Fabrication and Characterization of Zn Particle Incorporated Fibrous Scaffolds for Potential Application in Tissue Healing and Regeneration.

Zinc (Zn) metal and its alloys have received a lot of interest in biomedical applications due to their biodegradability, biocompatibility, antimicrobial activity, and ability to stimulate tissue regeneration. Bulk Zn has been successfully utilized in a variety of implant applications, most notably as bioabsorbable cardiac stents and orthopedic fixation devices, where it provides adequate mechanical properties while also releasing helpful Zn ions (Zn2+) during degradation. Such beneficial ions are dose-dependent and, when released in excess, can induce cellular toxicity. In this study, we hypothesize that embedding Zn metal particles into a polymer nanofibrous scaffold will enable control of the degradation and time release of the Zn2+. We designed and fabricated two polymer scaffolds, polycaprolactone (PCL) and polycaprolactone-chitosan (PCL-CH). Each scaffold had an increasing amount of Zn. Several physicochemical properties such as fiber morphology, crystallinity, mechanical strength, hydrophilicity, degradation and release of Zn2+, thermal properties, chemical compositions, and so forth were characterized and compared with the PCL fibrous scaffold. The biological properties of the scaffolds were evaluated in vitro utilizing direct and indirect cytotoxicity assays and cell viability. All the data show that the addition of Zn changed various physical properties of the PCL and PCL-CH scaffolds except their chemical structure. Further investigation reveals that the PCL-CH scaffolds degrade the Zn particles relatively faster than the PCL because the presence of the hydrophilic CH influences the faster release of Zn2+ in cell culture conditions as compared to the PCL fibrous scaffold. The combined advantages of CH and Zn in the PCL scaffold enriched 3T3 fibroblast cells' survival and proliferation except the ones with the higher concentration of Zn particles. These new composite scaffolds are promising and can be further considered for tissue healing and regeneration applications.

Chitin Nanofibrils Enabled Core-Shell Microcapsules of Alginate Hydrogel.

An engineered 3D architectural network of the biopolymeric hydrogel can mimic the native cell environment that promotes cell infiltration and growth. Among several bio-fabricated hydrogel structures, core-shell microcapsules inherit the potential of cell encapsulation to ensure the growth and transport of cells and cell metabolites. Herein, a co-axial electrostatic encapsulation strategy is used to create and encapsulate the cells into chitin nanofibrils integrated alginate hydrogel microcapsules. Three parameters that are critical in the electrostatic encapsulation process, hydrogel composition, flow rate, and voltage were optimized. The physicochemical characterization including structure, size, and stability of the core-shell microcapsules was analyzed by scanning electron microscope (SEM), FTIR, and mechanical tests. The cellular responses of the core-shell microcapsules were evaluated through in vitro cell studies by encapsulating NIH/3T3 fibroblast cells. Notably, the bioactive microcapsule showed that the cell viability was found excellent for more than 2 weeks. Thus, the results of this core-shell microcapsule showed a promising approach to creating 3D hydrogel networks suitable for different biomedical applications such as in vitro tissue models for toxicity studies, wound healing, and tissue repair.

L-cysteine aided polyaniline capped SrO2 nanoceramics: Assessment of MC3T3-E1-arbitrated osteogenesis and anti-bactericidal efficacy on the polyurethane 2D nanofibrous substrate.

