USE OF MITOCHONDRIA TO TREAT AND/OR PREVENT TENDON INJURY OR ITS RELATED DISEASE

Abstract

The present invention provides a second use of mitochondria, which can cure a tendon injury-related disease and prevent a disease caused by a tendon injury. Specifically, the mitochondria disclosed in the present invention have the effect of repairing injured tendon cells and accelerating the healing of the tendon cells. Therefore, by administering a predetermined amount of mitochondria or a composition containing a predetermined amount of mitochondria to a part with a tendon injury, wound healing of the part with the tendon injury can be promoted, thus achieving the effect of repairing the injured tendon and further preventing a joint disease caused by the tendon injury or inflammation.

Claims

1. A composition, comprising mitochondria and a blood product, wherein the blood product contains at least one of the following growth factors: PDGF-BB, IGF-1, TGF-β1, VEGF, and bFGF.

2. The composition of claim 1, wherein the blood product is platelet-rich plasma (PRP).

3. The composition of claim 1, wherein the dose of the mitochondria ranges from 5 .Math.g to 80 .Math.g.

4. A use of mitochondria to prepare a composition for treating a tendon injury-related disease.

5. The use of claim 4, wherein the tendon injury-related disease is tendonitis.

6. The use of claim 4, wherein the tendon injury-related disease has a symptom of tendon inflammation or swelling.

7. The use of claim 4, wherein the tendon injury-related disease is a tendon lesion.

8. The use of claim 4, wherein the composition further contains platelet-rich plasma (PRP).

9. A use of mitochondria to prepare a composition for preventing a joint disease or a ligament injury-related disease, wherein the joint disease or the ligament injury-related disease is caused by a tendon injury or tendon inflammation.

10. The use of claim 9, wherein the composition further contains platelet-rich plasma (PRP).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows an analysis result of a cell survival rate after the human tendon cells are treated with hydrogen peroxide and then cultured in different administration conditions;

[0019] FIG. 2 shows an analysis result of a cell survival rate after the human tendon cells are treated with tert-butyl hydroperoxide (tBHP) and then cultured in different administration conditions;

[0020] FIG. 3A shows a result of observing a cell migration assay after the human tendon cells are treated in different conditions; and

[0021] FIG. 3B shows a statistical analysis result of a cell migration assay after the human tendon cells are treated in different conditions.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention discloses a second use of mitochondria, which can treat a tendon injury-related disease and prevent a disease caused by a tendon injury. Specifically, the mitochondria disclosed in the present invention have the effect of repairing the injured tendon cells and accelerating the healing of the tendon cells. Therefore, by administering a predetermined amount of mitochondria or a composition containing a predetermined amount of mitochondria to a part with a tendon injury, the wound in this part can be healed, thus achieving the effect of repairing the injured tendon and preventing the occurrence of a joint disease caused by a tendon injury or inflammation.

[0023] Generally speaking, the administration dose of the mitochondria ranges from 5 .Math.g to 80 .Math.g, such as 5 .Math.g, 15 .Math.g, 20 .Math.g, 25 .Math.g, 30 .Math.g, 40 .Math.g, 55 .Math.g, 60 .Math.g, 70 .Math.g, or 80 .Math.g. Moreover, the mitochondria can be mixed with different components to prepare a composition, where the mitochondria are preferably mixed with a growth factor or a mixture containing a growth factor. For example, the mitochondria, and PRP or a component containing a growth factor, such as a blood product, are mixed to form a composition.

[0024] Further, the mitochondria disclosed in the present invention need to be mixed with another component before being administered to the injured part. For example, the mitochondria mixed with PRP can greatly accelerate the healing of the part with a tendon injury.

[0025] The “composition” mentioned in the present invention refers to a material containing an effective dose of mitochondria, and is prepared in different forms and dosage forms according to use requirements or methods, and is formed by necessarily mixing different components, carriers, excipients, or the like.

[0026] The “mitochondria” mentioned in the present invention are separated out from cells, and the used separation technique or method should be able to maintain the structural and functional integrity of the mitochondria. For those of ordinary skill in the art to which the present invention pertains, the separation technique or method may be physical or chemical.

[0027] The “cells” mentioned in the present invention refer to those having mitochondria, such as adipose-derived stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, blood cells, and the like.

