A medium and method for preparing hypertrophic cartilage tissue

Abstract

The present application relates to the technical field of cell and tissue engineering, and particularly relates to a culture medium and method for preparing hypertrophic cartilage tissue. The hypertrophic cartilage induction culture medium can effectively improve the induction and differentiation efficiency of hypertrophic cartilage cells or tissue, and the prepared hypertrophic cartilage tissue has high biocompatibility and activity and performs excellently in promoting bone defect repair. The culture medium group for inducing iPSC differentiation to prepare hypertrophic cartilage cell membrane pieces can accurately and efficiently induce iPSC to differentiate into hypertrophic cartilage cells, and can further form hypertrophic cartilage cell membrane pieces; the hypertrophic cartilage cell membrane pieces can quickly induce the regeneration of osteogenic centers, and provide a solution for treating large-area bone defects, and have good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell and tissue engineering, and in particular to a culture medium and method for preparing hypertrophic cartilage tissue. BACKGROUND

[0002] Although the skeleton has the ability of self-repair and remodeling, the treatment of large area bone defects is still an important clinical challenge. In current clinical practice, autologous and allogeneic bone transplantation is often used to treat large area bone defects. However, the wide application of these methods is restricted by multiple limiting factors, such as the need for secondary surgery, donor shortage, size mismatch and risk of immune rejection. Developmental tissue engineering based on stem cells provides a new solution for traditional orthopedic treatment technology.

[0003] Induced pluripotent stem cells (iPS cells, iPSC) are cells that are reprogrammed to a state similar to embryonic stem cell pluripotency by introducing a combination of transcription factors (such as OCT4, KLF4, SOX2 and MYC) into somatic cells (such as skin or blood cells) through gene reprogramming technology. The resulting iPSC has the ability to self-renew and differentiate into almost any tissue cell type, making it a powerful tool in regenerative medicine. This breakthrough technology has great potential in personalized treatment, disease modeling and drug development.

[0004] In the field of orthopedics, endochondral ossification is an important osteogenesis way for human bone development and bone repair; for example, after long bone (such as femur, tibia, humerus, etc.) fracture, especially when there is a certain gap or instability between the fracture ends, a cartilaginous callus will be formed during the repair process, and then the cartilage is replaced by bone tissue to complete bone healing. During endochondral ossification, chondrocytes will change from proliferative to hypertrophic chondrocytes with significantly increased volume. The hypertrophic cartilage zone is a prerequisite for the formation of ossification centers. Hypertrophic cartilage secretes specific signaling molecules to provide conditions for the invasion of blood vessels and osteoblasts, and finally the cartilage is replaced by bone tissue to complete bone formation or repair. As can be seen, hypertrophic cartilage tissue plays a very important role in the process of bone repair. However, there are still few reports on the preparation of hypertrophic cartilage cells or tissue, and the differentiation and preparation efficiency of hypertrophic cartilage cells and tissue still needs to be improved. SUMMARY

[0005] The present application provides a culture medium and method for preparing hypertrophic cartilage tissue.

[0006] The present application is found in the preparation of hypertrophic chondrocytes that Aloin-A and platelet lysate can significantly promote chondrocyte hypertrophy. Adding Aloin-A and platelet lysate during the induction and differentiation process can significantly improve the efficiency of inducing and differentiating chondrocyte precursors into hypertrophic chondrocytes or tissues, and based on this, an efficient induction and differentiation scheme of hypertrophic chondrocytes or tissues is developed. Aloin-A is a natural anti-tumor anthraquinone glycoside that plays an anti-tumor role by inhibiting the proliferation of tumor cells and inducing apoptosis, and also has neuroprotective, anti-inflammatory, osteogenic differentiation promoting, and antioxidant activities. Platelet lysate is an acellular derivative containing growth factors and cytokines extracted from human platelets, which is used as a nutritional supplement for cell culture. There is no report that the above two substances alone or in combination can promote chondrocyte hypertrophy.

[0007] Specifically, the present application provides the technical solutions described below.

[0008] In the first aspect, the present application provides a composition for promoting chondrocyte hypertrophy, which comprises Aloin-A and platelet lysate.

[0009] Preferably, in the composition, the ratio of Aloin-A to platelet lysate is 1-10 μM: 0.5%-5% v / v.

[0010] Preferably, in the composition, the working concentration of Aloin-A is 1-10 μM, and the working concentration of platelet lysate is 0.5%-5% v / v.

[0011] In the present application, the working concentration refers to the final concentration of the substance in the system when it is actually applied. Preferably, it is the final concentration in the culture medium.

[0012] In the above composition, Aloin-A and platelet lysate can exist in the form of a mixture, or can be packaged separately and mixed or added separately when used.

[0013] The above composition can consist only of Aloin-A and platelet lysate, or can further comprise other substances that promote chondrocyte hypertrophy.

[0014] In the present application, the platelet lysate is preferably human platelet lysate.

[0015] In the second aspect, the present application provides any of the following applications of Aloin-A and platelet lysate:

[0016] (1) promoting the hypertrophy of in vitro cultured chondrocytes;

[0017] (2) preparing a product for promoting chondrocyte hypertrophy;

[0018] (3) preparing hypertrophic chondrocytes or hypertrophic cartilage tissue.

[0019] Preferably, in the application, the working concentration of Aloin-A is 1-10 μM, and the working concentration of platelet lysate is 0.5%-5% v / v.

