Total flavonoids of Rhizoma drynariae promote angiogenesis and osteogenesis in bone defects.

Bone defects are difficult to heal, which conveys a heavy burden to patients' lives and their economy. The total flavonoids of Rhizoma drynariae (TFRD) can promote the osteogenesis of distraction osteogenesis. However, the dose effect is not clear, the treatment period is short, and the quality of bone formation is poor. In our study, we observed the long-term effects and dose effects of TFRD on bone defects, verified the main ingredients of TFRD in combination with network pharmacology for the first time, explored its potential mechanism, and verified these findings. We found that TFRD management for 12 weeks regulated osteogenesis and angiogenesis in rats with 4-mm tibial bone defects through the PI3K/AKT/HIF-1α/VEGF signaling pathway, especially at high doses (135 mg kg-1  d-1 ). The vascularization effect of TFRD in promoting human umbilical vein endothelial cells was inhibited by PI3K inhibitors. These results provide a reference for the clinical application of TFRD.

Exploring the treatment of COVID-19 with Yinqiao powder based on network pharmacology.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In China, Yinqiao powder is widely used to prevent and treat COVID-19 patients with Weifen syndrome. In this study, the screening and verification of active ingredients, target selection and DisGeNET scoring, drug-ingredient-gene network construction, protein-protein interaction network construction, molecular docking and surface plasmon resonance (SPR) analysis, gene ontology (GO) functional analysis, gene tissue analysis, and kyoto encyclopedia of genes and genomes (KEGG) pathway analysis were used to explore the active ingredients, targets, and potential mechanisms of Yinqiao powder in the treatment of COVID-19. We also predicted the therapeutic effect of Yinqiao powder using TCM anti-COVID-19 (TCMATCOV). Yinqiao powder has a certain therapeutic effect on COVID-19, with an intervention score of 20.16. Hesperetin, eriodictyol, luteolin, quercetin, and naringenin were the potentially effective active ingredients against COVID-19. The hub-proteins were interleukin-6 (IL-6), mitogen-activated protein kinase 3 (MAPK3), tumor necrosis factor (TNF), and tumor protein P53 (TP53). The potential mechanisms of Yinqiao powder in the treatment of COVID-19 are the TNF signaling pathway, T-cell receptor signaling pathway, Toll-like receptor signaling pathway, and MAPK signaling pathway. This study provides a new perspective for discovering potential drugs and mechanisms of COVID-19.

An anti-infection and biodegradable TFRD-loaded porous scaffold promotes bone regeneration in segmental bone defects: experimental studies.

BACKGROUND: Addressing segmental bone defects remains a complex task in orthopedics, and recent advancements have led to the development of novel drugs to enhance the bone regeneration. However, long-term oral administration can lead to malnutrition and poor patient compliance. Scaffolds loaded with medication are extensively employed to facilitate the restoration of bone defects. METHODS: Inspired by the local application of total flavonoids of Rhizoma Drynariae (TFRD) in the treatment of fracture, a novel 3D-printed HA/CMCS/PDA/TFRD scaffold with anti-infection, biodegradable and induced angiogenesis was designed, and to explore its preclinical value in segmental bone defect of tibia. RESULTS: The scaffold exhibited good degradation and drug release performance. In vitro, the scaffold extract promoted osteogenesis by enhancing bone-related gene/protein expression and mineral deposition in BMSCs. It also stimulated endothelial cell migration and promoted angiogenesis through the upregulation of specific genes and proteins associated with cell migration and tube formation. This may be attributed to the activation of the PI3k/AKT/HIF-1α pathway, facilitating the processes of osteogenesis and angiogenesis. Furthermore, the HA/CMCS/PDA/TFRD scaffold was demonstrated to alleviate infection, enhance angiogenesis, promote bone regeneration, and increase the maximum failure force of new formed bone in a rat model of segmental bone defects. CONCLUSION: Porous scaffolds loaded with TFRD can reduce infection, be biodegradable, and induce angiogenesis, presenting a novel approach for addressing tibial segmental bone defects.