[0028] The “blood product” mentioned in the present invention refers to a product prepared by using the blood as the raw material, and contains a certain amount of growth factors, such as PRP separated out from the whole blood, blood added with growth factors, or the like. The “certain amount” mentioned herein refers to an amount obtained according to well-known knowledge by persons of ordinary skill in the art to which the present invention pertains. For example, the number of platelets in the PRP disclosed in the present invention is at least 1,000,000/.Math.l and the PRP contains the following growth factors: PDGF-BB (155.2±57.67 ng/ml), IGF-1 (236.07±222.1 ng/ml), TGF-β1 (488.76±240.77 ng/ml), VEGF (242.29±97.64 ng/ml), and bFGF (82.24±64.51 ng/ml).For another example, the content of the growth factors in the blood product mentioned in the present invention is at least 522.54 ng/ml, and the blood product is required to contain platelets of which the number is at least 1,000,000/.Math.l.

[0029] The “administration” mentioned in the present invention refers to enabling the mitochondria disclosed in the present invention to contact the injured tendon part, and the way of contacting the injured tendon part is not limited to smearing, dripping, injecting, introducing, etc. Moreover, an external force, such as ultrasound waves, shockwaves, heating, or the like, is further utilized to strengthen or accelerate the uptake by the cells.

[0030] The “tendon injury-related disease” mentioned in the present invention refers to a disease resulting from tendon tears, reduced flexibility, and decreased force-bearing capacity, such as tendonitis. Because tendons exist between muscles and bones, the tendon injury-related disease can occur in various parts of the body having tendons. Clinically, there are different indication names according to different locations of the tendons, such as biceps tendinitis, rotator shoulder tendinitis, knee patellar tendinitis, Achilles tendinopathy, calcific tendinitis in a shoulder joint, etc.

[0031] The “joint disease” mentioned in the present invention refers to a disease caused by tendon inflammation or injury. That is, when the tendon is injured or inflamed, the ability to move where the tendon is located (i.e. the joint) may be impaired. Moreover, as the tendon injury or inflammation time increases, the chance of a joint lesion or joint disease is significantly improved. For example, if the tendon in the knee is injured, the knee movement ability, such as bending, walking, etc., may be affected. Therefore, if the part with a tendon injury can be rapidly repaired, the joint disease, such as a frozen shoulder, degenerative arthritis, knee arthritis, etc., caused by a long-term tendon injury can be effectively prevented.

[0032] In order to prove the effects of the technical features disclosed in the present invention, several examples are given below to describe the present invention in detail with reference to the accompanying drawings.

[0033] In the following examples, hydroxyurea (HU) is used as a cell proliferation inhibitor in a cell migration assay.

Example 1: Culture of Human Tendon Cells

[0034] The culture of human tendon cells was performed by using a TEN-1 growth medium (Tenocyte Growth Medium, zenbio). Before the culture of the human tendon cells, the culture dish was coated with 250 .Math.g/ml matrix gel (Brand: Corning, Model: 354234). After treatment for 1 hour, the matrix gel was removed and the phosphate buffer solution was used for cleaning; and then the human tendon cells were cultured in a 37° C. incubator (having 5% carbon dioxide) with the TEN-1 growth medium coated with the matrix gel. When the human tendon cells grew to the completeness of9, the cell culture medium was removed and the phosphate buffer solution was used for cleaning. Then, the phosphate buffer solution was removed and 0.25% trypsin was added in to react at 37° C. for 5 min. After reaction completion, the TEN-1 growth medium was added in to neutralize the trypsin, and centrifugation was performed at 1000 rpm for 5 min. The supernatant was removed after centrifugation; and a new TEN-1 growth medium was added and cell counting was performed, for use in subsequent examples.

Example 2: Preparation of PRP

[0035] Fresh blood was taken into a separator tube (Brand: BD, Model: REF362761) containing an anticoagulant, and was centrifuged at 1500-2000 g for 10 mins. Then, the blood sample was divided into four layers, which are a red blood cell layer, separating gel, a white buffy coat layer (containing monocytes and platelets), and a slightly-yellow transparent plasma layer from bottom to top. The buffy coat and the plasma were collected; and the collected buffy coat layer and plasma layer were transferred to another separator tube and centrifuged at 900 g for 10 min. After centrifugation, the top ⅔ of the plasma was removed, and the remaining product was uniformly mixed to obtain the PRP.