[0020] In the present application, the hypertrophic cartilage tissue includes a hypertrophic chondrocyte membrane.

[0021] In a third aspect, the present application provides a hypertrophic chondrocyte induction medium, which includes a basal medium and an additive component; wherein the additive component includes TGF-β3, dexamethasone, ascorbic acid-2-phosphate, L-proline, insulin, transferrin and sodium selenite, and further includes Aloin-A and platelet lysate; and in the medium, the concentration of Aloin-A is 1-10 μM, and the concentration of platelet lysate is 0.5%-5% v / v, based on the volume of the basal medium.

[0022] Preferably, in the hypertrophic chondrocyte induction medium, the concentrations of the respective components are as follows: TGF-β3 5-15 ng / mL, dexamethasone 5-15 nM, ascorbic acid-2-phosphate 50-150 μM, L-proline 0.2-1 mM, insulin 5-15 μg / mL, transferrin 3-10 μg / mL, sodium selenite 2-10 ng / mL, Aloin-A 1-10 μM, and platelet lysate 0.5%-5% v / v, based on the volume of the basal medium.

[0023] In the above induction medium, Aloin-A and platelet lysate can significantly promote the hypertrophy of chondrocytes, and in cooperation with other additive components, can significantly improve the efficiency of inducing mesenchymal-like chondrocyte precursor cells (iCPC) to differentiate into hypertrophic chondrocytes or tissue, and the prepared hypertrophic chondrocytes or tissue has a high survival rate, biocompatibility and biological activity, wherein the expression levels of hypertrophic chondrocyte-related genes, chondral ossification-related genes and osteogenesis-related genes are significantly improved, and the prepared hypertrophic chondrocytes or tissue performs significantly better in promoting the mineralization process of the ossification center and repairing bone defects.

[0024] In some embodiments of the present application, the concentrations of each of the added components in the hypertrophic cartilage induction medium are as follows, based on the volume of the basal medium: TGF-β3 7-12 ng / mL, dexamethasone 7-12 nM, ascorbic acid-2-phosphate 90-110 μΜ, L-proline 0.2-0.5 mM, insulin 7-12 μg / mL, transferrin 4-8 μg / mL, sodium selenite 4-8 ng / mL, Aloin-A 1-10 μΜ, and platelet lysate 0.5-5% v / v.

[0025] In some embodiments of the present application, the concentrations of each of the added components in the hypertrophic cartilage induction medium are as follows, based on the total volume of the basal medium: TGF-β3 9-11 ng / mL, dexamethasone 9-11 nM, ascorbic acid-2-phosphate 95-105 μΜ, L-proline 0.3-0.4 mM, insulin 9-11 μg / mL, transferrin 5-6 μg / mL, sodium selenite 4-6 ng / mL, Aloin-A 3-7 μΜ, and platelet lysate 0.5-2% v / v.

[0026] The basal medium described above comprises one or more selected from the group consisting of a-MEM, DMEM, IMEM, and DMEM / F12 medium.

[0027] Illustratively, the basal medium is a-MEM medium.

[0028] In the medium described above, the platelet lysate is preferably human platelet lysate (HPL).

[0029] In a fourth aspect, the present application provides a medium set for inducing iPSCs to differentiate into hypertrophic cartilage tissue, which comprises a medium for inducing iPSCs to differentiate into mesenchymal-like chondrocyte precursor cells and the hypertrophic cartilage induction medium described above in the third aspect.

[0030] Preferably, the medium for inducing iPSCs to differentiate into mesenchymal-like chondrocyte precursor cells comprises:

[0031] The first medium comprises a basal medium and the following components: B27 supplement, ITS-A, NEAA, β-mercaptoethanol, WNT3A, Activin-A, and FGF2;

[0032] The second medium comprises a basal medium and the following components: B27 supplement, ITS-A, NEAA, β-mercaptoethanol, WNT3A, Activin-A, FGF2, SB431542, and Noggin;

[0033] The third medium comprises a basal medium and the following components: B27 supplement, ITS-A, NEAA, β-mercaptoethanol, WNT3A, Activin-A, FGF2, SB431542, BMP4, Wnt-C59 and Noggin.

[0034] The fourth medium comprises a basal medium and the following components: B27 supplement, ITS-A, NEAA, β-mercaptoethanol, FGF2, BMP4, Follistatin, SHH and WNT-C59.

[0035] The first medium, the second medium, the third medium and the fourth medium described above are respectively used for four culture stages of inducing iPSCs to differentiate into mesenchymal-like chondrocyte precursor cells, wherein the differentiation factors used in the same medium and between the media can well cooperate to make the culture stages better connect and transition, significantly improve the differentiation efficiency of iPSCs into mesenchymal-like chondrocyte precursor cells, and efficiently produce mesenchymal-like chondrocyte precursor cells, and the obtained mesenchymal-like chondrocyte precursor cells exhibit better differentiation capacity into chondrocytes and osteoblasts.

[0036] Preferably, the first medium comprises a basal medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, WNT3A 20-30 ng / mL, Activin-A 40-60 ng / mL and FGF2 10-30 ng / mL.

[0037] The second medium comprises a basal medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, WNT3A 20-30 ng / mL, Activin-A 20-30 ng / mL, FGF2 10-30 ng / mL, SB431542 1-10 μM and Noggin 10-30 ng / mL.

