Biomineralized tetramethylpyrazine-loaded PCL/gelatin nanofibrous membrane promotes vascularization and bone regeneration of rat cranium defects.

Conventional electrospinning produces nanofibers with smooth surfaces that limit biomineralization ability. To overcome this disadvantage, we fabricated a tetramethylpyrazine (TMP)-loaded matrix-mimicking biomineralization in PCL/Gelatin composite electrospun membranes with bubble-shaped nanofibrous structures. PCL/Gelatin membranes (PG), PCL/Gelatin membranes containing biomineralized hydroxyapatite (HA) (PGH), and PCL/Gelatin membranes containing biomineralized HA and loaded TMP (PGHT) were tested. In vitro results indicated that the bubble-shaped nanofibrous surface increased the surface roughness of the nanofibers and promoted mineralization. Furthermore, sustained-release TMP had an excellent drug release efficiency. Initially released vigorously, it reached stabilization at day 7, and the slow-release rate stabilized at 61.0 ± 1.8% at 28 days. All membranes revealed an intact cytoskeleton, cell viability, and superior adhesion and proliferation when stained with Ghost Pen Cyclic Peptide, CCK-8, cell adhesion, and EdU. In PGHT membranes, the osteogenic and vascularized gene expression of BMSCs and human vascular endothelial cells was significantly upregulated compared with that in other groups, indicating the PGHT membranes exhibited an effective vascularization role. Subsequently, the membranes were implanted in a rat cranium defect model for 4 and 8 weeks. Micro-CT and histological analysis results showed that the PGHT membranes had better bone regenerative patterns. Additionally, the levels of CD31 and VEGF significantly increased in the PGHT membrane compared with those in other membranes. Thus, PGHT membranes could accelerate the repair of cranium defects in vivo via HA and TMP synergistic effects.

Polarization of macrophages in the trigeminal ganglion of rats with pulpitis.

BACKGROUND: Dental pulp tissues are rich in pain-related afferent nerve fibers, which originate from primary sensory neurons in the trigeminal ganglion (TG). The mechanisms of central nervous system (CNS) underlying ectopic pain following peripheral inflammation have been reported that the macrophages as inflammatory and immunologic mediators in the TG play an important role in the process of pulpitis and hyperalgesia. OBJECTIVE(S): To observe the polarization response and dynamic distribution of macrophages in the TG during the development of dental pulp inflammation. METHODS: A rat model of pulpitis was established using complete Freund's adjuvant (CFA). Hematoxylin-eosin (HE), immunohistochemistry (IHC), immunofluorescence (IF), toluidine blue (TB) staining, and RT-qPCR were performed to observe the expression of macrophage-related factors in the TG. RESULTS: The results of IHC staining showed that M2 macrophages labeled with CD206 were observed in the TG of both the control and CFA groups. The statistical analysis indicated that the number of CD206-positive macrophages in the TG increased significantly at 24 h after CFA-induced pulpitis, reached a peak at 2 weeks, and then returned to the normal level after 6 weeks. The ratio of M2/M1 in the CFA groups was significantly lower than that in the control group from 24 to 72 h, and this pattern was reversed at 2 weeks after CFA-induced pulpitis; then, the ratio increased significantly and was maintained at a high level for 4 weeks. RT-qPCR results showed that the expression of IL-10 in the TG increased significantly from 1 to 4 weeks after CFA-induced pulpitis. CONCLUSION: The trend of M2 macrophages was opposite to that of M1 macrophages in the TG during the process of pulpitis induced by CFA, which is consistent with the expression of macrophage-related cytokines. Macrophage polarization in the TG may participate in the neuroinflammation response induced by dental pulpitis.

Coaxial Electrospun Polycaprolactone/Gelatin Nanofiber Membrane Loaded with Salidroside and Cryptotanshinone Synergistically Promotes Vascularization and Osteogenesis.

