Composition for promoting osteogenic differentiation of mesenchymal stem cell, method, and bone repair use

Abstract

Provided are a composition for promoting osteogenic differentiation of a mesenchymal stem cell, a method, and bone repair use. Specifically, a treatment product obtained by treating collagen with a HtrA3 protein solves the problem of limitations to traditional osteanagenesis-related cytokines, and can specifically promote the differentiation of bone marrow mesenchymal stem cells at the early stage of bone defect, such that osteanagenesis or bone repair is effectively promoted.

Description

The present disclosure relates to the field of high-efficiency osteogenesis, and in particular to a composition and method for promoting osteogenic differentiation of a mesenchymal stem cell and use thereof in bone regeneration materials. BACKGROUND As life expectancy rises and global aging populations expand, the prevalence of diseases such as bone fractures, osteoporosis, and bone metastasis is rapidly increasing. Consequently, the medical care and costs related to bones are on the rise, making research on bone regeneration highly significant. Bone defect repair is a complex process regulated by multiple factors. One of the key steps involves the migration of mesenchymal stem cells to the target position, where they then initiate the synthesis of new bone. Thus, the migration efficiency of the mesenchymal stem cells determines the speed and quality of bone healing to some extent. The mesenchymal stem cells involved in bone repair are mainly derived from bone marrow and the inner layer of the periosteum. In their resting state, these cells are stably resident in the stem cell microenvironment around the vessel wall or other positions. When an injury occurs, the mesenchymal stem cells migrate to the defect repair area, where they undergo osteogenic differentiation and contribute to bone regeneration. At present, some classical cytokines that promote bone regeneration, such as BMP, VEGF, FGF, and PTH, have been used to promote the healing of bone defects. However, these conventional cytokines have many side effects such as ectopic bone formation and hemangioma due to their non-specific actions on various cells and organs, and some of these cytokines must be used with high accuracy in controlling the dosage. SUMMARY High-temperature requirement protein A3 (HtrA3) is a newly discovered pregnancy-associated protein and has been proven to be involved in physiological and pathological processes such as embryo implantation, embryo development, and tumor invasion. According to the research of the present disclosure, the HtrA3 protein has been found to be able to promote osteogenic differentiation of human bone marrow mesenchymal stem cells, thereby playing a role in promoting bone defect repair. Based at least in part on the finding described above, the present disclosure has been accomplished. Specifically, the present disclosure comprises the following. In a first aspect of the present disclosure, provided is a composition for promoting osteogenic differentiation of a mesenchymal stem cell, which comprises a collagen-derived product or a precursor capable of producing the collagen-derived product. In certain embodiments of the composition for promoting osteogenic differentiation of a mesenchymal stem cell according to the present disclosure, the precursor comprises HtrA3 protein or type IV collagen, or comprises HtrA3 protein and type IV collagen present in an independent or mixed state. In certain embodiments of the composition for promoting osteogenic differentiation of a mesenchymal stem cell according to the present disclosure, the precursor is in a gel state or a solid state. In certain embodiments of the composition for promoting osteogenic differentiation of a mesenchymal stem cell according to the present disclosure, the collagen-derived product is obtained by contacting a collagen raw material with HtrA3 protein under a condition suitable for culturing a mesenchymal stem cell. In certain embodiments, the composition for promoting osteogenic differentiation of a mesenchymal stem cell according to the present disclosure comprises collagen coating at least a culture surface of a culture substrate and HtrA3 protein dissolved in a culture medium, wherein when the culture medium is a working 2 culture medium directly used for culturing a mesenchymal stem cell, the concentration of the HtrA3 protein in the culture medium is 0.05-2 ng / mL. In certain embodiments of the composition for promoting osteogenic differentiation of a mesenchymal stem cell according to the present disclosure, the osteogenic differentiation comprises at least one of the following situations (1)-(5): (1) an increased transcription level of an osteogenesis-related gene or an increased expression level of the corresponding mRNA; (2) an increased amount or activity of an osteogenesis-related protein; (3) an increased activity of an alkaline phosphatase; (4) an increased amount of a calcium nodule or a mineralized nodule; and (5) an increased bone volume and / or bone mineral density. In a second aspect of the present disclosure, provided is a method for promoting osteogenic differentiation of a mesenchymal stem cell in vitro, which comprises a step of contacting the composition according to the first aspect, or at least a portion thereof, with a mesenchymal stem cell. In certain embodiments, the method for promoting osteogenic differentiation of a mesenchymal stem cell in vitro according to the present disclosure comprises the following steps: (1) adding a bone marrow mesenchymal stem cell and a first culture medium to a culture substrate having at least a culture surface coated with collagen, and leaving the mixture to stand for 5 hours to 5 days under a condition suitable for culture; and (2) then replacing the first culture medium with a second culture medium, continuing to culture until obtaining a differentiated cell; wherein the first culture medium comprises 0.05-2 ng / mL HtrA3 protein, and the second culture medium does not comprise the HtrA3 protein. In a third aspect of the present disclosure, provided is a bone marrow mesenchymal stem cell prepared by the method according to the second aspect. 