Method for inducing fibroblasts to be reprogrammed into osteoblasts

Abstract

The invention belongs to the technical field of cell small molecule compound reprogramming and bone tissue engineering material culture, and discloses a method for inducing fibroblasts to be reprogrammed into osteoblasts. According to the invention, small molecule compounds act on fibroblasts, each small molecule is endowed with characteristics of a set of characteristic gene expression mode, and the small molecules are divided into different modules according to the expression characteristic mode, so that the fibroblasts of the cells are converted into osteogenic differentiation, the osteogenic differentiation effect is enhanced and maintained, and the in-vivo development of the cells is simulated to a great extent; therefore, healthy osteoblasts can be prepared from the skin fibroblasts, cell stress pressure can be resisted, and the prepared cells are free of tumorigenicity, safe and healthy. The osteoblast disclosed by the invention can be applied to preparation of bone materials and medicines for treating bone defects, for example, the osteoblast and a traditional bone material are used for constructing an active bone tissue engineering material, so that the use of allogeneic bones commonly used in clinic at present is avoided, and the occurrence of adverse ethical events is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell small molecule compound reprogramming and bone tissue engineering material cultivation, and specifically relates to a method for inducing fibroblasts to reprogram into osteoblasts. Background Art

[0002] Skeletal aging has received extensive attention due to its high morbidity and serious health consequences. Although a certain degree of repair capacity is maintained in adulthood, bone regeneration capacity decreases significantly during aging. Autologous bone transplantation, as a traditional method for treating bone defects, has limited availability and cannot meet clinical needs. Autologous cell or tissue engineering bone treatments have not yet achieved the necessary major breakthroughs to meet clinical needs. Therefore, age-related bone diseases, such as osteoporotic fractures, remain difficult to treat. Osteoblasts are ideal candidates for treating bone diseases and engineering active materials because of their key role in bone formation; however, these cells and their source cells, bone and mesenchymal stem cells, show dysfunction with age.

[0003] Direct conversion of human fibroblasts into osteoblasts by small molecules has been reported. However, the application of these induced osteoblasts in bone defect repair has lagged behind. Although these cells contribute to mineralization and bone matrix production in bone defect repair, their survival rate is generally poor and their impact on endogenous cells after transplantation is limited. In addition, their dependence on material-based support may induce risks such as inflammation. Specifically, these cells may not be able to fully replicate the characteristics of natural osteoblasts in vivo, including interaction with the in vivo microenvironment, proper secretion of extracellular matrix proteins, and promotion of local angiogenesis; and the number of cells is limited. Summary of the invention

[0004] In view of the shortcomings and deficiencies in the prior art, the present invention aims to provide a method for inducing fibroblasts to reprogram into osteoblasts. The osteoblasts prepared by the present invention can be mass-produced with induced osteoblasts (iOBs) having the functional characteristics of young natural osteoblasts, regardless of the age of the donor, from a small amount of skin tissue (e.g., 1 cm 2 ) skin biopsy can obtain no less than 2 × 1011 iOBs with good functional activity, which is sufficient to repair no less than 1500cm 3 The osteoblasts prepared by the technology of the present invention survive well in vivo and in vitro, can differentiate into mature osteoblasts, have higher self-bone formation ability, secrete a variety of cytokines or matrix proteins, such as VEGF, COL1, IBSP, Osteocalcin, etc., and promote the osteogenesis of aging mesenchymal stem cells, promote the proliferation, angiogenesis and migration of vascular endothelial cells, and accelerate endogenous osteogenesis and local angiogenesis in vivo.

[0005] The first aspect of the present invention provides a method for inducing fibroblasts to reprogram into osteoblasts, comprising:

[0006] Placing the fibroblasts in an induction medium containing the small molecule composition for 1-30 days to obtain induced osteoblasts, and then replacing the culture medium and continuing to culture to obtain osteoblasts;

[0007] The small molecule composition adopts any of the following:

[0008] CHIR-99021+Forskolin;

[0009] CHIR-99021+Forskolin+Repsox;

[0010] CHIR-99021+Forskolin+Repsox+Valproic acid;

[0011] CHIR-99021+Forskolin+Repsox+Valproic acid+TRICHOSTATINA;

[0012] CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983;

[0013] CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125;

[0014] CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125+EPZ004777;

[0015] Repsox+EPZ004777;

[0016] Repsox+EPZ004777+bFGF.

