JP5521235B2 - High-strength fibrin molded body and artificial ligament - Google Patents

Description

  The present invention relates to a high-strength fibrin molded article having high breaking strength and useful as an artificial ligament for transplantation, and an artificial ligament using the same.

  Fibrinogen, a biological component, is known to have an effect of promoting the healing process of tissues, but there are problems of prion diseases such as hepatitis C virus infection by fibrinogen preparations, an increase in HIV virus domestic infection and mad cow disease. Based on the situation becoming more serious, the Ministry of Health, Labor and Welfare has identified blood coagulation factors derived from blood donations (specific organisms) in the revised Pharmaceutical Affairs Law, which came into effect in July 2003, and the “Law Concerning Ensuring the Stable Supply of Safe Blood”. It has been announced that the indication is strictly applied in use, and self-fibrin glue using fibrinogen derived from autologous blood has been attracting attention.

  In addition, platelets contain a high concentration of growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF) in intracellular granules. Gels obtained by coagulating platelet rich plasma (PRP), which is the plasma contained in, are gaining interest as tissue repair materials derived from autologous blood in general medical fields such as vascular surgery and orthopedic surgery, mainly in the dental field. Have already been clinically applied (Non-Patent Documents 1, 2, 3, 4, 5, 6). Although the growth factor in platelets is usually released immediately, it is said that this gel also has the effect of a drug delivery system that slowly releases the growth factor in platelets as a carrier (Non-patent Document 7). .

  However, although fibrin gel obtained by activating fibrinogen with thrombin is widely used in the medical field as a fibrin glue, its strength is very weak and it is easy for mechanical pulling. Because it tears, it has no physical properties that can be used as a ligament. For this reason, there is an attempt to produce a provisional scaffold for repairing the ligament by adding collagen to the PRP gel, but despite the use of collagen derived from a specific organism, its strength is It is not enough (Non-Patent Document 8).

By the way, the artificial ligament has a history of being widely used in the 1980s, but it is rarely used due to the fact that foreign matter reaction due to wear powder is likely to occur and deterioration over time is remarkable (Non-Patent Documents 9 and 10). ).
Mazzucco L, et al. The use of autologous platelet gel to treat difficult-to-heal wounds: a pilot study.Transfusion 2004; 44: 1013-10181. Senet P, et al. Randomized trial and local biological effect of autologous platelets used as adjuvant therapy for chronic venous leg ulcers.J Vasc Surg 2003; 38: 1342-1348. Iba O, et al. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation 2002; 106: 2019-2025. Anitua E, et al. Autologous platelets as a source of proteins for healing and tissue regeneration.Thromb Haemost 2004; 91: 4-15. Marlovits S, et al. A new simplified technique for producing platelet-rich plasma: a short technical note.Eur Spine J 2004; 1: S102-106. Ishida K, et al. The regenerative effects of platelet-rich plasma on meniscal cells in vitro and its in vivo application with biodegradable gelatin hydrogel.Tissue Eng. 2007; 13: 1103-1112. Yazawa M, et al. Basic studies on the clinical applications of platelet-rich plasma.Cell Transplant 2003; 12: 509-518. Murray MM, et al. Enhanced histologic repair in a central wound in the anterior cruciate ligament with a collagen-platelet-rich plasma scaffold.J Orthop Res 2007; 25: 1007-1017 Claes LE, et al. Biological response to ligament wear particles. J Appl Biomater 1995; 6: 35-41. Murray AW, et al. 10-16 year results of Leeds-Keio anterior cruciate ligament reconstruction.Knee 2004; 11: 9-14.

However, the number of operations requiring ligament reconstruction is increasing year by year as people's demands for quality of life increase, and in the case of anterior cruciate ligament reconstruction surgery, a typical example, more than 250,000 cases worldwide worldwide In Japan, there are about 16,000 cases.
In the case of Japan and Europe where allogeneic tissue transplantation from the cadaver is not common as in the United States, the reconstructed ligament used for transplantation (reconstruction) is mainly a sacrifice of the healthy tissue (ligament or tendon) of the transplanted tissue piece. Although it is generally used as a (graft), the functional loss of ligaments and tendons collected as tissue pieces is serious.
As described above, it is very important to develop a temporary scaffold type artificial ligament having both biocompatibility and excellent initial strength and high bioactivity.
Accordingly, an object of the present invention is to provide a molded article useful for an artificial ligament having biocompatibility and excellent initial strength, and an artificial ligament using the same.