3 In a fourth aspect of the present disclosure, provided is use of the composition according to the first aspect in the preparation of a bone regeneration material. The present disclosure solves the limitation of traditional bone regeneration-related cytokines, and provides a novel method for specifically promoting differentiation of bone marrow mesenchymal stem cells at the early stage of bone defect so as to effectively promote bone regeneration. In certain embodiments, experiments prove that the 100 ng / mL HtrA3 solution applied in vivo for three days can significantly promote bone regeneration or bone repair of the skull defect area in rats. The method of the present disclosure is novel, rapid, and convenient. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the expression levels of the osteogenesis-related genes on day 7, with a recombinant HtrA3 protein solution added to the culture medium during the first 3 days. The expression levels of the osteogenic genes were calculated from the Ct values, and histograms of ALP, RUNX2, OPN, and OPG in the control and experimental groups were statistically analyzed. FIG. 2 shows the protein expression levels of the osteogenesis-related genes on day 7, with the recombinant HtrA3 protein solution added to the culture medium during the first 3 days. The western blotting results show that the protein expression levels of RUNX2, BMP2, and SP7 in the experimental groups were significantly higher than those of the blank control groups. FIG. 3 shows ALP and ARS staining results, which indicate that the addition of the recombinant HtrA3 protein during the first 3 days of osteogenesis can significantly promote the osteogenic differentiation ability of bone marrow mesenchymal stem cells. FIG. 3A shows representative graphs of two groups of ALP staining results; FIG. 3B shows quantification of ALP activity by detecting alkaline phosphatase activity produced per gram of protein, and statistical analysis shows an increase in the ALP activity for the rhHtrA3 group; FIG. 3C shows representative graphs of two groups of ARS staining results, with more calcium nodules in the rhHtrA3 group; FIG. 3D shows quantification of alizarin red staining by measuring the absorbance at 562 nm, and statistical analysis shows that the rhHtrA3 group exhibited deeper alizarin red staining and greater osteogenic differentiation potential. FIG. 4 shows that 100 ng / mL HtrA3 solution applied in vivo for three days can significantly promote the skull defect repair in rats. FIG. 4A shows a CT scan image of the skull defect repair at 4 w, as well as quantitative statistical graphs of bone volume and bone mineral density; FIG. 4B shows a CT scan image of the skull defect repair at 8 w, as well as quantitative statistical graphs of bone volume and bone mineral density. DETAILED DESCRIPTION Various exemplary embodiments of the present disclosure are described in detail below. The detailed description should not be construed as limitations on the present disclosure but as a more detailed description of certain aspects, features, and embodiments of the present disclosure. It will be appreciated that the terms used herein are for the purpose of illustrating particular embodiments only, rather than limiting the present disclosure. In addition, for the numerical ranges in the present disclosure, it will be appreciated that the upper and lower limits of the ranges are specifically disclosed, as well as every intervening value between them. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the present disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art described in the present disclosure. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to 5 those described herein can be used in the practice or testing of the present disclosure. All documents described herein are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In case of conflict with any incorporated document, the specification shall prevail. Herein, the term “mesenchymal stem cell”, also abbreviated herein as MSC, refers to a totipotent stem cell having all the common properties of stem cells and having self-renewal and multi-directional differentiation ability or potential. For example, under specific conditions, it can differentiate into various cells such as muscle cells, hepatocytes, osteoblasts, adipocytes, chondrocytes, and stromal cells, which can further differentiate into various tissues such as bone, cartilage, muscle, or tendon. Sources of mesenchymal stem cells herein include, but are not limited to, bone marrow, skeletal muscle, periosteum, trabecular bone, blood (e.g., umbilical cord blood), and the like. Herein, the term “osteogenic differentiation” refers to the process of directional differentiation of mesenchymal stem cells having multi-directional differentiation ability or potential into osteocyte lineage. The osteocyte lineage includes osteogenic cells, osteoblast precursor cells, mature osteoblasts, and terminal osteocytes. There is no clear boundary among these types of cells, and osteogenic differentiation is a continuously occurring and progressive, time-ordered dynamic process. The osteogenic differentiation in the present disclosure can be characterized by characteristic markers or obtained osteogenic parameters, which may particularly include at least one of the following situations: (1) an increased transcription level of an osteogenesis-related gene or an increased expression level of the corresponding mRNA; (2) an increased amount or activity of an osteogenesis-related protein; (3) an increased activity of an alkaline phosphatase; (4) an increased amount of a calcium nodule or a mineralized nodule; and (5) an increased bone volume and / or bone mineral density. 