USE OF MITOCHONDRIA TO PROMOTE WOUND REPAIR AND/OR WOUND HEALING

TECHNICAL FIELD

The present invention relates to a second use of mitochondria, and in particular to a use of mitochondria to promote wound repair and/or wound healing.

BACKGROUND

Wound healing is a continuous and complicated biological response process, which can be roughly divided into several stages, including hemostatic, inflammatory, proliferative, and tissue remodeling phases. The length of the healing time is affected by external or internal factors. That is, when the wounded person has poor blood circulation, is old, is a diabetic, or has a bacterial infection, the time required for wound recovery is likely to increase.

As for treatment and repair of the wound, in addition to controlling the infection of the wound with antibiotics or other drugs in clinical practice, it is also necessary to consider the treatment of other complications, such as blood sugar control for diabetic patients. Current research points out that many growth factors are released after activation of the platelets. Therefore, administration of platelet-rich plasma (PRP) to the affected part that needs to be treated or improved can achieve the repair and alleviation effects. However, because anticoagulants are required during preparation of the PRP, these anticoagulants can actually affect wound repair.

In fact, when the wound fails to recover well, is difficult to heal, is repeatedly inflamed, or even persistently deteriorates, chronic wound infection is likely to occur, which may result in adverse outcomes, such as tissue necrosis, amputation, or threatening to life. Therefore, how to promote wound healing has always been a problem to be solved in clinical medicine.

SUMMARY

The present invention mainly aims to provide a use of mitochondria to promote wound repair and/or wound healing. Specifically, because of the mitochondria's ability to promote cell migration of fibroblasts and increase the expression level of collagen, by administering an effective amount of mitochondria or a composition containing the effective amount of mitochondria to a wound, the effect of promoting wound repair or healing can be effectively achieved, thus reducing or alleviating wound deterioration or persistent inflammation.

Another objective of the present invention is to provide a composition containing mitochondria and other substances containing growth factors, which can significantly improve cell repair and tissue regeneration, or alleviate cellular inflammation, so as to reduce the chance of inflammation-related complications.

Therefore, to achieve the foregoing objective, the present invention discloses a composition which includes mitochondria and a blood product, where the blood product contains at least one growth factor, such as platelet-rich plasma (PRP), plasma, serum, or platelet-rich fibrin (PRF).

In an embodiment, the present invention discloses a use of mitochondria for preparing a composition for repairing wounds or promoting wound healing, where the composition is administered to an affected part, thus improving the healing efficiency of the affected part.

In another embodiment of the present invention, a use of mitochondria for preparing a composition for promoting tissue regeneration is provided. Therefore, by administering an effective amount of mitochondria to a wound, inflammation of the wound can be inhibited or alleviated, thus avoiding inflammation-related complications.

In an embodiment of the present invention, the effective amount of the mitochondria in the composition at least ranges from 5 µg to 80 µg, and is preferably above 40 µg.

In another embodiment of the present invention, the mitochondria are separated out from cells, such as adipose-derived stem cells or mesenchymal stem cells, and the cells may be autologous or heterologous.

In an embodiment of the present invention, the composition further includes platelet-rich fibrin, and the effective amount of the mitochondria in the composition is at least 15 µg.

Another embodiment of the present invention discloses a composition, which includes mitochondria and platelet-rich fibrin (PRF). For example, the dose of the mitochondria ranges from 5 µg to 80 µg, and preferably ranges from 15 µg to 40 µg; and the concentration of the PRF is preferably above 5 volume percent (v/v %).

The beneficial effects of the present invention are:

By administering a certain amount of the mitochondria or the composition containing the mitochondria provided by the present invention to the wound, the wound healing can be promoted, so as to achieve the effect of accelerating wound repair and avoiding wound inflammation or related complications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a result of observing CCD-966SK cells subjected to treatments with different doses of mitochondria and a cell migration assay;

FIG. 1B shows cell migration ratios of the CCD-966SK cells subjected to different treatments in FIG. 1A after statistical analysis;

FIG. 2 shows a statistical analysis result regarding a collagen secretion amount after the CCD-966SK cells are treated with different doses of mitochondria;

FIG. 3A shows a result of observing CCD-966SK cells subjected to PRF treatments with different doses of mitochondria and a cell migration assay;

FIG. 3B shows cell migration ratios of the CCD-966SK cells subjected to different treatments in FIG. 3A after statistical analysis;

FIG. 4 is a statistical analysis result regarding a collagen secretion amount after the CCD-966SK cells are subjected to PRF treatments with different doses of mitochondria;

FIG. 5 shows results of observing wound recovery after PRF containing different doses of mitochondria is applied to the wound in a mouse; and

