Occlusal force orchestrates alveolar bone homeostasis via Piezo1 in female mice.

Healthy alveolar bone is the cornerstone of oral function and oral treatment. Alveolar bone is highly dynamic during the entire lifespan and is affected by both systemic and local factors. Importantly, alveolar bone is subjected to unique occlusal force in daily life, and mechanical force is a powerful trigger of bone remodeling, but the effect of occlusal force in maintaining alveolar bone mass remains ambiguous. In this study, the Piezo1 channel is identified as an occlusal force sensor. Activation of Piezo1 rescues alveolar bone loss caused by a loss of occlusal force. Moreover, we identify Piezo1 as the mediator of occlusal force in osteoblasts, maintaining alveolar bone homeostasis by directly promoting osteogenesis and by sequentially regulating catabolic metabolism through Fas ligand (FasL)-induced osteoclastic apoptosis. Interestingly, Piezo1 activation also exhibits remarkable efficacy in the treatment of alveolar bone osteoporosis caused by estrogen deficiency, which is highly prevalent among middle-aged and elderly women. Promisingly, Piezo1 may serve not only as a treatment target for occlusal force loss-induced alveolar bone loss but also as a potential target for metabolic bone loss, especially in older patients.

Daily occlusal force and estrogen synergistically maintain alveolar bone homeostasis. PIEZO1 in osteoblasts plays a critical role in sensing occlusal force and maintaining bone mass. PIEZO1 may promote osteoclastic apoptosis through osteoblast-secreted FasL through a PIEZO1-STAT3/ESR1-FasL pathway. Restoration of occlusal force with dental therapies as early as possible to prevent alveolar bone loss is the major priority in oral health care. PIEZO1 may serve as a potential target for bone metabolism disorders.

Early-Responsive Immunoregulation Therapy Improved Microenvironment for Bone Regeneration Via Engineered Extracellular Vesicles.

Overactivated inflammatory reactions hinder the bone regeneration process. Timely transformation of microenvironment from pro-inflammatory to anti-inflammatory after acute immune response is favorable for osteogenesis. Macrophages play an important role in the immune response to inflammation. Therefore, this study adopts TIM3 high expression extracellular vesicles (EVs) with immunosuppressive function to reshape the early immune microenvironment of bone injury, mainly by targeting macrophages. These EVs can be phagocytosed by macrophages, thereby increasing the infiltration of TIM3-positive macrophages (TIM3+ macrophages) and M2 subtypes. The TIM3+ macrophage group has some characteristics of M2 macrophages and secretes cytokines, such as IL-10 and TGF-ß1 to regulate inflammation. TIM3, which is highly expressed in the engineered EVs, mediates the release of anti-inflammatory cytokines by inhibiting the p38/MAPK pathway and promotes osseointegration by activating the Bmp2 promoter to enhance macrophage BMP2 secretion. After evenly loading the engineered EVs into the hydrogel, the continuous and slow release of EVsTIM3OE recruits more anti-inflammatory macrophages during the early stages of bone defect repair, regulating the immune microenvironment and eliminating the adverse effects of excessive inflammation. In summary, this study provides a new strategy for the treatment of refractory wounds through early inflammation control.

The odontoblastic differentiation of dental mesenchymal stem cells: molecular regulation mechanism and related genetic syndromes.

Dental mesenchymal stem cells (DMSCs) are multipotent progenitor cells that can differentiate into multiple lineages including odontoblasts, osteoblasts, chondrocytes, neural cells, myocytes, cardiomyocytes, adipocytes, endothelial cells, melanocytes, and hepatocytes. Odontoblastic differentiation of DMSCs is pivotal in dentinogenesis, a delicate and dynamic process regulated at the molecular level by signaling pathways, transcription factors, and posttranscriptional and epigenetic regulation. Mutations or dysregulation of related genes may contribute to genetic diseases with dentin defects caused by impaired odontoblastic differentiation, including tricho-dento-osseous (TDO) syndrome, X-linked hypophosphatemic rickets (XLH), Raine syndrome (RS), hypophosphatasia (HPP), Schimke immuno-osseous dysplasia (SIOD), and Elsahy-Waters syndrome (EWS). Herein, recent progress in the molecular regulation of the odontoblastic differentiation of DMSCs is summarized. In addition, genetic syndromes associated with disorders of odontoblastic differentiation of DMSCs are discussed. An improved understanding of the molecular regulation and related genetic syndromes may help clinicians better understand the etiology and pathogenesis of dentin lesions in systematic diseases and identify novel treatment targets.

