A method for screening human amniotic membrane mesenchymal stem cells with potential for promoting bone regeneration

Abstract

The application provides a screening method of human amniotic membrane mesenchymal stem cells (hAMSCs) with bone regeneration promoting potential, and uses the protein level of transmembrane protein 119 (TMEM119) as a functional index of the bone regeneration promoting ability of the hAMSCs, and uses an enzyme-linked immunosorbent assay (ELISA) to detect the TMEM119 protein concentration in a lysis solution prepared from 1×10 7 The application uses the TMEM119 protein concentration not lower than 15000 pg / mL as a screening standard of the hAMSCs with bone regeneration promoting potential, and screens the hAMSCs with significant bone regeneration promoting potential from different batches of hAMSCs, so that the hAMSCs meeting the screening standard of the application have obvious bone formation promoting potential, and the technical problem that the hAMSCs with bone regeneration promoting potential are difficult to be accurately identified and screened in clinical transformation is solved.

Description

Technical Field

[0001] This invention belongs to the field of stem cell screening technology, specifically a method for screening human amniotic mesenchymal stem cells with the potential to promote bone regeneration. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal and multi-lineage differentiation potential, widely found in various tissues. Among them, human amniotic mesenchymal stem cells (hAMSCs) are derived from the discarded placenta after childbirth, possessing advantages such as non-invasive sourcing, abundant sources, and low immunogenicity. hAMSCs can promote osteogenic differentiation of osteoblasts in bone defect areas through paracrine pathways, showing broad application prospects in bone regeneration and repair.

[0003] However, the clinical translation of hAMSCs still faces significant challenges. First, individual donor differences and isolation / culture methods lead to significant heterogeneity in the bone regeneration capacity of different batches of hAMSCs. Second, existing detection systems primarily focus on routine quality indicators such as cell viability and purity, as well as basic biological function evaluations, lacking specific screening criteria for bone regeneration efficacy, making it difficult to ensure the consistency and reliability of their therapeutic effects in bone defect treatment. Currently, only osteogenic differentiation induction methods can be used to assess the bone regeneration capacity of hAMSCs, but these methods are time-consuming, cumbersome, and resource-intensive. Furthermore, although MSC-E4 (CN117398525B) prepared using exogenous gene delivery technology has shown endochondrogenic effects, this method carries ethical and safety risks, and its preparation process is complex and costly. In summary, the heterogeneity of hAMSCs and related technological bottlenecks have become key obstacles restricting their standardized preparation and large-scale clinical application. Summary of the Invention

[0004] This invention provides a method for screening human amniotic mesenchymal stem cells with bone regeneration potential, in order to solve the technical problem of low accuracy in the identification and screening of human amniotic mesenchymal stem cells with bone regeneration potential in clinical translation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for screening human amniotic mesenchymal stem cells with bone regeneration potential, comprising the following steps:

[0006] S1. Acquisition and culture of hAMSCs: P0 primary hAMSCs are extracted from human placental amnion, or P1-P3 generation hAMSCs are revived from cell bank. The P0 primary hAMSCs or the P1-P3 generation hAMSCs are seeded at high density, cultured and passaged to P4-P6 generation hAMSCs.

[0007] S2. Detection of the content of the specific protein TMEM119 in hAMSCs: Extract 1×10 6 One P4-P6 generation of hAMSCs was prepared into 100 μL hAMSCs lysis buffer, and the TMEM119 protein content in the hAMSCs lysis buffer was absolutely quantitatively detected by enzyme-linked immunosorbent assay.

[0008] S3. Screening for hAMSCs with bone regeneration potential: 1×10 7 A batch of hAMSCs with a TMEM119 protein content of not less than 15,000 pg / mL in the lysate extracted from hAMSCs at a rate of 1 cell / mL is considered as hAMSCs with the potential to promote bone regeneration.

[0009] Preferably, the high-density inoculation density of the P1~P3 generation hAMSCs is 0.8~1.5×10⁻⁶. 4 cells / cm 2 .

[0010] Preferably, the screening method completes the evaluation of hAMSCs’ bone-promoting potential within 5 hours.

[0011] Preferably, the hAMSCs in S1 are derived from human placental amniotic tissue.

[0012] Preferably, the extraction of the PO primary hAMSCs from the human placental amnion in step S1 specifically involves: detaching the amnion from the placenta, removing blood and scraping off the chorionic membrane layer, washing with phosphate-buffered saline solution, and then chopping the amnion into small pieces; transferring an appropriate amount of the chopped amnion into a 50 mL centrifuge tube, adding 40 mL of 0.25% w / v trypsin containing 4 mg / mL neutral protease, and incubating in a 37°C water bath for 40 min of tissue digestion, shaking for 30 seconds every 10 min; washing again with phosphate-buffered saline solution, and then adding 40 mL of DMEM basal culture medium (containing 1 g / L collagenase I, 1 g / L collagenase IV, and 2.5 mg / mL DNase), and incubating in a 37°C water bath for a second tissue digestion of 40-60 min; after the second digestion, filtering the mixture through a 70 μm sieve, transferring the filtrate to a 15 mL centrifuge tube, centrifuging at 1500 rpm for 5 min, removing the supernatant, and adding 5 mL of... The cells were resuspended in phosphate buffer solution, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and 5 mL of complete MSC culture medium was added to resuspend the cells to obtain the P0 primary hAMSCs.

[0013] Preferably, the process of reviving hAMSCs from the cell bank in S1 specifically involves: taking frozen P1-P3 generation hAMSCs from the cell bank, heating them in a 37°C water bath for 3-10 min until the ice crystals are completely melted, centrifuging at 1000 rpm for 5 min, removing the supernatant, resuspending the hAMSCs in 1 mL of phosphate buffer solution, centrifuging again at 1000 rpm for 5 min, and removing the supernatant; resuspending the hAMSCs in 1 mL of complete MSC culture medium to obtain the revived P1-P3 generation hAMSCs.

