Early infiltrating NKT lymphocytes attenuate bone regeneration through secretion of CXCL2.

Trauma rapidly mobilizes the immune response of surrounding tissues and activates regeneration program. Manipulating immune response to promote tissue regeneration shows a broad application prospect. However, the understanding of bone healing dynamics at cellular level remains limited. Here, we characterize the landscape of immune cells after alveolar bone injury and reveal a pivotal role of infiltrating natural killer T (NKT) cells. We observe a rapid increase in NKT cells after injury, which inhibit osteogenic differentiation of mesenchymal stem cells (MSCs) and impair alveolar bone healing. Cxcl2 is up-regulated in NKT cells after injury. Systemic administration of CXCL2-neutralizing antibody or genetic deletion of Cxcl2 improves the bone healing process. In addition, we fabricate a gelatin-based porous hydrogel to deliver NK1.1 depletion antibody, which successfully promotes alveolar bone healing. In summary, our study highlights the importance of NKT cells in the early stage of bone healing and provides a potential therapeutic strategy for accelerating bone regeneration.

Interaction between immuno-stem dual lineages in jaw bone formation and injury repair.

The jawbone, a unique structure in the human body, undergoes faster remodeling than other bones due to the presence of stem cells and its distinct immune microenvironment. Long-term exposure of jawbones to an oral environment rich in microbes results in a complex immune balance, as shown by the higher proportion of activated macrophage in the jaw. Stem cells derived from the jawbone have a higher propensity to differentiate into osteoblasts than those derived from other bones. The unique immune microenvironment of the jaw also promotes osteogenic differentiation of jaw stem cells. Here, we summarize the various types of stem cells and immune cells involved in jawbone reconstruction. We describe the mechanism relationship between immune cells and stem cells, including through the production of inflammatory bodies, secretion of cytokines, activation of signaling pathways, etc. In addition, we also comb out cellular interaction of immune cells and stem cells within the jaw under jaw development, homeostasis maintenance and pathological conditions. This review aims to eclucidate the uniqueness of jawbone in the context of stem cell within immune microenvironment, hopefully advancing clinical regeneration of the jawbone.

Adult tissue-specific stem cell interaction: novel technologies and research advances.

Adult tissue-specific stem cells play a dominant role in tissue homeostasis and regeneration. Various in vivo markers of adult tissue-specific stem cells have been increasingly reported by lineage tracing in genetic mouse models, indicating that marked cells differentiation is crucial during homeostasis and regeneration. How adult tissue-specific stem cells with indicated markers contact the adjacent lineage with indicated markers is of significance to be studied. Novel methods bring future findings. Recent advances in lineage tracing, synthetic receptor systems, proximity labeling, and transcriptomics have enabled easier and more accurate cell behavior visualization and qualitative and quantitative analysis of cell-cell interactions than ever before. These technological innovations have prompted researchers to re-evaluate previous experimental results, providing increasingly compelling experimental results for understanding the mechanisms of cell-cell interactions. This review aimed to describe the recent methodological advances of dual enzyme lineage tracing system, the synthetic receptor system, proximity labeling, single-cell RNA sequencing and spatial transcriptomics in the study of adult tissue-specific stem cells interactions. An enhanced understanding of the mechanisms of adult tissue-specific stem cells interaction is important for tissue regeneration and maintenance of homeostasis in organisms.

[Application of Animal Model Training in Preclinical Skills Teaching for Graduate Students of Dentistry].

