Compositions comprising avenanthramide c for inducing differentiation of mesenchymal stem cells into chondrocytes and methods of use thereof

Abstract

The present invention discloses the use of Avn Anthramides C (Avn C) as an inducer of chondrocytic differentiation in Mesenchymal Stem Cells (MSCs), providing a promising approach for regenerative cartilage therapy. By employing Avn C treatment, MSCs exhibit enhanced chondrocytic differentiation and elevated expression of key specific chondrogenic extracellular matrix (ECM) factors, notably Collagen Type II (COL2A1). Furthermore, when Avn C is used in combination with Chondroitin Sulfate (CS), a synergistic effect is observed, leading to upregulation of the transcriptional activity state of SOX9, a key regulator of chondrogenesis. The disclosed invention shows significant improvements in mRNA gene expression (ACAN, LRP1, SOX9), cellular protein levels (COL2A1, SOX9, LRP1), and functional outcomes, as evidenced by the secretion of extracellular matrix proteins (FSTL-1, BGH3, CO1A1, CO1A2, and CO3A1).

Description

Technical Field

[0001] This invention generally relates to the use of avocado anthraquinone C (Avn C) as an inducer of chondrocyte differentiation in mesenchymal stem cells (MSCs). More specifically, embodiments of this disclosure relate to compositions comprising avocado anthraquinone C (Avn C) for the differentiation of mesenchymal stem cells (MSCs) into chondrocytes, and methods of using the same. Background Technology

[0002] Against the backdrop of an aging population, the reduction of functional chondrocytes, given their limited regenerative capacity, significantly contributes to the gradual worsening of joint discomfort over a long period. Chondrocytes are essential cells responsible for producing and releasing various components of the extracellular matrix, including various collagens, and are crucial for maintaining cartilage flexibility. The introduction of chondrocyte replacement therapy holds promise for significantly improving the quality of life for individuals suffering from joint-related problems.

[0003] Mesenchymal stem cells (MSCs) possess the ability to differentiate into various cell lineages, including chondrocytes, and have attracted considerable interest in developing MSC-based cartilage repair therapies. This innovative approach, by leveraging the inherent characteristics of patient-derived MSCs and modulating their directed differentiation into chondrocyte lineages, offers a safe alternative that effectively avoids the potential risks associated with immune system rejection of cell grafts.

[0004] Mesenchymal stem cells (MSCs) are adult stem cells derived from various tissues and hold great potential in regenerative cartilage therapy due to their multipotent differentiation capacity. However, practical and reliable methods for inducing chondrocyte differentiation in MSCs are needed. Current methods lack optimal efficacy, thus requiring novel inducers with superior results.

[0005] Avn (agar anthraquinone) is a polyphenolic compound mainly found in oats and has attracted attention due to its unique biological properties, including antioxidant, antiproliferative, antihistamine and anti-inflammatory effects.

[0006] Korean patent application KR20210091080A1, filed by RYU JE HWANG et al. and entitled “Pharmaceutical composition for treating or preventing arthritis,” discloses the use of Avn-C to reduce the expression of cartilage matrix degrading enzymes and cartilage degeneration inducers in chondrocytes for the treatment or prevention of arthritis.

[0007] Furthermore, a non-patent document titled "Avenanthramide C as a novel candidate to alleviate osteoarthritic pathogenesis," published by Thanh-Tam Tran et al., discloses the use of avenanthramide-C to prevent the progression of osteoarthritis (OA) and the prevention of matrix metalloproteinase (MMP) expression in the pathogenesis of OA. This non-patent document also emphasizes that administration of Avn-C does not affect the expression of inflammatory mediators (Ptgs2 and Nos) or anabolic factors (Col2a1, Aggrecan, and Sox9) in the pathogenesis of OA.

[0008] Another non-patented study, titled "Avenanthramides Prevent Osteoblast and Osteocyte Apoptosis and Induce Osteoclast Apoptosis in Vitroin an Nrf2-Independent Manner," published by Gretel G. Pellegrini et al., discloses the application of avenanthramides to enhance osteogenic differentiation by increasing COL1A expression. This study indicates that avenanthramides exert an anti-apoptotic effect on osteoblasts and a pro-apoptotic effect on osteoclasts.

[0009] A recent study by Trans et al. elucidated the inhibitory effect of avocado anthraquinone C (Avn C) on cartilage degradation. This study showed that Avn C effectively inhibited the expression of IL-1β-induced catabolic factors (particularly MMP-3, -12, and -13) in mouse articular chondrocytes. However, Avn C did not affect the reduction of IL-1β-mediated maturation factors such as type II collagen (COL2A1)—a major component of the chondrocyte extracellular matrix (ECM)—or SOX9—a key transcription factor involved in the regulation of type II collagen (collagen 2). Notably, in this study, Avn C was applied to the inflammatory response in fully differentiated chondrocytes of mature cartilage.

[0010] This specification recognizes the need for new applications of avocado anthraquinone C (Avn C) as an effective inducer of chondrocyte differentiation in mesenchymal stem cells (MSCs) and to establish more effective methods in this field. MSCs are adult stem cells derived from various tissues such as bone marrow, adipose tissue, and umbilical cord tissue, exhibiting significant pluripotency and the ability to differentiate into multiple cell types.

[0011] This article addresses the aforementioned shortcomings, disadvantages, and problems, which can be understood by reading and studying the following instructions. Summary of the Invention

[0012] Various embodiments described herein provide compositions containing avocado anthraquinone C (Avn C) for differentiating mesenchymal stem cells (MSCs) into chondrocytes and methods of using them.

[0013] One aspect of this disclosure relates to the novel application of avocado anthraquinone C (Avn C) as a potent inducer of chondrocyte differentiation in mesenchymal stem cells (MSCs), establishing a more efficient approach in this field. MSCs are adult stem cells derived from various tissues such as bone marrow, adipose tissue, and umbilical cord tissue, possessing remarkable pluripotency and the ability to differentiate into multiple cell types. This unique characteristic makes MSCs a valuable resource for regenerative cartilage applications.

[0014] Treatment with Avn C resulted in MSCs exhibiting impressive chondrocyte differentiation capacity, significantly enhanced by the upregulation of key chondrogenic extracellular matrix (ECM) factors, particularly chondrocyte-specific type II collagen (COL2A1). Furthermore, the synergistic effect of Avn C and chondroitin sulfate (CS) significantly improved the transcriptional activity of SOX9, a key regulator of chondrogenesis.