FIG. 6 shows a result of cell growth efficiency calculated after the CCD-966SK cells are cultured in cell culture media added with different doses of mitochondria.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention provides a use of mitochondria to promote wound repair and/or wound healing. Specifically, when a certain amount of mitochondria or a composition containing an effective amount of mitochondria is administered to a wound, the effect of promoting wound repair and healing can be effectively achieved, where the effective amount for administration of the mitochondria disclosed in the present invention ranges from 1 µg to 80 µg, such as 1 µg, 2 µg, 4 µg, 5 µg, 10 µg, 15 µg, 20 µg, 25 µg, 30 µg, 40 µg, 50 µg, 60 µg, 65 µg, 70 µg or 80 µg, and the effective amount of the mitochondria preferably ranges from 15 µg to 40 µg, thus achieving a better treatment effect or improving wound repair or promoting wound healing.

Further, the mitochondria disclosed in the present invention can be mixed with another component to prepare a composition, where the used component is preferably a material containing a growth factor and more preferably a blood product containing growth factors. For example, the blood product containing growth factors is platelet-rich fibrin (hereafter as PRF) which contains a variety of growth factors, such as PDGF-AA (15.6 to 1000 pg/ml), PDGF-AB (15.6 to 1000 pg/ml), PDGF-BB (31.2 to 2000 pg/ml), TGF-β1 (31.2 to 2000 pg/ml), VEGF (31.2 to 2000 pg/ml), EGF (31.2 to 2000 pg/ml), IGF (31.2 to 2000 pg/ml), etc.

The “mitochondria” disclosed in the present invention refer to mitochondria that have functional and structural integrity and are separated out from non-autologous or autologous cells. The types of the cells are not limited, including, but not limited to, adipose-derived stem cells, mesenchymal stem cells, skeletal muscle cells, liver cells, kidney cells, fibroblasts, nerve cells, skin cells, blood cells, and the like.

The “composition” mentioned in the present invention refers to which includes at least containing an effective amount of mitochondria, such as a pharmaceutical product, a pharmaceutical beauty product, etc. Moreover, the composition is prepared in different dosage forms, such as drops, emulsion, paste, etc., according to the mode of use or administration, and is formed by mixing different components, such as growth factors, PRF, or a blood product containing the foregoing substances.

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 PRF separated out from the whole blood, blood added with growth factors, platelet-rich plasma (hereafter as PRP), plasma, serum, 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.

The “administration” mentioned in the present invention refers to enabling the mitochondria disclosed in the present invention to contact the injured part, and the way of contacting the injured 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.

In order to prove the technical features disclosed in the present invention and the effects that can be achieved, several examples are given below to describe the present invention in detail with reference to the accompanying drawings.

According to studies, human skin fibroblasts (CCD-966SK) are usually used for verifying the in vitro cell model at the wound and injury. Therefore, CCD-966SK cells are used in the following examples as the in vitro cell model for wound repair.

Hydroxyurea (HU) is used as a cell proliferation inhibitor in the following examples.

Example 1: Culture of CCD-966SK cells

The culture of CCD-966SK cells was performed by using a Dulbecco's Modified Eagle's Medium (DMEM) added with 10% fetal calf serum and/or 2 mM L-glutamine in a 37° C. incubator having 5% carbon dioxide. When the cells grew to the completeness of 8-9, the cell culture medium was removed and the phosphate buffer solution was used for rinsing. Then, the phosphate buffer solution was removed and 0.25% trypsin was added in to react at 37° C. for 5 min. Afterwards, the cell culture medium was added in to neutralize the trypsin, and centrifugation was performed at 1000 rpmfor 5 min. The supernatant was removed after centrifugation; and a new cell culture fluid was added in and cell counting was performed. Cell subculture was performed according to the needs of subsequent examples.

Example 2: Preparation of PRF

10 ml blood sample was placed into a centrifuge tube without containing an anticoagulant and centrifuged at 700 rpm; and then, the supernatant was taken out, where the supernatant was liquid PRF. Through verification, the PRF contains a variety of growth factors. Among these contained and verified various growth factors, PDGF-AA (15.6-1000 pg/ml), PDGF-AB (15.6-1000 pg/ml), PDGF-BB (31.2-2000 pg/ml), TGF-β1 (31.2-2000 pg/ml), VEGF (31.2-2000 pg/ml), EGF (31.2-2000 pg/ml), and IGF (31.2-2000 pg/ml) are common.

