TW201827061A - Injectant of composition used to repair articular cartilage characterized by facilitating the regeneration of articular cartilage to achieve the efficacy of preventing and curing arthritis - Google Patents

Abstract

The present invention provides a composition capable of facalitating the regeneration of articular cartilage and a method of repairing the articular cartilage by using the same. The composition of repairing articular cartilage contains platelet-rich fibrin releasate and stem cells and can be used to facilitate the regeneration of articular cartilage, so as to achieve the efficacy of preventing and curing arthritis. The stem cells can be embryonic stem cells, adult stem cells, or induced pluripotent stem cells. The platelet-rich fibrin releasate contains at least one growth factor selected from the group consisting of TGF-[beta]1, VEGF, PDGF, EGF, FGF, NGF and IGF.

Description

Composition for repairing articular cartilage and method for repairing articular cartilage

The invention relates to a composition for promoting articular cartilage regeneration and a method for using the same.

The main function of articular cartilage is to absorb the impact force of the hard bone on the articular surface, transmit the stress of the hard bone on the articular surface, and reduce the friction between the articular surfaces. Articular cartilage has no blood vessels, lymphatic system, and nerves, and contains only a single type of cell, chondrocytes. The growth of chondrocytes is limited, so it is difficult to repair the articular cartilage after it is damaged.

Osteoarthritis (OA), also known as Degenerative Joint Disease (DJD), is a chronic joint disease with symptoms including joint pain, tenderness, stiffness, and locking ), Sometimes effusion occurs. It mainly occurs in middle-aged and elderly people, but with the westernization of diet and the popularity of sports in recent years, osteoarthritis has gradually become younger. Therefore, how to prevent and treat osteoarthritis has become one of the most important issues in the world .

Regarding the treatment of osteoarthritis, there are also many alternative medicines (Alternative medicine), and many nutritional supplements on the market are sold as OA treatment products. Studies have shown that glucosamine is a component of prochondral cartilage. However, the effectiveness of using glucosamine is controversial, and recent studies have found that it is only equal to or only slightly better than placebo (Burdett and McNeil 2012). Although it is determined that glucosamine is relatively ineffective, it is still a viable treatment option. Because its use as a treatment for osteoarthritis is generally safe (Henrotin, Mobasheri et al. 2012).

Phytodolor, SAMe, and SKI 306X (a mixture of Chinese herbal medicines) are also effective in improving arthritis pain, and there is preliminary evidence that avocado / soybean unsaponifiables (ASU) and rosehip oil are effective. In addition, Boswellia serrata extract slightly improves the pain and function in humans and osteoarthritis. (Rosenbaum, O'Mathuna et al. 2010)

Discussion on the treatment of osteoarthritis from traditional therapy to modern therapy. If osteoarthritis is a minor injury, you can use traditional therapy, inject painkillers and intra-articular injections to delay the symptoms, and if the effect cannot be achieved, treat it surgically The purpose of surgical treatment is to fix loose joint cartilage, reduce pain and improve joint function, for example: repairing loose cartilage and bones in joints, removing loose cartilage blocks and microfractures in joints. Among them, microfracture is the most common cartilage operation, which involves drilling multiple small holes in the bone to bleed blood from the bottom of the cartilage cavity, stimulate fibrocartilage to form scar tissue, and promote cartilage healing.

In addition, osteochondral transplantation and autologous chondrocyte transplantation. The osteochondral transplantation is to remove the cartilage that matches the size of the injured part from the secondary part of the knee joint and implant it into the cartilage defect. Although the surgery can be successful, the immune response can be ruled out. Risk, but the cartilage in the secondary area will be weak. Autologous chondrocyte transplantation (ACI) is to remove normal living cartilage tissue from secondary parts of the knee joint, then culture the chondrocytes in the laboratory, multiply to a sufficient number, and transplant the chondrocytes into the cartilage defect. The new chondrocyte status in the method will be similar to the natural articular cartilage. If the condition of the joint is very serious, the clinician will perform a rescue type operation, such as osteotomy or total joint replacement.

However, in the above-mentioned cartilage surgery, osteochondral transplantation, and autologous chondrocyte transplantation, patients must undergo a surgical procedure to achieve the effect of repair. For patients with osteoarthritis common in the elderly, it is nothing but a difficulty. Challenge.

In order to find effective and convenient treatments, the current research is based on stem cell-based treatments. Some studies have pointed out that platelet-rich plasma (PRP) is used to repair adipose mesenchymal stem cells after in vitro culture. Articular cartilage in mice; however, adipose mesenchymal stem cells may be rejected by the anticoagulant and bovine thrombin contained in PRP during in vitro culture, and there may be a risk of cross-infection, leading to therapeutic effects Not as expected.

In summary, although the above-mentioned treatment methods can repair articular cartilage, they all have problems to be solved. Therefore, it is urgent to develop a method that can solve the defects of the current articular cartilage treatment technology and can fully exert excellent medical effects. combination.

