Use of periosteum-derived stem / progenitor cells in the treatment of tendon and ligament injuries

Abstract

This invention provides a pharmaceutical composition comprising: i) isolated periosteum-derived stem / progenitor cells, wherein said cells have reduced PIEZO1 levels or activity; and ii) a pharmaceutically acceptable carrier. This invention also provides a method for inducing differentiation of periosteum-derived stem / progenitor cells into tendon tissue and a method for treating tendon or ligament injuries or defects with periosteum-derived stem / progenitor cells.

Description

Technical Field

[0001] This invention relates to cell biology and stem cell therapy. Specifically, this invention relates to a method for inducing the formation of tendon tissue from periosteum-derived stem / progenitor cells by reducing the level or activity of the PIEZO1 protein, and a method for treating tendon or ligament injuries or defects using periosteum-derived stem / progenitor cells. Background Technology

[0002] Tendons are structures in the musculoskeletal system that connect bones and skeletal muscles, anchoring skeletal muscles to bones and helping to transmit muscle contraction forces. Tendon cells secrete large amounts of extracellular matrix, primarily composed of collagen. Collagen fibers are tightly packed together, and glycoproteins secreted by tendon cells help assemble collagen structures. 1 Mature tendon tissue has a hierarchical structure: fascicles formed by collagen fibrils are wrapped by the endotenon, and multiple fascicles wrapped by the endotenon combine to be wrapped by the epitenon to form the tendon. 2 Tendon cells, tendon trunk / progenitor cells, nerves, blood vessels, and lymphatic vessels are located between these bundle-like structures composed of extracellular matrix.

[0003] Similar to the developmental origins of bones, tendon tissue cells also have a complex origin, including neural crest cells, paraaxial mesoderm, and lateral plate mesoderm. Craniofacial tendons develop from ectodermal neural crest cells. Tendons located along the body axis and in the limbs originate from the mesoderm. 3,4 .

[0004] Among the key transcription factors mediating tendon development, Scleraxis (SCX) has been the most studied. The Scx gene is crucial for mouse development; its absence leads to embryonic arrest at E8.5. 5 SCX is not specifically expressed in tendon tissue, but is also expressed in some other mesodermal-derived tissues and organs. 6 SCX regulates the aggregation of tendon stem / progenitor cells and their secretion of large amounts of extracellular matrix to build tendon cells and tissue structures and ensure the proper arrangement of cells within the tendon. As a transcription factor, SCX activates the expression of downstream genes such as Fibromodulin (Fmod) and Tenomodulin (Tnmd). 7 .

[0005] Tendons are dense connective tissues rich in collagen that transmit tension between muscles and bones. 8 Tendons lack cells and blood vessels, resulting in insufficient endogenous repair capacity. Approximately 30% of musculoskeletal diseases involve tendon injuries. 9Tendon injuries are a significant clinical challenge because damage to the tendon structure impairs tendon function and can lead to poor healing, such as heterotopic ossification. 10,11 While surgery can restore the integrity of tendon tissue, injured tendons often fail to return to their pre-injury state due to scarring and fibrosis, making them more prone to rupture. Various treatments have been proposed for tendon injuries, such as injections of platelet-rich plasma, hyaluronic acid, and corticosteroids; however, these methods primarily reduce inflammation and pain, with limited effectiveness in promoting tendon repair itself. 9,11,12 .

[0006] In addition, the morphology and function of tendons are highly dependent on the tightly and orderly arranged extracellular matrix. After repair of adult injured tendons, the cell density and extracellular matrix arrangement cannot be fully restored to the state before the injury. In addition, the formation of scars leads to the mechanical properties of the repaired tendons being inferior to those of the tendon tissue before the injury.

[0007] Stem cell-based therapies show great promise because exogenous cell transplantation or endogenous cell activation of injured or diseased tendons has demonstrated promising effects in promoting repair. 13 Currently studied cell sources include tendon stem cells (TSCs), bone marrow stem cells (BMSCs), adipose-derived stem cells (ADSCs), and embryonic stem cells (ESCs). Tendon stem cells are characterized by rapid proliferation and a greater tendency to form tendon cells, making them more suitable for stem cell therapy in tendon injuries. 14-16 However, the low cell content in tendons limits the source of tendon stem cells, thus restricting their application.

[0008] Therefore, new cell sources are needed to generate stem / progenitor cells that can be used for stem cell therapy for tendon injuries. Summary of the Invention

[0009] The periosteum, covering almost the entire bone surface, is one of the most regenerative bone tissues. Due to their unique location, periosteal-derived skeletal stem / progenitor cells, unlike bone marrow-derived cells, possess a stronger capacity for clonogenic formation and osteogenic differentiation. Because of the closer proximity of the periosteum to tendons, there may be interchangeability between the two. The inventors unexpectedly discovered that SCX+ skeletal stem / progenitor cells (periosteal-derived stem / progenitor cells) exist on the periosteum. The potential of these cells to form bone and tendons is regulated by PIEZO1, and the inactivation and activation of PIEZO1 can be applied to the regeneration and repair of bone, tendon, or ligament injuries.

[0010] In a first aspect, the present invention provides a pharmaceutical composition comprising:

[0011] i) Isolated stem / progenitor cells derived from the periosteum, wherein said cells have reduced PIEZO1 levels or activity; and

[0012] ii) Pharmaceutically acceptable carriers.

[0013] In some embodiments, the periosteum is human periosteum. In some embodiments, the cells have the ability to differentiate into tendon or ligament cells. In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels. In some embodiments, the cells are expanded in vitro.

[0014] In some embodiments, the pharmaceutical composition is formulated for topical application, such as at the site of tendon or ligament injury.

[0015] In a second aspect, the present invention provides a method for inducing the differentiation of periosteal-derived stem / progenitor cells into tendon or ligament tissue, the method comprising:

[0016] a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum;

[0017] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells; and

[0018] c) Optionally, amplify the cells obtained in step b) in a culture medium.

[0019] In some embodiments, the periosteum is human periosteum. In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon or ligament cells. In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels.

[0020] In a third aspect, the present invention provides a method for preparing a pharmaceutical composition for treating tendon or ligament injuries or defects in a subject of need, the method comprising:

[0021] a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum;

[0022] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells; and

[0023] c) Optionally, amplify the cells obtained in step b) in a culture medium.

[0024] In some embodiments, the periosteum is human periosteum. In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon or ligament cells. In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels.

[0025] In a fourth aspect, the present invention provides a method for treating tendon or ligament injuries or defects in a subject in need, the method comprising:

[0026] a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum;

[0027] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells;

[0028] c) Amplify the cells obtained in step b) in a culture medium; and

[0029] d) Apply the expanded cells from step c) to the recipient.

[0030] In some embodiments, the periosteum is an autologous periosteum of the subject in need. In some embodiments, the periosteum is an allogeneic periosteum. In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon or ligament cells. In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels.

[0031] In some implementations, step d) includes applying the expanded cells locally to the site of the tendon or ligament injury or defect.

[0032] The present invention also provides a method for treating tendon or ligament injuries or defects in a subject in need, the method comprising administering to the subject a pharmaceutical composition of the present invention (such as the pharmaceutical composition of the first aspect) or a pharmaceutical composition prepared by the method of the present invention (such as the method of the third aspect).

[0033] In some embodiments, the pharmaceutical composition is applied topically to the site of the tendon or ligament injury or defect.

[0034] In some implementations, the periosteum is autologous or allogeneic for the recipient.

[0035] The present invention also provides pharmaceutical compositions of the present invention (such as the pharmaceutical composition of the first aspect) for treating tendon or ligament injuries or defects in subjects in need.

[0036] The present invention also provides the use of the pharmaceutical compositions of the present invention (such as the pharmaceutical compositions of the first aspect) or pharmaceutical compositions prepared by the methods of the present invention (such as the methods of the third aspect) in the preparation of a medicament for treating tendon or ligament injuries or defects in subjects in need. Attached Figure Description

[0037] Figure 1 Display Prx1 Cre Piezo1 fl / fl Mice and Ocn Cre Piezo1 fl / fl Phenotypic comparison in mice. (A) Micro-CT scan images of the femur and (BF) quantification of cortical and cancellous bone in 6-week-old female mice of the corresponding genotypes. Ct.Th, cortical bone thickness; Tb.N, Tb.sp, Tb.Th, and BV / TV parameters represent the amount, spacing, thickness, and bone volume to tissue volume ratio of cancellous bone, respectively. n≥3. (G) Phenotypic comparison in 6-week-old wild-type and Prx1 mice. Cre Piezo1 fl / fl Imaging and (H, I) quantification of mineralization deposition rates in the periosteum and endosteum of the mouse tibia. n≥4. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0038] Figure 2 Piezo1 deficiency inhibits osteogenic differentiation of periosteal cells. (A) Illustration of periosteal cell isolation, culture, and differentiation experiments. (B) Images of alkaline phosphatase (ALP) and alizarin red staining in differentiated cells. (C, D) Quantitative detection of alkaline phosphatase activity and alizarin red region in differentiated cells. n≥4. (E) Quantitative detection of osteogenic marker gene expression in cells after osteogenic differentiation. n=4. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0039] Figure 3 Display and Ocn Cre Piezo1fl / fl Compared to mice, Prx1 Cre Piezo1 fl / fl Mice had a higher rate of long bone fractures. (A) Representative X-ray images of the hind limbs of mice with the corresponding genotype. (B) Statistics on the incidence of spontaneous long bone fractures in mice with the corresponding genotype. n≥20. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0040] Figure 4 Piezo1 deletion arrests periosteal cells in a stem / progenitor state. (A) Illustration of periosteal cell isolation and single-cell sequencing. (B) UMAP diagram of cell populations from single-cell sequencing results. (C) Expression of corresponding genes in each cell population. (D) Expression of Prx1 and Ocn in each cell population. (E) Pseudo-temporal analysis of single-cell transcriptome data.

[0041] Figure 5 This shows that Piezo1 deficiency arrests periosteal cells in a stem / progenitor state. (A) UMAP plot of pseudo-temporal analysis. (B) Density plot of pseudo-temporal analysis results. (C) Violin plot of gene expression in different cell populations.

[0042] Figure 6 This shows that Piezo1 deficiency upregulates SCX expression in periosteal cells. (A) SCX expression in pseudo-time series analysis of Prx1-positive periosteal cells of the corresponding genotype. (B) SCX expression in cluster 0 cell population. (C) SCX expression in each cell population. (D, E) Quantitative detection of SCX gene expression in periosteal cells of the corresponding genotype or in the virus-treated periosteal cells. n≥3. (F) Representative images of femoral sections from mice of the corresponding genotype. (G, H) Quantitative analysis of the proportion of OCN-positive and SCXGFP-positive cells in periosteal cells of mice of the corresponding genotype by flow cytometry. n=5. (I, J) Quantitative analysis of SCX expression in periosteal cells of the corresponding genotype after different treatments. n=4. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0043] Figure 7 The image shows that overexpression of SCX in periosteal cells inhibits osteogenic differentiation. (A) Images of alkaline phosphatase (ALP) and Alizarin red staining in differentiated cells. (B) Quantitative detection of alkaline phosphatase activity in differentiated cells and (C) Quantitative detection of Alizarin red regions. n=6. (D) Quantitative detection of osteogenic marker gene expression in cells after osteogenic differentiation. n=3. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0044] Figure 8 Scx-Cre shows the labeling of skeletal stem / progenitor cell populations in the periosteum. (A)Scx Cre Immunofluorescence staining of osteoblast markers on the periosteum of Ai9 / + mice. Flow cytometry analysis and quantification of skeletal stem / progenitor cell populations in the periosteum of ScxGFP mice. n=3. Immunofluorescence staining of CD200 in skeletal stem cells in the periosteum of ScxGFP mice. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0045] Figure 9 The image shows Lin-Scx+ periosteal cells containing skeletal stem / progenitor cells. (A) Illustration of a renal capsule transplantation experiment using periosteal cells. (B) Micro-CT scan of the kidney after renal capsule transplantation. n=6. (C) Immunofluorescence staining of osteoblasts in the kidney after renal capsule transplantation.