Objective: To explore the potential application value of animal model training in improving the comprehensive clinical ability of postgraduate students of dentistry and to provide reference for new methods of preclinical skills teaching. Methods: A total of 40 postgraduate students of dentistry were assigned to two groups, an experimental group and a control group. The control group took the routine teaching course on root canal treatment for the right mandibular first molar, using a simulated model of human head. The experimental group also took a teaching course on root canal therapy for the right mandibular first molar, but an animal model was used for the group. After the course was completed, the instructor conducted comprehensive evaluation of the students' psychological quality, patient communication skills, diagnosis and treatment logic, speed of performing procedures, and treatment plan design. A questionnaire survey was conducted to examine the students' attitudes toward and evaluation of animal model training. Results: The scores for psychological quality (0.430±0.024 vs. 0.115±0.036), patient communication skills (0.878±0.065 vs. 0.115±0.036), diagnosis and treatment logic (0.630±0.066 vs. 0.372±0.033), speed of performing procedures (0.8975±0.019 vs. 0.055±0.080), and treatment plan design (0.539±0.036 vs. 0.396±0.017) of the experimental group were significantly higher than those of the control group ( P<0.0001). The total score of the experimental group (3.374±0.184) was significantly higher than that of the control group (1.053±0.082) and the difference was statistically significant ( P<0.001). 95% of the students in the control group and 100% of those in the experimental group were willing to participate in animal model training to improve their level of diagnosis and treatment skills for dental and endodontic diseases, showing no statistically significant difference ( χ 2=1.026, P=0.3112). In the experimental group, 30% of the students believed that their psychological qualities had been improved, 50% believed that their procedure skills had been improved, and 20% believed that animal model training had expanded the scope of their theoretical knowledge. Conclusion: Adding animal model training can improve dentistry graduate students' comprehensive abilities, including their psychological quality, patient communication skills, diagnosis and treatment logic, speed of performing procedures, and treatment plan design. In addition, it helps students familiarize themselves in advance with animal experimental operations for basic research, thus helping them acquire dual professional skills.

Creating an atlas of the bone microenvironment during oral inflammatory-related bone disease using single-cell profiling.

Oral inflammatory diseases such as apical periodontitis are common bacterial infectious diseases that may affect the periapical alveolar bone tissues. A protective process occurs simultaneously with the inflammatory tissue destruction, in which mesenchymal stem cells (MSCs) play a primary role. However, a systematic and precise description of the cellular and molecular composition of the microenvironment of bone affected by inflammation is lacking. In this study, we created a single-cell atlas of cell populations that compose alveolar bone in healthy and inflammatory disease states. We investigated changes in expression frequency and patterns related to apical periodontitis, as well as the interactions between MSCs and immunocytes. Our results highlight an enhanced self-supporting network and osteogenic potential within MSCs during apical periodontitis-associated inflammation. MSCs not only differentiated toward osteoblast lineage cells but also expressed higher levels of osteogenic-related markers, including Sparc and Col1a1. This was confirmed by lineage tracing in transgenic mouse models and human samples from oral inflammatory-related alveolar bone lesions. In summary, the current study provides an in-depth description of the microenvironment of MSCs and immunocytes in both healthy and disease states. We also identified key apical periodontitis-associated MSC subclusters and their biomarkers, which could further our understanding of the protective process and the underlying mechanisms of oral inflammatory-related bone disease. Taken together, these results enhance our understanding of heterogeneity and cellular interactions of alveolar bone cells under pathogenic and inflammatory conditions. We provide these data as a tool for investigators not only to better appreciate the repertoire of progenitors that are stress responsive but importantly to help design new therapeutic targets to restore bone lesions caused by apical periodontitis and other inflammatory-related bone diseases.

Schwann Cells Contribute to Alveolar Bone Regeneration by Promoting Cell Proliferation.

The plasticity of Schwann cells (SCs) following nerve injury is a critical feature in the regeneration of peripheral nerves as well as surrounding tissues. Here, we show a pivotal role of Schwann cell-derived cells in alveolar bone regeneration through the specific ablation of proteolipid protein 1 (Plp)-expressing cells and the transplantation of teased nerve fibers and associated cells. With inducible Plp specific genetic tracing, we observe that Plp+ cells migrate into wounded alveolar defect and dedifferentiate into repair SCs. Notably, these cells barely transdifferentiate into osteogenic cell lineage in both SCs tracing model and transplant model, but secret factors to enhance the proliferation of alveolar skeletal stem cells (aSSCs). As to the mechanism, this effect is associated with the upregulation of extracellular matrix (ECM) receptors and receptor tyrosine kinases (RTKs) signaling and the downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and the phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) pathway. Collectively, our data demonstrate that SCs dedifferentiate after neighboring alveolar bone injury and contribute to bone regeneration mainly by a paracrine function. © 2022 American Society for Bone and Mineral Research (ASBMR).