Therefore, in view of this, the inventors have made intensive research on the problem points of the conventional technology and found that the combination of stem cells and platelet-fibrin-rich fluid GX Factor for repairing articular cartilage can not only provide beyond use The excellent effects of general articular cartilage repair technology, and stem cells do not need to be cultured in vitro. Because platelet-fibrin release solution GX Factor does not contain anticoagulants and bovine thrombin, it can reduce the possibility of rejection and cross infection Sex.

According to one aspect of the present invention, a joint cartilage repair composition can be provided, which includes stem cells and platelet-rich fibrin release fluid GX Factor; wherein the stem cell line is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell; The platelet-rich fibrin release solution GX Factor contains the following: growth factor), Nerve growth factor (NGF), and Insulin-like growth factor (IGF).

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided. The above platelet-rich fibrin release solution GX Factor is obtained by centrifuging mammalian autologous blood, taking out the intermediate layer of mass, and leaving it to stand for a while. Among them, a growth factor-rich release solution obtained after a specific time; the specific time is generally more than 1 hour; preferably more than 2 hours; more preferably 3 hours or more; particularly preferred is 4 hours or more; most preferably 5 hours the above.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of PDGF in the platelet-fibrin-rich solution GX Factor is above 15 pg / mL; preferably above 20 pg / mL; More preferably, it is 25 pg / mL or more; particularly preferred is 30 pg / mL or more; most preferred is 35 pg / mL or more.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of VEGF in the platelet-fibrin-rich solution GX Factor is above 0.5 pg / mL; preferably above 0.7 pg / mL; More preferably, it is above 0.9 pg / mL; most preferably, it is above 1.5 pg / mL.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of EGF in the platelet-rich fibrin release solution GX Factor is above 0.01 pg / mL; preferably above 0.03 pg / mL; more Preferably it is above 0.05 pg / mL; most preferably it is above 0.07 pg / mL.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of IGF in the platelet-fibrin-rich solution GX Factor is 1.0 pg / mL or more; preferably 3.0 pg / mL or more; More preferably, it is above 5.0 pg / mL; most preferably, it is above 7.0 pg / mL.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of FGF in the platelet-fibrin-rich solution GX Factor is above 0.01 pg / mL; preferably above 0.02 pg / mL; More preferably, it is above 0.05 pg / mL; most preferably, it is above 0.08 pg / mL.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of NGF in the platelet-rich fibrin release solution GX Factor is above 0.01 pg / mL; preferably above 0.05 pg / mL.

According to an aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the concentration of TGF-β1 in the platelet-rich fibrin release solution GX Factor is above 0.5 pg / mL; preferably 1.0 pg / mL Above; more preferably 1.5 pg / mL or more; most preferably 2.0 pg / mL or more.

According to one aspect of the present invention, a joint cartilage repairing composition can be provided. The stem cells can be any suitable stem cells, including, for example, embryonic stem cells, adult stem cells, or induced pluripotent stem cells; but the stem cells are not human. Totipotent stem cells.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided. The above-mentioned adult stem cells include, but are not limited to, umbilical cord blood stem cells, peripheral blood stem cells, neural stem cells, epidermal stem cells, muscle stem cells, adipose stem cells, bone marrow stem cells, eyes Corneal stem cells, liver stem cells, or intestinal epithelial stem cells; preferably umbilical cord blood stem cells, peripheral blood stem cells, neural stem cells, epidermal stem cells, muscle stem cells, adipose stem cells, or bone marrow stem cells; more preferably umbilical cord blood stem cells, peripheral blood stem cells, nerves Stem cells, muscle stem cells, adipose stem cells, or bone marrow stem cells; particularly preferred are umbilical cord blood stem cells, peripheral blood stem cells, adipose stem cells, or bone marrow stem cells; the most preferred are adipose stem cells.

According to one aspect of the present invention, a composition for repairing articular cartilage can be provided. The above-mentioned adipose stem cells are isolated from adipose tissues of humans or mammals.

According to still another aspect of the present invention, a joint cartilage repair composition can be provided, which can be used simultaneously or sequentially.

According to another aspect of the present invention, a composition for articular cartilage repair can be provided, wherein the carrier contains an excipient, a diluent, a thickener, a filler, a binding agent, a disintegrant, a lubricant, a grease, or a non-grease. Base, surfactant, suspending agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, colorant, etc.

According to another aspect of the present invention, a joint cartilage repair composition can also be provided, which is administered by injection.

According to another aspect of the present invention, a method for repairing articular cartilage can be provided, which comprises administering or administering a platelet fibrin-rich release solution to an individual in need of repairing articular cartilage, the platelet-fibrin-rich release solution GX Factor The line contains at least one growth factor selected from TGF-β1, VEGF, PDGF, EGF, FGF, NGF, and IGF; and a stem cell, which is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell.

