WO2015003623A1 - Composition for treating osteoarthritis - Google Patents

Abstract

Provided is a composition for treating osteoarthritis. Specifically provided are a biological product for treating osteoarthritis, a preparation method thereof, a preparation made by using the product, a reagent combination, and a kit. The product is made by using plasma that contains no red blood cell or white blood cell and is rich in blood platelet and a multifunctional fat-containing cell component in combination with other components.

Description

 Composition for treating osteoarthritis

 Technical field

 The present invention relates to the field of medical and biomedical engineering, and in particular to a biological product for treating osteoarthritis and a preparation method thereof. Background technique

 Osteoarthritis is a chronic joint disease characterized by degeneration, destruction and bone hyperplasia of articular cartilage. It has a high incidence and is very common in the elderly. The prevalence rate can reach 50% in people over 60 years old. 80% of people over the age of 75. Joint pain in patients with osteoarthritis often makes the patient intolerable, and the patient has a high rate of disability after onset. The existing drugs for treating osteoarthritis can delay the course of disease, improve the symptoms of patients, relieve the pain of patients, but can not reverse the pathological process, can not cure osteoarthritis, and most of the side effects of drugs are obvious. Surgical treatment is mainly through arthroscopy (special surgery), open surgery or artificial joint replacement. Although it can temporarily relieve pain, the long-term effect (> 10 years) is not ideal. Cell therapy such as stem cells is currently the most promising new approach to the treatment of osteoarthritis.

 Osteoarthritis often occurs in cartilage defects, but cartilage has limited ability to repair itself. Cartilage repair can often be used as the most important evaluation index for osteoarthritis treatment. In the past decade, stem cell transplantation has been reported in a large number of studies on osteoarthritis. After stem cells are injected into the joints of osteoarthritis, they can exert immunoregulatory effects, regulate inflammation and reduce pain; secrete anti-apoptotic factors and anti-fibrotic factors to inhibit disease progression; secrete TGF-β, ΒΜΡ-4 and other factors to promote endogenous Sexual stem cell cartilage repair; can also differentiate into chondrocytes to help repair cartilage.

 At present, stem cell therapy for osteoarthritis mostly uses bone marrow-derived mesenchymal stem cells. The treatment of osteoarthritis with fat pluripotent cells has also been reported in many literatures. Preclinical studies and clinical studies have shown mesenchymal stem cells and fat pluripotent stem cells. It is possible to improve the condition and report an increase in cartilage content, but there is still a certain distance from the full recovery of the joint. Bone marrow mesenchymal stem cells need to be worn through the bone marrow, causing greater damage to the donor. In contrast, obtaining pluripotent cells from fat is more advantageous, 1) taking less damage to the human body; 2) forming CFU-F in fat The pluripotent cell content of the ability is higher than 1%, and the bone marrow is less than 0.001%; 3) the fat source is rich, and the pluripotent cells are abundant; 4) the adipose-derived cells have stronger proliferative ability than the bone marrow mesenchymal stem cells. Therefore, fat pluripotent stem cells have an advantage in the treatment of osteoarthritis.

The stromal vascular component contains fat pluripotent cells and contains many other types of cells. It was first obtained from adipose tissue by enzymatic digestion, and can be extracted and purified to separate fatty pluripotent stem cells. The stromal vascular component also has a therapeutic effect on the treatment of osteoarthritis, and the stromal vascular component can be reduced from transplantation to transplantation for 2-3 hours, which can reduce the transplantation time, and is cheap, and can also reduce the risk in cell culture, but the fat contained therein can be The amount of cells is small. At the same time, stromal vascular components contain pluripotent cells of CD34+CD31- and CD34+CD31+, It has a strong ability to promote angiogenesis, and the expression of CD34+ in the pluripotent cells after culture is reduced or disappeared. At present, stromal vascular components or purified pluripotent cells of CD34+CD31- and CD34+CD31+ have been used in combination with fat pluripotent cells for the treatment of osteoarthritis, and the combination of the two may improve angiogenesis. Produce significant improvements in governance.

 Platelet-rich plasma (PRP) secretes a variety of cytokines that promote cartilage recovery, but the role of PRP in the treatment of osteoarthritis is controversial, and studies have reported negative effects on cartilage repair.

 At present, multiple components have been reported for the treatment of osteoarthritis. However, in the prior art, only the effective components are simply superimposed, and few components, ratios, active ingredients and impurities are selected. The parameters such as the content are optimized. Moreover, the injection volume of the existing intra-articular injection products is generally too large, such as injecting 5 ml, 8.5 ml of the liquid composition into the joint, and excessive fluid injection into the joint cavity causes an increase in pressure and causes discomfort to the patient.

 In summary, there is a lack of a liquid preparation for the treatment of osteoarthritis in which the composition is optimized, the impurity content is small, and the active ingredient content is high. Summary of the invention

 SUMMARY OF THE INVENTION An object of the present invention is to provide a liquid composition for the treatment of osteoarthritis which is optimized in composition, has a low content of impurities, and has a high content of active ingredients. In a first aspect of the invention, there is provided a composition for treating osteoarthritis, the composition comprising the following components:

 a fat-containing pluripotent cell component, the component comprising (al) a stromal vascular component; and/or (a2) a purified or cultured expanded pluripotent cell;

b. platelet-rich plasma, wherein the concentration of leukocytes in the plasma is 5 X 10 ⁵ /mL;

 c. polysaccharides; and

 d. An optional pharmaceutically acceptable buffer.

 In another preferred embodiment, the composition is a liquid composition.

 In another preferred embodiment, the stromal vascular component comprises a fat pluripotent cell.

 In another preferred embodiment, the fatty pluripotent cells in (a2) are selected from the group consisting of: a fatty pluripotent cell purified from a stromal vascular component, a culture-expanded fat pluripotent cell, or a combination thereof.

 In another preferred embodiment, the culture-amplified adipose pluripotent cells are expanded in serum-free medium or serum-containing medium.

 In another preferred embodiment, the fatty pluripotent cellular component is reconstituted immediately or cryopreserved.

In another preferred embodiment, the fat-containing pluripotent cell component comprises cells selected from the group consisting of: adipocytes, endothelial cells, smooth muscle cells, pericytes, fibroblasts, mast cells, nerve cells, fat precursors cell, Lymphocytes, blood cells, stromal cells, macrophages, or a combination thereof.