[0017] Optionally, the placing of the fibroblasts in an induction medium containing a small molecule composition for treatment for 1-30 days is replaced by: placing the fibroblasts in an induction medium containing a small molecule composition for treatment in the first stage for 1-30 days and in the second stage for 1-30 days; or in the first stage for 1-30 days, the second stage for 1-30 days and the third stage for 1-30 days; or in the first stage for 1-30 days, the second stage for 1-30 days, the third stage for 1-30 days and the fourth stage for 1-30 days;

[0018] The small molecule composition used in the first stage, the second stage, the third stage and the fourth stage is any of the following, or the small molecule composition is not added during the treatment process of one of the stages, and the small molecule composition used in each stage is different:

[0019] CHIR-99021+Forskolin+Repsox+Valproic acid+TRICHOSTATINA;

[0020] CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125+EPZ004777;

[0021] CHIR-99021+Forskolin+Repsox+Valproic acid;

[0022] Repsox+EPZ004777;

[0023] Repsox+EPZ004777+bFGF.

[0024] Preferably, in the induction medium containing the small molecule composition, the concentration of the small molecules is: CHIR-99021: 0.1-30 μM, Forskolin: 0.1-100 μM, Repsox: 0.1-10 μM, Valproic acid: 1-5000 μM, TRICHOSTATINA: 10 nM-10 μM, GO 6983: 0.1-50 μM, SP600125: 0.1-100 μM, EPZ004777: 0.1-50 μM, bFGF: 0.1-400 ng / mL.

[0025] Preferably, the fibroblasts are isolated from mammalian skin, for example, from the leg or abdominal skin of healthy donors of different ages.

[0026] Preferably, the separated fibroblasts are expanded in vitro to a generation before P30 for subsequent processing.

[0027] Preferably, the induction medium containing the small molecule composition further comprises DMEM or mesenchymal stem cell medium containing 1-20% fetal bovine serum.

[0028] Optionally, 100 U / mL penicillin and 100 μg / mL streptomycin antibiotics may also be added to the induction medium containing the small molecule composition.

[0029] Preferably, the step of replacing the culture medium and continuing the culture to obtain osteoblasts comprises:

[0030] The treated cells are cultured in a mesenchymal stem cell culture medium or a DMEM culture medium containing 1-20% fetal bovine serum for cell expansion to obtain a desired number of cells;

[0031] or

[0032] The treated cells were cultured in osteogenic medium to promote cell osteogenic maturation. The osteogenic medium contained DMEM containing 1-20% fetal bovine serum and supplemented with 10 mM sodium β-glycerophosphate and 0.2 mM L-ascorbic acid.

[0033] Preferably, the step of promoting osteogenic maturation of cells is performed by culturing in a humidified environment at 37° C. and 5% CO 2 for 0-100 days.

[0034] Optionally, 10-100 nM dexamethasone is also added to the osteogenic medium.

[0035] Optionally, the above-mentioned DMEM can be high-glucose DMEM or low-glucose DMEM; the mesenchymal stem cell culture medium can be a self-prepared or commercially available product.

[0036] The second aspect of the present invention provides osteoblasts obtained by the above method of inducing fibroblasts to reprogram into osteoblasts.

[0037] The third aspect of the present invention provides the use of the above-mentioned osteoblasts in the preparation of bone materials and drugs for treating bone defects.

[0038] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0039] (1) Currently, the sources of autologous or elderly autologous cells are limited, the quantity is insufficient, and the cell quality is poor (for example, previous literature only mentioned that the cells can promote bone mineralization, but cannot survive well in the body and cannot effectively interact with endogenous cells; the obtained osteoblasts do not have the characteristics of young and healthy cells). The osteoblasts prepared by the method of the present invention are not limited by the age of the donor, and even the elderly can obtain autologous functional osteoblasts.

[0040] (2) The present invention uses small molecule compounds to act on fibroblasts, giving each small molecule a set of characteristic gene expression pattern characteristics, and classifying the small molecules into different modules according to the expression characteristic patterns, so that the cells are diverted from fibroblast differentiation to osteoblast differentiation, the osteogenic differentiation effect is enhanced and maintained, and the cell development in vivo is simulated to a great extent, so that healthy osteoblasts can be prepared from skin fibroblasts and can resist cell stress.