[0014] Preferably, the high-density seeding, passage, and culture of human amniotic mesenchymal stem cells in S1 specifically involves: taking 20 μL of the P0 primary hAMSCs suspension or the revived P1-P3 generation hAMSCs suspension, mixing it with 20 μL of trypan blue and counting the cells, and then culturing the hAMSCs at a density of 0.8~1.5×10⁻⁶ cells / mL. 4 cells / cm 2 Add 25 cm 2 Culture flask, 75 cm 2 Culture flask, 175 cm 2 Culture flask, 225 cm 2 Culture flasks, 10 cm or 15 cm culture dishes, at a rate of 0.2 mL / cm 2 Add MSC complete medium and incubate at 37℃ in a 5% CO2 incubator. Change the MSC complete medium every 3 days until the cell confluence reaches 70-95%, then passage. Remove the medium from the culture flask or dish, add an appropriate amount of phosphate buffer solution to the culture flask, gently shake to rinse, then remove the rinse solution. Add an appropriate amount of recombinant trypsin TrypLE to the culture flask or dish, and incubate in a CO2 incubator for 3 min. Add 2 times the amount of recombinant trypsin TrypLE in MSC complete medium to stop the digestion. Transfer the digested cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, and resuspend the cells in 1-2 mL of MSC complete medium in a centrifuge tube. Count the cells using trypan blue and then pass them at a rate of 0.8-1.5 × 10⁻⁶ cells / mL. 4 cells / cm 2 Transfer to a new culture flask or dish, at a rate of 0.2 mL / cm³. 2 Add culture medium and place in a 37℃ 5% CO2 incubator to complete the subculture. For each subculture, add 1 to the previous generation.

[0015] Preferably, the method for preparing the hAMSCs lysis solution in S2 is one of the following: repeated freeze-thaw method, ultrasonic treatment method, permeation method, and lysis solution treatment method.

[0016] Preferably, in step S2, the enzyme-linked immunosorbent assay (ELISA) involves adding 100 μL of hAMSCs lysis buffer sample diluted 10-fold, along with controls and standards of different concentration gradients, to a microplate pre-coated with TMEM119 antibody. The plate is incubated at 37°C for 120 min, and the liquid is discarded and the plate is washed four times. Next, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody is added to each well, and the plate is incubated at 37°C for 40 min. The liquid is discarded and the plate is washed four times. Then, 100 μL of TMB chromogenic solution is added to each well, and the plate is incubated at 37°C in the dark for 20 min. Finally, 100 μL of stop solution is added to terminate the reaction. The absorbance value at 450 nm is immediately read on a microplate reader, and the concentration of TMEM119 protein in the hAMSCs lysis buffer sample is calculated using a standard curve and the dilution factor.

[0017] The present invention has the following advantages over the prior art:

[0018] This invention provides a method for screening human amniotic mesenchymal stem cells (hAMSCs) with bone regeneration potential. This method, for the first time, uses the protein level of transmembrane protein 119 (TMEM119) as a functional indicator of the bone regeneration capacity of hAMSCs, and employs enzyme-linked immunosorbent assay (ELISA) to detect 1×10⁻⁶ hAMSCs. 7 The concentration of TMEM119 protein in hAMSCs lysis buffer was measured at 15,000 pg / mL, and a screening criterion of not less than 15,000 pg / mL was used to achieve, for the first time, precise screening of hAMSCs with significant bone-promoting function was achieved in multiple batches of hAMSCs.

[0019] An absolute quantitative assay using enzyme-linked immunosorbent assay (ELISA) was employed for the screening of human amniotic mesenchymal stem cells (hAMSCs). Compared to relative quantitative methods such as qPCR, which rely on reference genes and control sample status, this method directly obtains the absolute content of target factors, avoiding result bias caused by system fluctuations, and offers significant advantages in data accuracy and reproducibility. Furthermore, ELISA requires smaller sample volumes, has a simpler operation procedure, and can improve screening efficiency while shortening the testing cycle, making it suitable for large-scale, standardized cell batch screening.

[0020] In cell culture and passage, this invention employs a high-density seeding strategy (0.8–1.5 × 10⁴ / cm²), which differs from conventional seeding methods. This method helps maintain the functional state of hAMSCs during passage, slows down cell senescence, and results in P4–P6 generation hAMSCs exhibiting more youthful characteristics. Simultaneously, this culture strategy improves the consistency and uniformity of hAMSCs during passage, thereby obtaining more stable P4–P6 generation hAMSCs, providing a more reliable guarantee for their subsequent use as a cell source to promote bone potential.

[0021] Compared to traditional methods requiring 21 days for osteogenic induction differentiation detection, this method can complete functional activity evaluation in just 5 hours, significantly improving efficiency. Furthermore, this invention establishes a highly reproducible, standardized, and industrially scalable screening system by clearly defining culture conditions, lysis buffer preparation methods, and standardized ELISA procedures, ensuring the stability and consistency of results. Verification has shown that batches of hAMSCs with bone regeneration potential obtained through this invention exhibit significantly improved osteogenic gene expression and mineralization function in osteoblasts through their conditioned medium, resulting in an osteogenic effect more than 30% higher than that of hAMSCs that do not meet the screening criteria. This effectively solves the technical challenge of accurately identifying and screening hAMSCs with bone regeneration potential in clinical translation. Attached Figure Description

[0022] Figure 1 shows the results of the difference analysis between human amniotic mesenchymal stem cells and chorionic mesenchymal stem cells in Example 1 of the present invention;

[0023] Figure 1A is a volcano plot showing the difference between hAMSCs and human chorionic mesenchymal stem cells (hPMSCs) in Example 1 of this invention. Each point represents a gene. The horizontal axis is Log2 (fold change), and the vertical axis is significance - Log10 (P value). The top 10 genes with the highest Log2 (fold change) among those significantly upregulated by hAMSCs compared to hPMSCs are marked.

[0024] In Figure 1, B represents the top 10 genes with Log2 (Fold change) among those genes that were significantly upregulated by hAMSCs compared to hPMSCs in Example 1 of this invention. The horizontal axis represents different hAMSCs and hPMSCs samples, and the vertical axis represents genes. The color of the grid in the figure is shown in the color scale on the right, which represents the Z-score value after the expression level is standardized. The lines above represent the consistency clustering tree of each sample.