Fabricating bioartificial bone graft ceramics retaining structural, mechanical, and bone induction properties akin to those of native stem-cell niches is a major challenge in the field of bone tissue engineering and regenerative medicine. Moreover, the developed materials are susceptible to microbial invasion leading to biomaterial-centered infections which might limit their clinical translation. Here, we successfully developed biomimetic porous scaffolds of polyurethane-reinforcedL-cysteine-anchored polyaniline capped strontium oxide nanoparticles to improve the scaffold's biocompatibility, osteo-regeneration, mechanical, and antibacterial properties. The engineered nanocomposite substrate PU/L-Cyst-SrO2 @PANI (0.4 wt%) significantly promotes bone repair and regeneration by modulating osteolysis and osteogenesis. ALP activity, collagen-I, ARS staining, as well as biomineralization of MC3T3-E1 cells, were used to assess the biocompatibility and cytocompatibility of the developed scaffolds in vitro, confirming that the scaffold provided a favorable microenvironment with a prominent effect on cell growth, proliferation, and differentiation. Furthermore, osteogenic protein markers were studied using qRT-PCR with expression levels of runt-related transcription factor 2 (RUNX2), secreted phosphoprotein 1 (Spp-I), and collagen type I (Col-I). The overall results suggest that PU/L-Cyst-SrO2 @PANI (0.4 wt%) scaffolds showed superior interfacial biocompatibility, antibacterial properties, load-bearing ability, and osteoinductivity as compared to pristine PU. Thus, prepared bioactive nanocomposite scaffolds perform as a promising biomaterial substrate for bone tissue regeneration.

A bimetallic load-bearing bioceramics of TiO2 @ ZrO2 integrated polycaprolactone fibrous tissue construct exhibits anti bactericidal effect and induces osteogenesis in MC3T3-E1 cells.

Bioactive mesoporous binary metal oxide nanoparticles allied with polymeric scaffolds can mimic natural extracellular matrix because of their self-mineralized functional matrix. Herein, we developed fibrous scaffolds of polycaprolactone (PCL) integrating well-dispersed TiO2@ZrO2 nanoparticles (NPs) via electrospinning for a tissue engineering approach. The scaffold with 0.1 wt% of bioceramic (TiO2@ZrO2) shows synergistic effects on physicochemical and bioactivity suited to stem cell attachment/proliferation. The bioceramics-based scaffold shows excellent antibacterial activity that can prevent implant-associated infections. In addition, the TiO2@ZrO2 in scaffold serves as a stem cell microenvironment to accelerate cell-to-cell interactions, including cell growth, morphology/orientation, differentiation, and regeneration. The NPs in PCL exert superior biocompatibility on MC3T3-E1 cells inducing osteogenic differentiation. The ALP activity and ARS staining confirm the upregulation of bone-related proteins and minerals suggesting the scaffolds exhibit osteoinductive abilities and contribute to bone cell regeneration. Based on this result, the bimetallic oxide could become a novel bone ceramic tailor TiO2@ZrO2 composite tissue-construct and keep potential nanomaterials-based scaffold for bone tissue engineering strategy.

Biomimetic Cell-Substrate of Chitosan-Cross-linked Polyaniline Patterning on TiO2 Nanotubes Enables hBM-MSCs to Differentiate the Osteoblast Cell Type.

Titanium-based substrates are widely used in orthopedic treatments and hard tissue engineering. However, many of these titanium (Ti) substrates fail to interact properly between the cell-to-implant interface, which can lead to loosening and dislocation from the implant site. As a result, scaffold implant-associated complications and the need for multiple surgeries lead to an increased clinical burden. To address these challenges, we engineered osteoconductive and osteoinductive biosubstrates of chitosan (CS)-cross-linked polyaniline (PANI) nanonets coated on titanium nanotubes (TiO2NTs) in an attempt to mimic bone tissue's major extracellular matrix. Inspired by the architectural and tunable mechanical properties of such tissue, the TiO2NTs-PANI@CS-based biofilm conferred strong anticorrosion, the ability to nucleate hydroxyapatite nanoparticles, and excellent biocompatibility with human bone marrow-derived mesenchymal stem cells (hBM-MSCs). An in vitro study showed that the substrate-supported cell activities induced greater cell proliferation and differentiation compared to cell-TiO2NTs alone. Notably, the bone-related genes (collagen-I, OPN, OCN, and RUNX 2) were highly expressed within TiO2NTs-PANI@CS over a period of 14 days, indicating greater bone cell differentiation. These findings demonstrate that the in vitro functionality of the cells on the osteoinductive-like platform of TiO2NTs-PANI@CS improves the efficiency for osteoblastic cell regeneration and that the substrate potentially has utility in bone tissue engineering applications.

Engineering 2D approaches fibrous platform incorporating turmeric and polyaniline nanoparticles to predict the expression of ßIII-Tubulin and TREK-1 through qRT-PCR to detect neuronal differentiation of PC12 cells.