6 [Composition] In a first aspect of the present disclosure, provided is a composition for promoting osteogenic differentiation of a mesenchymal stem cell, sometimes abbreviated herein as “the composition of the present disclosure”, which comprises a collagen-derived product or a precursor capable of producing the collagen-derived product. In the present disclosure, the collagen-derived product is a collagen degradation product or decomposition product obtained from collagen. Generally, the molecular weight of the treated product is lower than that of the collagen itself. In the present disclosure, the collagen may be sometimes understood to include collagen peptides, which generally refer to natural collagen or peptides obtained from animals, and the specific source is not limited. Preferably, the collagen refers to collagen IV or a mixture comprising at least collagen IV and a plurality of different types of collagen. In the present disclosure, the precursor for producing the collagen-derived product generally comprises collagen as a raw material and / or HtrA3 protein as a treatment agent. In the case where both are present, they may be present alone or in the form of a mixture. When present alone, the collagen and HtrA3 protein are mixed at least prior to use for the required period of time, for example, 5 h or more, preferably 10 h or more, 20 h or more, or 1 day or more such as 2 days, 3 days, and 4 days. In another aspect, this period of time is generally 5 days or less, preferably 4 days or less. The mixing temperature is generally room temperature. When the collagen and HtrA3 protein are present in the form of a mixture, the ratio of the two is not limited. For example, the ratio may be in the range of (1-10):(10-1) based on the weight. In certain embodiments, the precursor of the present disclosure comprises collagen and HtrA3 protein, and both are formed in a gel state or a solid state. In certain embodiments, the collagen-derived product is obtained by contacting a collagen raw material with HtrA3 protein. The conditions for the contact reaction 7 are not limited, and are generally mild conditions, including a temperature of 1-40 °C, preferably 5-35 °C, and more preferably 10-30 °C, or the required reaction time at room temperature, e.g., 5 h or more, preferably 10 h or more, 20 h or more, or 1 day or more such as 2 days, 3 days, and 4 days. In another aspect, this period of time is generally 5 days or less, preferably 4 days or less. If the time is too short, it may be insufficient to generate the desired treated product. In another aspect, if the time is too long, the resulting treated product has a weakened function of promoting osteogenic differentiation, and tends to differentiate in other directions, such as adipogenic differentiation. In a preferred embodiment, the conditions for the contact reaction of the collagen raw material and the HtrA3 protein are those suitable for culturing mesenchymal stem cells. These conditions are suitable for producing the treated product and also ensure the culture and differentiation of the mesenchymal stem cells. In this case, the production of the treated product and the culture and differentiation of the mesenchymal stem cells may be simultaneously performed. In certain embodiments, the collagen raw material and the HtrA3 protein are present in independent or separated forms. For example, at least a culture surface of a culture substrate is coated with the collagen, and the HtrA3 protein is dissolved in a culture medium, preferably a culture medium for differentiation of mesenchymal stem cells, the composition of which is known in the art. The concentration of the HtrA3 protein in the culture medium is not particularly limited, but is generally required to be such that the concentration in the resulting working culture medium reaches 0.05-2 ng / mL, preferably 0.01-1 ng / mL, and more preferably 0.5-1 ng / mL. [Osteogenic differentiation method] In a second direction of the present disclosure, provided is a method for promoting osteogenic differentiation of a mesenchymal stem cell in vitro, which comprises a step of contacting the composition according to the first aspect, or at least a portion thereof, with a mesenchymal stem cell. 8 In certain embodiments, the osteogenic differentiation of the present disclosure comprises the following steps: (1) adding a bone marrow mesenchymal stem cell and a first culture medium to a culture substrate having at least a culture surface coated with collagen, and leaving the mixture to stand for 5 hours to 5 days under a condition suitable for culture; and (2) then replacing the first culture medium with a second culture medium, continuing the culture, and even obtaining a differentiated cell; wherein the first culture medium comprises 0.05-2 ng / mL HtrA3 protein, and the second culture medium does not comprise the HtrA3 protein. In such embodiments, the culture