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF­BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney­tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co­immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow­derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet­derived growth factor (PDGF)­BB, VEGF, runt­related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription­quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF­BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF­BB. The protein and mRNA levels of PDGF­BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF­BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic­osteogenic coupling to regenerate bone defects during DO via the PDGF­BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Corrigendum: Total Flavonoids of Rhizoma Drynariae Promotes Differentiation of Osteoblasts and Growth of Bone Graft in Induced Membrane Partly by Activating Wnt/ß-Catenin Signaling Pathway.

[This corrects the article DOI: 10.3389/fphar.2021.675470.].

Total Flavonoids of Rhizoma Drynariae Promotes Differentiation of Osteoblasts and Growth of Bone Graft in Induced Membrane Partly by Activating Wnt/ß-Catenin Signaling Pathway.

Total flavonoids of Rhizoma drynariae (TFRD), a Chinese medicine, is widely used in the treatment of fracture, bone defect, osteoporosis and other orthopedic diseases, and has achieved good effects. Purpose of this trial was to explore efficacy of TFRD on bone graft's mineralization and osteoblasts' differentiation in Masquelet induced membrane technique in rats. Forty male Sprague-Dawley rats were randomly divided into high dose group (H-TFRD), middle dose group (M-TFRD), low dose group (L-TFRD) and control group (control). The critical size bone defect model of rats was established with 10 rats in each group. Polymethyl methacrylate (PMMA) spacer was implanted into the defect of right femur in rats. After the formation of the induced membrane, autogenous bone was implanted into the induced membrane. After 12 weeks of bone graft, bone tissues in the area of bone graft were examined by X-ray, Micro-CT, hematoxylin-eosin (HE) and Masson trichrome staining to evaluate the growth of the bone graft. The ß-catenin, c-myc, COL1A1, BMP-2 and OPN in bone graft were quantitatively analyzed by Western blot and Immunohistostaining. Osteoblasts were cultured in the medium containing TFRD. Cell Counting Kit-8 (CCK-8) method, Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining, Western blot, RT-PCR and other methods were used to detect the effects of TFRD on the proliferation of osteoblasts and the regulation of Wnt/ß-catenin signaling pathway. In vivo experiments showed that the growth and mineralization of bone graft in TFRD group was better. Moreover, the expression of Wnt/ß-catenin and osteogenesis-related proteins in bone tissue of TFRD group was more than that in other groups. In vitro experiments indicated that osteoblasts proliferated faster, activity of ALP was higher, number of mineralized nodules and proteins related to osteogenesis were more in TFRD group. But blocking Wnt/ß-catenin signaling pathway could limit these effects. Therefore, TFRD could promote mineralization of bone graft and differentiation of osteoblasts in a dose-dependent manner during growing period of the bone graft of induced membrane technique, which is partly related to the activation of Wnt/ß-catenin signaling pathway.

Exploring the Mechanism of Total Flavonoids of Drynariae Rhizoma to Improve Large Bone Defects by Network Pharmacology and Experimental Assessment.

Drynariae Rhizoma (DR) has been demonstrated to be effective in promoting fracture healing in clinical use. In the study, we tried to predicate potential signaling pathways and active ingredients of DR via network pharmacology, uncover its regulation mechanism to improve large bone defects by in vivo and in vitro experiment. We total discovered 18 potential active ingredients such as flavonoids and 81 corresponding targets, in which mitogen-activated protein kinase (MAPK) signaling pathway has the highest correlation with bone defects in pathway and functional enrichment analysis. Therefore, we hypothesized that flavonoids in DR improve large bone defects by activating MAPK signaling pathway. Animal experiments were carried out and all rats randomly divided into TFDR low, medium, and high dosage group, model group and control group. 12 weeks after treatment, according to X-ray and Micro-CT, TFDR medium dosage group significantly promote new bone mineralization compared with other groups. The results of HE and Masson staining and in vitro ALP level of BMSC also demonstrated the formation of bone matrix and mineralization in the TFDR groups. Also, angiographic imaging suggested that flavonoids in DR promoting angiogenesis in the defect area. Consistently, TFDR significantly enhanced the expression of BMP-2, RUNX-2, VEGF, HIF-1 in large bone defect rats based on ELISA and Real-Time PCR. Overall, we not only discover the active ingredients of DR in this study, but also explained how flavonoids in DR regulating MAPK signaling pathway to improve large bone defects.

