TWI475998B - A joint injection - Google Patents

Description

Joint injection

The present invention relates to an injection, and more particularly to a joint injection for repairing a cartilage matrix.

Degenerative arthritis (OA) is one of the common articular cartilage degenerative diseases, which occurs in the lower extremity joints (such as the knee joint or the ankle joint). It is characterized in that the articular cartilage in the joint cavity is damaged, resulting in joints. The cavity is shrunk, and due to the loss of the joint surface, the surface of the joint is rough, and in severe cases, local function is lost.

Because degenerative arthritis is still a disease that cannot be completely cured, it can only be clinically suppressed in a passive manner to aggravate or relieve pain, such as physical therapy to reduce the pressure on the joints and reduce inflammation of the joints; or use surgical treatment, Perform arthroscopic debridement and repair the rupture of joints.

In addition, it is also clinically possible to directly apply a conventional joint injection by an intra-articular injection method, which comprises hyaluronic acid, which can relieve a patient by injection of hyaluronic acid. Pain, and at the same time increase the mobility of the joint and restore the viscosity of the joint fluid; however, the application of the conventional joint injection is still unable to effectively cure degenerative arthritis.

The main object of the present invention is to provide a joint injection which can stimulate articular chondrocytes and repair cartilage matrix to improve joint damage.

In order to achieve the foregoing object, the technical means and the achievable effects of the present invention include: an articular injection comprising: 0.02 to 0.3 ppm of estradiol and 1 by weight percent; % hyaluronic acid, the remaining component is a pharmaceutically acceptable carrier, wherein the hyaluronic acid has a molecular weight of 500 to 1500 kDa.

The joint injection of the present invention, wherein the pharmaceutically acceptable carrier is preferably physiological saline.

The joint injection of the present invention, wherein the joint injection preferably contains 0.2 to 0.3 ppm of estradiol.

The joint injection of the invention not only enhances the viscosity of the joint synovial fluid by hyaluronic acid, but also enhances the function of synaptic vibration and lubrication of the joint synovial fluid; and enhances the chondrocyte by the estradiol content of 0.02-0.3 ppm. Active, repair damaged cartilage matrix, and thus improve the effect of joint damage symptoms.

Figure 1 shows the growth pattern of primary chondrocytes in group A0~A5.

Figure 2a shows the growth activity of chondrocytes in group B0~B2.

Figure 2b shows the proteoglycan content of chondrocytes in group B0~B2.

Figure 3a shows the amount of the second type of collagen gene in the chondrocytes of the C0~C2 group.

Figure 3b shows the amount of Aggrecan gene expression in chondrocytes from group C0 to C2.

Figure 3c shows the TIMP-3 gene expression in chondrocytes from group C0 to C2.

Figure 3d shows the MMP-3 gene expression of chondrocytes in the C0~C2 group.

Fig. 4 is a result of H&E staining and Alcian blue staining of the test animals of Group D0.

Fig. 5 is a result of H&E staining and Alcian blue staining of the test animals of Group D1.

Fig. 6 is a result of H&E staining and Alcian blue staining of the test animals of Group D2.

The above and other objects, features, and advantages of the present invention will become more apparent from the aspects of the invention. Acidic acid and estradiol improve the viscosity of joint synovial fluid by hyaluronic acid, and enhance the activity of chondrocytes by estradiol to improve the symptoms of joint damage.

In detail, the joint injection of the present invention comprises 1% by weight of hyaluronic acid to supplement the endogenous hyaluronic acid which loses its function due to degradation; and, in order to reduce the hyaluronic acid in the living body It is rapidly degraded, and the molecular weight of the hyaluronic acid is preferably 500 to 1500 kDa to enhance the elasticity, viscosity and duration of the hyaluronic acid.

Further, the joint injection of the present invention further comprises 0.02 to 0.3 ppm of estradiol, preferably 0.2 to 0.3 ppm of estradiol.

