IGF2 promotes alveolar bone regeneration in murine periodontitis via inhibiting cGAS/STING-mediated M1 macrophage polarization.

Periodontitis is a chronic inflammatory disease with the destruction of supporting periodontal tissue. This study evaluated the role of insulin-like growth factor 2 (IGF2) in periodontitis by inhibiting the polarization of M1 macrophages via the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. IGF2 was enriched in the gingival tissue of murine periodontitis model identified by RNA sequencing. IGF2 application alleviated the expression of pro-inflammatory factors and promoted osteogenesis and the expression of related genes and proteins in a dose-dependent manner in periodontitis. The result of micro-CT verified this finding. Both in vivo and in vitro results revealed that IGF2 decreased the polarization of M1 macrophages and pro-inflammatory factors by immunofluorescence staining, flow cytometry, western blotting and RT-PCR. IGF2 application promoted the osteogenic ability of periodontal ligament fibroblasts (PDLFs) indirectly via its inhibition of M1 polarization evaluated by alkaline phosphatase and alizarin red staining. Then, the cGAS/STING pathway was upregulated in periodontitis and macrophages challenged by LPS, the inhibition of which led to downregulation of M1 polarization. Furthermore, IGF2 could downregulate cGAS, STING and the phosphorylation of P65. Collectively, our study indicates IGF2 can regulate the polarization of M1 macrophages via the cGAS/STING pathway and highlights the promising future of IGF2 as a therapeutic treatment for periodontitis.

A bone-targeting near-infrared luminescence nanocarrier facilitates alpha-ketoglutarate efficacy enhancement for osteoporosis therapy.

Osteoporosis (OP), which largely increases the risk of fractures, is the most common chronic degenerative orthopedic disease in the elderly due to the imbalance of bone homeostasis. Alpha-ketoglutaric acid (AKG), an endogenous metabolic intermediate involved in osteogenesis, plays critical roles in osteogenic differentiation and mineralization and the inhibition of osteoclastogenic differentiation. However, the low bioavailability and poor bone-targeting efficiency of AKG seriously limit its efficacy in OP treatment. In this work, a bone-targeting, near-infrared emissive lanthanide luminescence nanocarrier loaded with AKG (ß-NaYF4:7%Yb, 60%Nd@NaLuF4@mSiO2-EDTA-AKG, abbreviated as LMEK) is developed for the enhancement of AKG efficacy in OP therapy. By utilizing the NIR-II luminescence (>1000 nm) of LMEK, whole-body bone imaging with high spatial resolution is achieved to confirm the bone enrichment of AKG noninvasively in vivo. The results reveal that LMEK exhibits a remarkable OP therapeutic effect in improving the osseointegration of the surrounding bone in the ovariectomized OP mice models, which is validated by the enhanced inhibition of osteoclast through hypoxia-inducible factor-1α suppression and promotion of osteogenic differentiation in osteoblast. Notably, the dose of AKG in LMEK can be reduced to only 0.2 % of the dose when pure AKG is used in therapy, which dramatically improves the bioavailability of AKG and mitigates the metabolism burden. This work provides a strategy to conquer the low utilization of AKG in OP therapy, which not only overcomes the challenges in AKG efficacy for OP treatment but also offers insights into the development and application of other potential drugs for skeletal diseases. STATEMENT OF SIGNIFICANCE: Alpha-ketoglutarate (AKG) is an intermediate within the Krebs cycle, participating in diverse metabolic and cellular processes, showing potential for osteoporosis (OP) therapy. However, AKG's limited bioavailability and inefficient bone-targeting hinder its effectiveness in treating OP. Herein, a near-infrared emissive nanocarrier is developed that precisely targets bones and delivers AKG, bolstering its effectiveness in OP therapy. Thanks to this efficient bone-targeting delivery, the AKG dosage is reduced to 0.2 % of the conventional treatment level. This marks the first utilization of a bone-targeting nanocarrier to amplify AKG's bioavailability and OP therapy efficacy. Furthermore, the mechanism of AKG-loaded nanocarrier regulating the biological behavior of osteoclasts and osteoblasts mediated is tentatively explored.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles restore Th17/Treg homeostasis in periodontitis via miR-1246.