[0041] (3) The cells prepared by the present invention are non-tumorigenic and safe and healthy.

[0042] (4) The osteoblasts of the present invention can be used to prepare bone materials, for example, to construct active bone tissue engineering materials together with traditional bone materials, thereby avoiding the use of allogeneic bones currently commonly used in clinical practice and reducing the occurrence of adverse ethical events; the cells themselves can also construct bone materials without the need for additional bioactive substances. The osteoblasts of the present invention or bone materials prepared by osteoblasts can be used to repair bone tissue damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 This is a schematic diagram of Example 46, in which skin fibroblasts are treated with small molecules and reprogrammed into osteoblasts.

[0045] Figure 2 The changes in cell morphology during the induction of the chemical mixture in Example 46, Bar = 100 μm.

[0046] Figure 3 This is the osteogenesis assay of induced osteoblasts (iOBs) in Example 46, Bar=100 μm.

[0047] Figure 4 Alizarin red staining of calcium deposition in osteoblasts induced by different molecular combinations in Examples 12, 13, and 14 at different treatment time points, Bar=100 μm.

[0048] Figure 5 The time-sequential effects of small molecule combinations on reprogramming in Examples 46, 47, 48, 49, 50, and 51 are shown. Alizarin red staining shows calcium deposition induced by treatment with different time-dependent chemical combinations. Bar=100 μm.

[0049] Figure 6 Alizarin red staining of calcium deposition in osteoblasts induced by different molecular combinations and staged treatments of Examples 1-4, 15-19, 27-45, 52-63 at different treatment time points.

[0050] Figure 7 The characteristics of induced osteoblasts from donors of different ages in Example 46;

[0051] (A) Alizarin red staining of iOBs and their parent fibroblasts derived from bone marrow mesenchymal stem cells (bMSCs, 31 years old), 8-year-old and 79-year-old donors after 14 days of culture in osteogenic medium; n ≥ 3 independent experiments; Y, young refers to individuals under 65 years old; O, old refers to individuals 65 years old and above, Bar = 100 μm;

[0052] (B) mRNA levels of ALP, OCN, SATB2, and IBSP after 14 days of culture in osteogenic medium; Y-bMSCs were derived from donors aged 26, 31, and 33 years; Y-Fib or Y-iOBs were derived from donors aged 8, 38, and 39 years; O-Fib or O-iOBs were derived from donors aged 67, 70, 74, 75, and 79 years; gene expression is expressed as log2 (fold change);

[0053] (C) Cell numbers of starting fibroblasts and resulting iOBs after 18 days of treatment;

[0054] (D) Schematic diagram of the extent of cell expansion during the conversion from skin fibroblasts to iOBs;

[0055] (E) Secretion of COL1 and VEGF in Y-bMSC-OB, iOBs and their parent fibroblasts; immunofluorescence images of Y-bMSC-OB are from a 31-year-old donor, and O-iOBs and their parent O-Fib are from a 79-year-old donor, Bar = 100 μm;

[0056] (F) SA-β-gal activity of Y-bMSC-OB, O-bMSC-OB, Y-iOB from a 39-year-old individual and O-iOB from a 79-year-old donor; n ≥ 3 independent experiments, Bar = 100 μm;

[0057] (G) IL-6 secretion in Y-bMSC-OBs, O-bMSC-OBs, Y-iOBs, and O-iOBs;

[0058] (H) mRNA levels of CDKN1A and CDKN2A in osteoblasts extracted from bMSCs and iOBs of young or old individuals; gene expression is expressed as log2 (fold change).

[0059] Figure 8 This is the staining of osteoblasts induced by Example 46 to differentiate into osteoblasts;

[0060] (A) Immunofluorescence staining of VEGF (red); Bar = 500 μm in the intact femur image; Bar = 200 μm in the high-resolution image;

[0061] (B) Immunofluorescence staining of CD31 (red) at the injury site; Bar = 500 μm in the intact femur image; Bar = 200 μm in the high-resolution image;

[0062] (C) Regeneration at the injury site is shown by IBSP staining (yellow); Bar = 500 μm in the intact femur image; Bar = 100 μm in the high-resolution image;

[0063] (D) Representative images and quantitative analysis of GFP- and IBSP-positive cells in bone defects.