 In another preferred embodiment, in the fat-containing pluripotent cell component, the fat pluripotent cell content is 10 to 100% of the total cell amount.

 In another preferred embodiment, the fatty pluripotent cellular component is autologous or allogeneic.

 In another preferred embodiment, the fatty pluripotent cellular component has a cartilage differentiation potential.

 In another preferred embodiment, the platelet-rich plasma is selected from the group consisting of activated PRP, unactivated PRP liquid, PRP gel, or a combination thereof.

 In another preferred embodiment, the stromal vascular component comprises a cell selected from the group consisting of:

CD45-CD235a-CD31-CD34+ cells, and/or CD34+CD31+ cells.

 In another preferred embodiment, the fat-containing pluripotent cell component is a CD45-CD235a-CD31-CD34+ cell, and/or a CD45-CD13+CD36+CD73+ cell-enriched fat-containing pluripotent cell component.

 In another preferred embodiment, the CD45-CD235a-CD31-CD34+ cells and/or

CD45-CD 13+CD36+CD73+ cell enrichment means that the content of CD45-CD235a-CD31-CD34+ cells and/or CD45-CD 13+CD36+CD73+ cells is 15-100% of the total cells in the fraction.

In another preferred embodiment, the concentration of red blood cells in the platelet rich plasma is 5 X 10 ⁵ /mL. In another preferred embodiment, the concentration of leukocytes in the platelet-rich plasma is 4 X 10 ⁵ /mL. In another preferred embodiment, the concentration of red blood cells in the platelet rich plasma is 4 X 10 ⁵ /mL. In another preferred embodiment, the platelet-rich plasma has a platelet concentration ranging from 0.5 X 10 ^{6 to} 1.5 X 10 1 ^Q /mL, and the total concentration of leukocytes and red blood cells in the plasma is 10 X 10 ⁵ /mL. ; and / or

The concentration of the fatty pluripotent cells in the composition is from 10 ^{5 to} 10 ⁸ /mL.

In another preferred embodiment, the platelet-rich plasma has a platelet concentration ranging from 5 X 10 ^{7 to} 5 X 10 ⁸ /mL.

In another preferred embodiment, the fat pluripotent cells are present in the composition at a concentration of 1 X 10 ⁶ -5 X 10 ⁷ /mL. In another preferred embodiment, the plasma further comprises a growth factor selected from the group consisting of TGF- ί PDGF, VEGF, IL-1, IL-6, TNF, IRAP, CTGF, EGF, IGF, BFGF, or The cytokine is further included in the composition of the fat-containing pluripotent cell, and the cytokine is selected from the group consisting of TGF-β, TIMPs, VEGF, HGF, PGE2, IGF-1, MIP. -la, IL-6, IDO, GM-CSF, IL-1RA, IL-12p40, IL-10, IL-13, or a combination thereof;

 The surface marker of the expression of the fatty pluripotent cell is selected from the group consisting of CD90, CD34, CD10, CD36, CDCD45, CD 105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD 13, CD63, CD166, CD31, CD106, CD71, or a combination thereof; and/or

 The surface marker not expressed by the fatty pluripotent cell is selected from the group consisting of CD133, CD146, CD14, CD117, CD1 lb, CD79a, CD19, HLA-DR, or a combination thereof.

In another preferred example: The polysaccharide is selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, dextran, alginic acid, chitin, or a combination thereof; and/or

 The pharmaceutically acceptable buffer is selected from the group consisting of sodium chloride buffer, calcium chloride buffer, calcium magnesium phosphate buffer, calcium magnesium sulfate buffer, calcium magnesium carboxylate buffer, glucose. Phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, serum-containing medium, serum-free medium, or a combination thereof.

 In another preferred embodiment, the polysaccharide has a mass percentage concentration of 0.001 to 30% based on the total mass of the solution.

 In another preferred embodiment, the pH of the buffer is 6.7-7.5.

 In another preferred embodiment, the composition is a unit dosage form, and the unit dosage form has a volume of 4 mL, preferably 3 mL.

 In another preferred embodiment, the composition has a single use volume of 0.15-3 mL.

 In another preferred embodiment, in the composition, the ratio of the fat pluripotent cell concentration to the platelet concentration is 100: 1-1: 100000.

 In another preferred embodiment, the ratio of the pluripotent cell concentration to the platelet concentration is 10: 1-1: 1000.

 In another preferred embodiment, the ratio of the pluripotent cell concentration to the platelet concentration is 1: 5-1: 50.

 According to a second aspect of the invention, there is provided a process for the preparation of a composition according to the first aspect of the invention, the process comprising the steps of: mixing components a, b, c and d to form a composition.

 In another preferred embodiment, the method includes:

 i separating fat-containing pluripotent cell components from adipose tissue;

 ϋ adding plasma-rich platelet-rich plasma from the blood;

 i i i added polysaccharide;

 Iv optionally adding a pharmaceutically acceptable buffer;

 Wherein, the step a. includes:

 (1) separating the stromal vascular component; and/or: separating and purifying the pluripotent cells of the vascular matrix component; and/or; culturing the expanded fat pluripotent cell;

 (2) mixing by one or a combination of stromal vascular components, purified fat pluripotent cells, and cultured expanded pluripotent cells.

 In another preferred embodiment, the stromal vascular component is derived from peripheral blood.

 In another preferred embodiment, the stromal vascular component is obtained using an enzymatic digestion method or an ultrasonic cavitation method. In another preferred embodiment, the step of isolating and purifying the pluripotent cells in the vascular matrix component comprises: separating CD45-CD235a-CD31-CD34+ cells and/or CD34+CD31+ cells by flow cytometry.

In another preferred embodiment, the step of isolating the platelet rich plasma comprises: separating the plasma with a fully automated and semi-automated device. In another preferred embodiment, the platelet-rich plasma removes red blood cells and white blood cells by centrifugation or filtration.

 According to a third aspect of the invention, there is provided a preparation for the treatment of osteoarthritis, which comprises the composition according to the first aspect of the invention as an active ingredient.

 In another preferred embodiment, the preparation is an injection.