[0046] Figure 10 Display Scx Cre Piezo1 fl / fl Bone mass was significantly reduced in mice. (A) WT and Scx of 6-week-old mice Cre Piezo1 fl / fl Micro-CT scan images of mouse femurs. (BG) Quantitative analysis of cortical and cancellous bone. Ct.Th, cortical bone thickness; Tb.N, Tb.sp, Tb.Th, and BV / TV parameters represent the amount, spacing, thickness, and bone volume to tissue volume ratio of cancellous bone, respectively. n=4. Data represent mean ± SD, *P<0.05. Data represent mean ± SD, *P<0.05, **P<0.01.

[0047] Figure 11 This indicates that bone repair requires the expression of Piezo1 in Scx+ cells of the periosteum. (A) Immunofluorescence staining of OCN in the periosteum of Scx fluorescent reporter mice and quantification of the corresponding cellular regions (B, C). n=3. (D) WT and Scx Cre Piezo1 fl / fl Micro-CT scan images and quantitative analysis of new bone (E, F) after bone puncture wound repair in mice. BV, bone volume; TV, tissue volume. n≥4. (G) Bone tissue sections and OPN immunofluorescence staining after wound repair in mice of corresponding genotypes. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0048] Figure 12The periosteum Scx+ cells are shown to participate in bone development and injury repair. (A)Scx CreERT2 ;Illustrations of the construction strategy and induction strategy under non-invasive conditions for Ai9 / + mice. (BC)Immunofluorescence staining of the OCN in the femur of P7 and P42 mice after P1 induction. (D)Scx CreERT2 ; Illustration of the induction strategy in Ai9 / + mice in a bone injury model. (E, F) Immunofluorescence staining of cartilage marker SOX9 and osteogenic marker OCN in bone tissue after injury repair following induction at 3 weeks of age.

[0049] Figure 13 This demonstrates how Piezo1-deficient periosteal stem / progenitor cells promote tendon injury repair. (A) Experimental illustration of periosteal cell transplantation in a patellar tendon injury model. (B, C) Micro-CT scans of cell-free transplantation and WT or Prx1... Cre Piezo1 fl / fl Detection of heterotopic ossification and quantification of bone mass in patellar tendon repair 2 months after transplantation of Prx1+ cells from the periosteum of mice. n≥5. (DF) Gait performance of mice after transplantation and statistics of maximum tensile strength of tendons in different treatment groups. n≥5. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0050] Figure 14 Piezo1-deficient periosteal stem / progenitor cells are shown to participate in tendon injury repair. (A) Fast green / Safranin O staining of patellar tendon tissue sections transplanted with labeled periosteal cells. (BH) Immunofluorescence staining of chondrogenic markers (COL 10, COL2) and tenogenic markers (THBS4, TNC) in patellar tendon tissue transplanted with labeled periosteal cells, and quantification of cartilage or tendon cells. n=3. Data are expressed as mean ± SD, *P<0.05. Data are expressed as mean ± SD, *P<0.05, **P<0.01.

[0051] Figure 15Inhibition of Piezo1 in periosteal stem / progenitor cells demonstrates their role in tendon repair. (A) Illustration of transplantation of MTMG;ScxGFP mouse periosteal cells treated with a Piezo1 inhibitor. (B, C) CT scans and quantification of ectopic osteogenic patellar tendon tissue transplanted with Piezo1 inhibitor-treated or control periosteal cells. n=4. (DK) Fastgreen / Safranin O staining and immunofluorescence staining and quantification of tendon markers (TNC) and osteogenic markers (OPN) in patellar tendon tissue sections transplanted with MTMG;ScxGFP mouse periosteal cells treated with a Piezo1 inhibitor or control cells. n≥3. (L, M) Micro-CT scans and quantification of ectopic ossification in damaged tendon tissue transplanted with human periosteal cells. (N) Fastgreen / Safranin O staining and immunofluorescence staining of COL 10 and OPN in tendon tissue transplanted with control or Piezo1 inhibitor-treated periosteal cells. (O, P) Gait assessment in mice transplanted with control or periosteal cells treated with a Piezo1 inhibitor. n = 4. Data are expressed as mean ± SD, *P < 0.05. Data are expressed as mean ± SD, *P < 0.05, **P < 0.01. Invention Details

[0052] definition

[0053] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. For example, the standard recombinant DNA and molecular cloning techniques used in this invention are well known to those skilled in the art and are described more fully in the following literature: Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0054] As used herein, the term “and / or” covers all combinations of items connected by the term and should be regarded as if each combination had been listed separately herein. For example, “A and / or B” covers “A,” “A and B,” and “B.” For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.”

[0055] The Piezo family is a family of mechanogated cation channels found in mammals, comprising Piezo1 and Piezo2. Previous studies have shown that changes in membrane tension when forces are applied to the lipid membrane can cause reversible deformation of the Piezo1 protein, changing it from a curved to a flattened state, leading to an expansion of the planar membrane area and driving the opening of the Piezo1 channel, thereby converting mechanical stimulation into cation flow. Existing techniques have also reported that the Piezo1 ion channel mediates the mechanosensitivity of osteoblasts and osteocytes and plays an important role in load-dependent bone formation, providing a new target for the treatment of bone loss caused by spaceflight weightlessness and prolonged bed rest. In this paper, when referring to the protein, Piezo1 can also be represented as "PIEZO1". An exemplary amino acid sequence of the human PIEZO1 protein is shown in GenBank accession number NP_001136336.2; an exemplary amino acid sequence of the mouse PIEZO1 protein is shown in GenBank accession number 15NP_001032375.

[0056] Stem cells are cells that retain the ability to renew themselves through mitosis, and their daughter cells can differentiate into a wide range of specialized cell types. The two main classes of mammalian cells are embryonic stem cells (ESCs), found in the blastocyst, and adult stem cells, found in adult tissues. In a developing embryo, ESCs can differentiate into all specialized embryonic tissues. In adult organisms, adult stem cells and progenitor cells act as the body's repair system, replenishing specialized cells and maintaining the normal functioning of regenerative organs such as blood, skin, and bone tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0057] As used herein, the term "stem cell" refers to an undifferentiated cell capable of proliferating and producing more progenitor cells, which in turn produce a large number of mother cells, which can then generate differentiated daughter cells, or known as stem / progenitor cells. The daughter cells themselves can be induced to proliferate and produce progeny, which subsequently differentiate into one or more mature cell types while retaining one or more cells with parental developmental potential.

[0058] The term "stem cell" also refers to a subpopulation of progenitor cells that have the ability or potential to differentiate into more specialized or differentiated phenotypes under certain conditions, and in some cases retain the ability to proliferate while remaining essentially undifferentiated.

[0059] Tendons are components of skeletal muscles, composed of dense connective tissue, white in color, relatively hard, and lacking contractile ability. The tendons of long muscles are mostly cord-like, while the tendons of broad muscles are wide and thin, membranous, and also called aponeurosis. The tendon sheath, also known as the synovial sheath, is a tubular synovial sac that surrounds the surface of long muscle tendons.

[0060] Ligaments are tissues that connect bones and are composed of dense connective tissue. The cellular composition of ligaments and tendons is similar. Therefore, the inventors believe that ligament injuries or defects can be treated using the same stem cell therapy used to treat tendon injuries or defects.

[0061] Periosteal-derived stem / progenitor cells play a vital role in bone development, homeostasis, and injury repair, possessing self-renewal capacity and clonal pluripotency. The inventors also discovered that inhibiting PIEZO1 can suppress the differentiation of periosteal-derived stem / progenitor cells into bone tissue and enable these cells to form tendon tissue.

[0062] In this paper, cell populations encompass heterogeneous or homogeneous populations of stem / progenitor cells derived from the periosteum. A population containing at least two different cell types is referred to as a "heterogeneous population" in this paper. A population containing only one cell type is referred to as a "homogeneous population" in this paper.

[0063] As used herein, “isolated cell” refers to a cell or a descendant of such a cell from which it was originally found in the organism, organ, or tissue. Optionally, the cell has been cultured in vitro.

[0064] "Self-renewal" refers to the ability of a cell to divide and produce at least one daughter cell with the same characteristics as the parent cell. The second daughter cell can enter a specific differentiation pathway. Daughter cells that enter the differentiation pathway typically have lost the ability to self-renew and, upon cell division, produce two daughter cells exhibiting a more differentiated (i.e., restricted) phenotype.

[0065] As used herein, the term “isolation” refers to the process of removing cells or populations of cells from the object or biological sample in which they were originally found. As used herein, the term “isolated population” refers to a population of cells removed and isolated from a biological sample, or a mixed or heterogeneous population of cells found in such a sample.

[0066] As used herein, the term "enrichment" means an increase in the amount, concentration, density, or proportion of a cell type relative to an initial biological sample, culture, or article, for example, an increase of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, preferably an increase of at least 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or higher. For example, if the proportion of a cell type in an initial sample is 3%, and the proportion of said cell type in the enriched sample is 30%, then the "enrichment" increases the proportion of said cell type by 1000%.

[0067] As used herein, a “marker” describes the characteristics and / or phenotype of a cell. Markers can be used to select cells containing the characteristics of interest. Markers vary from cell to cell. A marker is a cell type-specific characteristic, whether morphological, functional, or biochemical (enzymatic), or a molecule expressed by the cell type. Preferably, such markers are proteins, and more preferably, have epitopes of antibodies or other binding molecules available in the art. However, markers can consist of any molecules found in the cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, appearance (e.g., smooth, translucent), and nucleocytoplasmic ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a specific substrate, the ability to absorb or repel a specific dye, the ability to migrate under specific conditions, and the ability to differentiate along a specific lineage. Markers can be detected by any method available to those skilled in the art.

[0068] Therefore, as used herein, “cell surface marker” refers to any molecule expressed on the cell surface. Cell surface expression typically requires molecules to have transmembrane domains. Some molecules not normally found on cell surfaces can be engineered for expression on cell surfaces using recombinant technologies. Many naturally occurring cell surface markers are named “CD” or “differentiation cluster” molecules. Cell surface markers often provide antigenic determinants that antibodies can bind to.

[0069] As used herein, the term "antibody" refers to an intact immunoglobulin or a monoclonal or polyclonal antigen-binding fragment having an Fc (chip-binding) region or an FcRn-binding fragment of the Fc region. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. "Antigen-binding fragments" include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, single-domain antibodies, chimeric antibodies, bispecific antibodies, and polypeptides containing at least a portion of the immunoglobulin sufficient to confer specific antigen binding to the polypeptide. Such antibodies or antigen-binding fragments are commercially available from suppliers such as R&D Systems, BD Biosciences, e-Biosciences, Merck / millipore, Invitrogen, and ABCAM, or can be generated against these cell surface markers or other intracellular markers using methods known to those skilled in the art.

[0070] The term "object" refers to an animal, for example, a person to whom cells for the methods described herein can be obtained (i.e., a donor object) and / or a person to whom cells are provided for treatment (including prophylactic treatment) using the cells described herein (i.e., a recipient object). For the treatment of disease conditions or pathologies specific to a particular animal, such as a human object, the term "object" refers to that particular animal. The terms "non-human animal" and "non-human mammal," used interchangeably herein, include mammals such as rats, mice, rabbits, sheep, cats, dogs, cattle, pigs, and non-human primates. The term "object" also covers any vertebrate, including but not limited to mammals, reptiles, amphibians, and fish. However, advantageously, an object is a mammal such as a human, or other mammals such as domesticated animals, e.g., dogs, cats, horses, etc., or food-producing mammals, e.g., cattle, sheep, pigs, etc.

[0071] As used herein, the term “pharmaceuticalally acceptable” refers to molecular entities and compositions that are physiologically tolerable and that, when administered to humans, generally do not produce toxicity, allergic reactions, or similar adverse effects.