[0015] On one hand, Avn C at concentrations ranging from 10 nM to 50 µM (50,000 nM), co-administered with chondroitin sulfate at concentrations of 300–700 µg / mL (especially 500 µg / mL), was used as a highly efficient differentiation factor to promote the transformation of MSCs into chondrocyte-like cells. Using this optimized formulation, we observed morphological changes in MSCs, leading to chondrocyte-like characteristics, as evidenced by significant changes in mRNA gene expression (ACAN, LRP1, SOX9), cellular protein levels (COL2A1, SOX9, LRP1), and functional levels, exemplified by the secretion of extracellular matrix proteins (FSTL-1, BGH3, CO1A1, CO1A2, and CO3A1). These results clearly establish Avn C as a highly efficient differentiation factor, paving the way for its practical application in the differentiation of MSCs into chondrocytes.

[0016] One aspect of this disclosure relates to a composition for inducing mesenchymal stem cells (MSCs) to differentiate into chondrocytes, said composition comprising avocadothramide C (Avn C). In one embodiment, the term "composition" refers to a medium or hydrogel containing avocadothramide C (Avn C) for inducing stem cell differentiation into chondrocytes, and the term "chondrocyte-like cells" refers to chondrocyte-like cells in a two-dimensional manner.

[0017] In one respect, avocado anthraquinone C (Avn C) exists at concentrations ranging from 10 nM to 50,000 nM.

[0018] In one respect, avocado anthramide C (Avn C) is preferably present at a concentration of 50-300 nM.

[0019] In one aspect, the composition further comprises chondroitin sulfate (CS) as a bioactive factor.

[0020] In one aspect, the composition further comprises chondroitin sulfate (CS) as a bioactive factor.

[0021] In one respect, chondroitin sulfate (CS) exists at concentrations ranging from 300 to 700 µg / mL (particularly 500 µg / mL).

[0022] In one respect, mesenchymal stem cells are adipose-derived mesenchymal stem cells (aMSCs).

[0023] In one respect, chondrocytes are chondrocyte-like cells in a two-dimensional manner.

[0024] In one aspect, the composition is used to increase chondrocyte markers in chondrocytes, wherein the chondrocyte markers are selected from the group consisting of FSTL-1, LRP1, ACAN, COL2A, SOX9, and any combination thereof.

[0025] Another aspect of this disclosure relates to the use of a culture medium for inducing in vitro differentiation of mesenchymal stem cells (MSCs). The culture medium comprises a composition having anthraquinone C (Avn C) at a concentration ranging from 10 nM to 50,000 nM. Furthermore, such a composition further comprises chondroitin sulfate (CS) as a bioactive factor at a concentration ranging from 300 to 700 µg / mL, particularly 500 µg / mL.

[0026] Another aspect of this disclosure relates to a method for inducing differentiation of in vitro cultured mesenchymal stem cells (MSCs) into chondrocytes, the method comprising culturing the MSCs in a medium containing avothramide C (Avn C).

[0027] In one respect, avocado anthraquinone C (Avn C) exists at concentrations ranging from 10 nM to 50,000 nM.

[0028] In one respect, avocado anthramide C (Avn C) is preferably present at a concentration of 50 nM to 300 nM.

[0029] In one respect, chondroitin sulfate (CS) exists in the culture medium as a bioactive factor.

[0030] In one aspect, the method further includes chondroitin sulfate (CS) present in the culture medium as a bioactive factor.

[0031] In one respect, chondroitin sulfate (CS) is present in the culture medium at concentrations ranging from 300 to 700 µg / mL (particularly 500 µg / mL).

[0032] In one respect, mesenchymal stem cells are adipose-derived mesenchymal stem cells (aMSCs).

[0033] In one respect, chondrocytes are chondrocyte-like cells in a two-dimensional manner.

[0034] In one aspect, chondrocyte markers increased, and the chondrocyte markers were selected from the group consisting of FSTL-1, LRP1, ACAN, COL2A, SOX9 and any combination thereof.

[0035] Another aspect of this disclosure relates to pharmaceutical compositions for chondrocyte repair and regeneration.

[0036] These and other aspects of the embodiments described herein will be better understood when considered in conjunction with the following description and accompanying drawings. However, it should be understood that while the following description points out preferred embodiments and their numerous specific details, it is given by way of illustration rather than limitation. Many changes and modifications can be made within the scope of the embodiments described herein without departing from their spirit, and the embodiments described herein include all such modifications. Attached Figure Description

[0037] Other objects, features, and advantages will be understood by those skilled in the art from the following description and accompanying drawings of preferred embodiments, wherein:

[0038] Figure 1A-1C A perspective view is shown illustrating key aspects of the in vitro chondrocyte differentiation process of MSCs according to one embodiment of this article.

[0039] Figures 2A-2D A perspective view showing that avocadothramide (Avn) C, according to one embodiment of this article, exerts a potent effect on chondrogenesis differentiation by significantly increasing the expression of type II collagen.

[0040] Figures 3A-3H A perspective view showing the effective dose range for identifying the differentiation of MSCs into chondrocytes by avoanthramide C (Avn C) according to one embodiment.

[0041] Figures 4A-4EA perspective view showing the synergistic effect of Avn C and chondroitin sulfate (CS) on SOX9 protein expression according to one embodiment of this article.

[0042] Figures 5A-5F A perspective view showing the synergistic effect of Avn C and chondroitin sulfate (CS) on LRP1 protein expression according to one embodiment of this article.

[0043] Figure 6 A perspective view is shown showing the synergistic effect of Avn C and chondroitin sulfate (CS) on the expression of agglutinin protein, as revealed by alcine blue staining according to one embodiment of this article.

[0044] Figure 7 A perspective view is shown of an RT-PCR analysis of mRNA expression according to one embodiment of this paper: Avn C and chondroitin sulfate showed a synergistic effect on the expression of aggregate proteoglycan (ACAN), SOX9 and LRP1 genes, while maintaining the expression of COL2A1 gene.

[0045] Figure 8 A perspective view is shown illustrating the analysis of mRNA expression of chondrocyte markers in MSCs from multiple individuals using RT-PCR according to one embodiment of this paper.

[0046] Although specific features of the embodiments described herein are shown in some of the accompanying drawings but not in others, this is only for convenience, as each feature can be combined with any or all other features according to the embodiments described herein. Detailed Implementation

[0047] In the following detailed description, reference is made to the accompanying drawings, which form part of this specification, and specific embodiments that can be practiced are shown by way of example. These embodiments are described in sufficient detail to enable those skilled in the art to practice them, and it should be understood that logical, mechanical, and other changes can be made without departing from the scope of the embodiments. Therefore, the following detailed description should not be considered limiting.

[0048] Various embodiments described herein provide the application of avocado anthraquinone C (Avn C) as a highly effective inducer of chondrocyte differentiation in MSCs. Avn C treatment exhibits superior chondrogenic potential, thereby upregulating key chondrogenic extracellular matrix factors, including type II collagen (COL2A1). Furthermore, a synergistic effect was observed when Avn C was used in combination with chondroitin sulfate (CS), enhancing the transcriptional activity of SOX9, a key regulator of chondrogenesis.