 In another preferred embodiment, the formulation is for injecting a diseased joint of a patient.

 In another preferred embodiment, in the preparation,

The concentration of the fatty pluripotent cells is 10 ⁵ -10 ⁸ /mL;

The concentration of platelets is 5 X 10 ⁷ -5 X 10 ⁸ /mL;

The concentration of white blood cells is 5 X 10 ⁵ mL;

The concentration of red blood cells is 5 X 10 ⁵ mL;

 The volume of the preparation is 4 mL;

 The plasma further comprises a growth factor selected from the group consisting of TGF-i3, PDGF, VEGF, IL-1, IL-6, TNF, IRAP, CTGF, EGF, IGF, BFGF, or a combination thereof;

 The fat-containing pluripotent cell component further comprises a cytokine, and the cytokine is selected from the group consisting of:

TGF-β, TIMPs, VEGF, HGF, PGE2, IGF-1, MIP-la, IL-6, IDO, GM-CSF, IL-1RA, IL-12p40, IL-10, IL-13, or a combination thereof; And/or

 The surface marker for expression of the fat-containing pluripotent cell component is selected from the group consisting of CD90, CD34, CD10, CD36, CDCD45, CD105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD 13, CD63, CD166, CD31, CD106, CD71, or a combination thereof;

 The surface marker not expressed by the fat-containing pluripotent cellular component is selected from the group consisting of CD133, CD146, CD14, CD117, CD1 lb, CD79 a, CD19, HLA-DR, or a combination thereof.

 In another preferred embodiment, the formulation has a volume of 3 mL.

 In another preferred embodiment, the formulation has a volume of from 0.15 to 3 mL.

 According to a fourth aspect of the present invention, there is provided a reagent combination or kit comprising: a. a fat-containing pluripotent cell component, wherein said component comprises (al) a stromal vascular component, The stromal vascular component comprises fat pluripotent cells; and/or (a2) purified or cultured expanded pluripotent cells;

b. platelet-rich plasma, wherein the concentration of leukocytes in the plasma is 5 X 10 ⁵ /mL;

 c. polysaccharide;

 d. an optional pharmaceutically acceptable buffer;

 And e. instructions, the instructions described in the description;

 And the method of use comprises: mixing components a, b, c and d for treating patients with osteoarthritis.

In another preferred embodiment, the stromal vascular component comprises a fat pluripotent cell. In another preferred embodiment, the fatty pluripotent cells in (a2) are selected from the group consisting of: a fatty pluripotent cell purified from a stromal vascular component, a culture-expanded fat pluripotent cell, or a combination thereof.

 In another preferred embodiment, the treatment comprises promoting regeneration of cartilage in the patient with osteoarthritis.

 According to a fifth aspect of the invention, a method of treating osteoarthritis, the method comprising: administering to a patient an effective amount of the composition of the first aspect of the invention, or administering an effective amount of the patient to the patient The preparation of the second aspect of the invention.

 In another preferred embodiment, the treatment comprises promoting regeneration of cartilage in the patient with osteoarthritis. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS

 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a control diagram for treating the condition of repairing articular cartilage in Example 2 of the present invention.

 Fig. 2 is a comparison diagram of Mankin scores of each group in Example 2 of the present invention.

 Fig. 3 is a graph showing the detection of TNF-ci content in the joint fluid after treatment in Example 2 of the present invention.

 Fig. 4 is a graph showing the total amount of articular cartilage after treatment for 10 weeks in Example 2 of the present invention.

 Fig. 5 is a graph showing the pain relief score of knee osteoarthritis in Example 3 of the present invention.

 Fig. 6 is a graph showing the symptom relief score of knee osteoarthritis in Example 3 of the present invention.

 Fig. 7 is a graph showing the knee joint function (W0MAC) score in the third embodiment of the present invention.

 Fig. 8 is a view showing the volume of the knee joint cartilage in Example 3 of the present invention.

 Fig. 9 is a comparison diagram of evaluation results of treatment results of a subject to be treated in Example 4 of the present invention. detailed description

 After long-term and intensive research, the present inventors have unexpectedly discovered that a platelet-rich plasma-binding pluripotent cell which separates and purifies and removes white blood cells and red blood cells is used to prepare a biological composition for treating osteoarthritis patients. Unexpected therapeutic effects. And when the biological product is made or used as a joint injection, it is only necessary to inject a small amount of liquid to achieve a very good therapeutic effect. Based on the above findings, the inventors completed the present invention. Osteoarthritis

Osteoarthritis is a chronic joint disease. Its main changes are the degeneration of the articular cartilage surface and secondary bone hyperplasia, which is manifested in joint pain and inflexibility. X-ray shows narrowing of joint space, subchondral Dense bone, trabecular bone fracture, hardening and cystic changes; lip-like hyperplasia at the edge of the joint; The joint surface is uneven; the cartilage in the joint is spalled, the bone is broken into the joint, and the joint is free. In the present invention, the osteoarthritis may be osteoarthritis selected from the group consisting of knee osteoarthritis, spinal osteoarthritis, hip osteoarthritis, or a combination thereof. The osteoarthritis of the present invention is preferably knee osteoarthritis. fat

 Fat is an excellent source of plastic and anti-aging treatments. Adipose tissue materials can be derived from the waist, hips, abdomen, thighs, upper arms and more. Those skilled in the art can obtain adipose tissue using a general technical method including, but not limited to, aspiration, surgical separation, and the like.

 In the present invention, the adipose tissue or the fat raw material is not particularly limited, and may be an adipose tissue derived from any part of an animal or a human, preferably a human adipose tissue. Preferably, the adipose tissue may be a tissue of a part of the waist, the buttocks, the abdomen, the thigh, the upper arm, and the like. Matrix vascular component

 The stromal vascular component contains fat pluripotent cells and contains many other types of cells. It was first obtained from adipose tissue by enzymatic digestion, and can be extracted and purified to separate fatty pluripotent stem cells. The stromal vascular component also has a therapeutic effect on the treatment of osteoarthritis, and the stromal vascular component is isolated from transplantation to 2-3 hours, which can reduce the transplantation time, is inexpensive, and can reduce the risk in cell culture. At the same time, pluripotent cells containing CD34+CD31- and CD34+CD31+ in the vascular component of the stromal have strong ability to promote angiogenesis, and have obvious effects on the body. Fat-rich pluripotent cell component

 Fat pluripotent cells have been reported for the treatment of osteoarthritis. The pluripotent cells obtained from fat are simpler, more widely available, and have a higher pluripotent cell content with CFU-F formation ability in fat. Strong.