T-cell-mediated immunity is crucial in the immunopathology of periodontitis. The restoration of the homeostasis between the T helper cell 17 (Th17) and regulatory T cell (Treg) subsets by extracellular vesicles (EVs) obtained from human bone marrow stem cells (hBMSCs) promotes new bone formation and suppresses inflammation. Uncovering the functions of hBMSC-derived EVs in the immune microenvironment of periodontal tissue and their underlying regulatory mechanisms may shed new light on developing potential cell-free immunotherapies for periodontal regeneration. Here, we reported that the Th17/Treg ratio elevated in peripheral blood from periodontitis patients. Furthermore, we found that hBMSC-derived EVs could reduce the Th17/Treg ratio in CD4+ T cells from periodontitis patients in vitro and ameliorate conditions of experimental periodontitis in mice. Additionally, by investigating the differentially expressed miRNAs and target genes in EVs from hBMSCs stimulated with Porphyromonas gingivalis LPS using miRNA sequencing, we found that EV-miR-1246 is highly effective at downregulating the ratio of Th17/Treg in vitro. Mechanistically, EV-miR-1246 suppressed expression of its potential target angiotensin-converting enzyme 2 (ACE2) and increased the p-Yes-associated protein (YAP)1/YAP1 ratio in CD4+ T cells. Our results indicated that hBMSC-derived EVs improve periodontitis via miR-1246, consequently downregulating Th17/Treg ratio, and represented a promising therapeutic target for precision treatment in periodontitis.

Development of a novel patient-reported outcome measure for orthognathic surgery.

OBJECTIVES: This study aimed to develop a new patient-reported outcome measure (PROM) to systematically and scientifically evaluate patients' subjective feelings after orthognathic surgery. METHODS: A literature review and semi-structured interviews were conducted to construct a conceptual framework and an item pool, followed by expert and patient surveys for measure construction. We conducted a clinical investigation to test the feasibility, reliability, and content validity of this measure. RESULTS: The conceptual framework included four domains: psychological health, physiological health, social function, and satisfaction, and 33 items were included in the survey. Following the expert analysis, 31 items remained in the draft. The clinical investigation showed a 100% recovery and completion rate and good reliability, with Cronman-Brown formula coefficients of 0.893 and 0.944, respectively. CONCLUSIONS: A new outcome measure to evaluate patients' subjective feelings after orthognathic surgery was successfully developed, and the clinical investigation demonstrated that the PROM had satisfactory feasibility, reliability, and validity. Further studies are possible based on our PROM, and data on a larger scale may reveal more information on patients' subjective feelings about orthognathic surgery. CLINICAL SIGNIFICANCE: The novel PROM provides a systematic and scientific way to evaluate the patient's subjective feelings to help surgeons obtain complete patient-reported information after orthognathic surgery. Additionally, standardised multicentre research on patients' subjective feelings using our PROM is possible and could improve the effectiveness of the evaluation and help maintain treatment quality.

HIF-1α regulates osteoclastogenesis and alveolar bone resorption in periodontitis via ANGPTL4.

OBJECTIVE: The mechanism of alveolar bone resorption caused by periodontitis is not fully understood. We sought to investigate whether microenvironmental changes of local hypoxia are involved in these processes. METHODS: In this study, periodontitis models of control mice and knockout of Hypoxia Induced Factor 1α (HIF-1α) harboring Cathepsin K (CTSK) Cre mice were constructed to study the effect of osteoclasts affected by hypoxic environment on alveolar bone resorption. RAW264.7 cells were subsequently induced by CoCl2 to observe the effects of HIF-1α and Angiopoietin-like Protein 4 (ANGPTL4) on osteoblast differentiation and fusion. RESULTS: The degree of alveolar bone resorption in the periodontitis tissues was lesser in mice with conditional knockout of HIF-1α in osteoclasts than in wild-type mice. We also observed that HIF-1α conditional knockout mice had fewer osteoclasts on the alveolar bone surface than control mice. HIF-1α increases the expression of ANGPTL4 and promotes the differentiation of RAW264.7 cells into osteoblasts and cell fusion under chemically simulated hypoxic conditions. CONCLUSION: HIF-1α regulates osteoclastogenesis and participates in bone resorption in periodontitis through ANGPTL4.