[0038] the third medium comprises a basal medium and the following components: B27 supplement 0.5-10% v / v, ITS-A 0.5-5% v / v, NEAA 0.5-5% v / v, beta-mercaptoethanol 30-300 mM, WNT3A 20-30 ng / mL, Activin-A 5-15 ng / mL, FGF2 10-30 ng / mL, SB431542 1-10 mM, BMP4 30-50 ng / mL, Wnt-C59 0.5-3 mM, and Noggin 10-30 ng / mL;

[0039] the fourth medium comprises a basal medium and the following components: B27 supplement 0.5-10% v / v, ITS-A 0.5-5% v / v, NEAA 0.5-5% v / v, beta-mercaptoethanol 30-300 mM, FGF2 10-30 ng / mL, BMP4 30-50 ng / mL, Follistatin 80-120 ng / mL, SHH 10-30 ng / mL, and WNT-C59 0.5-3 mM.

[0040] In some embodiments of the present application, the first medium comprises a basal medium and the following components: B27 supplement 1-3% v / v, ITS-A 0.5-2% v / v, NEAA 0.5-2% v / v, β-mercaptoethanol 80-100 μM, WNT3A 23-27 ng / mL, Activin-A 48-52 ng / mL and FGF2 18-22 ng / mL; the second medium comprises a basal medium and the following components: B27 supplement 1-3% v / v, ITS-A 0.5-2% v / v, NEAA 0.5-2% v / v, β-mercaptoethanol 80-100 μM, WNT3A 23-27 ng / mL, Activin-A 23-27 ng / mL, FGF2 18-22 ng / mL, SB431542 3-7 μM and Noggin 18-22 ng / mL; the third medium comprises a basal medium and the following components: B27 supplement 1-3% v / v, ITS-A 0.5-2% v / v, NEAA 0.5-2% v / v, β-mercaptoethanol 80-100 μM, WNT3A 23-27 ng / mL, Activin-A 8-12 ng / mL, FGF2 18-22 ng / mL, SB431542 3-7 μM, BMP4 38-42 ng / mL, Wnt-C59 0.5-1.5 μM and Noggin 18-22 ng / mL; the fourth medium comprises a basal medium and the following components: B27 supplement 1-3% v / v, ITS-A 0.5-2% v / v, NEAA 0.5-2% v / v, β-mercaptoethanol 80-100 μM, FGF2 18-22 ng / mL, BMP4 38-42 ng / mL, Follistatin 95-105 ng / mL, SHH 18-22 ng / mL and WNT-C59 0.5-1.5 μM.

[0041] The basal medium described above comprises one or more selected from the group consisting of α-MEM, DMEM, IMEM and DMEM / F12 medium.

[0042] Exemplarily, the basal medium used in the medium for inducing iPSCs to differentiate into mesenchymal-like chondrocyte precursor cells is DMEM / F12 medium.

[0043] In a fifth aspect, the present application provides use of the hypertrophic cartilage induction medium or the medium set described above in preparation of hypertrophic chondrocytes or hypertrophic cartilage tissue.

[0044] In a sixth aspect, the present application provides a method for preparing hypertrophic chondrocytes or hypertrophic cartilage tissue, the method comprising: culturing mesenchymal-like chondrocyte precursor cells in the hypertrophic chondrocyte induction medium.

[0045] Preferably, the culturing time is 30-60 days. During the culturing, the medium is replaced with fresh hypertrophic chondrocyte induction medium every 1-3 days.

[0046] Preferably, the mesenchymal-like chondrocyte precursor cells are induced and differentiated from iPSCs; and the medium used for the induction and differentiation is the medium in the medium set described above for inducing and differentiating iPSCs into mesenchymal-like chondrocyte precursor cells.

[0047] Preferably, the induction and differentiation of iPSCs into mesenchymal-like chondrocyte precursor cells comprises:

[0048] In the first stage, the iPSCs are cultured in the first medium;

[0049] In the second stage, the iPSCs are cultured in the second medium;

[0050] In the third stage, the iPSCs are cultured in the third medium;

[0051] In the fourth stage, the iPSCs are cultured in the fourth medium.

[0052] Preferably, the culturing time in each stage is 1-3 days.

[0053] In the present application, the iPSCs are preferably human iPSCs. The hypertrophic chondrocytes or hypertrophic cartilage tissue are preferably human.

[0054] In a seventh aspect, the present application provides a hypertrophic cartilage tissue prepared by the method of the sixth aspect described above. The hypertrophic cartilage tissue is preferably a hypertrophic chondrocyte membrane.

[0055] In an eighth aspect, the present application provides a method for repairing or treating bone defects, the method comprising: transplanting the hypertrophic cartilage tissue prepared by the method of the sixth aspect described above or the hypertrophic cartilage tissue of the seventh aspect described above to a bone defect site.