 The stromal vascular component has a therapeutic effect on osteoarthritis, and the stromal vascular component is isolated from transplantation to 2-3 hours, which can reduce the transplantation time, and is inexpensive, and can also reduce the risk in cell culture, and the stromal vascular component contains CD34+. The pluripotent cells of CD31- and CD34+CD31+ have strong ability to promote angiogenesis. However, the vascular vascular component contains insufficient concentration of fat pluripotent cells, which affects the therapeutic effect. The expression of CD34+ in the pluripotent cells after culture is reduced or disappeared, which also leads to poor therapeutic effect.

 In the present invention, the stromal vascular component is used in combination with purified or cultured expanded pluripotent cells, thereby improving the respective defects of the above two, improving the angiogenic ability, and improving the effect more conspicuously.

Preferably, the surface marker of the expression of the fatty pluripotent cell is selected from the group consisting of CD90, CD34, CD10, CD36, CDCD45, CD105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD 13 , CD 63 , CD 166 , CD 31 , CD 106 , CD 71 , or a combination thereof; and/or

 The surface marker not expressed by the fatty pluripotent cell is selected from the group consisting of CD133, CD146, CD14, CD117, CD1 lb, CD79a, CD19, HLA-DR, or a combination thereof. Platelet-rich blood paddle (PRP)

 Platelet-rich plasma secretes a variety of cytokines that promote cartilage recovery, but studies have reported negative effects on cartilage repair. In the treatment of osteoarthritis, the effects of PRP are still controversial.

At present, platelet-rich plasma preparation is highly variable, with mononuclear cells ranging from 107ML to 10 ^lfl / ML, and there is currently no reported study on its optimal range of content.

The present invention employs platelet-rich plasma which separates and removes white blood cells and red blood cells, and unexpectedly obtains a better therapeutic effect. Preferably, in the PRP used in the present invention, the concentration of platelets is 5 X 10 ⁷ -5 X 10 ⁸ /mL, the leukocyte concentration is 5 X 10 ⁵ mL, and the concentration of red blood cells is 5 X 10 ⁵ mL.

 In the present invention, preferably, the plasma further comprises a growth factor selected from the group consisting of TGF- ί PDGF, VEGF, IL-1, IL-6, TNF, IRAP, CTGF, EGF, IGF, BFGF, or combination. Composition for treating osteoarthritis

 The present invention provides a composition for treating osteoarthritis, the composition comprising the following components: a. a fat-containing pluripotent cell component; the component comprising (al) a stromal vascular component, The stromal vascular component contains pluripotent cells; and (a2) purified or cultured expanded pluripotent cells;

 Wherein the fatty pluripotent cells are selected from the group consisting of: fat pluripotent cells purified from stromal vascular components, cultured expanded pluripotent cells, or a combination thereof; b. platelet-rich plasma;

 c polysaccharide; and

 Optional d. A pharmaceutically acceptable buffer.

 The composition is preferably a liquid composition.

 The polysaccharide component has buffering stress, shielding effect, anti-inflammatory, pain relief and protection of cartilage. The addition of a buffer last stabilizes the solution ra, and a component that promotes platelet-rich plasma release factor can be added. In the present invention, the polysaccharide includes (but is not limited to, hyaluronic acid, hyaluronic acid derivative, dextran, alginic acid, chitin, or a combination thereof.

 In another preferred embodiment, the polysaccharide has a mass percentage concentration of 0.001 to 30%.

The buffer is used to adjust the pH of the composition to reduce the pain of the patient during injection. The pharmaceutically acceptable buffers described in the present invention include, but are not limited to:: sodium chloride buffer, calcium chloride buffer, calcium magnesium phosphate buffer, calcium magnesium sulfate buffer, calcium Magnesium carboxylate buffer, glucose phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, serum-containing medium, serum-free medium, or a combination thereof. Preferably, the buffer is a calcium salt. In another preferred embodiment of the invention, the pH of the buffer is 6.7-7.5. Commercially available products for the treatment of osteoarthritis generally have a problem of large intra-injection volume, such as a total volume of 5 ml~8. 5 ml of liquid for a joint injection, a large amount of liquid injection not only makes the injection more likely to fail, but also Increase discomfort such as soreness and pain in patients. In the present invention, since the content and ratio of each component are optimized, the total injection volume can be reduced to 4 mL, which is advantageous for increasing the concentration of pluripotent cells and platelet-rich plasma release factors, and also promoting cell migration. Repair to the affected area.

 In a preferred embodiment of the invention, the composition is a unit dosage form, and the unit dosage form has a volume of 4 mL, preferably 3 mL.

 In another preferred embodiment, the unit dosage form has a volume of from 0.15 to 3 mL.

 In another preferred embodiment of the present invention, in the composition, the ratio of the fat pluripotent cell concentration to the platelet concentration is 100: 1-1: 100000;

 The ratio of the pluripotent cell concentration to the platelet concentration is 10: 1-1: 1000;

 The ratio of the pluripotent cell concentration to the platelet concentration is 1:5-1:50.

 The composition can be used directly for injection into a joint site of an osteoarthritic patient, or for the preparation of a preparation for treating osteoarthritis. The preparation may be in any dosage form, preferably an injection.