[0064] Fig. 9 μCT (micro-CT) imaging of the femur of a NOD-SCID mouse with femoral defect transplanted with fibroblasts, bone marrow mesenchymal stem cells and iOBs according to Example 46.

[0065] Fig.10 Example 46 shows SafO / FastG staining for bone remodeling 4 weeks after surgery and Alizarin red staining for observing ossification of the femoral bone injury tissue with iOB transplantation; SafO / FastG staining shows bone remodeling 4 weeks after surgery, and Alizarin red staining is used to observe ossification of the femoral bone injury tissue with iOB transplantation, Bar=100μm.

[0066] Fig.11 This is the observation of teratoma within 10 days after subcutaneous injection of iOB in Example 46. The teratoma formation test result of iOB was negative; HEK293T injection was used as a positive control, and teratoma was formed within 10 days after subcutaneous injection; Bar=1cm in the subcutaneous transplantation area, Bar=100μm in the H&E staining image. DETAILED DESCRIPTION

[0067] The embodiments of the present invention will be clearly and completely described below in conjunction with the examples, and the described examples are only some examples of the present invention, rather than all examples. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0068] Unless otherwise specified, the instruments and materials used in the following examples are all routinely used in laboratories, and the technical solutions described are all routine techniques in the art.

[0069] Example 1

[0070] 1. Isolate skin fibroblasts from skin tissue. Primary fibroblasts are obtained from the leg or abdominal skin of healthy donors of different ages with a size of about 0.2 to 5 cm. 2The skin was obtained from the skin of the patient. The dermis was cleared of hair, capillaries, and subcutaneous fat using a scalpel and ophthalmic forceps. The skin sample was cut into 1 mm 2 Small pieces of the culture medium were inoculated on a coated 10 cm culture dish (Corning) and cultured aseptically at 37°C in a 5% CO2 environment to allow skin fibroblasts to migrate out of the skin tissue. The fibroblast culture medium consists of DMEM (Dulbecco's Modified Eagle Medium, manufacturers: Corning, Cyagen, Gibco, sigma, Procell), 10% fetal bovine serum (FBS, manufacturers: ExCell, Hyclone, CellBox, Thermo), 100 U / ml penicillin and 100 μg / ml streptomycin antibiotics (these two antibiotics can be added or not). The obtained cells were expanded in vitro, and each generation was passaged every 2 to 7 days. The cells could be used for subsequent compound treatment before the P30 generation.

[0071] 2. When inducing osteoblasts, skin fibroblasts were seeded in coated 10 cm culture dishes (Corning) (100,000 to 3,000,000 cells / dish; equivalent to 1.8×10 3 ~5.4×10 4 / cm 2 The cells were inoculated at a density of 100 μg / ml and cultured in a fibroblast culture medium until the cells reached 80% confluence. The cells were then transferred to an iOB induction medium containing a chemical mixture. The iOB induction medium consists of DMEM, 10% FBS, 3 μM CHIR99021 (C) and 10 μM Forskolin (F). The induction solution may contain 100 U / ml penicillin and 100 μg / ml streptomycin antibiotics, or may not contain 100 U / ml penicillin and 100 μg / ml streptomycin antibiotics; or the DMEM containing 10% FBS may be replaced with a commercially available mesenchymal stem cell culture medium (manufacturers may be Cyagen, Gibco, Sigma). Fibroblasts were cultured aseptically in a humidified environment at 37°C and 5% CO2.

[0072] 3. Skin fibroblasts were cultured in the above culture system for 7 days.

[0073] 4. The cells treated in step 3 can be cultured in "DMEM, 10% FBS" medium for cell expansion to obtain the expected number of cells, or the high glucose DMEM containing 10% FBS can be replaced with a commercially available mesenchymal stem cell medium (manufacturers can be Cyagen, Gibco, Sigma); or cultured in "osteogenic medium, DMEM containing 10% fetal bovine serum, and added with 10mM β-sodium glycerophosphate, 100nM dexamethasone (dexamethasone can be omitted) and 0.2mM L-ascorbic acid" medium to promote maturation. The culture conditions are all cultured in a humidified environment at 37°C and 5% CO2, and the culture time is 0 to 100 days. The cells are then tested downstream.

