Pharmaceutical composition for bone regeneration

Abstract

The present invention provides a pharmaceutical composition for bone regeneration, the composition comprising, as an active ingredient, stem cells transplanted with a fusion-inducing liposome carrier in which osteoblast-derived mitochondria are encapsulated inside cationic liposomes. The pharmaceutical composition according to the present invention can be effectively used as a cell therapeutic agent for bone regeneration.

Description

Pharmaceutical composition for bone regeneration

[0001] The present invention relates to a pharmaceutical composition for bone regeneration. More specifically, it relates to a pharmaceutical composition for bone regeneration comprising, as an active ingredient, a stem cell transplanted with a fusion-inducing liposome delivery vehicle in which osteoblast-derived mitochondria are encapsulated inside a cationic liposome.

[0002] Mitochondrial delivery is an innovative biomedical strategy with the potential to enhance cellular function; thus, it can address various disease pathologies, alleviate chronic conditions, and improve overall health. Mitochondrial delivery technologies include mitochondrial gene transfer, pharmacologically induced mitochondrial biosynthesis, and direct mitochondrial transfer between cells or individuals. These strategies open up the possibility of treating diseases characterized by mitochondrial dysfunction or impaired cellular energy production.

[0003] Among these approaches, mitotherapy, a method of inducing self-healing by injecting mitochondria isolated from healthy donors into damaged cells and tissues, is attracting attention due to its potential. For example, in neurodegenerative diseases, mitochondrial delivery has demonstrated remarkable potential to improve cell survival and function [K. English, et al., Acta Neuropathol Commun 2020, 8(1), 36; Z. Geng, et al., CNS Neurosci Ther 2023, 29(11), 3121; K. Hayakawa, et al., Nature 2016, 535(7613), 551]. Similarly, in muscle diseases, mitochondrial therapy has significantly improved patient outcomes by restoring or protecting mitochondrial function [J. Sun, et al., Science Advances 2022, 8(33), eabp9245; SE Alway, et al.,J Cachexia Sarcopenia Muscle2023,14(1), 493; D. Bhattacharya, et al.,American Journal of Physiology-Cell Physiology2023,325(4), C862].

[0004] In addition, evidence is emerging that mitochondrial transfer can determine the fate of cells. For example, when osteoblast mitochondria are transferred to a progenitor cell, it can stimulate differentiation into a mature osteoblast [J. Suh, et al., Cell Metab 2023, 35(2), 345]. There are also cases where the donor cell and the recipient cell originate from different sources, and cases where the mitochondria of the donor cell reconfigure the metabolic activity of the recipient cell to transfer characteristics from the donor to the recipient [JL Spees, et al., Proceedings of the National Academy of Sciences 2006, 103(5), 1283; L. Cereceda, et al., Front Cell Dev Biol 2023, 11, 1324158; JG Baldwin, et al., Cell 2024; Y. Liu, T. et al., Ageing Res Rev 2023, 91, 102038]. For example, transferring macrophage mitochondria to MSCs reconfigures MSC metabolic processes [W. Cai, et al., Adv Sci (Weinh) 2023, 10(4), e2204871], and transferring MSC mitochondria to T cells induces differentiation into regulatory T cells [AC Court, et al., EMBO Rep 2020, 21(2), e48052]. These findings suggest the possibility that transferring mitochondria from a donor cell to a recipient cell can ultimately determine the fate of the recipient cell through mitochondrial remodeling.

[0005] The inventors conducted various tests to determine whether the transfer of mitochondria from osteoblasts to MSCs promotes osteoblast-like mitochondrial metabolism in MSCs; whether the recipient MSCs differentiate into osteogenic lineages; and if so, through what mechanism. The inventors discovered that when osteoblast mitochondria are introduced into stem cells using a specific carrier, namely a fusion-inducing liposome carrier, several pathways specifically associated with osteoblast mitochondrial metabolism and osteoblast differentiation are upregulated. In particular, the inventors discovered that stem cells transplanted with osteoblast mitochondria using a specific fusion-inducing liposome carrier exhibit excellent bone regeneration-promoting activity.

[0006] Accordingly, the present invention aims to provide a cell therapeutic agent for bone regeneration comprising stem cells transplanted with osteoblast mitochondria using a fusion-inducing liposome delivery vehicle as an active ingredient.

[0007] According to the present invention, a pharmaceutical composition for bone regeneration is provided, comprising as an active ingredient stem cells transplanted with a fusion-inducing liposome delivery vehicle in which osteoblast-derived mitochondria are encapsulated inside a cationic liposome.

[0008] The above cationic liposomes may be formed from a mixture of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane, and their molar ratio may be 1:1 to 2, preferably 1:2. The above fusion-inducing liposome delivery system may be formed by mixing osteoblast-derived mitochondria and cationic liposomes in a mass ratio of 1:1 to 2, preferably 1:1.

[0009] In the pharmaceutical composition of the present invention, the stem cells may be mesenchymal stem cells. The transplantation may be performed by culturing mesenchymal stem cells in a medium containing a fusion-inducing liposome delivery vehicle, and the culture may preferably be a spiroid-forming culture.

[0010] The present invention has revealed that stem cells transplanted with osteoblast mitochondria using a specific carrier, namely a fusion-inducing liposome carrier, exhibit excellent bone regeneration-promoting activity. Therefore, the pharmaceutical composition according to the present invention can be usefully employed as a cell therapeutic agent that promotes bone regeneration.

[0011] Figure 1 shows a comparative analysis of mitochondria in MG63 and MSC cells and mitochondrial formation using cationic liposomes.

[0012] Figures 1 (A) and (B) are transmission electron microscope (A) and confocal laser scanning microscope (B) images comparing mitochondrial morphological differences between MSCs and MG63 cells. Scale bar, 500 nm, 10 μm, and 2 μm.

[0013] Figure 1 (C) is the result of a Seahorse analysis showing the difference in cellular respiration between MSCs and MG63 cells.

[0014] Figure 1 (D) is a heatmap showing differences in mitochondrial characteristics using DE analysis.

[0015] Figure 1 (E) is a simple schematic diagram showing the experimental group.

[0016] Figures 1 (F) and (G) show the results of liposome size (F) and surface charge (G) analysis before and after mitochondrial loading.

[0017] Figure 1 (H) is the result of flow cytometry confirming the encapsulation of mitochondria within fusion-induced liposomes.

[0018] Figure 1 (I) is a TEM image showing MSCs and MG63 mitochondria being incorporated into a liposome. Scale bar, 500 nm.

[0019] Figure 2 shows the evaluation results of mitochondrial delivery and mitochondrial osteodifferentiation effects in MSCs.

[0020] Figure 2 (A) shows the fusion of cationic liposomes and cell membranes observed via confocal laser scanning microscopy and intensity profiling. Scale bar, 30 μm.

[0021] Figure 2 (B) is the result of mtDNA copy number analysis to determine the extent to which liposome-encapsulated mitochondria are delivered to MSCs.

[0022] (C) of Fig. 2 is mtMSC MSC and mtMSC MG63 This is a TEM image showing mitochondrial morphological changes. Scale bars, 10 μm and 1 μm.

[0023] Figure 2 (D) shows the results of observing changes in mitochondrial membrane potential via TMRE and JC-1 staining after mitochondrial delivery. Scale bar, 50 μm.

[0024] Figures 2 (E) and (F) are mtMSC MSC and mtMSC MG63 These are the results of qRT-PCR (E) and Western blotting (F) analysis showing the expression levels of bone formation-related genes.