[0049] According to one embodiment of this invention, the formulation of the present invention comprises Avn C at concentrations ranging from 10 nM to 50 µM (50,000 nM), co-administered with chondroitin sulfate at 300-700 µg / mL (particularly 500 µg / mL), serving as an optimized and highly efficient differentiation factor for MSCs, leading to their transformation into chondrocyte-like cells. The formulation of the present invention shows significant improvements in mRNA gene expression (ACAN, LRP1, SOX9), cellular protein levels (COL2A1, SOX9, LRP1), and functional outcomes, as demonstrated by the secretion of extracellular matrix proteins (FSTL-1, BGH3, CO1A1, CO1A2, and CO3A1). These results confirm the potential of Avn C as a valuable and highly efficient MSC-to-chondrocyte differentiation factor, which is suitable for patent protection.

[0050] Furthermore, certain terms and naming conventions are used in this description. As will be understood by those skilled in the art, different names may be used to refer to the same concept or feature, and this does not limit the scope of the invention unless otherwise specified. The effectiveness of each part of the embodiments should take precedence over its name, as the name itself is only for distinction. Unless otherwise specified, the numerical values ​​stated in this disclosure may be approximate or precise values, and various embodiments may deviate from the stated numerical values ​​without departing from the expected range.

[0051] Therefore, one advantage of the present invention is that it provides a unique and innovative use of avocado anthramide C (Avn C) as a chondrocyte differentiation inducer in MSCs, representing a significant advance in the field.

[0052] Therefore, another advantage of the present invention is that Avn C treatment exhibits remarkable efficacy in promoting chondrocyte differentiation, as demonstrated by the increased expression of key chondrogenic extracellular matrix factors, particularly type II collagen (COL2A1).

[0053] Therefore, another advantage of the present invention is that the combination of Avn C and chondroitin sulfate (CS) exhibits a synergistic effect, leading to an increase in the expression of the key cartilage formation regulator SOX9, thereby enhancing the differentiation potential of MSCs.

[0054] Therefore, another advantage of the present invention is that it provides an optimized formulation containing specific concentrations of Avn C and chondroitin sulfate, providing a reliable and efficient method for inducing chondrocyte-like features in MSCs.

[0055] In one embodiment, this disclosure presents avocado anthraquinone C (Avn C) as a novel and potent differentiation factor for inducing chondrocyte differentiation in mesenchymal stem cells (MSCs). The disclosed formulation, when used in combination with chondroitin sulfate, provides excellent results in terms of gene expression, protein levels, and functional characteristics of differentiated cells. This invention has great potential for practical applications in regenerative cartilage therapy and is worthy of patent protection.

[0056] According to one embodiment of this document, this disclosure further describes materials and methods for carrying out the invention. Cell culture: The mesenchymal stem cells used in this invention are adipose-derived mesenchymal stem cells (MSCs).

[0057] All MSCs were cultured and expanded in a 3:1:1 mixture of low-glucose Dulbecco modified Eagle medium (DMEM LG; Gibco #31600083), MCDB 201 (Sigma-Aldrich #M6770), and MCDB 131 (Sigma-Aldrich #M8537), supplemented with 0.5% human platelet lysate (HPL; Stemcell Technologies #06961), 1X insulin-transferrin-selenium-X (ITS-X; Gibo #51500056) or ITS+1 liquid medium supplement (Sigma-Aldrich #I2521), 0.2% bovine serum albumin (BSA; MP Biomedicals #0219989880), 1X linoleic acid BSA (Sigma-Aldrich #L9530), 100 μM L-ascorbic acid-2-phosphate (Sigma-Aldrich #49752), 100 μM β-mercaptoethanol (Gibco #21985023), 10 ng / ml recombinant human epidermal growth factor (rhEGF; Gibco #PHG0314), 1 nM dexamethasone (Sigma-Aldrich #D4902), 1% penicillin-strepmycin (pen-strep; Gibco #15140122), 250 ng / ml amphotericin B (Amp B; Gibco #15290026), and 20 μg / ml gentamicin solution (Sigma-Aldrich #G1272).

[0058] In vitro chondrogenic differentiation: In vitro chondrogenic differentiation was performed using monolayer culture. Fourth-generation (P4) MSCs were cultured at 6000 cells / cm². 2MSCs were seeded at a density in gelatin-coated 24-well plates. MSCs were expanded for 3 days and then cultured in chondrocyte differentiation medium (CDM) for 21 days (Figure 1A).

[0059] Standard chondrocyte differentiation medium (STD) consists of low-glucose DMEM (DMEM LG; Gibco #31600083) supplemented with 0.5 μg / ml hydrocortisone (Sigma-Aldrich #H0888), 1% fetal bovine serum (FBS; Biowest #S181B), 1X insulin-transferrin-selenium-X (ITS-X; Gibo #51500056) or ITS+1 liquid medium supplement (Sigma-Aldrich #I2521), 10 ng / ml recombinant human TGF-β3 (R&D Systems #8420-B3-025), 50 nM L-ascorbic acid-2-phosphate (Sigma-Aldrich #49752), 1% penicillin-strepmycin (pen-strep; Gibco #15140122), and 500 ng / ml amphotericin B (Amp B; Gibco #15290026). The standard CDM (STD) will be used as a reference (Figure 1A).

[0060] Standard chondrocyte differentiation medium (STD) or CDM1 was followed by three different treatment groups: CDM2 with 100 nM avenanthramide C (Avn C; Sigma-Aldrich #36465), CDM3 with 500 μg / mL chondroitin sulfate (5 x CS; Sigma-Aldrich #C4384), and CDM4 with a combination of 100 nM avenanthramide C and 500 μg / mL chondroitin sulfate (Figure 1A).

[0061] for Figure 7 The reverse transcription PCR (RT-PCR) performed included CDM3 containing standard chondrocyte differentiation medium and 500 μg / mL chondroitin sulfate (STD + 5X CS), and CDM4 containing standard chondrocyte differentiation medium, avenanthramide C, and 500 μg / mL chondroitin sulfate (STD + Avn C + 5X CS), supplemented with 0.1 μM dexamethasone (Sigma-Aldrich #D4902) instead of 0.5 μg / mL hydrocortisone (Sigma-Aldrich #H0888). Figure 7 ).

[0062] Furthermore, this disclosure describes immunofluorescence analysis performed during the experimental phase of the present invention.

[0063] On day 21 of chondrocyte differentiation, samples were washed with PBS (Sigma-Aldrich #56064C) and fixed with 4% formaldehyde (Fisher Chemical #F / 1501) at room temperature for 10 minutes or overnight at 4°C. The samples were then washed three times with PBS for 5 minutes each to remove the fixative. The samples were then permeabilized with 0.1% Triton-X (Sigma-Aldrich #X100) at room temperature for 15 minutes, followed by three washes with PBS for 5 minutes each.