[0056] The beneficial effects of the present application at least include: the present application finds that Aloin-A and platelet lysate can cooperate to promote chondrocyte hypertrophy, improve the induced differentiation efficiency of hypertrophic chondrocytes, the present application develops a hypertrophic cartilage induction medium, which can effectively promote mesenchymal-like chondrocyte precursor cells to differentiate into hypertrophic chondrocytes or tissues, and the prepared hypertrophic chondrocytes or tissues have high survival rate, biocompatibility and activity, and excellent performance in promoting bone defect repair. The medium group for preparing the hypertrophic cartilage cell membrane sheet provided by the present application can accurately and efficiently induce iPSC to differentiate into hypertrophic chondrocytes, and can further form a hypertrophic cartilage tissue membrane sheet; the hypertrophic cartilage tissue membrane sheet can quickly induce the regeneration of the ossification center, providing a new and effective solution for treating large-area bone defects, which has important significance for solving the clinical demand problem of large-area bone defect repair, and has good application prospect in bone defect repair. BRIEF DESCRIPTION OF DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0058] Figure 1 The iPSC differentiation-derived chondrocyte precursor cells and property identification in the experimental examples of the present application; wherein A is the morphology of chondrocyte precursor cells, bar = 100 μM; B is the genotype detection result of chondrocyte precursor cells (P5 and P8 generations).

[0059] Figure 2 The iPSC differentiation-derived hypertrophic cartilage cell membrane sheet and property identification in the experimental examples of the present application; wherein A is a representative picture of a large-size iPSC differentiation-derived iCDC-HA hypertrophic cartilage cell membrane sheet (diameter more than 20 cm); B is the general morphology of the hypertrophic cartilage cell membrane sheet, bar = 1 cm; C is the morphology of the hypertrophic cartilage cell, bar = 100 μM; D is the live / dead cell staining fluorescence image of the hypertrophic cartilage cell membrane sheet, green represents live cells, red represents dead cells, bar = 400 μM.

[0060] Figure 3 The gene expression level of the iPSC differentiation-derived hypertrophic cartilage cell membrane sheet in the experimental examples of the present application.

[0061] Figure 4Hypertrophic chondrocyte membrane sheet prepared by using Aloin-A and platelet lysate alone in the experimental example of the present application; wherein A is a digital image of the hypertrophic chondrocyte membrane sheet; B is a microscopic image of the hypertrophic chondrocyte membrane sheet, bar = 100 μM.

[0062] Figure 5 Representative digital images of hypertrophic chondrocyte membrane sheets prepared by using Aloin-A and platelet lysate in different concentrations in the experimental example of the present application.

[0063] Figure 6 Detection results of the experiment of promoting bone regeneration in a critical size femoral defect model by using the hypertrophic chondrocyte membrane sheet in the experimental example of the present application; wherein A is a digital image showing the surgical implantation of the hypertrophic chondrocyte membrane sheet in a critical size femoral metaphyseal defect of a Sprague-Dawley rat (marked by a yellow dotted line); B is a gross observation of the bone defect repair 4 weeks after the surgical implantation.

[0064] Figure 7 Imageological detection results 4 weeks after the transplantation of the hypertrophic chondrocyte membrane sheet in the experimental example of the present application; wherein A is a 2D μ-CT coronal view and axial view of the femoral defect area; B is a representative 3D reconstructed μ-CT image of the regenerated bone tissue around the femoral defect area.

[0065] Figure 8 Quantitative analysis results of the microstructure parameters of the regenerated bone tissue in the femoral defect area 4 weeks after the transplantation of the hypertrophic chondrocyte membrane sheet in the experimental example of the present application; wherein A is the bone volume / total volume ratio (BV / TV); B is the trabecular separation (Tb.Sp); C is the trabecular thickness (Tb.Th); D is the trabecular number (Tb.N).

[0066] Figure 9 Pathological analysis results of the femoral defect area 4 weeks after the transplantation of the hypertrophic chondrocyte membrane sheet in the experimental example of the present application; wherein A is a representative picture of HE staining and Goldner trichrome staining, bar = 2mM; B is the quantitative analysis results of the bone tissue area in the Goldner trichrome staining. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0068] In the following examples, the reagents used are as follows, unless otherwise specified: Aloin-A (MCE, HY-N0123), Human Platelet Lysate (UltraGRO-Advanced, Helios, HPCFDCRL50), B27 Supplement (Thermo Fisher), WNT3A (R&D), Activin-A (Peprotech), FGF2 (Invitrogen), SB431542 (Tocris), Noggin (R&D), BMP4 (Peprotech), Follistatin (Sigma), Wnt-C59 (Selleck), SHH (R&D), Vitronectin (Gibco), Mesenchymal Stem Cell Serum-Free Medium (Yocon, NC0103 and NC0103.S), mTESR1 (Stem Cell Technologies, 85850), Y27632 (Stem Cell Technologies, 72308), DMEM / F-12 Medium (Gibco), ITS-A (Gibco), NEAA (Gibco), β-mercaptoethanol (Gibco), α-MEM Medium (Gibco), Dexamethasone (Sigma, D4902), L-Ascorbic Acid (Sigma, A4403), TGF-β3 (EastMab), L-Proline (Sigma), Tissue Fixative (Solarbio, P1110), Hematoxylin-Eosin (HE, Solarbio, G1120), Goldner Trichrome Stain Kit (Solarbio, G3550), Trizol Reagent (Invitrogen), Reverse Transcription Kit PrimeScript RT (Takara), Fluorescent Dye Reagent TB Green® Premix Ex Taq™ II (Takara, RR820A).

[0069] Example 1

[0070] The present example provides a hypertrophic cartilage induction medium (i.e., iCDC-HA), which is obtained by supplementing the following components to an α-MEM medium: 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline, 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin, and 5 ng / mL sodium selenite), 5 μΜ Aloin-A, and 1% v / v human platelet lysate.

