Zanthoxylum bungeanum Maxim. (Rutaceae): A Systematic Review of Its Traditional Uses, Botany, Phytochemistry, Pharmacology, Pharmacokinetics, and Toxicology.

Zanthoxylum bungeanum Maxim. (Rutaceae) is a popular food additive and traditional Chinese herbal medicine commonly named HuaJiao in China. This plant is widely distributed in Asian countries. The aim of this paper is to provide a systematic review on the traditional usages, botany, phytochemistry, pharmacology, pharmacokinetics, and toxicology of this plant. Furthermore, the possible development and perspectives for future research on this plant are also discussed. To date, over 140 compounds have been isolated and identified from Z. bungeanum, including alkaloids, terpenoids, flavonoids, and free fatty acids. The extracts and compounds have been shown to possess wide-ranging biological activity, such as anti-inflammatory and analgesic effects, antioxidant and anti-tumor effects, antibacterial and antifungal effects, as well as regulatory effects on the gastrointestinal system and nervous system, and other effects. As a traditional herbal medicine, Z. bungeanum has been widely used to treat many diseases, especially digestive disorders, toothache, stomach ache, and diarrhea. Many traditional usages of this plant have been validated by present investigations. However, further research elucidating the structure-function relationship among chemical compounds, understanding the mechanism of unique sensation, as well as exploring new clinical effects and establishing criteria for quality control for Z. bungeanum should be further studied.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Hsp20 Promotes Endothelial Progenitor Cell Angiogenesis via Activation of PI3K/Akt Signaling Pathway under Hypoxia.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. METHODS: Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. RESULTS: CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. CONCLUSION: In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

ECFC-derived exosomal THBS1 mediates angiogenesis and osteogenesis in distraction osteogenesis via the PI3K/AKT/ERK pathway.

Background: Distraction osteogenesis (DO) is a widely used bone regenerative technique. However, the DO process is slow, and the consolidation phase is long. Therefore, it is of great clinical significance to explore the mechanism of DO, and shorten its duration. Recent studies reported that stem cell exosomes may play an important role in promoting angiogenesis related to DO, but the mechanism remains unclear. Methods: Canine endothelial colony-forming cells (ECFCs) were isolated and cultured, and the expression of THBS1 in canine ECFCs were inhibited using a lentiviral vector. The exosomes secreted by canine ECFCs were isolated and extracted, and the effect of exosomes on the angiogenic activity of Human umbilical vein endothelial cells (HUVECs) was detected by proliferation, migration, and tube formation experiments. WB and qRT-PCR were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on HUVECs angiogenesis. Then, a mandibular distraction osteogenesis (MDO) model was established in adult male beagles, and exosomes were injected into the canine peripheral blood. Micro-CT, H&E, Masson, and IHC staining were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on angiogenesis and osteogenesis in the DO area. Results: ECFC-Exo accelerated HUVECs proliferation, migration and tube formation, and this ability was enhanced by inhibiting the expression of THBS1 in ECFC-Exo. Using Western blot-mediated detection, we demonstrated that inhibiting THBS1 expression in ECFCs-Exo activated PI3K, AKT, and ERK phosphorylation levels in HUVECs, which promoted VEGF and bFGF expressions. In the DO model of the canine mandible, ECFCs-Exo injected into the peripheral blood aggregated into the DO gap, thus promoting angiogenesis and bone formation in the DO tissue by reducing THBS1 expression in ECFC-Exo. Conclusion: Our findings suggested that ECFC-Exos markedly enhances angiogenesis of endothelial cells, and promotes bone healing in canine MDO. Thus, THBS1 plays a crucial role in the ECFC-Exos-mediated regulation of canine MDO angiogenesis and bone remodeling. The translational potential of this article: This study reveals that the angiogenic promotion via THBS1 suppression in ECFC-Exos may be a promising strategy for shortening the DO duration.

Identification of the key exosomal lncRNAs/mRNAs in the serum during distraction osteogenesis.