 In another preferred embodiment of the invention, in the preparation:

The concentration of the fatty pluripotent cells is 10 ⁵ -10 ⁸ /mL;

The concentration of platelets is 5 X 10 ⁷ -5 X 10 ⁸ /mL;

The total concentration of white blood cells and red blood cells is 5 X 10 ⁵ mL;

 And the volume of the preparation is 4 mL;

 The plasma further comprises a growth factor selected from the group consisting of TGF-e, PDGF, VEGF, IL-1, IL-6,

TNF, IRAP, CTGF, EGF, IGF, BFGF, or a combination thereof;

 The fat-containing pluripotent cell component further comprises a cytokine, and the cytokine is selected from the group consisting of TGF-β, TIMPs, VEGF, HGF, PGE2, IGF-1, MIP-la, IL-6 , IDO, GM-CSF, IL-1RA, IL-12p40, IL-10, IL-13, or a combination thereof;

 The surface marker of the expression of the fatty pluripotent cell is selected from the group consisting of CD90, CD34, CD10, CD36,

CDCD45, CD 105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD 13, CD63, CD 166, CD31, CD 106, CD71, or a combination thereof; and/or

 The surface marker not expressed by the fatty pluripotent cell is selected from the group consisting of CD133, CD146, CD14, CD117, CD1 lb, CD79 a, CD19, HLA-DR, or a combination thereof.

 In another preferred embodiment, the preparation is administered in a single dose of 3 mL. The main advantages of the invention include:

1. The present invention employs PRP from which white blood cells and red blood cells are removed, so that the composition provided by the present invention has a better therapeutic effect. 2. Using a stromal vascular component in combination with a cultured pluripotent pluripotent cell, and using a lipid-containing pluripotent cellular component purified by CD34+CD31- and CD34+CD31+ pluripotent cells, the blood vessel of the composition of the present invention The regenerative capacity is stronger than the prior art, and the treatment effect is better.

 3. Optimized the content and ratio of pluripotent cells and platelets, with better therapeutic effect.

 4. Compared with the prior art, the preparation of the invention is smaller in volume, does not burden the patient's joint after injection, and has better therapeutic effect. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Example 1

 Preparation of biological products for treating osteoarthritis

 —, separation of fat-containing pluripotent cell components from adipose tissue

 1. Separation of stromal vascular components

30 ml of adipose tissue was taken from the patient's fat supply section, and the adipose tissue was centrifuged at 800 g X for 5 minutes. The supernatant was centrifuged for 45 minutes with PBS containing 0.075% collagenase, centrifuged, the supernatant was discarded, and the lower layer was resuspended in DMEM. Cell pellets. Collagenase was removed by washing 3 times. At the same time, 500 ml of aspirating fluid was collected, centrifuged at 800 g X for 5 minutes, the supernatant was discarded, and the mixture was whipped for 30 seconds by adding 200 ml of physiological saline. The addition of 22. 2 ml of 10 X PBS was resumed, centrifuged, and resuspended for 3 times. The two parts of the resuspension were mixed and filtered through a 100 mesh screen. The cell concentration was measured using a hemocytometer, resuspended in DMEM by centrifugation, and the cell concentration was adjusted to 3 × 10 ⁷ /ML.

 2. Isolation and purification of fat pluripotent cells from vascular matrix components

The isolated vascular matrix component cells were resuspended in FACS buffer (PBS containing 1% BSA and 0.05% sodium azide), the cells were adjusted to 10 ⁵ cells, the antibody was added, mixed, and incubated on ice. FACSvantageTM (Becton Di ckinson) sorting based on expression of cell surface marker levels

CD45-CD235a-CD31-CD34+ cells and CD34+CD31+ cells. The concentration of the cells was adjusted to 3 X 10^/ML by adding DMEM.

 3. Cultured adipose pluripotent cells

 The obtained stromal vascular fraction cells were centrifuged, resuspended in serum-free medium, and the cell concentration was adjusted to

3 X 10 ⁵ /cm ² , inoculated into a T75 flask, cultured, and the cells were proliferated to a degree of fusion of 80% and then subjected to cell passage. When the P3 generation cells were fused to 80%, the cells were digested with trypsin, resuspended in DMEM after centrifugation, and the cell concentration was adjusted to 3 10 ⁷ /]^.

4. Mix several pluripotent cellular components 5 ML。 Mixing a total of 1.5 ML in a centrifuge tube, a total of 1. 5 ML. Flow detection mixture

The pluripotent cell content of CD45-CD235a-CD31-CD34+ and CD45-CD13+CD36+CD73+, the final concentration of pluripotent cells was 2. 1 X 10^/ML.

 5. Flow surface marking determination

The mixed pluripotent cell sample was taken, and 5×10 ⁶ cells were placed in a 1.5 ml centrifuge tube and centrifuged at 3000 r/min for 5 minutes, and the supernatant was discarded. Add 100 μl of suspended cells to FACS buffer. 5 μL (0.5 mg/ml) was added to the closed cell surface Fc receptor, and the water was bathed for 3 minutes. One microliter (0.5 mg/ml) of the fluorescent antibody was added and the mixture was bathed for 30 minutes. Add 350 μl of FACS buffer, mix gently, centrifuge at 3000 r/min for 5 minutes, discard the supernatant, and repeat the wash twice. 100 μl of 1 instrument buffer was added to the obtained cell pellet, and the suspension cells were gently mixed, and the cell suspension was transferred to a FACS-dedicated tube, and instrument detection and analysis were performed on a BD flow cytometer.

 The surface markers detected by the mixed pluripotent cells were: CD90, CD34, CD10, CD36, CD45, CD73, CD13, CD31, CD106, CD71, and the surface markers not expressed were: CD133, CD14, CD79, CD19, HLA - DR.

 6. Detection of cartilage differentiation ability

 The mixed pluripotent cell samples were inoculated into 24-well plates, fused to 80%, and replaced with cartilage-inducing solution (10 ng/mL TGF, 10 mmol/mL dexamethasone, 50 mg/mL vitamin C, high glucose DMEM). Cheng), change the liquid once every 3 days, induce 1 1 day, the control group is used. In the culture, the coverslips were pre-placed in the culture wells, and the cells were covered with coverslips. The cells were removed from the slides at 11 days, and the volume fraction was fixed in 10% formaldehyde for 1 hour, washed in PBS for 15 minutes, rinsed once with distilled water, and added dropwise. g/L a new blue staining, dyeing for 3 hours, adding 95% ethanol, washing off the excess dye solution, drying, neutral gum seal.

 The pluripotent cells were able to differentiate into chondrocytes, and the results of the differentiation experiment in the control group were negative.