The bioengineering electroactive construct of a nerve-guided conduit for repairing and restoring injured nerves is an exciting biomedical endeavor that has implications for the treatment of peripheral nerve injury. In this study, we report the development the polycaprolactone (PCL) nanofibrous substrate consisting of turmeric (TUR) and polyaniline nanoparticles (PANINPs) exhibits topological and biological features that mimics the natural extracellular matrix (ECM) for nerve cells. We evaluated the morphology of 2-dimensional (2D) fibrous substrates, and their ability of stem cell adhesion, growth and proliferation rate were influenced by use of various concentrations of turmeric in PCL-TUR substrates. The results showed that 0.62 wt% of TUR and 0.28 wt% of PANINPs in PCL nanofibers substrate exhibited the optimal cellular microenvironment to accelerate PC12 cellular activities. The in vitro experiments revealed that PCL-TUR@PANI substrates significantly stimulated the proliferation, differentiation, and spontaneous outgrowth and extension of neurites from the cells. The substrate has the capacity to respond directly to neuronal markers with significant upregulation of ßIII-Tubulin and TREK-1 through myelination, and also trigger neurotrophic protein expression, which was confirmed via immunocytochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. This study provides a new technique to design substrate of nerve tissue-specific microenvironment for peripheral nerve cell regeneration and could offer promising biomaterials for in vivo peripheral nerve repair.

A Comparative Study of Raja Isteri Pengiran Anak Saleha and Alvarado Scores to Diagnose Acute Appendicitis.

BACKGROUND: Acute Appendicitis is a clinical diagnosis with atypical presentation in young, elderly, females, genitourinary and gynecological conditions. Delayed appendectomy increases the risk of appendicular perforation, sepsis morbidity and mortality. Literature reports as high as 20-40% negative appendectomy. Raja Isteri Pengiran Anak Saleha score has come with higher sensitivity and diagnostic accuracy than Alvarado score in Asian population. This study aims to compare RIPASA and Alvarado score for diagnostic accuracy. METHODS: Appendectomy patients at Patan Hospital from April to September 2014 were compared on raja isteri pengiran anak saleha (cut-off value 7.5 out of 15) and Alvarado score (cut-off value 7 out of 10). Final diagnosis was histopathology based. Microsoft Excel and SPSS 17 were used for analysing sensitivity, specificity and diagnostic accuracy of both scores. The study included patients who underwent appendectomy with histopathology report and excluded those with conservative management, generalized peritonitis, appendicular lump and abscess. RESULTS: There were 88 appendectomy patients with median age 26 (18.25, 35) years, and male 52 (59.1%). Negative appendectomy was 10 (11.36%). Sensitivity and specificity of Raja Isteri Pengiran Anak Saleha 98.71% and 80.00% respectively, and for Alvarado 52.56% and 70%.The Raja Isteri Pengiran Anak Saleha score had statistically significant sensitivity (p=0.000). Positive Predictive value, Negative Predictive Value and diagnostic accuracy were 97.46%, 88.89% and 96.6% for RIPASA and 93.18%, 15.19% and 54.4% for Alvarado respectively. CONCLUSIONS: The Raja Isteri Pengiran Anak Saleha score had better diagnostic accuracy compared to Alvarado score for diagnosis of Acute Appendicitis.

Para-substituted sulfonic acid-doped protonated emeraldine salt nanobuds: a potent neural interface targeting PC12 cell interactions and promotes neuronal cell differentiation.