[0025] In Figure 1, C represents the significantly upregulated genes of hAMSCs compared to hPMSCs in Example 1 of this invention. The gene ontology biological function (GO_BP) enrichment is shown on the horizontal axis as the normalized enrichment score and the vertical axis as the top 10 entries with the enrichment score. The box is labeled "Skeletal System Morphogenesis".

[0026] Figure 2 shows the results of the difference analysis between human amniotic mesenchymal stem cells and umbilical cord mesenchymal stem cells in Example 1 of the present invention;

[0027] Figure 2A is a volcano plot showing the difference between hAMSCs and human umbilical cord mesenchymal stem cells (hUCMSCs) in Example 1 of this invention. Each point represents a gene. The horizontal axis is Log2 (Fold change), and the vertical axis is significance - Log10 (P value). The top 10 genes with the highest Log2 (Fold change) among those significantly upregulated by hAMSCs compared to hUCMSCs are marked.

[0028] In Figure 2, B represents the top 10 genes with Log2 (Fold change) among those genes that were significantly upregulated by hAMSCs compared to hUCMSCs in Example 1 of this invention. The horizontal axis represents different hAMSCs and hUCMSCs samples, and the vertical axis represents genes. The color of the grid in the figure is shown in the color scale on the right, which represents the Z-score value after the expression level is standardized. The lines above represent the consistency clustering tree of each sample.

[0029] In Figure 2, C represents the significantly upregulated genes of hAMSCs compared to hUCMSCs in Example 1 of this invention. The gene ontology biological function (GO_BP) enrichment is shown on the horizontal axis as the normalized enrichment score and the vertical axis as the top 10 entries with the enrichment score. The "bone mineralization" entry is marked with a box.

[0030] Figure 3 shows the trilineage differentiation and flow cytometry results of human amniotic mesenchymal stem cells in Example 2 of the present invention;

[0031] Figure 3A shows the results of adipogenic (left), osteogenic (middle), and chondrogenic (right) induction differentiation of human amniotic mesenchymal stem cells in Example 2 of the present invention;

[0032] Figure 3B shows the flow cytometry results of surface markers CD29, CD73, CD90, CD105, CD34, CD45, CD324 and HLA-DR of human amniotic mesenchymal stem cells in Example 2 of the present invention. The horizontal axis represents fluorescence signal intensity and the vertical axis represents the normalized cell proportion.

[0033] Figure 4 shows the experimental results of determining the content of TMEM119 mRNA and protein in hAMSCs lysate and the content of TMEM119 protein in hAMSCs conditioned medium in Example 3 of the present invention.

[0034] Figure 4A shows the TMEM119 expression level of four batches of hAMSCs relative to human embryonic lung fibroblasts (HFL1) in Example 3 of this invention. The horizontal axis represents HFL1 and batches hAMSC1~4, and the vertical axis represents the relative expression level of TMEM119. In the figure, a, b, and c represent the significance of the difference between the batch of hAMSCs and HFL1, hAMSC3, and hAMSC4, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (b and c are similar).

[0035] Figure 4B shows the TMEM119 protein content in the lysates of four batches of hAMSCs and human embryonic lung fibroblasts (HFL1) in Example 3 of this invention. The horizontal axis represents HFL1 and batches of hAMSC1~4, and the vertical axis represents the protein concentration of TMEM119. In the figure, a, b, and c represent the significance of the differences between the batch of hAMSCs and HFL1, hAMSC3, and hAMSC4, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (b and c are similar).

[0036] Figure 5 shows the results of TMEM119 mRNA and protein detection experiments of osteoblasts hFOB 1.19 in Example 4 of the present invention;

[0037] Figure 5A shows the relative expression level of TMEM119 in osteoblasts (hFOB 1.19) co-cultured for 24 hours in untreated control osteoblasts (hFOB 1.19), HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cell conditioned medium, as described in Example 4 of this invention. The horizontal axis represents the control group, the HFL1 conditioned medium co-culture group, and the batches of hAMSC1~4 conditioned medium co-culture groups. The vertical axis represents the relative expression level of TMEM119. In the figure, a, b, c, and d represent the significance of the differences between this group and the control group, the HFL1-CM co-culture group, the hAMSC3-CM co-culture group, and the hAMSC4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0038] Figure 5B shows the TMEM119 protein concentration in hFOB 1.19 osteoblasts co-cultured for 24 hours with untreated control hFOB 1.19, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cell conditioned medium according to Example 4 of this invention. The horizontal axis represents the control group, the HFL1 conditioned medium co-culture group, and the batches of hAMSC1~4 conditioned medium co-culture group, and the vertical axis represents the TMEM119 protein concentration. In the figure, a, b, c, and d represent the significance of the differences between the group and the control group, the HFL1-CM co-culture group, the hAMSC3-CM co-culture group, and the hAMSC4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0039] Figure 6 shows the results of further verification experiments on the bone-promoting potential of hAMSCs when the TMEM119 protein content is not less than 15000 pg / mL in Example 5 of the present invention.