METTL5 regulates cranial suture fusion via Wnt signaling.

METTL5 is a methyltransferase that mediates eukaryotic 18S ribosomal RNA m6A modification, and its mutations lead to intellectual disability, microcephaly, and facial dysmorphism in patients. However, the role of METTL5 in craniofacial development remains poorly understood. This study demonstrates that Mettl5 knockout mice exhibit poor ossification, widened cranial sutures, and a cleidocranial dysplasia-like phenotype. Deletion of Mettl5 leads to increased proliferation and decreased osteogenic differentiation of suture mesenchymal stem cells. Mechanistically, we find that Wnt signaling is significantly downregulated after Mettl5 knockout. Overall, we reveal an essential role of METTL5 in craniofacial development and osteogenic differentiation of suture mesenchymal stem cells, making METTL5 a potential diagnostic and therapeutic target for craniofacial developmental diseases.

Insights into skeletal stem cells.

The tissue-resident skeletal stem cells (SSCs), which are self-renewal and multipotent, continuously provide cells (including chondrocytes, bone cells, marrow adipocytes, and stromal cells) for the development and homeostasis of the skeletal system. In recent decade, utilizing fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing, studies have identified various types of SSCs, plotted the lineage commitment trajectory, and partially revealed their properties under physiological and pathological conditions. In this review, we retrospect to SSCs identification and functional studies. We discuss the principles and approaches to identify bona fide SSCs, highlighting pioneering findings that plot the lineage atlas of SSCs. The roles of SSCs and progenitors in long bone, craniofacial tissues, and periosteum are systematically discussed. We further focus on disputes and challenges in SSC research.

Mechanisms of bone remodeling and therapeutic strategies in chronic apical periodontitis.

Chronic periapical periodontitis (CAP) is a typical oral disease in which periodontal inflammation caused by an odontogenic infection eventually leads to bone loss. Uncontrolled infections often lead to extensive bone loss around the root tip, which ultimately leads to tooth loss. The main clinical issue in the treatment of periapical periodontitis is the repair of jawbone defects, and infection control is the first priority. However, the oral cavity is an open environment, and the distribution of microorganisms through the mouth in jawbone defects is inevitable. The subversion of host cell metabolism by oral microorganisms initiates disease. The presence of microorganisms stimulates a series of immune responses, which in turn stimulates bone healing. Given the above background, we intended to examine the paradoxes and connections between microorganisms and jaw defect repair in anticipation of new ideas for jaw defect repair. To this end, we reviewed the microbial factors, human signaling pathways, immune cells, and cytokines involved in the development of CAP, as well as concentrated growth factor (CGF) and stem cells in bone defect repair, with the aim of understanding the impact of microbial factors on host cell metabolism to inform the etiology and clinical management of CAP.

METTL3-mediated m6A RNA methylation regulates dorsal lingual epithelium homeostasis.

The dorsal lingual epithelium, which is composed of taste buds and keratinocytes differentiated from K14+ basal cells, discriminates taste compounds and maintains the epithelial barrier. N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotic cells. How METTL3-mediated m6A modification regulates K14+ basal cell fate during dorsal lingual epithelium formation and regeneration remains unclear. Here we show knockout of Mettl3 in K14+ cells reduced the taste buds and enhanced keratinocytes. Deletion of Mettl3 led to increased basal cell proliferation and decreased cell division in taste buds. Conditional Mettl3 knock-in mice showed little impact on taste buds or keratinization, but displayed increased proliferation of cells around taste buds in a protective manner during post-irradiation recovery. Mechanically, we revealed that the most frequent m6A modifications were enriched in Hippo and Wnt signaling, and specific peaks were observed near the stop codons of Lats1 and FZD7. Our study elucidates that METTL3 is essential for taste bud formation and could promote the quantity recovery of taste bud after radiation.

Klotho in Osx+-mesenchymal progenitors exerts pro-osteogenic and anti-inflammatory effects during mandibular alveolar bone formation and repair.