[0074] Example 2-35

[0075] Example 2-35 Osteoblasts were prepared according to the method of Example 1, and the remaining differences are shown in the following table:

[0076]

[0077]

[0078] Embodiment 36

[0079] Same as Example 1, except that 10% FBS was replaced by 1% FBS.

[0080] Embodiment 37

[0081] Same as Example 1, wherein 10% FBS was replaced by 20% FBS.

[0082] Embodiment 38

[0083] Same as Example 1, wherein 100 nM dexamethasone is replaced by 10 nM dexamethasone.

[0084] Embodiment 39

[0085] 1. Isolate and culture skin fibroblasts according to the method of Example 1.

[0086] 2. When inducing osteoblasts, skin fibroblasts were seeded in coated 10 cm culture dishes (Corning) (100,000 to 3,000,000 cells / dish; equivalent to 1.8×103 to 5.4×10 4 / cm 2The cells were inoculated at a density of 100 μg / ml and cultured in a fibroblast medium until the cells reached 80% confluence. The cells were then transferred to an iOB induction medium containing a chemical mixture. The iOB induction medium consists of DMEM, 10% FBS, 1 μM Repsox (R) and 5 μM EPZ004777 (E). The induction solution may or may not contain 100 U / ml penicillin and 100 μg / ml streptomycin antibiotics; or the high glucose DMEM containing 10% FBS may be replaced with a commercially available mesenchymal stem cell medium (manufacturers may be Cyagen, Gibco, Sigma). Fibroblasts were cultured aseptically in a humidified environment at 37°C and 5% CO2.

[0087] 3. The processing time for the first stage is 10 days.

[0088] 4. The cells treated in step 3 are subjected to the second stage of treatment, and the iOB induction medium of the second stage is used. The iOB induction medium consists of DMEM, 10% FBS, 3μM CHIR99021 (C), 10μM Forskolin (F), 1μM Repsox (R) and 500μM Valproic acid (V). The induction solution may contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics, or may not contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics; or the DMEM containing 10% FBS can be replaced with a commercially available mesenchymal stem cell culture medium (manufacturer may be Cyagen, Gibco, Sigma). Fibroblasts are cultured aseptically in a humidified environment at 37°C and 5% CO2.

[0089] 5. The processing time for the second stage is 6 days.

[0090] 6. The cells treated in step 5 are subjected to the third stage of treatment, and the iOB induction medium of the third stage is used. The iOB induction medium is composed of DMEM, 10% FBS, 3μM CHIR99021 (C), 10μM Forskolin (F), 1μM Repsox (R), 500μM Valproic acid (V), 5μM Go6983 (G), 10μM SP600125 (S) and 5μM EPZ004777 (E). The induction solution may contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics, or may not contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics; or the DMEM containing 10% FBS may be replaced with a commercially available mesenchymal stem cell culture medium (manufacturers may be Cyagen, Gibco, Sigma). Fibroblasts are cultured aseptically in a humidified environment at 37°C and 5% CO2.

[0091] 7. The processing time for the third stage is 3 days.

[0092] 8. The cells treated in step 7 are subjected to the fourth stage treatment, and the fourth stage iOB induction medium. The iOB induction medium consists of DMEM, 10% FBS, 3μM CHIR99021 (C), 10μM Forskolin (F), 1μM Repsox (R), 500μM Valproic acid (V) and 0.1μM Trichostatin A (T). The induction solution may contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics, or may not contain 100U / ml penicillin and 100μg / ml streptomycin antibiotics; or DMEM containing 10% FBS can be replaced with a commercially available mesenchymal stem cell culture medium (manufacturers can be Cyagen, Gibco, Sigma). Fibroblasts are cultured aseptically in a humidified environment at 37°C and 5% CO2.

[0093] 9. The processing time for the third stage is 1 day.