Hypoxia-induced factor 1α promotes osteotomy-induced regional acceleratory phenomenon via DC-STAMP mediated membrane fusion.

OBJECTIVES: The mechanisms of regional acceleratory phenomenon (RAP) induced by orthognathic osteotomy are unclear. It was not known if locally hypoxic microenvironment changes were involved in this phenomenon. METHODS: Hypoxia-induced factor-1α knockout mice harboring Cathepsin K (CTSK) Cre were used to investigate the effect of hypoxia-driven osteoclasts on alveolar bone remodeling. RAW264.7 cells were induced by CoCl2 to observe the effects of dendritic cell-specific transmembrane protein (DC-STAMP) on the fusion and differentiation of osteoclasts. RESULTS: We found mandibular osteotomy of C57 mice induced active alveolar osteoclasts and increased hypoxia-induced factor-1α (HIF-1α) positive staining areas. Alveolar bone density of the 10-week-old HIF-1α conditional knockout (CKO) mouse was increased at 10 and 14 days after bilateral mandibular osteotomy. Moreover, decreased numbers of osteoclasts and matrix metalloproteinase 9 (MMP-9)-positive cells were observed on the surface of bone resorption lacunae in the CKO group. HIF-1α could increase the expression level of DC-STAMP to enhance osteoclastogenesis and cell fusion in active RAW264.7 cells. CONCLUSION: Our data considered hypoxia-driven osteoclasts resorption to be an adaptive mechanism to permit alveolar bone loss after bilateral mandibular osteotomy of mice.

HIF-1α mediates osteoclast-induced disuse osteoporosis via cytoophidia in the femur of mice.

Osteoporosis induced by disuse because of bed rest or the aerospace industry has become one of the most common skeletal disorders. However, mechanisms underlying the disuse osteoporosis remain largely unknown. We validated the tail-suspended model in mice and demonstrated that there is bone loss in the trabecular and cortical bones of the femur. Importantly, we showed that genetical deletion of hypoxia-inducible factor-1α (HIF-1α) in osteoclasts ameliorated osteoclastic bone resorption in the trabecular bone whereas pharmacological treatment with HIF-1α inhibitor protected the hindlimb-unloaded mice from disuse-induced osteoporosis in the trabecular and cortical bones. The HIF-1α knockout RAW264.7 cells and RNA-sequencing proved that HIF-1α is vital for osteoclastogenesis and bone resorption because it regulated the level of inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase (CTPS) via cellular myelocytomatosis (c-Myc) oncogene. The IMPDH and CTPS are vital nucleotide metabolic enzymes which have an important functional role in cell metabolism, and they can assemble into intracellular linear or ring-shaped structures to cope with cell stress. Interestingly, both in vitro and in vivo, the IMPDH and CTPS cytoophidia were found in osteoclasts, and the level of HIF-1α correlated with osteoclastogenesis and bone-resorbing activity. Our data revealed that HIF-1α/c-Myc/cytoophidia signalling might be required for osteoclasts to mediate cell metabolism in disuse-induced osteoporosis. Overall, our results revealed a new role of HIF-1α/c-Myc/cytoophidia in supporting osteoclastogenesis and bone resorption and exposed evidence for its role in the pathogenesis of disuse osteoporosis, which might provide promising therapeutic targets.

LncRNA-mRNA Expression Profiles of Osteoclast After Conditional Knockout HIF-1α.

Background: Osteoclasts, which are multinucleated cells formed by monocyte fusion, play a key role in bone resorption. Hypoxia-inducible factor (HIF)-1α is vital for the development of osteoclasts in hypoxic environments and during bone resorption. However, additional research is required to further study the HIF-1α-dependent regulation of osteoclast differentiation at the genetic level. Methods: In our study, RNA sequencing (RNA-seq) was used to identify the expression profiles of long noncoding RNAs (lncRNAs) and mRNAs in conditional HIF-1α-knockout osteoclasts. Results: A total of 1,320 mRNAs and 95 lncRNAs were differentially expressed. The expression of lncRNAs MSTRG.7566.12 and MSTRG.31769.2 were strongly negatively correlated with that of Mmp9, Ctsk, etc. Conclusion: Our research provides a basis for further understanding the role of mRNAs and lncRNAs in conditional HIF-1α-knockout osteoclasts, and many of these molecules may be potential targets for treating bone diseases related to HIF-1α.