Further, in order to maintain the activity of the joint injection of the present invention, the joint injection further comprises a pharmaceutically acceptable carrier, which may be selected from the group consisting of a dispersant, an electrolyte, a tissue penetrant, and a tissue. a group consisting of a softener, a pH adjuster, a surfactant, a sustained release agent, a viscosity improver, a comfort enhancer, a solubilizer, a stabilizer, a softener, and a lubricant. In the present embodiment, the medicine The acceptable carrier is selected as physiological saline to avoid the discomfort of the organism due to the difference in osmotic pressure when the joint injection is administered to the organism.

In order to confirm that the joint injection of the present invention does have a function of improving the symptoms of joint damage, the following tests were carried out:

(A) Effect of estradiol on the growth pattern of primary chondrocytes

This test is a Japanese white rabbit with a weekly age of 2~4 weeks. Rabbit), after sacrifice, remove the femur and soak it in protease (protease XIV) at a concentration of 2 mg/mL for 2 hours to dissolve the proteoglycan and continue to soak in the collagenase type II (concentration type II). 2 mg/mL) of serum-free F12 medium (F12 medium, purchased from Gibco) was reacted for 3 hours to dissolve collagen fibers, and a hydrolysis solution was obtained; after the centrifugation solution was centrifuged at 2000 rpm for 5 minutes, the upper body was removed. Serum, and culture the primary chondrocytes with F12 medium (including 10% fetal bovine serum and 200 U/mL penicillin/streptomycin, purchased from Gibco) After the primary chondrocytes are cultured to the second generation, the following cell tests can be performed.

Please refer to the first table. In this test, the primary chondrocytes were treated with estradiol at a concentration of 10 ^-4 ~ 10 ^-8 M. The primary chondrocytes were cultured in 24-well plates (each well contained 1 × 10). Among the ⁵ cells, the cells were cultured in F12 medium containing different concentrations of estradiol, and the new culture solution was changed every two days, and the growth patterns of the cells in each group were observed on the third day.

The test was observed with an Olympus IX70 optical microscope, and the result of magnification 100× is shown in Fig. 1. The results of the test showed that the growth of primary chondrocytes in group A1 was seriously affected, and the cells died. It indicated that the concentration of 10 ^-4 M estradiol would cause the death of primary chondrocytes; in addition, the remaining A2 The primary chondrocytes of the ~A5 group increased with the number of culture days. The growth space of the primary chondrocytes at the bottom of the 24-well plate was saturated, and there was no significant difference in the growth patterns of each group.

(B) Effect of estradiol on cell proliferation and biochemical function of primary chondrocytes

Please refer to the second table. In this test, each group of cells was suspended in a bioreactor, and different concentrations of estradiol were added to the culture solution for treatment, and continued on days 0, 1, 3 and 7. MTT cell activity assays were performed.

Referring to Fig. 2a, the cell activity of the B1 group in which the concentration of 10 ^-6 M estradiol was added to the culture solution was better, indicating that the concentration of the estradiol line was beneficial for the growth of chondrocytes.

Chondrogenic lineage may be secreted into the extracellular matrix proteoglycans in the cartilage tissue to absorb large amounts of water, providing shock, is continued using Blyscan ^TM Sulfated Glycosaminoglycan Assay kit (available from Biocolor) of cartilage B0 ~ B2 test group of The amount of proteoglycan secreted by the cells to confirm whether each group of chondrocytes has normal biochemical functions.

Referring to Fig. 2b, the amount of proteoglycan secreted in group B1 of estradiol at a concentration of 10 ^-6 M in the culture solution was higher than that in groups B0 and B2, indicating that the concentration of estradiol has Promotes the ability of chondrocytes to secrete proteoglycans.

(C) Effect of estradiol on gene expression of primary chondrocytes

Please refer to the third table. In this test, each group of cells is suspended in a bioreactor, and different concentrations of estradiol are added to the culture solution for treatment. The RNA of the primary chondrocytes of each group was extracted at 1, 3 and 7 days, and then reverse transcribed into cDNA, and then analyzed by quantitative real-time PCR.

