CN-114555783-B - Method for producing hyaline cartilage tissue in vitro

Abstract

The present invention relates to a novel method for producing cartilage tissue in vitro, and therapeutic uses and screening methods using the cartilage tissue produced thereby.

Inventors

• V. Dion

Assignees

• 瓦纳里克斯股份公司

Dates

Publication Date

20260505

Application Date

20200807

Priority Date

20190814

Claims (18)

1. 1. A method of producing hyaline cartilage tissue in vitro, the method comprising: i) Culturing chondrocytes on an adherent culture system in a FGF-2-containing dedifferentiating medium that activates the Wnt signaling pathway to obtain chondrocytes having a fibroblast-like cell morphology; ii) culturing the fibroblast-like chondrocytes on an adherent culture system in FGF-2-free redifferentiation medium that inactivates the Wnt signaling pathway for 2 to 10 days to obtain chondrocytes with complete capacity to resynthesize the hyaline matrix; iii) Culturing said chondrocytes obtained in step ii) in a three-dimensional culture system in an induction/maturation medium without FGF-2 maintaining inactivation of the Wnt signaling pathway for 10 to 20 days.
2. 2. The method of claim 1, wherein the dedifferentiating medium further comprises at least one growth factor selected from the group consisting of PDGF-BB, TGF-beta and EGF.
3. 3. The method of claim 1, wherein the redifferentiation medium and the induction/maturation medium comprise TGF- β.
4. 4. The method of claim 1, wherein the redifferentiation medium and the induction/maturation medium comprise TGF- β3.
5. 5. The method of claim 1, wherein the redifferentiation medium and the induction/maturation medium comprise TGF- β3 and FGF7.
6. 6. The method of any one of claims 1 to 5, wherein the redifferentiation medium comprises platelet lysate.
7. 7. The method of any one of claims 1 to 5, wherein the dedifferentiating, redifferentiating and/or inducing/maturing medium comprises serum.
8. 8. The method of any one of claims 1 to 5, wherein the induction/maturation medium comprises at least one component selected from the group consisting of insulin, IGF-1, BMP-2, selenium, transferrin, and ethanolamine.
9. 9. The method of any one of claims 1 to 5, wherein the chondrocytes are cultured in step iii) in an anoxic environment comprising less than 10% O 2 (v/v).
10. 10. The method of any one of claims 1 to 5, wherein the chondrocytes are cultured in step i) for 10 to 15 days, in step ii) for 4 to 8 days, and/or in step iii) for 10 to 15 days.
11. 11. The method of any one of claims 1 to 5, wherein the chondrocytes of step i) are isolated from a subject.
12. 12. The method of any one of claims 1 to 5, wherein the chondrocytes of step i) are isolated from cartilage tissue of a human or equine subject.
13. 13. An engineered hyaline cartilage tissue in the form of spheroids exhibiting a glycosaminoglycan (GAG) content per spheroid of 10 to 100 μg/spheroid, wherein the hyaline cartilage tissue is obtained by the method of any one of claims 1 to 12.
14. 14. The engineered hyaline cartilage tissue of claim 13, wherein the hyaline cartilage tissue has a spheroid diameter of 1 to 2mm and comprises 50000 to 250000 cells.
15. 15. Use of the engineered hyaline cartilage tissue of claim 13 or 14 in the manufacture of a medicament for treating cartilage defects and cartilage degenerative diseases in a subject in need thereof.
16. 16. The use of claim 15, wherein the medicament is for autograft.
17. 17. The use of claim 15, wherein the medicament is for allograft.
18. 18. A method of screening for molecules that inhibit cartilage degeneration processes, the method comprising: i) Contacting the engineered hyaline cartilage tissue of claim 13 or 14 with one or more candidate molecules, and Ii) selecting a molecule that inhibits the cartilage degenerative process.