[0025] Figure 2 (G) shows MG63 and mtMSC using DE analysis. MG63 This is the result of comparing the oncogene expression level of MSCs with the oncogene expression level of the tumor gene.

[0026] Figure 3 shows the osteogenic signaling pathway and gene expression changes induced by MG63-derived mitochondria in MSCs.

[0027] Figure 3 (A) shows MG63 vs. MSC and mtMSC MG63This is a Volcano plot showing transcriptome differences between vs. MSCs.

[0028] Figure 3 (B) is a Venn diagram showing DEGs that are equally upregulated in comparison to (A).

[0029] Figure 3 (C) is the result of gene network analysis showing the BMP2-Wnt / β-catenin-calcium inflow axis.

[0030] Figure 3 (D) shows BMP2 signaling and β-catenin markers over time after each mitochondrial delivery.

[0031] Figure 3 (E) is the result of qRT-PCR analysis comparing the expression levels of major markers of calcium influx.

[0032] Figure 3 (F) shows the results of confocal laser scanning microscope image analysis of calcium influx occurring during mitochondrial delivery. Scale bar, 200 μm.

[0033] Figure 4 shows the results of DE analysis and evaluation in 3D MSC spiroids after MG63-derived mitochondria delivery.

[0034] Figure 4 (A) is mtMSC MG63 This is a schematic diagram of the analysis performed according to bone differentiation.

[0035] Figure 4 (B) shows mtMSCs 7 days after mitochondrial delivery MG63 This is a Volcano plot showing transcriptome differences between MSC spiroids.

[0036] (C) of Fig. 4 is mtMSC MG63 This is the result of the GO function analysis of DEGs upregulated in spiroids.

[0037] (D) of Fig. 4 is mtMSC MG63 This is the result of an analysis of gene set enrichment of genes related to the osteogenic differentiation pathway in spiroids.

[0038] (E) of Fig. 4 is mtMSC MG63This is the result of the bone differentiation gene network analysis of spiroids.

[0039] Figure 5 shows the results of measuring bone morphogenetic protein expression in 3D MSC spiroids after MG63-derived mitochondria delivery.

[0040] (A) of Fig. 5 is MSC, mtMSC Lipo and mtMSC MG63 This is the result of Western blot analysis of bone formation differentiation protein expression in spiroids.

[0041] Figure 5 (B) shows MSC, mtMSC Lipo and mtMSC MG63 These are the results of the immunohistochemistry analysis of osteogenic differentiation protein expression in spiroid sections. Scale bars, 500 μm and 100 μm.

[0042] (C) of Fig. 5 is MSC, mtMSC Lipo and mtMSC MG63 This is the histological staining result of a spiroid section. Scale bar, 500 μm.

[0043] Figure 6 shows the results of evaluating in vivo bone regeneration and bone formation differentiation protein expression after transplanting MG63-derived mitochondria into a bone defect site.

[0044] Figure 6 (A) is a schematic diagram of an in vivo experimental protocol for implanting each spiroid into a bone defect rat model.

[0045] Figure 6 (B) shows MSCs and mtMSCs MG63 This is an image of the surgical procedure for implanting a spiroid into a rat femoral defect.

[0046] Figure 6 (C) shows MSCs and mtMSCs MG63 These are micro-CT images and quantitative analysis results of the femur after spiroid implantation.

[0047] Figure 6 (D) shows MSCs and mtMSCs MG63 This is the result of the histological staining analysis of the femur after spiroid implantation. Scale bar, 100 μm.

[0048] Figure 6 (E) shows MSCs and mtMSCs MG63 This is the result of immunohistochemical analysis of osteogenic differentiation protein expression in the femur after spiroid implantation. Scale bars, 1 mm and 200 μm.

[0049] Figure 7 shows the transcriptome analysis results for the difference between MSC and MG63.

[0050] Figures 7 (A) and (B) are the results of a Volcano plot (A) and a GO function analysis (B) of DEG showing transcriptome differences between MSC and MG63.

[0051] Figure 7 (C) shows a heatmap of the difference in mitochondrial dynamics between MSCs and MG63 cells.

[0052] Figure 8 shows the results of flow cytometry analysis of mitochondria and fusion-inducing capsules.

[0053] Figure 9 shows the results of the characterization of the synthesized fusion liposome.

[0054] Figure 9 (A) shows the results of the WST-1 analysis of the cytotoxic release capsule. Figure 9 (B) shows the results of the quantitative analysis of PCR and rat mtDNA copy numbers used to compare the delivery efficiency of nMTs and FMCs.

[0055] Figure 10 is a low-magnification image of the recipient cell after MT delivery.

[0056] Figures 10 (A) and (B) show the results of tetramethylrhodamine ethyl ester (A) and JC-1 analysis (B) on changes in MMP activity in recipient cells. Scale bar, 100 μm.

[0057] Figure 11 shows the results of measuring changes in cellular respiration after mitochondrial delivery. Figure 11 (A) shows the results of cellular mitochondrial stress analysis, and Figure 11 (B) shows the results of glycolytic rate analysis after MG63 mitochondrial delivery.

[0058] Figure 12 shows the results of qRT-PCR analysis to confirm the osteogenic differentiation effect of liposome treatment alone.

[0059] Figure 13 shows the results of qRT-PCR analysis comparing protein expression levels of the BMP-Wnt / β-catenin axis.

[0060] Figure 14 shows the results of Western blot analysis after mitochondrial delivery under β-catenin activation and inhibition conditions. Figure 14 (A) shows FMC pretreated with IWP2 for 48 hours and FMC MG63 This is the result of analyzing the expression levels of β-catenin and bone formation-related proteins after treatment for 0.5 hours. Figure 14 (B) shows FMC after pretreatment with FSK for 0.5 hours. MG63 In the case where it is processed for 0.5 hours, (C) of Fig. 14 is FMC MG63 This is the result of analyzing the expression levels of β-catenin and bone formation-related proteins 24 hours after treatment.

[0061] Figure 15 shows the results of an ELISA analysis of BMP2 secretion over time after MSC and MG63 mitochondrial delivery.

[0062] Fig. 16 is a sequential confocal laser scanning microscope image analyzing calcium influx during mitochondrial transport. Scale bar, 200 μm.

[0063] Figure 17 shows mtMSC MG63 This is the result of an analysis of gene set enrichment of genes related to bone differentiation pathways in spiroids.

[0064] Figure 18 shows mtMSC using DE analysis MG63 This shows the results of comparing the tumor gene expression levels of spiroids with the tumor gene expression levels of MSC spiroids.

[0065] Fig. 19 shows mtMSC MSC and mtMSC MG63The results of qRT-PCR analysis to confirm the effect on other differentiation pathways (adipogenesis and chondrogenesis) are shown. Figure 19 (A) shows the results 24 hours after each mitochondria delivery, and Figure 19 (B) shows the results performed after 7 days.

[0066] Figure 20 shows MSC, mtMSC Lipo and mtMSC MG63 Shows the results of immunostaining analysis for ATF4 and BSP protein expression in spiroid sections. Scale bars, 500 μm and 100 μm.