[0072] PIEZO1-lowering periosteum-derived stem / progenitor cells

[0073] As is well known, stem cells generate all the cells and tissues in the body due to their properties. Therefore, stem cells can be used to repair or accelerate the repair of damaged and / or defective tendons or ligaments. If a sufficient quantity of stem cells capable of forming tendon or ligament tissue is available, damaged and / or defective tendons or ligaments can be repaired by building new tissue within the tendon or ligament. In defective and / or damaged tendons or ligaments, stem cells may be scarce or absent. Because adult stem cells self-renew, implanted adult stem cells will colonize at the site of tendon or ligament injury or defect. Through cloning, self-renewal, and differentiation, the implanted stem cells will generate new tendon or ligament tissue. Therefore, isolated populations of stem cells or compositions containing isolated populations of stem cells can be used to treat tendon or ligament injuries or defects in a subject.

[0074] The low cellularity of tendons limits the availability of tendon or ligament stem cells, thus restricting the application of stem cell therapy in treating tendon or ligament injuries. The inventors unexpectedly discovered that by regulating PIEZO1, SCX+ skeletal stem / progenitor cells (periosteal-derived stem / progenitor cells) on the periosteum can be induced to form tendons, providing a broader source of seed cells for tendon or ligament regeneration and repair of tendon or ligament injuries.

[0075] Therefore, the present invention provides isolated periosteum-derived stem / progenitor cells, wherein said cells have reduced PIEZO1 levels or activity. The present invention also provides isolated periosteum-derived cell populations, wherein the cells in said populations have reduced PIEZO1 levels or activity, and said populations are enriched in stem / progenitor cells, for example, said populations predominantly comprising stem / progenitor cells (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, said populations consist of stem / progenitor cells.

[0076] In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels. In some embodiments, the stem / progenitor cells are Lin-Scx+ cells (e.g., for mouse CD45 / CD31 / Ter119 negative SCX-positive cells, for human CD45 / CD235a / CD31 / Tie2 negative SCX-positive cells) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells).

[0077] In some embodiments, the stem / progenitor cells have the ability to differentiate into tendon or ligament cells.

[0078] In some implementations, the stem / progenitor cell population is self-renewing and clonal.

[0079] In some implementations, the stem / progenitor cells are derived from periosteal tissue samples.

[0080] In some implementations, the cell population is expanded in vitro.

[0081] The level or activity of PIEZO1 can be reduced using methods known in the art. For example, the gene encoding PIEZO1 can be knocked down or eliminated using antisense oligonucleotides, RNA interference (such as siRNA and shRNA), or gene editing (such as broad-spectrum nucleases, zinc finger nucleases, TALEN, and CRISPR). The expression of the PIEZO1-encoding gene can also be reduced using CRISPR-dCas-based methods, such as CRISPR-dCas systems targeting the PIEZO1-encoding gene or its regulatory sequences, which contain transcriptional repression domains or domains that increase locus methylation. Cells can also be treated with PIEZO1 inhibitors (such as GsMTx4, Gd...). 3+ Ions and Dooku1) come into contact.

[0082] Inducing periosteal-derived stem / progenitor cells to form tendon tissue

[0083] The inventors discovered that periosteum-derived stem / progenitor cells can be made capable of forming tendon tissue by reducing the level or activity of PIEZO1.

[0084] Therefore, the present invention provides a method for inducing the differentiation of periosteal-derived stem / progenitor cells into tendon or ligament tissue, the method comprising:

[0085] a) Provide a cell pool from the periosteum containing periosteum-derived stem / progenitor cells; and

[0086] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells.

[0087] The level or activity of PIEZO1 can be reduced using methods known in the art. For example, the gene encoding PIEZO1 can be knocked down or eliminated using antisense oligonucleotides, RNA interference (such as siRNA and shRNA), or gene editing (such as broad-spectrum nucleases, zinc finger nucleases, TALEN, and CRISPR). The expression of the PIEZO1-encoding gene can also be reduced using CRISPR-dCas-based methods, such as CRISPR-dCas systems targeting the PIEZO1-encoding gene or its regulatory sequences, which contain, for example, transcriptional repression domains or domains that increase locus methylation. Cells can also be treated with PIEZO1 inhibitors (such as GsMTx4, Gd...). 3+ Ions and Dooku1) come into contact.

[0088] In some embodiments, the periosteum is a human periosteum or a mouse periosteum.

[0089] In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon cells. In some embodiments, the method further includes selecting SCX+ cells. In some embodiments, the method further includes selecting cells with elevated SCX levels. This selection may be performed before or after step b).

[0090] In some embodiments, the method further includes selecting Lin-Scx+ cells (e.g., mouse CD45 / CD31 / Ter119 negative SCX positive, or human CD45 / CD235a / CD31 / Tie2 negative SCX positive) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells). This selection can be performed before or after step b) or c).

[0091] In some embodiments, step a) includes obtaining periosteal tissue from the subject. In some embodiments, the subject is a person, including adolescents, adults, and the elderly. In some embodiments, the periosteal tissue is external periosteum tissue.

[0092] In some embodiments, the method further includes digesting the periosteum tissue with an enzyme, for example, removing non-cellular structures in the tissue to obtain a pool of cells from the periosteum tissue. In some embodiments, the method further includes removing red blood cells, for example, by lysing red blood cells. In some embodiments, the method further includes filtering to remove cell aggregates.

[0093] In some embodiments, the method further includes selection with a binding molecule targeting SCX, such as an anti-SCX antibody or its antigen-binding fragment.

[0094] In some embodiments, the method further includes selection using binding molecules against CD45, CD31, CD235a, and / or Tie2, such as anti-CD45, anti-CD31, anti-CD235a, and / or anti-Tie2 antibodies or antigen-binding fragments thereof. In some embodiments, the method further includes selection using binding molecules against CD45, CD31, and / or Ter119, such as anti-CD45, anti-CD31, and / or anti-Ter119 antibodies or antigen-binding fragments thereof.

[0095] In some embodiments, the binding molecule is conjugated to a solid particle, such as a magnetic bead. In some embodiments, the binding molecule is labeled, for example, with a fluorescent dye.

[0096] In some embodiments, the selection includes selection by flow cytometry. In some embodiments, the selection includes magnetic selection.

[0097] In one specific embodiment, the method includes: obtaining periosteal tissue from a subject; digesting the periosteal tissue with a collagenase (such as collagenase II) and a dispersant (such as dispersant II) to obtain single cells; centrifuging to collect the single cells and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; and reducing the level or activity of PIEZO1 in the obtained cells, for example by reacting the obtained cells with a PIEZO1 inhibitor (such as GsMTx4, Gd). 3+ Ions and Dooku1) come into contact.

[0098] In some embodiments, the method further includes c) amplifying the cells obtained in step b) in a culture medium. In some embodiments, the cells are cultured on a biocompatible scaffold, preferably a 3D scaffold.

[0099] In some embodiments, cell selection is performed before or after step c), such as selecting SCX+ cells. In some embodiments, cells with elevated SCX levels are selected. In some embodiments, the method further includes selecting Lin-Scx+ cells (e.g., for mouse CD45 / CD31 / Ter119 negative SCX-positive cells, for human CD45 / CD235a / CD31 / Tie2 negative SCX-positive cells) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells).

[0100] In some embodiments, the method further includes selection with a binding molecule targeting SCX, such as an anti-SCX antibody or its antigen-binding fragment.

[0101] In some embodiments, the method further includes selection using binding molecules against CD45, CD31, CD235a, and / or Tie2, such as anti-CD45, anti-CD31, anti-CD235a, and / or anti-Tie2 antibodies or antigen-binding fragments thereof. In some embodiments, the method further includes selection using binding molecules against CD45, CD31, and / or Ter119, such as anti-CD45, anti-CD31, and / or anti-Ter119 antibodies or antigen-binding fragments thereof.

[0102] In some embodiments, the binding molecule is conjugated to a solid particle, such as a magnetic bead. In some embodiments, the binding molecule is labeled, for example, with a fluorescent dye.

[0103] In some embodiments, the selection includes selection by flow cytometry. In some embodiments, the selection includes magnetic selection.

[0104] In some implementations, after amplification, the number of cells is at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or more times the number before amplification.

[0105] The number of cells in a culture can be determined by any method known in the art, for example, by using a Coulter counter. These methods are well known to those skilled in the art.

[0106] In some implementations, the cells obtained in step b) are stored at low temperatures (e.g., -80°C).

[0107] In some implementations, the expanded cells are stored at low temperatures (e.g., -80°C) for storage purposes. When needed, the frozen cells are thawed and then, for example, implanted into a recipient.

[0108] In some embodiments, the cell suspension is mixed with a cryoprotectant prior to cryopreservation. Methods for cryopreserving tissues and cells using cryoprotectants are well known in the art. Frozen samples can be prepared in the presence of one or more different cryoprotectants to minimize cell damage during the freeze-thaw process. For example, dimethyl sulfoxide (DMSO), trehalose, or sucrose can be used.

[0109] The method further includes: d) culturing the cells obtained in step b) or the cells expanded in step c) under conditions that allow for the formation of tendon tissue.

[0110] The present invention also provides a method for preparing a pharmaceutical composition for treating tendon or ligament injuries or defects in subjects in need, the method comprising:

[0111] a) Provide a cell pool from the periosteum containing periosteum-derived stem / progenitor cells; and

[0112] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells.

[0113] The level or activity of PIEZO1 can be reduced using methods known in the art. For example, the gene encoding PIEZO1 can be knocked down or eliminated using antisense oligonucleotides, RNA interference (such as siRNA and shRNA), or gene editing (such as broad-spectrum nucleases, zinc finger nucleases, TALEN, and CRISPR). The expression of the PIEZO1-encoding gene can also be reduced using CRISPR-dCas-based methods, such as CRISPR-dCas systems targeting the PIEZO1-encoding gene or its regulatory sequences, which contain, for example, transcriptional repression domains or domains that increase locus methylation. Cells can also be treated with PIEZO1 inhibitors (such as GsMTx4, Gd...). 3+ Ions and Dooku1) come into contact.

[0114] In some embodiments, the periosteum is human periosteum. In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon or ligament cells. In some embodiments, the method further includes selecting SCX+ cells. In some embodiments, the method further includes selecting cells with elevated SCX levels. This selection may be performed before or after step b).

[0115] In some embodiments, the method further includes selecting Lin-Scx+ cells (e.g., mouse CD45 / CD31 / Ter119 negative SCX positive, or human CD45 / CD235a / CD31 / Tie2 negative SCX positive) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells). This selection can be performed before or after step b) or c).

[0116] In some embodiments, step a) includes obtaining periosteal tissue from the subject. In some embodiments, the subject is a person, including adolescents, adults, and the elderly. In some embodiments, the periosteal tissue is external periosteum tissue.

[0117] In some embodiments, the method further includes digesting the periosteum tissue with an enzyme, for example, removing non-cellular structures in the tissue to obtain a pool of cells from the periosteum tissue. In some embodiments, the method further includes removing red blood cells, for example, by lysing red blood cells. In some embodiments, the method further includes filtering to remove cell aggregates.

[0118] In some embodiments, the method further includes selection with a binding molecule targeting SCX, such as an anti-SCX antibody or its antigen-binding fragment.

[0119] In some embodiments, the method further includes selection using binding molecules against CD45, CD31, CD235a, and / or Tie2, such as anti-CD45, anti-CD31, anti-CD235a, and / or anti-Tie2 antibodies or antigen-binding fragments thereof. In some embodiments, the method further includes selection using binding molecules against CD45, CD31, and / or Ter119, such as anti-CD45, anti-CD31, and / or anti-Ter119 antibodies or antigen-binding fragments thereof.

[0120] In some embodiments, the binding molecule is conjugated to a solid particle, such as a magnetic bead. In some embodiments, the binding molecule is labeled, for example, with a fluorescent dye.