  Therefore, the present inventor has been studying focusing on self-fibrin from the viewpoint of biocompatibility, suspending the plasma components and platelets and calcium ions that have been concentrated in advance, solidifying this into a predetermined shape, and then washing Thus, it was found that an extremely high strength fibrin molded body having a breaking strength of 0.01 MPa or more can be obtained without adding collagen or the like. Furthermore, when this fibrin molded body was transplanted as a scaffold-type artificial ligament, it was found that it had excellent engraftment and a ligament similar to a normal ligament tissue was regenerated, and the present invention was completed.

That is, the present invention is to solidify and mold a suspension containing a plasma component, platelets and calcium ions concentrated to 1 to 4 times for 0.5 to 72 hours. A high-strength fibrin molded body obtained by washing is provided.
Moreover, this invention provides the artificial ligament containing the said high intensity | strength fibrin molded object.

  The high-strength fibrin molded body of the present invention has excellent initial strength with a breaking strength of 0.01 MPa or more and is composed of only biological components, so that it has excellent biocompatibility and is useful as a scaffold artificial ligament. This artificial ligament is useful as a material for reconstruction surgery of ligaments and tendons such as knees, elbows, shoulders, wrist joints, ankle joints, and finger joints. In addition, since the high-strength fibrin molded body of the present invention can be manufactured from plasma and platelets derived from the blood of the person undergoing treatment without using complicated procedures such as cell culture, virus infection, bacterial infection, Compatibility issues are also eliminated.

  The high-strength fibrin molded product of the present invention is obtained by solidifying and molding a suspension containing a plasma component concentrated to 1.2 to 4 times, platelets and calcium ions for 0.5 to 72 hours. It can be obtained by washing the molded body. Here, the plasma component and platelets are preferably prepared separately from the same blood. That is, first, blood is centrifuged at 90 to 4500 × g in one or two stages, and the platelet-poor plasma (PPP) and the fraction of buffy coat and red blood cell components, which are platelet fractions, or platelet-rich plasma. Separate into two fractions of (PRP) and other blood cell components and fractionate PPP, platelets or PRP. A buffy coat method etc. are mentioned as a method of centrifuging in two steps. These operations are preferably performed under physiological conditions, for example, 4 to 40 ° C.

  The plasma component is concentrated 1.2 to 4 times, more preferably 2 to 3 times. If the concentration ratio is less than 1.2 times, a fibrin molded product having sufficient strength cannot be obtained. On the other hand, if the concentration ratio exceeds 4 times, the viscosity becomes extremely high, which is not preferable. Concentration means include ultrafiltration, desiccant, and freeze-drying. Ultrafiltration, and further ultrafiltration with a pore size of 10,000 to 300,000 in molecular weight, especially tangential flow type ultrafiltration. It is preferable to adopt it because it is simple and performs the concentration by a clean operation in a short time. These concentration operations are preferably performed under physiological conditions, for example, 4 to 40 ° C, particularly 20 to 24 ° C.

The platelet concentration may be adjusted by adding a buffy coat after concentrating the PPP, but it is also possible to concentrate the platelets by concentrating the PRP separated from the blood by ultrafiltration or the like. It may be used by adjusting the concentration by precipitating platelets by light centrifugation at ˜400 × g. The platelet content in the suspension is presumed to be higher when considering the effective use of various growth factors contained in the platelets. Taking into account the reduction in strength, the amount of blood remaining as a plasma raw material from the concentration that remains without being separated from plasma by centrifugation, for example, 1/10 of the normal platelet amount However, it is realistic that the amount is preferably about 5 times. Individual differences in platelets are large, but in the case of human blood, the normal range is about 15 × 10 ⁴ to 40 × 10 ⁴ / μL. Therefore, platelet content of specific suspension, 1 × 10 ⁴ / μL~400 × about 10 ⁴ / [mu] L are preferred.

