Elucidating osseointegration in vivo in 3D printed scaffolds eliciting different foreign body responses.

Osseointegration between biomaterial and bone is critical for the clinical success of many orthopaedic and dental implants. However, the mechanisms of in vivo interfacial bonding formation and the role of immune cells in this process remain unclear. In this study, we investigated the bone-scaffold material interfaces in two different 3D printed porous scaffolds (polymer/hydroxyapatite and sintered hydroxyapatite) that elicited different levels of foreign body response (FBR). The polymer/hydroxyapatite composite scaffolds elicited more intensive FBR, which was evidenced by more FBR components, such as macrophages/foreign body giant cells and fibrous tissue, surrounding the material surface. Sintered hydroxyapatite scaffolds showed less intensive FBR compared to the composite scaffolds. The interfacial bonding appeared to form via new bone first forming within the pores of the scaffolds followed by growing towards strut surfaces. In contrast, it was previously thought that bone regeneration starts at biomaterial surfaces via osteogenic stem/progenitor cells first attaching to them. The material-bone interface of the less immunogenic hydroxyapatite scaffolds was heterogenous across all samples, evidenced by the coexistence of osseointegration and FBR components. The presence of FBR components appeared to inhibit osseointegration. Where FBR components were present there was no osseointegration. Our results offer new insight on the in vivo formation of bone-material interface, which highlights the importance of minimizing FBR to facilitate osseointegration for the development of better orthopaedic and dental biomaterials.

Negative impact of smoking on lung function: comparing FEV1/FVC values in smokers and non-smokers.

Background: Cigarette smoking has been reported as the significant adverse effects on lung function, which can be evaluated by measuring forced expiratory volume in one second (FEV1), forced vital capacity (FVC), and the ratio of FEV1 to FVC (FEV1/FVC) values. Objective: This study investigated the prevalence of tobacco use and nicotine dependence among college students and their stress levels to inform targeted interventions for smoking prevention and cessation. Material and methods: A total of 429 participants were interviewed face-to-face, of which 71.6% were female and 28.4% were male. Pulmonary function tests were exclusively administered to the 9.8% of participants who reported using tobacco. Results: Our findings revealed a low prevalence of nicotine dependence among college students, with only 6.8% exhibiting moderate or high levels of dependence. Students who reported tobacco use were found to have moderate stress levels,suggesting a potential association between smoking and stress. Chi-square tests revealed that gender, school affiliation,and nicotine dependence were significantly associated with smoking behavior. Long-term smoking (>5 years) was found to be associated with negative health outcomes, such as higher BMI, and increased smoking per day. The analysis of lung function parameters showed that smoking frequency and duration were negatively associated with lung function, while nicotine dependence increased with smoking frequency and duration. Conclusion: Our study suggests that targeted prevention and cessation programs should address these factors to reduce smoking rates among college students.

Inhibition of α-Glucosidase and Pancreatic Lipase Properties of Mitragyna speciosa (Korth.) Havil. (Kratom) Leaves.

Kratom (Mitragyna speciosa (Korth.) Havil.) has been used to reduce blood sugar and lipid profiles in traditional medicine, and mitragynine is a major constituent in kratom leaves. Previous data on the blood sugar and lipid-altering effects of kratom are limited. In this study, phytochemical analyses of mitragynine, 7-hydroxymitragynine, quercetin, and rutin were performed in kratom extracts. The effects on α-glucosidase and pancreatic lipase activities were investigated in kratom extracts and mitragynine. The LC-MS/MS analysis showed that the mitragynine, quercetin, and rutin contents from kratom extracts were different. The ethanol extract exhibited the highest total phenolic content (TPC), total flavonoid content (TFC), and total alkaloid content (TAC). Additionally, compared to methanol and aqueous extracts, the ethanol extract showed the strongest inhibition activity against α-glucosidase and pancreatic lipase. Compared with the anti-diabetic agent acarbose, mitragynine showed the most potent α-glucosidase inhibition, with less potent activity of pancreatic lipase inhibition. Analysis of α-glucosidase and pancreatic lipase kinetics revealed that mitragynine inhibited noncompetitive and competitive effects, respectively. Combining mitragynine with acarbose resulted in a synergistic interaction with α-glucosidase inhibition. These results have established the potential of mitragynine from kratom as a herbal supplement for the treatment and prevention of diabetes mellitus.