HIF-1α regulates osteoclast activation and mediates osteogenesis during mandibular bone repair via CT-1.

OBJECTIVES: Hypoxia is one of the characteristics of microenvironmental changes after orthognathic surgery for fractures. HIF-1α is a main regulator of the hypoxic response and plays a crucial role in bone formation, remodelling, and homeostasis. Osteoclasts participate in bone absorption and affect osteogenesis, and osteoclasts differentiate in a path from the oxygen-rich bone marrow to oxygen-deficient bone lesions. Thus, we aimed to study the key functions of HIF-1α in osteoclasts during mandibular healing after osteotomy. MATERIALS AND METHODS: The function of HIF-1α in osteoclasts during fracture healing in osteoclast-specific HIF-1α-conditional-knockout mice was investigated in mandibular osteotomy. Primary osteoclasts were used to explore the expression of HIF-1α and cardiotrophin-1 (CT-1) at both the mRNA and protein levels. The ability of BMSCs co-cultured with conditioned media from osteoclast-specific HIF-1α-knockout primary osteoclasts was detected using osteoclast-mediated osteogenesis experiments. RESULTS: Hypoxia-inducible factor-1α increased osteoclastogenesis and bone resorption, and a delay in bone healing was found in osteoclast-specific HIF-1α-conditional-knockout mice compared with normal mice. HIF-1α-knockout primary osteoclasts inhibited bone resorption and CT-1 expression, and HIF-1α enhanced the osteoclast-mediated stimulation of BMSC differentiation by secreting CT-1. CONCLUSIONS: Hypoxia-inducible factor-1α can play a key role in the physiology and pathogenesis of bone resorption by promoting osteoclastogenesis during fracture and influencing osteogenesis through CT-1 during bone healing.

[Effect of zoledronic acid on mandibular socket healing process of mice at an early stage].

PURPOSE: To investigate how bisphosphonates affect early process of socket healing in mice model. METHODS: Eighteen 8-9 weeks C57BL/6 male mice were randomly divided into control and experimental group. Bisphosphonate-related osteonecrosis of the jaw（BRONJ）model was conducted by intraperitoneal injection of zoledronate（ZOL） and extraction of lower left first molar in mice. Three, five, seven day after surgery, the mice were sacrificed and paraffin-embedded slides were made. H-E staining was used to evaluate the gross condition. The distribution and amounts of osteoclasts were measured by TRAP staining. Finally, immunochemical staining was used to detect RUNX2 and CTSK level. All experiments were duplicated thrice, ImageJ was used for transformation of pictures and SPSS 20.0 software package was used for statistical analysis. RESULTS: In comparison with the control group, early process of socket healing in the experimental group was generally delayed. RUNX2,CTSK,TRAP expression level was decreased. On the whole, early bone remodeling process in ZOL injection group was suppressed. CONCLUSIONS: Zoledronic acid inhibited the migration of fibroblasts into socket, and reduced the expression of Runx2, hindering new bone formation. It also can reduce the expression of TRAP 3，5，7 days after tooth extraction and CTSK expression three days after operation, thus inhibiting the bone resorption function of osteoclasts.

[Expression of hypoxia inducible factor-1alpha during mouse mandibular first molar eruption and its significance].

PURPOSE: To investigate the expression of hypoxia inducible factor-1alpha (HIF-1α) during the eruption of mouse lower first molar, and to explore the correlation between its expression and the activity of osteoclasts. METHODS: Mouse mandibles were dissected from mice at day 1.5 to 14.5 after birth. They were then serially sectioned and stained respectively using hemotoxylin, tartrate-resistant acid phosphatase (TRAP) staining, and immunohistochemical staining. SPSS 25.0 software package was used for data analysis. RESULTS: During the eruption of mouse lower first molar, the thickness of the crown alveolar bone gradually decreased, so did the number of osteoclasts and the expression of HIF-1α. CONCLUSIONS: During the eruption of mouse lower first molar, the less expression of HIF-1α, the fewer osteoclasts there were. HIF-1α may be involved in the eruption of mouse lower first molar by regulating the activity of osteoclasts.

