Exosome‐shuttled mitochondrial transcription factor A mRNA promotes the osteogenesis of dental pulp stem cells through mitochondrial oxidative phosphorylation activation

Abstract Objectives The treatment of bone defects by stem cells (MSCs) has achieved limited success over the recent few decades. The emergence of exosomes provides a new strategy for bone regeneration. Here, we aimed to investigate the effect and mechanisms of exosomes combined with dental pulp stem cells (DPSCs) on bone regeneration. Materials and Methods We isolated exosomes from stem cells from human exfoliated deciduous teeth (SHED) aggregates and evaluated the efficacy of exosomes combined with DPSCs in a cranial bone defect model. The potential mechanisms were further investigated. Results The effect of exosomes combined with DPSCs was remarkable on bone regeneration in vivo and exosomes promoted osteogenic differentiation of DPSCs in vitro. Mechanistically, exosomes increased the expression of mitochondrial transcription factor A (TFAM) in DPSCs by transferring TFAM mRNA. Moreover, highly expressed TFAM in DPSCs enhanced glutamate metabolism and oxidative phosphorylation (OXPHOS) activity. Conclusions Consequently, exosomes strengthened bone regeneration of DPSCs through the activation of mitochondrial aerobic metabolism. Our study provides a new potential strategy to improve DPSC‐based bone regenerative treatment.

Due to the ability of efficient ATP production to sustain cell function, mitochondria are often considered the "powerhouses of the cell." 4 Mitochondria maintain relatively low activity in undifferentiated MSCs and are activated in differentiated MSCs. 5 Bone marrow stem cells 6,7 and skull osteoblasts 8 have been reported to have increased mitochondrial oxidative phosphorylation (OXPHOS) when undergoing osteogenic differentiation. Moreover, both nuclear DNA and mtDNA encode the mitochondrial OXPHOS subunits, where 13 subunits of the OXPHOS complexes are controlled by mtDNA. TFAM, a nuclearcoding protein transported into mitochondria from the cytoplasm, binds specifically to the mtDNA promoter, recruits RNA polymerase to enable the transcription of mtDNA and thus enhances upregulations of five mitochondrial complexes. 9 The deficiency of TFAM causes mitochondrial metabolic reprogramming and predominantly depends on glycolysis through the increased activity of lactate dehydrogenase. 10 Thus, TFAM may be the key to influence osteogenesis through mitochondrial metabolism.
As a newly recognized mechanism of bone formation and bone homeostasis, exosomes shuttle selective proteins, lipids, nucleic acids and glycoconjugates, 11 which are necessary for cell communication. It has been confirmed that exosomes are important stimulators of the osteogenic differentiation of MSCs. 12 A previous study demonstrated that SHED-derived conditioned exosomes enhanced osteogenic differentiation of MSCs in vitro. 13 In addition, exosomes often play an inducing role in metabolic reprogramming in physiological and pathological activities. For example, oligodendrocytes enhance axonal energy metabolism, 14 and tumour-derived exosomes activate glycolysis to promote bone metastasis. 15 It remains unclear whether exosomes from SHED aggregates promote osteogenic differentiation of DPSCs, and the underlying mechanisms by which exosomes could regulate mitochondrial metabolism require in-depth exploration.
In the present study, we investigated the effects of DPSCs com-  Western blot analysis showed that exosomes expressed surface markers, including CD9, CD63, CD81 and TSG101, except for a negative marker Golgin 84 ( Figure S1G). To investigate whether exosomes could be internalized into DPSCs, exosomes labelled with PKH67 were incubated with DPSCs for 24 h. Confocal fluorescence microscopy analysis showed not only exosomes (green) internalized by DPSCs but also colocalization between exosomes and mitochondria (MitoTracker-red) in DPSCs ( Figure 1A).
We performed a functional assay to assess whether exosomes derived from SHED aggregates contribute to the osteogenic differentiation of DPSCs. First, the osteogenic effects of exosomes at different concentrations (0, 30, 60, 90, 120 and 150 μg/ml) were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). In comparison with groups with other exosomes' concentrations (0, 30, 60, 120 and 150 μg/mL), the expression of Runt-related transcription factor 2 (Runx2) was significantly increased in the 90 μg/ml exosomes group ( Figure S2A). DPSCs treated with 90 μg/ml exosomes showed enhanced proliferation ability compared with those treated with other concentrations of exosomes (0 and 30 μg/ml) after 6 days ( Figure 1B). We then evaluated the osteogenic differentiation properties of DPSCs treated with 90 μg/ml exosomes (EXO group) and equal phosphate buffer saline (PBS group). Higher mRNA levels of alkaline phosphatase (ALP), Runx2 and bone morphogenetic protein 2 (BMP2) were found in the EXO group compared with the PBS group as analysed by qRT-PCR ( Figure 1C). Consistently, the EXO group expressed more osteogenic proteins, including ALP, Runx2 and BMP2, than the PBS group ( Figure 1D, Figure S2B). The alizarin red staining of the EXO group showed more mineralized nodules than the PBS group ( Figure 1E, F). Hence, 90 μg/ml exosomes significantly promoted osteogenic differentiation of DPSCs in vitro, and this dose was selected for the following experiments. In addition, we adopted a rat model of mandibular bone defect.