[0067] While mitochondria are known to be essential for intracellular energy production and overall function, new evidence highlights their importance in influencing cellular behavior within cells through mitochondrial delivery. This phenomenon provides a potential basis for developing therapeutic strategies for tissue damage and degeneration, as well as for inhibiting tumor progression. The inventors evaluated whether mitochondria isolated from osteoblasts could promote osteogenic differentiation in mesenchymal stem cells (MSCs). First, mitochondria from MSCs, which primarily utilize glycolysis, were compared with mitochondria from MG63 cells, which rely on OXPHOS. Mitochondria from both cell types were encapsulated in cationic liposomes and delivered to MSCs, and their effects on differentiation were evaluated. In 2D and 3D cultures, the delivery of mitochondria from MG63 cells to MSCs resulted in increased expression of osteogenic markers, including RUNX2, OSX, and OPN, as well as the upregulation of genes involved in BMP2 signaling and calcium influx. This occurred due to increased calcium influx and was regulated by the Wnt / β-catenin signaling pathway. In animal models of bone defects, the transplantation of spiroids containing MSCs with MG63-derived mitochondria improved bone regeneration. These results suggest that delivering MG63-derived mitochondria effectively induces MSCs to osseoform, leading to the development of mitochondria-based therapies.

[0068] The present invention provides a pharmaceutical composition for bone regeneration comprising, as an active ingredient, stem cells transplanted with a fusion-inducing liposome delivery vehicle in which osteoblast-derived mitochondria are encapsulated inside a cationic liposome.

[0069] Mitochondria derived from osteoblasts can be isolated from various osteoblasts by conventional methods. For example, mitochondria derived from osteoblasts can be isolated from human osteosarcoma MG63 cells by differential centrifugation using a commercially available mitochondrial isolation kit.

[0070] The above cationic liposomes can be formed using various lipids used for liposome formation. When a specific lipid mixture is used as a component of the above cationic liposomes, the encapsulation efficiency of osteoblast-derived mitochondria can be increased. That is, the above cationic liposomes can be formed from a mixture of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane. The molar ratio of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane may be 1:1 to 2, preferably 1:2.

[0071] The above fusogenic liposomal delivery system can be formed by mixing osteoblast-derived mitochondria and cationic liposomes. For example, the above fusogenic liposomal delivery system can be formed by mixing osteoblast-derived mitochondria and cationic liposomes in a mass ratio of 1:1 to 2, preferably 1:1.

[0072] In the pharmaceutical composition of the present invention, the stem cells may be mesenchymal stem cells. The transplantation may be performed by culturing mesenchymal stem cells in a medium containing a fusion-inducing liposome delivery vehicle. The culture may preferably be a spiroid-forming culture. For example, a spiroid-forming culture may be performed by culturing 5.0 x 10⁶ cells in a medium containing about 4 μg of a fusion-inducing liposome delivery vehicle. 5This can be performed by culturing mesenchymal stem cells for about 7 days.

[0073] The pharmaceutical composition of the present invention may be formulated into various formulations comprising a pharmaceutically acceptable carrier in addition to stem cells implanted with the aforementioned fusion-inducing liposome delivery vehicle as an active ingredient. That is, the pharmaceutical composition of the present invention may be in the form of a parenteral administration formulation, such as an injection, suitable for intramuscular, intraperitoneal, subcutaneous, or intravenous administration, and may be in the form of a single dose or a multi-dose administration. The injection is typically prepared by preparing a sterile solution of the stem cells implanted with the aforementioned fusion-inducing liposome delivery vehicle of the present invention, and the pH of the solution must be appropriately adjusted and buffered. In the case of intravenous administration, the total concentration of the solute must be adjusted to impart isotonicity to the formulation. The pharmaceutical composition may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier, such as saline solution with a pH of 7.4. The pharmaceutical composition of the present invention comprises 1 x 10⁶ stem cells implanted with the aforementioned fusion-inducing liposome delivery vehicle 6 ~ 5 x 10 6 It may be administered once or multiple times a day at a dosage of cells / kg. Of course, the above dosage and frequency of administration may be appropriately adjusted according to the patient's condition, age, severity, etc.

[0074] The present invention will be explained in more detail below through examples. However, these examples are illustrative of the present invention and are not limited thereto.

[0075] Examples

[0076] 1. Test Method

[0077] (1) Materials

[0078] Neutral lipid 18:0 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), cationic lipid 18:1 DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), and fluorescently labeled lipid 18:1 Liss Rhod PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lisamine rhodamine B sulfonyl) (ammonium salt)) were purchased from Avanti Polar Lipids (Alabama, USA). Dulbecco's modified Eagle's medium, high glucose (DMEM-high), fetal bovine serum (FBS), and Dulbecco's phosphate-buffered saline were purchased from HyClone (Logan, Canada). Antibiotic-antifungal solution and trypsin-EDTA were purchased from Gibco (Massachusetts, USA). Mitotracker Green FM, Mitotracker Deep Red FM, Tetramethylrhodamine (TMRE), JC-1 dye, and Fluo-4 AM were purchased from Invitrogen (CA, USA).

[0079] (2) Cell culture and isolation of mitochondria from donor cells

[0080] Human MSCs were obtained from Lonza (Basel, Switzerland), and human osteosarcoma MG63 cells and rat myoblast L6 cells were obtained from ATCC (Virginia, USA). All cells were cultured in DMEM-high glucose (SH30243.01, HyClone) supplemented with 10% FBS and 1% antibiotic-antifungal solution at 37°C in 5% CO2. The culture medium was changed every 2–3 days, and experiments were mainly performed using cultured cells from the 7th–9th passages. Mitochondria (MT) were isolated from each cell by differential centrifugation using the MT Isolation Kit (89874, Thermo Fisher Scientific).

[0081] (3) RNA isolation

[0082] Total RNA was isolated using Trizol reagent according to the manufacturer's instructions (Invitrogen), and its quality was evaluated using a TapeStation 4000 system (Agilent Technologies, Amstelveen, The Netherlands). RNA quantification was performed using an ND-2000 spectrophotometer (Thermo Fisher Scientific, USA).

[0083] (4) Library preparation and sequencing

[0084] Libraries were prepared from total RNA using the CORALL RNA-Seq V2 Library Prep Kit (LEXOGEN, Austria). mRNA was isolated using the Poly(A) RNA Selection Kit (LEXOGEN, Austria), and the generated mRNA was used for cDNA synthesis and shearing according to the manufacturer's instructions. Indexing was performed using Illumina indexes 1-12, and the enrichment step was carried out using PCR. Subsequently, the libraries were verified by evaluating the average fragment size using TapeStation HS D1000 Screen Tape (Agilent Technologies, Amstelveen, The Netherlands). Quantification was performed using the library quantification kit on the StepOne Real-Time PCR System (Life Technologies, USA). High-throughput sequencing was performed using paired-end 100 sequencing with the NovaSeq 6000 (Illumina, USA).

[0085] (5) RNA-seq data processing and analysis

[0086] Fastq files containing raw sequencing data from RNA-sequencing underwent raw read quality control using FastQC (v0.11.9) and MultiQC (v1.18). The files were processed with Fastp (v0.23.1) to remove low-quality reads and adapters. Subsequently, paired-end reads were mapped to the human genome (GRCh38) using STAR (v2.7.10.b), and the number of reads mapped at the gene level was calculated using Salmon (v1.10.0). The raw count data was normalized using the DESeq2 R package (v1.42.1).

[0087] DE analysis was performed using the DESeq2 R package. Significant DEGs were selected based on the criteria of p < 0.05 and absolute log2 fold change (|Log2FC|) > 1. To elucidate the molecular functions, biological processes, and cellular components of the identified DEGs, GO analysis was performed using g:Profiler. Additionally, GSEA was conducted using the ClusterProfiler R package (v4.10.1) with normalized expression data to identify significant pathways in all genes. Specifically, to observe interactions between key genes, a protein-PPI network was constructed using Cytoscape (v3.10.2) based on the STRING database (v12.0). Based on the genes in the PPI network, hub genes were screened using cytoHubba and ranked using the maximum clique centrality method. Statistical analysis and plotting were performed using R (v4.1.3) supplemented with the ggplot2 R package (v3.5.1).