Structural parameters, such as metal-like semiconductor and electrochemical properties of functionalized polyaniline, hold great potential especially for the development of the cell-substrate interface due to its ion/electron transfer ability. We report the one-step synthesis of sulfonic acid-doped polyaniline nanobuds (s-PANINbs) with controlled shape/size under various oxidation potentials. The different oxidation states of s-PANINbs are used to investigate the cell-specific platform for the induction of neuronal networks in PC12 cells, including the growth, proliferation, and differentiation of cells. The unique structure of one-dimensional (1-D) s-PANINbs enhances its intrinsic conductive properties, and facilitates the dispersibility and electrochemical activity via covalent bonding with dopants. The protonated emeraldine salt nanobuds of s-PANINbs synthesized at 0.18 V anodic potential demonstrated low resistivity (∼81.18 mΩ) and charge transfer resistance (∼3253 Ω). The most biologically compatible protonated emeraldine salt was used in vitro to induce PC12 cells associated with neurite outgrowth, contributing to the electrophysiology of neuronal cells under an external electrical stimulation. The western blotting analysis and qRT-PCR results show that ß-III Tubulin, synapsin I, and TREK-1 are highly expressed in PC12 cells, confirming their successful differentiation into neural-specific cells. Our approach demonstrates the promising role of the self-standing framework based on the s-PANINbs of the protonated emeraldine salt in peripheral nerve repair for the future in vivo cell-interface.

Trimethoprim-sulfamethoxazole Versus Azithromycin for the Treatment of Undifferentiated Febrile Illness in Nepal: A Double-blind, Randomized, Placebo-controlled Trial.

BACKGROUND: Azithromycin and trimethoprim-sulfamethoxazole (SXT) are widely used to treat undifferentiated febrile illness (UFI). We hypothesized that azithromycin is superior to SXT for UFI treatment, but the drugs are noninferior to each other for culture-confirmed enteric fever treatment. METHODS: We conducted a double-blind, randomized, placebo-controlled trial of azithromycin (20 mg/kg/day) or SXT (trimethoprim 10 mg/kg/day plus sulfamethoxazole 50 mg/kg/day) orally for 7 days for UFI treatment in Nepal. We enrolled patients >2 years and <65 years of age presenting to 2 Kathmandu hospitals with temperature ≥38.0°C for ≥4 days without localizing signs. The primary endpoint was fever clearance time (FCT); secondary endpoints were treatment failure and adverse events. RESULTS: From June 2016 to May 2019, we randomized 326 participants (163 in each arm); 87 (26.7%) had blood culture-confirmed enteric fever. In all participants, the median FCT was 2.7 days (95% confidence interval [CI], 2.6-3.3 days) in the SXT arm and 2.1 days (95% CI, 1.6-3.2 days) in the azithromycin arm (hazard ratio [HR], 1.25 [95% CI, .99-1.58]; P = .059). The HR of treatment failures by 28 days between azithromycin and SXT was 0.62 (95% CI, .37-1.05; P = .073). Planned subgroup analysis showed that azithromycin resulted in faster FCT in those with sterile blood cultures and fewer relapses in culture-confirmed enteric fever. Nausea, vomiting, constipation, and headache were more common in the SXT arm. CONCLUSIONS: Despite similar FCT and treatment failure in the 2 arms, significantly fewer complications and relapses make azithromycin a better choice for empirical treatment of UFI in Nepal. CLINICAL TRIALS REGISTRATION: NCT02773407.

Engineered cellular microenvironments from functionalized multiwalled carbon nanotubes integrating Zein/Chitosan @Polyurethane for bone cell regeneration.

A biomimetic-based approaches, especially with artificial scaffolding, have established great potential to provide tissue regeneration capacity and an effective way to bridge the gap between host cell responses and organ demands. However, the synthesis of biomaterial is most efficient when the functional behavior involved most resembles the natural extracellular matrix. Here, a fibrous scaffold was engineered by integrating zein and chitosan (CS) in to polyurethane (PU) associated with functionalized multiwalled carbon nanotubes (fMWCNTs) as a bone cell repair material. The chitosan-based, tissue-engineered scaffold containing 0.1 mg/mL fMWCNTs shows potent synergistic results where improved biomechanical strength, hydrophilicity and antibacterial efficacy produce a scaffold akin to a truly natural extracellular matrix found in the bone cell microenvironments. The scaffold enables rapid cell-to-cell communication through a bio-interface and greatly promotes the regenerative effect of pre-osteoblast (MC3T3-E1) which is reflected in terms of cell growth, proliferation, and differentiation in our in vitro experiments. Alizarin red staining analysis, alkaline phosphatase activity, and Western blotting also confirm the nucleation of hydroxyapatite (HA) nanocrystals and the expression of osteogenic protein markers, all of which indicate the scaffold's excellent osteoinductive properties. These results suggest that this precisely engineered PU/Zein/CS-fMWCNTs fibrous scaffold possesses suitable biological behavior to act as an artificial bone extracellular matrix that will ensure bone cell regeneration while contributing numerous benefits to the field of artificial bone grafts.