This study was to analyze the gene expression of type II collagen, aggrecan, TIMP-3 (tissue inhibitor of metalloproteinase) and MMP3 (matrix metalloproteinase) in chondrocytes. GAPDH is used as an internal control group; wherein the primer pair for analyzing the expression of the second type collagen gene has the sequence shown in SEQ ID NOs: 1 and 2, and the primer pair for analyzing the expression of the aggrecan gene has the SEQ ID The sequence shown by NOs: 3 and 4, the primer pair for analyzing the expression of the TIMP-3 gene has the sequence shown in SEQ ID NOs: 5 and 6, and the primer pair for analyzing the expression of the MMP3 gene has the SEQ ID. The sequence shown by NOs: 7 and 8, and the primer pair for analyzing the expression of the GAPDH gene has the sequences shown in SEQ ID NOs: 9 and 10.

Please refer to Figure 3a for the results of the second type of collagen mRNA in each group of the experiment. Among them, the type II collagen mRNA of group B1 (equol concentration of 10 ^-6 M) is obvious. There was a higher amount of performance, while Group B2 was not significantly different from Group B0 of untreated estradiol. The second type of collagen is the main component of articular cartilage, which accounts for 85-90% of the total protein of articular cartilage. The second type of collagen provides the necessary tensile strength of the joint by the help of proteoglycan. Therefore, estradiol promotes the strength of joints by promoting the production of type 2 collagen and proteoglycans.

Continued to refer to Figure 3b, which is a quantitative real-time polymerase chain reaction test of the aggrecan mRNA performance of each group of the test, similar to the test results of the second type of collagen, the treatment of the concentration of 10 ^-6 M of the second In group B1 of alcohol, aggrecan mRNA was significantly higher in performance. Aggrecan is an important component of cartilage and can be combined with hyaluronic acid to provide interaction between chondrocytes and between chondrocytes and extracellular matrices.

Further, referring to Figures 3c and 3d, Group B1 also had lower MMP-3 and higher TIMP-3 mRNA expression, and the results of Group B2 were not significantly different from Group B0. MMP-3 has the ability to degrade extracellular matrix, and it degrades proteins important to joints such as type II collagen and proteoglycan, while TIMP-3 is an important inhibitor of MMP family proteins. The alcohol system protects the joint by inhibiting the performance of MMP-3 and improving the performance of TIMP-3.

Based on the results of the 3rd to 3d images, estradiol can induce the expression of type 2 collagen and aggrecan in chondrocytes, and estradiol can also promote TIMP-3 gene expression and inhibit MMP-3 gene expression. Therefore, it has the functions of promoting the synthesis of extracellular matrix of chondrocytes and inhibiting the destruction of extracellular matrix.

(D) Effect of joint injection of the present invention on repair of cartilage defects

In this test, mature Japanese white rabbits (average body weight of about 2.0 to 2.5 kg) were selected. During the test period, the activities of each group of test animals were not restricted, and the same amount of food was provided daily. The test animals of each group were treated with Zoletil 50. Anesthetic (amount of anesthetic with a dose of 1 mL per kilogram of body weight), after anesthesia, wipe the knee joint with alcohol, continue shaving and disinfection, and apply type 2 collagenase (5mg/mL, intra-articular injection) 0.4 mL) was administered, and the same dose of type 2 collagenase was continuously administered on the fourth day, and test animals having degenerative arthritis in each group of the test were obtained after 4 weeks.

Please refer to the fourth table. This experiment uses physiological test water as the negative control group (D0 group), hyaluronic acid as the positive control group (D1 group), and the joint injection of the present invention as the test group (the first) Group D2); wherein the joint injection of the present invention comprises 1% by weight of hyaluronic acid and 0.2 to 0.3 ppm (ie, 10 ^-6 M) of estradiol, and the molecular weight of the hyaluronic acid is It is 900kDa.