[0064] Samples were blocked with blocking buffer at room temperature for at least 1 hour, or overnight at 4°C. The blocking buffer consisted of 0.02% Tween-20 (Sigma-Aldrich #P1379) and 1% BSA (MP Biomedicals #0219989880) in PBS. Samples were then incubated overnight at 4°C with primary antibodies. The primary antibodies used were rabbit polyclonal anti-type II collagen antibody (Abcam #ab34712) and rabbit monoclonal anti-SOX9 antibody (Abcam #ab185230), both diluted 1:50 in blocking buffer. Rabbit monoclonal anti-LRP1 antibody (Abcam #ab92544) was used at a 1:200 dilution in blocking buffer. Mouse anti-human CD166 IgG1 monoclonal antibody (R&D Systems #SC017) was used at a 1:50 dilution.

[0065] Primary antibodies were removed by washing three times with PBS for 5 minutes each time, followed by incubation with secondary antibodies at room temperature for 1 hour. The secondary antibodies used were goat polyclonal anti-rabbit IgG H&L secondary antibody (Alexa Fluor® 555) (Abcam #ab150078) for SOX9, type II collagen, and LRP1 antibodies, and goat polyclonal anti-mouse IgG H&L secondary antibody (Alexa Fluor® 488) (Abcam #ab150113) for CD166 antibody, both diluted 1:500 in blocking buffer. Secondary antibodies were removed by washing three times with PBS for 5 minutes each time, followed by counterstaining with DAPI (Tocris #5748 / 10). DAPI was removed from the samples by rinsing with PBS. Samples were then stored in PBS and wrapped in aluminum foil at 4°C to prevent fluorophore bleaching. Samples were observed within 48–72 hours.

[0066] Alecine blue staining: On day 21 of chondrocyte differentiation, alecine blue staining was performed as described. Samples were washed with PBS and fixed with 4% formaldehyde for 45 minutes. After fixation, the wells were rinsed twice with DPBS and incubated overnight in 1% alecine blue staining solution (pH 2.5) prepared in 0.1 N HCl. The next day, the wells were rinsed three times with 0.1 N HCl, followed by rinsing with DPBS to neutralize the acidity. The wells were examined under an optical microscope. result:

[0067] MSCs exhibit mesenchymal stem cell characteristics and can be effectively induced to exhibit chondrocyte-like features.

[0068] A comprehensive evaluation was conducted to ensure the quality of mesenchymal stem cells (MSCs), focusing on the preservation of their proliferative and differentiation capabilities, as well as the expression of their MSC markers. Following this validation process, MSCs were differentiated into chondrocyte-like cells, as shown in Figure 1A. Cells were treated with standard chondrocyte differentiation medium, commonly known as STD medium.

[0069] The results demonstrated the high quality of the MSCs, characterized by robust proliferation rates and maintenance of MSC markers. Therefore, we successfully induced chondrocyte-like features in our MSCs. Figure 1A visually outlines the experimental design, depicting the expression of MSC markers on day 0 (D0) and their gradual decrease as differentiation progresses to day 21 (D21), as shown in Figure 1B. During this transition, the cells exhibited a more rounded morphology, deviating from the typical fibroblast-like appearance of MSCs (Figure 1C).

[0070] To confirm successful chondrocyte differentiation, allicin blue staining, a technique widely used to detect chondrocyte proteoglycans, was employed. The staining showed a positive result 21 days after chondrocyte differentiation (Figure 1C), further demonstrating the superior quality of our MSCs and their ability to exhibit differentiation potential by acquiring chondrocyte-like characteristics.

[0071] Figure 1A-1C A perspective view 100 illustrates key aspects of the in vitro chondrocyte differentiation process of MSCs according to one embodiment of this paper. Figure 1A presents a schematic diagram outlining the experimental procedures. In Figure 1B (scale bar = 50 µm), the expression of MSC markers at day 0 and day 21 of chondrocyte differentiation using STD medium is depicted. Furthermore, Figure 1C (scale bar = 100 µm) shows optical microscopic images of cells at day 0 and day 21 of chondrocyte differentiation using STD medium, with alecnin blue staining.

[0072] Furthermore, compared with the standard MSC-to-chondrocyte differentiation method, avocado anthraquinone C (Avn C) showed superior efficacy in promoting the expression of chondrocyte-specific characteristics.

[0073] Chondrocytes are responsible for synthesizing the extracellular matrix (ECM) in cartilage, which consists of large proteoglycans for compressive stiffness and collagen for tensile strength. Of the various collagen types, type II collagen (COL2A1) is found only in chondrocytes and accounts for 90-95% of the collagen present in the cartilage ECM. The functions of type II collagen (COL2A1) include promoting cell proliferation within chondrocytes, ECM deposition, and wound healing processes. Therefore, COL2A1 is commonly used as a biomarker for chondrocyte identification.

[0074] Notably, the introduction of avocado anthramide C (Avn C) enhanced chondrocyte differentiation by significantly increasing the expression of the COL2A1 protein. This enhancement was clearly visible in differentiated MSCs after 21 days of treatment with Avn C in combination with standard differentiation medium conditions (denoted as CDM2 (Avn C)). This effect was significantly more pronounced compared to cells treated with standard differentiation medium alone (denoted as CDM1 (STD)), as shown in Figures 2B and 2C.

[0075] Figure 2A-2D A perspective view 200 illustrates the potent effect of avenanthramide (Avn) C on chondrogenic differentiation by significantly enhancing type II collagen expression according to one embodiment of this article. In Figure 2A, MSCs are stained for type II collagen on day 0 of the MSC differentiation process into chondrocytes. Figure 2B shows MSCs cultured for 21 days in CDM1 (STD) differentiation medium. Conversely, in Figure 2C (scale bar = 50 µm), MSCs were cultured in CDM2 containing Avn C in addition to standard differentiation medium, also for 21 days. Figure 2D represents the quantification of intracellular COL2A1 protein immunostaining positivity derived from the intensity and area of ​​the immunofluorescence microscopy images. Effects of oat anthraquinone C dosage range on MSC differentiation into chondrocytes: a comprehensive immunofluorescence staining analysis of collagen 2A1 expression

[0076] To establish the optimal dosage range of avocado anthraquinone C (Avn C) for promoting the differentiation of mesenchymal stem cells (MSCs) into chondrocyte-like cells, a detailed analysis was performed using collagen 2A1 immunofluorescence staining. Figure 3 presents the remarkable results of this study, with Figure 3A depicting the staining of MSCs on day 0 (D0) when initially incubated in differentiation medium. Subsequently, MSCs were cultured for 21 days in standard differentiation medium supplemented with different concentrations of Avn C, including 0 nM (Figure 3B), 10 nM (Figure 3C), 100 nM (Figure 3D), 1 µM (Figure 3E), 10 µM (Figure 3F), and 50 µM (Figure 3G). Figure 3H highlights the quantitative assessment of COL2A1 immunogenicity derived from these experimental conditions.