[0040] Figure 6A shows the relative expression level of RUNX2 in hFOB 1.19 osteoblasts (untreated control hFOB 1.19), HFL1 conditioned medium, and conditioned medium from four batches of amniotic mesenchymal stem cells (AMSCs) after 24 hours of co-culture. The horizontal axis represents the control group and the co-culture groups of hAMSCs 1-4 batches, and the vertical axis represents the relative expression level of TMEM119. In the figure, a, b, c, and d represent the significance of the differences between this group and the control group, the HFL1-CM co-culture group, the hAMSCs 3-CM co-culture group, and the hAMSCs 4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0041] Figure 6B shows the relative expression level of BMP2 in osteoblasts hFOB 1.19 (untreated control), HFL1 conditioned medium, and conditioned medium from four batches of amniotic mesenchymal stem cells (AMMSCs) after 24 hours of co-culture. The horizontal axis represents the control group and the co-culture groups in conditioned medium from batches hAMSC1-4, and the vertical axis represents the relative expression level of TMEM119. In the figure, a, b, c, and d represent the significance of the differences between this group and the control group, HFL1-CM co-culture group, hAMSC3-CM co-culture group, and hAMSC4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0042] In Figure 6, C represents the relative ALP expression level in osteoblasts (hFOB 1.19) co-cultured for 24 hours in the untreated control, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cell conditioned medium according to Example 5 of this invention. The horizontal axis represents the control group, the co-culture group in HFL1 conditioned medium, and the co-culture group in batches of hAMSC1~4 conditioned medium, and the vertical axis represents the relative expression level of TMEM119. In the figure, a, b, c, and d represent the significance of the differences between this group and the control group, the HFL1-CM co-culture group, the hAMSC3-CM co-culture group, and the hAMSC4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0043] In Figure 6, D represents the ALP activity in hFOB 1.19 osteoblasts co-cultured with the untreated control, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cell conditioned medium at 0, 1, 3, 7, and 14 days after osteogenic differentiation. The horizontal axis represents the detection results at 0, 1, 3, 7, and 14 days, and the colors are shown in the color scale on the right, representing the control group and the co-culture groups of batches hAMSC1-4 conditioned medium. The vertical axis represents ALP activity. In the figure, a, b, c, and d represent the significance of the differences between this group and the control group, the HFL1-CM co-culture group, the hAMSC3-CM co-culture group, and the hAMSC4-CM co-culture group, respectively. The number of letters represents the significance level, such as a indicating P<0.05, aa indicating P<0.01, and aaa indicating P<0.001 (the same applies to b, c, and d).

[0044] Figure 7 shows the verification results of hAMSCs screened using the screening method of the present invention in Example 6 of the present invention promoting osteoblast viability;

[0045] Figure 7A shows the panoramic (left) and microscopic (right) results of hFOB 1.19 Alizarin Red (ARS) staining of osteoblasts co-cultured with untreated control, HFL1 conditioned medium and four batches of amniotic mesenchymal stem cell conditioned medium and induced osteogenic differentiation for 21 days in Example 6 of the present invention.

[0046] Figure 7B shows the proportion of calcium nodules stained with alizarin red (hFOB 1.19) in osteoblasts co-cultured with untreated control, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cell conditioned medium for 21 days after osteogenic differentiation, compared to the control group. The horizontal axis represents the hFOB 1.19 of osteoblasts in each group, and the vertical axis represents the proportion of calcium nodules compared to the control group. In the figure, a and b represent the significant differences between this group and the control group and the HFL1-CM co-culture group, respectively (P<0.05).

[0047] Figure 7C shows the semi-quantitative analysis of hFOB 1.19 Alizarin Red staining of osteoblasts co-cultured with untreated control, HFL1 conditioned medium and 4 batches of amniotic mesenchymal stem cell conditioned medium for 21 days after induction of osteogenic differentiation in Example 6 of the present invention; in the figure, a and b represent the significant differences between this group and the control group and the HFL1-CM co-culture group, respectively (P<0.05). Detailed Implementation

[0048] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it is not limited to a specific instance, and this method can be applied to similar situations. Example 1

[0049] Please refer to Figures 1 and 2. In this embodiment, the dataset GSE118808 was obtained from the Gene Expression Database (GEO) of the National Center for Biotechnology Information (NCBI). This dataset includes four cases each of hAMSCs, human chorionic mesenchymal stem cells (hPMSCs), and human umbilical cord mesenchymal stem cells (hUCMSCs). Differential analysis was performed using the "limma" package in R. The screening criteria for significantly differentially expressed genes were set as |log2(Fold change)|>1 and P<0.05. Gene ontology (GO) enrichment was performed using the "clusterProfiler" package, and plotting was performed using the "ggplot2" package.

[0050] The analysis results showed that TMEM119 ranked 5th among the genes that were significantly upregulated by hAMSCs compared to hPMSCs. Figure 1 China A Figure 1 In the B group, hAMSCs upregulated genes (including TMEM119) were significantly enriched in the biological function of "skeletal system morphogenesis" compared to hPMSCs. Figure 1 (C). Among the genes significantly upregulated by hAMSCs compared to hUCMSCs, TMEM119 ranked 9th ( Figure 2 China A Figure 2 In the middle B group, hAMSCs upregulated genes (including TMEM119) were significantly enriched in the biological function of "bone mineralization" compared to hUCMSCs. Figure 2 (C). TMEM119 promotes osteoblast proliferation and differentiation by activating the bone morphogenetic protein (BMP)-Runt-related transcription factor 2 (RUNX2) pathway, thereby accelerating bone formation. These results suggest that TMEM119 is highly expressed in hAMSCs compared to hPMSCs and hUCMSCs, and may be closely related to the osteogenic potential of hAMSCs. Example 2

[0051] Please refer to Figure 3. This embodiment is an experiment that extracts hAMSCs with trilineage differentiation potential from human placental amnion tissue.

[0052] Adipogenic differentiation induction: hAMSCs were induced at a rate of 1×10⁻⁶. 5 Seed cells / well into 6-well plates, add 2 mL of MSC complete medium, and incubate at 37°C in a 5% CO2 incubator until confluence reaches 85%. Discard the medium, add 2 mL of hMSC adipogenic differentiation complete medium (adipogenic differentiation basal medium: adipogenic differentiation supplement medium ratio of 8:2), transfer the cells to a 37°C, 5% CO2 incubator, change the medium every 3 days, and continue culturing until day 21. On day 21, aspirate the supernatant, add fixative (4% paraformaldehyde) and fix for 30 min, aspirate the fixative, add 2 mL of 0.216 mg / mL Oil Red O staining solution, incubate at room temperature in the dark for 20-60 min, then aspirate the staining solution, rinse twice with 2 mL of phosphate-buffered saline (PBS), add 2 mL of PBS to each well for further soaking, observe and photograph under a microscope.