BACKGROUND: Distraction osteogenesis (DO), a kind of bone regenerative process, is not only extremely effective, but the osteogenesis rate is far beyond ordinary bone fracture (BF) healing. Exosomes (Exo) are thought to play a part in bone regeneration and healing as key players in cell-to-cell contact. The object of this work was to determine whether exosomes derived from DO and BF serum could stimulate the Osteogenic Differentiation in these two processes, and if so, which genes could be involved. METHODS: The osteogenesis in DO-gap or BF-gap was evaluated using radiographic analysis and histological analysis. On the 14th postoperative day, DO-Exos and BF-Exos were isolated and cocultured with the jaw of bone marrow mesenchymal stem cells (JBMMSCs). Proliferation, migration and osteogenic differentiation of JBMMSCs were ascertained, after which exosomes RNA-seq was performed to identify the relevant gene. RESULTS: Radiographic and histological analyses manifested that osteogenesis was remarkably accelerated in DO-gap in comparison with BF-gap. Both of the two types of Exos were taken up by JBMMSCs, and their migration and osteogenic differentiation were also seen to improve. However, the proliferation showed no significant difference. Finally, exosome RNA-seq revealed that the lncRNA MSTRG.532277.1 and the mRNA F-box and leucine-rich repeat protein 14(FBXL14) may play a key role in DO. CONCLUSIONS: Our findings suggest that exosomes from serum exert a critical effect on the rapid osteogenesis in DO. This promoting effect might have relevance with the co-expression of MSTRG.532277.1 and FBXL14. On the whole, these findings provide new insights into bone regeneration, thereby outlining possible therapeutic targets for clinical intervention.

microRNA-146a mediates distraction osteogenesis via bone mesenchymal stem cell inflammatory response.

Distraction osteogenesis (DO) is a widely used surgical technique to repair bone defects, partly owing to its high efficiency in inducing osteogenesis; however, the process of osteogenesis is complex, and the precise mechanism is still unclear. Among the factors identified for an effective DO procedure, well-controlled inflammation is essential. We aimed to explore how microRNA(miR)-146a, a negative regulator of inflammation, influences osteogenesis in DO. First, we established canine right mandibular DO and bone fracture models to evaluate the expression level of miR-146a in response to these procedures. Second, bone marrow mesenchymal stem cells (BMSCs) were isolated from healthy puppies and cultured with lipopolysaccharide (LPS) to observe how inflammation affects osteogenesis. Finally, the osteogenesis activity of BMSCs transfected with lentiviral vector either overexpressing (miR-146a-up) or inhibited for miR-146a expression was evaluated. miR-146a-up-transfected BMSCs were injected locally into the distraction gaps of the DO model canines. On days 42 and 56 post-surgery, the bone volume/tissue volume and bone mineral density values were evaluated via using micro-computed tomography, and newly formed tissues were harvested and evaluated via histological staining. The expression of miR-146a in both the DO canine model and LPS-stimulated BMSCs increased. Overexpression of miR-146a enhanced cell proliferation, migration, and osteogenic differentiation. Additionally, the newly formed callus was improved in canine mandibles injected with miR-146a-up-transfected BMSCs. In summary, miR-146a regulates mandibular DO by improving osteogenesis, and can serve as a potential target to shorten the therapy period of DO.

Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-ß1 Signaling Pathway.

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TßR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-ß1/Smad pathway. Consistently, the overexpression or silencing of TßR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TßR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-ß1/Smad pathway.

The RNA Methyltransferase METTL3 Promotes Endothelial Progenitor Cell Angiogenesis in Mandibular Distraction Osteogenesis via the PI3K/AKT Pathway.