Characterisation of bone regeneration in 3D printed ductile PCL/PEG/hydroxyapatite scaffolds with high ceramic microparticle concentrations.

3D printed bioactive glass or bioceramic particle reinforced composite scaffolds for bone tissue engineering currently suffer from low particle concentration (<50 wt%) hence low osteoconductivity. Meanwhile, composites with very high inorganic particle concentrations are very brittle. Scaffolds combining high particle content and ductility are urgently required for bone tissue engineering. Herein, 3D printed PCL/hydroxyapatite (HA) scaffolds with high ceramic concentration (up to 90 wt%) are made ductile (>100% breaking strain) by adding poly(ethylene glycol) which is biocompatible and FDA approved. The scaffolds require no post-printing washing to remove hazardous components. More exposure of HA microparticles on strut surfaces is enabled by incorporating higher HA concentrations. Compared to scaffolds with 72 wt% HA, scaffolds with higher HA content (90 wt%) enhance matrix formation but not new bone volume after 12 weeks implantation in rat calvarial defects. Histological analyses demonstrate that bone regeneration within the 3D printed scaffolds is via intramembranous ossification and starts in the central region of pores. Fibrous tissue that resembles non-union tissue within bone fractures is formed within pores that do not have new bone. The amount of blood vessels is similar between scaffolds with mainly fibrous tissue and those with more bone tissue, suggesting vascularization is not a deciding factor for determining the type of tissues regenerated within the pores of 3D printed scaffolds. Multinucleated immune cells are commonly present in all scaffolds surrounding the struts, suggesting a role of managing inflammation in bone regeneration within 3D printed scaffolds.

Three dimensional printed degradable and conductive polymer scaffolds promote chondrogenic differentiation of chondroprogenitor cells.

Conductive polymers have been used for various biomedical applications including biosensors, tissue engineering and regenerative medicine. However, the poor processability and brittleness of these polymers hinder the fabrication of three-dimensional structures with desirable geometries. Moreover, their application in tissue engineering and regenerative medicine has been so far limited to excitable cells such as neurons and muscle cells. To enable their wider adoption in tissue engineering and regenerative medicine, new materials and formulations that overcome current limitations are required. Herein, a biodegradable conductive block copolymer, tetraaniline-b-polycaprolactone-b-tetraaniline (TPT), is synthesised and 3D printed for the first time into porous scaffolds with defined geometries. Inks are formulated by combining TPT with PCL in solutions which are then directly 3D printed to generate porous scaffolds. TPT and PCL are both biodegradable. The combination of TPT with PCL increases the flexibility of the hybrid material compared to pure TPT, which is critical for applications that need mechanical robustness of the scaffolds. The highest TPT content shows the lowest tensile failure strain. Moreover, the absorption of a cell adhesion-promoting protein (fibronectin) and chondrogenic differentiation of chondroprogenitor cells are found to be dependent on the amount of TPT in the blends. Higher content of TPT in the blends increases both fibronectin adsorption and chondrogenic differentiation, though the highest concentration of TPT in the blends is limited by its solubility in the ink. Despite the contradicting effects of TPT concentration on flexibility and chondrogenic differentiation, a concentration that strikes a balance between the two factors is still available. It is worth noting that the effect on chondrogenic differentiation is found in scaffolds without external electric stimulation. Our work demonstrates the possibility of 3D printing flexible conductive and biodegradable scaffolds and their potential use in cartilage tissue regeneration, and opens up future opportunities in using electric stimulation to control chondrogenesis in these scaffolds.

Structure-activity relationship and in vitro inhibition of human cytochrome CYP2A6 and CYP2A13 by flavonoids.