[0071] The concentration of each component is a final concentration based on the volume of the α-MEM medium.

[0072] Example 2

[0073] This example provides a hypertrophic cartilage induction medium, which is obtained by supplementing an a-MEM medium with 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline, 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin, and 5 ng / mL sodium selenite), 1 μΜ Aloin-A, and 0.5% v / v human platelet lysate.

[0074] The concentration of each of the above components is a final concentration based on the volume of the a-MEM medium.

[0075] Example 3

[0076] This example provides a hypertrophic cartilage induction medium, which is obtained by supplementing an a-MEM medium with 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline, 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin, and 5 ng / mL sodium selenite), 10 μΜ Aloin-A, and 5% v / v human platelet lysate.

[0077] The concentration of each of the above components is a final concentration based on the volume of the a-MEM medium.

[0078] Example 4

[0079] This example provides a medium set for inducing iPSC differentiation to produce mesenchymal-like cartilage precursor cells (iCPCs), which includes:

[0080] a first medium, which is a DMEM / F12 medium supplemented with 2% (v / v) B27 supplement, 1% (v / v) ITS-A, 1% (v / v) NEAA, 90 μΜ β-mercaptoethanol, 25 ng / mL WNT3A, 50 ng / mL Activin-A, and 20 ng / mL FGF2;

[0081] a second medium, which is a DMEM / F12 medium supplemented with 2% (v / v) B27 supplement, 1% (v / v) ITS-A, 1% (v / v) NEAA, 90 μΜ β-mercaptoethanol, 25 ng / mL WNT3A, 25 ng / mL Activin-A, 20 ng / mL FGF2, 5 μΜ SB431542, and 20 ng / mL Noggin;

[0082] Third medium, in DMEM / F12 medium added the following components: B27 supplement 2% (v / v), ITS-A 1% (v / v), NEAA 1% (v / v), β-mercaptoethanol 90 μΜ, WNT3A 25 ng / mL, Activin-A 10 ng / mL, FGF2 20 ng / mL, SB431542 5 μΜ, BMP4 40 ng / mL, Wnt-C59 1 μΜ and Noggin 20 ng / mL;

[0083] Fourth medium, in DMEM / F12 medium added the following components: B27 supplement 2% (v / v), ITS-A 1% (v / v), NEAA 1% (v / v), β-mercaptoethanol 90 μΜ, FGF2 20 ng / mL, BMP4 40 ng / mL, Follistatin 100 ng / mL, SHH 20 ng / mL and WNT-C59 1 μΜ.

[0084] Example 5

[0085] The present example provides a method for preparing hypertrophic chondrocyte sheet from human iPSCs, comprising the following steps:

[0086] (1) Differentiation of human iPSCs into chondrocyte precursor cells (iCPCs)

[0087] First, iPSCs were seeded into 10 μg / mL Vitronectin-coated cell culture plates at a cell concentration of 10,000 cells per square centimeter for 24 h, and the culture medium was mTESR1 and 10 μM Y27632. Subsequently, using the medium group for inducing the differentiation of iPSCs to prepare mesenchymal-like chondrocyte precursor cells (iCPCs) in Example 4, the medium for inducing the differentiation of chondrocyte precursor cells was gradually replaced according to the following steps: on days 1-2, the first medium was used for induction; on days 3-4, the second medium was used for induction; on days 5-6, the third medium was used for induction; on days 7-8, the fourth medium was used for induction, and at this time the chondrocyte precursor cells obtained can be subcultured for at least 10 passages using mesenchymal stem cell serum-free medium.

[0088] (2) Preparation of iPSC-derived hypertrophic chondrocyte sheet

[0089] The iCPC cells obtained in step (1) were seeded into 10 μg / mL Vitronectin-coated cell culture plates at a cell concentration of 3 x 10 6The cells were seeded at a density of 1 x 105cells / cm2in 10 cm tissue culture dishes and cultured for 6 weeks using the hypertrophic chondroinductive medium of Examples 1, 2 or 3, and observed and photographed under a microscope every 2-3 days with fresh medium being exchanged. The hypertrophic chondrocyte sheet prepared using the medium of Example 1, iCDC-HA, was designated iCDC-HA hypertrophic chondrocyte sheet.

[0090] Comparative Example 1

[0091] This comparative example provides a hypertrophic chondroinductive medium (i.e. iCDC) which is prepared by supplementing an a-MEM medium with 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline and 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium selenite).

[0092] The concentrations of the above components are the final concentrations based on the volume of the a-MEM medium.

[0093] Comparative Example 2

[0094] This comparative example provides a hypertrophic chondroinductive medium (i.e. iCDC-A) which is prepared by supplementing an a-MEM medium with 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline, 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium selenite) and 5 μΜ Aloin-A.

[0095] The concentrations of the above components are the final concentrations based on the volume of the a-MEM medium.

[0096] Comparative Example 3

[0097] This comparative example provides a hypertrophic chondroinductive medium (i.e. iCDC-H) which is prepared by supplementing an a-MEM medium with 10 ng / mL TGF-β3, 10 nM dexamethasone, 100 μΜ ascorbic acid-2-phosphate, 0.35 mM L-proline, 1x ITS supplement (10 μg / mL insulin, 5.5 μg / mL transferrin and 5 ng / mL sodium selenite) and 1% v / v human platelet lysate.

[0098] The concentrations of the above components are the final concentrations based on the volume of the a-MEM medium.