Distraction osteogenesis (DO) is used to treat large bone defects in the field of oral and maxillofacial surgery. Successful DO-mediated bone regeneration is dependent upon angiogenesis, and endothelial progenitor cells (EPCs) are key mediators of angiogenic processes. The N6-methyladenosine (m6A) methyltransferase has been identified as an important regulator of diverse biological processes, but its role in EPC-mediated angiogenesis during DO remains to be clarified. In the present study, we found that the level of m6A modification was significantly elevated during the process of DO and that it was also increased in the context of EPC angiogenesis under hypoxic conditions, which was characterized by increased METTL3 levels. After knocking down METTL3 in EPCs, m6A RNA methylation, proliferation, tube formation, migration, and chicken embryo chorioallantoic membrane (CAM) angiogenic activity were inhibited, whereas the opposite was observed upon the overexpression of METTL3. Mechanistically, METTL3 silencing reduced the levels of VEGF and PI3Kp110 as well as the phosphorylation of AKT, whereas METTL3 overexpression reduced these levels. SC79-mediated AKT phosphorylation was also able to restore the angiogenic capabilities of METTL3-deficient EPCs in vitro and ex vivo. In vivo, METTL3-overexpressing EPCs were additionally transplanted into the DO callus, significantly enhancing bone regeneration as evidenced by improved radiological and histological manifestations in a canine mandibular DO model after consolidation over a 4-week period. Overall, these results indicate that METTL3 accelerates bone regeneration during DO by enhancing EPC angiogenesis via the PI3K/AKT pathway.

MicroRNA-205 mediates endothelial progenitor functions in distraction osteogenesis by targeting the transcription regulator NOTCH2.

BACKGROUND: Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure. METHODS: A mandibular DO (MDO) canine model was established, after which small RNA sequencing was performed to identify relevant molecular targets genes. Putative miRNA target genes were identified through bioinformatics and confirmed through qPCR, Western blotting, and dual-luciferase reporter assays. Peripheral blood samples were collected to isolate serum and endothelial colony-forming cells (ECFCs) in order to measure miR-205, NOTCH2, and angiogenic cytokines expression levels. Lentiviral constructs were then used to inhibit or overexpress miR-205 and NOTCH2 in isolated ECFCs, after which the angiogenic activity of these cells was evaluated in migration, wound healing, proliferation, tube formation, and chick chorioallantoic membrane (CAM) assay. Autologous ECFCs transfected to knockdown miR-205 and were injected directly into the distraction callus. On days 14, 28, 35 and 42 after surgery, bone density was evaluated via CBCT, and callus samples were collected and evaluated via histological staining to analyze bone regeneration and remodeling. RESULTS: MiR-205 was identified as being one of the miRNAs that was most significantly downregulated in MDO callus samples. Downregulation of miR-205 was also observed in DO-ECFCs and serum of animals undergoing MDO. Inhibiting miR-205 markedly enhanced angiogenesis, whereas overexpressing miR-205 had the opposite effect in vitro. Importantly, NOTCH2, which is a unique regulator in bone angiogenesis, was identified as a miR-205 target gene. Consistent with this regulatory relationship, knocking down NOTCH2 suppressed angiogenesis, and transduction with a miR-205 inhibitor lentivirus was sufficient to rescue angiogenic activity. When ECFCs in which miR-205 had been inhibited were transplanted into the MDO callus, this significantly bolstered osteogenesis, and remodeling in vivo. CONCLUSIONS: MiR-205 is a significant regulator of the MDO process, and inhibiting this miRNA can accelerate MDO-related mineralization. Overall, these results offer new insights into the mechanistic basis for this procedure, highlighting potential targets for therapeutic clinical intervention.

Hypoxia improved vasculogenesis in distraction osteogenesis through Mesenchymal-Epithelial transition (MET), Wnt/ß-catenin signaling pathway, and autophagy.

