CN112029710A - Screening method of direct mechanical response cell subset and application thereof - Google Patents
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Abstract
The invention discloses a screening method of direct mechanical response cell subsets and application thereof, wherein the screened cell subsets comprise a Fat4+ subset, an Fgfr3+ subset and a Scara5+ subset, and the application of the mechanical response cell subsets in preparation of drugs for local bone defect repair and regeneration and stress-related bone metabolic diseases is also disclosed. The invention screens direct mechanical response cells by a single cell sequencing technology, finely analyzes a cell gene expression spectrum under stress stimulation by a single cell transcriptome level, searches a key cell subset with life transport change under stress regulation and control, carries out in vitro flow type sorting and in vitro stress loading by a marker gene, and verifies the mechanical responsiveness by combining with the construction of a CreER-loxP inducible reporter gene system to carry out in vivo lineage tracing, thereby achieving the purpose of identifying the direct mechanical response cells.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for screening a direct mechanical response cell subset and application thereof.
Background
Biomechanical phenomena are widespread in many organs and tissues of the body, such as the skin, heart, alveoli, bones, etc. The skeletal system is a 'steel bar iron bone' for protecting and supporting human body and is responsible for bearing, sensing and transduction of stress stimulation by the body. The skeletal system senses mechanical stress and adapts its structure to environmental changes by responding to mechanical stimuli. The clinical phenomena of bone traction, fracture repair, orthodontic tooth movement, osteoporosis caused by weightlessness and the like show that the mechanical stress is an important factor for regulating and controlling bone metabolism and reconstruction. Therefore, revealing the mechanical response mechanism of bone tissue from the cellular molecular level is an important frontier in the field of biomechanical research. Clarifies that the mechanical response mechanism of the bone tissue has important significance for screening key targets of biomechanical intervention and gene adjuvant therapy of bone tissue diseases.
At present, a plurality of documents report that the stress stimulation can promote the osteogenic differentiation enhancement of bone tissue cells. However, as a core rule of skeletal physiology, cells that directly sense and respond to mechanical factors within bone tissue have not been determined. This is due to the limitations of the existing mechanical response cell technology — most studies employ a method of directly performing in vitro stress loading on osteoblast cell lines such as C3H10T1/2, MC3T3-E1, or primary mesenchymal stem cells (BMSCs). But the cell line can not reflect the mechanical response condition of the primary cells; the primary BMSCs are heterogeneous cell populations containing various differentiation states, different lineage differentiation tendencies and different functions, and different BMSCs subpopulations can have different response states and functional effects under the action of stress; besides BMSCs, researchers also consider that mature osteoblasts or osteocytes in the skeletal system can also be mechanically sensitive cells, and traditional methods are difficult to directly obtain homogeneous primary osteoblasts and osteocytes, and still cannot clearly define true mechanically responsive cells in a heterogeneous population. Other studies have used the Cre-loxP system to construct model animals for lineage tracing to find mechano-responsive cells, however due to the complexity of Cre expression profiling and the heterogeneity of most cells, true subsets of mechano-responsive cells cannot be precisely identified.
Cells are the basic building blocks of the body, each cell of which has its uniqueness. Single-cell RNA sequencing (scRNA-seq) technology is used as a revolutionary tool, and achieves unprecedented precision in revealing cell uniqueness. Currently, the single cell sequencing technology is mainly applied to the research fields of tumors, immunity, development, microorganisms and the like, and most researches redefine cell types by carrying out single cell transcriptome sequencing on cells, namely constructing single cell maps of species individuals/organs/tissues or identifying tumor molecular markers. However, reports on the level of single-cell transcriptome have not been found in the field of biomechanics. Based on the superiority of single cell sequencing in cell heterogeneity analysis and quasi-time sequence analysis, whether the single cell sequencing technology can be used as a powerful tool for screening direct mechanical response cells or not, whether a cell subset directly responding to mechanical stress exists in a mixed bone tissue cell population or not, and how the cell fate of the mixed bone tissue cell population is transferred under the action of stress.