[0036] The number of platelets in the PRP prepared in this example was more than 1,000,000/.Math.l, and the PRP contained a variety of growth factors, such as PDGF-BB (155.2±57.67 ng/ml), IGF-1 (236.07±222.1 ng/ml), TGF-β1 (488.76±240.77 ng/ml), VEGF (242.29±97.64 ng/ml), and bFGF (82.24±64.51 ng/ml).

[0037] For use in the following examples, the prepared PRP was added to a cell culture medium at 5 percent by volume or to an animal solvent to be injected, to prepare a PRP solution with a volume percentage concentration of 5%.

Example 3: Preparation of Mitochondria

[0038] The human adipose-derived stem cells were cultured to obtain 1.5 × 10.sup.8 cells, and the Duchenne phosphate buffer solution (DPBS) was used to flush the cells and then was removed. Trypsin was added in to react for 3 min, and then a stem cell culture liquid (Keratinocyte SFM (1X) liquid, bovine pituitary extract, or 10 wt% fetal calf serum) was added in to terminate the reaction. Afterwards, the cells were collected and centrifuged (600 g for 10 min), and the supernatant was removed. Then, 80ml IBC-1 buffer solution (the buffer solution is compounded of 225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA, and 30 mM Tris-HCl with pH of 7.4) was added to the cells, and centrifugation was conducted after homogenization, to obtain a precipitate that was the mitochondria (referred to as a mitochondrial precipitate in the following description). 1.5 ml IBC-1 buffer solution and a proteolytic enzyme inhibitor were added to the mitochondrial precipitate, and then the mitochondrial precipitate was placed aside in a 4° C. environment, for use in the following examples.

Example 4: Injury Test for Tendon Cells (1)

[0039] The human tendon cells cultured in Example 1 were subcultured in a 24-well plate, where the concentration per well was 5 × 10.sup.4 cells/500 .Math.L. After 8-hour culturing, the supernatant was removed and the phosphate buffer solution was used for cleaning. Afterwards, the phosphate buffer solution was removed, and 500.Math.LTEN-1 growth medium was added to each well to perform culturing for 8 hours. After culturing, hydrogen peroxide with a concentration of 300 .Math.M was used for treatment. The supernatant was removed after 4-hour reaction, and the phosphate buffer solution was used for cleaning. Afterwards, the cells in different groups were treated according to the following different administration conditions: adding the PRP, adding the mitochondria (40 .Math.g), and adding the mitochondria (40 .Math.g) and the PRP. Afterwards, each group was cultured separately for 24 hours. After culturing completion, the phosphate buffer solution was used for cleaning, and then the TEN-1 growth medium (250 .Math.L/well) containing 10% alamar Blue was added in to perform culturing for 3-4 hours in a 37° C. environment. After culturing completion, fluorescent signal measurement (Excitation/Emission: 560/590 nm) was performed, to obtain a result shown in FIG. 1.

[0040] It can be known from the result of FIG. 1 that, the survival rate of the human tendon cells (referred to as a blank group in the following description) not treated with hydrogen peroxide is 100.4±1.83%; the survival rate of the human tendon cells (referred to as an H.sub.2O.sub.2 group in the following description) that are treated with hydrogen peroxide but not administered with the mitochondria and/or PRP is 59.97±12.83%; the survival rate of the human tendon cells (referred to as an H.sub.2O.sub.2/PRP group in the following description) that are treated with hydrogen peroxide and further administered with the PRP is 80.12±9.19%; the survival rate of the human tendon cells (referred to as an H.sub.2O.sub.2/ mitochondrial group in the following description) that are treated with hydrogen peroxide and further administered with the mitochondria is 85.22±11.46%; and the survival rate of the human tendon cells (referred to as an H.sub.2O.sub.2/PRP/mitochondrial group in the following description) that are treated with hydrogen peroxide and mitochondria and further administered with the mitochondria and PRP is 99.16±11.83%.

