Platelet-derived mitochondria transfer accelerates fracture healing by promoting osteogenic differentiation and metabolic reprogramming of adipose-derived stem cells

Figure 1 Characteristics of rat adipose-derived stem cells. A: Adipogenic differentiation capability of adipose-derived stem cells (ASCs) was analyzed using Oil Red O staining. Scale bar = 50 μm; B and C: Alizarin Red S staining (B) and alkaline phosphatase staining (C) assays were both performed to analyze osteogenic differentiation capability of ASCs. Scale bar = 50 μm; D: Flow cytometry was conducted to determine CD31, CD44, and CD105 levels in ASCs. n = 3.

Figure 2 Platelets facilitate metabolic reprogramming and osteogenic differentiation. Adipose-derived stem cells were treated with platelets. A: Cell proliferation was detected using carboxyfluorescein diacetate succinimidyl ester; B: Glucose consumption was detected using a commercial kit; C and D: Extracellular acidification rate (C) and oxygen consumption rate (D) were measured using the Seahorse experiments; E: Lactate content was detected using a commercial kit; F and G: Alkaline phosphatase staining (F) and Alizarin Red S staining (G) assays were performed to assess osteogenic differentiation. H and I: Quantitative real-time polymerase chain reaction (H) and western blotting (I) were performed to determine the expression levels of collagen I, BMP2, and RUNX2. n = 3. Data were analyzed using Student’s t-test and are presented as mean ± SD. NS: Not significant; CFSE: Carboxyfluorescein diacetate succinimidyl ester; ECAR: Extracellular acidification rate; OCR: Oxygen consumption rate; 2-DG: 2-deoxy-D-glucose; Rote/AA: Rotenone/antimycin A; ALP: Alkaline phosphatase; ARS: Alizarin Red S.

Figure 3 Platelets facilitate metabolic reprogramming and osteogenic differentiation via transferring mitochondria. Adipose-derived stem cells were treated with platelets without mitochondria or mitochondria from the platelets. A and B: Osteogenic differentiation was evaluated using alkaline phosphatase (A) and Alizarin Red S (B) staining; C-E: The expression of collagen I (C), BMP2 (D), and RUNX2 (E) was measured using quantitative real-time polymerase chain reaction; F: Protein levels of collagen I, BMP2, and RUNX2 were measured using western blotting; G and H: Extracellular acidification rate (G) and oxygen consumption rate (H) were measured using Seahorse experiments; I and J: Glucose consumption (I) and lactate content (J) were detected using their corresponding commercial kits. n = 3. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and are presented as mean ± SD. NS: Not significant; ALP: Alkaline phosphatase; ARS: Alizarin Red S; ECAR: Extracellular acidification rate; OCR: Oxygen consumption rate; 2-DG: 2-deoxy-D-glucose; Rote/AA: Rotenone/antimycin A.

Figure 4 The transfer of mitochondria to adipose-derived stem cells promotes histone lactylation. A: After mitochondria and adipose-derived stem cells (ASCs) were co-cultured in a series of ratios, the fluorescence intensity of mitochondrial-labeled ASCs was detected by flow cytometry; B: The linear relationship between mean fluorescence intensity value and mitochondria/ASCs ratio was analyzed using linear regression; C: In the untreated, platelet-treated and mitochondrial-treated groups of ASCs, mitochondria in ASCs were labeled with MitoTracker dye. 4’,6-diamidino-2-phenylindole was used to label the cell nuclei (200 × magnification); D: After treating with mitochondria, the levels of total lactylation (pan-kla), H3K27 lactylation (H3K27 La), H3K18 La, and H3K9 La were examined using western blotting. n = 3. Data were analyzed using Student’s t-test and are presented as mean ± SD. NS: Not significant; MFI: Mean fluorescence intensity; ASC: Adipose-derived stem cell; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 5 H3K18 lactylation facilitates osteogenesis-related gene transcription. A: The heatmap showed the differentially expressed RNAs in adipose-derived stem cells (ASCs) in the control and mitochondria-treated groups; B: The volcano plot showed differentially expressed RNAs in ASCs in the control and mitochondria-treated groups; C: Gene Set Enrichment Analysis results showed the functions of differentially expressed genes; D: The bubble plot showed the Kyoto Encyclopedia of Genes and Genomes enrichment analysis of RNAs in different pathways; E-H: ASCs were treated with mitochondria and 2-deoxy-D-glucose. The expression of AXIN2, BMPR1B, COL1A1, and OSTN was measured using quantitative real-time polymerase chain reaction (E). Chromatin immunoprecipitation was conducted to determine H3K18 lactylation in the gene promoter (F). The transcriptional activity was evaluated by luciferase reporter assay (G). Protein levels of AXIN2, BMPR1B, COL1A1, and OSTN were examined by western blotting (H). n = 3. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and are presented as mean ± SD. 2-DG: 2-deoxy-D-glucose.

Figure 6 Mitochondria facilitated osteogenic differentiation and glycolysis via histone lactylation. Adipose-derived stem cells were treated with mitochondria and 2-deoxy-D-glucose. A and B: Osteogenic differentiation was evaluated using alkaline phosphatase (A) and Alizarin Red S (B) staining; C-E: The expression of collagen I (C), BMP2 (D), and RUNX2 (E) was measured using quantitative real-time polymerase chain reaction; F: Protein levels of collagen I, BMP2, and RUNX2 were measured using western blotting; G and H: Extracellular acidification rate (G) and oxygen consumption rate (H) were measured using Seahorse experiments; I and J: Glucose consumption (I) and lactate content (J) were detected using their corresponding commercial kits. n = 3. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and are presented as mean ± SD. 2-DG: 2-deoxy-D-glucose; ALP: Alkaline phosphatase; ARS: Alizarin Red S; ECAR: Extracellular acidification rate; OCR: Oxygen consumption rate; Rote/AA: Rotenone/antimycin A.

Figure 7 Mitochondria enhance the effect of adipose-derived stem cells on fracture healing by promoting histone lactylation. A: Micro-computed tomography three-dimensional reconstruction image of the fracture location surface; B: Bone microstructure parameters, including bone mineral density, bone volume/total volume, trabecular number, and trabecular thickness were detected using micro-computed tomography; C: Bone histological evaluation was performed using hematoxylin and eosin staining (20 × magnification); D: Cartilage injury was evaluated using saffron O-fast green staining (20 × magnification). n = 6. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and are presented as mean ± SD. ASC: Adipose-derived stem cell; BMD: Bone mineral density; BV/TV: Bone volume/total volume; Tb.N: Trabecular number; Tb.Th: Trabecular thickness; 2-DG: 2-deoxy-D-glucose.