[0077] The striking results depicted in Figure 3 highlight that the optimal Avn C concentration for enhancing collagen 2A1 protein signaling is approximately 100 nM. Notably, in this experiment, we used primary MSC cell lines from different individuals, further reinforcing the consistency of our observations regarding the effect of Avn C on enhancing MSC differentiation into chondrocyte-like cells, as shown in Figure 2. Avn C dosages ranging from 10 nM to 50 µM (50,000 nM) demonstrated its significant efficacy in driving differentiation, as evidenced by significant immunofluorescence staining of collagen 2A1.

[0078] These findings highlight the strong potential of Avn C as a transformant that promotes the conversion of MSCs into chondrocyte-like cells, thus offering a promising prospect for regenerative cartilage therapy.

[0079] Figures 3A-3H A perspective view 300 shows the effective dose range for identifying the differentiation of MSCs into chondrocytes by avocado anthraquinone C (Avn C) according to embodiments described herein.

[0080] Figure 3A shows the immunofluorescence staining of type II collagen on day 0 of MSC differentiation into chondrocytes.

[0081] Figure 3B shows MSCs cultured in standard chondrocyte differentiation medium (STD) supplemented with 0 nM Avn C.

[0082] Figure 3C shows MSCs cultured in standard chondrocyte differentiation medium (STD) supplemented with 10 nM Avn C.

[0083] Figure 3D shows MSCs cultured in standard chondrocyte differentiation medium (STD) supplemented with 100 nM Avn C.

[0084] Figure 3E shows MSCs cultured in standard chondrocyte differentiation medium (STD) supplemented with 1 µM Avn C.

[0085] Figure 3F shows MSCs cultured in standard chondrocyte differentiation medium (STD) supplemented with 10 µM Avn C.

[0086] Figure 3G shows MSCs cultured for 21 days in standard chondrocyte differentiation medium (STD) supplemented with 50 µM (50,000 nM) Avn C (scale bar = 50 µm).

[0087] Figure 3H shows the quantification of intracellular COL2A1 protein determined by assessing the intensity and corresponding area of ​​immunostaining using immunofluorescence microscopy.

[0088] The objective of this invention is to identify the effective dose range of avocado anthraquinone C (Avn C) for inducing MSC differentiation into chondrocytes. Immunofluorescence staining for type II collagen was performed on day 0 of the differentiation process to provide baseline comparison. MSCs were then cultured in standard chondrocyte differentiation medium (STD) supplemented with different concentrations of Avn C, including 0 nM, 10 nM, 100 nM, 1 µM, 10 µM, and 50 µM (50,000 nM). Culture was maintained for 21 days to assess the effect of Avn C on chondrocyte differentiation. Synergistic enhancement of chondrogenic effects: Avocado anthraquinone C (Avn C) and chondroitin sulfate (CS) for MSCs

[0089] The chondrogenic effects of avocado anthraquinone C (Avn C) and chondroitin sulfate (CS) on mesenchymal stem cells (MSCs) are demonstrated by their ability to upregulate key proteins involved in chondrocyte differentiation and maturation. In particular, transcription factor SOX9 and protein LRP1 play crucial roles in promoting chondrogenic differentiation and regulating chondrocyte maturation.

[0090] LRP1 (low-density lipoprotein receptor-associated protein 1) plays a crucial role in chondrocyte maturation, during which chondrocytes acquire their mature phenotype and function. LRP1 regulates important signaling pathways and extracellular matrix (ECM) components involved in cartilage formation.

[0091] Nuclear localization of SOX9 (a key transcription factor) plays a crucial role in chondrogenesis, the process by which chondrocytes develop and differentiate into chondrocytes. Proper nuclear localization of SOX9 is essential for its transcriptional activity and the activation of chondrogenesis gene expression. During chondrogenesis, SOX9 is transported to the nucleus, where it binds to specific DNA sequences called enhancer elements within the regulatory regions of chondrogenesis genes. This binding activates the transcription of these genes, leading to the synthesis of proteins essential for chondrocyte function and chondrocyte tissue formation. Nuclear localization of SOX9 is tightly regulated and influenced by a variety of factors, including signaling pathways and co-regulatory factors. These factors regulate the activity and stability of SOX9, ensuring its proper function in chondrogenesis. Nuclear localization of SOX9 is critical for the characteristics and function of chondrocytes. It ensures the precise regulation of gene expression necessary for chondrocyte proliferation, extracellular matrix synthesis, and the maintenance of mature chondrocyte characteristics.

[0092] Figures 4A-4E A perspective view 400 illustrates the synergistic effect of Avn C and chondroitin sulfate (CS) on SOX9 protein expression according to one embodiment of this document. Figure 4A shows immunofluorescence staining of SOX9 in MSCs on day 0 of MSC differentiation into chondrocytes. In Figure 4B, MSCs were cultured in CDM1 (STD) differentiation medium for 21 days. In Figure 4C, MSCs were cultured in CDM2 (STD + Avn C) differentiation medium for 21 days. In Figure 4D, MSCs were cultured in CDM3 (STD + 5X CS) differentiation medium for 21 days. In Figure 4E, MSCs were cultured in CDM4 (STD + Avn C + 5X CS) differentiation medium for 21 days. Figures 4A-4E This study demonstrates the synergistic enhancement of chondrogenic effects in mesenchymal stem cells (MSCs) achieved through the combined use of avocado anthraquinone C (Avn C) and chondroitin sulfate (CS). These figures show increased nuclear localization of the transcription factor SOX9 in Figure 4 and upregulation of the protein LRP1 in Figure 5, both of which play crucial roles in chondrocyte differentiation and maturation. In Figure 4, MSCs treated with the combined Avn C and 5X CS (CDM4) exhibited enhanced SOX9 immunofluorescence signal compared to standard differentiation medium (CDM1) or CS alone (CDM3). Notably, the immunofluorescence signals in Figures 4C and 4E indicate the presence of functionally active SOX9 with nuclear localization, suggesting its key role in chondrogenesis when Avn C is present.

[0093] Furthermore, LRP1, a protein known to play a crucial role in coordinating chondrocyte function and maintaining cartilage tissue homeostasis, is significantly affected by combined Avn C and CS treatment. LRP1 interacts with growth factors such as TGF-β, IGF-1, and PDGF, which are essential for chondrocyte proliferation, differentiation, and ECM synthesis. By modulating the activity of these growth factors, LRP1 influences the synthesis and assembly of ECM components, including collagen and proteoglycans, and contributes to cartilage homeostasis. In addition to ECM regulation, LRP1 interacts with intracellular signaling molecules and transcription factors, including SOX9, RUNX2, and components of the Wnt / β-catenin pathway. These interactions fine-tune gene expression programs involved in chondrocyte function and ECM synthesis.