[0041] The foregoing results show that the cell survival rate of the H.sub.2O.sub.2 group is obviously reduced as compared with that of the blank group, which indicates that the hydrogen peroxide can indeed injure the human tendon cells and lead to death of the tendon cells. The cell survival rate of the group administered with the mitochondria or PRP is obviously higher than that of the group using the hydrogen peroxide, and the cell survival rate of the H.sub.2O.sub.2/mitochondrial group is higher than that of the H.sub.2O.sub.2/PRP group. That is, the mitochondria and the PRP can both alleviate the injury in tendon cells and reduce the cell death, but the mitochondria have a better alleviation effect for the injury in tendon cells. Moreover, the cell survival rate of the H.sub.2O.sub.2/PRP/mitochondrial group is almost equal to that of the blank group. It indicates that, when the human tendon cells are injured, the simultaneous administration of the mitochondria and the PRP can alleviate the injury in the human tendon cells and further can repair the injured human tendon cells, thus achieving the effect of treating the injured human tendon cells or the related diseases.

Example 5: Injury Test for Tendon Cells (2)

[0042] The process of this example was substantially identical with that in Example 4, but had the following differences. In this example, except the blank group, the human tendon cells in other groups were first treated with tBHP with a concentration of 300 .Math.M, and were then cultured according to different administration conditions for different groups. After culturing completion, fluorescent signal measurement (Excitation/Emission: 560/590 nm) was performed, to obtain a result shown in FIG. 2.

[0043] It can be known from the result of FIG. 2 that, the survival rate of the human tendon cells (referred to as a blank group in the following description) not treated with tBHP is 93.38±7.65%; the survival rate of the human tendon cells (referred to as a tBHP group in the following description) that are treated with tBHP but not administered with the mitochondria and/or PRP is 63.3±16.03%; the survival rate of the human tendon cells (referred to as a tBHP/PRP group in the following description) that are treated with tBHP and further administered with the PRP is 76.4±19.55%; the survival rate of the human tendon cells (referred to as a tBHP/mitochondrial group in the following description) that are treated with tBHP and further administered with the mitochondria is 90.2±11.09%; and the survival rate of the human tendon cells (referred to as a tBHP/PRP/mitochondrial group in the following description) that are treated with tBHP/mitochondria and further administered with the mitochondria and PRP is 97±2.29%.

[0044] The cell survival rate of the tBHP group is obviously reduced as compared with that of the blank group, which indicates that tBHP can indeed injure the human tendon cells and lead to death of the tendon cells. The cell survival rate of the group administered with the mitochondria and/or PRP is obviously higher than that of the tBHP group, which indicates that the mitochondria and the PRP can both alleviate the injury in the human tendon cells induced by tBHP. In this case, the alleviation effect achieved by administration of only the mitochondria is superior to that achieved by administration of only the PRP. When the human tendon cells are injured after treatment with tBHP, the simultaneous administration of the mitochondria and the PRP can almost eliminate the injury in the human tendon cells, and the cell survival rate is almost equal to that of the blank group. That is, when the human tendon cells are injured, the simultaneous administration of the mitochondria and the PRP can effectively alleviate and repair the injury in the human tendon cells, thus promoting or accelerating the treatment of the injury in the human tendon cells or the related diseases.

Example 6: Migration Assay of Tendon Cells

[0045] The tendon cells were cultured in a 24-well plate at a concentration of 5 × 10.sup.4 cells/500 .Math.L for 24 hours. When the cell completeness reached 9, the phosphate buffer solution was first used to clean the cells, and then a straight wound of a fixed width was scraped in the middle of the cell. The TEN-1 growth medium and cells in suspension were removed and a mixed cell culture liquid (90%DMEM/F12+10% TEN-1 growth medium) containing 10 .Math.M hydroxyurea was used to replace the original medium, and then the cells were cultured for 24 hours in the following different conditions separately according to an assay design: adding 40 .Math.g mitochondria, adding 5% PRP, and adding 5% PRP and 40 .Math.g mitochondria. After culturing, the repair and healing of the wound was observed and analyzed, to obtain a result shown in FIG. 3.

[0046] It can be known from the result of FIG. 3A that, as compared with the blank group not administered with any medicine, the human tendon cells administered with the mitochondria, the PRP, or the PRP and mitochondria have a better repair effect. Moreover, it can be known from the result of FIG. 3B that, the cell migration number in the blank group is 100±27.58; the cell migration number in the group treated with the mitochondria is 211.31±42.18; the cell migration number in the group treated with the PRP is 221.6±56.61; and the cell migration number in the group treated with both the PRP and the mitochondria is 302.06±84.97.