[0094] 10. The cells treated in step 7 can be cultured in "DMEM, 10% FBS" medium for cell expansion to obtain the expected number of cells, or DMEM containing 10% FBS can be replaced with commercially available mesenchymal stem cell medium (manufacturers can be Cyagen, Gibco, Sigma); or cultured in "osteogenic medium, DMEM containing 10% fetal bovine serum, and added with 10mM β-sodium glycerophosphate, 100nM dexamethasone (dexamethasone can be omitted) and 0.2mM L-ascorbic acid" medium to promote maturation. The culture conditions are all cultured in a humidified environment at 37°C and 5% CO2, and the culture time is 0 to 100 days. Then the cells are tested downstream.

[0095] Examples 40-63

[0096] Examples 40-63 prepared osteoblasts according to the method of Example 39, with other differences as shown in the following table:

[0097]

[0098]

[0099]

[0100] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A method for inducing fibroblasts to reprogram into osteoblasts, characterized in that: include: Placing the fibroblasts in an induction medium containing the small molecule composition for 1-30 days to obtain induced osteoblasts, and then replacing the culture medium and continuing to culture to obtain osteoblasts; The small molecule composition adopts any of the following: CHIR-99021+Forskolin; CHIR-99021+Forskolin+Repsox; CHIR-99021+Forskolin+Repsox+Valproic acid; CHIR-99021+Forskolin+Repsox+Valproic acid+TRICHOSTATINA; CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983; CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125; CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125+EPZ004777; Repsox+EPZ004777; Repsox+EPZ004777+bFGF.

2. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 1, characterized in that: The step of placing the fibroblasts in an induction medium containing a small molecule composition for treatment for 1-30 days is replaced by: placing the fibroblasts in an induction medium containing a small molecule composition for treatment in the first stage for 1-30 days and in the second stage for 1-30 days; or in the first stage for 1-30 days, the second stage for 1-30 days and the third stage for 1-30 days; or in the first stage for 1-30 days, the second stage for 1-30 days, the third stage for 1-30 days and the fourth stage for 1-30 days; The small molecule composition used in the first stage, the second stage, the third stage and the fourth stage is any of the following, or the small molecule composition is not added during the treatment process of one of the stages, and the small molecule composition used in each stage is different: CHIR-99021+Forskolin+Repsox+Valproic acid+TRICHOSTATINA; CHIR-99021+Forskolin+Repsox+Valproic acid+GO 6983+SP600125+EPZ004777; CHIR-99021+Forskolin+Repsox+Valproic acid; Repsox+EPZ004777; Repsox+EPZ004777+bFGF.

3. The method for inducing fibroblasts to reprogram into osteoblasts according to any one of claims 1 to 2, characterized in that: In the induction culture medium containing the small molecule composition, the concentrations of the small molecules are: CHIR-99021: 0.1-30 μM, Forskolin: 0.1-100 μM, Repsox: 0.1-10 μM, Valproic acid: 1-5000 μM, TRICHOSTATIN A: 10 nM-10 μM, GO 6983: 0.1-50 μM, SP600125: 0.1-100 μM, EPZ004777: 0.1-50 μM, bFGF: 0.1-400 ng / mL.

4. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 1, characterized in that: The fibroblasts are isolated from mammalian skin.

5. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 4, characterized in that: The isolated fibroblasts were expanded in vitro to a generation before P30 for subsequent treatment.

6. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 1, characterized in that: The induction culture medium containing the small molecule composition also comprises DMEM or mesenchymal stem cell culture medium containing 1-20% fetal bovine serum.

7. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 1, characterized in that: The method of replacing the culture medium and continuing to culture to obtain osteoblasts specifically comprises: The treated cells are cultured in a mesenchymal stem cell culture medium or a DMEM culture medium containing 1-20% fetal bovine serum for cell expansion to obtain a desired number of cells; or The treated cells were cultured in osteogenic medium to promote cell osteogenic maturation. The osteogenic medium contained DMEM containing 1-20% fetal bovine serum and supplemented with 10 mM sodium β-glycerophosphate and 0.2 mM L-ascorbic acid.

8. The method for inducing fibroblasts to reprogram into osteoblasts according to claim 7, characterized in that: 10-100 nM dexamethasone was also added to the osteogenic medium.

9. Osteoblasts obtained by the method for inducing fibroblasts to reprogram into osteoblasts according to any one of claims 1 to 8.

10. Use of the osteoblasts according to claim 9 in preparing bone materials and drugs for treating bone defects.

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