As the calcium ion source, water-soluble calcium salts such as calcium chloride, calcium gluconate, calcium L-aspartate, calcium lactate, calcium hydrogen phosphate, calcium glycerophosphate, calcium carbonate, calcium citrate and calcium malate are used. Particularly preferred are calcium chloride and calcium gluconate. The concentration of calcium ions in the suspension is, for example, the amount of calcium ion added to the suspension when the suspension is made from blood using a mixture of 1 volume of 5% sodium citrate and 9 volumes of blood. The final concentration of calcium ions is preferably 5 to 35 mM , more preferably 10 to 30 mM , and particularly preferably 15 to 25 mM in terms of improving the strength of the fibrin molded body.

  The suspension contains collagen, gelatin, tranexamic acid, aprotinin, soybean trypsin inhibitor, etc. for the purpose of improving the strength of the fibrin molded body, adjusting the strength, or delaying dissolution by the fibrinolytic system. Can be made.

  The suspension is solidified by allowing to stand for 0.5 to 72 hours. Here, the molding may be performed by filling the suspension into a container that matches the shape of the fibrin molded body and allowing to stand and solidify. That is, when a columnar fibrin molded body is obtained, it may be solidified in a cylindrical tube. Examples of the shape include a thin film shape, a prismatic shape, and a cylindrical shape. The preferred standing time is 1 to 48 hours, more preferably 3 to 24 hours. The standing conditions are preferably physiological conditions in which a blood coagulation reaction that is an enzyme reaction can occur, for example, 25 to 40 ° C.

In the present invention, the strength of the fibrin molded body obtained by standing is not sufficient, and is washed. Strength is improved by washing operation. Cleaning is preferably performed by immersing the fibrin molded body in a cleaning solution. Here, it is preferable to use a 0-30 mM aqueous solution containing an inorganic salt as the cleaning solution. The inorganic salt-containing aqueous solution is preferably a 0.1-30 mM calcium ion-containing aqueous solution. 10-25 mM calcium chloride aqueous solution is especially preferable.

  In the cleaning step, it is particularly preferable to apply a load of 0.5 to 10 kPa to the molded body from the viewpoint of improving the strength. Here, the load may be a pressing force or a tensile force. A more preferable load is 1 to 8 kPa, and a more preferable load is 1 to 5 kPa. Specifically, it is preferable to apply a load by pulling the entire molded body with the force of rubber or a spring in the major axis direction, pressing with a force perpendicular to the major axis direction, applying a hydrostatic pressure, or the like. . It is preferable that the load is continued for 0.5 to 72 hours, further 1 to 48 hours, particularly 6 to 24 hours.

  A more preferable load loading means is a means for pressing with a compression plate, such as a compression means such as a pickled container. According to this means, a constant load can be uniformly applied to the entire molded body.

  The reason why the strength is improved by washing and loading is not clear, but impurities such as albumin in the molded body are removed, and a certain amount of moisture in the molded body structure is removed. This is considered to be due to the progress of cross-linking of fibrin by coagulation factor 13. Therefore, cryoprecipitate, which is a precipitate containing fibrinogen at a high concentration, is obtained by freezing and thawing the plasma concentrated by the above method, and then removing a part of this supernatant containing albumin at a high concentration. By reducing the relative albumin content in the suspension and solidifying it, the time required for this washing process can be shortened.

  In addition, the loading process of washing | cleaning and a load does not need to be physiological conditions, It can carry out at 0-40 degreeC, More preferably, 4-30 degreeC, More preferably, it is 4-25 degreeC.

  By the above operation, the high-strength fibrin molded body of the present invention is obtained. The obtained fibrin molded product is a molded product having white flexibility. Its breaking strength is 0.01 MPa or more, which is high enough that it cannot be predicted from the strength of conventionally known fibrin bodies. A preferred breaking strength is 0.1 MPa or more, and a more preferred breaking strength is 0.5 MPa or more.

  Thus, since the fibrin molded body of the present invention has flexibility and high strength, it is useful as an artificial ligament, an artificial dura mater, an artificial periosteum, a wound dressing material, a filling material for a tissue defect portion, etc. Useful as a ligament. Ordinary fibrin gel is brittle and cannot be fixed with a suture, but the molded article of the present invention has a strength that does not tear even when the suture is applied. When the fibrin molded body of the present invention is used as a scaffold for transplantation of ligaments and tendons, it is preferable to use plasma and platelets derived from patients.