[0121] In some embodiments, the selection includes selection by flow cytometry. In some embodiments, the selection includes magnetic selection.

[0122] In one specific embodiment, the method includes: obtaining periosteal tissue from a subject; digesting the periosteal tissue with a collagenase (such as collagenase II) and a dispersant (such as dispersant II) to obtain single cells; centrifuging to collect the single cells and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; and reducing the level or activity of PIEZO1 in the obtained cells, for example by reacting the obtained cells with a PIEZO1 inhibitor (such as GsMTx4, Gd). 3+ Ions and Dooku1) come into contact.

[0123] In some embodiments, the method further includes c) amplifying the cells obtained in step b) in a culture medium. In some embodiments, the cells are cultured on a biocompatible scaffold, preferably a 3D scaffold.

[0124] In some embodiments, cell selection is performed before or after step c), such as selecting SCX+ cells. In some embodiments, cells with elevated SCX levels are selected. In some embodiments, the method further includes selecting Lin-Scx+ cells (e.g., for mouse CD45 / CD31 / Ter119 negative SCX-positive cells, for human CD45 / CD235a / CD31 / Tie2 negative SCX-positive cells) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells).

[0125] In some embodiments, the method further includes selection with a binding molecule targeting SCX, such as an anti-SCX antibody or its antigen-binding fragment.

[0126] In some embodiments, the method further includes selection using binding molecules against CD45, CD31, CD235a, and / or Tie2, such as anti-CD45, anti-CD31, anti-CD235a, and / or anti-Tie2 antibodies or antigen-binding fragments thereof. In some embodiments, the method further includes selection using binding molecules against CD45, CD31, and / or Ter119, such as anti-CD45, anti-CD31, and / or anti-Ter119 antibodies or antigen-binding fragments thereof.

[0127] In some embodiments, the binding molecule is conjugated to a solid particle, such as a magnetic bead. In some embodiments, the binding molecule is labeled, for example, with a fluorescent dye.

[0128] In some embodiments, the selection includes selection by flow cytometry. In some embodiments, the selection includes magnetic selection.

[0129] In some implementations, after amplification, the number of cells is at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or more times the number before amplification.

[0130] The number of cells in a culture can be determined by any method known in the art, for example, by using a Coulter counter. These methods are well known to those skilled in the art.

[0131] In some implementations, the cells obtained in step b) are stored at low temperatures (e.g., -80°C).

[0132] In some implementations, the expanded cells are stored at low temperatures (e.g., -80°C) for storage purposes. When needed, the frozen cells are thawed and then, for example, implanted into a recipient.

[0133] In some embodiments, the cell suspension is mixed with a cryoprotectant prior to cryopreservation. Methods for cryopreserving tissues and cells using cryoprotectants are well known in the art. Frozen samples can be prepared in the presence of one or more different cryoprotectants to minimize cell damage during the freeze-thaw process. For example, dimethyl sulfoxide (DMSO), trehalose, or sucrose can be used.

[0134] Pharmaceutical Composition

[0135] This invention provides a pharmaceutical composition comprising:

[0136] i) Isolated stem / progenitor cells derived from the periosteum, wherein said cells have reduced PIEZO1 levels or activity; and

[0137] ii) Pharmaceutically acceptable carriers.

[0138] In some of the embodiments, the pharmaceutical composition comprises an isolated periosteum-derived cell population having reduced PIEZO1 levels or activity, and the population is enriched with stem / progenitor cells, for example, the population mainly comprising stem / progenitor cells (e.g., at least 60%, 70%, 80%, 90% or more). In some embodiments, the population consists entirely of stem / progenitor cells.

[0139] In some embodiments, the stem / progenitor cells are SCX+ cells. In some embodiments, the stem / progenitor cells have elevated SCX levels. In some embodiments, the stem / progenitor cells are Lin-Scx+ cells (e.g., for mouse CD45 / CD31 / Ter119 negative SCX-positive cells, for human CD45 / CD235a / CD31 / Tie2 negative SCX-positive cells) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells).

[0140] In some implementations, the stem / progenitor cells have the ability to differentiate into tendon cells.

[0141] In some implementations, the stem / progenitor cell population is self-renewing and clonal.

[0142] In some implementations, the stem / progenitor cells are derived from periosteal tissue samples.

[0143] In some implementations, the cell population is expanded in vitro.

[0144] The level or activity of PIEZO1 can be reduced using methods known in the art. For example, the gene encoding PIEZO1 can be knocked down or eliminated using antisense oligonucleotides, RNA interference (such as siRNA and shRNA), or gene editing (such as broad-spectrum nucleases, zinc finger nucleases, TALEN, and CRISPR). The expression of the PIEZO1-encoding gene can also be reduced using CRISPR-dCas-based methods, such as CRISPR-dCas systems targeting the PIEZO1-encoding gene or its regulatory sequences, which contain transcriptional repression domains or domains that increase locus methylation. Cells can also be treated with PIEZO1 inhibitors (such as GsMTx4, Gd...). 3+ Ions and Dooku1) come into contact.

[0145] In some embodiments, the pharmaceutical composition is formulated for topical application, such as at the site of tendon or ligament injury or defect.

[0146] In some embodiments, the pharmaceutical composition comprises a 3D culture of the cell population. The 3D culture can be obtained by culturing the cell population on a biocompatible 3D tissue scaffold.

[0147] In some embodiments, the pharmaceutical composition is formulated as a gel.

[0148] The composition may contain additional bioactive agents, including but not limited to pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, or combinations thereof.

[0149] In some embodiments, the bioactive agents include, but are not limited to, bFGF, GDF5, GDF6 (BMP13), GDF7 (BMP12), IGF1, PDGF, TGF-b1, TGF-b2, and VEGF.

[0150] Treatment of tendon or ligament injuries or defects

[0151] This invention provides a method for treating tendon or ligament injuries or defects in a patient, the method comprising:

[0152] a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum;

[0153] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells;

[0154] c) Amplify the cells obtained in step b) in a culture medium; and

[0155] d) Apply the expanded cells from step c) to the recipient.

[0156] Furthermore, tendon or ligament injuries can lead to complications such as tendon or ligament adhesions and heterotopic ossification, resulting in pain and impaired tendon recovery. Improving tendon or ligament repair can prevent and treat complications of tendon or ligament injuries.

[0157] Therefore, the present invention also provides a method for preventing or treating complications (such as tendon adhesions and heterotopic ossification) of tendon or ligament injuries or defects in subjects of need, the method comprising:

[0158] a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum;

[0159] b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells;

[0160] c) Amplify the cells obtained in step b) in a culture medium; and

[0161] d) Apply the expanded cells from step c) to the recipient.

[0162] In some embodiments, the periosteum is the autologous periosteum of the recipient. In some embodiments, the periosteum is an allogeneic periosteum. In some embodiments, the recipient is a person, including adolescents, adults, and the elderly. In some embodiments, the periosteum is the periosteum.

[0163] The level or activity of PIEZO1 can be reduced using methods known in the art. For example, the gene encoding PIEZO1 can be knocked down or eliminated by antisense oligonucleotides, RNA interference (such as siRNA and shRNA), or gene editing (such as broad-spectrum nucleases, zinc finger nucleases, TALEN, and CRISPR). The expression of the PIEZO1-encoding gene can also be reduced using CRISPR-dCas-based methods, such as CRISPR-dCas systems targeting the PIEZO1-encoding gene or its regulatory sequences, which contain, for example, transcriptional repression domains or domains that increase locus methylation. Cells can also be exposed to PIEZO1 inhibitors (such as GsMTx4).

[0164] In some embodiments, the method further includes digesting the periosteum tissue with an enzyme, for example, removing non-cellular structures in the tissue to obtain a pool of cells from the periosteum tissue. In some embodiments, the method further includes removing red blood cells, for example, by lysing red blood cells. In some embodiments, the method further includes filtering to remove cell aggregates.

[0165] In some embodiments, the cells obtained in step b) have the ability to differentiate into tendon tissue. In some embodiments, the method further includes selecting SCX+ cells. In some embodiments, the method further includes selecting cells with elevated SCX levels. This selection may be performed before or after step b).

[0166] In some embodiments, the method further includes selecting Lin-Scx+ cells (e.g., mouse CD45 / CD31 / Ter119 negative SCX positive, or human CD45 / CD235a / CD31 / Tie2 negative SCX positive) (e.g., mouse CD45-CD31-Ter119-Scx+ cells, or human CD45-CD235a-CD31-Tie2-Scx+ cells).

[0167] In some embodiments, the method further includes selection with a binding molecule targeting SCX, such as an anti-SCX antibody or its antigen-binding fragment.

[0168] In some embodiments, the method further includes selection using binding molecules against CD45, CD31, CD235a, and / or Tie2, such as anti-CD45, anti-CD31, anti-CD235a, and / or anti-Tie2 antibodies or antigen-binding fragments thereof. In some embodiments, the method further includes selection using binding molecules against CD45, CD31, and / or Ter119, such as anti-CD45, anti-CD31, and / or anti-Ter119 antibodies or antigen-binding fragments thereof.

[0169] In some embodiments, the binding molecule is conjugated to a solid particle, such as a magnetic bead. In some embodiments, the binding molecule is labeled, for example, with a fluorescent dye.

[0170] In some embodiments, the selection includes selection by flow cytometry. In some embodiments, the selection includes magnetic selection.

[0171] In one specific embodiment, the method includes: obtaining periosteal tissue from a subject; digesting the periosteal tissue with a collagenase (such as collagenase II) and a dispersant (such as dispersant II) to obtain single cells; centrifuging to collect the single cells and preparing a suspension containing the single cells; optionally, filtering the suspension to remove cell aggregates; and reducing the level or activity of PIEZO1 in the obtained cells, for example by reacting the obtained cells with a PIEZO1 inhibitor (such as GsMTx4, Gd). 3+ Ions and Dooku1) come into contact.

[0172] In some embodiments, after expansion, the number of cells is at least 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, 50,000, or more times the number before expansion. The number of cells in the culture can be determined by any method known in the art, for example, by using a Coulter counter. These methods are well known to those skilled in the art.

[0173] In some implementations, the cells obtained in step b) are stored at low temperatures (e.g., -80°C).

[0174] In some implementations, step c) includes culturing the cells on a biocompatible scaffold, preferably a 3D scaffold.

[0175] In some implementations, the expanded cells are stored at low temperatures (e.g., -80°C) for storage purposes. When needed, the frozen cells are thawed and then implanted into the recipient.

[0176] In some embodiments, the cell suspension is mixed with a cryoprotectant prior to cryopreservation. Methods for cryopreserving tissues and cells using cryoprotectants are well known in the art. Frozen samples can be prepared in the presence of one or more different cryoprotectants to minimize cell damage during the freeze-thaw process. For example, dimethyl sulfoxide (DMSO), trehalose, or sucrose can be used.

[0177] In some embodiments, the expanded cells are applied topically to the site of tendon or ligament injury or defect. In some embodiments, the cells are applied in gel form. The cells may be applied in combination with additional bioactive agents, including but not limited to pharmaceutically active compounds, hormones, growth factors, enzymes, DNA, RNA, siRNA, hyaluronic acid, antibodies, antibiotics, anti-inflammatory molecules, or combinations thereof.

[0178] In some embodiments, the bioactive agent comprises "angiogenic factors," including but not limited to epidermal growth factor (EGF), E-cadherin, VEGF, angiogenic protein, angiopoietin-1, fibroblast growth factor (FGF, including aFGF and bFGF), hepatocyte growth factor (HGF), angiopoietin, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), and inflammatory cytokines and chemokines (which are inducers of angiogenesis and increased angiogenesis), such as interleukin-3 (IL-3), interleukin-8 (IL-8), CCL2 (MCP-1), interleukin-8 (IL-8), and CCL5 (RANTES).

[0179] The present invention also provides a method for treating tendon or ligament injuries or defects in a subject in need, the method comprising administering the pharmaceutical composition of the present invention to the subject in need.