The features and advantages of the present invention can be further understood from the following description. Please refer to FIGS. 1 to 5 when reading.

In the following, different specific embodiments are listed and described in more detail for the implementation aspects of the present invention in order to make the spirit and content of the present invention more complete and easy to understand; however, those with ordinary knowledge in this technology should understand The invention is of course not limited to these examples, and other identical or equal functions and sequence of steps can be used to achieve the invention.

First, a specific term or noun used in this specification is described descriptively.

Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and used by those having ordinary knowledge in the technical field to which the present invention pertains.

As used herein, the term "treatment" refers to the implementation of a preventive, pharmacological and / or physiological effect on an individual or patient with a medical condition, symptom, disease, disorder, or a prior condition. Curative or mitigating treatment, an action that partially or completely reduces the severity, delays the onset of progress, and / or suppresses the occurrence of one or more symptoms, abnormalities, and / or diseases of the medical condition. The aforementioned symptoms, diseases, abnormalities and / or medical conditions may be articular cartilage damage.

As used herein, the term "an effective amount" refers to a medical drug that is administered directly or indirectly (administered, administering, or administration) to damaged articular cartilage and is capable of achieving repair after an appropriate period of administration The specific amount used for the effect of articular cartilage tissue or for the purpose of treating articular cartilage damage.

Herein, the aforementioned "medical drug" refers to a pharmaceutically active substance capable of inducing a desired pharmacological and / or physiological response through local and / or systemic effects, and generally includes a compound, a formulation (formulation), composition (composition), medicament (agent), pharmaceutical (medicine or medicament), or prodrug, derivative, or analog of a pharmaceutically active compound.

In this article, "subject" or "patient" may be used interchangeably with each other. The "individual" or "patient" respectively refers to animals that can be treated with the compounds and / or methods, including but not limited to any mammals including, for example, dogs, cats, horses, sheep, pigs, cattle, etc., and humans, Non-human primates. Unless specifically stated otherwise, the "individual" or "patient" may include both male and female genders. The individual or patient suitable for treatment with the composition and / or method of the present invention is preferably a human.

In this article, the numerical values and parameters used to define the scope of the present invention inherently inevitably contain the standard deviation due to individual test methods, and are therefore mostly expressed as approximate numerical values. However, in specific embodiments, Relevant values presented as accurately as possible. In this context, "about" usually depends on the consideration of those with ordinary knowledge in the technical field to which the present invention belongs, and generally refers to the fact that the actual value falls within the acceptable standard error of the average value, for example, the actual value is within one Within ± 10%, ± 5%, ± 1%, or ± 0.5% of a specific value or range.

In this article, the data obtained in each of the embodiments are determined by statistical analysis to determine whether there are differences between different groups. One-way ANOVA (Analysis of variance) test was used to compare the groups. When the p-value was less than 0.05, it was considered to have statistically significant difference.

That is, the disclosure of the present invention can provide a composition for treatment and repair, which includes stem cells and platelet fibrin release solution; wherein the stem cell line is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell; the The platelet fibrin release solution contains at least one growth factor selected from the group consisting of TGF-β1, VEGF, PDGF, EGF, FGF, NGF, and IGF.

Furthermore, according to another embodiment of the present invention, a composition for repairing articular cartilage may be provided. The aforementioned carrier includes an excipient, a diluent, a thickener, a filler, a binder, a disintegrant, and a lubricant. , Grease or non-grease base, surfactant, suspending agent, gelling agent, adjuvant, preservative, antioxidant, stabilizer, colorant, etc.

Furthermore, according to another embodiment of the present invention, a joint cartilage repairing composition can be provided, which is a liquid, lyophilized powder, crystal ball, or slow release agent; the liquid, crystal ball is preferred Or a slow release agent; more preferred is a crystal ball or a slow release agent; the most preferred is a slow release agent.

In addition, according to another aspect of the present invention, a composition for repairing articular cartilage can be provided, wherein the above-mentioned excipients are those that are compatible with other ingredients in the pharmaceutical preparation and compatible with the organism, for example, an encapsulation material or such as Absorption enhancers, antioxidants, adhesives, buffers, coatings, colorants, diluents, disintegrants, emulsifiers, supplements, fillers, flavoring agents, humectants, lubricants, preservatives, propellants , Release agents, fungicides, sweeteners, solubilizers, humectants and mixtures of additives.