Due to the limitation of the existing research mechanical response cell technology, cells which directly sense and respond to mechanical factors in bone tissues are difficult to detect, so that the research has not been determined yet. The prior art mainly comprises two methods of in vitro stress loading and model animal research. In vitro stress loading mostly adopts a method of directly carrying out in vitro stress loading on osteoblast lines such as C3H10T1/2 and MC3T3-E1, but the cell lines cannot reflect the mechanical response condition of cells in vivo. Secondly, other researches adopt primary BMSCs to apply force, but the BMSCs are heterogeneous cell populations with various differentiation states, different lineage differentiation tendencies and different functions, under the action of stress, different BMSCs subsets can have different response states and functional effects, and the real mechanical response subsets cannot be identified. Besides BMSCs, researchers think that mature osteoblasts or osteocytes in a skeletal system can also be mechanically sensitive cells, but the traditional method is difficult to directly obtain homogeneous primary osteoblasts and osteocytes, and still cannot clearly determine real mechanical response cells in the mixed population. Model animal research adopts Cre-loxP system to construct specific fluorescence expression mouse for lineage tracing to search for mechanical response cells, however, due to the complexity of Cre expression spectrum and the heterogeneity of most cells, the mechanical response cell subset can not be accurately identified.
In conclusion, none of the prior art essentially screens and identifies true subpopulations of mechanically responsive cells from heterogeneous cell populations.
Disclosure of Invention
In order to solve the problems of the prior art, the present invention aims to provide a method for screening a direct mechanical response cell subset and an application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for screening a subpopulation of directly mechanically responsive cells, comprising the steps of:
a. constructing an osteoblast line specific fluorescence expression mouse by using a Cre-loxP system, and establishing an in vivo stress loading model;
b. obtaining positive expression stress application group and control group bone tissue single cell suspension by using flow sorting;
c. single cell sorting, library construction, high-throughput sequencing and data analysis, and constructing an osteogenic cell grouping map and a pseudo-time sequence cell differentiation path;
d. comparing the cell population proportion of the stress group and the cell population proportion of the control group and performing quasi-time sequence analysis, and screening cell subsets mainly subjected to proportion and quasi-time sequence change;
e. in-vitro verification, namely performing flow sorting on the membrane protein coded by the marker gene of the candidate population, culturing the subgroup in vitro, and performing in-vitro stress loading;
f. and (3) in vivo verification, namely performing lineage tracing and cell elimination verification on the CreER mice driven by the constructed marker gene promoters of the candidate population.
The direct mechanical response cell subsets obtained by the preparation method comprise a Fat4+ subset, an Fgfr3+ subset and a Scara5+ subset.
The cell subset is applied to preparing medicines for repairing and regenerating local bone defects and stress-related bone metabolic diseases.
Has the advantages that: the invention provides a screening method of a direct mechanical response cell subset and application thereof, the invention screens the direct mechanical response cell by a single cell sequencing technology, carries out fine analysis of cell gene expression profile under stress stimulation by a single cell transcriptome level, searches a key cell subset with life operation change under stress regulation and control, carries out in-vitro flow type sorting and in-vitro stress loading by a marker gene, and combines the construction of a CreER-loxP inducible reporter gene system to carry out in-vivo lineage tracing and verify the mechanical responsiveness of the cell subset, thereby achieving the purpose of identifying the direct mechanical response cell. The mechanical response cells obtained by identification and sorting can be stably cultured in vitro and have osteogenic effect under stress stimulation, so the mechanical response cells can be used as seed cells for local bone defect repair and regeneration, can also be used for preparing medicines for force-related bone metabolic diseases such as osteoarthritis and the like, and are potential targets for gene therapy of the bone metabolic diseases.
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FIG. 1A shows Osx-Cre mouse and R26tdTomatoBreeding the mice to obtain an osteoblast line cell specificity tdTomato fluorescence expression mouse pattern diagram; b is a schematic diagram of a fluorescence expression mouse orthodontic stress loading model; c is a tdTomato fluorescence map of a force application side and a contrast side of the tooth movement modeling for 7 days; d is a tooth movement modeling 7-day stress side and control side tdTomato fluorescence expression histogram,
**P<0.05。
FIG. 2A is a schematic diagram showing preparation, flow sorting and single cell sequencing of alveolar bone osteogenic single cell suspensions on the force application side and the control side; b is a diagram for analyzing and displaying the grouping condition of alveolar bone osteogenic line cells on a stress application side and a control side by virtue of single cell sequencing tSNE; c is a tSNE grouping diagram showing the distribution of each subgroup O1-O8 in alveolar bone osteogenic lineage cells; d is a heat map of relative expression significant gene characteristics of each subgroup O1-O8.