[0053] Osteogenic differentiation induction: hAMSCs were induced at a rate of 1×10⁻⁶. 5Seed cells per well into 6-well plates, add 2 mL of MSC complete medium, and incubate at 37°C in a 5% CO2 incubator until confluence reaches 85%. Remove the medium, add 2 mL of hMSC osteogenic differentiation complete medium (osteogenic differentiation basal medium: osteogenic differentiation supplement medium at a ratio of 8:2), and transfer the cells to a 37°C, 5% CO2 incubator. Change the medium every 3 days and continue culturing until day 21. On day 21, aspirate the supernatant, add fixative (4% paraformaldehyde) and fix for 30 minutes. Aspirate the fixative, add 2 mL of 0.1% alizarin red working solution, and incubate at room temperature in the dark for 20-60 minutes. Then aspirate the staining solution, rinse twice with 2 mL of PBS, add 2 mL of PBS to each well for further soaking, and observe and photograph under a microscope.

[0054] Chondrogenic differentiation induction: hAMSCs were induced at a rate of 2 × 10⁻⁶. 5 Add cells to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, resuspend the cells in 1 mL of MSC complete medium, and centrifuge again at 1000 rpm for 5 min. Gently loosen the centrifuge tube cap and incubate at 37°C in a 5% CO2 incubator for 16 hours. Carefully remove the medium, and slowly add 2 mL of chondrogenic differentiation complete medium (chondrogenic differentiation basal medium: chondrogenic differentiation supplement medium ratio of 8:2) along the wall. Transfer the cells to a 37°C 5% CO2 incubator and culture, changing the medium every 2 days for 14 consecutive days. Discard the medium, add 2 mL of PBS to rinse, remove the supernatant, add 2 mL of 4% paraformaldehyde solution, and fix at 25°C for 10 min. Embed the hAMSCs particles with embedding agent to a thickness of 10 μm and section them. Immerse the slides in PBS twice for washing, shaking for 10 min each time at 60 rpm. After the slides were thoroughly dried, 50 μL of OriCell Alcian Blue emulsion was added to each slide. The slides were placed in a humidified chamber and stained in a 37°C drying oven for 30 min. The slides were then slowly rinsed with running water for 3 min. After the slides were thoroughly dried, they were observed and photographed under a microscope.

[0055] Detection of surface markers in hAMSCs: The concentration of hAMSCs cells was adjusted to 2 × 10⁻⁶ cells using FACS staining buffer. 6Cells / mL: Take 50 μL of hAMSCs cell suspension and add it to a 15 mL centrifuge tube. Add 45 μL of PBS and 5 μL of flow cytometry antibodies labeled with FITC or PE (including CD29, CD73, CD90, CD105, CD34, CD45, CD324, and HLA-DR), mix thoroughly, and incubate at 4°C in the dark for 30 min. After centrifugation at 1000 rpm for 5 min, remove the supernatant, wash 3 times with PBS, and centrifuge at 1000 rpm for 5 min after each wash to remove the supernatant. Resuspend the cells in 400 μL of FACS staining buffer and analyze them using a flow cytometer.

[0056] The results showed that hAMSCs were extracted from human placental amnion tissue, and the obtained hAMSCs had the potential for differentiation into adipocytes, osteoblasts, and chondrocytes. Figure 3 (A) The positive rates of CD29, CD73, CD90, and CD105 in the obtained hAMSCs were ≥95%, and the positive rates of CD34, CD45, CD324, and HLA-DR were ≤2%. Figure 3 (B), meaning the obtained hAMSCs conform to the characteristics of MSCs. Example 3

[0057] Please refer to Figure 4. This embodiment provides an experimental procedure for determining the screening criteria for TMEM119 mRNA content in hAMSCs and TMEM119 protein content in lysis buffer.

[0058] qPCR detection of TMEM119 mRNA:

[0059] 1×10⁻⁶ cells were taken from P5 generation hAMSCs, human embryonic lung fibroblast lines HFL1 and hFOB 1.19, respectively, when the confluence reached 90%. 6 Total RNA was extracted from cells, and 200 ng of total RNA was reverse transcribed into cDNA using a reverse transcription kit. Based on TMEM119-specific primers, the mRNA expression of the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TMEM119 was detected by qPCR. The specific primer sequences for TMEM119 and GAPDH are shown below, with GAPDH used as the internal control.

[0060] Human TMEM119 upstream primer F: 5'-CGATGGGTAATAGGCCGAGG-3' (SEQ ID NO.1);

[0061] Human TMEM119 downstream primer R: 5'-TTCCGCCATGACTTCTCTCC-3' (SEQ ID NO.2);

[0062] Human GAPDH upstream primer F: 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO.3);

[0063] Human GAPDH downstream primer R: 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO.4).

[0064] The relative expression level of qPCR was calculated using the 2^(-ΔΔCt) method. The TMEM119 protein content was detected by enzyme-linked immunosorbent assay (ELISA).

[0065] Four batches of hAMSCs were isolated and extracted from four different placental amniotic tissues and cultured to passage P5. When the hFOB1.19, HFL1, and P5 generation hAMSCs reached 90% confluence, cell suspensions were obtained by digestion, centrifugation, and resuspending in PBS. The cell suspensions were then transferred to 1.5 mL EP tubes, and the cell suspension volume was adjusted to 1×10⁻⁶. 6 Centrifuge again to remove supernatant, add 100 µL of cell lysis buffer (concentration 1×10⁻⁶ cells / tube) to lyse cells. 7(cells / mL), the collected lysate was centrifuged at 4℃ and 10000×g for 10 min to remove cell debris, and the supernatant was collected; the lysate sample was diluted 10 times with the provided sample diluent. The human TMEM119 ELISA kit was used to quantify TMEM119 protein in the samples according to the instructions. After equilibration of the kit to room temperature for 30 min, 100 µL of standard, quality control, and diluted test sample were added to the corresponding wells of a 96-well microplate pre-coated with antibody. The plate was covered with a sealing membrane and incubated at 37°C for 120 min. After incubation, the liquid was discarded and the plate was washed four times. 100 μL of horseradish peroxidase (HRP) labeled antibody was added to each well and incubated at 37°C for 40 min. After incubation, the liquid was discarded and the plate was washed four times. 100 μL of TMB chromogenic solution was added to each well and the plate was incubated at 37°C in the dark for 20 min. 100 μL of stop solution was added to terminate the reaction, and the absorbance value at 450 nm was immediately read on a microplate reader. A standard curve was plotted based on the standard concentration and its corresponding absorbance value (OD). The TMEM119 concentration in each sample was calculated using a four-parameter logic (4-PL) curve fitting equation. The absolute concentration of the protein (pg / mL) was calculated, and the TMEM119 concentration was calculated based on the sample dilution factor.