INTRODUCTION: The osteogenesis rate of distraction osteogenesis is 4-6 times faster than that of infants, far beyond fracture healing. However, the osteogenesis mechanism of DO is complicated and inconclusive owing to two significant elements: mechanical tension which is well explored and trauma caused by bone fracture. Vasculogenesis and EPCs are critical for successful bone regeneration during DO. Thus, this study aimed to explore the effects of hypoxia caused by trauma or CoCl2 on the vasculogenesis of DO and EPCs. MATERIAL AND METHODS: Mandibular DO and BF models were generated using 6 beagle dogs with a distraction rate of 1 mm per day for 7 days or acute lengthening for 7 mm. The vasculogenesis in DO-gap or BF-gap were assessed via histological analyses, qRT-PCR and immunofluorescence staining. Dog bone marrow EPCs were isolated and cultured with or without 0.1 mM CoCl2. The effect of hypoxia caused by CoCl2 were subsequently valuated via in vitro assays including Cell Counting Kit-8, transwell assay, qRT-PCR, western blot, and immunofluorescence staining. RESULTS: Histological analyses, qRT-PCR and immunofluorescence staining revealed that vasculogenesis markedly accelerated in DO-gap compared with BF-gap, and the DO-gap displayed more positive to CD133, CD34, HIF-1α, E-cadherin, beclin1, ß-catenin, VEGF, bFGF, and less positive to ZEB1 than BF-gap. In addition, in vitro analyses revealed CoCl2 treatment enhanced EPCs proliferation and migration, and the levels of HIF-1α, E-cadherin, ß-catenin, beclin1, VEGF, bFGF of EPCs were increased, but the level of ZEB1 was decreased. CONCLUSION: Our studies showed that hypoxia promoted vasculogenesis in DO and EPCs, and the mechanism may involve autophagy, Wnt/ß-catenin signaling pathway, and Mesenchymal-Epithelial transition (MET).

[Clinical application of custom-made key-key attachment in the restoration of maxillofacial defect].

PURPOSE: To evaluate the usefulness of custom-made key-key attachment in restoration of maxillofacial defect. METHODS: Using key-key attachment, facial prostheses and intraoral prostheses were combined to restore dentition, maxilla and right facial defects in a female patient. RESULTS: Good retention of maxillofacial prostheses and intraoral prostheses were obtained. The appearance of the patient was remarkably improved. The function of mastication was improved. CONCLUSIONS: Good result can be obtained by using custom-made key-key attachment in restoration of complex maxillofacial defect. Supported by Shanghai Leading Academic Discipline Project(Grant No.T0202).

[Application of stud structure to sectional upper complete denture for a patient with microstomia].

PURPOSE: Prosthetic rehabilitation with stud structure to sectional complete denture for a patient with microstomia was done and the treatment result was evaluated. METHODS: A sectional impression tray technique was used. And a custom-made palatal hinge mechanism with stud structure on the left and right side of the upper complete denture along the middle line were also designed and fabricated. Half metal abutment was fabricated on the left and right side of the denture on the position of the upper central incisor, and a PFM was made according to the metal abutment. After the sectional upper complete denture was inserted, the PFM was set to connect the sectional denture as a whole and provide stability. RESULTS: The sectional upper complete denture was successfully and easily inserted and provided adequate function in the patient's mouth. CONCLUSIONS: Application of stud structure to sectional upper complete denture for patient with microstomia is feasible.

[The effects of different welding wires on the mechanical properties of laser welding joints].