[0094] The immunofluorescence analysis depicted in Figure 5 provides valuable insights into LRP1 expression during MSC differentiation into chondrocytes. Figure 5A shows that no positive LRP1 signal is present in MSCs at the initial stage of differentiation. However, a strong LRP1 immunofluorescence signal was detected in MSCs cultured in CDM1 (STD) (Figure 5B). Notably, the strongest LRP1 immunofluorescence signal was observed in MSCs cultured in CDM4 (STD + AvnC + 5X CS), exceeding the signal observed in CDM3 (STD + 5XCS) or CDM2 (STD + AvnC) (Figures 5C and 5D). Figure 5F represents the quantitative signal of LRP1 under the stated conditions and highlights the statistical significance of the combined treatment of AvnC and CS on LRP1 protein expression. These findings clearly demonstrate the synergistic effect of AvnC and 5X CS in enhancing LRP1 protein expression, which further promotes chondrocyte differentiation and has a positive impact on its maturation.

[0095] In summary, LRP1 coordinates chondrocyte function and maintains cartilage tissue homeostasis. Its role in regulating ECM turnover, growth factor signaling, and transcriptional regulation of chondrogenesis genes highlights its importance in chondrocyte maturation. Enhanced LRP1 expression through the synergistic effect of avocadothramide C (Avn C) and chondroitin sulfate (CS), as demonstrated in our study, further supports the crucial role of LRP1 in promoting chondrocyte differentiation and maturation.

[0096] In addition to increased expression of specific proteins, the presence of specific chondrocyte proteoglycans detected by alecnin blue staining confirmed that MSCs successfully differentiated into chondrocyte-like cells under the influence of Avn C and CS. Alecnin blue staining is a method for detecting chondrocyte proteoglycans. Figure 6Significant findings were revealed. Positive staining observed after treatment with Avn C and CS indicated successful synthesis and deposition of chondrocyte-specific proteoglycans, enhancing the chondrogenic potential of the combined treatment. Notably, MSCs cultured in CDM4 (STD + Avn C + 5 X CS) exhibited the strongest blue staining, surpassing those in CDM3 (STD + 5 X CS) or CDM2 (STD + Avn C). Figure 6 These results strongly suggest that the synergistic effect achieved through the combined application of Avn C and 500 µg / mL chondroitin sulfate (5X CS) leads to enhanced proteoglycan production. This finding highlights the crucial role of proteoglycans in promoting the compressive stiffness of the chondrocyte extracellular matrix (ECM).

[0097] In summary, administration of Avn C and CS enhanced the chondrogenic effect of MSCs, as evidenced by increased nuclear localization of the transcription factor SOX9 and upregulation of the protein LRP1. Upregulation of SOX9 indicates the induction of chondrogenic differentiation, while increased LRP1 expression highlights the promotion of chondrocyte maturation. These findings underscore the potential of Avn C and CS as co-factors driving chondrogenic differentiation in MSCs, making them highly promising candidates for regenerative medicine and tissue engineering strategies focused on cartilage repair and regeneration.

[0098] Figures 5A-5F A perspective view 500 illustrates the synergistic effect of Avn C and chondroitin sulfate (CS) on LRP1 protein expression according to the embodiments. Figure 5A shows immunofluorescence staining of LRP1 in MSCs at day 0 of MSC differentiation into chondrocytes. In Figure 5B, MSCs were cultured in CDM1 (STD) differentiation medium for 21 days. In Figure 5C, MSCs were cultured in CDM2 (STD + Avn C) differentiation medium for 21 days. In Figure 5D, MSCs were cultured in CDM3 (STD + 5X CS) differentiation medium for 21 days. In Figure 5E, MSCs were cultured in CDM4 (STD + Avn C + 5X CS) differentiation medium for 21 days. The brightest and more uniformly distributed LRP1 immunofluorescence signal was observed in MSCs cultured in CDM4, indicating a synergistic effect of Avn C and 500 µg / mL chondroitin sulfate (5X CS) in enhancing LRP1 protein expression. Figure 5F shows the quantification of intracellular LRP1 protein levels as assessed by measuring immunostaining intensity and corresponding area using immunofluorescence microscopy.

[0099] Figure 6A perspective view 600 shows the synergistic effect of Avn C and chondroitin sulfate (CS) on protein expression of agglutinin, as revealed by alixin blue staining according to one embodiment of this article. Figure 6 A shows the Alixin Blue staining of MSCs on day 0 of MSC differentiation into chondrocytes. Figure 6 In B, MSCs were cultured in CDM1 (STD) differentiation medium for 21 days. Figure 6 In C, MSCs were cultured for 21 days in CDM2 (STD + Avn C) differentiation medium. Figure 6 In D, MSCs were cultured for 21 days in CDM3 (STD+ 5X CS) differentiation medium. Figure 6 In E, MSCs were cultured for 21 days in CDM4 (STD + Avn C + 5X CS) differentiation medium. The bluest staining was observed in MSCs cultured in CDM4, indicating enhanced proteoglycan production due to the synergistic effect of Avn C and 500 µg / mL chondroitin sulfate (5X CS). Upregulation of mRNA expression for chondrocyte characteristics by Avn C and chondroitin sulfate: mRNA analysis by RT-PCR

[0100] Investigating the effects of Avn C on transcriptionally regulated chondrocyte properties is crucial, whether used alone or in combination with the optimal dose of chondroitin sulfate (5X CS). To assess this, RT-PCR mRNA expression analysis was employed, providing precise quantitative data on relative mRNA expression. Aggregate proteoglycans (ACANs) are important proteoglycans in the chondrocyte extracellular matrix (ECM) and are essential for cartilage repair, serving as key chondrocyte markers. Aggregate proteoglycans play a crucial role in the function and integrity of chondrocytes. As a major proteoglycan component of the ECM, it provides essential structural support and contributes to the unique biomechanical properties of cartilage. Aggregate proteoglycan molecules consist of a core protein and numerous glycosaminoglycan (GAG) chains. The interactions between aggregate proteoglycans and other ECM components, such as collagen fibers, form a complex network that maintains the structural integrity of cartilage tissue. Aggregate proteoglycans act as "water sponges," enabling cartilage to resist compressive forces and maintain its shock-absorbing capacity. This unique property is crucial for cartilage's ability to withstand mechanical loads and provide cushioning between joints. Imbalances in the metabolism and degradation of proteoglycans are associated with cartilage degeneration and the development of osteoarthritis.

[0101] It is worth noting that the intensity of the GAPDH (internal control) band remained consistent across the CDM1-4 samples. Figure 7These findings demonstrate that the differences observed among samples (CDM1–4) in the expression of agglutinin, SOX9, LRP1, and COL2A1 are real. Specifically, CDM2 (STD + Avn C) showed a significantly stronger ACAN band compared to CDM1 (STD), providing strong evidence that Avn C alone effectively enhances ACAN gene expression.