 Second, the separation of platelet-rich plasma

 The patient's 20ML peripheral blood was drawn, shaken, placed in a centrifuge tube, and centrifuged twice. The first centrifugation speed was 1500 rpm, which was 10 minutes. After centrifugation, it is divided into upper layer plasma and lower layer red blood cells. Discard the red blood cells located in the lower part of the centrifuge tube and pipet all the plasma to another centrifuge tube. The leukocytes were removed by filtering the plasma using a leukocyte filter. The filtered plasma was subjected to a second centrifugation at a speed of 3000 rpm and centrifuged for 10 minutes. The plasma was divided into upper platelet-poor plasma and lower platelet-rich plasma, and the platelet-rich plasma was taken.

Using a pocH-lOOi blood cell analyzer (Sysmex, Japan) to detect the platelet content of the isolated platelet-rich 9. 7 X 10 ⁷ / ML, white blood cell content 1. IX 10 ⁵ / ML, red blood cell content 4. 3 X 10 ⁵ /ML.

3. ELISA for detection of cytokine expression in pluripotent cell culture supernatants and platelet-rich plasma The mixed pluripotent cell samples were inoculated into the culture dish at 105 cells/cm 2 , and the culture supernatant was taken after adherent culture for 36 hours, and the expression of TGF-β, HGF, IGF-1, and VEGF was detected by ELISA.

 Platelet-rich plasma was added to the activator at a volume ratio of 1:9 (500 U thrombin lyophilized powder dissolved in 1 mL of 10% calcium chloride) to activate platelet-rich plasma, allowed to stand at room temperature for 24 hours, 4 000 rpm The extract was extracted by centrifugation for 15 minutes, and the expression of cytokines was measured by ELISA.

 The results showed that: pluripotent cell culture supernatant expressed TGF-β, HGF, IGF-U VEGF, cytokine expression in platelet-rich plasma TGF-β, PDGF, VEGF, EGF, IGF, BFGF

 4. Mixed pluripotent cell components, platelet-rich plasma, hyaluronic acid, buffer

 5ML。 10% calcium chloride buffer 0. 5ML. 5ML, 10% calcium chloride buffer 0. 5ML. A total of 3ML, after shaking for 5 minutes.

 V. Cryopreservation of biological products

 The pluripotent stem cell component of the biological product was added with 10% DMS0, cooled in a program desuperheater, stored in liquid nitrogen at -196 °C, and rapidly resuscitated in a 37 °C water bath during use, and the cell viability after resuscitation was >90%. Platelet-rich plasma was added to 5% DMS0 and stored in a -80 °C freezer, and rewarmed at room temperature. Hyaluronic acid and 10% calcium chloride buffer are stored at 4 ° C and rewarmed at room temperature. Example 2:

 Preparation of biological products and experiments for treating rabbit osteoarthritis

 1. Preparation of biological products:

1) Isolation of rabbit skin fat 10ML, using matrix digestion to obtain stromal vascular components, adjusted cell concentration to 1 X 10 ⁷ / ML. The expanded fat pluripotent cells were cultured, and the cell concentration was adjusted to 1 × 10 ⁷ /ML. Take 1 ML matrix vascular component and 0.1 ML amplified fat pluripotent cells mixed, flow detection mixture

CD45-CD235a-CD31-CD34+ and CD45-CD13+CD36+CD73+ pluripotent cell content. The final concentration of pluripotent cells is 6. 5 X 10 ⁶ / ML.

2) 10 mL of rabbit femoral artery blood was drawn, the first centrifugation speed was 1500 rpm, centrifuged for 10 minutes, and the upper plasma was aspirated to another centrifuge tube. The platelet-rich plasma was centrifuged at a rate of 3000 rpm, centrifuged for 10 minutes, the upper layer of white blood cells and red blood cell liquid were removed, and the platelet-rich plasma was taken, resuspended in physiological saline, and centrifuged three times. The platelet content of the isolated platelet-rich plasma was measured by a pocH-100i blood cell analyzer (Sysmex, Japan) at a rate of 5 X 10 ⁷ /ML, a white blood cell content of 5 X 10 ⁴ /ML, and a red blood cell content of 1. 2 X 10 ⁵ / ML.

 3 ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML ML .

 Second, rabbit osteoarthritis modeling

Choose healthy adult New Zealand white rabbits weighing 4 to 5 kg. Carrying the rabbit's left knee joint forward The ligamentum ligament was combined with medial meniscectomy, and the right knee was used as the control side. After 6 weeks of surgery, the osteochondal model was evaluated pathologically.

 Third, rabbit osteoarthritis treatment

 In the 4th week after the successful modeling, the syringe was used to inject 0.3 ml of biological products into the joints with osteoarthritis under ultrasound guidance, and the bolus was slowly injected.

 Fourth, the evaluation of treatment effect

 At the 10th week after receiving treatment, the test is performed:

 1) Joint pathological observation

 After removing the distal femur and tibial plateau, the joint was fixed with 10% formaldehyde. After the sample was decalcified, the paraffin was embedded and sliced for HE staining. The results are shown in Fig. 1. The normal knee joint cartilage surface is intact and the chondrocytes are normal. The untreated knee joint cartilage surface is obviously damaged, the exfoliation phenomenon is obvious, the superficial cartilage vacuolar degeneration, the mature chondrocyte column line disappears, and the structure is destroyed. Obviously; after cartilage treatment, the damaged cartilage surface recovered intact, the cartilage did not undergo vacuolar degeneration, and the mature chondrocyte column line recovered.

 2) Mankin score

 The distal femur and tibial plateau were removed and fixed with 10% formaldehyde. After decalcification, the samples were embedded in paraffin and sectioned for HE staining, toluidine blue and Safranin 0 staining. The joints were scored using the Mankin scoring method and the results are shown in Figure 2.

 3) Take joint fluid and detect the expression of inflammatory factors such as TNF-d, IL-6 and MMP-13 by ELISA. The detection of TNF-α in synovial fluid after treatment is shown in Figure 3.