[0099] Comparative Example 4

[0100] The present comparative example provides a method for inducing human iPSC differentiation to prepare hypertrophic cartilage cell sheets, which is only different from the method of Example 5 in that the hypertrophic cartilage induction medium of Comparative Example 1, 2 or 3 is used in step (2). Among them, the hypertrophic cartilage cell sheet prepared by using the medium iCDC of Comparative Example 1 is named iCDC hypertrophic cartilage cell sheet.

[0101] Experimental Example

[0102] The hypertrophic cartilage cell sheets prepared by using the medium and method in the above examples and comparative examples were evaluated for performance, and the specific methods and results are described below.

[0103] 1. Experimental method

[0104] (1) In order to evaluate the cell viability in the hypertrophic cartilage cell sheet sample, the hypertrophic cartilage cell sheet was collected and stained with calcein-Am (green fluorescence) and propidium iodide (PI, red fluorescence), and images were taken using an Olympus IX81 microscope and a UC90 camera.

[0105] (2) Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR)

[0106] Total RNA was extracted from iPSC, iCPC, and hypertrophic cartilage tissue using Trizol reagent, and the RNA concentration was determined using NanoDrop. The mRNA was reverse transcribed into cDNA using the reverse transcription kit PrimeScript RT. The qRT-PCR reaction was performed using the fluorescent dye reagent TBGreen® Premix Ex Taq™ II, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference gene, and the relevant gene primer sequences are shown in Table 1. Finally, the relative expression of the target gene was calculated using the 2 -△△ct Method. All experiments were repeated 3 times.

[0107] Table 1. List of primers

[0108]

[0109] (3) Evaluation of hypertrophic cartilage cell sheets promoting bone regeneration in a critical-sized femoral defect model

[0110] The experimental animals used were 8-week-old male Sprague-Dawley rats (SPF) weighing approximately 200-300 g. All animal experiments were approved by the Research Ethics Committee of the Sixth People's Hospital of Shanghai Jiaotong University. Anesthesia was induced by intraperitoneal injection of 8% chloral hydrate (dose of 400 mg / kg body weight). Subsequently, the femur was carefully exposed by dissection surgery, and an artificial bone cavity was created on the cortical bone after the periosteum was stripped using a dental handle equipped with a trephine, with a transverse length and longitudinal length of 2.5 mM and 5.0 mM, respectively. After thoroughly and carefully cleaning the bone cavity, the administration group implanted the hypertrophic chondrocyte membrane in the defect site; the control group only modeled a critical bone defect without treatment. Finally, the periosteum and skin were sutured to close the incision. After 4 weeks of in vivo implantation of the hypertrophic chondrocyte membrane, the rats were euthanized. Immediately after sampling, the femur was subjected to μ-CT scanning, and the femur specimen was collected and stored in 4% tissue fixative for histological staining.

[0111] The collected femur samples were scanned by a μ-CT scanner (Skyscan, Kontich, Belgium). According to the established experimental protocol, the samples were evaluated at a resolution of 18 μM / voxel. The μ-CT images of the femur were reconstructed and analyzed in three dimensions, and the tissue morphological parameters such as the volume fraction of new bone tissue (BV / TV), trabecular spacing (Tb.Sp), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were calculated using the auxiliary software of the CT-An system.

[0112] After sampling, the femur tissue samples were dehydrated, embedded in hard plastic resin, and carefully sectioned to study the generation of new bone at the femur defect site. Subsequently, these sections were stained with hematoxylin-eosin (HE) staining solution and Goldner trichrome staining solution. Briefly, for HE staining, the sections were stained with hematoxylin and eosin for 10 min, respectively; for Goldner trichrome staining, the sections were incubated in Weigert hematoxylin solution and Acid ponceau staining solution for 30 minutes, and then sequentially incubated in Orange G staining solution for 5 minutes and in Bright green staining solution for 15 minutes. The proportion of bone tissue staining positive area in Goldner staining was quantitatively analyzed by Image J software.

[0113] 2、Experimental results

[0114] This invention simulates the endochondral ossification pathway of embryonic osteoblasts during in vivo development and develops a novel induction protocol (i.e., using the culture medium group for preparing mesenchymal-like chondrocyte progenitor cells (iCPCs) induced by iPSC differentiation in Example 4, and employing the method of step (1) in Example 5) to generate mesenchymal-like chondrocyte progenitor cells (iCPCs). The chondrocyte progenitor cells prepared using this induction protocol exhibit strong proliferative capacity and are mostly round or irregular in shape. Figure 1 qRT-PCR analysis showed that these chondrogenic progenitor cells expressed the COL2A1, SOX5, SOX6, and SOX9 genes (A). Figure 1 (B). These data indicate that the characteristics of chondrocyte precursor cells differ from those of traditional mesenchymal stem cells; these chondrocyte precursor cells exhibit a tendency to differentiate into chondrocytes and osteoblasts.

