 1: 10000 (mass ratio), and is 5000: 1 to 1: 5000 (mass ratio). More preferably, it is 15: 1 to 1:15 (mass ratio). It is preferable to use collagen previously dissolved in dilute hydrochloric acid (concentration of about 5 to 50 mM) at a concentration of 0.1 to 20% by mass. Hyaluronic acid is preferably dissolved in sterile water (water for injection, etc.) at a concentration of 0.1 to 20% by mass in advance. Mixing of the aqueous solution of hyaluronic acid and collagen is preferably performed at 3 to 25 ° C.

  In the step of obtaining the second composition, the mixing ratio of proteoglycan and collagen is preferably 10000: 1 to 1: 10000 (mass ratio), and is 5000: 1 to 1: 5000 (mass ratio). Is more preferable, and 10: 1 to 1:10 (mass ratio) is most preferable. It is preferable to use collagen previously dissolved in dilute hydrochloric acid (concentration of about 5 to 50 mM) at a concentration of 0.1 to 20% by mass. In addition, it is preferable to use proteoglycan previously dissolved in sterile water (water for injection etc.) at a concentration of 0.1 to 20% by mass. The mixing of the aqueous solution of proteoglycan and collagen is preferably performed at 3 to 25 ° C.

  The mixing of the aqueous solution of hyaluronic acid and collagen (first composition) and the mixing of the aqueous solution of proteoglycan and collagen (second composition) does not require particularly high shear, so that a commonly used stirrer, It can be performed using an instrument such as a mixer. The mixing is performed at 3 to 25 ° C. for about 1 second to 3 minutes so that hyaluronic acid and collagen or proteoglycan and collagen are uniformly mixed.

  The mixing ratio of the first and second compositions is such that the composition contains 15 to 95 mass% collagen, 4.9 to 70 mass% proteoglycan and 0.1 to 20 mass% hyaluronic acid after mixing. The mixing of the first and second compositions is preferably performed by a method having shearing force using an instrument such as a homogenizer or a dissolver. For example, when using a homogenizer, it is preferable to repeat the stirring for 30 seconds to 3 minutes 1 to 5 times at a rotation speed of 1,000 to 12,000 rpm. The sample at the time of mixing is preferably kept at a temperature of about 3 to 25 ° C. The progress of the synthesis can be further promoted by preparing the first and second compositions separately and then mixing them.

  In addition, the mixture of collagen, proteoglycan and hyaluronic acid is not a method of preparing the first and second compositions and mixing them as described above, but directly mixing collagen, proteoglycan and hyaluronic acid as described below. May be produced.

  The mixing ratio of collagen, proteoglycan and hyaluronic acid is such that the composition contains 15 to 95% by mass of collagen, 4.9 to 70% by mass of proteoglycan and 0.1 to 20% by mass of hyaluronic acid after mixing. It is preferable to use collagen previously dissolved in water or dilute hydrochloric acid (concentration of about 5 to 50 mM) at a concentration of 0.1 to 20% by mass. It is preferable to use proteoglycan previously dissolved in sterile water (water for injection, etc.) at a concentration of 0.1 to 20% by mass. It is preferable to use hyaluronic acid previously dissolved in sterile water (water for injection, etc.) at a concentration of 0.1 to 20% by mass.

  Each solution of collagen, proteoglycan and hyaluronic acid is preferably mixed by applying a shearing force using an instrument such as a homogenizer or a dissolver. For example, when using a homogenizer, it is preferable to repeat the stirring for 30 seconds to 3 minutes 1 to 5 times at a rotation speed of 1,000 to 12,000 rpm. Preparation and mixing of an aqueous solution of collagen, proteoglycan and hyaluronic acid is preferably carried out while keeping the temperature at 3 to 25 ° C.

(b) First freeze-drying The obtained mixture is put in a container (such as a metal vat) having good heat conductivity and frozen at -80 to -60 ° C for one night or more. The frozen mixture is evacuated for about 10 hours to 10 days at the shelf temperature of about -50 to -5 ° C (preferably -40 to -5 ° C) until the water (ice) of the mixture is almost gone (first drying). The shelf temperature is raised to about 20 to 40 ° C. (preferably 25 to 40 ° C.) while being evacuated, and further dried (second drying) for 3 to 24 hours. Thus, by freeze-drying by changing the temperature in two stages, even the bound water is removed and the product is further dried, and the resulting freeze-dried product has excellent storage stability.