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

Example 3: Extraction of Mitochondria

The human adipose-derived stem cells were cultured to obtain 1.5 × 10⁸ 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, 80 ml 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: Cell Migration Assay (1)

The CCD-966SK cells cultured in Example 1 were cultured in a 24-well plate at a concentration of 2×10⁴ cells/0.25 ml per well, for 24 hours. After it was confirmed that the cell completeness reached 90%, the phosphate buffer solution was used to clean the cells and a cell culture medium without the addition of 10% fetal calf serum was used to replace the original medium to continuously culture the cells for 8 hours. Afterwards, a straight wound of a fixed width was scraped in the middle of the cell. The cell culture liquid and the cells in suspension were removed, a cell culture liquid containing 10 µM HU was used to replace the original liquid, and mitochondria with different concentrations (15 µg and 40 µg) were separately added in to perform culturing for 24 hours. After 24-hour culturing, cell migration was performed, to obtain results shown in FIGS. 1A and 1B through observation and analysis.

It can be known from FIGS. 1A and 1B that, the administration of the mitochondria can effectively promote the movement of the fibroblasts towards the injured part, and as the administration dose of the mitochondria increases, the fibroblasts move faster. Further, as shown in FIG. 1B, by using a blank group not subjected to a treatment with any mitochondria as a reference (100%) for calculation of the cell migration proportion, the cell migration proportion of the CCD-966SK cells subjected to a treatment with 15 µg mitochondria is 149.4±40.9% and the cell migration proportion of those subjected to a treatment with 40 µg mitochondria is 160.4±26.1% through calculation.

The results of this example show that the mitochondria can indeed promote the movement of the fibroblasts towards the wound, thereby accelerating wound healing or promoting wound repair.

Example 5: Collagen Secretion Assay (1)

The process of this example was substantially identical with that in Example 4, but had the following differences. In this example, after 24-hour culturing by addition of different concentrations (15 µg and 40 µg) of mitochondria, the cell supernatant was collected and a collagen secretion assay was performed by using the Sircol™ Soluble Collagen Assay Kit, to obtain a result shown in FIG. 2.

It can be known from the result of FIG. 2 that, a collagen expression amount of the blank group is 5.85±0.1 µg/ml; a collagen expression amount of the CCD-966SK cells subjected to a treatment with 15 µg mitochondria is 15.1±0.3 µg/ml; and a collagen expression amount of the CCD-966SK cells subjected to a treatment with 40 µg mitochondria is 25.3±0.3 µg/ml. Thus, the result indicates that in the case of simulation of cell damage, the CCD-966SK cells subjected to a treatment with mitochondria can secrete more collagen; and moreover, the expression level of the collagen rises as the administration dose of the mitochondria increases.

It can be known from the result of this example that, the mitochondria can indeed improve a collagen expression amount of the fibroblasts, thereby accelerating wound healing or promoting wound repair.

Example 6: Cell Migration Assay (2)

The process of this example was substantially identical with that in Example 4, but had the following differences. In this example, a cell culture liquid containing 10 µM HU was used; and the PRF (prepared in Example 2), 15 µg mitochondria and 5 vol% PRF, and 40 µg mitochondria and 5 vol% PRF were separately added in to perform culturing for 24 hours. After culture completion, cell migration was observed and analyzed, to obtain results shown in FIGS. 3A and 3B.

It can be known from FIGS. 3A and 3B that, a cell migration proportion of the CCD-966SK cells in the blank group is 100%; a cell migration proportion of the CCD-966SK cells subjected to a treatment with the PRF is 156.8±16.0%; a cell migration proportion of the CCD-966SK cells subjected to a treatment with the PRF and 15 µg mitochondria is 185.4±40.9%; and a cell migration proportion of the CCD-966SK cells subjected to a treatment with the PRF and 40 µg mitochondria is 202.0±30.9%.

The foregoing result shows that, although the administration of only the PRF can promote the migration of the fibroblasts, the administration of both the PRF and the mitochondria can obviously improve the migration proportion of the fibroblasts. Moreover, as the dose of the mitochondria increases, a better cell migration effect can be achieved. That is, by administering a certain amount of mitochondria disclosed in the present invention to the wound or injured tissue, the effect of promoting wound recovery can be achieved.

Example 7: Collagen Secretion Assay (2)

The process of this example was substantially identical with that in Example 5, but had the following differences. In this example, after completion of 24-hour culturing by addition of the PRF, the cell supernatant was collected and a collagen secretion assay was performed by using a soluble collagen assay kit, to obtain a result shown in FIG. 4.

It can be known from the result in FIG. 4 that, a collagen expression amount of the blank group is 5.85±0.1 µg/ml; a collagen expression amount of the CCD-966SK cells subjected to a treatment with the PRF is 342.51±15.84 µg/ml; a collagen expression amount of the CCD-966SK cells subjected to a treatment with the PRF and 15 µg mitochondria is 1107.33±87.97 µg/ml; and a collagen expression amount of the CCD-966SK cells subjected to a treatment with the PRF and 40 µg mitochondria is 1413.4±158.72 µg/ml.