Furthermore, in some preferred embodiments of the present invention, a joint cartilage repairing composition may be provided, wherein the above-mentioned adjuvant may include, but is not limited to, microcrystalline cellulose, calcium carbonate, dicalcium phosphate, or glycine Etc .; the above disintegrating agent may include, but is not limited to, starch, alginic acid, or a specific silicate, etc .; the above-mentioned granular adhesive may include, but is not limited to, polyvinylpyrrolidone, sucrose, gelatin, or acacia ); Wherein the above-mentioned lubricant may include, but is not limited to, magnesium stearate, sodium lauryl sulfate or talc; etc .; the above-mentioned excipient may include, but is not limited to, lactose, sucrose, mannitol, sorbitol, corn starch , Wheat starch, rice starch, potato starch, gelatin, or tragacanth, etc.

Furthermore, in other preferred embodiments of the present invention, the liquid formulation containing the composition of the present invention is made into a sterile injection solution or suspension; for example, it is made suitable for intravenous injection, intramuscular injection, and subcutaneous injection. Or a solution administered by intraperitoneal injection; a diluent suitable for use in the sterile injection solution or suspension described above, for example, it may include, but is not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution; fatty acids such as oleic acid, glyceride derivatives, or pharmaceutically acceptable natural oils such as olive oil or rapeseed oil can also be used.

Furthermore, in another preferred embodiment of the present invention, the above-mentioned composition of the present invention and a pharmaceutically acceptable polymer adjuvant can also be made into a sustained-release dosage form. The above-mentioned polymer adjuvant includes a hydrophilic polymer. For example, for example, it can be used including, but not limited to, methyl cellulose (Methyl Cellulose), hydroxypropyl cellulose (Hydroxypropyl Cellulose), and hydroxypropyl methyl fiber. Hydroxypropyl Methyl Cellulose, Carboxypolymethylene, Carbopol, Cabomer, Polyethylene oxide, Carboxymethyl Cellulose Sodium, Alginin, Hyaluronic Acid, Gelatin, etc.

Hereinafter, specific implementation aspects of the present invention will be described by examples, but the content scope of the present invention is not limited to these examples.

First, a standard operation method for each test in the comparative examples and examples of the present invention will be described.

"Appraisal Method of Appearance and Tissue Section of Regenerating Joint Cartilage"

The rabbits used in each of the comparative examples and the examples were sacrificed, and the joint at the surgical site of the right hind knee was removed using a saw blade, and then scored by the International Cartilage Repair Society (ICRS) scoring standard, as shown in the table. As shown in Figure 1, protocol A (subchondral drilling) is used. According to the assessment of the degree of defect repair, the integration of the boundary area, and the macroscopic appearance (ICRS Cartilage Injury Evaluation Package, 2000), more than 6 evaluations can be performed. Scoring results to assess treatment effectiveness.

Table 1

Next, the bone samples were packed in plastic cans and placed in decalcifying solution. They were replaced once every three to four days, and replaced about four to five times. The degree of decalcification was checked as appropriate, and the end point of decalcification was determined by physical acupuncture. If the fine needle used can penetrate the bone tissue smoothly, the section can be successfully performed.

The decalcification solution was prepared by mixing an equal volume of 50% formic acid (250ml 88% formic acid + 250ml ddH ₂ O) with 20% sodium citrate (100g sodium citrate + 500ml ddH ₂ O).

After decalcification, the bone sample was sliced with a tissue trimmer, and the fixed tissue was sent to the section room of the Taiwan Institute of Animal Science and Technology for sectioning. The tissue was cut into tissue sections with a thickness of about 4 μm, and stained with Hematoxylin & Eosin (H & E) and Toluidiue blue, respectively, and observed under a light microscope to understand the articular cartilage tissue regeneration, and Take a picture.

HE staining can observe the complete and clear structure of bone tissue. The nucleus was purple-red in different degrees, bright, clear, and strong in contrast, and it would not fade after long-term storage. The toluidine blue staining can stain the collagen content in the sample, and the collagen content can be judged according to the depth of the staining.

Then, in accordance with the histological scoring system of the ICRS International Cartilage Repair Organization (THE INTERNATIONAL CARTILAGE REPAIR SOCIETY, ICRS) (Mainil-Varlet, Aigner et al. 2003), as shown in Table 2, the cartilage surface and matrix under histology were respectively targeted. , Cell distribution, cell population survival rate and the degree of mineralization of subchondral hard bone and cartilage. After 6 or more evaluations, the results of the treatment can be evaluated.

Table 2

Comparative Example 1

Female New Zealand white rabbits were selected as experimental animals. Each rabbit was about 10 weeks old and weighed about 2 kg. The rabbits were raised to 16 weeks of age according to the standard operating procedures of the Experimental Animal Breeding Center, and the weight was increased to about 3 kg, so that their bones were fully developed, and then cartilage defect surgery was performed.

The cartilage defect surgery was performed on the femoral condyle of the right hind limb of the rabbit to create a wound with a diameter of 3mm and a depth of 2mm. After suture the wound, apply an appropriate amount of Oxineomycin Ointment (manufactured by China Chemical Pharmaceutical Co., Ltd.) on the wound, and give an analgesic Carprofen (4mg / kg) and antibiotic Cephazolin (25 mg / kg, China Chemical Pharmaceutical Co., Ltd.) Co., Ltd.).