[0180] The present invention also provides a method for preventing or treating complications (such as tendon adhesions and heterotopic ossification) of tendon or ligament injury or defects in a subject in need, the method comprising administering the pharmaceutical composition of the present invention to the subject in need.

[0181] In some embodiments, the pharmaceutical composition is applied topically to the site of tendon or ligament injury or defect.

[0182] In some implementations, the periosteum-derived stem / progenitor cells are autologous or allogeneic for the subject in need.

[0183] The present invention also provides isolated periosteal-derived stem / progenitor cell populations or pharmaceutical compositions comprising said populations for treating tendon or ligament injuries or defects in subjects of need, wherein said stem / progenitor cells have reduced levels or activity of PIEZO1.

[0184] The present invention also provides isolated periosteal-derived stem / progenitor cell populations or pharmaceutical compositions comprising said populations for the prevention or treatment of complications (such as tendon adhesions and heterotopic ossification of tendons) of tendon or ligament injury or defect in subjects of need, wherein said stem / progenitor cells have reduced levels or activity of PIEZO1.

[0185] The present invention also provides the use of isolated periosteal-derived stem / progenitor cell populations or pharmaceutical compositions comprising said populations in the preparation of a medicament for treating tendon or ligament injuries or defects in subjects of need, wherein said stem / progenitor cells have reduced levels or activity of PIEZO1.

[0186] The present invention also provides the use of isolated periosteal-derived stem / progenitor cell populations or pharmaceutical compositions comprising said populations in the preparation of medicaments for the prevention or treatment of complications (such as tendon adhesions and heterotopic ossification of tendons) of tendon or ligament injury or defects in subjects of need, wherein said stem / progenitor cells have reduced levels or activity of PIEZO1.

[0187] The present invention also provides a method for screening agents for inducing periosteal-derived stem / progenitor cells to differentiate into tendon cells or form tendon tissue, the method comprising:

[0188] i) Contacting the compound with periosteum-derived stem / progenitor cells, for example, culturing the cells in a culture medium containing the compound;

[0189] ii) Detect the level or activity of PIEZO1 in the cells;

[0190] iii) Compare the level or activity of PIEZO1 with that in control periosteum-derived stem / progenitor cells; and

[0191] iv) Select compounds that reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells.

[0192] The control periosteum-derived stem / progenitor cells are periosteum-derived stem / progenitor cells that have not been exposed to the compound. The compound may be a small molecule compound or a protein. In some embodiments, the compound promotes the differentiation of the periosteum-derived stem / progenitor cells into tendon cells. In some embodiments, the periosteum-derived stem / progenitor cells are isolated cells.

[0193] In some implementations, the detection in step ii) is a detection at the nucleic acid level or the protein level. Examples of nucleic acid level detection include, but are not limited to, quantitative PCR and RNA-seq. Examples of protein level detection include, but are not limited to, Western blotting and ELISA. PIEZO1 activity can also be detected by patch clamp.

[0194] The present invention also provides a method for screening agents for inducing periosteal-derived stem / progenitor cells to differentiate into tendon cells or form tendon tissue, the method comprising:

[0195] i) Applying a compound to the object;

[0196] ii) Isolate periosteal-derived stem / progenitor cells or populations thereof from the subject and detect the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells or populations thereof;

[0197] iii) Compare the level or activity of PIEZO1 in periosteal-derived stem / progenitor cells or their populations isolated from control subjects; and

[0198] iv) Select compounds that reduce the level or activity of PIEZO1.

[0199] The control group is a sample that has not been treated with the compound. The compound may be a small molecule compound or a protein.

[0200] In some implementations, the detection in step ii) is a detection at the nucleic acid level or the protein level. Examples of nucleic acid level detection include, but are not limited to, quantitative PCR and RNAseq. Examples of protein level detection include, but are not limited to, Western blotting and ELISA.

[0201] Beneficial effects of the present invention

[0202] The inventors discovered that periosteum-derived stem / progenitor cells possess excellent proliferative (self-renewal) and differentiation capabilities; they can inhibit PIEZO1 in periosteum-derived stem / progenitor cells, i.e., reduce the level or activity of PIEZO1, thereby enabling these cells to differentiate into tendon tissue. The periosteum-derived stem / progenitor cells provided by this invention can be obtained from the patient's own body and used for transplantation, significantly reducing the risk of rejection during cell / tissue transplantation. Therefore, this invention provides suitable seed cells for tendon or ligament injury repair and offers a broader source of seed cells for stem cell therapy of tendon or ligament injuries. Example

[0203] The examples provided below are for illustrative purposes only and are not intended to limit the scope of this application in any way.

[0204] Example 1, Materials and Methods

[0205] 1.1 Experimental Materials

[0206] 1.1.1 Laboratory Animals

[0207] The mice used in the embodiments of this application were all C57BL6 background mice, and sex-matched littermate mice were used as controls in all analyses.

[0208] Mice were housed at the SPF-grade Laboratory Animal Center of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, while wild-type mice were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0209] Prx1-Cre mice were purchased from Jackson Laboratory.

[0210] The Ai9 mice were provided by Researcher Qiu Zilong from the Institute of Neurobiology, Chinese Academy of Sciences.

[0211] Piezo1 fl / fl mice were purchased from UCDAVIS KOMP.

[0212] The ScxGFP and ScxCre mice were provided by Professor Ouyang Hongwei of Zhejiang University.

[0213] The ScxCreERT2 mice were provided by Professor Zheng Qiujian of Guangdong Provincial People's Hospital.

[0214] The Ocn-Cre mice were provided by Southern Medical University.

[0215] Mice used in the experiment were obtained through hybridization.

[0216] ScxCreERT2;Ai9 mice were induced to take tamoxifen by intraperitoneal injection of 2 mg of tamoxifen dissolved in corn oil, with one day between injections, for a total of three injections over one week.

[0217] 1.1.2 Cell lines

[0218] The 293FT cell line was purchased from the American Terminology Collection Center (CRL-3216) in the United States. It is derived from human embryonic kidney epithelial cells, expresses SV40T antigen, and is used for experiments such as lentiviral packaging and protein overexpression.

[0219] 1.1.3 Experimental Instruments and Analysis Software

[0220] i) Experimental apparatus:

[0221] The Biomomentum MACH-1V500C tendon tensile strength tester is used to test tendon tensile strength.

[0222] Paraffin microtome (Yidi YD335A), Leica paraffin embedding machine, Leica CM3050S cryostat, Thermo NX70 cryostat and hard tissue microtome (Leica RM2265) are used to section tissue samples.

[0223] The Real-time CFX96 PCR instrument is used for real-time quantitative PCR analysis;

[0224] The Nanodrop spectrophotometer is used to determine nucleic acid concentration;

[0225] Perkin Elmer Envision multimode spectrophotometer was used for the quantification of Alamar Blue and ALP activity;

[0226] Olympus BX53 upright fluorescence microscope; Olympus IX73 inverted fluorescence microscope; Olympus FV3000 laser confocal microscope; Leica SP8 STED nanomicroscope and Olympus VS120 fully automated slide scanning system for microscopic imaging;

[0227] The SkyScan 1176 microCT scanner is used for microCT examinations.

[0228] CytoFlex five- or three-laser flow cytometers are used for cell information detection.

[0229] The SONY MA900 or BD Fusion sorter is used for cell sorting.

[0230] Atomic force microscopy (Multimode-8, BRUKER NANO INC) is used to detect the elastic modulus of cortical bone.

[0231] ii) Software used for analysis:

[0232] Primer Premier 5.0 is used for primer design;

[0233] SnapGene Viewer is used for sequence analysis;

[0234] ImageJ is used for image analysis;

[0235] Adobe Photoshop and Adobe Illustrator are used for image processing;

[0236] Microsoft Office and GraphPad are used for graphing;

[0237] CTAn and CTVox are used for the analysis of Micro-CT.

[0238] 1.1.4 Reagents

[0239] The reagents used in the experiment are shown in Table 1.

[0240] Table 1. Reagents used in the experiment

[0241]

[0242]

[0243] 1.1.5 Antibodies

[0244] The antibodies used in the experiment are shown in Table 2.

[0245] Table 2. Antibodies used in the experiment

[0246]

[0247] 1.2 Experimental Methods

[0248] 1.2.1 Molecular Cloning

[0249] For molecular cloning experiments such as polymerase chain reaction, agarose gel electrophoresis, restriction endonuclease digestion, vector ligation, and transformation into *E. coli*, refer to *Molecular Cloning: A Laboratory Manual* (2nd edition, Cold Spring Harbor Laboratory Press) and the relevant reagent instructions. For experiments such as gel recovery of enzyme digested fragments and small / large-scale plasmid extraction, follow the instructions of the corresponding kits.

[0250] 1.2.2 RNA extraction, reverse transcription, and quantitative real-time PCR

[0251] Tissue samples were flash-frozen in liquid nitrogen and then homogenized. Adherent cells were discarded from the culture medium and washed with PBS. 500 μL of Trizol was added for lysis, and the mixture was transferred to a 1.5 mL RNase-Free centrifuge tube and vortexed to ensure complete lysis. 20% (by volume) chloroform of the Trizol solution was added and vortexed thoroughly. After standing for 3-5 min, the mixture was centrifuged at 12000 rpm for 20 min at 4 °C. The aqueous phase was transferred to a new 1.5 mL RNase-Free centrifuge tube, an equal volume of isopropanol was added, and the mixture was vortexed thoroughly and incubated at -20 °C for 3 h. Then, the mixture was centrifuged at 12000 rpm for 20 min at 4 °C, the supernatant was discarded, and the mixture was washed twice (1 mL of pre-chilled 75% ethanol prepared with DEPC water was added, the mixture was inverted and centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was discarded). Afterward, the mixture was briefly centrifuged, and the supernatant was discarded. The mixture was then air-dried at room temperature, dissolved in DEPC water, and the concentration was determined using Nanodrop.

[0252] Reverse transcription was performed according to the reverse transcription kit instructions at 37°C for 20 min and 85°C for 5 s. The reaction mixture (10 μL) contained 2 μL of 5×PrimeScript Buffer; 0.5 μL of PrimeScript RT Enzyme I; 0.5 μL of Oligo dT Primer (50 μM); 0.5 μL of Random Primer; 500 ng of RNA; and DEPC water.

[0253] The real-time qPCR reaction was performed as follows.

[0254] Prepare a 10 μL real-time PCR reaction system containing 2 μL of 5×SYBR Green qPCR reaction solution; 1.0 μL each of forward and reverse primers (5 μM); 0.1 μL of Hotstart Taq enzyme; 0.2 μL of cDNA; and sterile ddH2O.

[0255] After adding samples to a 96-well plate, centrifuge at 1,000g (unless otherwise specified, centrifugation is at room temperature) for 1 min, then place the plate in a real-time PCR instrument and perform the reaction according to the following program: 95℃, 2 min; then 95℃, 15 s; and 60℃, 30 s, for 40 cycles. Using Hprt as an internal control, the relative quantification method (ΔΔCt) was used for calculation.

[0256] 1.2.3. Mouse Genotyping

[0257] First, prepare the Tail lysis buffer by adding 1 mL of 20 mg / mL proteinase K to 50 mL of Tail Lysis Buffer (1 M Tris-Cl (pH 8.5), 0.5 M EDTA, 10% SDS, 5 M NaCl). Mix well and use directly or store at 4°C. Preheat the Tail lysis buffer to 37°C before use. Add 500 μL of Tail lysis buffer to each mouse tail and incubate overnight at 55°C to ensure complete lysis. Add an equal volume of isopropanol and vortex; flocculent DNA precipitates will be visible. Centrifuge at 13000 rpm for 2 min, discard the supernatant, and add 300 μL of TE buffer to dissolve the precipitate and obtain the genomic DNA solution. The PCR reaction mixture (20 μL) was then prepared, containing 10 μL of 2×Taq Mix, 6 μL of ddH2O, 0.8 μL of Primer 1 (10 μM), 0.8 μL of Primer 2 (10 μM), and 2.4 μL of genomic DNA solution. The PCR program was set up based on the primer information for mouse genotyping. PCR products were analyzed by agarose gel electrophoresis.