  The obtained freeze-dried product is subjected to crosslinking and sterilization described below, and may be used as an artificial cartilage as it is, but may be further subjected to grinding, dispersion, and freeze-drying described below. Thus, a high-density artificial cartilage is obtained by going through the grinding step.

(c) Grinding The obtained freeze-dried product is ground with a solid grinder such as a mill. The pulverization method is not particularly limited, but it is preferable to carry out the lyophilized product so as not to reach a very high temperature.

(d) Dispersion The pulverized lyophilized product is mixed with physiological saline so as to have a concentration of 3 to 20% by mass, and using a device such as a homogenizer, the conditions are 3 to 25 ° C. and 1,000 to 15,000 rpm. Disperse 1 to 5 times for seconds to 3 minutes.

(e) Gelation The obtained dispersion is put into a container such as a petri dish, capped, and allowed to stand at 30 to 40 ° C. for 1 to 5 hours for gelation.

(f) Second lyophilization The gelled dispersion is lyophilized again to form a molded product. The gelled dispersion is refrigerated at 2 to 10 ° C. for 1 to 20 hours and further frozen at about −20 to −60 ° C. overnight. When freezing, it is preferable to place the container on a mesh pan placed in a stainless steel bat. The frozen dispersion is dried in the same manner as in the first lyophilization described above.

(g) Crosslinking The dispersion after freeze-drying is subjected to thermal dehydration cross-linking treatment to increase the mechanical strength and to hold the artificial cartilage inserted into the body for a long period of time. Cross-links proteoglycans. Thermal dehydration crosslinking is performed by holding the dispersion after freeze-drying in a vacuum oven at 105 to 110 ° C. and 0 Pa to atmospheric pressure for 16 to 23 hours. The holding time is preferably 16 to 20 hours.

  Artificial cartilage coloring occurs when heat dehydration is performed at a high temperature of over 110 ° C or a long time of over 23 hours, and crosslinking is insufficient under conditions of a low temperature of less than 105 ° C or a short time of less than 16 hours Therefore, the artificial cartilage contracts due to water content. The degree of cross-linking of the artificial cartilage can be evaluated by, for example, the degree to which the elastic modulus after hydration decreases with time. It can be said that the lower the modulus of elasticity, the higher the degree of crosslinking.

(h) Sterilization treatment Artificial cartilage is preferably sterilized by ultraviolet rays, γ rays, electron beams, drying and heating, and the like. In particular, it is preferable to sterilize by irradiation with gamma rays of 25 kGy or less.

  The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
(1) Preparation of raw material solution
Collagen was dissolved in 5 mM hydrochloric acid to prepare a 1% by mass collagen solution. Further, proteoglycan was dissolved in water for injection to prepare a 1% by mass proteoglycan solution, and further hyaluronic acid was dissolved in water for injection to prepare a 0.2% by mass hyaluronic acid solution. All of these preparations were performed at 4 ° C.

(2) Mixing of raw materials The collagen solution and the proteoglycan solution were mixed at 1: 1 (mass ratio) and stirred with a mixer to obtain a mixed solution A. Similarly, the collagen solution and the hyaluronic acid solution were mixed at a ratio of 2: 1 (mass ratio), and stirred with a mixer to obtain a mixed solution B. Mixtures A and B were mixed at a ratio of 1.5: 1 (mass ratio), and stirred for 1 minute at a rotation speed of 2, OOOO rpm with a homogenizer three times at intervals of 30 seconds. The stirring was performed while keeping the temperature of the sample at 5 ° C.

(3) First freeze-drying The obtained mixture was poured into a vat and frozen at −80 ° C. for 6 days, and then vacuum-evacuated at a shelf temperature of −5 ° C. for 8 days for first drying. With this first drying, there was almost no moisture (ice) in the mixture. Subsequently, the shelf temperature was raised to 25 ° C. while evacuating, and the second drying was further performed for 4 hours to obtain a freeze-dried product.