[0102] Furthermore, regarding ACAN expression, CDM4 (STD + Avn C + 5X CS) showed a more prominent band than CDM3 (5X CS). This finding highlights the synergistic effect of 5X CS and Avn C in promoting ACAN gene expression. Notably, CDM4 (STD + Avn C + 5X CS) showed the strongest bands for both SOX9 and LRP1. These compelling results further confirm the synergistic effect of the optimal dose of chondroitin sulfate (5X CS) and Avn C in enhancing SOX9 and LRP1 gene expression. We observed that Avn C treatment alone affected COL2A1 at the protein level (Figures 2 and 3) rather than the transcriptional level. Avn C may exert an enhancing effect on the post-translational protein, rather than directly regulating the transcription of COL2A1.

[0103] In summary, mRNA expression analysis by RT-PCR reinforces the view that when Avn C is used in combination with 500 µg / mL chondroitin sulfate (5X CS), it significantly drives MSCs to exhibit chondrocyte-specific properties with enhanced efficacy. The quantitative nature of this analysis provides valuable insights into the role of Avn C in promoting chondrocyte-specific characteristics.

[0104] Figure 7 A perspective view 700 illustrates an RT-PCR analysis of mRNA expression according to one embodiment of this paper: Avn C and chondroitin sulfate (CS) exhibit synergistic effects on the expression of agglutinin (ACAN), SOX9, and LRP1 genes, while maintaining COL2A1 gene expression. The expression levels of the target genes were assessed using RT-PCR in MSCs cultured under different culture conditions. The combination of Avn C and CS enhanced the expression of ACAN, SOX9, and LRP1 genes, indicating their synergistic effect on the expression of chondrocyte-related genes. Furthermore, COL2A1 gene expression was maintained, indicating the preservation of chondrocyte-specific characteristics. GAPDH was used as an internal control to ensure accurate normalization of mRNA expression levels between samples. The combined action of Avn C and chondroitin sulfate continuously enhanced chondrocyte differentiation in MSCs from multiple biological samples.

[0105] To further validate and enhance the effectiveness of our innovative approach, which involves a combination of Avn C and a high concentration of chondroitin sulfate (5X CS) at 500 µg / mL, we conducted extensive experiments using multiple biological samples. The aim was to investigate the reproducibility and consistency of the effects observed on adult mesenchymal stem cells (MSCs) when implementing chondrocyte differentiation protocols.

[0106] To assess the robustness of our method, we applied CDM4 (STD + Avn C + 5X CS) differentiation medium to MSCs obtained from different individuals. This experimental design allowed us to evaluate the performance of our method across a variety of biological samples, ensuring that our findings are not limited to a specific cell source. Figure 8 ).

[0107] Notably, our results consistently demonstrate enhanced chondrocyte differentiation in response to the combination of Avn C and chondroitin sulfate. The observed effects were reproducible and remained highly consistent across all tested biological samples. This validation provides strong evidence supporting the effectiveness and reliability of this method in driving MSCs toward a chondrocyte-like phenotype.

[0108] By using multiple biological samples from different ages and sexes, we were able to determine that positive results were independent of individual differences or experimental abnormalities. The consistent enhancement of chondrocyte differentiation across different cell sources further strengthens the potential translational applications of our method in regenerative medicine and cartilage tissue engineering.

[0109] In summary, our comprehensive validation of the concept involving the combination of Avn C with high concentrations of chondroitin sulfate, conducted on adult MSCs from diverse individuals, clearly demonstrates the reproducibility and reliability of our method. These findings lay a solid foundation for future research and the development of advanced strategies to effectively promote chondrocyte differentiation and chondrocyte regeneration.

[0110] Figure 8A perspective view 800 illustrates the analysis of mRNA expression of chondrocyte markers in multiple individuals' MSCs using RT-PCR according to one embodiment of this paper. The effects of AvnC alone (i.e., CDM2 compared to CDM1), and AvnC combined with 500 µg / mL (5X) chondroitin sulfate (CS) treatment (particularly CDM4 compared to CDM1), on the expression levels of aggregates, SOX9, and LRP1 genes relative to GAPDH in adult MSCs (MSC0, MSC1, MSC2, and MSC4) from different individuals are shown. The results indicate that the expression of aggregates, SOX9, and LRP1 genes significantly increased in response to Avn C and 5X CS treatment. Notably, these findings are consistent across multiple MSC lines, demonstrating the reproducibility and reliability of the observed effects. Table AD: MSC differentiation into chondrocytes: A. Top 15 most abundant proteins in MSC conditioned medium, B. CDM1 (STD) conditioned medium, and C. CDM4 (STD + Avn C + 5 X CS) conditioned medium. D. Comparison of noteworthy chondrocyte marker proteins among the top 15 most abundant proteins in CDM1, CDM4, and MSC conditioned media. Secretome analysis: Revealing the full picture of functional proteins in the conditioned medium for MSC differentiation into chondrocytes and validating the Avn C and chondroitin sulfate methods.

[0111] Table AD above provides a comprehensive analysis of the secreted protein profiles across different treatment groups, highlighting the importance of validating the function of chondrocytes in cartilage repair. A detailed breakdown list is provided, showcasing the top 15 most abundant proteins in the conditioned media of MSCs, CDM1 (STD), and CDM4 (STD + Avn C + 5X CS). Notably, the presence of chondrocyte marker proteins among those identified in CDM1 and CDM4 is crucial for confirming the native chondrocyte-like functional behavior of differentiated cells and their ability to contribute to cartilage repair. Secreted proteins play a vital role in validating the function of chondrocytes in cartilage repair. Chondrocytes are essential for cartilage development and maintenance, and the identification of chondrocyte marker proteins is crucial for monitoring and validating the differentiation of MSCs into a chondrocyte-like phenotype. By comparing the proteomic profiles, we gain insight into the unique characterization of the conditioned media of MSCs, CDM1, and CDM4. This comprehensive analysis allows us to identify proteins that are particularly enriched under various conditions, revealing key players involved in the differentiation process.

[0112] Furthermore, the validation of chondrocyte marker proteins in the treatment group provides valuable information on the effectiveness of the Avn C and chondroitin sulfate combination in cartilage repair. Examining secreted proteins allows us to assess the effects of Avn C and CS treatment, not only on intracellular gene and protein markers, but more importantly on secreted proteins, which indicate the ability of differentiated cells to successfully function, similar to native chondrocytes.