Corrigendum: Exploring the Mechanism of Total Flavonoids of Drynariae Rhizoma to Improve Large Bone Defects by Network Pharmacology and Experimental Assessment.

[This corrects the article DOI: 10.3389/fphar.2021.603734.].

Total flavonoids of rhizoma drynariae ameliorates bone formation and mineralization in BMP-Smad signaling pathway induced large tibial defect rats.

Osteogenesis and angiogenesis acts as an essential role in repairing large tibial defects (LTDs). Total flavonoids of rhizoma drynariae (TFRD), a traditional Chinese medicinal herb, is reported to show anabolic effects on fracture healing. However, whether TFRD could improve the bone formation and angiogenesis in LTDs remains unknown. The purpose of this study was to evaluate the effect of TFRD on bone formation and angiogenesis in LTDs in distraction osteogenesis (DO). Using a previously established fracture model, LTD rats was established with circular external fixator (CEF). All rats then randomly divided into TFRD low dosage group (with DO), TFRD medium dosage group (with DO), TFRD high dosage group (with DO), model group (with DO) and blank group (without DO). Twelve weeks after treatment, according to X-ray and Micro-CT, TFRD groups (especially in medium dosage group) can significantly promote the formation of a large number of epiphyses and improve new bone mineralization compared with model group, and the results of HE and Masson staining and in vitro ALP level of BMSC also demonstrated the formation of bone matrix and mineralization in the TFRD groups. Also, angiographic imaging suggested that total flavonoids of TFRD was able to promote angiogenesis in the defect area. Consistently, TFRD significantly increased the levels of BMP-2, SMAD1, SMAD4, RUNX-2, OSX and VEGF in LTD rats based on ELISA and Real-Time PCR. In addition, we found that ALP activity of TFRD medium dosage group reached a peak after 10 days of induction through BMSC cell culture in vitro experiment. TFRD promoted bone formation in LTD through activation of BMP-Smad signaling pathway, which provides a promising new strategy for repairing bone defects in DO surgeries.

Total Flavonoids of Rhizoma Drynariae Enhances Angiogenic-Osteogenic Coupling During Distraction Osteogenesis by Promoting Type H Vessel Formation Through PDGF-BB/PDGFR-ß Instead of HIF-1α/ VEGF Axis.

Background: Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney-tonifying traditional Chinese medicine Rhizoma Rrynariae, has been proved to be effective in treating osteoporosis, bone fractures and defects. However, pharmacological effects of TFRD on type H vessels, angiogenic-osteogenic coupling in distraction osteogenesis (DO) and the mechanism remain unclear. This study aims at investigating whether type H vessels exist in the DO model, effects of TFRD on angiogenic-osteogenic coupling and further elucidating the underlying mechanism. Methods: Rats models of DO and bone fracture (FR) were established, and then were separately divided into TFRD and control subgroups. Imageological and histological analyses were performed to assess bone and vessel formation. Immunofluorescent staining of CD31 and endomucin (Emcn) was conducted to determine type H vessel formation. Matrigel tube formation, ALP and Alizarin Red S staining assays were performed to test the effects of TFRD on angiogenesis or osteogenesis of endothelial precursor cells (EPCs) or bone marrow-derived mesenchymal stem cells (BMSCs). Additionally, expression levels of HIF-1α, VEGF, PDGF-BB, RUNX2 and OSX were determined by ELISA, qPCR or western blot, respectively. Results: The in vivo results indicated more formed type H vessels in DO groups than in FR groups and TFRD obviously increased the abundance of type H vessels. Moreover, groups with higher abundance of type H vessels showed better angiogenesis and osteogenesis outcomes. Further in vitro experiments showed that TFRD significantly promoted while blocking PDGF-BB remarkably suppressed the angiogenic activity of EPCs under stress conditions. The levels of p-AKT and p-ERK1/2, downstream mediators of the PDGF-BB pathway, were up-regulated by TFRD but blocked by function blocking anti-PDGF-BB antibody. In contrast, the activated AKT and ERK1/2 and corresponding tube formation were not affected by the HIF-1α inhibitor. Besides, blocking PDGF-BB inhibited the osteogenic differentiation of the stretched BMSCs, but TFRD enhanced the osteogenic activity of BMSCs and ameliorated the inhibition, with more calcium nodes, higher ALP activity and mRNA and protein levels of RUNX2 and OSX. Conclusion: Type H vessels exist in the DO model and TFRD enhances angiogenic-osteogenic coupling during DO by promoting type H vessel formation via PDGF-BB/PDGFR-ß instead of HIF-1α/VEGF axis.