[0047] The results of FIGS. 3A and 3B show that, the administration of only the mitochondria to the injured human tendon cells can promote the wound healing of the tendon, and its healing effect is equivalent or even superior to that achieved by the administration of only the PRP. If the mitochondria and the PRP are simultaneously administered to the injured human tendon cells, the wound healing rate can be accelerated. That is, the mitochondria or the composition containing the mitochondria disclosed in the present invention can indeed promote or accelerate the healing of the tendon wound, thus achieving the effect of accelerating the repair of the injured tendon cells or treating the tendon lesion.

Example 7: Experiment on Animals

[0048] 12-week-old female Sprague-Dawley (SD) mice were raised in a 22±2° C. environment with the humidity ranging from 50% to 70%, and a tendon injury was induced in each mouse by using type 2 collagenase. Then, their tendon strength was observed and analyzed after treatment in different conditions.

[0049] In detail, the mice in each group were first anesthetized, the hair around their shoulder joints was shaved, and disinfection was performed with 70% alcohol. Then, the type 2 collagenase was injected into the supraspinatus tendon between coracoids and clavicle of each mouse with a needle and a syringe at an angle of 45 degrees, where the injection was required to be completed at a concentration/dose of 80 U/8 ul within 1 min. The day when the injection of type 2 collagenase was performed and completed was set to the 0.sup.th day of the experiment, and the tendon injury status of each mouse was observed and estimated from the 0.sup.thday of the experiment. On the 3.sup.rdday of the experiment, the injury was treated in different administration conditions for the different groups, including no administration, administering the mitochondria (15 .Math.g), administering the PRP (5 vol%), and administering the mitochondria (15 .Math.g) and the PRP (5 vol%).The tendon strength was analyzed on the 7.sup.thday and the 14.sup.th day of the experiment, and in this example, the tendon injury status was estimated and the tendon strength was analyzed by using a vertical automatic tester (JSV-H1000).That is, after the mice that have completed the experiment were sacrificed, the supraspinatus tendons together with the humeri were taken down and placed on the vertical automatic tester to take a test at a rate of 10 mm/min, where a force that broke the tendon was the maximum strength of the mouse tendon. The results of this example are shown in tables 1 and 2.

[0050] It can be known from the result of table 1 that, on the 3.sup.rd day after the type 2 collagenase is administered, the tendon inflammation and injury can be observed and the tendon strength is obviously reduced, which indicates that the type 2 collagenase can indeed induce the tendon inflammation and swelling. On the 7.sup.th day and the 14.sup.th day, it is observed that the tendon inflammation and swelling are both alleviated, and the tendon strength is gradually improved but still less than half of the strength on the day (the 0.sup.th day) of injection. As shown in table 2, it can be known from the results of the tendon strength on the 7.sup.th day and 14.sup.th day of the experiment that, the mouse tendon inflammation and injury status in which the mitochondria and/or PRP are/is administered is better than that without administration of any medicine, and moreover, the tendon strength recovers better. Further, the tendon strength has the best recovery effect in the case where the PRP and the mitochondria are simultaneously administered.

[0051] The results indicate that, the mitochondria or a composition containing the mitochondria, such as a composition compounded of the mitochondria and PRP, disclosed in the present invention can indeed alleviate the tendon inflammation and swelling and further can promote the tendon repair efficiency. Therefore, the injured tendon can be restored to its strength and elasticity in a short period of time, thus achieving the effect of treating or preventing the tendon injury-related diseases.

TABLE-US-00001 Estimation results of tendon strength of mice in a tendon injury pattern (type 2 collagenase is only injected and mitochondria and/or PRP are/is not administered) Experiment day the 0.sup.th day the 3.sup.rd day the 7.sup.th day the 14.sup.th day Maximum tendon strength (N) 33.32±2.13 5.32±1.21 9.13±1.19 16.13±2.06

TABLE-US-00002 Results of a tendon strength test during the experiment for mice in each group Tendon strength (N) Experiment day the 3.sup.rd day the 7.sup.th day the 9.sup.th day Group administered with collagenase (the blank group) 5.32±1.21 9.13±1.19 16.13±2.06 Group administered with PRP 12.83±1.28 17.96±1.73 Group administered with mitochondria 13.27±1.36 18.28±1.94 Group administered with mitochondria and PRP 15.31±2.21 21.36±2.26