[0113] The presence and abundance of these markers in CDM1 and CDM4 conditioned media indicate successful induction of MSCs into a chondrocyte-like state. By delving into the full proteomic picture, we gained insights into the proteomic profile associated with MSC differentiation and validated the expression of chondrocyte marker proteins. This thorough analysis not only enhances our understanding of the underlying mechanisms driving MSC differentiation into chondrocytes but also reinforces the idea of ​​using Avn C in combination with high concentrations of chondroitin sulfate to achieve more efficient and effective differentiation results.

[0114] Tables A and C summarize the proteomic profiles of MSC, CDM1, and CDM4 conditioned media, highlighting the top 15 most abundant proteins. Notably, chondrocyte markers were selected from the top 15 proteins identified in CDM1 and CDM4 conditioned media. One of these markers is folliculorin-associated protein 1 (FSTL-1), which is found in both MSCs and cartilage, but showed significantly higher abundance in CDM4-treated cells. FSTL-1 is believed to be involved in regulating chondrocyte proliferation, MSC differentiation into chondrocytes, and the expression of extracellular matrix (ECM) proteins in chondrocytes. Table D shows that FSTL-1 protein was upregulated in CDM1 and CDM4-treated MSC conditioned media compared to MSC conditioned media alone, thus validating FSTL-1 as an effective regulator of MSC differentiation into chondrocytes. Furthermore, the higher abundance of FSTL-1 in CDM4 conditioned medium compared to CDM1 conditioned medium provides evidence that the combined treatment with Avn C and 500 µg / mL (5X CS) chondroitin sulfate is more effective in driving MSC differentiation into chondrocytes. Transforming growth factor-β (TGF-β) superfamily BGH3, which induces MSC differentiation into chondrocytes in vitro, and ECM proteins typically found in the chondrocyte secretome, such as chondrocyte oligomeric matrix protein (COMP), type I collagen α-1 (CO1A1), type I collagen α-2 (CO1A2), type III collagen α-1 (CO3A1), and COMP, serve as marker genes for chondrocytes. The higher protein abundance of these markers in the conditioned media of MSCs treated with CDM1 and CDM4 compared to MSC conditioned media alone confirms the successful differentiation of MSCs into chondrocytes.

[0115] The presence and abundance of chondrocyte marker proteins in the conditioned medium of CDM1 and CDM4-treated MSCs indicate successful induction of MSCs into a chondrocyte-like state. Validation of this chondrocyte function is crucial in the context of cartilage repair, as the secretion of specific proteins demonstrates the ability of differentiated cells to promote and maintain cartilage regeneration. By exploring the full spectrum of secreted proteins, we revealed the proteomic profile associated with MSC differentiation into chondrocytes and validated the presence of chondrocyte marker proteins. This comprehensive analysis not only enhances our understanding of the underlying mechanisms driving MSC differentiation into chondrocytes but also underscores the importance of utilizing the combination of Avn C with high concentrations of chondroitin sulfate to obtain more efficient and effective differentiation outcomes.

[0116] In summary, the analyses presented in Table AD provide a comprehensive overview of the secreted protein profiles across different treatment groups, confirming that differentiated cells can secrete proteins important for chondrocyte function in cartilage repair. The identification and validation of chondrocyte marker proteins underscore the importance of evaluating the full spectrum of functional proteins to assess the success of MSC differentiation into chondrocytes and ensure their potential to promote cartilage regeneration and repair.

[0117] The embodiments described herein provide compositions for inducing the differentiation of mesenchymal stem cells (MSCs) into chondrocytes, said compositions comprising avocadothramide C (Avn C). The foregoing description of specific embodiments will so fully reveal the general nature of the embodiments herein that others may readily modify and / or adjust various applications (such as the specific embodiments) by applying existing knowledge without departing from the overall conception, and therefore, such modifications and adjustments should and are intended to be understood within the meaning and scope of equivalents of the disclosed embodiments.

[0118] It should be understood that the wording or terminology used herein is for descriptive purposes and not for limitation. Therefore, while embodiments described herein have been presented with reference to preferred embodiments, those skilled in the art will recognize that the embodiments described herein can be practiced with modifications. However, all such modifications are considered to be within the scope of the claims.

Claims

1. A composition for inducing mesenchymal stem cells (MSCs) to differentiate into chondrocytes, said composition comprising avocado anthraquinone C (Avn C).

2. The composition according to claim 1, wherein, The oat anthraquinone C (Avn C) is present at concentrations ranging from 10 nM to 50,000 nM.

3. The composition according to claim 2, wherein, The oat anthramide C (Avn C) is preferably present at a concentration of 50-300 nM.

4. The composition according to claim 1, further comprising chondroitin sulfate (CS) as a bioactive factor.

5. The composition according to claim 3, further comprising chondroitin sulfate (CS) as a bioactive factor.

6. The composition according to claim 5, wherein, The chondroitin sulfate (CS) is present at a concentration in the range of 300-700 µg / mL.

7. The composition according to claim 1, wherein, The mesenchymal stem cells mentioned are adipose-derived mesenchymal stem cells (aMSCs).

8. The composition according to any one of claims 1-7, wherein, The chondrocytes are two-dimensional chondrocyte-like cells.

9. The composition according to any one of claims 1-7, for increasing chondrocyte markers in the chondrocytes, wherein the chondrocyte markers are selected from the group consisting of FSTL-1, LRP1, ACAN, COL2A, SOX9, and any combination thereof.

10. Use of a culture medium for inducing in vitro differentiation of mesenchymal stem cells (MSCs), said culture medium comprising the composition according to any one of claims 1-7.

11. A method for inducing differentiation of in vitro cultured mesenchymal stem cells (MSCs) into chondrocytes, the method comprising culturing the MSCs in a medium containing avothramide C (Avn C).

12. The method according to claim 11, wherein, The oat anthraquinone C (Avn C) is present at concentrations ranging from 10 nM to 50,000 nM.

13. The method according to claim 12, wherein, The oat anthraquinone C (Avn C) is preferably present at a concentration of 50 nM to 300 nM.

14. The method according to claim 11, wherein, Chondroitin sulfate (CS) exists in culture media as a bioactive factor.

15. The method of claim 13, further comprising chondroitin sulfate (CS) present in the culture medium as a bioactive factor.

16. The method according to claim 15, wherein, The chondroitin sulfate (CS) was present in the culture medium at a concentration in the range of 300-700 µg / mL.

17. The method according to claim 11, wherein, The mesenchymal stem cells mentioned are adipose-derived mesenchymal stem cells (aMSCs).

18. The method according to any one of claims 11-17, wherein, The chondrocytes are two-dimensional chondrocyte-like cells.

19. The method according to any one of claims 11-17, wherein, The chondrocyte markers of the chondrocytes are increased, and the chondrocyte markers are selected from the group consisting of FSTL-1, LRP1, ACAN, COL2A, SOX9 and any combination thereof.

20. A pharmaceutical composition for chondrocyte repair and regeneration, said pharmaceutical composition comprising chondrocytes obtained by the method according to any one of claims 11-17.

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