| Exosomes enhance DPSC-mediated repair of cranial and mandibular bone defects
As shown by micro-CT, there was obvious bone regeneration in the EXO + DPSCs + HA group compared with the Control, HA and DPSCs + HA groups ( Figure S2C, D). HE staining and Masson trichrome staining showed increased new bone formation and more collagen deposition in the EXO + DPSCs + HA group ( Figure S2E). Therefore, the results indicated that the combination of DPSCs with exosomes increased cranial and mandibular bone formation in vivo.

| Exosomes improve the expression of TFAM and enhance OXPHOS in DPSCs
Based on our observation of colocalization between exosomes and mitochondria in DPSCs ( Figure 1A), we assumed that the improvement of osteogenic differentiation was related to the mitochondrial function of DPSCs. Consistent with previous reports, we also found that the oxygen consumption rate (OCR) of DPSCs was increased after osteogenic induction ( Figure S3A respiratory capacity ( Figure 3B). We also confirmed by luminescent assay that the EXO group produced more ATP than the PBS group ( Figure 3C). Exosomes enhanced the expression of mitochondrial complexes I-V, as detected by western blot ( Figure 3D). As a regulator of mtDNA transcription, TFAM expression analogously increased ( Figure 3D). Nicotinamide adenine dinucleotide (NADH) produced by the TCA cycle drives the electronic respiratory chain to produce ATP. 16 TFAM knockdown could decrease the NADH/NAD ratio. 17 The NADH/NAD ratio in DPSCs supplemented with exosomes was elevated at 24, 48 and 72 h ( Figure 3E).
As exosomes significantly heightened the OCR of DPSCs, DPSCs might consume a certain nutrient increase, such as glucose, amino acids and fatty acids. However, the extracellular acidification rate (ECAR) of glucose metabolism did not differ between the EXO group and the PBS group ( Figure 3F reserve ( Figure 3G). Glutamate dehydrogenase (GDH), mainly localized in the mitochondrial matrix, catalyses oxidative deamination of L-glutamate to α-ketoglutarate (α-KG) into the TCA cycle. 18 Carnitine palmitoyltransferase (CPT) is a key enzyme of mitochondrial fatty acid oxidation. 19 Here, we found that exosomes slightly reduced the expression of glucose transporter 1 (Glut1) and increased the expression of GDH, and there was no change in the expression of CPT1A ( Figure 3H). To explore whether the mitochondrial function was in a Reactive oxygen species (ROS) level of DPSCs did not change significantly ( Figure S3C). Hence, we showed that exosomes promoted TFAM and GDH expression and enhanced mitochondrial OXPHOS in DPSCs.

| Exosomes strengthen mitochondrial OXPHOS and osteogenic differentiation of DPSCs through TFAM
As we observed the augmentation of TFAM mediated by exosomes, we examined whether this effect would enhance mitochondrial

| Exosomes shuttle TFAM mRNA to heighten osteogenic differentiation of DPSCs
We then investigated the underlying mechanism by which exosomes increase the TFAM expression of DPSCs. Exosomes contained more TFAM mRNA than DPSCs ( Figure 6A) but had no TFAM protein F I G U R E 5 Exosomes strengthen the oxidative phosphorylation (OXPHOS) activity of dental pulp stem cells (DPSCs) through mitochondrial transcription factor A (TFAM). (A) The expression of TFAM and total OXPHOS complexes by western blot analysis. N = 3 independent experiments. (B) Oxygen consumption rate (OCR) from seahorse analysis in DPSCs with supplementation of exosomes. (C) Basal respiration, ATP production, proton leak, maximal respiration and spare capacity in OCR assay. N = 5 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars are mean ± SD.
The alizarin red staining showed that mineralization of the EXO-KD group was reduced ( Figure 6G).
In conclusion, TFAM mRNA was shuttled by exosomes and then expressed in DPSCs. TFAM directly enhanced the expression of