  In order to reconstruct a ligament or tendon using the fibrin molded body of the present invention, the fibrin molded body may be transplanted to a site requiring reconstruction such as a knee or an elbow. At this time, the fibrin molded body can be cut into a desired shape, for example, a thin string shape, a flat shape imitating the shape of a physiological ligament, and the like.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

Example 1
900 mL of bovine blood to which 100 mL of 5% aqueous sodium citrate solution was added as an anticoagulant was centrifuged at 3000 × g for 15 minutes, and the plasma component and the buffy coat layer were fractionated. The plasma component was further centrifuged at 3000 × g for 15 minutes to remove red blood cells mixed in the plasma layer, and further ultrafiltered with a molecular weight cut off of 10,000 (using Vivaflow 50 of Vivascience AG, Hanover, Germany). Concentrated twice. On the other hand, the buffy coat was centrifuged twice at 3000 × g for 15 minutes to remove red blood cells mixed in the buffy coat layer, and plasma was added thereto to adjust to 10 mL. 11.6 mL of concentrated plasma, 0.12 mL of buffy coat corresponding to the amount of platelets corresponding to this amount of plasma, and 0.27 mL of 1M CaCl ₂ were added to a polypropylene (PP) or PET tube and mixed (FIG. 1). . The obtained suspension was allowed to stand in a 37 ° C. incubator for 3 hours. After 3 hours, it was confirmed that the suspension was gelled (FIG. 2). The obtained molded body was taken out from the tube and immersed in a 10 mM CaCl ₂ aqueous solution (FIG. 3). In that state, in a pickle container (Pikure K-10, Shinki-ku, Shinagawa-ku, Tokyo) overnight (about 15 hours), pressurize 3.3 KPa at the start of pressurization and 1.3 KPa at the end of pressurization. As a result, a high-strength fibrin molded body was obtained (FIG. 4). The molded body had a width of 14 mm, a length of 69 to 73 mm, and a thickness of 1 mm.
The tensile strength (breaking strength) of the obtained fibrin molded body was measured. The test method was performed using a mechanical tensile tester EZ-TEST manufactured by Shimadzu Corporation with a crosshead speed of 10 mm / min and a distance between ratings of 30 mm. As a result, the maximum load was 7 to 17 N and the maximum stress was 0.50 to 1.22 MPa.

Example 2
(1) A fibrin molded body was obtained in the same manner as in Example 1 except that the amount of buffy coat added was changed. The amount of buffy coat added was 0.06 mL corresponding to a half of Example 1, 0.36 mL corresponding to 3 times, 0.60 mL corresponding to 5 times, 0.84 mL corresponding to 7 times, The amount was 1.20 mL corresponding to 10 times. Furthermore, the case where no buffy coat was added was also performed. As a result, when no buffy coat was added, it took 3 hours or more from the addition of calcium to the suspension prepared in the same manner as in Example 1 from plasma, and it did not coagulate even after 72 hours. It was. The strength of the high-strength fibrin-formed product obtained by pressurizing the obtained molded body in the same manner as in Example 1 was 0.40 to 0.80 MPa. Further, as the amount of buffy coat added increased, the time from calcium and buffy coat addition to solidification was shortened, but the strength of the high strength fibrin molded product finally obtained in the same manner as in Example 1 was When added 5 times or more, the pressure dropped to approximately 0.50 MPa or less. That is, when the amount of buffy coat (as the amount of platelets) exceeds 5 times the amount of platelets corresponding to the amount of blood corresponding to the suspension, the strength generally tends to decrease.

(2) A fibrin molded body was obtained in the same manner as in Example 1 except that the concentration of plasma components by ultrafiltration was changed from 1 to 4 times. As a result, it was found that the concentration of plasma components was 2 to 3 times better for improving the strength of the fibrin molded article.

(3) A fibrin molded body was obtained in the same manner as in Example 1 except that the addition amount of the 1M CaCl ₂ aqueous solution was changed. The final concentration of calcium ions added to the suspension was 0.25 mM corresponding to the amount added in Example 1, and 0.10 mM, 0.20 mM, 0.30 mM, and 0.45 mM. There was a sample that did not gel at 0.10 mM. Solidification did not occur at 0.45 mM. Gelation was observed reliably at 0.20 mM and 0.30 mM. About the high intensity | strength fibrin molded object obtained by pressurizing the molded object obtained by solidification in the way of Example 1, breaking strength became 0.58-1.14MPa.