[0115] In the methods of Example 5 and Comparative Example 4, iPSC-derived mesenchymal-like chondrocyte precursor cells were cultured for 3 weeks using hypertrophic chondrocyte induction medium, which resulted in the formation of chondrocyte sheets. Subsequent 3 weeks of continuous culture yielded hypertrophic chondrocyte sheets. The results of using the culture media from Example 1 and Comparative Example 1 for hypertrophic chondrocyte induction were as follows: Figure 2 As shown in A and B, the iPSC-derived hypertrophic chondrocyte tissue constructs exhibit excellent scalability, enabling the fabrication of large-area hypertrophic chondrocyte membrane sheets with diameters exceeding 20 cm. Figure 2 A). iPSC-derived hypertrophic chondrocyte tissue constructs exhibit a translucent, glassy appearance and contain numerous well-defined cavities of hypertrophic chondrocytes (A). Figure 2 The cell viability of hypertrophic chondrocyte flaps was assessed using calcein-AM and propidium iodide (PI) staining. Live cells showed green fluorescence, while dead cells showed red fluorescence. The live / dead cell staining reagents showed that iPSC-derived hypertrophic chondrocytes had a high viability (green). Figure 2 The above results confirm that iPSC-derived hypertrophic chondrocyte membranes possess good biological activity. Using different hypertrophic chondrocyte induction media, iCDC and iCDC-HA, cell membrane tissues with different cell properties were obtained. qRT-PCR data showed that cell membranes prepared in both iCDC and iCDC-HA media exhibited high levels of cartilage-related genes (such as COL2A1 and ACAN); however, compared to cell membranes prepared using iCDC, cell membrane tissues prepared using iCDC-HA showed higher expression levels of hypertrophic chondrocyte-related genes (such as COL10A1), higher expression levels of genes regulating intrachondral ossification (such as ALPL, IHH, and MATN3), and higher expression levels of osteogenic genes (such as RUNX2 and COL1A2). Figure 3 ).

[0116] The effects of Aloin-A and human platelet lysate alone (Comparative Examples 2 and 3) and in combination (Example 1) on the differentiation of iCPC into mast chondrocytes were compared. The results showed that the mast chondrocyte membranes prepared by using 5 μM Aloin-A alone (i.e., using iCDC-A, iCPC cells cultured in the medium of Comparative Example 2) curled inward at the edges, making it difficult to obtain large cell membranes, and the shape was random and difficult to control; the mast chondrocyte membranes prepared by using 1% v / v human platelet lysate alone (i.e., using iCDC-H, iCPC cells cultured in the medium of Comparative Example 3) showed a certain degree of shrinkage and curling at the edges, with a softer texture and irregular shape; in comparison, the mast chondrocyte membranes prepared by using 5 μM Aloin-A + 1% v / v human platelet lysate in combination (i.e., using iCDC-HA, iCPC cells cultured in the medium of Example 1) had larger internal cell volume and exhibited a typical mast chondrocyte morphology; the membranes in the iCDC-HA group were milky white or translucent, with slight curling at the edges, a certain thickness, uniformity and tensile strength, a dense structure, and were rich in extracellular matrix components. Figure 4 (A and B). The results of this comparative experiment show that the combined use of Aloin-A and human platelet lysate significantly enhances the promoting effect of iCPC differentiation into mast chondrocyte membranes compared to the effect of using each component alone.

[0117] To investigate the effects of different concentrations of Aloin-A and human platelet lysate on the culture of mast chondrocyte membranes, we further tested the use of 1 μM Aloin-A + 0.5% v / v human platelet lysate (i.e., the culture medium in Example 2) and 10 μM Aloin-A + 5% v / v human platelet lysate (i.e., the culture medium in Example 3) to prepare mast chondrocyte membranes. The results showed that all three conditions could induce iCPC cells to differentiate into mast chondrocyte membranes with relatively controllable morphology and size. Figure 5 ).

[0118] As previously mentioned, the hypertrophic chondrocyte membranes prepared using the culture media of Examples 1-4 and the method of Example 5 all exhibited good hypertrophic chondrocyte characteristics in vitro. Further experiments were conducted using 8-week-old male Sprague-Dawley rats (SD rats) to create a femoral critical defect model. Hypertrophic chondrocyte membranes prepared with iCDC and iCDC-HA were implanted, respectively, and observations were performed (hereinafter referred to as the iCDC group and the iCDC-HA group). Figure 6A-B). The bone defect repair was observed by 3D reconstruction and μ-CT scanning Figure 7 A-B). In the control group, there was no obvious bone healing at 4 weeks after operation. Compared with the control group, the bone defect area of the iCDC and iCDC-HA prepared hypertrophic chondrocyte membrane sheet implantation was significantly reduced, showing a significant bone repair trend. Among them, the iCDC group cell membrane sheet implantation for 4 weeks, the bone defect area depth and defect area were reduced, and a small amount of fresh bone tissue regeneration was observed in the central region of the defect. Encouragingly, the iCDC-HA group of hypertrophic chondrocyte membrane sheet implantation for 4 weeks, the bone defect gap was significantly reduced, and active partial mineralized tissue and new bone formation appeared, the defect area was completely filled with mature mineralized bone tissue, and almost no modeling gap was observed Figure 6 B and Figure 7 A-B).

[0119] The bone defect repair was evaluated by quantitative analysis of basic histomorphometric parameters Figure 8 ). The results showed that the bone volume / total volume (BV / TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N) of the iCDC group and the iCDC-HA group were significantly increased, while the trabecular separation (Tb.Sp) was correspondingly reduced.