[0066] qPCR and ELISA assays showed that different batches of hAMSCs had significantly different TMEM119 mRNA and protein levels, and the TMEM119 expression in all batches of hAMSCs was higher than that in HFL1. The expression in batches hAMSC1 and hAMSC2 was significantly higher than that in batches hAMSC3 and hAMSC4. Figure 4 China A Figure 4 (See Example B). The lysate of hAMSC1 and hAMSC2 from these batches, containing TMEM119 protein with a concentration exceeding 15000 pg / mL, indicates that these batches possess osteogenic potential. Furthermore, this method screens hAMSCs with osteogenic potential, requiring only 5 hours from P5 generation confluence reaching 90% to obtaining results, while the traditional method, as shown in Example 2, requires 21 days to induce osteogenic differentiation in hAMSCs. Example 4

[0067] Please refer to Figure 5. This embodiment provides the experimental results of TMEM119 mRNA and protein detection of osteoblasts hFOB 1.19 co-cultured with control osteoblasts hFOB 1.19, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cells conditioned medium for 24 hours.

[0068] Four batches of hAMSCs were isolated and extracted from four different placental amniotic tissues and cultured to passage P5. When HFL1 cells and P5 passage hAMSCs reached 50% confluence, the medium was replaced with DMEM / F12. After 48 hours of culture, the culture medium was collected, centrifuged at 5000 rpm for 10 min, and the supernatant was aspirated to remove cell debris. The supernatant was then filtered through a 0.22 μm filter for sterilization to obtain conditioned medium for HFL1 (HFL1-CM) and conditioned medium for hAMSCs (hAMSC-CM). Each batch was subjected to three biological replicates. Human osteoblast cell line hFOB1.19 was cultured at a ratio of 3 × 10⁻⁶ cells / mL. 5 Cells were seeded per well in 6-well plates. A hFOB 1.19 conjugate medium (90% DMEM / F12 + 10% fetal bovine serum (FBS) + 0.3 mg / mL genistein (G418)) was prepared. 2 mL of hFOB 1.19 conjugate medium was added to each well. After 24 hours of culture, cells were observed under a microscope to confirm adherence and growth. The medium was removed from the wells, and the cells were rinsed with 1 mL of phosphate-buffered saline solution. The rinse solution was then removed. 2 mL of hFOB 1.19 conjugate medium was added to the wells of the control group. 2 mL of a 1:1 mixture of the corresponding batch of HFL1-CM and hFOB 1.19 conjugate medium was added to the wells of the HFL1-CM co-culture group. 2 mL of the same mixture was added to the wells of the hAMSC-CM co-culture group. After 24 hours of culture, cells were harvested for subsequent experiments. Details of each group are as follows:

[0069] Control group: Human osteoblast cell line hFOB 1.19 cultured in combined medium;

[0070] HFL1-CM co-culture group: After culturing hFOB 1.19 in the combined medium for 24 hours, the conditioned medium (HFL1-CM) derived from HFL1 was replaced with the hFOB 1.19 combined medium and co-cultured for 24 hours.

[0071] hAMSC1-CM co-culture group: After culturing hFOB 1.19 in the combined medium for 24 hours, hAMSC-CM from a different batch of hAMSC1 was cultured in the combined medium of hFOB 1.19 and co-cultured for another 24 hours.

[0072] hAMSC2-CM co-culture group: After culturing hFOB 1.19 in the combined medium for 24 hours, hAMSC-CM from a different batch of hAMSC2 was cultured in the combined medium of hFOB 1.19 and co-cultured for another 24 hours.

[0073] hAMSC3-CM co-culture group: After culturing hFOB 1.19 in the combined medium for 24 hours, hAMSC-CM from a different batch of hAMSC3 was cultured in the combined medium of hFOB 1.19 and co-cultured for another 24 hours.

[0074] hAMSC4-CM co-culture group: After culturing hFOB 1.19 in the combined medium for 24 hours, hAMSC-CM from a different batch of hAMSC4 was replaced with a mixture of hFOB 1.19 and cultured for another 24 hours.

[0075] The results of this study show that hAMSCs (hAMSC1-CM co-culture group and hAMSC2-CM co-culture group) that meet the screening criteria of this invention can significantly increase the mRNA and protein content of TMEM119 in hFOB 1.19. Figure 5 China A Figure 5 hAMSCs (hAMSC3-CM co-culture group and hAMSC4-CM co-culture group) that did not meet the screening criteria of this invention failed to increase the mRNA and protein content of TMEM119 in hFOB 1.19. Example 5

[0076] Please refer to Figure 6. This embodiment provides the results of osteogenic-related gene mRNA detection experiments and ALP activity detection results of osteoblast hFOB 1.19 cells co-cultured with control osteoblast hFOB 1.19 cells, HFL1 conditioned medium, and conditioned medium from four batches of amniotic mesenchymal stem cells for 24 hours.

[0077] The qPCR detection method is the same as in Example 3. The specific primer sequences for osteogenic-related genes RUNX2, bone morphogenetic protein 2 (BMP2), and alkaline phosphatase (ALP) are as follows, with GAPDH as an internal control.