Maxillofacial bone defects are commonly seen in clinical practice. A clearer understanding of the regulatory network directing maxillofacial bone formation will promote the development of novel therapeutic approaches for bone regeneration. The fibroblast growth factor (FGF) signalling pathway is critical for the development of maxillofacial bone. Klotho, a type I transmembrane protein, is an important components of FGF receptor complexes. Recent studies have reported the presence of Klotho expression in bone. However, the role of Klotho in cranioskeletal development and repair remains unknown. Here, we use a genetic strategy to report that deletion of Klotho in Osx-positive mesenchymal progenitors leads to a significant reduction in osteogenesis under physiological and pathological conditions. Klotho-deficient mensenchymal progenitors also suppress osteoclastogenesis in vitro and in vivo. Under conditions of inflammation and trauma-induced bone loss, we find that Klotho exerts an inhibitory function on inflammation-induced TNFR signaling by attenuating Rankl expression. More importantly, we show for the first time that Klotho is present in human alveolar bone, with a distinct expression pattern under both normal and pathological conditions. In summary, our results identify the mechanism whereby Klotho expressed in Osx+-mensenchymal progenitors controls osteoblast differentiation and osteoclastogenesis during mandibular alveolar bone formation and repair. Klotho-mediated signaling is an important component of alveolar bone remodeling and regeneration. It may also be a target for future therapeutics.

A self-amplifying loop of YAP and SHH drives formation and expansion of heterotopic ossification.

Heterotopic ossification (HO) occurs as a common complication after injury or in genetic disorders. The mechanisms underlying HO remain incompletely understood, and there are no approved prophylactic or secondary treatments available. Here, we identify a self-amplifying, self-propagating loop of Yes-associated protein (YAP)-Sonic hedgehog (SHH) as a core molecular mechanism underlying diverse forms of HO. In mouse models of progressive osseous heteroplasia (POH), a disease caused by null mutations in GNAS, we found that Gnas-/- mesenchymal cells secreted SHH, which induced osteoblast differentiation of the surrounding wild-type cells. We further showed that loss of Gnas led to activation of YAP transcription activity, which directly drove Shh expression. Secreted SHH further induced YAP activation, Shh expression, and osteoblast differentiation in surrounding wild-type cells. This self-propagating positive feedback loop was both necessary and sufficient for HO expansion and could act independently of Gnas in fibrodysplasia ossificans progressiva (FOP), another genetic HO, and nonhereditary HO mouse models. Genetic or pharmacological inhibition of YAP or SHH abolished HO in POH and FOP and acquired HO mouse models without affecting normal bone homeostasis, providing a previously unrecognized therapeutic rationale to prevent, reduce, and shrink HO.

Challenges of Stem-cell-based Craniofacial Regeneration.

Oral diseases, such as dental caries, pulpitis, periodontitis, and craniofacial trauma, are common. Some individuals suffer from oral cancer or congenital craniofacial defects. The oral-systemic disease link reveals that a dental disorder is not a minor problem. Tissue loss is an inevitable consequence of most oral diseases, and repairing the tissue loss and restoring craniofacial function are highly expected by patients and are terminal targets of dental treatment. The current clinical approach for tissue loss due to dental caries, pulpitis, periodontitis, oral cancer, trauma, and developmental diseases depends on the filling of corresponding material, allograft, or autograft bone after lesion removal. Repair of the tissue volume is expectedly followed by promising functional restoration using regenerative dental tissue or tissue engineering, which has currently aroused the interest of clinicians and researchers. This review focuses on the ideas and recent findings on newly identified skeletal stem cells (SSCs) as candidates for craniofacial regeneration, signaling regulation of SSCs extended from embryonic development, and signal molecule delivery for the repair of the craniofacial defect, sincerely hoping that the hypothesis of craniofacial self-healing is true in the future.

AFF4 regulates osteogenic differentiation of human dental follicle cells.

As a member of the AFF (AF4/FMR2) family, AFF4 is a transcription elongation factor that is a component of the super elongation complex. AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling. Here, we investigated the effect of AFF4 on human dental follicle cells (DFCs) in osteogenic differentiation. In this study, we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase (ALP) activity and impaired mineralization. In addition, the expression of osteogenic-related genes (DLX5, SP7, RUNX2 and BGLAP) was significantly downregulated. In contrast, lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs. Mechanistically, we found that both the mRNA and protein levels of ALKBH1, a critical regulator of epigenetics, changed in accordance with AFF4 expression levels. Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation. Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.