FIG. 3A is a graph showing a comparison of the ratio of each subpopulation of alveolar bone osteoblast lineage cells on the force application side and the control side; b is a coloring chart of O1, O2 and O4 cell subset marker genes; c is O1, O2, O4 cell subset marker gene heat map; d is a diagram showing the differentiation traces of the O1, O2 and O4 cell subsets through pseudo-time sequence analysis; e is a gene expression pattern diagram in differentiation tracks of O1, O2 and O4; f is a tree comparison chart of quasi-time sequence analysis of the stress group and the control group. FIG. 4A is a flow sort Fat4+, DAPI cell profile; a morphological feature map under Fat4+ cell light mirror; c is a Fat4+ cell monoclonal colony crystal violet staining pattern; d is an oil red O staining pattern; e is alizarin red staining diagram; f is Fat4+ cell in vitro force 7 days alkaline phosphatase (ALP) staining pattern; g is a histogram of the q-PCR analysis of Fat4+ cell in vitro force for 7 days osteogenesis related genes,. P < 0.05.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The invention provides a research method for screening direct mechanical response cells based on a single cell sequencing technology, which comprises the following steps:
a. an osteoblast line specific tdTomato fluorescence expression mouse is constructed by using a Cre-loxP system, and an in vivo stress loading model is established.
Constructing a fluorescence expression mouse: Osx-Cre mouse and R26 specifically expressed by osteoblaststdTomatoThe mice can obtain conditional fluorescent mice R26 through matingtdTomato,Osx-Cre；
Secondly, the internal stress loading model can be a mouse upper jaw first molar mesial moving model, and the upper jaw incisor is used as an anchorage to enable the upper jaw left first molar mesial to tilt and move by using a nickel-titanium tension spring;
and thirdly, the mouse is sacrificed 7 days after the molar is moved by orthodontic force of 10g, the left molar of the upper jaw and the tissues around the molar are taken as a stress application side, and the right side is taken as a control group.
FIG. 1A shows Osx-Cre mouse and R26tdTomatoBreeding the mice to obtain an osteoblast line cell specificity tdTomato fluorescence expression mouse pattern diagram; FIG. 1B shows fluorescenceSchematic representation of an orthodontic stress loading model of an expression mouse; FIG. 1C is a 7 day force side and control side tdTomato fluorescence plot for dental movement modeling; FIG. 1D is a histogram of the fluorescence expression of tdTomato on the force side and control side of tooth movement modeling for 7 days<0.05. As can be seen from the graphs C and D, the number of osteoblastic lineage cells (i.e., tdTomato fluorescence expressing cells) on the force side was significantly increased after 7 days of orthodontic stress loading.
b. Obtaining bone tissue single cell suspensions of a tdTomato positive expression stress application group and a control group by utilizing flow sorting, wherein FIG. 2A is a schematic diagram of preparation, flow sorting and single cell sequencing of alveolar bone osteogenic single cell suspensions at a stress application side and a control side.
Conditional fluorescent mouse R26tdTomatoOsx-Cre establishes orthodontic tooth moving model, kills mouse by breaking neck after applying force for 7d, and soaks in 75% ethanol solution. Using a toothed forceps to pull out upper jaw molars under a stereomicroscope, separating alveolar bones acutely, removing attached soft tissues, and repeatedly washing and cleaning without calcium and magnesium ion phosphate buffer solution DPBS. The ophthalmic scissors cut the alveolar bone into pieces with the size of 1mm x 1mm, and the pieces are digested by Dispase/collagen at 37 ℃ for 2h under oscillation. The cell suspension was filtered through a 40 μm cell sieve, centrifuged at 200g for 5min at4 ℃, the supernatant was discarded, and the DPBS resuspended the cell pellet, placed on ice.