[0088] (6) MT and FMC Characterization

[0089] To observe the morphology of MTs in each cell, cells were harvested and fixed in 4% paraformaldehyde (PFA) for 0.5 hours. After fixation, the cells were embedded in resin. Subsequently, the blocks were sectioned, stained with uranyl acetate, washed, and observed using TEM (Hitachi, Tokyo, Japan). For fluorescence imaging, MTs were labeled with Mitotracker Green FM (M7514, Invitrogen) or Deep Red (M22426, Invitrogen) and visualized using CLSM (Olympus FV3000, Tokyo, Japan). For the Seahorse assay (103015-100, Agilent), MSCs were 4.0 x 10⁶ 3 Dog, MG63 cells 6.0 x 10 3 The cells were inoculated onto Seahorse cell culture plates (103774-100, Agilent), and the protocol was performed according to the manufacturer's instructions.

[0090] Fusogenic liposomes (FCs) were synthesized using the film hydration method. Lipid components including PE (850715P, Avanti Polar Lipids), DOTAP (890890P, Avanti Polar Lipids), and Liss Rhod PE (810150P, Avanti Polar Lipids) were mixed in chloroform. The molar ratio of PE / DOTAP / LissRhod PE was 1:2:0.1. The chloroform was evaporated overnight in air, and the lipids were dispersed in distilled water (total lipid concentration, 6 mg / mL). The FCs were stored at 4°C prior to use. FMCs were formed by multiple pipetting the isolated MT dispersed in DPBS with the 6 mg / mL FCs prepared by hydration in distilled water at a mass ratio of 1:1 and a volume ratio of 1:5. The size and ZP of each MT and FMC were measured using dynamic light scattering (DLS; Zetasizer Nano ZS, Malvern Panalytical, Malvern, UK). To measure the MT encapsulation efficiency, isolated MTs and FCs were detected using VSSC on a CytoFLEX flow cytometer (Beckman Coulter, CA, USA). Each isolated MT was stained with Mitotracker Deep Red (M22426, Invitrogen), and non-fluorescent FCs were synthesized by removing Liss Rhod PE from the FCs. Subsequently, the isolated MTs and FCs were zoned using non-stained groups, regions where MTs and FCs simultaneously emitted fluorescence were characterized as FMCs, and the MT encapsulation efficiency of the FMCs was quantified.

[0091] (7) Evaluation of MT uptake and delivered MT effects

[0092] To perform imaging of cell membrane fusion for fusion-inducing liposomes to analyze intracellular delivery of MT cells, 8.0 x 10 3MSCs were seeded into 96-well cell culture black plates (33396, SPL), and 0.16 μg of FMC labeled with Mitotracker Green FM was delivered to the cells. Time-course observations were performed for 0.5 hours using CLSM.

[0093] To observe the presence of mitochondria transferred from cells, MSCs were placed in a 6-well cell culture plate (140675, Thermo Fisher Scientific) at a depth of 2.0 x 10⁶ 5 Cells were inoculated, and each FMC was delivered at a rate of 4 μg for 0.5 hours. Afterward, cells were harvested and observed using TEM, and the sampling process was carried out as described above.

[0094] To quantify MT delivery using the xenograft method, L6 cells were placed in a 6-well cell culture plate (140675, Thermo Fisher Scientific) at a density of 2.0 x 10⁶ 5 L6 cells were inoculated, and 4 μg of MSC and MG63 FMC was delivered to the cells for 0.5 hours. After preparing gDNA (106-101, GeneAll), the gDNA was analyzed by PCR and visualized using a gel documentation imaging system (BR170-8265; Bio-Rad Laboratories, Korea).

[0095] (8) TMRE and JC-1 staining analysis

[0096] For TMRE (T669, Invitrogen) and JC-1 (T3168, Invitrogen) staining, MSCs were placed on 8.0 x 10⁶ 96-well black plates (33396, SPL). 3The cells were inoculated with [the appropriate amount]. Subsequently, 0.16 μg of each FMC was administered over 0.5 hours. After 4 hours, the cells were stained with TMRE and JC-1 and observed using CLSM. In this experiment, non-fluorescent FCs were used; TMRE was stained at 1 μm for 0.5 hours, and JC-1 was stained at 1 μg / ml for 0.5 hours. For quantitative data, the same experiment was performed on 96-well black plates (30296, SPL), and fluorescence was measured using a microplate reader (SpectraMax-ID5, Molecule Devices, CA, USA).

[0097] (9) Fluo-4 staining analysis

[0098] For Fluo-4 (F14201, Invitrogen) staining, MSCs were placed on 96-well black plates (33396, SPL) at a depth of 8.0 x 10⁻⁶ 3 After inoculation, 0.16 μg of each FMC was delivered for 0.5 hours. Time-lapse CLSM images were acquired every 3 minutes during the delivery of each FMC for 0.5 hours. Subsequently, the intensity of fluo-4 was quantified using CellSens software (Olympus, Japan).

[0099] (10) Analysis of mRNA and protein levels of mtMSCs in 2D culture

[0100] 2.0 x 10⁶ MSCs in a 6-well cell culture plate (140675, Thermo Fisher Scientific) 5Cells were inoculated, and each FMC was delivered at a rate of 4 μg for 0.5 hours. After 24 hours, cells were harvested, and qRT-PCR was performed to analyze the mRNA levels of each marker. Total RNA was extracted from each group using an RNA prep kit (K-3140, Bioneer, Korea), and cDNA was synthesized using M-MLV reverse transcriptase (28025013, Invitrogen). Real-time PCR was performed under conditions suitable for each primer set using SYBR Green master mix (Takara, Japan) and the primers listed in Table 1 below.

[0101]

[0102]

[0103] For Western blotting analysis, cells were lysed in radioimmunoprecipitation (RIPA) buffer and quantified using a BCA assay (23225, Thermo Fisher Scientific). Protein lysate (30 μg) was separated from an 8-12% SDS-PAGE gel and transferred to a PVDF membrane. After blocking, the membrane was incubated with a primary antibody against the target protein, then with an HRP-conjugated secondary antibody corresponding to the species from which the primary antibody was produced, and the protein signal was detected using a gel documentation imaging system (BR170-8265; Bio-Rad Laboratories, Korea).

[0104] (11) Spheroid culture and staining

[0105] Cells in appropriate liposomes (FC or FMC) MG63 After treatment with ) at a concentration of 4 μg for 0.5 hours, 5.0 x 10 per group 5Spiroid cultures were performed with dog cells for 7 days. To evaluate osteogenic differentiation, Western blotting was performed as described above.

[0106] For staining of spiroid sections, each spiroid was fixed with 4% PFA, dehydrated with 20% sucrose, embedded in an OCT compound, frozen, and then frozen sections were prepared (Leica, Wetzlar, Germany).

[0107] For immunofluorescence, each fragment was permeated with 0.1% Triton X-100, blocked with 1% BSA, and incubated with a primary antibody against the target protein. After incubation with a secondary antibody, the DNA was stained with DAPI. The fragments were placed on glass slides using Canadian balsam (C0249, SAMCHUN chemical), observed under CLSM, and then quantified using CellSens software.