[0258] 1.2.4 Isolation, digestion, and culture of mouse periosteal cells and tendon cells

[0259] i) The mouse hind limbs were separated using ophthalmic scissors and pointed forceps. The pointed forceps were used to separate the patellar tendon located outside the knee joint and the Achilles tendon located outside the ankle joint from the surrounding connective tissue. The mucosa surrounding the tendons was removed, and the tendons were severed from their connections to the muscles and bones at both ends using scissors. The obtained tendon tissue was then temporarily stored in PBS. The peripheral skeletal muscles of the femur and tibia were removed using scissors to obtain intact long bones without damaging the periosteum, and were then temporarily stored in PBS.

[0260] ii) Heat a 10% low-melting-point agarose gel solution in a microwave oven until it becomes a completely homogeneous liquid. Place the long bone obtained in step i) on absorbent paper to remove moisture, then use pointed forceps to pick it up and evenly coat it with the slightly viscous agarose gel, ensuring the gel coats both ends of the long bone in a spherical shape. Place it in serum-free medium on ice to allow the gel to solidify and solidify at low temperature. Place the coated long bone in a 15 ml centrifuge tube containing 5 ml of digestion solution (antibiotic-free, serum-free MEMα medium containing 1 mg / ml collagenase and 2 mg / ml dispersin II, filtered through a 0.22 μm filter). Place the centrifuge tube in a shaker at 37°C and digest at 100 rpm for 5 min to initially remove residual connective tissue from the periphery. Discard the liquid in the centrifuge tube, add 5 ml of digestion solution again, and digest under the same conditions on a shaker for 40 min, repeating once. Each time, the digestion suspension was separated and neutralized with 5 ml of MEMα medium containing 10% FBS, and then centrifuged (room temperature, 1200 rpm, 5 min).

[0261] Wash the tendon tissue obtained in step i) three times with PBS, soak it in alcohol, and then mince it with ophthalmic scissors. Place the minced tissue in a 1.5 ml centrifuge tube and add 1 ml of digestion solution (antibiotic-free and serum-free MEMα medium containing 1 mg / ml collagenase and 2 mg / ml dispersin II, filtered through a 0.22 μm membrane). Place the centrifuge tube in a 37°C shaker and digest at 100 rpm for 40 min. After no visible particles remain in the centrifuge tube, transfer the digestion suspension to a 15 ml centrifuge tube, add 5 ml of MEMα medium containing 10% FBS for neutralization, and then centrifuge (room temperature, 1200 rpm, 5 min).

[0262] iii) After centrifuging the digestion suspension from step ii), remove the supernatant. Add 2 ml of complete culture medium to each centrifuge tube, resuspend, centrifuge (room temperature, 1200 rpm, 5 min), and remove the supernatant to wash away any residual digestion solution. Resuspend the final precipitate in 2 ml of complete culture medium and transfer it to a culture dish. Incubate at 37°C for 72 h, then observe cell adhesion and proliferation under a microscope.

[0263] 1.2.5 Flow cytometry analysis and cell sorting

[0264] i) Obtain a single-cell suspension of the tissue to be tested according to the method in 1.2.4.

[0265] ii) Filter the cell suspension through a 70 μm filter membrane, then centrifuge at 1000 g for 5 min at 4 °C and remove the supernatant. Add 1 ml of erythrocyte lysis buffer and incubate at room temperature for 5 min to lyse the erythrocytes in the cell suspension. Then add 5 ml of MEMα medium to neutralize the erythrocyte lysis buffer. Centrifuge at 1000 g for 5 min at 4 °C and remove the supernatant.

[0266] iii) Resuspend the cells in 100 μl of flow cytometry buffer (PBS solution containing 2% FBS) for each sample loaded into the centrifuge tube, add the antibody mixture for flow cytometry (prepared according to cell grouping requirements), and mix thoroughly. Incubate the centrifuge tubes on ice in the dark for 20-30 min, mixing thoroughly every 10 min to avoid cell precipitation.

[0267] iv) Add 1 ml of flow cytometry buffer to each tube and mix well. Centrifuge at 1000 g for 5 min at 4 °C. Remove the supernatant and add 250 μl of flow cytometry buffer, then mix well.

[0268] v) Cell information detection is performed using a CytoFlex five-laser or three-laser flow cytometer; or cell sorting is performed using a SONY MA900 or BD Fusion sorter.

[0269] 1.2.6 Lentiviral Packaging and Cell Infection

[0270] 293FT cells were divided into two groups of 2.5 × 10⁻⁶ cells. 6 Inoculate the culture medium at a density of 10 cm per dish into 10 cm diameter dishes and incubate at 37°C for 16-18 h. Then replace the medium with preheated 1 / 10 anti-medium medium (450 mL DMEM + 50 mL FBS + 0.5 mL P / S) and continue incubation for 3 h.

[0271] Cell transfection was performed according to the instructions of the Effectene Transfection Reagent kit. Specifically, 300 μL of EC buffer, 1 μg of target plasmid, 0.2 μg of lentiviral packaging helper plasmid VSVG, and 1 μg of lentiviral packaging helper plasmid Δ8.9 were added to a 1.5 mL centrifuge tube. Finally, 17.6 μL of Enhancer Buffer was added, vortexed to mix, and incubated at room temperature for 5 min. 60 μL of Effectene Reagent was added, vortexed to mix for 5 s, and incubated at room temperature for 5–10 min. 1 mL of 1 / 10 antibiotic culture medium was added and mixed. The mixture was then added dropwise to a culture dish containing 293FT cells and gently mixed. After incubation at 37°C overnight, the culture medium was removed, and 8 mL of pre-warmed 293FT complete culture medium (450 mL DMEM + 50 mL FBS + 5 mL P / S) was added. Incubation continued at 37°C until the afternoon of the following day. The culture supernatant was filtered through a 0.45 μm filter to obtain a solution containing lentivirus.

[0272] Cells to be infected were cultured in culture dishes at 37°C for 24 h. Then, the virus solution was mixed with fresh culture medium at a ratio of 1:4, and polybrene was added to the cells to be infected. The mixture was then incubated at 37°C. The cell seeding density was 5 x 10^5 cells / dish, and the total culture medium volume was 10 ml. 24 h post-infection, the supernatant was removed, and fresh culture medium was added. Fluorescence was observed under a fluorescence microscope 48 h post-infection, or virus-infected cells were screened using a lentiviral selectable marker, puromycin (final concentration 2 μg / mL, until all uninfected control cells died).

[0273] 1.2.7 Osteogenic Differentiation of Mouse Periosteal Cells

[0274] i) Prepare the digestion solution (50 mL MEM-α medium, 50 mg collagenase, 100 mg dispersin II, 1 mL P / S), dissolve in a 37°C water bath, and then filter through a 0.22 μm filter membrane.

[0275] ii) Genotyping of mice aged 4-6 weeks was performed, followed by euthanasia with CO2. The femur and tibia, after muscle removal, were then placed in pre-cooled PBS.

[0276] iii) Wash the trimmed long bones with sterile PBS, then transfer them to 1.5 mL centrifuge tubes. Add 1 mL of preheated digestion solution and incubate on a shaker at 37°C and 275 rpm for 5 min. Remove the digestion solution, then add 1 mL of fresh digestion solution and incubate for 15 min under the same conditions. Transfer the digestion solution to 4 mL of pre-chilled fresh C3H10 medium (10% FBS, 1% P / S in MEM-α medium) and store temporarily at 4°C. Add 1 mL of fresh digestion solution to the centrifuge tube containing the long bones and continue incubating for 30 min under the same conditions. After digestion, mix the digestion solution and combine it with the digestion product stored at 4°C.

[0277] iv) Mix the combined digestion products and centrifuge at 1200 rpm for 3 min at 4 °C. Remove the supernatant, resuspend the pellet in C3H10 medium and count the cells. Transfer the cell suspension to 10 cm culture dishes (1-3*10^6 cells / 10 cm culture dish) and incubate at 37 °C.

[0278] v) Once the cells have adhered and reached confluence, re-digest them with 0.5% trypsin and collect the cells. Then resuspend them in fresh C3H10 medium and dilute to 2.0 × 10⁻⁶. 4 The cell suspension was set at a density of cells / mL. The cell suspension was transferred to a new culture dish and incubated overnight at 37°C. The culture medium was removed and osteogenic differentiation medium (C3H10 medium containing 0.05 mg / mL L-ascorbic acid and 1.08 mg / mL sodium β-glycerophosphate) was added. Fresh osteogenic induction differentiation medium was added every 2 days.

[0279] vi) After 4-7 days of induction, osteoblasts were quantified for Alamar Blue and ALP activity and stained with ALP.

[0280] For quantification of Alamar Blue and ALP activities, 1 / 10 volume of Alamar Blue was added to the culture medium 2 hours before the test, and the mixture was incubated at 37°C for 2 hours. Envision readings were then performed (maximum absorption wavelength 540 nm, maximum emission wavelength 590 nm). The Alamar Blue-containing culture medium was removed, and the plate was washed once with PBS. 50 μL of substrate reaction solution (5 mL of substrate reaction solution contains 4.5 mL ddH₂O + 65 μL 0.5 M Na₂CO₃ + 185 μL 0.5 M NaHCO₃ + 10 μL 1 M MgCl₂ + 1 tablet of phosphatase substrate) was added to each well of a 96-well plate. The plate was incubated at room temperature in the dark for 30 minutes, and Envision readings were performed (maximum absorption wavelength 405 nm).

[0281] For ALP staining of osteoblasts, remove the culture medium and wash the cells with PBS. Fix with pre-cooled 10% neutral formalin solution at room temperature for 5 min, remove the fixative, wash with ddH2O for 10 min, and completely remove the liquid. Add the reaction substrate according to the Beyotime BCIP / NBT alkaline phosphatase colorimetric kit instructions, react at room temperature in the dark for 10-30 min, remove the reaction substrate, wash the cells with ddH2O, and completely remove the liquid. After air drying, image and photograph using an inverted microscope.

[0282] vii) Alizarin red staining was performed at the late osteogenic differentiation stage (21 days after induction) to detect calcium nodule formation. Specifically, the culture medium was removed, and the cells were washed with PBS. The cells were fixed with pre-cooled 10% neutral formalin solution at room temperature for 5 min, the fixative was removed, and the cells were washed with ddH2O for 10 min, and the liquid was completely removed. Alizarin red staining solution was added to the cells, and the cells were incubated at room temperature for 20 min. The reaction substrate was removed, the cells were washed with ddH2O, and the liquid was completely removed. After drying, the cells were imaged and photographed using an inverted microscope.

[0283] 1.2.8 Preparation of GelMA soft and hard matrices and cell culture

[0284] i) GelMA preparation

[0285] Dissolve 10g of gelatin in 100ml of ddH2O, and fully dissolve in a 50℃ water bath. Add 6ml of methacrylate (MA) in a fume hood to obtain a mixture of gelatin and MA. Place the mixture in a 50℃ water bath and stir for about 3 hours until homogeneous, changing from a turbid state to a uniform, viscous white gel-like liquid. Centrifuge the liquid at 3500rpm for 3 minutes, then transfer the supernatant to a beaker, add 5 times the volume of ddH2O, and mix well to dilute. Place the diluted mixture in a dialysis bag (molecular weight cutoff: 14KD) and dialyze in deionized water at 37℃ for one week, changing the water daily. Freeze-dry the dialyzed liquid to obtain a white, fluffy GelMA solid material.

[0286] ii) Preparation of GelMA gel for cell culture:

[0287] Add 0.25% (m / V) of photocrosslinking initiator LAP to 15 ml of deionized water. Add the appropriate mass of GelMA material according to the required concentration (5% or 20%) and dissolve at 60°C. Filter the prepared gel solution through a 0.22 μm filter membrane.