Description

Method for producing hyaline cartilage tissue in vitro Technical Field The present invention relates to a novel method for producing hyaline cartilage tissue ('Cartibead') in vitro, and therapeutic uses and screening methods using the cartilage tissue produced thereby. Background Hyaline cartilage (HYALINE CARTILAGE) consists of specialized cells called chondrocytes (chondrocyte) and surrounding extracellular matrix (extracellular matrix). The matrix is synthesized and secreted by chondrocytes and mainly consists of type II collagen fibers, glycosaminoglycans (GAGs) and 60-80% water. Hyaline cartilage has four layers above subchondral bone (subchondral bone), a superficial layer (superficial layer), an intermediate layer (INTERMEDIATE LAYER), a deep layer (DEEP LAYER), and a calcified layer (CALCIFIED LAYER). The biomechanical properties of articular cartilage are largely dependent on the composition and integrity of the extracellular matrix. Cartilage has very limited ability to repair itself, and once injured, it often develops into Osteoarthritis (OA). Aging and repetitive wounds (e.g., that occur during high intensity physical exercise) are the primary risk factors for cartilage degradation in the knee. OA affects a large population and most commonly occurs in the elderly (prevalence > 10% of people over 60 years old), while young people are often affected by OA after joint injury. Surgical treatment includes joint replacement with a prosthesis (prosthesis). But the service life of the prosthesis is limited to 15-20 years. In addition, pain relief is not completely reduced in most patients, and 20% to 30% of patients fitted with knee prostheses continue to feel discomfort or pain. The treatment is primarily palliative and is intended to alleviate pain. Some biological agents, such as mesenchymal stem cells, hyaluronic acid or platelet-rich plasma injection, can delay joint degeneration, but they cannot promote tissue regeneration. Currently, strategies to fill defects using external synthetic scaffolds are not satisfactory and do not fully mimic the biomechanical properties of articular cartilage. Classical treatments include micro-fracture surgery (micro-fracture surgery) to stimulate stem cell migration to the damaged area, or direct implantation of chondrocytes. Microfracture involves making small holes into the subchondral bone in order to stimulate the migration of mesenchymal stem cells from bone marrow (bone medulla) to coagulum (coagulum) to form new chondrocytes and replace damaged tissue. In autologous chondrocyte implantation (autologous chondrocytes implantation, ACI), chondrocytes are extracted from cartilage, cultured a limited number of times, and transplanted into the lesion. To improve the method, matrices such as porcine collagen type I/III or hyaluronic acid may be used for culturing chondrocytes (Kon E, et al., 2009, The American journal of sports medicine 37(1):33-41;Hettrich CM, Crawford D, & Rodeo SA, 2008, 16(4):230-235). either directly after microfracture surgery for injury or in vitro for culturing chondrocytes prior to re-implantation (Ekkers JE et al 2013,Osteoarthritis Research Society 21 (7): 950-956). Cell-based therapies using autologous chondrocyte transplantation (ACI) are suitable for cartilage lesions greater than 2 square centimeters (Armoiry x et al 2019, pharmacoeconomics 37, 879-886). Chondrocytes represent an inevitable source of cells for cartilage regeneration. Indeed, only this type of chondrocyte is involved in maintaining hyaline cartilage matrix. The major challenge is that chondrocytes tend to de-differentiate into fibroblast-like cells in culture, resulting in a rapid loss of function, which typically occurs in a two-dimensional cell culture system in either the second or third cell generation (Munirah, s. Et al 2010. Tissue & cell 42, 282-292). Dedifferentiated chondrocytes are characterized by no longer producing glycosaminoglycans (GAGs) and type II collagen, i.e. the main component (Wu, L. et al. 2014. Tissue Eng Part C Methods 20, 160-168;Benya, P. D et al. 1978. Cell. 15, 1313-1321), of hyaline cartilage, which is replaced by type I collagen in fibrocartilage (fibrocartilage). Indeed, during the expansion step, chondrocytes tend to dedifferentiate by losing their original phenotype and become fibroblast-like cells (elongated cells (elongated cells)) with stem cell-like characteristics in terms of gene expression (expression of cell surface markers such as CD73, CD90, CD105 used to characterize mesenchymal stem cells (MESENCHYMAL STEM CELL, MSC). For example, chondrocytes extracted from articular cartilage of a patient rapidly lose their cartilage-forming ability to synthesize specific hyaline matrix cartilage after several generations of in vitro culture. Fibrocartilage differs biomechanically from hyaline cartilage (HYALINE CARTILAGE) and is not considered an effective long-term treatment due to the resulting dysfunctional repair (dysfunctional re