[0108] H&E, alizarin red S, Von Kossa, and aniline blue staining were all performed according to the manufacturer's protocol after removing OCT compounds from each section. The stained samples were examined under an optical microscope (EVOS). TM Visualized using XL Core, AMEX1000, Invitrogen, USA.

[0109] (12) In vivo experiment

[0110] The animal testing was approved by the Institutional Animal Care and Use Committee (IACUC) of CHA University (Approval No.: IACUC230175). 8-week-old SD rats were purchased from Raonbio (Yongin, Korea).

[0111] To create a femoral defect, a hole 2 mm long, 2 mm wide, and 2 mm deep was drilled in the femur of rats anesthetized with isoflurane. In the hole, No cells (Null), MSC spiroids, or mtMSCs MG63 Spiroids were implanted. Two weeks after surgery, the rats were sacrificed and the femurs were excised. The excised femurs were fixed with 4% PFA, and micro-CT imaging and histological analysis were performed. Micro-CT imaging and bone density analysis were performed using a micro-CT scanner (skyscan 1173; Bruker, Massachusetts, USA).

[0112] For histological analysis, the femur was embedded in a paraffin block. After paraffin sectioning and deparaffinization, immunofluorescence, H&E, and Masson's trichrome staining were performed as described above.

[0113] (13) Statistical analysis

[0114] Statistical analysis was performed using Student's t-test with GraphPad Prism 8.0 software. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 were considered statistically significant.

[0115] 2. Results and Discussion

[0116] 2.1. Mitochondria in MSCs and MG63 cells exhibit different characteristics.

[0117] To validate our approach, we first characterized the mitochondria of donor cells (MG63) and recipient cells (MSC) to determine if there were distinct and measurable differences. Transmission electron microscopy (TEM) analysis revealed clear morphological differences between the two mitochondria (Fig. 1A). While MSC mitochondria were elongated, MG63 mitochondria exhibited a much more spherical shape. These morphological differences were further confirmed using confocal laser scanning microscopy (CLSM) analysis of mitochondria stained with the mitochondrial probe Mitotracker (Fig. 1B). Next, mitochondrial activity in the cells was investigated using the Seahorse assay to measure the mitochondrial oxygen consumption rate (OCR) (Fig. 1C). The mitochondria of MG63 cells exhibited higher OCR and greater aerobic metabolism than those of MSCs, suggesting that MG63 cells possess higher mitochondrial activity than MSCs. These results are consistent with previous studies indicating that undifferentiated MSCs generate energy primarily through glycolysis rather than oxidative phosphorylation (OXPHOS). Furthermore, the increased mitochondrial OCR and aerobic metabolism observed in MG63 cells suggest that mitochondria in these cells may promote osteogenic differentiation.

[0118] Aerobic metabolism, which primarily uses oxygen for energy production, is a core function of mitochondria, and OCR values ​​may also represent a measure of OXPHOS activity. Therefore, these data indicate that the mitochondria of MG63 cells have higher OXPHOS activity than those of MSCs. This feature provides evidence that replacing mitochondria in MSCs can promote osteogenic differentiation.

[0119] Next, transcriptome analysis was performed to investigate the mitochondrial characteristics of MG63 and MSC cells. Analysis of Volcano plots revealed various differences in the expression of differentially expressed genes (DEGs) between MSCs and MG63 cells (Fig. 7). To more accurately identify the relevant differences between the two cell types, gene ontology (GO) analysis was performed on DEGs that reached significance using the following criteria: OXPHOS, mitochondrial DNA (mtDNA), glycolysis, mitochondrial fusion, and cleavage (Figs. 1D and 7). This analysis demonstrated that DEGs associated with mtDNA and OXPHOS were upregulated in MG63 cells compared to MSCs, whereas DEGs associated with glycolysis and mitochondrial fusion / decomposition showed significant differences between the two cell types.

[0120] 2.2. Mitochondria formulated into cationic liposomes can be delivered to MSCs through fusion with the cell membrane.

[0121] The inventors aimed to develop a method for stably and effectively delivering mitochondria extracted from MSCs and MG63 cells to MSCs. The inventors encapsulated MG63-derived mitochondria in fusion-inducing liposomes and delivered them to MSCs, after which they evaluated the function of the transplanted mitochondria (Fig. 1E).

[0122] Mitochondrial encapsulation was indirectly confirmed by measuring changes in size and zeta potential (ZP) of each naked mitochondria (nMT), fusogenic capsule (FC), and fusogenic mito-capsule (FMC) using dynamic light scattering (Figs. 1F and 1G). As a result, FMCs were approximately 100 nm larger than FCs, indicating successful mitochondrial encapsulation. When ZP was measured, nMTs exhibited a negative charge of approximately -38 mV, while FCs exhibited a positive charge of approximately +50 mV. FMCs exhibited a positive charge of approximately +35 mV, which is consistent with the encapsulation of negatively charged mitochondria within positively charged liposomes. Subsequently, flow cytometry was used to confirm that mitochondria were successfully encapsulated within fusogenic liposomes. In this analysis, mitochondria and fusion-inducing liposomes in MSCs and MG63 cells were measured using a violet side scatter (VSSC) filter, which enables the detection and degradation of nano-sized particles (Figs. 1H and 8). Unstained MTs and non-fluorescent fusion-inducing liposomes were used to define the negative zone, while stained MTs and fluorescent FCs were used to define the gates of MTs, FCs, and FMCs (Fig. 8). FMC MSC and FMC MG63 Flow cytometry analysis showed that 94% and 92% of mitochondria were successfully encapsulated, respectively, confirming efficient mitochondrial loading into liposomes. TEM imaging enabled the visualization of liposome-encapsulated mitochondria, further confirming these results (Fig. 1I).

[0123] Analysis of the cytotoxicity of liposome-encapsulated mitochondria and the capacity for mitochondrial delivery to MSCs demonstrated that delivery at a concentration of 4 μg for 0.5 hours was non-cytotoxic and mitochondrial delivery was achieved with approximately twice the efficiency of nMTs (Fig. 9). Therefore, unless otherwise specified, these conditions were used for mitochondrial delivery in all additional experiments.

[0124] The purpose of encapsulating mitochondria in FC is to deliver mitochondria (MT) into the cell through fusion with the cell membrane. In CLSM and subsequent cell quantification analyses, the nucleus and cell membrane were stained with DAPI and FITC, respectively, and cells treated with FC derived from the fluorescently labeled lipid Liss Rhod PE showed good colocalization between the FC and the cell membrane (Fig. 2A). These results suggest that FC can efficiently deliver MT by fusing with the cell membrane.

[0125] Next, rat-derived cell lines were treated with MSC-derived mitochondria (mtMSC MSC ) or MG63-derived mitochondria (mtMSC) MG63 ) was treated with equal amounts, and mtDNA expression was confirmed by PCR (Fig. 2B). As a result, no significant difference in mtDNA expression was observed between recipient cells, indicating that mitochondria-encapsulated liposomes were uniformly delivered to the recipient cells.

[0126] Further analysis of mitochondria using TEM imaging after delivery to MSCs revealed clear morphological differences between mitochondria derived from MSCs and those derived from MG63 cells (Fig. 2C). mtMSC MSC In the cells, only mitochondria with an elongated shape characteristic of MSCs were observed, and mtMSCs MG63In this study, both elongated mitochondria of MSCs and short, rounded mitochondria of MG63 cells were observed. These data confirm that mitochondria can be successfully and safely delivered to recipient cells.