Intelligent rehabilitation assistant system to promote the early functional recovery of the elderly patients with femoral neck fracture after hemiarthroplasty (HA): A protocol for a randomized controlled trial.

BACKGROUND: Femoral neck fracture is a common type of hip fracture, which has a high morbidity and mortality. Surgical treatment is the first choice. However, the functional rehabilitation after operation has not been paid enough attention. In addition, the quality of exercise is difficult to quantify, and the rehabilitation is lack of standards. Therefore, the intelligent rehabilitation assistant system which could record exercise details, might be used to evaluate the quality and adherence to the prescribed exercise to this fragile group of patients has great relevance, so as to provide new ideas for postoperative rehabilitation of hip fracture. METHODS: This is an opening, prospective, double-dummy RCT. Fifty femoral neck fractures patients, older than 65 years and are about to hospitalize for HA, will be invited to study. The sample will be divided into monitoring group and control group randomly at a 1:1 ratio. The prescribed exercises need to be done continuously for 2 weeks. The monitoring group needs additional use intelligent rehabilitation assistant system. Each subject will receive a total of 4 follow-up visits at the designated time (2 weeks, 4 weeks, 12 weeks, and 24 weeks). The following factors will be talked as dependent variables:Each subject will receive a total of 4 follow-up visits at the designated time, and the findings will be analyzed statistically considering a 5% significance level (P < .05). DISCUSSION: Exercise under monitor may improve patients compliance and exercise quality, and accelerate the rehabilitation process. This protocol reported in accordance with the CONSORT 2010 checklist and SPIRIT 2013 Checklist. TRIAL REGISTRATION: The trial is registered at Chinese Clinical Trials Registry (ChiCTR2000033213, May 24, 2020).

Epidemiological characteristics of dengue in mainland China from 1990 to 2019: A descriptive analysis.

In the past 30 years, dengue has undergone dramatic changes in China every year. This study explores the epidemiological trend of dengue in China during this period to identify high-risk seasons, regions, ages, susceptible populations, and provide information for dengue prevention and control activities.Dengue data from 1990 to 2019 were derived from the Public Health Science Data Center, Web of Science, China National Knowledge Infrastructure, PubMed, and Centers for Disease Control and Prevention of the corresponding province. GraphPad Prism 7 was conducted to generate disease evolution maps, occupational heat maps, and monthly heat maps of dengue cases and deaths in mainland China and Guangdong Province. Excel 2016 was used to create a cyclone map of age and gender distribution. Powerpoint 2016 was performed to create geographic maps.From 1990 to 2019, the annual number of dengue cases showed an increasing trend and reaching a peak in 2014, with 46,864 dengue cases (incidence rate: 3.4582/100,000), mainly contributed by Guangdong Province (45,189 cases, accounting for 96.43%). Dengue pandemics occurred every 4 to 6 years. The prevalence of dengue fever was Autumn, which was generally prevalent from June to December and reached its peak from September to November. The provinces reporting dengue cases each year have expanded from the southeastern coastal region to the southwest, central, northeast, and northwest regions, and the provinces with a high incidence were Guangdong, Guangxi, Yunnan, Fujian, and Zhejiang. People aged 25 to 44 years were more susceptible to dengue virus infection. And most of them were male patients. Dengue mainly occurs in the following groups: students, business service staffs, workers, farmers, retired staffs, housewives, and the unemployed. Four provinces reported deaths from dengue, namely Guangdong Province, Zhejiang Province, Henan Province, and Hunan Province.The dengue fever epidemic occurred every 4 to 6 years, mostly in autumn. The endemic areas were Guangdong, Guangxi, Yunnan, Fujian, and Zhejiang provinces. People aged 25 to 44 years, men, students, business service staffs, workers, farmers, retired staffs, housewives, and the unemployed were more susceptible to dengue fever. These findings help to develop targeted public health prevention and control measures.