Partial deficiency of HIF-1α in chondrocytes effected bone repair of mandibular condylar neck.

OBJECTIVES: This study aimed to explore the expression of hypoxia-inducible factor 1α (HIF-1α) in chondrocytes with the healing process after unilateral mandibular condylar neck osteotomy and to verify its effect on bone repair. METHODS: Models of mandibular condylar neck osteotomy were established in mice. Transgenic mice with heterozygous deficiency in HIF-1α gene in chondrocytes were used. Radiographic evaluation, quantitative reverse transcription polymerase chain reaction and histomorphometric analyses were used to compare the difference in capacities of chondrogenesis, vasifaction, osteogenesis, and bone resorption. RESULTS: HIF-1α was expressed in the chondrocytes of calluses. Decreased expression of HIF-1α in chondrocytes promoted the proliferation of chondrocytes and upregulated the expression of apoptosis markers. However, the density and thickness of newly formed trabecula in transgenic mice were reduced on post-osteotomy day 28, and some expression of angiogenic, osteogenic, and osteoclastogenic markers was impaired. CONCLUSIONS: These results demonstrated the importance of HIF-1α to chondrocytes and bone repair during the healing process after osteotomy of the mandibular condylar neck. Decreased HIF-1α promoted the chondrocyte proliferation, and effected endochondral ossification.

The effects of mandibular osteotomy on maxillary orthodontic tooth movement and bone remodelling in a rat model.

OBJECTIVES: The accelerated tooth movement phenomenon after orthognathic surgery has been observed. However, the underlying mechanism remains unclear. There is no experimental study showing the effect of orthognathic surgery on orthodontic tooth movement of the opposing jaw. Therefore, the present study aimed at investigating if mandibular osteotomy enhances maxillary tooth movement and bone remodelling. MATERIALS AND METHODS: Fifty-four male Sprague-Dawley rats were randomly divided into two groups: maxillary tooth movement (TM) and maxillary tooth movement + mandibular surgery (TM + MS). The orthodontic force was delivered to move the left maxillary first molar mesially. The surgical intervention was performed on the left mandible. Microcomputed tomography, histological analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction were used to assess changes at 3, 7, and 21 days after surgery. RESULTS: The mandibular osteotomy accelerates the rate of maxillary tooth movement with decreased bone volume fraction on the seventh day. Bone resorption was observed on the third and seventh day after mandibular osteotomy. It was found that serum interleukin-1ß level increased significantly in the TM + MS group compared with the TM group, as well as the high expression level of cathepsin K and tumour necrosis factor receptor-associated factor 5 of the orthodontic tooth on the third and seventh day after mandibular osteotomy. CONCLUSION: Data from the present study suggested that mandibular osteotomy accelerates maxillary osteoclast activity and post-operative tooth movement, providing evidence for accelerated tooth movement phenomenon after orthognathic surgery.

HIF-1α induces hypoxic apoptosis of MLO-Y4 osteocytes via JNK/caspase-3 pathway and the apoptotic-osteocyte-mediated osteoclastogenesis in vitro.

Apoptotic osteocytes were found in the hypoxic bone microenvironment in osteoporosis, osteotomy, orthodontic tooth movement and periodontitis, and played a key role in bone remolding and the differentiation of osteoclasts. Hypoxia inducible factor-1α(HIF-1α), as a transcription factor under hypoxic conditions, has been confirmed to participate in cell apoptosis. However, the effect of HIF-1α on osteocytes apoptosis and the osteocyte-mediated osteoclast formation remains elusive. Here, we hypothesized that HIF-1α was involved in osteocytes apoptosis. Our results showed that CoCl2 increased the MLO-Y4 cells apoptosis by upregulating the proapoptotic gene expression of caspase-3. Moreover, siRNA-mediated knockdown of HIF-1α decreased the phosphorylation by JNK and the activation of caspase-3 to inhibit the cell apoptosis in MLO-Y4. Furthermore, SP600125, an inhibitor of JNK, reversed CoCl2-induced the increased apoptosis of MLO-Y4 cells in term of reducing the expression of caspase-3. These findings revealed that HIF-1α served as a pro-apoptotic factor in the apoptosis of MLO-Y4 cells cultured with CoCl2, by activating the JNK/caspase-3 signaling pathway. Besides, the osteocyte-mediated osteoclastogenesis was reduced with the decline of the expression of HIF-1α and caspase-3 in MLO-Y4 cells. Our study provided an idea for a more comprehensive understanding of HIF-1α and the process of bone remodeling.