[0127] The inventors hypothesized that delivering osteoblast-derived mitochondria to stem cells would have a significant effect on the function, differentiation, and growth of the stem cells, thereby altering energy metabolism, mitochondrial activity, and the regulation of cell signaling pathways, which would influence cell differentiation. Furthermore, they hypothesized that delivering mitochondria to stem cells could confer characteristics similar to those of donor cells. Accordingly, after developing and characterizing a mitochondria delivery technology, they characterized the changes induced by delivering osteoblast-derived mitochondria to MSCs.

[0128] 2.3. MG63-derived mitochondria induced changes in MSC metabolism and protein expression.

[0129] To evaluate changes in liposome-loaded mitochondria after transfer to MSCs, tetramethylrhodamine ethyl ester (TMRE) and the JC-1 assay, which are widely used to measure mitochondrial membrane potential (MMP), were used. TMRE fluorescence intensity increases proportionally with increasing membrane potential, while JC-1 fluorescence is predominantly green at low MMP and red at high MMP (Figs. 2D and 10). Fluorescence intensity measurements using CLSM and a plate reader were performed on mtMSCs MSC and mtMSC MG63 It exhibited TMRE intensity approximately 30% higher than that of MSC cells, indicating that mitochondrial delivery increased MMPs. Additionally, JC-1 fluorescence in mtMSC MSC and mtMSC MG63The cells were predominantly red, indicating that mitochondrial delivery led to a healthier and more active state. Taken together, these data suggest that this approach results in the successful delivery of functional mitochondria and that mitochondrial delivery increases MMPs.

[0130] Next, the delivered MG63 mitochondria were analyzed using the Seahorse assay on mtMSCs MG63 The effect on cellular respiration was investigated (Fig. 11). MSC mitochondria are generally inactive, but after the delivery of MG63-derived mitochondria, mtMSC MG63 Cells transitioned from glycolytic metabolism to aerobic metabolism and exhibited a change in the extracellular acidification rate consistent with this transition. This change was not observed in cells treated only with FC, indicating that this change depends on the introduction of mitochondria. Taken together, these data suggest that introducing MG63 mitochondria into MSCs converts the cellular respiration process to the characteristics of MG63 cells.

[0131] In addition, the effect on osteogenic markers in the liposome group treated alone under the same conditions as mitochondrial delivery (MT) was evaluated via qRT-PCR analysis (Fig. 12), and it was confirmed that liposomes did not have a statistically significant effect on osteogenic markers. Through these results, it was confirmed that liposomes alone do not affect MSCs.

[0132] To investigate the effects of mitochondrial transfer from osteocytes to stem cells in more detail, the expression of various proteins related to bone differentiation was examined. Gene expression analysis via RT-qPCR of bone morphology genes, including osteopontin (OPN), alkaline phosphatase (ALP), osteocalcin (OCN), and osterix (OSX), was performed on mtMSC MSC or compared to MSCs, mtMSCs MG63 It showed that the expression patterns of these genes in cells were clearly upregulated (Fig. 2E). In addition, changes in these gene expression patterns were mtMSC MG63 Protein expression in the cells was found to increase (Fig. 2F). These results indicate that introducing mitochondria derived from osteocytes into stem cells can change cell behavior and improve differentiation ability.

[0133] Considering that the mitochondrial donor cell line originated from osteosarcoma, mtMSC MG63 The expression of oncogenic genes was also evaluated. The expression of functional genes (MSH2, MSH6, POLD1, POLE, and RIOK1) that maintain the osteosarcoma genome was compared in MSCs and MG63 cells (Fig. 2G). Although the expression of these genes was much higher in MG63 cells than in MSCs, mtMSC MG63 It was not higher than in MSCs. Therefore, it is unlikely that transplanting mitochondria from MG63 cells will increase the risk of carcinogenesis in MSCs.

[0134] 2.4. MG63-derived mitochondria promote bone formation through the BMP2-Wnt / β-catenin-calcium influx axis.

[0135] To identify the pathways through which mitochondrial delivery can promote bone formation, differential expression (DE) analysis was performed using transcriptome data on MG63 cells, MSCs, and mtMSCs MG63 Expression profiles between cells and MSCs were compared, and genes commonly expressed in both comparisons were identified. A significant number of DEGs were detected in the comparison between MG63 and MSCs, with 2,965 upregulated and 3,573 downregulated. mtMSC MG63 In the comparison with MSCs, 27 upregulated DEGs and 41 downregulated DEGs were identified, and the bone formation-related genes PTHLT, IDO1, CEMIP, ACAN, HSBP7, TMEFF1, U2AF1, and ATF3 were found in mtMSCs compared to MSC cells. MG63 We observed upregulation or downregulation in (Fig. 3A). The Venn diagram (Fig. 3B) shows the overlap of upregulated DEGs between these two comparisons. Among the six commonly upregulated DEGs, we excluded two DEGs unrelated to osteogenicity and noncoding DEGs. Through this, we focused on two genes associated with osteogenicity: MICOS10-NBL1 and Plastin 1 (PLS1). Analysis of the Protein Interaction Initiative (PPI) subnetwork using the STRING database revealed that MICOS10-NBL1 and PLS1, along with the first neighbor nodes, are involved in BMP2-SMAD signaling (blue cluster) and calcium influx (green cluster), respectively. Additionally, we discovered the presence of a Wnt / β-catenin signaling cluster between these two clusters (Fig. 3C).

[0136] Key genes involved in BMP-2 (bone morphogenetic protein-2) signaling include SMAD, a key mediator of the BMP-2 pathway, and RUNX2 (Runt-related transcription factor 2), which is essential for osteocyte differentiation. Along with OSX, RUNX2 plays a crucial role in the differentiation and maturation of osteocytes. In the Wnt / β-catenin signaling pathway, β-catenin regulates gene expression related to osteoblast differentiation and bone formation, and its role may vary depending on the phosphorylation status of β-catenin. The combined action of BMP-2 and the Wnt / β-catenin signaling pathway contributes to the differentiation of MSCs into mature osteoblasts and the subsequent formation of mineralized bone tissue.

[0137] First, we investigated the differences in expression levels of key factors corresponding to the NBL1-BMP2 axis and the Wnt / β-catenin cluster at the mRNA and protein levels, and mtMSC MSC and mtMSC MG63 Their expression was compared between (Figs. 13 and 3D). The mRNA expression level of NBL1 was mtMSC MG63 It significantly increased in, and the expression levels of related proteins BMP2, BMP2r, phospho-SMAD1 / 5, RUNX2, and OSX were all mtMSC MG63 It increased in the cells. Since BMP2 is a representative growth factor that promotes bone formation, the secreted BMP2 levels were also analyzed, taking into account that the expression of its receptor (BMP2r) was also increased. Consistent with the Western blotting results, mtMSC MG63 The amount of BMP2 secreted from cells is mtMSC MSC It was significantly higher than the amount secreted from the cell and increased in a time-dependent manner (Fig. 15).

[0138] The Wnt / β-catenin signaling pathway, which is closely associated with the NBL1-BMP2 axis, was also investigated. The expression levels of WNT2 and WNT3 were mtMSC MG63In cells, mtMSC MSC It was significantly higher than in cells and MSCs, and the expression of phosphorylated β-catenin (Ser675) was also higher in mtMSCs MG63 It was higher in cells. Phosphorylated β-catenin (Ser675) translocates to the nucleus and acts as a transcription factor that promotes the transcription of osteogenic factors such as RUNX2 and OSX. Therefore, these findings suggest that MG63-derived mitochondria have the potential to promote osteodifferentiation in MSCs by promoting the expression of osteodifferentiation-related genes through the BMP-2 and WNT / β-catenin signaling pathways.