One week after cartilage defect surgery, the wound healed. Observe if the rabbit is not abnormal and immediately start to remove the suture without any treatment. Sacrifice the rabbit after 12 weeks of continuous breeding. Evaluate the rabbit's regeneration according to the above "Evaluation Method of Regenerating Joint Cartilage Appearance and Tissue Section". Articular cartilage appearance and tissue. As a result, the appearance of articular cartilage without any treatment in Comparative Example 1 after continuous feeding for 12 weeks is shown in the photograph of FIG. 1, and the articular cartilage tissue section is shown in the optical microscope photograph of FIG. 3.

Comparative Example 2

First, adipose mesenchymal stem cells R are prepared.

Rabbit adipose tissue was soaked in a serum bottle containing iced PBS (containing 1X-2X P / S / A; 250 ml) and kept on ice to maintain the temperature. Shake the bottle to wash the adipose tissue, remove the liquid, add 200 mL of a solution containing PBS and P / S / A, and wash 2-3 times. Pour the adipose tissue into a 10 or 15 cm petri dish, remove the red fascial meat, and cut the adipose tissue. The cut adipose tissue was placed in a centrifuge tube (10 ml adipose tissue / 50 ml centrifuge tube), filled with PBS, and then shaken for 30 minutes, during which a new PBS or a new centrifuge tube can be replaced.

Clip the washed fragmented adipose tissue into a new centrifuge tube (25ml adipose tissue / centrifuge tube), and pour collagenase type I (Sigma) at a concentration of 0.1-0.2%. The volume ratio is at least 1: 1, and then the centrifuge tube is placed in a 37 ° C water bath, shaken every 5 minutes, and the reaction is stopped for more than 15 minutes or by performing this digestion procedure to the remaining 5% of the fat tissue.

Let the centrifuge tube stand, wait for the oil droplets to float between fat and water, take the water layer (25ml / tube), fill it with PBS, and then centrifuge at 500xg for 5 minutes, then suck off the top white oil and divide it in half Fill with PBS and centrifuge for 10 minutes. Tissues can be washed with PBS, and the residues are centrifuged together. Both the cells and the red blood cells will precipitate, but the red blood cells will not stick to the culture dish. Remove the supernatant from the centrifuge tube, leaving about 5-10 ml in the centrifuge tube, mix with a pipet, and then filter through a 100 µm nylon sieve. PBS was added again, and the supernatant was removed after centrifugation for 10 minutes. Then, the medium was added and cultured on a petri dish. After the cells were attached to the petri dish for 1-2 days, half of the medium was replaced. After that, the medium was changed every two days while checking the cells' differentiation ability.

The above-mentioned primary cultured adipose mesenchymal stem cells were purified by flow cytometry (FACSAria ^™ , Becton Dickinson), and cultured in αMEM culture medium in a 37 ° C incubator to make the cell type more specialized and relatively Consistent spindle-like appearance, and adipose mesenchymal stem cells R were obtained.

Next, female New Zealand white rabbits were selected as experimental animals. Each rabbit was about 10 weeks old and weighed about 2 kg. The rabbits were raised to 16 weeks of age according to the standard operating procedures of the Experimental Animal Breeding Center, and the weight was increased to about 3 kg, so that their bones were fully developed, and then cartilage defect surgery was performed.

Cartilage defect surgery is to create a wound with a diameter of 3mm and a depth of 2mm at the femoral condyle of the femur of the right hind limb of the rabbit. After suture the wound, apply an appropriate amount of Wanlingsu Ointment (Oxineomycin Ointment, manufactured by China Chemical Pharmaceutical Co., Ltd.), and give an analgesic Carprofen (4 mg / kg) and antibiotic Cephazolin (25 mg / kg, China Chemical Pharmaceutical) Co., Ltd.).

One week after the surgery wound healing cartilage defect, if the rabbit was observed no abnormal starts stitches, then, adipose stem cells R (in an amount of 1x10 ⁶ th) and 1.0mL of phosphate buffered saline (PBS) mixed right knee from a rabbit The joint was injected into the joint capsule, and the same dose was repeated every other week.

Rabbits were sacrificed after continuous feeding for 12 weeks, and the appearance and tissue of the regenerated articular cartilage of the rabbits were evaluated according to the above-mentioned "Appraisal Method of Reappeared Articular Cartilage and Tissue Section". As a result, the appearance of articular cartilage treated with adipose mesenchymal stem cells R after continuous feeding for 12 weeks in this comparative example 2 is shown in the photograph of FIG. 1, and the articular cartilage tissue section is shown in the optical microscope photograph of FIG. 3.

Comparative Example 3

First, a platelet-rich fibrin release solution GX Factor was prepared.