Background: Salidroside (SAL) is the most effective component of Rhodiola rosea, a traditional Chinese medicine. Cryptotanshinone (CT) is the main fat-soluble extract of Salvia miltiorrhiza, exhibiting considerable potential for application in osteogenesis. Herein, a polycaprolactone/gelatin nanofiber membrane loaded with CT and SAL (PSGC membrane) was successfully fabricated via coaxial electrospinning and characterized. Methods and Results: This membrane capable of sustained and controlled drug release was employed in this study. Co-culturing the membrane with bone marrow mesenchymal stem cells and human umbilical vein endothelial cells revealed excellent biocompatibility and demonstrated osteogenic and angiogenic capabilities. Furthermore, drug release from the PSGC membrane activated the Wnt/ß-catenin signaling pathway and promoted osteogenic differentiation and vascularization. Evaluation of the membrane's vascularization and osteogenic capacities involved transplantation onto a rat's subcutaneous area and assessing rat cranium defects for bone regeneration, respectively. Microcomputed tomography, histological tests, immunohistochemistry, and immunofluorescence staining confirmed the membrane's outstanding angiogenic capacity two weeks post-operation, with a higher incidence of osteogenesis observed in rat cranial defects eight weeks post-surgery. Conclusion: Overall, the SAL- and CT-loaded coaxial electrospun nanofiber membrane synergistically enhances bone repair and regeneration.

Fabrication of a three-dimensional printed gelatin/sodium alginate/nano-attapulgite composite polymer scaffold loaded with leonurine hydrochloride and its effects on osteogenesis and vascularization.

Bone tissue engineering scaffolds have made significant progress in treating bone defects in recent decades. However, the lack of a vascular network within the scaffold limits bone formation after implantation in vivo. Recent research suggests that leonurine hydrochloride (LH) can promote healing in full-thickness cutaneous wounds by increasing vessel formation and collagen deposition. Gelatin and Sodium Alginate are both polymers. ATP is a magnesium silicate chain mineral. In this study, a Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel was used as the base material first, and the Gelatin/Sodium Alginate/Nano-Attapulgite composite polymer scaffold loaded with LH was then created using 3D printing technology. Finally, LH was grafted onto the base material by an amide reaction to construct a scaffold loaded with LH to achieve long-term LH release. When compared to pure polymer scaffolds, in vitro results showed that LH-loaded scaffolds promoted the differentiation of BMSCs into osteoblasts, as evidenced by increased expression of osteogenic key genes. The results of in vivo tissue staining revealed that the drug-loaded scaffold promoted both angiogenesis and bone formation. Collectively, these findings suggest that LH-loaded Gelatin/Sodium Alginate/Nano-Attapulgite composite hydrogel scaffolds are a potential therapeutic strategy and can assist bone regeneration.

Polycaprolactone/Gelatin/Hydroxyapatite Electrospun Nanomembrane Materials Incorporated with Different Proportions of Attapulgite Synergistically Promote Bone Formation.

Purpose: To enhance the osteoinductive effect of Hydroxyapatite (HA) in bone tissue engineering, this study manufactured polycaprolactone (PCL)/gelatin (GEL)/HA nanofibrous scaffolds incorporated with different ratios of attapulgite (ATP): HA (0:3, 0:0, 1:1, 2:1 and 3:0) by high-voltage electrospinning. The synergistic effect exerted by ATP and HA on bone formation was explored both in vivo and in vitro. Methods and Results: First, we determined the group composition and crystal structure of the nanosheets by Fourier transform infrared (FTIR) and X-ray diffraction (XRD) analyses. Then, the physical properties of the scaffolds, including the modulus of elasticity, porosity and water absorption were evaluated. Moreover, the surface microstructure of the nanofibrous scaffolds was captured by Scanning electron microscopy (SEM) and Transmission Electron Microscope (TEM). The biocompatibility of the fabricated scaffolds represented by cell counting kit 8 (CCK-8) and phalloidin staining was also assessed. Next, in vitro osteogenesis was evaluated. Real-time PCR, alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining results showed that the materials incorporated with HA and ATP at a ratio of 2:1 synergistically promoted more osteoblastic differentiation and extracellular mineralization than scaffolds doped with HA and ATP alone. Last, in vivo, Hematoxylin-Eosin staining (HE staining) and Masson staining showed that groups treated with HA and ATP acquired optimal patterns of bone regeneration. Conclusion: This study clarified for the first time that the combination of HA and ATP orchestrated biomaterial-induced osseointegration, and the synergistic effect was more significant when the ratio of ATP/HA was 2:1. This conclusion also provides new ideas and a scientific basis for the development of functionalized nanomaterials in bone tissue engineering.