AFF1 inhibits adipogenic differentiation via targeting TGM2 transcription.

OBJECTIVES: AF4/FMR2 family member 1 (AFF1), known as a central scaffolding protein of super elongation complex (SEC), regulates gene transcription. We previously reported that AFF1 inhibited osteogenic differentiation of human mesenchymal stromal/stem cells (hMSCs). However, its role in adipogenic differentiation has not been elucidated. MATERIALS AND METHODS: hMSCs and 3T3-L1 pre-adipocytes were cultured and induced for adipogenic differentiation. Small interfering RNAs (siRNAs) were applied to deplete AFF1 while lentiviruses expressing HA-Aff1 were used for overexpression. Oil Red O staining, triglyceride (TAG) quantification, quantitative real-time PCR (qPCR), Western blot analysis, immunofluorescence staining, RNA sequencing (RNA-seq) analysis and ChIP-qPCR were performed. To evaluate the adipogenesis in vivo, BALB/c nude mice were subcutaneously injected with Aff1-overexpressed 3T3-L1 pre-adipocytes. RESULTS: AFF1 depletion leads to an enhanced adipogenesis in both hMSCs and 3T3-L1 pre-adipocytes. Overexpression of Aff1 in 3T3-L1 cells results in the reduction of adipogenic differentiation and less adipose tissue formation in vivo. Mechanistically, AFF1 binds to the promoter region of Tgm2 gene and regulates its transcription. Overexpression of Tgm2 largely rescues adipogenic differentiation of Aff1-deficient cells. CONCLUSIONS: Our data indicate that AFF1 inhibits adipogenic differentiation by regulating the transcription of TGM2.

Saliva: potential diagnostic value and transmission of 2019-nCoV.

2019-nCoV epidemic was firstly reported at late December of 2019 and has caused a global outbreak of COVID-19 now. Saliva, a biofluid largely generated from salivary glands in oral cavity, has been reported 2019-nCoV nucleic acid positive. Besides lungs, salivary glands and tongue are possibly another hosts of 2019-nCoV due to expression of ACE2. Close contact or short-range transmission of infectious saliva droplets is a primary mode for 2019-nCoV to disseminate as claimed by WHO, while long-distance saliva aerosol transmission is highly environment dependent within indoor space with aerosol-generating procedures such as dental practice. So far, no direct evidence has been found that 2019-nCoV is vital in air flow for long time. Therefore, to prevent formation of infectious saliva droplets, to thoroughly disinfect indoor air and to block acquisition of saliva droplets could slow down 2019-nCoV dissemination. This review summarizes diagnostic value of saliva for 2019-nCoV, possibly direct invasion into oral tissues, and close contact transmission of 2019-nCoV by saliva droplets, expecting to contribute to 2019-nCoV epidemic control.

Ubiquitin-Specific Protease 34 Inhibits Osteoclast Differentiation by Regulating NF-κB Signaling.

The ubiquitination and deubiquitination enzymes ensure the stability and proper function of most cellular proteins. Disturbance of either enzyme compromises tissue homeostasis. We recently have identified that the ubiquitin-specific protease 34 (USP34) contributes to bone formation by promoting osteogenic differentiation of mesenchymal stem cells. However, its role in bone resorption, which couples bone formation, remains unknown. Here we show that knockdown of Usp34 promotes osteoclast differentiation of RAW264.7 cells. Conditional knockout of Usp34 in bone marrow-derived macrophages (BMMs) or in osteoclasts leads to elevated osteoclast function and low bone mass. Mechanically, we identify that USP34 restrains NF-κB signaling by deubiquitinating and stabilizing the NF-κB inhibitor alpha (IκBα). Overexpression of IκBα represses osteoclastic hyperfunction of Usp34-deficient RAW264.7 cells. Collectively, our results show that USP34 inhibits osteoclastogenesis by regulating NF-κB signaling. © 2020 American Society for Bone and Mineral Research.