[0288] A non-stick membrane was placed on the curing light source plate, a curing ring with a diameter of 1.5 cm was placed on it, 200 μl of filtered gel solution was added, and the gel was irradiated with ultraviolet light for 1 min to solidify the gel. Then the entire curing ring containing gel was removed from the non-stick membrane and placed into the well of a 24-well plate. After washing twice with PBS, it was used for cell seeding.

[0289] 1.2.9 Transplantation of periosteal cells in a tendon injury model

[0290] i) Obtaining periosteal cells: Obtain periosteal cells from 5-6 week old mice of the desired genotype or wild type according to the method in 1.2.4.

[0291] ii) Modeling of patellar tendon hemisection injury:

[0292] The hair around the knee joint of anesthetized mice was shaved, and the skin around the knee joint was disinfected with 75% ethanol. A longitudinal incision of approximately 5 mm was made in the skin directly above the knee joint using ophthalmic scissors. The transparent mucosal connective tissue surrounding the patellar tendon was removed using pointed forceps to fully expose the tendon. The patellar tendon tissue was then freed using pointed forceps, and the tendon was halved from the medial side using ophthalmic scissors.

[0293] iii) Cell transplantation:

[0294] Thoroughly mix the cells to be transplanted with the melted Matrigel. In a patellar tendon hemisection model, inject 10 μl of cell-containing Matrigel into the space between the patellar tendon and the knee joint using a microsyringe. The gel will overflow and coat the patellar tendon. Allow the Matrigel to solidify completely at room temperature for 5 minutes, then suture the skin.

[0295] 1.2.10. Frozen tissue sections and immunofluorescence staining and imaging

[0296] i) Tissue sampling and pretreatment: Joint tissue was completely immersed in 4% PFA and fixed at 4°C for 48 hours. PBS was then soaked in a shaker at room temperature for 20 minutes, followed by three washes. Decalcification solution (BBI, EDTA decalcification solution, E671001-0500) was used for 3-7 days at room temperature until the hard bone tissue became elastic. Afterward, it was dehydrated overnight in 30% sucrose PBS solution.

[0297] ii) Tissue embedding: After dehydration, absorb excess liquid with absorbent paper and place the tissue into an OCT embedding mold. Cover with plastic wrap to prevent moisture loss from the OCT. Let stand at room temperature for 2 hours.

[0298] iii) After adjusting the position of the tissue in the OCT, freeze the sample at -80°C, and then section it using a cryostat (thickness 16-20 μm).

[0299] iv) Incubate frozen sections in PBS at room temperature for 5 min to wash away OCT. Soak the sample in blocking buffer (PBS containing 10% horse serum and 0.3% Triton X100) and incubate at room temperature for 1 h. Remove the blocking buffer and add primary antibody diluted with the blocking buffer; the dilution concentration depends on the antibody's instructions. Incubate overnight at 4°C. Remove the primary antibody and immerse the sections in PBS at room temperature for 15 min; repeat three times. Incubate the sample with a 1:1000 dilution of fluorescently labeled secondary antibody at room temperature for 1 h. Remove the secondary antibody and incubate the sample with a 1:1000 dilution of DAPI staining solution at room temperature for 5 min.

[0300] v) Remove the DAPI staining solution and soak the sections in PBS, incubate at room temperature for 15 min, repeat three times. Remove moisture around the sample, then mount with fluorescent mounting medium. Perform fluorescence imaging using confocal microscopy.

[0301] 1.2.11 Sample Preparation and Data Analysis for Single-Cell Sequencing

[0302] As described in 1.2.4, from 8-week-old Prx1 Cre; Ai9 / + and Prx1 Cre; Piezo1 fl / fl Single-cell suspensions of the periosteum were obtained from Ai9 / + mice. Prx1-positive cells were sorted using a BD Aria Fusion flow cytometer. After AO / PI staining, cell viability was assessed using a Countstar Fluorescence Cell Analyzer. The single-cell suspensions were finally captured using a BD Phapsody system.

[0303] Labeled and amplified cDNA libraries were obtained from lysed single cells processed using a microwell system. After processing with a highly sensitive DNA microarray and quantification, sequencing was performed in an Illumina system.

[0304] Initial data presentation was completed by NovelBio and the NovelBrain cloud platform. Data cleaning was performed using a FastQ workflow with default parameters. The cleaned UMI-based data was then aligned with the STAR genome.

[0305] 1.2.12. Sorting of periosteal cells and renal capsule transplantation

[0306] Periosteal cells were obtained from 5-6 week old ScxCre;Ai9 / + mice and wild-type mice without fluorescent labels, as described in 1.2.4.

[0307] The cells were resuspended in liquid Matrigel, with 10 μl of cell suspension injected into one kidney of each mouse, containing approximately 20,000 cells.

[0308] Nude mice (Lingchang Company) aged 8-10 weeks were anesthetized. A 5-10 mm longitudinal incision was made in the skin above the kidney on the dorsal side to expose the kidney. A 1-2 mm opening was made in the renal capsule. Matrigel containing cells was then injected into the renal capsule using a microsyringe. The kidney was then returned to the abdominal cavity. The muscle and skin wounds were sutured in sequence.

[0309] 1.2.13. Micro-CT Scanning and Analysis of Mouse Bone Tissue

[0310] Mice were euthanized with CO2, and bone tissue from the desired sites was then preserved by immersion in 70% ethanol. Scanning and analysis were performed using a SkyScan 1272 microCT instrument with a resolution of 9-18 μm. The analysis region for cancellous bone was a 2 mm area extending downwards from 0.7 mm below the end of the growth plate; the grayscale threshold ranged from 80 to 255. The analysis region for cortical bone was a 0.5 mm area extending downwards from 4.7 mm below the end of the growth plate; the grayscale threshold ranged from 120 to 255. After reconstruction, analyzable 2D images were obtained from the original images. CTAn software was used for 2D image analysis and 3D reconstruction, and CTVox software was used to obtain the 3D reconstructed tissue model.

[0311] 1.2.14 Paraffin sections

[0312] Bone samples were fixed in fresh 4% PFA at 4°C for 24-48 h. They were washed three times with PBS and then decalcified in decalcification solution (see 1.2.10). After washing three times with PBS, they were soaked overnight in 75% ethanol, followed by dehydration and paraffin infusion sequentially in 95% ethanol (1 h × 2 times), 100% ethanol (1 h × 2 times), xylene (1 h × 2 times), 1 / 2 xylene / 1 / 2 paraffin (30 min), and paraffin (30 min × 2 times). The samples were then embedded using a paraffin embedding machine. Paraffin sections were 5-8 μm thick and incubated overnight at 42°C.

[0313] 1.2.15. Detection of the elastic modulus of cortical bone using atomic force microscopy

[0314] Femurs were harvested from 8-week-old wild-type mice, and peripheral skeletal muscle and bone marrow were removed. The cortical bone was trimmed into pieces approximately 1 mm in length and width using ophthalmic scissors and fixed to the sample platform with double-sided tape, with the periosteal side facing upwards towards the probe. The elastic modulus of the cortical bone surface was measured using a liquid chromatography method under atomic force microscopy, after obtaining a stable mechanical curve. At least five measurements were repeated at the same site within a 10-20 μm radius to obtain the average value.

[0315] 1.2.16. Safranin O staining

[0316] Paraffin sections were placed at 65°C for 30 min, then dewaxed and rehydrated sequentially with xylene, 100% ethanol, 95% ethanol, 75% ethanol, and ddH2O, 5 min x 2 times per step, followed by washing with PBS for 10 min. Next, the sections were stained with 0.05% Fast Green dye for 2 min, differentiated with acetic acid for 10 s, stained with 0.5% safranin solution for 1 min, differentiated with 95% ethanol for 10 s, and then rinsed with running water. Subsequently, they were dehydrated sequentially with 75% ethanol, 95% ethanol, 100% ethanol, and xylene, air-dried, and mounted with neutral resin.

[0317] 1.2.17 Tendon Strength Test

[0318] The Achilles tendon sample with the tibia and hind limb foot attached (after cutting the connection between the Achilles tendon and skeletal muscles such as the gastrocnemius muscle) was removed and then soaked in PBS without fixation.

[0319] A 100N sensor was installed on the Biomomentum MACH-1V500C tendon tension meter. The fixation assembly was installed below the sensor. The zero-point position was located using the software's "find contact" function. The basic opening length was set to 10μm. The distance between the upper and lower fixation units was manually controlled using the manual control function. The calcaneus was secured to the foot using the lower fixation unit. Approximately 20μl of bio-adhesive was added to the upper fixation unit to secure the severed tendon end. Starting from the zero point (where the tissue is not under tension), the upper and lower fixation units were slowly pulled, and the force at which the tendon ruptured was recorded.

[0320] 1.2.18 Immunofluorescence of paraffin sections

[0321] Paraffin sections were incubated at 65°C for 2 hours, followed by a gradient dewaxing and rehydration with xylene, 100%, 95%, 70% ethanol, and ddH2O, 5 min x 2 per step, and then washed with PBS for 10 min. The sections were then heated in a 0.01 M sodium citrate buffer (pH 6.0) at 95°C for 20 min or incubated in a 10 μg / ml proteinase K solution (50 mM Tris-HCl, 5 mM EDTA, pH 8.0) at 37°C for 15 min. The sections were washed three times with PBS for 5 min each time. The samples were then immersed in antibody blocking buffer and incubated at room temperature for 45-60 min. The primary antibody was diluted with antibody dilution buffer according to the product instructions and incubated overnight at 4°C. The sections were washed three times with PBS for 10 min each time. A fluorescently labeled secondary antibody (1:1000) diluted with PBS corresponding to the primary antibody was added and incubated at room temperature in the dark for 1 hour. The sections were washed three times with PBS for 10 min each time. Stain with DAPI (1:1000 dilution) at room temperature in the dark for 10 min. Wash twice with PBS, mount with fluorescent mounting medium, and store at 4°C in the dark until photographed using a fluorescence microscope or confocal microscope.

[0322] 1.5 mL antibody blocking solution: 150 μL non-primary antibody species serum, 90 μL 5% Triton X-100, 1260 μL PBS.

[0323] 1.5 mL antibody dilution buffer: 75 μL non-primary antibody species serum, 90 μL 5% Triton X-100, 1335 μL PBS.

[0324] 1.2.19. Preparation of the mouse femoral drilling model

[0325] Mice were anesthetized by intraperitoneal injection of chloral hydrate (400 mg / kg). Exposure of the lateral femur: After anesthesia, the hair around the femur and tibia was shaved, and the skin surface was wiped with alcohol swabs. A 5-10 mm longitudinal incision was made along the femur, and the skeletal muscles on the anterior and posterior sides of the femur were separated with pointed forceps to expose the lateral femur. While fixing the femur with curved forceps, a penetrating injury was made to the mid-section of the lateral cortical bone using a 0.5 mm diameter drill, creating a regular circular defect. The drill was immediately withdrawn after penetrating the cortical bone. A small amount of bleeding was absorbed with dry cotton balls, and the muscles and skin were sutured. The mice were placed in warm cages and, after recovery, transferred to IVC cages for culture. Postoperatively, micro-CT (SKYSCAN 1272, Bruker) was used to detect new bone formation at the drill site on day 10.

[0326] 1.2.20 Statistical Methods

[0327] All data in the figures and tables of this paper are expressed as mean ± standard deviation (mean ± SD). Statistical tests: Student's t-test was used for comparisons between two groups. One-way analysis of variance (ANOVA) was used for comparisons of multiple groups, followed by Tukery's posthoc test. In all data, "*" represents P < 0.05, and "**" represents P < 0.01, indicating a significant difference.

[0328] Example 2: Piezo1 affects the osteogenic capacity of periosteal osteoblasts / progenitor cells.