Using Inducible Osteoblastic Lineage-Specific Stat3 Knockout Mice to Study Alveolar Bone Remodeling During Orthodontic Tooth Movement.

The alveolar bone, with a high turnover rate, is the most actively-remodeling bone in the body. Orthodontic tooth movement (OTM) is a common artificial process of alveolar bone remodeling in response to mechanical force, but the underlying mechanism remains elusive. Previous studies have been unable to reveal the precise mechanism of bone remodeling in any time and space due to animal model-related restrictions. The signal transducer and activator of transcription 3 (STAT3) is important in bone metabolism, but its role in osteoblasts during OTM is unclear. To provide in vivo evidence that STAT3 participates in OTM at specific time points and in particular cells during OTM, we generated a tamoxifen-inducible osteoblast lineage-specific Stat3 knockout mouse model, applied orthodontic force, and analyzed the alveolar bone phenotype. Micro-computed tomography (Micro-CT) and stereo microscopy were used to access OTM distance. Histological analysis selected the area located within three roots of the first molar (M1) in the cross-section of the maxillary bone as the region of interest (ROI) to evaluate the metabolic activity of osteoblasts and osteoclasts, indicating the effect of orthodontic force on alveolar bone. In short, we provide a protocol for using inducible osteoblast lineage-specific Stat3 knockout mice to study bone remodeling under orthodontic force and describe methods for analyzing alveolar bone remodeling during OTM, thus shedding new light on skeletal mechanical biology.

Exercise maintains bone homeostasis by promoting osteogenesis through STAT3.

Bone exhibits changes in density, strength, and microarchitecture in relation to mechanical loading mediated by exercise. Appropriate exercise maintains bone homeostasis, while the absence of exercise leads to disuse bone loss. However, the acting mechanism of mechanotransduction in bone remains unclear. We performed the running-wheel exercise and tail suspension model to study the effects of exercise on bone metabolism, and found that osteoblastic Signal transducer and activator of transcription 3 (STAT3) activity was closely related to exercise-induced bone mass and metabolism changes. With the Flexcell tension-loading system in vitro, mechanical force promoted STAT3 activity, which was accompanied by increased osteoblastic differentiation of the bone marrow mesenchymal stem cells (BMSCs). In contrast, the inhibition of STAT3 phosphorylation blocked force-induced osteoblastic differentiation. Furthermore, pharmacological inactivation of STAT3 impaired the increase in exercise-induced bone mass and osteogenesis. With an inducible conditional deletion mouse model, we found that the osteoblast lineage-specific Stat3 deletion could also block force-induced osteoblastic differentiation in vitro and impair exercise-promoted bone mass and osteogenesis in vivo. This confirmed the crucial role of osteoblastic STAT3 in exercise-mediated bone metabolism. Finally, colivelin, a STAT3 agonist, promoted osteoblastic differentiation in vitro and partly rescued exercise loss-induced disuse bone loss by improving osteogenesis in the tail suspension model. Taken together, our study revealed the essential role of STAT3 in maintaining exercise-mediated bone homeostasis. In addition, STAT3 might act as a potential target for osteoporosis caused by exercise loss.

ScRNA-seq links dental fibroblasts heterogeneity with mechanoresponsiveness.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) and dental pulp (DP) share a common origin but have distinct biological and mechanical functions. To what extent the mechanoresponsive property of PDL can be attributed to its unique transcriptional profiles of cellular heterogeneity is unclear. This study aims to decipher cellular heterogeneity and distinct mechanoresponsive characteristics of odontogenic soft tissues and their underlying molecular mechanisms. MATERIALS AND METHODS: A single-cell comparison of digested human periodontal ligament (PDL) and dental pulp (DP) was performed using scRNA-seq. An in vitro loading model was constructed to measure mechanoresponsive ability. Dual-luciferase assay, overexpression, and shRNA knockdown were used to investigate the molecular mechanism. RESULTS: Our results demonstrate striking fibroblast heterogeneity across and within human PDL and DP. We demonstrated that a tissue-specific subset of fibroblasts existed in PDL exhibiting high expression of mechanoresponsive extracellular matrix (ECM) genes, which was verified by an in vitro loading model. ScRNA-seq analysis indicated a particularly enriched regulator in PDL-specific fibroblast subtype, Jun Dimerization Protein 2 (JDP2). Overexpression and knockdown of JDP2 extensively regulated the downstream mechanoresponsive ECM genes in human PDL cells. The force loading model demonstrated that JDP2 responded to tension and that knockdown of JDP2 effectively inhibited the mechanical force-induced ECM remodeling. CONCLUSIONS: Our study constructed the PDL and DP ScRNA-seq atlas to demonstrate PDL and DP fibroblast cellular heterogeneity and identify a PDL-specific mechanoresponsive fibroblast subtype and its underlying mechanism.