(4) A fibrin molded body was obtained in the same manner as in Example 1 by changing the standing time of the suspension. As a result, when the standing time was 3 hours or less, solidification was often insufficient, and when standing for 12 hours or more, the effect of increasing the strength was not particularly observed.

(5) It is considered that the strength of the high-strength fibrin molded body finally obtained becomes stronger when albumin contained in a high concentration in the suspension is relatively reduced and then calcium ions are added and solidified. Therefore, the suspension prepared by the method of Example 1 was freeze-thawed, thereby obtaining a cryoprecipitate containing a high concentration of fibrinogen and its supernatant. The obtained supernatant was ultrafiltered with a filter having a molecular weight of 300,000 (Pellicon XL manufactured by Millipore, USA; the type of filter membrane was Biomax). By this operation, most of the albumin was separated without being filtered. When the obtained filtrate was mixed with cryoprecipitate and made into a suspension to produce a high-strength fibrin molded body by the method of Example 1, its breaking strength was further improved to 1.27 to 1.55 MPa. It was done. The time required for pressurization was shortened.

Example 3
Using the fibrin molded body obtained from rabbit blood in the same manner as in Example 1, ligament reconstruction of the rabbit knee was performed. That is, the medial collateral ligaments of both knees of Japanese white rabbits were completely excised, and the fibrin molded product of the present invention prepared from rabbit allogeneic blood only on the right knee was transplanted, and the left knee was left excised.
Eight weeks after the implantation (reconstruction) surgery, the right knee with the fibrin molded body had a medial collateral ligament-like repaired tissue (Fig. 5), but the left knee with only the ligament removed had a ligamentous appearance. There was no organization.
In the HE staining of the above ligament-like tissue (Fig. 6, left), there are many cells with spindle-shaped nuclei that appear to be fibroblasts in the fibrous tissue that has a running property in the long axis direction. Staining (FIG. 6, right) contained abundant collagen fibers regularly arranged in the long axis direction.
In addition, in the decalcified specimen at the boundary between the fibrin molded body and the embedded bone hole and the fibrin molded body, the boundary between the ligament and the bone has a similar image to the ligament-bone transition (direct insertion) found in normal tissue. It was present (Fig. 7).

Example 4
One half of the central part of both knee patellar ligaments of five Japanese white rabbits was excised, and a fibrin molded body prepared from rabbit allogeneic blood was transplanted only to the right knee, while the left knee was left excised.
The patella ligaments of both knees were removed 12 weeks after the implantation operation. The thickness of the central part of the repaired tissue seen in the part where the tendon was excised was 2.1 mm on the right knee transplanted with the fibrin molded body on average and 1.5 mm on average on the left knee. Using a Shimadzu mechanical tensile tester EZ-TEST, a rupture strength test of the patella tendon taken out at a crosshead speed of 10 mm / min was performed. The rupture strength of the right patellar ligament grafted with the fibrin molded body was stronger.

It is a figure which shows the state which added the suspension liquid to the tube. It is a figure which shows the state which gelatinized after stationary. It is a figure which shows the taken-out fibrin molded object. It is a figure which shows the fibrin molded object after a pressurization. It is a figure which shows the state 8 weeks after a transplant. It is a figure which shows the HE stained tissue (left) and Sirius red staining (right) 8 weeks after transplantation. It is a figure which shows the HE dyeing | staining structure | tissue 3 weeks after a transplant.

Claims (5)

High-strength fibrin molding obtained by allowing a suspension containing plasma components, platelets and calcium ions concentrated 2-3 times to stand for 3 to 24 hours to solidify and then wash the resulting molded body In the solidification stage, the suspension contains platelets in an amount equivalent to 5 times the amount of blood used as a plasma raw material, from 1/10 the normal platelet concentration to the platelets. The final concentration of calcium ions is 20 to 30 mM , and the washing is performed with an aqueous solution containing 10 to 30 mM calcium ions while applying a load of 1.3 to 3.3 kPa to the obtained molded body. High strength fibrin molded body.   The high-strength fibrin molded article according to claim 1, wherein the standing is performed at 4 to 40 ° C.   The high-strength fibrin molded body according to claim 1 or 2, wherein solidification molding by standing is performed by solidifying in a predetermined container.   The high-strength fibrin molded article according to any one of claims 1 to 3, which has a breaking strength of 0.5 MPa or more.   An artificial ligament containing the high-strength fibrin molded article according to any one of claims 1 to 4.