[0139] We hypothesized that MG63-derived mitochondria could act as activators promoting osteodifferentiation in MSCs by activating p-β-catenin Ser675, thereby stimulating the BMP-2 signaling pathway and enhancing related factors. To verify this, we used IWP2, a Wnt inhibitor that suppresses Wnt production and reduces the expression levels of β-catenin. Cells were pretreated with IWP2 for 48 hours and then treated with MG63 mitochondria for 1 hour; subsequently, phospho-β-catenin S675 and components of the BMP2 signaling pathway were analyzed. In the IWP2-treated group, the levels of phospho-β-catenin S675, BMP2, p-SMAD1 / 5, and RUNX2 were reduced, suggesting that phospho-β-catenin S675 acts as a transcription factor to stimulate the BMP2 pathway and enhance the transcription and expression of related markers. On the other hand, the expression of these factors increased in the MG63 mitochondria-treated group, but no increase in expression was observed in the group treated with MG63 mitochondria and IWP2 together (Fig. 14A).

[0140] Next, an experiment was conducted to activate p-β-catenin S675 using FSK (forskolin) as an activator. Cells were treated with FSK for 30 minutes followed by treatment with MG63 mitochondria, and the results were evaluated. In the FSK-treated group, p-β-catenin S675 levels increased, and the expression of BMP2-related markers also increased. This indicates a positive correlation between p-β-catenin S675 activation and the promotion of osteogenic differentiation. Similarly, the MG63 mitochondria-treated group showed effects similar to those of FSK, and the same trend was observed in the group treated simultaneously with FSK and MG63 mitochondria. Similar results were confirmed in the same experiment with the analysis time extended to 24 hours (Fig. 14B, C). These results demonstrate that MG63-derived mitochondria play a functional role in promoting osteogenic differentiation by activating phospho-β-catenin S675.

[0141] Next, the PLS1-calcium influx axis (green cluster), which was found to be closely associated with the Wnt / β-catenin signaling pathway in the DE analysis, was investigated. Initial analysis showed increased expression of β-catenin and its first neighbor, CDC42, at the mRNA level (Fig. 13). Therefore, the expression levels of the linked genes, CORO1A and PLS1, were analyzed. The expression levels of CORO1A and PLS1 were compared between MSCs and mtMSCs MSC mtMSCs are better than cells MG63 It was higher in the cells. In addition, the expression levels of TRPV4, another first neighbor of β-catenin, and its neighbors TRPV5 and 6 were higher in mtMSCs than in other groups. MG63 It was higher in cells (Fig. 3E).

[0142] Considering that TRPV4 (transient receptor potential vanilloid 4) is a calcium-permeable ion channel and that there were significant differences in the expression of genes related to calcium influx in the above analysis, calcium ion influx into cells was investigated. To this end, mitochondria were delivered to cells stained with Flou-4, and changes in intracellular calcium ion influx occurring during mitochondrial delivery were observed in real time (Figs. 3F and 16). After mitochondrial delivery, Flou-4 intensity was higher in mtMSCs compared to MSCs MSC and mtMSC MG63 It increased in cells, and this increase is mtMSC MG63 This was particularly pronounced in cells, suggesting that calcium influx occurs in mitochondria, specifically when MG63-derived mitochondria are delivered to cells. mtMSC MG63 If calcium influx increases in cells, there is a possibility that TRPV expression will also increase.

[0143] These data confirm that MG63-derived mitochondria (rather than MSC-derived mitochondria) regulate the complex action of the BMP2 pathway centered on Wnt / β-catenin signaling and calcium influx, ultimately causing MSCs to differentiate into osteogenic lineages.

[0144] 2.5. MG63-derived mitochondria promote bone-forming transcript expression in a 3D spiroid state.

[0145] mtMSC so far MG63 It has been confirmed that while cells undergo significant changes during the process of bone differentiation, MSCs and mtMSCs do not. Therefore, mtMSCs MG63 MSCs were used as a control in additional experiments involving cells. To compare the degree of bone differentiation more directly, the expression of osteogenic transcriptome genes was investigated in cells grown into three-dimensional (3D) spiroids (Fig. 4A).

[0146] DE analysis results showed that the bone formation genes FN1, TET2, RICTOR, IGF2, PDGFB, FLT1, WNT4, and S100A4 were present in mtMSCs compared to MSCs. MG63 It was upregulated or downregulated in cells (Fig. 4B). When abundant GO terms were investigated in 230 upregulated DEGs, pathways associated with osteodifferentiation, including collagen binding, regulation of cell differentiation, insulin-like growth factor receptor signaling, and bone mineralization, were found in mtMSCs MG63 It was abundant in cells (Fig. 4C). In addition, by gene set enrichment assay (GSEA), ossification, BMP signaling, bone mineralization, osteoblast differentiation, and Wnt signaling pathways were found in mtMSCs. MG63 It was found that it was significantly upregulated in cells (Fig. 4D). Electron transport combined with mitochondrial ATP synthesis, mitochondrial proteins (including complexes), and nitric oxide metabolism were also upregulated (Fig. 17). These results support the hypothesis that mitochondria in MG63 cells can induce osteodifferentiation genes in MSCs.

[0147] mtMSC MG63 The osteogenic gene network was mapped based on DEGs upregulated in cells (Fig. 4E). This network consisted of four major categories: ossification, bone mineralization, osteoblast differentiation, and extracellular matrix (ECM). This analysis showed that the DEGs within these categories were interconnected, representing a coordinated network. These results suggest that MG63-derived mitochondria influence osteogenic differentiation through a complex gene network involving ossification, bone mineralization, and ECM formation.

[0148] mtMSC MG63 Analysis of the expression of functional genes related to the osteosarcoma genome in 3D spiroids derived from cells was performed (Fig. 18). mtMSCs in 2D cultured cells MG63No significant difference in osteosarcoma gene expression was observed between spiroids and MSC spiroids. This suggests that there is no delayed upregulation of osteosarcoma-related genes after the delivery of MG63-derived mitochondria.

[0149] MSCs are known to be capable of differentiating into three major lineages: osteogenic, chondrogenic, and adipogenic. To investigate the effect of mitochondrial delivery on alternative differentiation pathways, the degree of chondrogenic and adipogenic differentiation was evaluated 24 hours and 7 days after the delivery of mitochondria derived from MG63 cells and MSCs (Fig. 19). At the 24-hour mark, mtMSC MSC Compared to the control group MSC, it did not show statistically significant differences in chondrogenic differentiation markers (COLII, SOX9, COMP) and adipogenic differentiation markers (C / EBPα, SREBP1, adiponectin). On the other hand, mtMSC MG63 MSC and mtMSC MSC Compared to, the expression of all these markers was found to be significantly reduced. These results contrast with the trend observed in osteogenic differentiation markers, suggesting that mitochondrial delivery selectively promotes osteogenic differentiation. Furthermore, mtMSCs cultured for 7 days MG63 In this case, the expression levels of previously reduced markers were restored to levels similar to those of MSCs. Based on these results, it can be concluded that osteocyte-derived mitochondria strongly induce osteodifferentiation in MSCs in the early stages and have minimal effect on alternative differentiation pathways.

[0150] 2.6. MG63-derived mitochondria promote ECM expression and guide MSCs to the osteogenic linease.