Osteoblastic STAT3 Is Crucial for Orthodontic Force Driving Alveolar Bone Remodeling and Tooth Movement.

Mechanical force is essential to shape the internal architecture and external form of the skeleton by regulating the bone remodeling process. However, the underlying mechanism of how the bone responds to mechanical force remains elusive. Here, we generated both orthodontic tooth movement (OTM) model in vivo and a cyclic stretch-loading model in vitro to investigate biomechanical regulation of the alveolar bone. In this study, signal transducer and activator of transcription 3 (STAT3) was screened as one of the mechanosensitive proteins by protein array analysis of cyclic stretch-loaded bone mesenchymal stem cells (BMSCs) and was also proven to be activated in osteoblasts in response to the mechanical force during OTM. With an inducible osteoblast linage-specific Stat3 knockout model, we found that Stat3 deletion decelerated the OTM rate and reduced orthodontic force-induced bone remodeling, as indicated by both decreased bone resorption and formation. Both genetic deletion and pharmacological inhibition of STAT3 in BMSCs directly inhibited mechanical force-induced osteoblast differentiation and impaired osteoclast formation via osteoblast-osteoclast cross-talk under mechanical force loading. According to RNA-seq analysis of Stat3-deleted BMSCs under mechanical force, matrix metalloproteinase 3 (Mmp3) was screened and predicted to be a downstream target of STAT3. The luciferase and ChIP assays identified that Stat3 could bind to the Mmp3 promotor and upregulate its transcription activity. Furthermore, STAT3-inhibitor decelerated tooth movement through inhibition of the bone resorption activity, as well as MMP3 expression. In summary, our study identified the mechanosensitive characteristics of STAT3 in osteoblasts and highlighted its critical role in force-induced bone remodeling during orthodontic tooth movement via osteoblast-osteoclast cross-talk. © 2022 American Society for Bone and Mineral Research (ASBMR).

Reducing relapse and accelerating osteogenesis in rapid maxillary expansion using an injectable mesoporous bioactive glass/fibrin glue composite hydrogel.

Rapid maxillary expansion (RME), as a common treatment for craniomaxillofacial deformity, faces the challenge of high relapse rates and unsatisfactory therapeutic effects. In this study, a standardized Sprague-Dawley (SD) rat RME model was first established with a modified expander as well as retainer design and optimized anterior maxillary expanding force of 100 g which exerted the most synchronized mobility of mid-palatal suture and incisors. Via the standardized model, the high relapse rate was proven to be attributed to insufficient osteogenesis in expanded suture, requiring long-term retainer wearing in clinical situations. To reduce the relapse rate, mesoporous bioactive glass/fibrin glue (MBG/FG) composite hydrogels were developed for an in situ minimal invasive injection that enhance osteogenesis in the expanded palate. The component of 1 wt% MBG was adopted for enhanced mechanical strength, matched degradation rate and ion dissolution, excellent in vitro biocompatibility and osteoinductivity. Effects of 1%MBG/FG composite hydrogel on osteogenesis in expanded mid-palatal sutures with/without retention were evaluated in the standardized model. The results demonstrated that injection of 1%MBG/FG composite hydrogel significantly promoted bone formation within the expanded mid-palatal suture, inhibited osteoclastogenesis and benefited the balance of bone remodeling towards osteogenesis. Combination of retainer and injectable biomaterial was demonstrated as a promising treatment to reduce relapse rate and enhance osteogenesis after RME. The model establishment and the composite hydrogel development in this article might provide new insight to other craniomaxillofacial deformity treatment and design of bone-repairing biomaterials with higher regenerative efficiency.

STAT3 is critical for skeletal development and bone homeostasis by regulating osteogenesis.