substrate may be in the form of a culture dish, a culture flask, a culture plate, or the like, as is known in the art. At least the culture surface of the culture substrate that is in contact with the bone marrow mesenchymal stem cells is coated with collagen, preferably collagen IV or a mixture comprising collagen IV, e.g., matrigel. Example 1 Human bone marrow mesenchymal stem cells and a culture medium were added to a culture plate pre-coated with matrigel. The experimental group was cultured by using a culture medium containing a recombinant HtrA3 protein solution (0.1 ng / mL) during the first 3 days, and then this culture medium was replaced with a normal culture medium. After 7 days of culture, RNA samples were extracted by using Trizo, and after reverse transcription, the osteogenesis-related genes were detected by qRT-PCR. As shown in FIG. 1, FIG. 1 shows the expression levels of the osteogenesis-related genes on day 7, with the recombinant HtrA3 protein solution added to the culture medium during the first 3 days. The expression levels of the osteogenic genes were calculated from the Ct values, and histograms of ALP, RUNX2, OPN, and OPG in the control and experimental groups were statistically analyzed. The results show that the addition of the recombinant 9 HtrA3 protein solution during the first 3 days could significantly increase the expression levels of the osteogenesis-related genes of the bone marrow mesenchymal stem cells. After 7 days, a cell lysis buffer (containing 1% PMSF) was added, and the protein was extracted. The protein concentration was measured, and the expression levels of the osteogenesis-related proteins were detected by western blotting. The results show that the addition of the recombinant HtrA3 protein solution during the first 3 days significantly promoted the expression levels of the osteogenesis-related proteins of the bone marrow mesenchymal stem cells. FIG. 2 shows the protein expression levels of the osteogenesis-related genes on day 7, with the recombinant HtrA3 protein solution added to the culture medium during the first 3 days. The western blotting results show that the protein expression levels of RUNX2, BMP2, and SP7 in the experimental groups were significantly higher than those of the blank control groups. Example 2 Human bone marrow mesenchymal stem cells and a culture medium were added to a culture plate pre-coated with matrigel, and in the experimental group, a recombinant HtrA3 protein solution (0.1 ng / mL) was added during the first 3 days. Alkaline phosphatase (ALP) staining and ALP activity quantitative analysis were performed on day 14, and alizarin red (ARS) mineralized nodule staining was performed on day 21. After photographing, 100 nM cetylpyridinium was added to dissolve the alizarin red dye chelated with calcium, and the absorbance was measured at 562 nm on a microplate reader for ARS quantification. The results show that ALP activity was 1.3 times that of the control group after the addition of the recombinant HtrA3 protein solution during the first 3 days, and ARS quantification results show that the experimental group was improved by 5 times as compared to the control group. The results in FIG. 3 show ALP and ARS staining results, which indicate that the addition of the recombinant HtrA3 protein during the first 3 days of osteogenesis can significantly promote the io osteogenic differentiation ability of bone marrow mesenchymal stem cells. FIG. 3A shows representative graphs of two groups of ALP staining results; FIG. 3B shows quantification of ALP activity by detecting alkaline phosphatase activity produced per gram of protein, and statistical analysis shows an increase in the ALP activity for the rhHtrA3 group; FIG. 3C shows representative graphs of two groups of ARS staining results, with more calcium nodules in the rhHtrA3 group; FIG. 3D shows quantification of alizarin red staining by measuring the absorbance at 562 nm, and statistical analysis shows that the rhHtrA3 group exhibited deeper alizarin red staining and greater osteogenic differentiation potential. Example 3 An SD rat skull defect model was constructed, and matrigel containing 100 ng / mL HtrA3 solution was applied to the left-side defect, while matrigel alone was used as a blank control for the right-side defect. The samples were collected at 4 w and 8 w after the model was constructed, and micro-CT scanning analysis was performed after the samples were fixed in paraformaldehyde for 24-48 h. The results in FIG. 4 show that 100 ng / mL HtrA3 solution applied in vivo for three days could significantly promote the skull defect repair in rats. FIG. 4A shows a CT scan image of the skull defect repair at 4 w, as well as quantitative statistical graphs of bone volume and bone mineral density; FIG. 4B shows a CT scan image of the skull defect repair at 8 w, as well as quantitative statistical graphs of bone volume and bone mineral density. The results indicate that the 100 ng / mL HtrA3 solution applied in vivo for three days can significantly promote bone regeneration of the skull defect area in rats. Although the present disclosure has been described with reference to exemplary embodiments, it is to be understood that the present disclosure is not limited to the disclosed exemplary embodiments. Various modifications and variations can be made to the exemplary embodiments in the specification of the present ii disclosure without departing from the scope or spirit of the present disclosure. The scope of the claims should be based on the broadest interpretation to encompass all modifications and equivalent structures and functions.