[0151] We investigated the changes occurring during the late stages of MSC osteodifferentiation. To evaluate the effects of MG63-derived mitochondria, the experimental group was compared with two control groups. The first control group consisted solely of MSCs, and the second control group consisted of MSCs treated with liposomes lacking mitochondria (MSC Lipo It was composed of ). Through this, the specific effects of MG63-derived mitochondria on osteogenic differentiation can be distinguished from the changes occurring in liposomes used for mitochondrial delivery.

[0152] After mitochondrial delivery, recipient MSCs were cultured in 3D spiroids for 7 days, and the degree of differentiation into osteocytes was evaluated using Western blot analysis. The degree of differentiation was assessed by analyzing the expression of major differentiation-related proteins, including ALP, collagen type I (COL I), activating transcription factor 4 (ATF4), and OSX, as described in previous experiments. In all cases, the expression of major osteogenic differentiation proteins was mtMSC MG63 It was significantly higher than MSC and MSCLipo (Fig. 5A). This finding demonstrates that the osteogenic differentiation potential of stem cells can be enhanced through mitochondrial delivery by aligning the metabolic profiles of donor and recipient stem cells.

[0153] To further validate these findings, the expression of the aforementioned differentiation-related proteins was investigated using immunohistochemistry. mtMSC MG63 , 3D spiroid analysis results derived from MSC or MSCLipo cells, mtMSC compared to controls (MSC and MSCLipo) MG63 The expression of ALP, COL I, OSX, and ATF4 was found to increase 1.5 to 5-fold (Figs. 5B and 20). Next, cell morphology, degree of calcification, and ECM expression were investigated to obtain additional evidence for bone formation (Fig. 5C).

[0154] Hematoxylin and eosin (H&E) staining, which distinguishes the nucleus and cytoplasm, showed no abnormalities in the three 3D spiroids, which indicates MSC Lipo and mtMSC MG63 This suggests that the cells are healthy and did not experience side effects such as cellular stress or apoptosis that could be induced by the activation of immune responses or other complications. When calcium salts were detected in 3D spiroids using Von Kossa staining, the level of the black precipitate representing calcium salts was mtMSC MG63 The values ​​in the samples were significantly higher than those in the control samples, indicating advanced bone differentiation. Similarly, by Alizarin Red S staining, mtMSCs were higher than control spiroids. MG63 Abundant and higher levels of calcium ions were detected in the spiroids. Aniline blue staining for the quantitative evaluation of COL I was used on mtMSCs MG63 Spiroids showed higher levels of COL I expression than the control group. Taken together, these data indicate that both increased calcification and ECM formation occurred as a result of MG63 mitochondrial delivery.

[0155] 2.7. MG63-derived mitochondria promote bone regeneration in a bone defect model.

[0156] It was demonstrated that delivering MG63-derived mitochondria to stem cells enhanced differentiation in vitro and within 3D spiroids. Furthermore, these changes were dependent on the presence of mitochondria and were not caused by the FC itself. Next, we evaluated whether MG63-derived mitochondria could promote tissue regeneration in vivo using a rat model with an artificially generated femoral defect.

[0157] An artificial 2mm defect was created in the femur of rats, and three experimental groups were formed: a control group with an untreated defect (null), a group transplanted with only MSC spiroids (MSC), and a group transplanted with MSC spiroids delivered with MG63-derived mitochondria (mtMSC MG63 ) was established (Fig. 6A). Fig. 6B shows an overview of the surgical procedure and the grouping of experimental rats.

[0158] Micro-CT analysis results, mtMSC MG63 Bone regeneration in this group was significantly higher than in the null group and the MSC group (Fig. 6C). Indicators such as bone mineral density, bone volume, and bone surface density were significantly higher in this group than in the MSC-alone group and the control group. This indicates that MG63-derived mitochondria increased bone formation and repair more effectively than MSCs alone.

[0159] mtMSCs in histological examination using H&E and Masson's trichrome staining MG63 The group exhibited extensive tissue repair and collagen formation, in contrast to the minimal repair of the null group and partial repair of the MSC group (Fig. 6D). mtMSC MG63 The group's extensive collagen-rich fibrous matrix supports the fact that MG63-derived mitochondria significantly increase the osteogenic potential of MSCs, leading to excellent tissue repair.

[0160] Immunofluorescence staining of mtMSC MG63The group showed strong expression of major bone differentiation proteins such as bone sialoprotein (BSP), collagen type I (COL I), and osteopontin (OPN) (Fig. 6E). This suggests that MG63-derived mitochondria not only promote tissue repair but also promote bone differentiation of MSCs. These results support the conclusion that MG63-derived mitochondria significantly contribute to effective bone regeneration and suggest potential for clinical applications in bone tissue engineering.

[0161] These results highlight the significant benefits of using MG63-derived mitochondria in enhancing bone regeneration. Compared to the partial or minimal recovery of the null and MSC groups, mtMSC MG63 The nearly complete recovery observed in the group suggests that MG63 mitochondria play a crucial role in promoting bone repair and bone differentiation. Enhanced collagen and ECM formation, along with increased expression of bone differentiation markers, further support the efficacy of MG63-derived mitochondria in promoting bone formation.

[0162] 3. Conclusion

[0163] This study effectively demonstrated that the transplantation of mitochondria from MG63 osteoblast progenitor cells into MSCs significantly enhances bone differentiation and tissue regeneration. MG63-derived mitochondria, which have higher metabolic activity, actively influenced the energy metabolism and gene expression of recipient MSCs, thereby promoting differentiation into osteocytes. Key osteogenic pathways, including BMP and Wnt / β-catenin signaling, were activated, leading to the upregulation of essential osteogenic markers. Importantly, the safety of this approach was demonstrated as mitochondrial delivery did not induce oncogene expression. In vivo experiments using a rat femoral defect model showed that MSCs containing MG63-derived mitochondria promoted near-complete bone regeneration and improved bone density. These findings highlight the potential of mitochondrial transplantation as an effective strategy for optimizing stem cell-based bone tissue engineering. Furthermore, mitochondrial delivery not only enhances the bioenergetic capacity of the recipient cell but also drives specific cell fates by inducing key metabolic and epigenetic changes.

Claims

1. A pharmaceutical composition for bone regeneration comprising, as an active ingredient, stem cells transplanted into a fusion-inducing liposome delivery vehicle in which osteoblast-derived mitochondria are encapsulated inside a cationic liposome.

2. A pharmaceutical composition according to claim 1, characterized in that the cationic liposome is formed from a mixture of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane.

3. A pharmaceutical composition according to claim 2, characterized in that the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane is 1:1 to 2.

4. A pharmaceutical composition according to claim 2, characterized in that the molar ratio of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleo-3-trimethylammonium-propane is 1:

2.

5. A pharmaceutical composition according to claim 1, characterized in that the fusion-inducing liposome delivery vehicle is formed by mixing osteoblast-derived mitochondria and cationic liposomes in a mass ratio of 1:1 to 2.

6. A pharmaceutical composition according to claim 1, characterized in that the fusion-inducing liposome delivery vehicle is formed by mixing osteoblast-derived mitochondria and cationic liposomes in a mass ratio of 1:

1.

7. A pharmaceutical composition according to claim 1, characterized in that the stem cells are mesenchymal stem cells.

8. A pharmaceutical composition according to claim 7, characterized in that the transplantation is performed by culturing mesenchymal stem cells in a medium containing a fusion-inducing liposome delivery vehicle.

9. A pharmaceutical composition according to claim 8, characterized in that the culture is a spiroid-forming culture.

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