[Mechanism of participation of osteocytes in the formation of osteoclasts under hypoxia].

OBJECTIVE: To investigate the mechanism of the participation of osteocytes in the formation of osteoclasts under hypoxia. METHODS: The hypoxia culture system of osteocyte-like cell line MLO-Y4 was established by deferoxamine mesylate (DFO) in vitro. The proliferation of MLO-Y4 cells was examined by CCK-8 cell proliferation/toxicity assay. RAW264.7 cells were induced to osteoclasts by the conditioned medium containing the cultured MLO-Y4. Tartrate-resistant acid phosphatase (TRAP) staining was performed on day 7. Quantitative real-time fluorescence polymerase chain reaction, immunofluorescence, and Western blot were used to detect the expression levels of hypoxia-inducible factor (HIF)-1α and receptor activator of nuclear factor-κB ligand (RANKL) in MLO-Y4 under hypoxia. The effects of siHIF-1α on the expression levels of HIF-1α and RANKL in MLO-Y4 under the same conditions were detected. RESULTS: DFO (100 µmol·L⁻¹) promoted the proliferation of MLO-Y4 at 24 h, which decreased with time (P<0.01). After the addition of soluble sRANKL, the formation of osteoclasts was significantly increased in the DFO group (P<0.001). The expression of RANKL mRNA in MLO-Y4 under 100 µmol·L⁻¹ DFO increased first and then decreased with the duration of hypoxia. This expression reached a peak at 24 h (P<0.01). Hypoxia up-regulated the expression of HIF-1α and RANKL protein (P<0.01). Under hypoxia, siHIF-1α downregulated the expression of HIF-1α and RANKL (P<0.01). siHIF-1α also decreased the number of osteoclasts (P<0.01). CONCLUSIONS: Under hypoxia, MLO-Y4 could facilitate the formation of RANKL through upre-gulating the expression of HIF-1α protein, thereby accelerate the differentiation of RAW264.7 cells into osteoclasts.

Coronectomy: A Useful Approach in Minimizing Nerve Injury Compared With Traditional Extraction of Deeply Impacted Mandibular Third Molars.

PURPOSE: A surgical procedure to minimize the incidence of inferior alveolar nerve injury (IANI) in deeply impacted mandibular third molars (IMTMs) has been proposed. Our study compared the near-term outcomes between coronectomy and traditional extraction of IMTMs and evaluated the long-term complications after coronectomy using cone-beam computed tomography (CBCT). PATIENTS AND METHODS: A prospective study was performed of patients with IMTMs at high-risk of IANI using radiographic examination and CBCT. The patients were divided into 2 groups: a coronectomy group and an extraction group. The short-term outcomes, including IANI and other conditions, such as bleeding, pain, and swelling, were assessed in both groups 1 week after surgery. The coronectomy patients were evaluated at 3, 6, 12, and 36 months after the procedure. The primary long-term complications assessed included root migration, secondary included inflammation, socket healing, and eruption. Relevant factors affecting the outcomes (ie, age, gender, root morphology, impacted depth, impacted angle) were also analyzed. The data were analyzed using SPSS Statistics, version 20.0 (IBM Corp, Armonk, NY). RESULTS: A total of 110 IMTMs (55 in the coronectomy group and 55 in the extraction group) in 92 patients (49 men and 43 women) were included in CBCT assessment. IANI was found in 6 patients in the extraction group and no patient in the coronectomy group (P < .05). After 6 months, 2 patients still presented with light numbness. After coronectomy, the roots had migrated quickly during the initial 6 months and had become stable 1 year after surgery; 90.9% of the roots had migrated away from the mandibular nerve canal at 6 months postoperatively. No infection had occurred within the 3-year follow-up period. CONCLUSIONS: Coronectomy should be considered superior to traditional extraction in the management of the risk of IANI, with few additional complications occurring during follow-up. It could be used as a useful and safe clinical treatment of IMTMs with a high risk of IANI.