Skeletal deformities are typical AD-HIES manifestations, which are mainly caused by heterozygous and loss-of-function mutations in Signal transducer and activator of transcription 3 (STAT3). However, the mechanism is still unclear and the treatment strategy is limited. Herein, we reported that the mice with Stat3 deletion in osteoblasts, but not in osteoclasts, induced AD-HIES-like skeletal defects, including craniofacial malformation, osteoporosis, and spontaneous bone fracture. Mechanistic analyses revealed that STAT3 in cooperation with Msh homeobox 1(MSX1) drove osteoblast differentiation by promoting Distal-less homeobox 5(Dlx5) transcription. Furthermore, pharmacological activation of STAT3 partially rescued skeletal deformities in heterozygous knockout mice, while inhibition of STAT3 aggravated bone loss. Taken together, these data show that STAT3 is critical for modulating skeletal development and maintaining bone homeostasis through STAT3-indcued osteogenesis and suggest it may be a potential target for treatments.

Napabucasin Induces Mouse Bone Loss by Impairing Bone Formation via STAT3.

The novel small molecule Napabucasin (also known as BBI608) was shown to inhibit gene transcription driven by Signal Transducer and Activator of Transcription 3 (STAT3), which is considered a promising anticancer target. Many preclinical studies have been conducted in cancer patients examining the selective targeting of cancer stem cells by Napabucasin, but few studies have examined side effects of Napabucasin in the skeleton system. In the present study, we found treating bone marrow mesenchymal stem cells (BMSCs) with Napabucasin in vitro impaired their osteogenic differentiation. In terms of mechanisms, Napabucasin disrupted differentiation of BMSCs by inhibiting the transcription of osteogenic gene osteocalcin (Ocn) through STAT3. Moreover, through micro-CT analysis we found 4 weeks of Napabucasin injections induced mouse bone loss. Histological analysis revealed that Napabucasin-induced bone loss in mice was the result of impaired osteogenesis. In conclusion, this study provided evidence for the effect of Napabucasin on mouse bone homeostasis and revealed its underlying mechanisms in vivo and in vitro.

[Generation and validation of inducible osteoblast-specific Stat3 knockout mice].

PURPOSE: Based on the Cre-Loxp gene knockout system, this study intended to construct tamoxifen-inducible STAT3 conditional knockout mice and verify the knockout efficiency. METHODS: The inducible osteoblasts-specific Stat3 knockout mice Stat3Col1ERT2 were obtained by hybridization through C57 mice of Stat3fl/fl and Col1 creERT2. Bone mesenchymal stem cells(BMSCs) of these mice were isolated and cultured with or without 4-hydroxytamoxin(4-OTH), to verify the effect of Stat3 knockout in vitro by real-time quantitative PCR and Western blotting in the level of mRNA and protein. Meanwhile, wild type and Stat3Col1ERT2 mice were both intraperitoneally injected with tamoxifen, the expression of STAT3 in the maxillary alveolar bone was observed by immunofluorescent staining to confirm the knockout effect in vivo. Statistical analysis was conducted with SPSS 24.0 software package. RESULTS: Real-time quantitative PCR and Western blotting results demonstrated that mRNA(P<0.05) and protein levels of STAT3 were significantly decreased (P<0.05) in BMSCs derived from Stat3Col1ERT2 mice by 4-OHT induced knockout in vitro. Immunofluorescent staining indicated that STAT3 expression was significantly reduced(P<0.05) in osteoblasts of the maxillary alveolar bone in Stat3Col1ERT2 mice. CONCLUSIONS: This study successfully constructed the inducible osteoblasts-specific Stat3 gene knockout mice, which helped investigators control the time and space of gene knockout, therefore providing new insights and guidance for research fields of orthodontic tooth movement, distraction osteogenesis and jaw fractures in the future.

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats.

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers. In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45- mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

Nanoparticles modified by polydopamine: Working as "drug" carriers.

Inspired by the mechanism of mussel adhesion, polydopamine (PDA), a versatile polymer for surface modification has been discovered. Owing to its unique properties like extraordinary adhesiveness, excellent biocompatibility, mild synthesis requirements, as well as distinctive drug loading approach, strong photothermal conversion capacity and reactive oxygen species (ROS) scavenging facility, various PDA-modified nanoparticles have been desired as drug carriers. These nanoparticles with diverse nanostructures are exploited in multifunctions, consisting of targeting, imaging, chemical treatment (CT), photodynamic therapy (PDT), photothermal therapy (PTT), tissue regeneration ability, therefore have attracted great attentions in plenty biomedical applications. Herein, recent progress of PDA-modified nanoparticle drug carriers in cancer therapy, antibiosis, prevention of inflammation, theranostics, vaccine delivery and adjuvant, tissue repair and implant materials are reviewed, including preparation of PDA-modified nanoparticle drug carriers with various nanostructures and their drug loading strategies, basic roles of PDA surface modification, etc. The advantages of PDA modification in overcoming the existing limitations of cancer therapy, antibiosis, tissue repair and the developing trends in the future of PDA-modified nanoparticle drug carriers are also discussed.