Parathyroid hormone ameliorates temporomandibular joint osteoarthritic-like changes related to age.

OBJECTIVES: Ageing could be a contributing factor to the progression of temporomandibular joint osteoarthritis (TMJ OA), whereas its pathogenesis and potential therapeutic strategy have not been comprehensively investigated. MATERIALS AND METHODS: We generated ageing mouse models (45-week and 60-week; 12-week mice as control) and intermittently injected 45-week mice with parathyroid hormone (PTH(1-34)) or vehicle for 4 weeks. Cartilage and subchondral bone of TMJ were analysed by microCT, histological and immunostaining. Western blot, qRT-PCR, ChIP, ELISA and immunohistochemical analysis were utilized to examination the mechanism of PTH(1-34)'s function. RESULTS: We showed apparent OA-like phenotypes in ageing mice. PTH treatment could ameliorate the degenerative changes and improve bone microarchitecture in the subchondral bone by activating bone remodelling. Moreover, PTH inhibited phosphorylation level of Smad3, which can combine with p16ink4a gene promoter region, resulting in reduced senescent cells accumulation and increased cellular proliferation of marrow mesenchymal stem cells (MSCs). ELISA also showed relieved levels of specific senescent-associated secretory phenotype (SASP) in ageing mice after PTH treatment. CONCLUSIONS: In summary, PTH may reduce the accumulation of senescent cells in subchondral bone by inhibiting p16ink4a and improve bone marrow microenvironment to active bone remodelling process, indicating PTH administration could be a potential preventative and therapeutic treatment for age-related TMJ OA.

AFF4 enhances odontogenic differentiation of human dental pulp cells.

AFF4 is a component of super elongation complex (SECs) and functions as a scaffold protein to bridge the transcription elongation factors. It is associated with leukemia, HIV transcription, and head neck cancer. However, its role in odontogenic differentiation of dental pulp cells (DPCs) is unclear. Here, we show the expression of AFF4 is increased during odontogenesis. Depletion of AFF4 in human DPCs leads to a decrease of alkaline phosphatase (ALP) activity, calcium mineralization and odontogenic-related genes expression. On the contrary, Lentivirus-mediated overexpression of AFF4 induces the odontogenic potential of DPCs. Mechanistically, we found AFF4 regulates the transcription of NFIC, a key factor for tooth root formation. Overexpression of NFIC successfully rescues the restricted differentiation of AFF4-depleted cells. Our data demonstrate that AFF4 serves as a previously unknown regulator of odontogenesis.

Disturbed bone remodelling activity varies in different stages of experimental, gradually progressive apical periodontitis in rats.

Bone remodelling keeps going through the lifespan of human by bone formation and bone resorption. In the craniofacial region, mandibles act as the main force for biting and chewing, and also become susceptible to a common bone-loss disease, namely, apical periodontitis, once infected dental pulp is not treated timely, during which bone resorption occurs from the apical foramen to the apical bone area. Although conventional root canal treatment (RCT) can remove the most of the infection, chronical apical periodontitis due to incomplete removal of dental pulp and subsequent microleakage will become refractory and more challenging, and this process has scarcely been specifically studied as a bone remodelling issue in rat models. Therefore, to study chronical and refractory apical periodontitis owing to incomplete cleaning of infected dental pulp and microleackage in vivo, we establish a modified rat model of gradually progressive apical periodontitis by sealing residual necrotic dental pulp and introducing limited saliva, which simulates gradually progressive apical periodontitis, as observed in the clinical treatment of chronical and refractory apical periodontitis. We show that bone-loss is inevitable and progressive in this case of apical periodontitis, which confirms again that complete and sound root canal treatment is crucial to halt the progression of chronical and refractory apical periodontitis and promote bone formation. Interestingly, bone remodelling was enhanced at the initial stage of apical periodontitis in this model while reduced with a high osteoblast number afterwards, as shown by the time course study of the modified model. Suggesting that the pathological apical microenvironment reserve its hard tissue formation ability to some degree but in a disturbed manner. Hopefully, our findings can provide insights for future bone regenerative treatment for apical periodontitis-associated bone loss.