[0329] 6-week-old Prx1 Cre Piezo1 fl / fl With Ocn Cre Piezo1 fl / fl Micro-CT scan analysis of the femur of mice (CKO mice) showed that, compared with wild-type controls, Prx1 Cre Piezo1 fl / fl The degree of bone loss in the cortical femur of mice was greater than that in Ocn CrePiezo1 fl / fl Mice severely ( Figure 1 A, B). However, there was no significant difference in cancellous bone between the wild-type control and CKO mice. Figure 1 Bone formation marker experiments showed that the loss of Piezo1 primarily affected cortical bone mass at the periosteal level rather than the endoosteal level. Figure 1 HJ).

[0330] By separating WT and Prx1 Cre Piezo1 fl / fl Osteogenic differentiation of mouse periosteal cells, alkaline phosphatase activity of differentiated cells, and Alizarin Red staining revealed that Piezo1 deficiency inhibited the osteogenic differentiation capacity of periosteal osteoblasts / progenitor cells. Figure 2 AE).

[0331] For Prx1 Cre Piezo1 fl / fl and Ocn Cre Piezo1 fl / fl Statistical analysis of spontaneous fractures of long bones in mice showed that Prx1 Cre Piezo1 fl / fl The spontaneous fracture rate of long bones in mice exceeded 90%, while Ocn Cre Piezo1 fl / fl Spontaneous fractures are almost nonexistent in the long bones of mice. Figure 3 A, B).

[0332] Separate Prx1 Cre Ai9 / + and Prx1 Cre Piezo1 fl / fl Prx1+ cells were extracted from the periosteal cells of Ai9 / + mice, and single-cell transcriptome sequencing, cell population, and pseudo-time series analysis were performed. Results showed that Piezo1 deletion arrested periosteal skeletal stem / progenitor cells at the stem / progenitor stage. Figure 4 AE and Figure 5 AC).

[0333] Example 3: Piezo1 inhibits Scx expression in periosteal cells via the YAP signaling pathway

[0334] 3.1 PIEZO1 regulates SCX expression via the YAP pathway

[0335] Analysis of single-cell transcriptome data showed that the cluster 0 cell population is likely a stem / progenitor cell group of the periosteum, and the loss of Piezo1 increased the number of this cell group. Analysis of the top 10 significantly altered genes revealed that Scx was the gene most upregulated after piezo1 loss. Figure 6 AC). RT-qPCR analysis of Scx gene expression in periosteal cells also showed that Scx gene expression was significantly upregulated after Piezo1 deletion. Figure 6 D). Conversely, activation of Piezo1 in periosteal cells using Yoda1 (MCE, HY-18723) significantly reduced Scx expression. Figure 6 E). For WT ScxGFP and Prx1 Cre Piezo1 fl / fl Histological imaging of femur sections from ScxGFP mice also showed increased Scx expression after Piezo1 knockout. Figure 6 F). Flow cytometry analysis of periosteal cells showed that after Piezo1 deletion, the number of Ocn-positive cells in Lin- cells was significantly reduced, while the number of ScxGFP-positive cells was significantly increased. Figure 6 G, H) indicates a decreased level of cellular osteogenic calcification. The MST1 / 2 inhibitor XMUMP1 promotes YAP nuclear translocation, while XMUMP1 treatment of periosteal cells inhibits Piezo1-induced upregulation of Scx expression (G, H). Figure 6 I). Treatment with lentiviruses overexpressing YAP significantly upregulated Scx expression in periosteal cells. Figure 6 J).

[0336] 3.2 Overexpression of SCX in periosteal cells inhibits osteogenic differentiation

[0337] Periosteum cells were overexpressed via lentiviral infection. Results showed that, compared to control cells, periosteal cells overexpressing Scx exhibited significantly reduced osteogenic differentiation capacity. Figure 7 AD).

[0338] Example 4: SCX can label periosteal stem / progenitor cell populations

[0339] 4.1 Analysis of the properties of SCX-positive cells in the periosteum

[0340] For Scx Cre Imaging analysis of Ai9 / + mouse tissue sections revealed that SCX-Cre was able to label cells in the periosteal membrane ( Figure 8A). Simultaneously, flow cytometry analysis and tissue section imaging of periosteal cells revealed that both ScxGFP-positive and negative cell populations contained three skeletal stem / progenitor cell populations (SSC: CD105-CD200+; Pre-BCSP: CD105-CD200-; BCSP: CD105+). 17 ( Figure 8 BF).

[0341] 4.2. SCX-Cre labeled periosteal cells have osteogenic capacity.

[0342] To further demonstrate that Scx-labeled cells have the ability to label skeletal stem / progenitor cells, Scx CreERT2 In Ai9 / + mice, Lin-Scx+ cells from the periosteal membrane were sorted by flow cytometry after induction and then transplanted into the renal capsule of nude mice using a renal capsule transplantation model. Figure 9 A). Two months after cell transplantation, kidney examination showed that Scx+ cells had the ability to form bone and medullary cavities within the renal capsule. Figure 9 (B, C). This further illustrates that the Lin-Scx+ cell population in the periosteum contains skeletal stem / progenitor cells.

[0343] Example 5: Piezo1 helps maintain bone formation of SCX+ periosteal skeletal stem / progenitor cells.

[0344] 5.1, SCX Cre Piezo1 fl / fl Mice had significantly lower bone mass than controls.

[0345] With Scx Cre Piezo1 fl / fl Mice were used as a model to study the response of periosteal Scx+ cells to mechanical stimulation. Six-week-old mice were used as a model. Cre Piezo1 fl / fl Miro-CT analysis of the femur in mice and controls showed that Piezo1 deficiency in SCX+ cells led to a significant decrease in cortical bone mass, while trabecular bone showed no significant change. Figure 10 AG).

[0346] 5.2 Bone injury repair requires the expression of Piezo1 in SCX+ cells.

[0347] Previous experimental results have shown that the loss of Piezo1 in periosteal cells upregulates Scx expression. Immunofluorescence staining of OCN in osteoblasts of fluorescent reporter mice is consistent with previous results, and OCN expression is regulated by Scx. Cre Less expression in Ai9 / + cells ( Figure 11 AC). WT and Scx Cre Piezo1 fl / fl A mouse model of femoral drill-hole injury revealed that the absence of Piezo1 in SCX+ cells led to a decrease in the rate of bone regeneration during the repair process. Figure 11 DG).

[0348] 5.3. Periosteal SCX+ cells participate in periosteal development and cortical bone injury repair.

[0349] Using inducible fluorescent labeling of mice ( Figure 12 A) Cell tracing of Scx+ cells in the periosteum under early and adult non-injury and post-adult injury conditions revealed the presence of Scx+ cells in the periosteum during both early development (P7) and adulthood (P42). Figure 12 B, C). SCX+ cells derived from the periosteum can participate in bone regeneration after injury. Figure 12 DF).

[0350] Example 6: Piezo1-deficient or inhibited periosteal stem / progenitor cells can mediate tendon tissue regeneration.

[0351] 6.1. Piezo1-deficient periosteal stem / progenitor cells promote patellar tendon injury repair.

[0352] As shown in Example 2, Piezo1 deficiency arrests periosteal cells at the stem / progenitor stage and upregulates Scx expression in periosteal cells, suggesting that Piezo1-deficient periosteal cells have a stronger tendonogenic capacity compared to the control. The tendonogenic capacity of digested and sorted control cells and Piezo1-deficient periosteal cells can be tested in a tendon injury repair model. Figure 13 A). Cell-free transplantation and transplantation of WT and Prx1 respectively. Cre Piezo1 fl / fl Statistical analysis of ectopic osteogenic formation in the patellar tendon of mice using Prx1+ cells. Results showed that in cell-free transplanted mice, ectopic ossification occurred at the patellar tendon site two months after hemisection injury, and transplantation of WT periosteal Prx1+ cells yielded similar results. However, mice transplanted with Piezo1-deficient periosteal cells showed almost no ectopic ossification following patellar tendon injury. Figure 13 B, C). Gait and tensile strength tests on transplanted tendon tissue yielded results consistent with heterotopic ossification, indicating that Piezo1-deficient periosteal cells significantly optimized gait and tendon tensile strength in mice with patellar tendon injury, and there was no significant difference between mice treated with Piezo1-deficient periosteal cells and sham-operated controls. Figure 13 DF).

[0353] 6.2 Detection of cell properties when periosteal cells are transplanted into injured tendons

[0354] Fluorescently labeled periosteal cells, after transplantation into damaged tendon tissue, exhibit different morphologies and express different tissue markers due to changes in their cellular properties. (Prx1 transplantation) Cre Ai9 / + or Prx1 Cre Piezo1 fl / fl Prx1+ cells from Ai9 / + mice were used to repair a patellar tendon hemisection injury model. Immunofluorescence staining of the repaired tendon tissue sections showed that the transplanted wild-type periosteal cells highly expressed cartilage-related markers, and the cell morphology also exhibited polygonal or nearly spherical chondrocyte shapes. In contrast, Piezo1-deficient cells highly expressed tendon tissue markers, and the cell morphology also showed a neatly arranged fibrous structure. Figure 14 AH).

[0355] 6.3. Piezo1 inhibitor treatment can promote tendon repair by stimulating periosteal cells.

[0356] Periosteal cells from MTMG;ScxGFP mice were treated with the Piezo1 inhibitor GsMTx4 and then transplanted into the injured tendon. Figure 15 A) Micro-CT analysis of repaired tendons showed that treatment with Piezo1 inhibitors significantly inhibited ectopic osteoogenesis during tendon repair. Figure 15 B, C). Sectional analysis of the repaired tendon tissue showed that after Piezo1 inhibition, periosteal cells were more likely to form tendon tissue, while in the control group, periosteal cells were more likely to form bone tissue. Figure 15 DK).

[0357] Similar conclusions were obtained from transplantation and property analysis using periosteal cells isolated from and cultured from the human body. Legs transplanted with Piezo1-inhibited periosteal cells had a larger contact area and contact pressure when walking, demonstrating better tendon recovery in vivo. Figure 15 In summary, inhibition of Piezo1 can encourage periosteal stem / progenitor cells to participate in tendon tissue formation rather than osteogenic formation, thereby promoting tendon repair.

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Claims

1. A pharmaceutical composition comprising: i) Isolated stem / progenitor cells derived from the periosteum, wherein said cells have reduced PIEZO1 levels or activity; and ii) Pharmaceutically acceptable carriers.

2. The pharmaceutical composition of claim 1, wherein the periosteum is a human periosteum.

3. The pharmaceutical composition of claim 1 or 2, wherein the cells have the ability to differentiate into tendon cells.

4. The pharmaceutical composition of any one of claims 1-3, wherein the stem / progenitor cells are SCX+ cells.

5. The pharmaceutical composition of any one of claims 1-4, wherein the stem / progenitor cells have an increased SCX level.

6. The pharmaceutical composition of claim 5, wherein the cells are expanded in vitro.

7. The pharmaceutical composition of any one of claims 1-6, wherein the pharmaceutical composition is formulated for topical application.

8. A method for inducing differentiation of periosteal-derived stem / progenitor cells into tendon tissue, or for preparing a pharmaceutical composition for treating tendon or ligament injuries or defects in subjects of need, said method comprising: a) Provide a cell pool from the periosteum containing stem / progenitor cells derived from the periosteum; b) Reduce the level or activity of PIEZO1 in the periosteal-derived stem / progenitor cells; and c) Optionally, amplify the cells obtained in step b) in a culture medium.

9. The method of claim 8, wherein the periosteum is a human periosteum.

10. The method of claim 9, wherein the cells obtained in step b) have the ability to differentiate into tendon cells.

11. The method of any one of claims 8-10, wherein the stem / progenitor cells are SCX+ cells.

12. The method of any one of claims 8-11, wherein the stem / progenitor cells have an increased SCX level.

13. Use of a pharmaceutical composition of any one of claims 1-7 or a pharmaceutical composition prepared by any one of claims 8-12 in the preparation of a medicament for treating tendon or ligament injury or defect in a subject in need.

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