 4) Magnetic resonance imaging of the rabbit joint MRI observation, joint recovery and cartilage volume were detected. The total amount of articular cartilage after 10 weeks of treatment is shown in Figure 4. Example 3:

 Preparation of biological products and treatment for patients with osteoarthritis

 1. Preparation of biological products:

1) Separate the patient's subcutaneous fat by 30ML, obtain the stromal vascular component by enzymatic digestion, inoculate the stromal vascular component into the Τ75 culture flask, expand and culture the fat pluripotent cells, grow to the degree of fusion 80%, enzymatically digest the cells, adjust the cell concentration to 5 X 10 ⁷ / ML. Take 0. 1 ML matrix vascular component and 0.1 ML amplified fat pluripotent cells, flow detection of CD45-CD235a-CD31-CD34+ in the mixture

CD45-CD13+CD36+CD73+ pluripotent cell content. The final concentration of pluripotent cells was 4.97 X 10 ⁷ cells/ML.

2) 20ML of human peripheral blood was drawn, the first centrifugation speed was 1500r/min, centrifuged for 10 minutes, and the upper layer of plasma was aspirated to another centrifuge tube. The platelet-rich plasma was centrifuged at a rate of 3000 r/min and centrifuged for 10 minutes to remove the upper white blood cells and red blood cell liquid. The platelet-rich plasma was taken, resuspended in physiological saline, and centrifuged three times. Using pocH-100i blood cell analyzer (Sysmex, Japan) The platelet content in the isolated platelet-rich plasma was measured to be 5 X 10 ⁸ /ML, the white blood cell content was 9 X 10 ⁴ /ML, and the red blood cell content was ⁴ ×10 ⁵ /ML.

 3) Take 1ML of fat pluripotent cell component, 1 ML of platelet-rich plasma, and 1 ML of hyaluronic acid 1 ML after shaking and mixing.

 Second, biological products for the treatment of patients with knee osteoarthritis

 Twenty patients with knee osteoarthritis were divided into two groups. Group 1 patients underwent ultrasound-guided slow injection of 3 ml of biological products into the joint cavity of osteoarthritis; Group 2 patients received injected biologics with hyaluronic acid to increase the volume to 8 ML, under ultrasound-guided slow injection into osteoarthritis joint. Follow-up was performed at 2, 5, 10, 18, and 52 weeks after surgery.

 Third, the evaluation of treatment effect

 The biologic product was injected into the joint cavity of osteoarthritis, and the 3 ml volume of the biological product showed a feeling of soreness and pain of less than 8 ml of the biological product during the injection.

 1. Knee osteoarthritis pain relief score

 The pain relief score is shown in Figure 5, and the symptom relief score is shown in Figure 6. The results showed that a 3 ml biologic product was superior to 8 ml of biologics in terms of pain and symptom relief.

 2. Knee joint function (W0MAC) score

 The knee function (W0MAC) score is shown in Figure 7. The results show that a 3 ml volume of biological products can effectively improve knee function.

 3. Ultrasound determination of cartilage thickness (mm, n=10)

 Table 1. Ultrasonic determination of cartilage thickness

 3ml injection joint group 8ml injection joint group lateral cartilage

Thickness Before injection 2. 48±0· 97 2. 50±0· 92

 After injection for 6 months 2. 86 + 0. 81 2. 78±0· 80

 Volume change 0. 38±0· 61 0. 28±0· 57

Central cartilage

Thickness Before injection 3. 30±0· 97 3. 32±0· 93

 After injection, 6 months later 3. 40+1. 02 3. 35±1· 01

 Volume change 0. 10±0· 32 0. 03±0· 32

Medial cartilage

Thickness Before injection 2. 53±0· 78 2. 54±0· 74

 After 6 months of injection 2. 53±0· 63 2. 54±0· 75

Volume change 0. 00±0· 70 0. 00±0· 64 SD (mm), n=10

 Ultrasound measurements showed that the recovery of cartilage in a volume of 3 ml was superior to that of a biomass of 8 ml.

4. MRI measures knee cartilage volume (匪³ , n=10)

 The knee cartilage volume measured by MRI is shown in Fig. 8. The results showed that a 3 ml biologic product significantly increased knee cartilage volume at 6 months after treatment. Example 4:

 Platelet-rich plasma is used for osteoarthritis treatment after removing leukocytes and red blood cells — platelet-rich plasma separation and reduction of white blood cell and red blood cell content

The dog femoral artery blood was extracted 12 mL, the first centrifugation speed was 1500 r/min, and the cells were centrifuged for 10 minutes, and the upper layer of plasma was aspirated to another centrifuge tube. The plasma was removed using a leukocyte filter to remove white blood cells. The platelet-rich plasma was centrifuged at a rate of 3000 r/min and centrifuged for 10 minutes to remove the upper white blood cells and red blood cell liquid. The platelet-rich plasma was taken, resuspended in physiological saline, and centrifuged three times. The pecH-100i blood cell analyzer (Sysmex, Japan) was used to detect the platelet content of the isolated platelet-rich plasma by 3 X 10 ⁸ /ML, the white blood cell content of 3 X 10 ³ /ML, and the red blood cell content of 7 X 10 ⁴ /ML.

The platelet content of the platelet-rich plasma without red blood cell and white blood cell removal was 2. 8 X 10 ⁸ /ML, the white blood cell content was IX 10 ⁶ /ML, and the red blood cell content was 5 X 10 ⁶ /ML.

 Second, platelet-rich plasma for the treatment of patients with osteoarthritis

 The posterior cruciate ligamentectomy and the medial meniscus resection were performed in the posterior left knee joint of the dog. Model dogs with osteoarthritis were divided into two groups of 10 animals each. The sputum of the group 1 was slowly injected with 0.5 ml of platelet-rich plasma to remove the red blood cells and white blood cells to the osteoarthritis joint; the dog of group 2 was slowly injected under ultrasound guidance 0. 5 ml of the red blood cells and white blood cells were not removed. Platelet plasma to osteoarthritic joints were followed up at 30, 60 and 90 days postoperatively.