HIF-1α facilitates osteocyte-mediated osteoclastogenesis by activating JAK2/STAT3 pathway in vitro.

Osteocytes, entrapped within the mineralized bone matrix, has been found to have numerous functions such as acting as an orchestrator of bone remodeling through regulation of both osteoclast and osteoblast activity and also functioning as an endocrine cell. Due to a specialized morphology and surrounding structure, osteocytes are more tolerant to hypoxia during osteoporosis, fracture, osteoarthritis, and orthodontic-orthognathic combination therapy. Hypoxia-inducible factor-1α (HIF-1α) is one of the master regulators of hypoxia reactions, playing an important role in bone modeling, remodeling, and homeostasis. This study aimed to investigate the pivotal functional role of HIF-1α in osteocytes initiating of bone remodeling under hypoxia. In the present study, the osteoclasts formation induced by RAW264.7 was significantly promoted in conditioned media (CM) from osteocytic MLO-Y4 exposed to hypoxia in vitro. Therefore, hypoxic MLO-Y4 cells simulated by 100 µmol/L CoCl2 or 2% O2 stably expressed HIF-1α proteins and upregulated the expression of receptor activator of nuclear factor-κB ligand (RANKL) at both the messenger RNA (mRNA) and protein level. Furthermore, with the Knockdown of HIF-1α, the expression of RANKL mRNA and protein decreased after transient transfection. In addition, the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription (STAT3) was also correlated with HIF-1α and RANKL levels under hypoxia. Then AG490, a JAK2 inhibitor, inhibited p-JAK2, p-STAT3 and RANKL expression. It was possible that AG490 disturbed the contact of HIF-1α and RANKL by JAK2/STAT3 pathway, influencing osteoclastogenesis. Our findings suggested that HIF-1α promoted the expression of RANKL by activating JAK2/STAT3 pathway in MLO-Y4 cells, and enhanced osteocyte-mediated osteoclastic differentiation in vitro.

Mandibular osteotomy-induced hypoxia enhances osteoclast activation and acid secretion by increasing glycolysis.

The rapid bone remodeling after osteotomy has been reported for a long time. However, the underlying mechanism promoting the active bone reconstruction was still to be elucidated. Since not only the bone, blood vessels, and supportive tissues, but also the local microenvironment were destroyed, if the changes on the cell metabolism was contributed to the accelerated bone remodeling came into sight. In present study, we found that the mandibular osteotomy in rabbit activated osteoclasts, as well as the expression of hypoxia-inducible factor 1α (HIF-1α) in alveolar bone. Hypoxia or HIF-1α could enhanced osteoclastogenesis, bone absorption, and lactic acid concentration in receptor activator of nuclear factor κΒ ligand-induced RAW264.7 cells. Coincided with the upregulated HIF-1α expression, HIF-driven glycolytic enzymes, such as lactate dehydrogenase A (LDHA), glucokinase (GCK), pyruvate kinase M2 (PKM2), and phosphofructokinase1 (PFK1), were found massively increased in both hypoxic RAW264.7 cells and the alveolar HIF-1α-positive osteoclasts after mandibular osteotomy. Knockdown of HIF-1α suppressed not only the hypoxia-mediated glycolysis, but also the hypoxia-induced acid secretion and bone resorption in RAW264.7 cells. Application of inhibitor on glycolysis gave rise to the similar results as HIF-1α knockdown. Our findings suggested that hypoxia-driven glycolysis in osteoclasts was an adaptive mechanism to permit alveolar bone remodeling after mandibular osteotomy.

Activity and morphologic changes in the mandible after mandibular osteotomy.