② the nucleic acid dye DAPI (0.05-0.2 mu g/mL) is used for live-dead staining: preparing a negative control tube, a single staining tube and a sample tube of each channel, and arranging a gate on the negative control tube to debug the flow type sorting parameters. Respectively collecting and sorting complete culture medium to obtain tdTomato+Single cell suspensions of DAPI stress and control.
c. Single cell sorting, library construction, high-throughput sequencing and data analysis, and the construction of an osteogenic cell grouping map and a pseudo-time sequence cell differentiation path.
Normalization (CellNormalization): SeuratPackage was used to remove dead cells, double cells, low quality cells and batch effects from bias on subsequent analyses. And performing dimensionality reduction processing and information display on the data by adopting a tSNE (t-partitioned stored geometrical carbon Embedding) algorithm and a PCA (principal components analysis) algorithm.
② Cell clustering (Cell Cluster): and for the cells after tSNE clustering, carrying out unsupervised clustering based on Graphcluster or KMean algorithm according to the gene expression condition, and further partitioning the single cell population. Cell regrouping (cluster): after the preliminary division of the single cell population is completed, a Marker Gene population of the single cell Cluster is obtained by further adopting a differential screening algorithm, and through the Marker genes, the cell class group to which each cell population belongs can be presumed and identified, and the interested population is selected from the cell class group for further subdivision.
③ rdplot analysis: and selecting specific genes and specific cell groups by adopting an R language, and drawing a single-cell UMAP/tsne diagram, a violin diagram and a bubble diagram.
(iv) pseudo timing analysis (Pseudotime): the machine learning strategy (Reversed Graph Embedding) is used for learning the explicit main Graph constructed by the unicellular genomics data to arrange the cell sequence, and the trend and the development of the cell fate under the mechanical action are stably and accurately explained.
Gene function analysis (GO analysis) and signal path analysis (Pathway analysis): based on a database, performing GO annotation on the genes falling into each cluster from three levels of BP, MF and CC to obtain all GO involved in the genes, and calculating the significance level (P-Value) of each GO by adopting Fisher test, thereby screening out the significance of gene enrichment. Based on the gene annotation database, signal pathways significantly enriched for differentially expressed genes were detected. Pathway annotation is carried out on the genes falling into each cluster based on a KEGG database to obtain all Pathway Terms in which the genes participate, and Fisher test is adopted to calculate the significance level (P-Value) of Pathway, so that the significance Pathway Term enriched by the genes is screened out.
Sixthly, (single Cell regulated network interference and Cluster): the single Cell data is used as a basis for deducing a transcription factor regulatory network (Gene regulatory network) and a related Cell State (Cell State) thereof. Based on the transcription factor target database (or the transcription factor Motif database), the expression of the transcription factor and the target gene thereof in the target cell population, the regulatory gene of each transcription factor in the cell and the regulatory intensity thereof (AUCelScore) are calculated.
Analysis of QuSAGE: and (3) carrying out GSEA-like enrichment degree analysis on the gene set by adopting a Variance expansion Factor algorithm (Variance initialization Factor), and comparing the enrichment degree difference of different Clusters of the same gene.
(viii) cellular traffic analysis (CellPhoneAnalysis): understanding the most common Ligand-Receptor relationship pairs between different cell populations, and understanding how the cell populations interact with each other.
Ninthly, ScGeneModule analysis: based on the single cell GeneModule analysis, the possible functions of genes in cell populations and even some subdivided cell types can be predicted more reliably based on the expression pattern grouping of the genes.
Through the steps of single cell sorting, library construction, high-throughput sequencing and data analysis, the osteoblast lineage cell grouping map and the quasi-time sequence cell differentiation path are successfully constructed, and the alveolar osteoblast lineage cell map is successfully drawn. As shown in fig. 2B-D, osteoblast lineage cells were divided into 8 cell subsets (O1-O8), where O2(Fgfr3+ subset) was likely the osteoblast precursor cell, O4(Bglap + subset) was likely the mature osteoblast subset, and O1, O3, O5, O8 were likely different subsets of BMSCs.
d. Comparing the cell population proportion of the Force application group (Force) and the Control group (Control) and performing quasi-time sequence analysis, and screening the cell subsets mainly subjected to the proportional and quasi-time sequence changes.
As shown in fig. 3, the screening resulted in O1(Fat4+ subset), O2(Fgfr3+ subset), and O3(Scara5+ subset) as candidate direct mechanical responsive cell subsets.
e. And (3) in-vitro verification, namely performing flow sorting on the membrane protein coded by the marker gene of the candidate population, culturing the subset in vitro, and performing in-vitro stress loading.