The result of FIG. 4 shows that a collagen expression amount of the CCD-966SK cells subjected to the treatment with both the PRF and the mitochondria is obviously higher than that of the secretion from the CCD-966SK cells subjected to the treatment with only the PRF; and moreover, a collagen expression amount rises as the administration dose of the mitochondria increases. It can be known from the above that, the administering the mitochondria disclosed in the present invention to the wound or the injured tissue can indeed promote wound recovery, and its ability to promote wound recovery is obviously higher than the PRF.

Example 8: Experiment on Animals

Several 8-week-old C57BL/6 male mice were used and separately anaesthetized; their back hair was shaved and their backs were disinfected with 75% alcohol. After disinfection, a wound of about 1 cm in diameter was created on the back of each male mouse with a disinfected instrument, and then the wound was treated according to the following different conditions: The blank group was not subjected to any wound treatment; for the PRF group, PRF was dripped to the wound for two consecutive days; and for the PRF + mitochondria group, PRF and 15 µg mitochondria were directly dripped to the wound for two consecutive days. In the medicine administration process, it is required to avoid dripping the medicine on the normal skin as much as possible. After dripping, the mice were put aside for 5 to 10 min to determine that the wound absorbed the composition for treatment. Photographs were taken on the 0^th day and the 10^th day of the experiment to observe wound repair in each mouse, as shown in FIG. 5.

It can be known from the result of FIG. 5 that, as compared with the blank group without administration of any medicine, the wound to which only the PRF is administered and the wound to which the PRF and the mitochondria are simultaneously administered both have a better recovery status. As compared with the wound to which only the PRF is administered, the wound to which the PRF and the mitochondria are simultaneously administered has a better recovery status. That is, on the 10^th day of the experiment, the wound is almost completely healed.

Example 9: Test of Cell Proliferation Rates

First, the CCD-966SK cells were used and cultured under the same conditions for 4 hours. Then, different doses (0 µg, 1 µg, 15 µg, and 40 µg) of mitochondria were administered in the cell culture process as a supplement to the cell culture medium to perform cell culturing for 24 hours. Afterwards, a culture medium containing alamar blue was used to continue culturing for 3 hours, and then a cell growth efficiency of each group was estimated by means of a wavelength of OD 530/595 nm, to obtain a result shown in FIG. 6.

The cell culture medium of each group is shown in the following table 1.

Table 1

Composition of the cell culture medium in each group

Groups
Control group (0 µg mitochondria)
Group of using 1 µg mitochondria
Group of using 15 µg mitochondria
Group of using 40 µg mitochondria

Cell culture media
DEME/2 mM L-glutamine; 10%fetal calf serum
DEME/2 mM L-glutamine; 10%fetal calf serum; 1 µg mitochondria
DEME/2 mM L-glutamine; 10%fetal calf serum; 15 µg mitochondria
DEME/2 mM L-glutamine; 10%fetal calf serum; 40 µg mitochondria

It can be known from the result of FIG. 6 that, with the number of the initially cultured cells in different conditions as the basis (1 fold), after 24-hour culturing, the cell growth efficiency of the group not added with the mitochondria achieves a ratio of 1.16±0.08, the cell growth efficiency of the group added with 1 µg mitochondria achieves a ratio of 1.17±0.06, the cell growth efficiency of the group added with 15 µg mitochondria achieves a ratio of 1.28±0.08, and the cell growth efficiency of the group added with 40 µg mitochondria achieves a ratio of 1.31±0.07. After 48-hour culturing, the cell growth efficiency of the group not added with the mitochondria achieves a ratio of 1.77±0.06, the cell growth efficiency of the group added with 1 µg mitochondria achieves a ratio of 1.77±0.07, the cell growth efficiency of the group added with 15 µg mitochondria achieves a ratio of 1.90±0.12, and the cell growth efficiency of the group added with 40 µg mitochondria achieves a ratio of 2.20±0.16.The result of FIG. 6 shows that the addition of the mitochondria in the culture process of the CCD-966SK cells can improve the cell growth efficiency, and the growth efficiency rises as the concentration of the mitochondria increases, thus achieving the effect of promoting cell proliferation, tissue regeneration and repair.

The results of the cell assays and animal experiment in the foregoing examples show that, the administration of a certain amount of mitochondria or a composition containing the mitochondria disclosed in the present invention to a wound can indeed promote wound healing, thus accelerating wound repair and avoiding wound inflammation or related complications.

USE OF MITOCHONDRIA TO PROMOTE WOUND REPAIR AND/OR WOUND HEALING

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