INTRODUCTION: Orthognathic surgery accelerates orthodontic tooth movement, and tooth movement accelerates with demineralized bone and accelerated bone remodeling. The purpose of this study was to ascertain whether orthognathic surgery induces accelerated bone remodeling. The research design included a human model and an animal model. METHODS: The levels of serum tartrate resistant acid phosphatase-5b (TRAP) and bone alkaline phosphatase (BALP) were measured in 15 patients after sagittal split ramus osteotomy. For the animal study, 18 rabbits were divided into 6 groups: a control group and 5 surgery groups. The rabbits in the surgery groups had osteotomies in the molar regions of the mandible. Changes in bone mass of the anterior mandibles were examined by microcomputed tomography, and changes in osteoblast and osteoclast numbers were analyzed by real-time polymerase chain reaction, hematoxylin and eosin staining, TRAP staining, and alkaline phosphatase staining. RESULTS: In the 15 patients, TRAP-5b increased from 1 to 8 weeks postoperatively, and BALP increased significantly in 2 weeks postoperatively. In the rabbits, the levels of mRNA expression of TRAP were increased at 3 weeks, and matrix metalloproteinase 9 was increased at 4 and 8 weeks, whereas mRNA expression of BALP and bone morphogenetic protein 2 were increased at 4 weeks. Bone loss was detected from 1 week postoperatively and reached the maximum at 3 weeks; and bone mass and mechanical structure did not recoverer to preoperative levels until 8 weeks postoperatively. CONCLUSIONS: These findings show active bone remodeling induced by osteotomy.

IL-6 Enhances Osteocyte-Mediated Osteoclastogenesis by Promoting JAK2 and RANKL Activity In Vitro.

BACKGROUND/AIMS: Evidence suggests that IL-6 affects bone mass by modulating osteocyte communication towards osteoclasts. However, the mechanism by which IL-6 enhances osteocyte-mediated osteoclastogenesis is unclear. We aimed to investigate the inflammatory factors in serum after orthodontic surgery and their relationship between osteocytes and osteoclasts. METHODS: Serum was obtained from 10 orthognathic surgery patients, and inflammatory factors were detected by ELISA. We treated the osteocyte-like cell line MLO-Y4 with recombinant mouse IL-6 and IL-6 receptor (IL-6R), and used quantitative RT-PCR and Western blotting to explore Receptor activator of nuclear factor-κB ligand (RANKL) expression at both the mRNA and protein level. MLO-Y4 cells were co-cultured with osteoclast precursor cells, and the formation of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. To explore the role of JAK2 in the osteocyte-mediated osteoclastogenesis, AG490, a JAK2 inhibitor, was used to inhibit the JAK2-STAT3 pathway in osteocytes. RESULTS: In our study, we found that IL-6 and RANKL were stimulated in serum 3-7 days after orthognathic surgery. Therefore, IL-6 and IL-6 receptor enhanced the expression of RANKL at both the mRNA and protein level in MLO-Y4. Furthermore, when MLO-Y4 cells were co-cultured with osteoclast precursor cells, it significantly stimulated osteoclastogenesis. Our study indicated that osteocytes could promote osteoclastic differentiation and the formation of TRAP-positive multinucleated cells after stimulation with IL-6 and IL-6R. Our results also indicated that treatment with IL-6 and IL-6R increased RANKL mRNA expression and the RANKL/OPG expression ratio. Meanwhile, the phosphorylation of Janus kinase 2 (JAK2) and Signal transducer and activator of transcription (STAT3) also correlated with RANKL levels. Furthermore, we investigated the effects of a specific JAK2 inhibitor, AG490, on the expression of RANKL in osteocyte-like MLO-Y4 cells and osteocyte-mediated osteoclastogenesis. The results showed that AG490 inhibited (p)-JAK2 and RANKL expression. Osteoclastic differentiation was decreased after pretreatment in MLO-Y4 with mouse IL-6/IL-6R and AG490; therefore, we concluded that IL-6 increased osteocyte-mediated osteoclastic differentiation by activating JAK2 and RANKL. CONCLUSION: The effects of IL-6/il-6R and AG490 on osteocyte-mediated osteoclastogenesis contribute to our understanding of the role of inflammatory factors in the interaction between osteocytes and osteoclast precursors. IL-6 and RANKL are key factors for bone remodelling after the orthodontic surgery, and their roles in bone remodelling may be fundamental mechanisms accelerating tooth movement by orthodontic surgery.