(4) Grinding and dispersion After the obtained lyophilized product was pulverized with a mill, the pulverized lyophilized product was mixed with physiological saline so as to be 1O.7% by mass, and the homogenizer was used under conditions of 10, 00 rpm. Dispersion for 1 minute was performed 5 times (interval: 1 minute). In addition, the dispersion | distribution by a homogenizer was carried out keeping 5 degreeC.

(5) Defoaming The obtained dispersion was stirred for 1 minute with a rotation / revolution mixer (manufactured by Shinky Co., Ltd., Awatori Nertaro ARE-250) to remove bubbles contained in the dispersion.

(6) Gelation The obtained dispersion was put into a glass petri dish, capped, left to stand at 37.5 ° C for 3 hours to gel, and then refrigerated at 5 ° C for 3 hours.

(6) Second freeze-drying Place the refrigerated material together with the petri dish on a mesh pan placed in a stainless steel pad, freeze at -60 ° C for 3 days, and then vacuum for 7 days at a shelf temperature of -40 ° C. Was dried. With this first drying, there was almost no moisture (ice) in the mixture. Subsequently, the shelf temperature was raised to 25 ° C. while evacuating, and the second drying was further performed for 4 hours to obtain a freeze-dried product.

(7) Crosslinking treatment The obtained lyophilized product was molded into a diameter of 4.2 mm and a thickness of 4 mm, and 105 ° C, 110 ° C, 115 ° C under vacuum (0 Pa) in a vacuum oven, and the times shown in Table 1. Artificial cartilage was obtained by thermal dehydration crosslinking under the conditions.

  The resulting artificial cartilage was evaluated for coloring and crosslinking degree as follows. The coloring was judged by visual observation, and evaluation was made with ○ indicating that there was no coloring, Δ indicating slightly coloring, and × indicating coloring. The degree of cross-linking was measured by measuring the elastic modulus of the obtained artificial cartilage immediately after hydration and after 4 weeks with a viscoelasticity measuring device (wave cyber, Vesmeter, E-200DT). Immediately after the evaluation, 70% or more was evaluated as ○, 50% or more and less than 70% as Δ, and less than 50% as ×. When the coloring and the degree of crosslinking are both evaluated as ◯, it has practicality as an artificial cartilage. The results are shown in Table 1.

  As is clear from Table 1, when the thermal dehydration crosslinking treatment was carried out at 105 to 110 ° C. for 16 hours or more and less than 24 hours, an artificial cartilage having a sufficient degree of crosslinking without being colored was obtained.

Example 2
The freeze-dried product obtained in the same manner as in Example 1 was molded into a diameter of 4.2 mm and a thickness of 4 mm, and subjected to thermal dehydration crosslinking at 105 ° C. for 20 hours in a vacuum oven at atmospheric pressure to obtain an artificial cartilage. . The obtained artificial cartilage had the same coloration and evaluation of the degree of crosslinking as artificial cartilage subjected to thermal dehydration crosslinking treatment at 105 ° C. for 20 hours under vacuum.

Claims (4)

  A method for producing an artificial cartilage comprising collagen, proteoglycan and hyaluronic acid, comprising a step of thermally dehydrating and crosslinking a molded article comprising collagen, proteoglycan and hyaluronic acid at 105 to 110 ° C. and 0 Pa to atmospheric pressure for 16 to 23 hours. A method for producing an artificial cartilage, comprising:   The method for producing artificial cartilage according to claim 1, wherein the molded body is obtained by freeze-drying a composition comprising collagen, proteoglycan and hyaluronic acid.   The method for producing an artificial cartilage according to claim 2, wherein the composition contains 15 to 95% by mass of collagen, 4.9 to 70% by mass of proteoglycan and 0.1 to 20% by mass of hyaluronic acid. .   The method for producing an artificial cartilage according to any one of claims 1 to 3, wherein the crosslinked artificial cartilage is subjected to gamma ray irradiation treatment.