Heterojunction formation between copper(II) oxide nanoparticles and single-walled carbon nanotubes to enhance antibacterial performance.

We delineate the excellent bactericidal efficacy of stable heterojunction nanocomposites composed of single-walled carbon nanotubes (SWCNTs) and copper(II) oxide (CuO) synthesized via facile recrystallization and calcination. The bactericidal effectiveness of the fabricated nanocomposites was examined using the standard broth-dilution method and the growth-inhibition-zone analysis method, in which bacteria cultured in an incubator in tryptic soy broth medium were subjected to the prepared samples. The bactericidal activity of all of the as-synthesized samples is evident in both methods, displaying a substantial decrease in bacterial colonies and resulting in clear inhibition zones, respectively. Among the CuO-SWCNT nanocomposites, the sample subjected to calcination at 500 °C for 5 h was found to exhibit the best performance against Staphylococcus aureus and Escherichia coli, forming inhibition zones 182% and 162% larger than those formed by pure CuO, respectively.

Nanographene enfolded AuNPs sophisticatedly synchronized polycaprolactone based electrospun nanofibre scaffold for peripheral nerve regeneration.

Herein, we report the bioactivity of monodispersed nanosized reduced graphene oxide (RGO) enfolded gold nanoparticles (AuNPs) engineered polycaprolactone (PCL) based electrospun composite scaffolds. The 2D patterns of PCL based nanofibers prepared by the homogenous distribution of RGO-AuNPs exhibited unique topological and biological features such as mechanical properties, porous structure, large surface area, high electrical conductivity, biodegradability, and resemble the natural extracellular matrix (ECM) that supports the adhesion, growth, proliferation, and differentiation of stem cells. The prepared composite nanofibers based scaffolds containing RGO-AuNPs accelerated neuronal cell functions and confirmed that the optimized concentration showed cytocompatibility to PC12 and S42 cells. The 0.0005 wt% loading of RGO-AuNPs on PCL has a huge impact on neurite growth which leads to an almost one-fold increase in neurite length growth. The present study provides a new strategic design of highly efficient scaffolds that have a significant direct impact on cell activity and could be a potential bioimplant for peripheral nerve repair.

Covalent Surface Functionalization of Bovine Serum Albumin to Magnesium Surface to Provide Robust Corrosion Inhibition and Enhance In Vitro Osteo-Inductivity.

Herein, we describe precisely a covalent modification of pure magnesium (Mg) surface and its application to induce in vitro osteogenic differentiation. The new concept of a chemical bonding method is proposed for developing stable chemical bonds on the Mg surface through the serial assembly of bioactive additives that include ascorbic acid (AA) and bovine serum albumin (BSA). We studied both the physicochemical and electrochemical properties using scanning electron microscopy and other techniques to confirm how the covalent bonding of BSA on Mg can, after coating, significantly enhance the chemical stability of the substrate. The modified Mg-OH-AA-BSA exhibits better anti-corrosion behavior with high corrosion potential (Ecorr = -0.96 V) and low corrosion current density (Icorr = 0.2 µA cm-2) as compared to the pure Mg (Ecorr = -1.46 V, Icorr = 10.42 µA cm-2). The outer layer of BSA on Mg protects the fast degradation rate of Mg, which is the consequence of the strong chemicals bonds between amine groups on BSA with carboxylic groups on AA as the possible mechanism of peptide bonds. Collectively, the results suggest that the surface-modified Mg provides a strong bio-interface, and enhances the proliferation and differentiation of pre-osteoblast (MC3T3-E1) cells through a protein-lipid interaction. We therefore conclude that the technique we describe provides a cost-effective and scalable way to generate chemically stable Mg surface that inherits a biological advantage in orthopedic and dental implants in clinical applications.