Research hotspots and trends of bone defects based on Web of Science: a bibliometric analysis.

BACKGROUND: Bone defects can be seen everywhere in the clinic, but it is still a challenge for clinicians. Bibliometrics tool CiteSpace is based on the principle of "co-citation analysis theory" to reveal new technologies, hotspots, and trends in the medical field. In this study, CiteSpace was used to perform co-citation analysis on authors, countries (regions) and institutions, journals and cited journals, authors and cited literature, as well as keywords to reveal leaders, cooperative institutions, and research hotspots of bone defects and predict development trends. METHOD: Data related to bone defect from 1994 to 2019 were retrieved from the Web of Science core collection; then, we use Excel to construct an exponential function to predict the number of annual publications; conduct a descriptive analysis on the top 10 journals with the largest number of publications; and perform co-citation analysis on authors, countries (regions) and institutions, journals and cited journals, authors and cited reference, and keywords using CiteSpace V5.5 and use the Burst Detection Algorithm to perform analysis on the countries (regions) and institutions and keywords, as well as cluster the keywords using log-likelihood ratio. RESULTS: A total of 5193 studies were retrieved, and the number of annual publications of bone defects showed an exponential function Y = 1×10- 70e0.0829x (R2 = 0.9778). The high-yield author was Choi Seong-Ho at Yonsei University in South Korea. The high-yielding countries were the USA and Germany, and the high-yielding institutions were the Sao Paulo University and China and the Chinese Academy of Sciences which were the emerging research countries and institutions. The research results were mainly published in the fields of dentistry, bone, and metabolism. Among them, the Journal of Dental Research and Journal of Bone and Mineral Research were high-quality journals that report bone defect research, but the most cited journal was the Clinical Orthopaedics and Related Research. Hot keywords were regeneration, repair, in vitro, bone regeneration, reconstruction, and graft. The keywords that were strongly cited in 2010-2019 were transportation, osteogenic differentiation, proliferation, and biomaterials. After 2018, proliferation, osteogenic differentiation, stromal cells, transmission, and mechanical properties have become new vocabulary. The drug delivery, vascularization, osteogenic differentiation and biomaterial properties of bone defects were expected to be further studied. CONCLUSION: The application of CiteSpace can reveal the leaders, cooperating institutions and research hotspots of bone defects and provide references for new technologies and further research directions.

Comparison between the induced membrane technique and distraction osteogenesis in treating segmental bone defects: An experimental study in a rat model.

Previous studies have suggested that treatment plans for segmental bone defects (SBDs) are affected by the bone defect sizes. If the selected treatment was not the most appropriate, it would not contribute to bone healing, but increase complications. The induced membrane technique (IM) and distraction osteogenesis (DO) have been proved to be effective in treating SBDs. However, the differences between the two in therapeutic effects on SBDs with different sizes are still unclear. Thus, we aimed to observe the effects of IM and DO on different sizes of SBDs and to further determine what method is more appropriate for what defect size. Rat models of 4-, 6-and 8-mm mid-diaphyseal defects using IM and DO techniques were established. X-rays, micro-CT, histological and immunohistochemical examinations were performed to assess bone repair. Faster bone formation rate, shorter treatment duration, higher expressions of OPN and OCN and higher parameters of bone properties including bone mineral density (BMD), bone volume/total tissue volume (BV/TV), mineral apposition rate (MAR) and mineral surface/bone surface (MS/BS) were found in 4-mm SBDs treated with DO than in those with IM treatment. However, the results were reversed and IM outperformed DO in bone repair capacity for 8-mm SBDs, while no significant difference emerges in the case of 6-mm SBDs. This study suggests that the therapeutic effects of IM and DO may be subjected to sizes of bone defects and the best treatment size of defects is different between the two. For small-sized SBDs, DO may be more suitable and efficient than IM, but IM has advantages over DO for over-sized SBDs, while DO and IM show similar bone repair capability in moderate-sized SBDs, which would offer a new insight into how to choose DO and IM for SBDs in clinical practice and provide references for further clinical research.