[Experimental study on the construction of telmisartan/collagen/polycaprolactone nerve conduit and its repair effect on rat sciatic nerve defect].

Objective: To construction the telmisartan/collagen/polycaprolactone (PCL) nerve conduit and assess its effect on repairing sciatic nerve defect in rats. Methods: The 60% collagen/hexafluoroisopropanol (HFIP) solution and 40% PCL/HFIP solution were prepared and mixed (collagen/PCL solution). Then the 0, 5, 10, and 20 mg of telmisartan were mixed with the 10 mL collagen/PCL solution, respectively. Telmisartan/collagen/PCL nerve conduits were fabricated via high voltage electrospinning technology. The structure of nerve conduit before and after crosslinking was observed by using scanning electron microscope (SEM). The drug release efficiency was detected by in vitro sustained release method. RAW264.7 cells were cultured with lipopolysaccharide to induce inflammation, and then co-cultured with nerve conduits loaded with different concentrations of telmisartan for 24 hours. The mRNA expressions of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg-1) were detected by using real-time fluorescence quantitative PCR. Forty adult Wistar rats were randomly divided into 4 groups ( n=10). After preparing 15-mm-long sciatic nerve defect, the defect was repaired by cross-linked nerve conduits loaded with 0, 5, 10, and 20 mg telmisartan in groups A, B, C, and D, respectively. After operation, the general condition of rats was observed after operation; the sciatic function index (SFI) was tested; the bridging between the nerve conduit and sciatic nerve, and the integrity of nerve conduit were observed; the tissue growth in nerve conduit and material degradation were observed by HE staining; the expressions of CD86 (M1 macrophage marker), CD206 (M2 macrophage marker), myelin basic protein (MBP), and myelin protein 0 (P0) in new tissues were also observed by immunohistochemical staining; the expressions of neurofilament 200 (NF-200) and S-100ß in new tissues were assessed by immunofluorescence staining. Results: The general observation showed that the inner diameter of the nerve conduit was 1.8 mm and the outer diameter was 2.0 mm. After cross-linking by genipin, the nanofiber became thicker and denser. The drug release test showed that the telmisartan loaded nerve conduit could be released gradually. With the increase of telmisartan content in nerve conduit, the iNOS mRNA expression decreased and the Arg-1 mRNA expression increased; and the differences between 20 mg group and other groups were significant ( P<0.05). In vivo experiment showed that all animals in each group survived until the completion of the experiment. The SFI was significantly higher in groups C and D than in groups A and B at different time points ( P<0.05) and in group D than in group C at 6 months after operation ( P<0.05). HE staining showed that there were significantly more new tissues in the middle of the nerve conduit in group D after operation than in other groups. Immunohistochemical staining showed that CD86 and CD206 stainings were positive in each group at 1 month after operation, among which group D had the lowest positive rate of CD86 and the highest positive rate of CD206, and there were significant differences in the positive rate of CD206 between group D and groups A, B, and C ( P<0.05); the MBP and P0 stainings were positive in groups C and D at 6 months, and the positive rate in group D was significantly higher than that in group C ( P<0.05). Immunofluorescence staining showed that the NF-200 and S-100ß expressions in group D were significantly higher than those in other groups. Conclusion: Telmisartan/collagen/PLC nerve conduit can promote the sciatic nerve defect repair in rats through promoting the polarization of M1 macrophages to M2 macrophages, and the nerve conduit loaded with20 mg telmisartan has the most significant effect.

An Optical Smartphone-Based Inspection Platform for Identification of Diseased Orchids.