PURPOSE: To evaluate the mechanical properties and microstructure of laser-welded joints with different welding wires for clinical use of welding wire. METHODS: The standard tensile test and three-point bending test rods were made from Co-Cr and Ni-Cr alloy, and were laser-welded with different welding wire (commercially welding wire and casting wire). Then the tensile rods were tested for the ultimate tensile strength (UTS), and the bending rods for the ultimate bending strength (UBS). The results was analyzed by one-way ANOVA. The tensile fracture surface were examined by scanning electron microscopy (SEM). Metallurgical analysis were also performed on polished longitudinal sectioned samples. RESULTS: For Co-Cr alloy, the UTS of casting wire group and commercially welding wire group was respectively (606.40+/-82.53)MPa and (693.61+/-47.68)MPa; the UBS was respectively (997.95+/-88.89)MPa and (1160.76+/-91.59)MPa. ANOVA showed a significant difference of UTS and UBS between the two groups at the 0.05 level (P<0.05). For Ni-Cr alloy, the UTS of casting wire group and commercially welding wire group was respectively (558.14+/-46.75)MPa and (582.32+/-35.43)MPa; the UBS was respectively (1084.75+/-46.02)MPa and (1078.29+/-36.25)MPa. There was no significant difference between the two groups (P>0.05). SEM and metallurgical examination showed the welded zone exhibiting more cracks in the casting wire group than in the commercially welding wire group. CONCLUSION: It would be advisable to work with commercially welding wire for the joints that need better strength.

[Design and application of the framework to prevent fracture of the magnetic denture].

PURPOSE: To investigate the methods to prevent magnetic denture from fracture. METHODS: 17 cases were selected and divided into group A and group B. 9 cases in group A only used resin base, 8 cases in group B were strengthened with frameworks. The rates of fracture or fissure in denture base were observed for 5 years, Chi(2) test was used for statistical analysis. RESULTS: In group A, 3 cases were found with damaged base, there was no fracture happened in group B. Chi(2) test showed that there was significant difference between the two groups (P<0.05). CONCLUSION: Frameworks should be designed to improve the strength of the magnetic denture base. It is easy to manufacture and apply with satisfactory clinical effects.

[An experimental study on laser-welded dissimilar alloys in dentistry.].

PURPOSE: This study evaluated the mechanical properties and microstructure of the joints of laser-welded dissimilar alloys usually used in clinical nowadays. METHODS: The dissimilar alloys for Co-Cr alloy,Ni-Cr alloy and pure titanium were laser-welded. The joints were tested for the ultimate tensile strength (UTS) and the ultimate bending strength (UBS). The tensile fracture surfaces were examined by scanning electron microscopy (SEM). Metallurgical analysis was also performed on polished longitudinal sectioned samples. RESULTS: Laser-welded dissimilar alloys between Co-Cr and Ni-Cr alloy can yield satisfactory mechanical properties, and there was no significant difference of UTS and UBS between Co-Cr welding wire group and Ni-Cr welding wire group (P>0.05). The laser-welded joints for dissimilar metal of pure titanium and Co-Cr,pure titanium and Ni-Cr, showed more brittle characteristics and severe defects (pores and cracks). CONCLUSIONS: With Co-Cr or Ni-Cr welding wire, the laser-welded dissimilar alloys of Co-Cr alloy and Ni-Cr alloy can yield satisfactory joints. But pure titanium and Co-Cr alloy, pure titanium and Ni-Cr alloy cannot be directly melted together.

[Effect of laser irradiation power on the mechanical properties of Co-Cr and Ni-Cr laser-welded joints.].

PURPOSE: To investigate the effect of different irradiation power on the mechanical properties of laser-welded joints. METHODS: The standard tensile test and three-point bending test rods made from Co-Cr and Ni-Cr alloy were laser-welded with different irradiation powers. Then the tensile rods were tested for the ultimate tensile strength (UTS), and the bending rods for the ultimate bending strength (UBS). The tensile fracture surface was examined by scanning electron microscopy (SEM). Metallurgical analysis and microhardness test were also performed on polished longitudinal sectioned samples. RESULTS: For Co-Cr alloy, greater irradiation power resulted in greater UTS, minor UBS. For Ni-Cr alloy, there was no significant difference of UTS and UBS between the four groups. SEM and metallurgical examination showed that the welded zone exhibited more gas pores and cracks as the irradiation power increased. Microhardness test showed that hardness increased toward the center of the welded zone and as the irradiation power increased, the range of hardness increased was expanded. CONCLUSION: The optimal laser irradiation power was the key to achieved superior mechanical properties joints.