Cigarette smoking is one of the major risk factors of various diseases including respiratory diseases and lung cancer. While the liver-specific CYP2A6 is associated with the nicotine clearance and smoking addiction, the metabolic activation of the tobacco-specific nitrosamine by lung-specific CYP2A13 can lead to lung tumorigenesis.It has been reported that inhibition of CYP2A6 and CYP2A13 enzymes by flavonoids constituents could be an aids in smoking cessation. This study demonstrates the inhibition activity of kaempferol and myricetin and the structure-function relationship of these two flavonoids and previously isolated flavonoids from Vernonia cinerea and Pluchea indica against both enzymes.Kaempferol could inhibit CYP2A6 with Kic value of 1.77 ± 0.47 µM while inhibit CYP2A13 with Kic value of 0.12 ± 0.01 µM. Myricetin could inhibit CYP2A6 with Kic value of 4.06 ± 0.52 µM while inhibit CYP2A13 with Kic value of 1.88 ± 0.03 µM.Molecular docking indicated that CYP2A13 enzyme has strong hydrophobic interaction with ring B of flavonoids compared to CYP2A6 enzyme. The presence of the hydroxyl group at C3 position of ring C and the hydroxyl group at C5' of ring B affected inhibitory activity on both enzymes.

Spatially-offset Raman spectroscopy for monitoring mineralization of bone tissue engineering scaffolds: feasibility study based on phantom samples.

Using phantom samples, we investigated the feasibility of spatially-offset Raman spectroscopy (SORS) as a tool for monitoring non-invasively the mineralization of bone tissue engineering scaffold in-vivo. The phantom samples consisted of 3D-printed scaffolds of poly-caprolactone (PCL) and hydroxyapatite (HA) blends, with varying concentrations of HA, to mimic the mineralisation process. The scaffolds were covered by a 4 mm layer of skin to simulate the real in-vivo measurement conditions. At a concentration of HA approximately 1/3 that of bone (~0.6 g/cm3), the characteristic Raman band of HA (960 cm-1) was detectable when the PCL:HA layer was located at 4 mm depth within the scaffold (i.e. 8 mm below the skin surface). For the layers of the PCL:HA immediately under the skin (i.e. top of the scaffold), the detection limit of HA was 0.18 g/cm3, which is approximately one order of magnitude lower than that of bone. Similar results were also found for the phantoms simulating uniform and inward gradual mineralisation of the scaffold, indicating the suitability of SORS to detect early stages of mineralisation. Nevertheless, the results also show that the contribution of the materials surrounding the scaffold can be significant and methods for subtraction need to be investigated in the future. In conclusion, these results indicate that spatially-offset Raman spectroscopy is a promising technique for in-vivo longitudinal monitoring scaffold mineralization and bone re-growth.

Three-Dimensional Printed Scaffolds with Controlled Micro-/Nanoporous Surface Topography Direct Chondrogenic and Osteogenic Differentiation of Mesenchymal Stem Cells.

The effect of topography in three-dimensional (3D) printed polymer scaffolds on stem cell differentiation is a significantly underexplored area. Compared to two-dimensional (2D) biomaterials on which various well-defined topographies have been incorporated and shown to direct a range of cell behaviors including adhesion, cytoskeleton organization, and differentiation, incorporating topographical features to 3D polymer scaffolds is challenging due to the difficulty of accessing the inside of a porous scaffold. Only the roughened strut surface has been introduced to 3D printed porous scaffolds. Here, a rapid, single-step 3D printing method to fabricate polymeric scaffolds consisting of microstruts (ca. 60 µm) with micro-/nanosurface pores (0.2-2.4 µm) has been developed based on direct ink writing of an agitated viscous polymer solution. The density, size, and alignment of these pores can be controlled by changing the degree of agitation or the speed of printing. Three-dimensional printed scaffolds with micro-/nanoporous struts enhanced chondrogenic and osteogenic differentiation of mesenchymal stem cells (MSCs) without soluble differentiation factors. The topography also selectively affected adhesion, morphology, and differentiation of MSC to chondrogenic and osteogenic lineages depending on the composition of the differentiation medium. This fabrication method can potentially be used for a wide range of polymers where desirable architecture and topography are required.

Direct three-dimensional printing of polymeric scaffolds with nanofibrous topography.