| DISCUSSION
In this study, we found that the combination of DPSCs and exosomes repaired defective cranial bone in mice and mandibular bone in rats.
Our study revealed a previously unrecognized mechanism of exo- Smad5. 22 Moreover, optimized osteoinductive exosomes can be immobilized in a hierarchical scaffold for bone repair through the Bmpr2/Acvr2b competitive receptor-activated Smad pathway. 23 In our previous study, exosomes derived from SHED aggregates promoted angiogenesis in dental pulp regeneration. 24 In the present study, we found that exosomes derived from SHED aggregates strengthened the osteogenic properties of DPSCs, and significantly promoted bone regeneration based on HA as a scaffold.
Exosomes prompted the rise of TFAM, which aroused our interest.
TFAM binds to mtDNA to regulate the transcription of subunits of the mitochondrial electronic respiration chain so as to affect aerobic respiration of mitochondria, 25 which is consistent with our results. Suppression of TFAM has been reported to cause decreased mitochondrial activity and function, impaired mitochondrial respiration and restrained osteogenesis. 26 In our study, TFAM-enhanced mitochondrial OXPHOS and relied on the catabolism of glutamate to support mitochondrial aerobic respiration. Glutamate is oxidized and deaminated to α-KG and NADH by GDH and then flows into the TCA cycle. 16 Importantly, we found that the deletion of TFAM caused a decrease in GDH, suggesting that TFAM might support glutamate metabolites for the TCA cycle to enhance mitochondrial OXPHOS. There have been no other similar reports about this, and we will further investigate the underlying mechanisms by which TFAM affects GDH.
Mitochondrial metabolism is emerging as an instructive signal for cell fate programs, 27 and activated oxidative respiration has been demonstrated to be crucial for differentiation. 28 Inhibiting the entry of pyruvate into the TCA cycle reduces the OXPHOS and differentiation ability of MSCs, while supplementation with α-KG into the TCA cycle can restore OXPHOS and differentiation capacity. 29 Key enzymes that regulate chromatin (both DNA and histones) and protein modifications (i.e., acetylation and methylation) rely on mitochondrial metabolic intermediates as cofactors. 30 Hence, mitochondrial metabolism is inextricably coupled to gene expression and even regulates stem cell fate decisions. 31 α-KG subsequently upregulates BMP signalling through the decrease in H3K9me3 and H3K27me3. 32,33 We found that TFAM was necessary for osteogenesis differentiation in DPSCs. It has been reported that probiotic treatment increases TFAM expression in osteoblasts by promoting Kdm6b/Jmjd3 histone demethylase, which inhibits H3K27me3 epigenetic methylation at the TFAM promoter. 34 Furthermore, TFAM-transgenic mice fed a high-fat diet did not experience an obesity-linked reduction in glucose uptake, mitochondrial biogenesis and mineralization in osteoblasts. 34 Moreover, we found that the knockdown of TFAM caused a decrease in GDH expression, but the mechanism needs to be further explored. Generally, exosomes could induce osteogenic differentiation by using their cargos. 35 In our study, exosomes were rich in TFAM mRNA but did not contain TFAM protein compared with DPSCs. TFAM mRNA shuttled by exosomes was a probable element to promote osteogenic differentiation of DPSCs. Cargo nucleic acids in exosomes could be selectively carried by specific nucleotide sequences or RNA-binding proteins. 36 Recent studies have shown that stem cell-derived exosomes can restore damaged OXPHOS processes by the transfer of TFAM mRNA, thereby repairing mitochondrial damage to some extent and exerting anti-inflammatory effects. 37

| Cell isolation
The experimental protocols were approved by the Hospital Ethics Com- SHED was isolated and cultivated under the protocol previously described. SHED used for each experiment was at passage 2nd-5th. The uptake of exosomes was visualized by a confocal fluorescence microscope (Nikon, Japan).

| qRT-PCR analysis
Total RNA was extracted with TRIzol reagent (15,596,026,Invitrogen) and converted to cDNA using PrimeScript™ RT Master Mix Kit (RR036A, Takara, Japan). Then, qRT-PCR was conducted with TB Green ® Premix Ex Taq™ II (RB820A, Takara) using the quantitative PCR System (Bio-Rad, USA). The primers are shown in Table S1.

| Alizarin red staining
Alizarin red staining (Sigma, USA) was used to assess calcium deposits, and 10% cetylpyridinium chloride was added for quantitative analysis.
The absorbance values were measured at 562 nm.

| Histological analysis
After micro-CT analysis, the mandibles were decalcified with 17% ethylenediaminetetraacetic acid (EDTA) (EDTA0500, MP Biomedicals) for 1 month and then embedded in paraffin. Paraffin sections (3-mm-thick) were stained using HE, as described previously. 39 The percentage of new bone area to the total area was evaluated quantitatively from three randomly-selected sections by ImageJ 1.53c.  In the ECAR assay, glucose (10 mM), oligomycin (1 μM) and 2-DG (50 mM) were subsequently added into the medium.

| Statistical analysis
All results were presented as the mean ± SD of at least three independent experiments. Two-group comparisons were analysed by Student's t tests. Comparisons among three or four groups were evaluated by one-way ANOVA followed by an LSD post hoc test.
A p-value less than 0.05 was considered statistically significant.

| CONCLUSION
Exosomes derived from SHED aggregates promote DPSCs osteogenic differentiation, which contribute to cranial bone defect reparation.
Moreover, the exosomes transfer TFAM mRNA to DPSCs and might activate mitochondrial OXPHOS to increase osteogenic differentiation. Our study provides a new potential strategy to improve the clinical therapy with DPSCs in bone regenerative medicine.

AUTHOR CONTRIBUTIONS
Jia Guo contributed to the study design, execution, data acquisition,