After the rabbit was under general anesthesia, the neck hair was removed, and 6 ml of blood was collected from the external jugular vein and directly placed in a vacuum blood collection tube without anticoagulant (BD® Vacutainer SST ™ REF 367988 Sterile BD, Franklin Lakes NJ USA), of which The bottom of the tube contains a gel that allows red blood cells to penetrate and completely separate the red blood cells at the bottom. Next, after centrifuging at 3000 rpm for 10 minutes, the gel in the middle layer was taken out, and then it was left in a sterilization tube for 5 hours, and the release solution was collected, which is the platelet fibrin-rich release solution GX Factor.

Then, the concentration of the growth factor in the platelet fibrin release solution GX Factor was analyzed and recorded in Table 3.

table 3

The platelet-rich fibrin release solution GX Factor prepared by the present invention has the following advantages over the conventional technology: (1) no anticoagulant and thrombin need to be added in the preparation process; (2) a shorter process time; (3) It is an autologous product and has no problems such as immune response. (4) Only a single centrifugation can naturally form a dense fibrin matrix. This structure is more tolerant and can trap white blood cells in between, so it has anti-infective properties, etc. Effect, and can slowly release growth factors and active proteins to increase the effect time; (5) the concentration of growth factors and cytokines is higher, and the healing effect is better.

Next, female New Zealand white rabbits were selected as experimental animals. Each rabbit was about 10 weeks old and weighed about 2 kg. The rabbits were raised to 16 weeks of age according to the standard operating procedures of the Experimental Animal Breeding Center, and the weight was increased to about 3 kg, so that their bones were fully developed, and then cartilage defect surgery was performed.

Cartilage defect surgery is to create a wound with a diameter of 3mm and a depth of 2mm at the femoral condyle of the femur of the right hind limb of the rabbit. After suture the wound, apply an appropriate amount of Wanlingsu Ointment (Oxineomycin Ointment, manufactured by China Chemical Pharmaceutical Co., Ltd.), and give an analgesic Carprofen (4 mg / kg) and antibiotic Cephazolin (25 mg / kg, China Chemical Pharmaceutical) Co., Ltd.).

One week after the cartilage defect operation, the wound healed. If the rabbit is not abnormal, start to remove the suture. Then, 1 mL of platelet fibrin release solution GX Factor is injected from the rabbit right knee joint into the joint capsule, and the same dose is repeated every other week.

Rabbits were sacrificed after continuous feeding for 12 weeks, and the appearance and tissue of the regenerated articular cartilage of the rabbits were evaluated according to the above-mentioned "Appraisal Method of Reappeared Articular Cartilage and Tissue Section". As a result, the appearance of articular cartilage treated with platelet fibrin release solution GX Factor after continuous rearing for 12 weeks in this comparative example 3 is shown in the photograph of FIG. 1, and the articular cartilage tissue section is shown in the optical microscope photograph of FIG. 3. Show.

Comparative Example 4

First, the adipose mesenchymal stem cells R obtained in Comparative Example 3 were induced into chondrocytes C.

The adipose mesenchymal stem cells R were centrifuged at 1500 rpm for 10 minutes. After removing the supernatant, the cells were reconstituted with chondrogenic medium (SH30889.02) at 1 × 10 ⁶ cells / ml, and 1 mL of the cell solution was placed in a 15 mL test tube and centrifuged. Remove the supernatant and add 5 mL of culture solution. Release the lid and incubate for 28 days. Change the medium every three days. The differentiated chondrocytes were identified by Toluidine Staining, and it was confirmed that the adipose mesenchymal stem cells R have been induced into chondrocytes C.

Next, female New Zealand white rabbits were selected as experimental animals. Each rabbit was about 10 weeks old and weighed about 2 kg. The rabbits were raised to 16 weeks of age according to the standard operating procedures of the Experimental Animal Breeding Center, and the weight was increased to about 3 kg, so that their bones were fully developed, and then cartilage defect surgery was performed.

Cartilage defect surgery is to create a wound with a diameter of 3mm and a depth of 2mm at the femoral condyle of the femur of the right hind limb of the rabbit. After suture the wound, apply an appropriate amount of Wanlingsu Ointment (Oxineomycin Ointment, manufactured by China Chemical Pharmaceutical Co., Ltd.), and give an analgesic Carprofen (4 mg / kg) and antibiotic Cephazolin (25 mg / kg, China Chemical Pharmaceutical) Co., Ltd.).

One week after the surgery wound healing cartilage defect was observed no abnormal rabbit starts stitches, then, chondrocytes C (in an amount of 1x10 ⁶ th) arranged in 0.2mL of PBS and with 0.8mL of Comparative Example 3 prepared The obtained platelet fibrin release solution GX Factor was mixed uniformly and injected into the joint capsule from the rabbit right knee joint, and the same dose was repeatedly administered every other week.