[0078] Human RUNX2 upstream primer F: 5'-TGTCATGGCGGGTAACGAT-3' (SEQ ID NO.5);

[0079] Human RUNX2 downstream primer R: 5'-AAGACGGTTATGGTCAAGGTGAA-3' (SEQ ID NO.6);

[0080] Human BMP2 upstream primer F: 5'-CAGACCACCGGTTGGAGA-3' (SEQ ID NO.7);

[0081] Human BMP2 downstream primer R: 5'-CCACTCGTTTCTGGTAGTTCTTC-3' (SEQ ID NO.8);

[0082] Human ALP upstream primer F: 5'-TCAGGGCAATGAGGTCACATC-3' (SEQ ID NO.9);

[0083] Human ALP downstream primer R: 5'-CACAATGCCCACGGACTTC-3' (SEQ ID NO.10).

[0084] Induced hFOB 1.19 osteogenic differentiation and ALP detection

[0085] Prepare a combined hFOB1.19 medium of 90% DMEM / F12 + 10% fetal bovine serum (FBS) + 0.3 mg / mL genimycin (G418), and prepare an osteogenic differentiation medium for hFOB 1.19 (90% DMEM / F12 + 10% fetal bovine serum + 0.3 mg / mL genimycin (G418) + 10 mM β-glycerophosphate sodium + 50 μg / mL L-ascorbic acid). Inoculate the human osteoblast line hFOB 1.19 at a rate of 3 × 10⁻⁶ cells / mL. 5Cells were seeded per well in 6-well plates, with 2 mL of hFOB 1.19 combined culture medium added to each well. The plates were cultured until confluence reached 90%. In the control group wells, 2 mL of hFOB 1.19 osteogenic differentiation medium was added again. In the HFL1-CM co-culture group wells, 2 mL of a 1:1 mixture of the corresponding batch of HFL1-CM and hFOB 1.19 osteogenic differentiation medium was added to each well. In the hAMSC-CM co-culture group wells, 2 mL of a 1:1 mixture of the corresponding batch of hAMSC-CM and hFOB 1.19 osteogenic differentiation medium was added to each well. The plates were incubated at 39.5℃, with the medium changed every 3 days. Cells were harvested and lysed at 0, 1, 3, 7, and 14 days to obtain lysate. 50 μL of the lysate was taken and 50 μL of ALP substrate was added. The mixture was incubated at 37°C for 10 min. The reaction was terminated by adding 100 μL of stop solution. The OD value was detected at 405 nm using a microplate reader, and ALP activity was calculated.

[0086] The expression levels of osteogenic-related genes RUNX2, BMP2, and ALP in hFOB 1.19 of each group were detected by qPCR. Figure 6 (From A to C) The results showed that co-culturing hAMSCs (hAMSC1-CM co-culture group and hAMSC2-CM co-culture group) with conditioned medium that met the screening criteria of this invention significantly increased the mRNA expression levels of RUNX2, BMP2 and ALP in hFOB 1.19. However, the expression of osteogenic-related genes in hFOB 1.19 co-cultured with hAMSCs (hAMSC3-CM co-culture group and hAMSC4-CM co-culture group) that did not meet the screening criteria of this invention was not different from that in the control group. This suggests that co-culturing hAMSCs with conditioned medium that met the screening criteria of this invention promoted the osteogenic differentiation potential of hFOB 1.19. ALP activity assays showed that ALP activity in hFOB 1.19 increased with induction differentiation time in all groups. hFOB 1.19 co-cultured with hAMSCs (hAMSC1-CM co-culture group and hAMSC2-CM co-culture group) meeting the screening criteria of this invention exhibited higher ALP activity than other groups at 7 and 14 days. Figure 6 (D). The above results indicate that hAMSCs meeting the screening criteria of this invention can significantly promote the expression of osteoblast-related genes in hFOB 1.19, thereby promoting early osteoogenesis. Example 6

[0087] Please refer to Figure 7. This example provides the results of co-culturing untreated control osteoblasts hFOB 1.19, HFL1 conditioned medium, and four batches of amniotic mesenchymal stem cells conditioned medium with hFOB 1.19 and inducing osteogenic differentiation for 21 days.

[0088] The osteogenic differentiation induction method for hFOB 1.19 in this embodiment is the same as in Example 5. After 21 days of culture, the culture medium was discarded, and the cells were washed twice with PBS. 2 mL of 4% paraformaldehyde fixative was added to each well, and the cells were fixed at room temperature for 30 min. The fixative was discarded, and the cells were washed twice more with PBS. Then, 2 mL of 0.1% Alizarin Red S working solution was added, and the cells were incubated at room temperature in the dark for 30 min. After discarding the staining solution, the cells were washed three times with PBS to remove unbound dye. 2 mL of PBS was added to each well to cover the cells, and the cells were observed and photographed under a microscope. To perform semi-quantitative analysis of the Alizarin Red staining results, after discarding the PBS, 400 µL of 10% (w / v) cetylpyridinium chloride (CPC) solution was added to each well to dissolve the bound dye, and the cells were incubated at room temperature for 30 min. After eluting Alizarin Red S, collect the eluted Alizarin Red S solution and add 100 μL to each well of a 96-well plate. Measure the absorbance at 560 nm using a microplate reader.

[0089] The results showed that co-culturing hAMSCs (hAMSC1-CM or hAMSC2-CM co-culture group) with conditioned medium according to the screening criteria of this invention resulted in hFOB1.19 forming approximately 30% more calcified nodules at 21 days of induced osteogenic differentiation compared to the control group and the HFL1-CM co-culture group. Figure 7 China A Figure 7 Semi-quantitative analysis showed that its absorbance value was significantly higher than that of the control group and the HFL1-CM co-culture group (B). Figure 7 The hFOB1.19 cells co-cultured with conditioned medium containing hAMSCs (C) that did not meet the screening criteria of this invention showed no significant difference from the control group and the HFL1-CM co-culture group. These results further demonstrate that hAMSCs meeting the screening criteria of this invention can significantly enhance the osteogenic differentiation capacity of osteoblasts hFOB1.19.

