Mechanical force induces macrophage-derived exosomal UCHL3 promoting bone marrow mesenchymal stem cell osteogenesis by targeting SMAD1.

BACKGROUND: Orthodontic tooth movement (OTM), a process of alveolar bone remodelling, is induced by mechanical force and regulated by local inflammation. Bone marrow-derived mesenchymal stem cells (BMSCs) play a fundamental role in osteogenesis during OTM. Macrophages are mechanosensitive cells that can regulate local inflammatory microenvironment and promote BMSCs osteogenesis by secreting diverse mediators. However, whether and how mechanical force regulates osteogenesis during OTM via macrophage-derived exosomes remains elusive. RESULTS: Mechanical stimulation (MS) promoted bone marrow-derived macrophage (BMDM)-mediated BMSCs osteogenesis. Importantly, when exosomes from mechanically stimulated BMDMs (MS-BMDM-EXOs) were blocked, the pro-osteogenic effect was suppressed. Additionally, compared with exosomes derived from BMDMs (BMDM-EXOs), MS-BMDM-EXOs exhibited a stronger ability to enhance BMSCs osteogenesis. At in vivo, mechanical force-induced alveolar bone formation was impaired during OTM when exosomes were blocked, and MS-BMDM-EXOs were more effective in promoting alveolar bone formation than BMDM-EXOs. Further proteomic analysis revealed that ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) was enriched in MS-BMDM-EXOs compared with BMDM-EXOs. We went on to show that BMSCs osteogenesis and mechanical force-induced bone formation were impaired when UCHL3 was inhibited. Furthermore, mothers against decapentaplegic homologue 1 (SMAD1) was identified as the target protein of UCHL3. At the mechanistic level, we showed that SMAD1 interacted with UCHL3 in BMSCs and was downregulated when UCHL3 was suppressed. Consistently, overexpression of SMAD1 rescued the adverse effect of inhibiting UCHL3 on BMSCs osteogenesis. CONCLUSIONS: This study suggests that mechanical force-induced macrophage-derived exosomal UCHL3 promotes BMSCs osteogenesis by targeting SMAD1, thereby promoting alveolar bone formation during OTM.

Mechanical force modulates macrophage proliferation via Piezo1-AKT-Cyclin D1 axis.

Orthodontic tooth movement (OTM) is induced by biomechanical stimuli and facilitated by periodontal tissue remodeling, where multiple immune cells participate in this progression. It has been demonstrated that macrophage is essential for mechanical force-induced tissue remodeling. In this study, we first found that mechanical force significantly induced macrophage proliferation in human periodontal samples and murine OTM models. Yet, how macrophages perceive mechanical stimuli and thereby modulate their biological behaviors remain elusive. To illustrate the mechanisms of mechanical force-induced macrophage proliferation, we subsequently identified Piezo1, a novel mechanosensory ion channel, to modulate macrophage response subjected to mechanical stimuli. Mechanical force upregulates Piezo1 expression in periodontal tissues and cultured bone-marrow-derived macrophages (BMDMs). Remarkably, suppressing Piezo1 with GsMTx4 retarded OTM through reduced macrophage proliferation. Moreover, knockdown of Piezo1 effectively inhibited mechanical force-induced BMDMs proliferation. RNA sequencing was further performed to dissect the underlying mechanisms of Piezo1-mediated mechanotransduction utilizing mechanical stretch system. We revealed that Piezo1-activated AKT/GSK3ß signaling was closely associated with macrophage proliferation upon mechanical stimuli. Importantly, Cyclin D1 (Ccnd1) was authenticated as a critical downstream factor of Piezo1 that facilitated proliferation by enhancing Rb phosphorylation. We generated genetically modified mice in which Ccnd1 could be deleted in macrophages in an inducible manner. Conditional ablation of Ccnd1 inhibited periodontal macrophage proliferation and therefore delayed OTM. Overall, our findings highlight that proliferation driven by mechanical force is a key process by which macrophages infiltrate in periodontal tissue during OTM, where Piezo1-AKT-Ccnd1 axis plays a pivotal role.

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling.

During mechanical force-induced alveolar bone remodeling, macrophage-mediated local inflammation plays a critical role. Yet, the detailed heterogeneity of macrophages is still unknown. Single-cell RNA sequencing was used to study the transcriptome heterogeneity of macrophages during alveolar bone remodeling. We identified macrophage subclusters with specific gene expression profiles and functions. CellChat and trajectory analysis revealed a central role of the Ccr2 cluster during development, with the CCL signaling pathway playing a crucial role. We further demonstrated that the Ccr2 cluster modulated bone remodeling associated inflammation through an NF-κB dependent pathway. Blocking CCR2 could significantly reduce the Orthodontic tooth movement (OTM) progression. In addition, we confirmed the variation of CCR2+ macrophages in human periodontal tissues. Our findings reveal that mechanical force-induced functional shift of the Ccr2 macrophages cluster mediated by NF-κB pathway, leading to a pro-inflammatory response and bone remodeling. This macrophage cluster may represent a potential target for the manipulation of OTM.

Biodegradable nanoparticles for improved kidney bioavailability of rhein: preparation, characterization, plasma, and kidney pharmacokinetics.

The aim of this work is to develop biodegradable nanoparticles for improved kidney bioavailability of rhein (RH). RH-loaded nanoparticles were prepared using an emulsification solvent evaporation method and fully characterized by several techniques. Kidney pharmacokinetics was assessed by implanting a microdialysis probe in rat's kidney cortex. Blood samples were simultaneously collected (via femoral artery) for assessing plasma pharmacokinetics. Optimized nanoparticles were small, with a mean particle size of 132.6 ± 5.95 nm, and homogeneously dispersed. The charge on the particles was nearly zero, the encapsulation efficiency was 62.71 ± 3.02%, and the drug loading was 1.56 ± 0.15%. In vitro release of RH from the nanoparticles showed an initial burst release followed by a sustained release. Plasma and kidney pharmacokinetics showed that encapsulation of RH into nanoparticles significantly increased its kidney bioavailability (AUCkidney/AUCplasma = 0.586 ± 0.072), clearly indicating that nanoparticles are a promising strategy for kidney drug delivery.

Effect of glycyrrhizic acid on rhein renal penetration: a microdialysis study in rats.

1. Rhein (RH), a primary active component isolated from rhubarb, is effective in protecting against the progression of diabetic nephropathy (DN) progression. Glycyrrhizic acid (GA), an active constituent of liquorice, is also considered to be a protective agent against DN. Here, we evaluated the effect of GA on the renal penetration of RH in rats. 2. Plasma and renal pharmacokinetics were profiled to estimate kidney penetration. After rats were anesthetized, the carotid artery was used for blood collection and a microdialysis probe was inserted into the kidney cortex to collect dialysate samples. 3. When co-administered with GA, the Vss and CL values of RH in plasma increased by 25% and 34%, respectively. The Cmax in kidney dialysates significantly increased 1.3-fold (p<0.05). There was no change in AUC0-∞ in kidney dialysates, but a significant decrease (2×fold) in the plasma was observed. The AUC0-∞kidney/AUC0-∞plasma ratio of RH, representing kidney penetration, increased by 1.4-fold in the group pre-treated with GA compared to the RH alone group. 4. These results demonstrate that GA increases the renal penetration of RH efficiently and may exert a synergistic effect, although the molecular mechanism of interaction requires further investigation.