Rabbits were sacrificed after continuous feeding for 12 weeks, and the appearance and tissue of the regenerated articular cartilage of the rabbits were evaluated according to the above-mentioned "Appraisal Method of Reappeared Articular Cartilage and Tissue Section". As a result, the appearance of articular cartilage treated with chondrocyte C mixed with platelet fibrin release solution GX Factor after continuous feeding for 12 weeks in this comparative example 4 is shown in the photograph of FIG. 1, and the articular cartilage tissue section is shown in FIG. 3. As shown in the photomicrograph.

<< Example 1 >>

Female New Zealand white rabbits were selected as experimental animals. Each rabbit was about 10 weeks old and weighed about 2 kg. The rabbits were raised to 16 weeks of age according to the standard operating procedures of the Experimental Animal Breeding Center, and the weight was increased to about 3 kg, so that their bones were fully developed, and then cartilage defect surgery was performed.

Cartilage defect surgery is to create a wound with a diameter of 3mm and a depth of 2mm at the femoral condyle of the femur of the right hind limb of the rabbit. After suture the wound, apply an appropriate amount of Wanlingsu Ointment (Oxineomycin Ointment, manufactured by China Chemical Pharmaceutical Co., Ltd.), and give an analgesic Carprofen (4 mg / kg) and antibiotic Cephazolin (25 mg / kg, China Chemical Pharmaceutical) Co., Ltd.).

One week after the cartilage defect surgery, the wound healed. Observed that if the rabbit was not abnormal, the suture was started. Then, the adipose-derived stem cells R (the number of cells was 1 × 10 ⁶ ) prepared in the above Comparative Example 2 were prepared in 0.2 mL of PBS, and It was mixed with 0.8 mL of the platelet fibrin release solution GX Factor prepared in the above Comparative Example 3, and then injected into the articular capsule from the right knee joint of the rabbit, and the same dose was repeatedly administered every other week.

Rabbits were sacrificed after continuous feeding for 12 weeks, and the appearance and tissue of the regenerated articular cartilage of the rabbits were evaluated according to the above-mentioned "Appraisal Method of Reappeared Articular Cartilage and Tissue Section". As a result, the appearance of the articular cartilage treated with adipose-derived stem cells R and platelet fibrin release solution GX Factor after continuous feeding for 12 weeks in this comparative example is shown in the photo of FIG. 1, and the articular cartilage tissue section is shown in FIG. 3. As shown in the photomicrograph.

《Results of Evaluation of Regenerating Joint Cartilage Appearance》

FIG. 1 shows the appearance of rabbit articular cartilage in Comparative Examples 1 to 4 and Example 1. The surface of knee cartilage in Comparative Example 1 was defective, rough, had obvious wound margins, and appeared osteoarthritis (OA). Features include cartilage wear, fibrillation, erosion, and osteophyte formation. Compared with Comparative Example 1, the cartilage defect area of Comparative Example 2 was partially repaired, and its repair covered about 60% of the volume, but fractured tissues and small cavities were still observed. For the cartilage defect sites of Comparative Example 3, Example 1 and Comparative Example 4, it can be clearly seen that there is a repair phenomenon, and the surface is smoother and smoother than Comparative Example 1. Among them, the appearance of Example 1 is closest to that of normal cartilage. The cartilage defect site is filled with tissues similar to hyaline cartilage, the wound edge boundary at the defect site is less obvious, and it is well combined with the surrounding native cartilage. From this, it can be seen that the composition of the present invention has better ability to promote cartilage tissue regeneration.

Then, the average score results of the appearance of regenerated articular cartilage in Comparative Examples 1-4 and Example 4 are recorded in Table 4.

Table 4

In the ICRS appearance scoring system, the highest score is 12 divided into normal cartilage. As can be seen from Table 4, the average score of Example 1 is the highest (9.1905 ± 1.38701), which is the group with the best treatment effect and is closer to normal cartilage. Situation.

Next, the ICRS appearance score is divided into detailed items such as defect repair degree, boundary area integration degree, and macro appearance to observe. As shown in Figure 2, it can also be seen that whether it is in defect repair degree, boundary area integration degree, macro In appearance, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Example 1 all have higher scores than Comparative Example 1, and Example 1 has a better degree of defect repair.

《Assessment Results of Regenerating Joint Cartilage Tissues》

Please refer to Figure 3, Comparative Example 1 without any treatment, the regenerating tissue is a thin layer of new cartilage, which contains immature cartilage tissue and a small amount of cell aggregation, its cartilage structure is chaotic, too few chondrocytes, and tissue type Inconsistent, and there are various osteoarthritis symptoms such as the appearance of small cracks. It is worth noting that the cartilage layer was not observed in the repair tissue of Comparative Example 1, and the hard layer was exposed at the outermost layer of the joint.