Each group of experimental animals was administered with 0.5 mL of the treatment agent (administered once a week), and sacrificed one month after the continuous administration of the treatment agent (four times in total), and the femur of each group of test animals was taken out and soaked in 4 % of formaldehyde is fixed for 24 hours, and then the femur is immersed in decalcifying solution to soften the bone tissue. After washing three times with phosphate buffer solution, it is soaked in 30%, 50%, 70%, 90%, 95. % and 100% alcohol solution is dehydrated for 15~30 minutes, and the dehydrated bone tissue is soaked in xylene to remove residual alcohol solution; the above bone tissue is immersed in liquid paraffin at 56~60 °C for 2-3 hours. After cooling, a paraffin block is formed and cut into a sheet having a thickness of about 5 to 7 μm, and then dried on a carrier sheet containing protein glycerin, and subjected to H&E staining (hematoxylin-eosin) and Alcian blue staining (Alcian Blue), respectively. . [0043]

Please refer to Figure 4 for the test results of Group D0 of the physiological test water. Among them, (a) and (b) are the H&E of the right ankle and the medial aspect of the right foot of the group D0 test animals. The results of the staining, and (c) and (d) are the results of the indigo blue staining of the right ankle and the medial aspect of the right ankle in the group D0 test animals, respectively.

In addition, as shown in Fig. 5, it is the test result of the D1 group of the physiological test water, wherein (a) and (b) are the right ankle and the right medial side of the D1 test animals, respectively. The H&E staining results, and (c) and (d) are the results of the indigo blue staining of the right ankle bone surface and the right medial aspect of the D1 group of test animals, respectively. Compared with the staining result of the D0 group, the D1 group administered with hyaluronic acid made the humeral surface and the medial ridge appear smoother and smoother, indicating that administration of hyaluronic acid can indeed maintain the cartilage matrix of the test animal.

Continued to refer to Figure 6 for the results of the D2 group in the treatment of physiological test water. Among them, the (a) and (b) diagrams are the right ankle and the medial aspect of the right foot of the D2 group. The H&E staining results, and (c) and (d) are the results of the indigo blue staining of the right ankle bone surface and the right medial aspect of the D2 group of test animals, respectively. Similar to the staining result of the group D1, the group D2 of the joint injection of the present invention can also make the humeral surface and the medial ridge appear smoother and smoother, that is, the joint injection of the present invention can be administered to the organism, and can be improved. The joint damage symptoms of the organism.

In summary, the joint injection of the present invention not only enhances the viscosity of the joint synovial fluid by hyaluronic acid, but also enhances the function of synaptic vibration and lubrication of the joint synovial fluid; and further enhances the estradiol content of 0.02-0.3 ppm. The activity of chondrocytes repairs the damaged cartilage matrix and thus improves the symptoms of joint damage.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

<110> Yishou University

<120> Joint injection

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<223> Type II collagen forward primer for quantitative polymerase chain reaction

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<223> A second type of collagen reverse primer for quantifying polymerase chain reaction

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<223> aggrecan forward primer for quantifying polymerase chain reaction

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<223> TIMP-3 forward primer for quantifying polymerase chain reaction

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<223> MMP-3 forward primer for quantifying polymerase chain reaction

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<223> MMP-3 reverse primer for quantifying polymerase chain reaction

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Claims (3)

A joint injection comprising: 0.02 to 0.3 ppm of estradiol and 1% by weight of hyaluronic acid, and the remaining component is a pharmaceutically acceptable carrier, wherein the molecular weight of the hyaluronic acid The system is 500~1500kDa. The joint injection according to claim 1, wherein the pharmaceutically acceptable carrier is physiological saline. The joint injection according to claim 1 or 2, wherein the joint injection contains 0.2 to 0.3 ppm of estradiol.