Three-dimensional (3D) printing is a powerful manufacturing tool for making 3D structures with well-defined architectures for a wide range of applications. The field of tissue engineering has also adopted this technology to fabricate scaffolds for tissue regeneration. The ability to control architecture of scaffolds, e.g. matching anatomical shapes and having defined pore size, has since been improved significantly. However, the material surface of these scaffolds is smooth and does not resemble that found in natural extracellular matrix (ECM), in particular, the nanofibrous morphology of collagen. This natural nanoscale morphology plays a critical role in cell behaviour. Here, we have developed a new approach to directly fabricate polymeric scaffolds with an ECM-like nanofibrous topography and defined architectures using extrusion-based 3D printing. 3D printed tall scaffolds with interconnected pores were created with disparate features spanning from nanometres to centimetres. Our approach removes the need for a sacrificial mould and subsequent mould removal compared to previous methods. Moreover, the nanofibrous topography of the 3D printed scaffolds significantly enhanced protein absorption, cell adhesion and differentiation of human mesenchymal stem cells when compared to those with smooth material surfaces. These 3D printed scaffolds with both defined architectures and nanoscale ECM-mimicking morphologies have potential applications in cartilage and bone regeneration.

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea.

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1-4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5-8 from V. cinerea, and acetylenic thiophenes 9-11 from P. indica that inhibited CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic KI values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32-15.4 and 0.92-8.67 µM, respectively, while those of thiophenes were 0.11-1.01 and 0.67-0.97 µM, respectively.

Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes.

The cytochrome P450 monooxygenases play a major role in insecticide detoxification and become a target for development of insecticide synergists. In this study, a collection of rhinacanthins (rhinacanthin-D, -E, -G, -N, -Q, and -H/I) purified from Rhinacanthus nasutus, in addition to previously purified rhinacanthin-B and -C, were isolated. These compounds displayed various degrees of inhibition against benzyloxyresorufin-O-debenzylation mediated by CYP6AA3 and CYP6P7 which were implicated in pyrethroid resistance in Anopheles minimus malaria vector. Inhibition modes and kinetics were determined for each of rhinacanthins. Cell-based inhibition assays by rhinacanthins employing 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity test were explored their synergistic effects with cypermethrin toxicity on CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells. Rhinacanthin-B, -D, -E, -G, and -N exhibited mechanism-based inhibition against CYP6AA3, an indication of irreversible inhibition, while rhinacanthin-B, -D, -G, and -N were mechanism-based inhibitors of CYP6P7. There was structure-function relationship of these rhinacanthins in inhibition effects against both enzymes. In vitro enzymatic inhibition assays revealed that there were synergistic interactions among rhinacanthins, except rhinacanthin-B and -Q, in inhibition against both enzymes. These rhinacanthins exerted synergism with cypermethrin toxicity on Sf9 cells expressing each of the two P450 enzymes via P450 inhibition and in addition could interact in synergy to further increase cypermethrin toxicity. The inhibition potentials, synergy among rhinacanthins in inhibition against the P450 detoxification enzymes, and synergism with cypermethrin toxicity of the R. nasutus constituents of reported herein could be beneficial to implement effective resistance management of mosquito vector control.

Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.

  Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition. Among the three rhinacanthins, rhinacanthin-B possessed highest inhibitory potency against CYP2A13 with apparent KI and kinact of 0.16 µM and 0.1 min(-1), respectively, while values of 0.44 µM and 0.12 min(-1) were found against CYP2A6. Rhinacanthin-C had least inhibition potency, with apparent KI and kinact of 0.97 µM and 0.07 min(-1) for CYP2A6, respectively, and values of 1.68 µM and 0.05 min(-1) for CYP2A13. Rhinacanthin-A inhibited CYP2A6 and CYP2A13 with apparent KI values of 0.69 and 0.42 µM, respectively and apparent kinact of 0.18 and 0.06 min(-1), respectively. The inhibition of both enzymes by rhinacanthins A-C could not be prevented by addition of trapping agents or reversed by dialysis or potassium ferricyanide. These findings demonstrated that rhinacanthins A-C, which are 1,4-naphthoquinone derivatives, irreversibly inhibited CYP2A6 and CYP2A13 in a mechanism-based inhibition mode.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis.

AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.