[0090] Of course, the above description is not intended to limit the invention, nor is the invention limited to the examples given above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also fall within the protection scope of the invention. The parts of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A method for screening human amniotic mesenchymal stem cells with bone regeneration potential, characterized in that, Includes the following steps: S1. Acquisition and Culture of hAMSCs: P0 primary hAMSCs were extracted from human placental amnion, or P1-P3 generation hAMSCs were revived from a cell bank. The P0 primary hAMSCs or the P1-P3 generation hAMSCs were seeded at high density, cultured, and passaged to P4-P6 generation hAMSCs. The high-density seeding density of the P1-P3 generation hAMSCs was 0.8-1.5 × 10⁻⁶. 4 cells / cm 2 ; S2. Detection of the content of the specific protein TMEM119 in hAMSCs: Extract 1×10 6 One P4-P6 generation of hAMSCs was prepared into 100 μL hAMSCs lysis buffer, and the TMEM119 protein content in the hAMSCs lysis buffer was absolutely quantitatively detected by enzyme-linked immunosorbent assay. S3. Screening for hAMSCs with bone regeneration potential: 1×10 7 A batch of hAMSCs with a TMEM119 protein content of not less than 15,000 pg / mL in the lysate extracted from hAMSCs at a rate of 1 cell / mL is considered as hAMSCs with the potential to promote bone regeneration.

2. The method for screening human amniotic mesenchymal stem cells with bone regeneration potential according to claim 1, characterized in that, The extraction of P0 primary hAMSCs from human placental amnion in S1 specifically involves: detaching the amnion from the placenta, removing blood and scraping off the chorionic membrane layer, washing with phosphate-buffered saline solution, and then mincing the amnion; transferring an appropriate amount of the minced amnion to a 50 mL centrifuge tube, adding 40 mL of 0.25% w / v trypsin containing 4 mg / mL neutral protease, and incubating in a 37°C water bath for 40 min of tissue digestion, shaking for 30 seconds every 10 min; washing again with phosphate-buffered saline solution, and then adding 40 mL of DMEM basal medium containing 1 g / L collagenase I, 1 g / L collagenase IV, and 2.5 mg / mL DNase; and incubating in a 37°C water bath for a second tissue digestion of 40-60 min; after the second digestion, filtering the mixture through a 70 μm sieve, transferring the filtrate to a 15 mL centrifuge tube, centrifuging at 1500 rpm for 5 min, removing the supernatant, and adding 5 mL of... The cells were resuspended in phosphate buffer solution, centrifuged at 1500 rpm for 5 min, the supernatant was removed, and 5 mL of complete MSC culture medium was added to resuspend the cells to obtain the P0 primary hAMSCs.

3. The method for screening human amniotic mesenchymal stem cells with bone regeneration potential according to claim 1, characterized in that, The specific steps of S1 in reviving hAMSCs from the cell bank are as follows: Take frozen P1-P3 generation hAMSCs from the cell bank, place them in a 37℃ water bath for 3-10 minutes until the ice crystals are completely melted, then centrifuge at 1000 rpm for 5 minutes, remove the supernatant, add 1 mL of phosphate buffer solution to resuspend the hAMSCs, centrifuge again at 1000 rpm for 5 minutes, and remove the supernatant; add 1 mL of complete MSC culture medium to resuspend the hAMSCs, thus obtaining reviving P1-P3 generation hAMSCs.

4. The method for screening human amniotic mesenchymal stem cells with bone regeneration potential according to claim 1, characterized in that, The high-density seeding, passage, and culture of human amniotic mesenchymal stem cells in S1 are specifically as follows: 20 μL of the P0 primary hAMSCs suspension or the revived P1-P3 generation hAMSCs suspension is mixed with 20 μL of trypan blue and counted. The hAMSCs are then cultured at a density of 0.8~1.5×10⁻⁶ cells / mL. 4 cells / cm 2 Add 25 cm 2 Culture flask, 75 cm 2 Culture flask, 175cm 2 Culture flask, 225 cm 2 Culture flasks, 10 cm culture dishes, or 15 cm culture dishes, at a rate of 0.2 mL / cm. 2 Add MSC complete medium and incubate at 37°C in a 5% CO2 incubator. Change the MSC complete medium every 3 days until the cell confluence reaches 70-95%, then passage. Remove the medium from the culture flask or dish, add an appropriate amount of phosphate buffer solution to the culture flask, shake to rinse the flask, then remove the rinse solution. Add an appropriate amount of recombinant trypsin TrypLE to the culture flask or dish, and incubate in a CO2 incubator for 3 min. Add 2 times the amount of recombinant trypsin TrypLE in MSC complete medium to stop the digestion. Transfer the digested cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, and resuspend the cells in 1-2 mL of MSC complete medium in a centrifuge tube. Count the cells using trypan blue and then fill at a rate of 0.8-1.5 × 10⁻⁶ cells / mL. 4 cells / cm 2 Transfer to a new culture flask or dish, at a rate of 0.2 mL / cm³. 2 Add culture medium and place in a 37℃ 5% CO2 incubator to complete the subculture. For each subculture, add 1 to the previous generation.

5. The method for screening human amniotic mesenchymal stem cells with bone regeneration potential according to claim 1, characterized in that, The method for preparing the hAMSCs lysis buffer in S2 is one of the following: repeated freeze-thaw method, ultrasonic treatment method, permeation method, and lysis buffer treatment method.

6. The method for screening human amniotic mesenchymal stem cells with bone regeneration potential according to claim 1, characterized in that, The enzyme-linked immunosorbent assay (ELISA) in S2 involves adding 100 μL of hAMSCs lysis buffer (diluted 10-fold) along with controls and standards of different concentration gradients to a microplate pre-coated with TMEM119 antibody. The plate is incubated at 37°C for 120 min, after which the liquid is discarded and the plate is washed four times. Next, 100 μL of horseradish peroxidase (HRP)-labeled antibody is added to each well, and the plate is incubated at 37°C for 40 min. After incubation, the liquid is discarded and the plate is washed four times. Then, 100 μL of TMB chromogenic buffer is added to each well, and the plate is incubated at 37°C in the dark for 20 min. Finally, 100 μL of stop solution is added to terminate the reaction. The absorbance at 450 nm is immediately read on a microplate reader, and the concentration of TMEM119 protein in the hAMSCs lysis buffer is calculated using a standard curve and the dilution factor.

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