Claims

1. A composition for promoting osteogenic differentiation of a mesenchymal stem cell comprising a collagen-derived product or a precursor capable of producing the collagen-derived product.

2. The composition for promoting osteogenic differentiation of a mesenchymal stem cell according to claim 1, wherein the precursor comprises HtrA3 protein or type IV collagen, or comprises HtrA3 protein and type IV collagen present in an independent or mixed state.

3. The composition for promoting osteogenic differentiation of a mesenchymal stem cell according to claim 1, wherein the precursor is in a gel state or a solid state.

4. The composition for promoting osteogenic differentiation of a mesenchymal stem cell according to claim 1, wherein the collagen-derived product is obtained by contacting a collagen raw material with HtrA3 protein under a condition suitable for culturing a mesenchymal stem cell.

5. The composition for promoting osteogenic differentiation of a mesenchymal stem cell according to claim 1, comprising collagen coating at least a culture surface of a culture substrate and HtrA3 protein dissolved in a culture medium, wherein when the culture medium is a working culture medium directly used for culturing a mesenchymal stem cell, the concentration of the HtrA3 protein in the culture medium is 0.05-2 ng / mL.

6. The composition for promoting osteogenic differentiation of a mesenchymal stem cell according to claim 1, wherein the osteogenic differentiation comprises at least one of the following situations (1)-(5):(1) an increased transcription amount of an osteogenesis-related gene or an increased expression amount of the corresponding mRNA;(2) an increased amount or activity of an osteogenesis-related protein;(3) an increased activity of an alkaline phosphatase;(4) an increased amount of a calcium nodule or a mineralized nodule; and(5) an increased bone volume and / or bone mineral density.CLAIMS7. A method for promoting osteogenic differentiation of a mesenchymal stem cell in vitro, comprising a step of contacting the composition according to any one of claims 1-6, or at least a portion thereof, with a mesenchymal stem cell.

8. The method for promoting osteogenic differentiation of a mesenchymal stem cell in vitro according to claim 7, comprising the following steps:(1) adding a bone marrow mesenchymal stem cell and a first culture medium to a culture substrate having at least a culture surface coated with collagen, and leaving the mixture to stand for 5 hours to 5 days under a condition suitable for culture; and(2) then replacing the first culture medium with a second culture medium, continuing to culture until obtaining a differentiated cell;wherein the first culture medium comprises 0.05-2 ng / mL HtrA3 protein, and the second culture medium does not comprise the HtrA3 protein.

9. A bone marrow mesenchymal stem cell prepared by the method according to claim 7 or 8.

10. Use of the composition according to any one of claims 1-6 for the preparation of a bone regeneration or bone repair material.

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