Icariin prevents oestrogen deficiency-induced alveolar bone loss through promoting osteogenesis via STAT3.

OBJECTIVES: Alveolar bone osteoporosis has attracted more and more attention because of its profound impact on stomatognathic function and treatment, but current treatments have not been targeted to alveolar bone and might even cause severe side effects. Thus, identifying the effects of anti-osteoporosis agents on alveolar bone is essential. Icariin ameliorates metabolic dysfunction of long bones, but its effects on alveolar bone remain unclarified. MATERIALS AND METHODS: BMSCs were isolated from rat mandibles (mBMSCs). The osteogenic potential of mBMSCs and the signalling pathway involved under icariin treatment were measured by ALP and alizarin red staining, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence. Dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation were used to investigate the molecular mechanism. Ovariectomized and sham-operated rats treated with or without icariin were analysed by micro-CT, TRAP staining and calcein double labelling. RESULTS: We found that icariin promoted osteoblast differentiation of mBMSCs. Furthermore, STAT3 was critical for icariin-promoted osteoblast differentiation, as indicated by increased phosphorylation levels in icariin-treated mBMSCs, while preventing STAT3 activation blocked icariin-induced osteoblast differentiation. Mechanistically, icariin-promoted transcription of the downstream osteogenic gene osteocalcin (Ocn) through STAT3 and STAT3 bound to the promoter of Ocn. Notably, icariin prevented the alveolar bone osteoporosis induced by oestrogen deficiency through promoting bone formation. CONCLUSIONS: For the first time, our work provides evidence supporting the potential application of icariin in promoting osteogenesis and treating alveolar bone osteoporosis.

STAT3 controls osteoclast differentiation and bone homeostasis by regulating NFATc1 transcription.

The transcription factor signal transducer and activator of transcription 3 (STAT3) plays a central role in cell survival and function. STAT3 has been demonstrated to participate in the maintenance of bone homeostasis in osteoblasts, but its role in osteoclasts in vivo remains poorly defined. Here, we generated a conditional knockout mouse model in which Stat3 was deleted in osteoclasts using a cathepsin K-Cre (Ctsk-Cre) driver. We observed that osteoclast-specific Stat3 deficiency caused increased bone mass in mice, which we attributed to impaired bone catabolism by osteoclasts. Stat3-deficient bone marrow macrophages (BMMs) showed decreased expression of nuclear factor of activated T cells, cytoplasm 1 (NFATc1), and reduced osteoclast differentiation determined by decreases in osteoclast number, tartrate-resistant acid phosphatase activity, and expression of osteoclast marker genes. Enforced expression of NFATc1 in Stat3-deficient BMMs rescued the impaired osteoclast differentiation. Mechanistically, we revealed that STAT3 could drive the transcription of NFATc1 by binding to its promoter. Furthermore, preventing STAT3 activation by using an inhibitor of upstream phosphorylases, AG490, also impaired osteoclast differentiation and formation in a similar way as gene deletion of Stat3 In summary, our data provide the first evidence that STAT3 is significant in osteoclast differentiation and bone homeostasis in vivo, and it may be identified as a potential pharmacological target for the treatment of bone metabolic diseases through regulation of osteoclast activity.

Sequential traction of a labio-palatal horizontally impacted maxillary canine with a custom three-directional force device in the space of a missing ipsilateral first premolar.

Orthodontic treatment is more complicated when both soft and hard tissues must be considered because an impacted maxillary canine has important effects on function and esthetics. Compared with extraction of impacted maxillary canines, exposure followed by orthodontic traction can improve esthetics and better protect the patient's teeth and alveolar bone. Therefore, in order to achieve desirable tooth movement with minimal unexpected complications, a precise diagnosis is indispensable to establish an effective and efficient force system. In this report, we describe the case of a 31-year-old patient who had a labio-palatal horizontally impacted maxillary left canine with a severe occlusal alveolar bone defect and a missing maxillary left first premolar. Herein, with the aid of three-dimensional imaging, sequential traction was performed with a three-directional force device that finally achieved acceptable occlusion by bringing the horizontally impacted maxillary left canine into alignment. The maxillary left canine had normal gingival contours and was surrounded by a substantial amount of regenerated alveolar bone. The 1-year follow-up stability assessment demonstrated that the esthetic and functional outcomes were successful.