WT mouse enzyme digestion method to obtain bone tissue cell. CD16/CD32 was subjected to cell surface Fc receptor blocking, fluorescein-conjugated membrane protein antibodies (encoded by marker genes of candidate cell subsets) were subjected to cell surface marker staining, DAPI (0.05-0.2. mu.g/mL) was subjected to dying and the membrane protein-highly expressed viable cell population was flow sorted.
② in vitro culture. The suspension was resuspended in MEM (20% fetal calf serum, 100U/ml penicillin, 100ug/ml streptomycin) and then placed in a cell culture incubator (37 ℃ C., 5% CO)2) Incubation was performed. The first liquid change at 5 days, the suspension cells are discarded, and the liquid change is carried out every 3 days. After 80% cell growth and fusion, passage was performed with 0.25% pancreatin. Well-growing cells were taken for study at passage 1.
Detecting the characteristics of the stem cells:
osteogenic differentiation: replacement of osteogenic induction solution was performed when the cells reached 80% confluence, and every 3 days. The change of related protein and mRNA was detected at 7 days of induction, and alkaline phosphatase enzymatic chemical staining and alizarin red staining were performed at 7 days and 14 days, respectively.
Chondrogenic differentiation: 3-4X 105The cells were resuspended in 0.5ml of complete chondrogenic differentiation medium of C57BL/6 mouse mesenchymal stem cells, placed at 37 ℃ in 5% CO2Culturing in the incubator until the cell mass is gathered, replacing the cells with fresh chondroblast inducing and differentiating complete culture medium every 2-3 days until cartilage balls are formed, and carrying out Alixin blue staining identification.
Adipogenic differentiation: according to 2X 104cells/cm2The cell density of (2) is inoculated in a six-hole plate, after 80% of cells are fused, 2ml of adipogenic induction differentiation culture medium A liquid is added into the six-hole plate, after 3 days of induction, the A liquid in the six-hole plate is sucked away, 2ml of adipogenic induction differentiation culture medium B liquid is added, after 24 hours, the B liquid is sucked away, the A liquid is changed back for induction, and after 3-5 times of alternate action of the A liquid and the B liquid (12-20 days), lipid drops are observed by a microscope.
Alkaline phosphatase enzymatic chemical staining: inducing bone formation at 7 days Fat4+Cells were rinsed 1 time with PBS. 4% PFA was fixed for 3 min. PFA was removed and PBS rinsed for 3 min. ALP staining reagent (formulated according to kit instructions) was added. Incubate at 37 ℃ for 20min in the dark. Rinsing with double distilled water for 5min, and air drying at room temperature.
Alizarin red staining, Fat4 at 14 days of osteogenesis induction+Cells were rinsed 1 time with PBS. 4% PFA was fixed for 3 min. PFA was removed and PBS rinsed for 3 min. Adding saturated alizarin red staining solution, and incubating for 10 min. Rinsing with double distilled water for 5min, and air drying at room temperature.
Crystal violet dyeing: after fixation with 4% paraformaldehyde, washing with distilled water, the monoclonal colonies formed were observed microscopically after staining with crystal violet staining solution.
And fourthly, detecting the mechanical responsiveness by external stress loading. The stress condition of cells in vivo is simulated by periodic tensile stress loading respectively by using an American Flexcell cell strain loading device (Flexcell 6000).
The tensile strain is divided into: 5%, 10%, 15%.
Grouping according to load size, frequency, duration as follows:
blank control group;
continuous tensile stress set: amplitude-5%, 10%, 15%; mode-sustained; the maximum time is-48 h, and the frequency is-1 hz; intermittent tensile stress set: intermittent tensile stress set: amplitude-5%, 10%, 15%; the mode is intermittent, twice a day, 4 hours each time, and 8 hours of pause in the middle; the maximum time is 7d, and the frequency is-0.5 Hz.