A conducting neural interface of polyurethane/silk-functionalized multiwall carbon nanotubes with enhanced mechanical strength for neuroregeneration.

A fibrous scaffold, fully assimilating polyurethane (PU) and silk fibroin associated with functionalized multi-walled carbon nanotubes (fMWCNTs) was developed by electrospinning technique. Herein, we engineered the PU/Silk fibroin-fMWCNTs-based biomaterial that shows great promise as electrospun scaffolds for neuronal growth and differentiation, because of its unique mechanical properties, hydrophilicity, and biodegradability, with outstanding biocompatibility in nerve tissue engineering. The morphology and structural properties of the scaffolds were studied using various techniques. In particular, the presence of fMWCNTs enhances the electrical conductivity and plausible absorption of sufficient extracellular matrix (ECM). The in vitro tests revealed that the aligned scaffolds (PU/Silk-fMWCNTs) significantly stimulated the growth and proliferation of Schwann cells (S42), together with the differentiation and spontaneous neurite outgrowth of rat pheochromocytoma (PC12) cells that were particularly guided along the axis of fiber alignment. The conductive PU/Silk-fMWCNTs scaffold significantly improves neural expression in vitro with successful axonal regrowth, which was confirmed by immunocytochemistry and qRT-PCR analysis. Inspired by the comprehensive experimental results, the fMWCNTs-based scaffold affords new insight into nerve-guided conduit design from both conductive and protein rich standpoints, and opens a new perspective on peripheral nerve restoration in preclinical applications.

Globular Shaped Polypyrrole Doped Well-Dispersed Functionalized Multiwall Carbon Nanotubes/Nafion Composite for Enzymatic Glucose Biosensor Application.

Herein, we report preparation of a bio-nanohybrid material of homogenously dispersed functionalized multiwall carbon nanotubes (fMWCNTs) in Nafion (Nf) doped with polypyrrole (PPy) and followed by one-step in situ electrochemical polymerization along with glucose oxidase (GOx) on a platinum (Pt) electrode. The bioengineered Nf-GOx-fMWCNTs-PPy/Pt electrode showed excellent electrocatalytic performance to detect glucose with a high sensitivity (54.2 µAmM-1 cm-2) in linear range of up to 4.1 mM as well as a low detection limit of 5 µM (S/N = 3), response time within 4 s, good selectivity, stability, and practical applicability. It is our hope that the comprehensive results will contribute to design an efficient glucose biosensor with practical prospects for biomedical applications.

In situ synthesis of cylindrical spongy polypyrrole doped protonated graphitic carbon nitride for cholesterol sensing application.

Herein, we demonstrate the exfoliation of bulk graphitic carbon nitrides (g-C3N4) into ultra-thin (~3.4nm) two-dimensional (2D) nanosheets and their functionalization with proton (g-C3N4H+). The layered semiconductor g-C3N4H+ nanosheets were doped with cylindrical spongy shaped polypyrrole (CSPPy-g-C3N4H+) using chemical polymerization method. The as-prepared nanohybrid composite was utilized to fabricate cholesterol biosensors after immobilization of cholesterol oxidase (ChOx) at physiological pH. Large specific surface area and positive charge nature of CSPPy-g-C3N4H+ composite has tendency to generate strong electrostatic attraction with negatively charged ChOx, and as a result they formed stable bionanohybrid composite with high enzyme loading. A detailed electrochemical characterization of as-fabricated biosensor electrode (ChOx-CSPPy-g-C3N4H+/GCE) exhibited high-sensitivity (645.7 µAmM-1 cm-2) in wide-linear range of 0.02-5.0mM, low detection limit (8.0µM), fast response time (~3s), long-term stability, and good selectivity during cholesterol detection. To the best of our knowledge, this novel nanocomposite was utilized for the first time for cholesterol biosensor fabrication that resulted in high sensing performance. Hence, this approach opens a new prospective to utilize CSPPy-g-C3N4H+ composite as cost-effective, biocompatible, eco-friendly, and superior electrocatalytic as well as electroconductive having great application potentials that could pave the ways to explore many other new sensors fabrication and biomedical applications.