Infections of orchids by the Odontoglossum ringspot virus or Cymbidium mosaic virus cause orchid disfiguration and are a substantial source of economic loss for orchid farms. Although immunoassays can identify these infections, immunoassays are expensive, time consuming, and labor consuming and limited to sampling-based testing methods. This study proposes a noncontact inspection platform that uses a spectrometer and Android smartphone. When orchid leaves are illuminated with a handheld optical probe, the Android app based on the Internet of Things and artificial intelligence can display the measured florescence spectrum and determine the infection status within 3 s by using an algorithm hosted on a remote server. The algorithm was trained on optical data and the results of polymerase chain reaction assays. The testing accuracy of the algorithm was 89%. The area under the receiver operating characteristic curve was 91%; thus, the platform with the algorithm was accurate and convenient for infection screening in orchids.

Epidemiological Features of Hand, Foot and Mouth Disease Outbreaks among Chinese Preschool Children: A Meta-analysis.

BACKGROUND: Hand-foot-mouth disease (HFMD) is a widespread communicable disease and has caused large epidemics in many countries. This meta-analysis aimed to analyze and evaluate the epidemiological features of HFMD outbreaks in Chinese preschools. METHODS: Literature review was based on PubMed, Chinese National Knowledge Infrastructure (CNKI) and Wanfang databases from 2008 to 2015. The temporal, spatial and demographic parameters were summarized and analyzed. RESULTS: Overall, 19 studies with a total of 11269 HFMD cases were selected for data synthesis and analysis. April, May, June and July were detected as the peak months of HFMD outbreaks, with the pooled rate of 21% (95% CI: 12%-34%), 23% (95% CI: 19%-27%), 20% (95% CI: 17%-24%) and 11% (95% CI: 7%-15%). Urban areas were at a higher risk of suffering from HFMD outbreaks than rural areas, with the pooled rate of 65% (95% CI: 48%-78%) and 35% (95% CI: 22%-52%) respectively. The constituent ratio of children aged 37-48 months is the highest, accounting for 46% (95% CI: 39%-53%) of the total cases during HFMD outbreaks. The pooled rate of male cases (60%) was higher than that of female cases (40%). CONCLUSION: Month, residence, age, and gender may be early risk factors for potential HFMD outbreaks. Before the advent of peak months from Apr to Jul each year, measures should be taken to prevent the HFMD outbreaks among preschool children in China. Preschools located in urban areas should take priority over special prevention. HFMD surveillance should preferentially focus on children aged 37-48 months, especially boys in preschools.

Reconstruction of lymph vessel by lymphatic endothelial cells combined with polyglycolic acid scaffolds: a pilot study.

Restoration of lymphatic drainage using lymph vessels or tissue grafting is becoming an efficient method for alleviating obstructive lymphedema. However, the lack of ideal lymphatic grafts is the key problem that limits the application of lymphatic transplantation, but now that may be resolved with tissue-engineered lymph vessels. In this study, the feasibility of reconstructing lymph vessels was explored using lymphatic endothelial cells (LECs) combined with polyglycolic acid (PGA) scaffolds. The highly purified human dermal LECs can be isolated from human dermis by immunomagnetic bead sorting and multiplied in culture. The viability and growth potential of subcultured LECs make it possible to obtain large amount of cells in vitro. Light and scanning electron microscopy (SEM) showed that the prefabricated PGA scaffolds, with three-dimensional structure, can support cell adhesion, growth and spreading. The constructs formed with LECs combined with PGA scaffolds were cultured in vitro for 10 days and then implanted subcutaneously into nude mice. Six weeks after implantation, the portions of implanted tubules were harvested. Gross and histological observation demonstrated that the tubular structure still remained in the experimental groups but not in the control groups. Immunohistochemical staining and RT-PCR assay of the implanted vessels revealed positive staining in experimental groups for the lymphatic specific markers Podoplanin, VEGFR-3 and LYVE-1. The results indicate that LECs can serve as seed cells and be successfully combined with PGA scaffolds, and the tissue-engineered tubular structure using implanted LECs-PGA compounds showed preliminary characteristics of lymph vessels. A gap between the nearly normal or functional lymph vessel still exists as we have only the endothelial cell-lined duct, but this study demonstrates that it is feasible to construct tissue-engineered lymph vessels using LECs combined with a biodegradable material.