 Third, the evaluation of treatment effect

 Overall rating

The dog's treatment, mobility limitation, and dysfunction were evaluated. The results showed that the overall condition of the treatment group was significantly improved compared with the control group (total score). Among them, the condition of the limp was improved obviously, and the walking was significantly improved after 30 days (P<0.05), and further improved to 60 days after 60 days; the improvement of the leap in the jump was more obvious, about 30 days. There was a significant improvement (P < 0.01) and remained until about 90 days. The improvement in joint mobility was also quite noticeable, with a significant improvement in about 30 days (P < 0.01), and it was not repeated until the end of the experiment. The overall scores of the treatment group and the control group were compared, and the results are shown in Fig. 9. The biologic product composition provided by the invention has obvious recovery of cartilage in the treatment of osteoarthritis and animal experimental treatment, and has significant difference compared with the control group, and the recovery condition is similar to that of the normal group. The neovascularization of the treatment group was better than that of the control group; the inflammatory factor of the joint fluid was significantly down-regulated after treatment, and there was no significant difference from the normal value. Clinical patients received treatment, pain and discomfort were weak, knee function recovered well, and the score was significantly higher than the control group. The results show that the composition provided by the present invention can promote the regeneration of cartilage in patients with osteoarthritis and is very effective in treating osteoarthritis. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request A composition for treating osteoarthritis, comprising the following components:  a fat-containing pluripotent cell component, wherein said component comprises (al) a stromal vascular component; and/or (a2) purified or cultured expanded pluripotent cells; b. Platelet-rich plasma, wherein the concentration of leukocytes in the plasma is 5 X 10 ⁵ /mL;  c. polysaccharide; and  d. An optional pharmaceutically acceptable buffer. 2. The composition according to claim 1, wherein the concentration of red blood cells in the platelet rich plasma is 5 X 10 ⁵ /mL. The composition according to claim 1, wherein the platelet-rich plasma has a platelet concentration ranging from 0.5 X 10 ^{6 to} 1.5 X 10 1 ^Q /mL, and the plasma is white blood cells and red blood cells. The total concentration is 10 X 10 ⁵ /mL; and / or The concentration of the fatty pluripotent cells in the composition is from 10 ^{5 to} 10 ⁸ /mL.  The composition according to claim 1, wherein the plasma further comprises a growth factor selected from the group consisting of TGF-e, PDGF, VEGF, IL-1, IL-6, TNF, IRAP, CTGF, EGF, IGF, BFGF, or a combination thereof; and/or  The fat-containing pluripotent cell component further comprises a cytokine, and the cytokine is selected from the group consisting of TGF-β, TIMPs, VEGF, HGF, PGE2, IGF-1, MIP-la, IL-6 , IDO, GM-CSF, IL-1RA, IL-12p40, IL-10, IL-13, or a combination thereof;  The surface marker of the expression of the fatty pluripotent cell is selected from the group consisting of CD90, CD34, CD10, CD36, CDCD45, CD 105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD 13, CD63, CD 166, CD31, CD 106, CD71, or a combination thereof; and/or  The surface marker not expressed by the fatty pluripotent cell is selected from the group consisting of CD133, CD146, CD 14, CD117, CD1 lb, CD79 a, CD19, HLA-DR, or a combination thereof.  5. The composition of claim 1 wherein:  The polysaccharide is selected from the group consisting of hyaluronic acid, hyaluronic acid derivatives, dextran, alginic acid, chitin, or a combination thereof; and/or  The pharmaceutically acceptable buffer is selected from the group consisting of sodium chloride buffer, calcium chloride buffer, calcium magnesium phosphate buffer, calcium magnesium sulfate buffer, calcium magnesium carboxylate buffer, glucose. Phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, serum-containing medium, serum-free medium, or a combination thereof.  The composition according to claim 1, wherein the composition is a unit dosage form, and the volume of the unit dosage form is 4 mL, preferably 3 mL. 7. A method of preparing a composition according to any of claims 1-6, wherein the method comprises Procedure: The components a, b, c and c were mixed to prepare a composition.  The composition according to any one of claims 1 to 6, wherein the composition is for promoting regeneration of cartilage in a patient with osteoarthritis.  A preparation for treating osteoarthritis, characterized in that the preparation contains the composition according to claims 1-6 as an active ingredient.  10. The preparation according to claim 9, wherein in the preparation, The concentration of the fatty pluripotent cells is 10 ⁵ -10 ⁸ /mL; The concentration of platelets is 5 X 10 ⁷ -5 X 10 ⁸ /mL; The concentration of white blood cells is 5 X 10 ⁵ mL; The concentration of red blood cells is 5 X 10 ⁵ mL;  The volume of the preparation is 4 mL;  The plasma further comprises a growth factor selected from the group consisting of TGF-i3, PDGF, VEGF, IL-1, IL-6, TNF, IRAP, CTGF, EGF, IGF, BFGF, or a combination thereof;  The fat-containing pluripotent cell component further comprises a cytokine, and the cytokine is selected from the group consisting of TGF-β, TIMPs, VEGF, HGF, PGE2, IGF-1, MIP-la, IL-6 , IDO, GM-CSF, IL-1RA, IL-12p40, IL-10, IL-13, or a combination thereof;  The surface marker for expression of the fat-containing pluripotent cell component is selected from the group consisting of CD90, CD34, CD10, CD36, CDCD45, CD105, CD29, CD44, CD49b, CD49e, CD58, HLA-ABC, CD73, CD13, CD63 , CD166, CD31, CD106, CD71, or a combination thereof; and/or  The surface marker not expressed by the fat-containing pluripotent cell component is selected from the group consisting of CD133, CD146, CD14, CD117, CD1 lb, CD79 alpha, CD19, HLA-DR, or a combination thereof.  The preparation according to claim 9, wherein the preparation is for promoting regeneration of cartilage in a patient with osteoarthritis.  12. A reagent combination or kit, comprising:  a fat-containing pluripotent cell component, wherein said component comprises (al) a stromal vascular component, said stromal vascular component comprising an aliphatic pluripotent cell; and/or (a2) purified or cultured expanded fat Pluripotent cell b. platelet-rich plasma, wherein the concentration of leukocytes in the plasma is 5 X 10 ⁵ /mL;  c. polysaccharide;  d. an optional pharmaceutically acceptable buffer;  And e. instructions, the instructions described in the description;  And the method of use comprises: mixing components a, b, c and d for treating patients with osteoarthritis. The reagent combination or kit according to claim 12, wherein the reagent combination or kit is for promoting regeneration of cartilage in a patient with osteoarthritis. 14. A method of treating osteoarthritis, the method comprising: administering to a patient an effective amount of a composition according to the first aspect of the invention, or administering to the patient an effective amount of a second aspect of the invention preparation.