In vitro validation experiments, the study of the Fat4+ subgroup was first conducted with emphasis on the significant changes of the Fat4+ subgroup in the stress and control groups, and its expression of BMSCs surface marker molecules. The FAT4+ cell subset was isolated by flow sorting using fluorescein-coupled direct-labeled FAT 4-anti-labeled cell membrane FAT4 protein (fig. 4A). In vitro culture showed that this subpopulation has clonogenic and multipotent differentiation capacity, and it was preliminarily verified that it has stem cell characteristics (FIGS. 4B-E). After the Fat4+ cells are cultured, the uniform Fat4 positive characteristics are still maintained, and in vitro stress loading experiments show that ALP activity and osteogenic marker gene expression of Fat4+ cell subsets are increased under the action of stress (figures 4F-G), which shows that the stress can promote osteogenic differentiation of Fat4+ cell subsets, and direct mechanical responsiveness of the cells is verified.
In vitro flow sorting technology is carried out by adopting fluorescein-coupled direct-labeled FGFR3 primary anti-labeled cell membrane FGFR3 protein and SCARA5 protein, and the Fgfr3+ subgroup and the Scara5+ subgroup are also verified to have direct mechanical responsiveness.
f. And (3) in vivo verification, namely performing lineage tracing and cell elimination verification on the CreER mice driven by the constructed marker gene promoters of the candidate population.
Modifying candidate marker gene by CRISPR/Cas9 technology, and inserting P2A-Cre before stop codonERT2Obtaining a tamoxifen-induced inducible conditional Cre expression mouse model, and constructing a creER mouse driven by a marker gene promoter of a candidate population.
Constructing an induction type conditional fluorescent expression mouse model: marker gene-CreERT2Mouse and R26tdTomatoThe mice are hybridized for the first generation to obtain the induction type conditional fluorescence expression mice.
Constructing an induced cell removal mouse model: marker gene-CreERT2And R26DTA(Rosa26-loxP-STOP-loxP-DTR) mice were mated to obtain a knockout model mouse.
And fourthly, analyzing the osteogenic fate determination and the lineage generation of the candidate cell subset by using an induced conditional fluorescent expression mouse construct internal stress loading model through lineage tracing, and analyzing the osteogenic fate determination and the lineage generation change of the cell subset under the action of stress to clarify the direct mechanical responsiveness of the cell subset.
And analyzing the bone reconstruction speed, alveolar bone microstructure parameters and bone metabolism parameter changes after the candidate cell subset is removed by utilizing an internal stress loading model of the small inducible cell removal construct.
The following results were obtained by in vivo validation: the lineage tracing proves that the Fat4+ subgroup is mainly located around periosteum, marrow cavity and blood vessel cavity under physiological state, while the Fat4+ subgroup is finally differentiated into mature osteoblasts and osteocytes under stress loading, and it is shown that new bones generated in a stress action area contain a large amount of progeny cells of the Fat4+ subgroup. Fgfr3-CreERT2 and Scara5-CreERT2 mice are constructed in vivo, and the Fgfr3+ subgroup and the Scara5+ subgroup are also verified to have direct mechanical responsiveness.
Claims (3)
1. A method for screening a subpopulation of directly mechanically responsive cells, comprising the steps of:
a. constructing an osteoblast line specific fluorescence expression mouse by using a Cre-loxP system, and establishing an in vivo stress loading model;
b. obtaining positive expression stress application group and control group bone tissue single cell suspension by using flow sorting;
c. single cell sorting, library construction, high-throughput sequencing and data analysis, and constructing an osteogenic cell grouping map and a pseudo-time sequence cell differentiation path;
d. comparing the cell population proportion of the stress group and the cell population proportion of the control group and performing quasi-time sequence analysis, and screening cell subsets mainly subjected to proportion and quasi-time sequence change;
e. in-vitro verification, namely performing flow sorting on the membrane protein coded by the marker gene of the candidate population, culturing the subgroup in vitro, and performing in-vitro stress loading;
f. and (3) in vivo verification, namely performing lineage tracing and cell elimination verification on the CreER mice driven by the constructed marker gene promoters of the candidate population.
2. The subpopulation of direct mechanical response cells obtained by the method of claim 1, comprising a Fat4+ subpopulation, a Fgfr3+ subpopulation and a Scara5+ subpopulation.
3. Use of the cell subpopulation according to claim 2 for the preparation of a medicament for the regeneration of local bone defect repair and stress-related bone metabolic disorders.
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