In Comparative Example 2, the surface cartilage showed irregular thickness, the subchondral bone was not vertically integrated, and the formation was insufficient. The surface of the regenerated cartilage can also be observed to be irregular and broken. The defect site is filled with a mixture of fibrocartilage tissue and cartilage-like transparent cartilage tissue, so fibrous tissue will still form.

Although the repair tissue of Comparative Example 3 was well integrated with surrounding normal cartilage, and the cartilage surface layer also formed a smooth and gentle tissue repair, and the formation of subchondral bone was good, fibrous tissue was still observed in the superficial cartilage. In addition, the phenomenon of irregularity and fracture was also observed.

Although the defect sites of Example 1 and Comparative Example 4 are covered with hyaline cartilage-like tissue. However, the cartilage fracture in Example 1 is rare, and the conditions of the defect filling rate and the surface layer of the defect are also good. The articular cartilage structure is normal, and the cartilage surface shows very mild irregularities, and is smooth and irregular. Intermittently, the cartilage tissue has a proper thickness, the cells are distributed normally, and the cartilage matrix is consistent and good. The growth of chondrocytes in the inner layer of columnar tissue and the surface layer of hyaline cartilage, the regeneration tissue and the surrounding cartilage are well integrated.

The results in FIG. 3 show that although cartilage-like repair tissues appeared in the defect sites of each group, the integrity of the cartilage grown was inconsistent, and Example 1 was used to give the composition of the present invention. Regenerated cartilage, more similar to the characteristics of native cartilage.

Then, the repair score was performed according to the histological scoring system of the International Cartilage Repair Organization (THE INTERNATIONAL CARTILAGE REPAIR SOCIETY, ICRS), and the score results are recorded in Table 5.

table 5

Figure 4 shows the average score of articular cartilage sections in each group. In the ICRS histological scoring system, the highest score is 18 cases of normal cartilage. The average score of Example 1 is the highest (14.8333 ± 1.72713), which is the best treatment effect. Group, and closer to normal cartilage.

Next, the ICRS slice score was divided into sub-chondral surface, matrix, cell distribution, cell population survival rate, subchondral bone and cartilage mineralization and other detailed items to observe, as shown in Figure 5, it can also be seen whether or not In terms of cartilage surface, matrix, cell distribution, cell population survival rate, mineralization of subchondral bone and cartilage, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Example 1 were all higher than Comparative Example 1. The score of Example 1, which has a higher score in Example 1, shows that the composition of the present invention has good cartilage repair and regeneration ability, can slow down the development process of osteoarthritis, and has the effect of preventing and treating osteoarthritis.

The above embodiments disclose specific embodiments of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains may, without departing from the principles and spirit of the present invention, Various changes and modifications are made, so the protection scope of the present invention shall be defined by the scope of the accompanying patent application.

no.

FIG. 1 shows the appearance of the articular cartilage of Comparative Examples 1 to 4 and Example 1 in the present invention. FIG. 2 shows the ICRS sub-scores of the articular cartilage appearance of Comparative Examples 1 to 4 and Example 1 in the present invention. FIG. 3 is a tissue section staining diagram of the articular cartilage of Comparative Examples 1 to 4 and Example 1 in the present invention. FIG. 4 is the average ICRS score of articular cartilage sections of Comparative Examples 1 to 4 and Example 1 in the present invention. FIG. 5 shows the ICRS item scores of the articular cartilage sections of Comparative Examples 1 to 4 and Example 1 in the present invention.

Claims (10)

A composition for repairing articular cartilage, comprising stem cells and a platelet-rich fibrin release solution; wherein the stem cells are embryonic stem cells, adult stem cells, or induced pluripotent stem cells, and non-human totipotent stem cells; The platelet fibrin release solution contains at least one growth factor selected from the group consisting of TGF-β1, VEGF, PDGF, EGF, FGF, NGF, and IGF. The composition according to claim 1, wherein the platelet-fibrin-rich release solution is enriched by centrifugation of mammalian autologous blood, taking out the middle layer of block material, and leaving it for a certain period of time. Growth factor release fluid. The composition according to claim 2, wherein the specific time is 1 hour or more. The composition according to claim 1, wherein the PDGF has a concentration of 15 pg / mL or more. The composition according to claim 1, wherein the concentration of the VEGF is 0.5 pg / mL or more. The composition according to claim 1, wherein the concentration of the EGF is 0.01 pg / mL or more. The composition according to claim 1, wherein the concentration of the IGF-1 is 1.0 pg / mL or more. The composition according to claim 1, wherein the concentration of the FGF and the NGF is 0.01 pg / mL or more. The composition according to claim 1, wherein the concentration of the TGF-β1 is 0.5 pg / mL or more. A method for repairing articular cartilage, which comprises administering or administering the articular cartilage to an individual in need of repair: the articular cartilage repair composition according to any one of claims 1 to 9.