[0120] Histology provides a deeper perspective for in-depth understanding of the behavior of osteoblasts in the defect area Figure 9 ). A large number of new bone formation was observed in the defect femur of the iCDC-HA group of hypertrophic chondrocyte membrane sheet implantation for 4 weeks Figure 9 A). The semi-quantitative analysis of bone tissue area supports the beneficial effect of the iCDC-HA prepared hypertrophic chondrocyte membrane sheet on bone regeneration, and compared with the iCDC prepared hypertrophic chondrocyte membrane sheet implanted for 4 weeks, the highest proportion of new bone tissue was detected after the implantation of the iCDC-HA prepared hypertrophic chondrocyte membrane sheet Figure 9 B).

[0121] The above results show that the iCDC-HA prepared hypertrophic chondrocyte membrane sheet implantation can promote the repair of the critical defect of the rat femur. And compared with the iCDC prepared hypertrophic chondrocyte membrane sheet, the iCDC-HA prepared hypertrophic chondrocyte membrane sheet can significantly improve the effect of bone defect repair.

[0122] In the present application, the mechanism of action (MOA) of the iCDC-HA prepared hypertrophic chondrocyte membrane sheet for repairing bone defects may be that when exposed to the bone defect microenvironment stimulation in vivo, the (hypertrophic) chondrocytes on the iCDC-HA prepared hypertrophic chondrocyte membrane sheet can also be successfully transformed into bone cells to form new bone tissue, and ultimately achieve the effect of repairing bone defects.

[0123] It should be pointed out finally that the above embodiments are only used to illustrate the technical solutions of the present application, but not to limit the same; and although the present application has been described in detail with reference to the foregoing embodiments, it should be appreciated by those skilled in the art that the technical solutions recorded in the foregoing embodiments can be modified, or some technical features thereof can be replaced equivalently; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A culture medium for inducing hypertrophic chondrocytes, characterized in that, The culture medium consists of a basal medium and additives; wherein the additives are: TGF-β3, dexamethasone, ascorbate-2-phosphate, L-proline, insulin, transferrin and sodium selenite, as well as Aloin-A and human platelet lysate. In the hypertrophic chondrocyte induction culture medium, based on the volume of the basal culture medium, the concentrations of each added component are as follows: TGF-β3 5-15 ng / mL, dexamethasone 5-15 nM, ascorbic acid-2-phosphate 50-150 μM, L-proline 0.2-1 mM, insulin 5-15 μg / mL, transferrin 3-10 μg / mL, sodium selenite 2-10 ng / mL, Aloin-A 1-10 μM, and human platelet lysate 0.5%-5% v / v; The basal culture medium includes one or more selected from α-MEM, DMEM, IMEM, and DMEM / F12 culture medium.

2. A culture medium group for inducing iPSC differentiation to prepare hypertrophic cartilage tissue, characterized in that, The culture medium group includes a culture medium for inducing iPSC differentiation into mesenchymal chondrocyte precursor cells and the hypertrophic chondrocyte induction culture medium as described in claim 1. The culture medium for inducing iPSC differentiation into mesenchymal-like chondrocyte precursor cells includes: The first culture medium consists of basal medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, WNT3A 20-30 ng / mL and Activin-A 40-60 ng / mL, FGF2 10-30 ng / mL; The second culture medium consists of the basal culture medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, WNT3A 20-30 ng / mL, Activin-A 20-30 ng / mL, FGF2 10-30 ng / mL, and SB431542 1-10 μM, Noggin 10-30 ng / mL; The third culture medium consists of the basal medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, WNT3A 20-30 ng / mL, Activin-A 5-15 ng / mL, FGF2 10-30 ng / mL, SB431542 1-10 μM, BMP4 30-50 ng / mL, Wnt-C59 0.5-3 μM, and Noggin 10-30 ng / mL; The fourth culture medium consists of the basal medium and the following components: B27 supplement 0.5%-10% v / v, ITS-A 0.5%-5% v / v, NEAA 0.5%-5% v / v, β-mercaptoethanol 30-300 μM, FGF2 10-30 ng / mL, BMP4 30-50 ng / mL, Follistatin 80-120 ng / mL, SHH 10-30 ng / mL and WNT-C59 0.5-3 μM; The basal culture medium used in the culture medium for inducing iPSC differentiation into mesenchymal-like chondrocyte precursor cells includes one or more selected from α-MEM, DMEM, IMEM, and DMEM / F12 media.

3. The use of the hypertrophic chondrocyte induction culture medium of claim 1 or the culture medium group of claim 2 in the preparation of hypertrophic chondrocytes.

4. The use of the hypertrophic cartilage induction culture medium of claim 1 or the culture medium group of claim 2 in the preparation of hypertrophic cartilage tissue.

5. A method for preparing hypertrophic chondrocytes or hypertrophic cartilage tissue, characterized in that, The method includes culturing mesenchymal-like chondrocyte precursor cells using the hypertrophic chondrocyte induction medium described in claim 1; The mesenchymal-like chondrocyte precursor cells were obtained by iPSC-induced differentiation; The culture medium used for inducing differentiation is the culture medium for inducing iPSC differentiation into mesenchymal-like chondrocyte precursor cells in the culture medium group described in claim 2; Among them, the mesenchymal-like chondrocyte precursor cells induced by iPSC differentiation include: In the first stage, the culture is carried out using the first culture medium; In the second stage, the culture is carried out using the second culture medium; In the third stage, the culture is carried out using the third culture medium. In the fourth stage, the culture is carried out using the fourth culture medium; The cultivation time for each of the above stages is 1-3 days.

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