Oriented Antibody Coupling to an Antifouling Polymer Using Glycan Remodeling for Biosensing by Particle Motion.

Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.

Sustained Release of Antifungal and Antibacterial Agents from Novel Hybrid Degradable Nanofibers for the Treatment of Polymicrobial Osteomyelitis.

This study aimed to develop a drug delivery system with hybrid biodegradable antifungal and antibacterial agents incorporated into poly lactic-co-glycolic acid (PLGA) nanofibers, facilitating an extended release of fluconazole, vancomycin, and ceftazidime to treat polymicrobial osteomyelitis. The nanofibers were assessed using scanning electron microscopy, tensile testing, water contact angle analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy. The in vitro release of the antimicrobial agents was assessed using an elution method and a high-performance liquid chromatography assay. The in vivo elution pattern of nanofibrous mats was assessed using a rat femoral model. The experimental results demonstrated that the antimicrobial agent-loaded nanofibers released high levels of fluconazole, vancomycin, and ceftazidime for 30 and 56 days in vitro and in vivo, respectively. Histological assays revealed no notable tissue inflammation. Therefore, hybrid biodegradable PLGA nanofibers with a sustainable release of antifungal and antibacterial agents may be employed for the treatment of polymicrobial osteomyelitis.

Clinical T4b Squamous Cell Carcinoma Resection With Hemimandibulectomy and Temporomandibular Joint Reconstruction With Bridging Plate and Modified Dautrey Procedure.

ABSTRACT: Clinical T4b oral squamous cell carcinoma is traditionally considered nonoperable. We present a case of right mandibular squamous cell carcinoma (PT4bN0M0, stage IVB). The tumor had extended to the right mandibular condylar head and masticatory space. The patient received right modified radical neck dissection and radical operation with hemimandibulectomy to remove the right mandibular condyle. Then, we reconstructed the resected portion with an anterior lateral thigh free flap and bridging plate, which was bent to form an artificial condyle and fixed using the modified Dautrey procedure. The patient had stable postoperative occlusion and normal hinge movement of the temporomandibular joint. After postoperative concurrent chemotherapy and radiotherapy, the patient is under regular out patient department follow-up with satisfactory jaw function and oral intake.

Clinical evaluation of two-piece zirconia abutments with bonded titanium inserts for implant-supported restorations.

STATEMENT OF PROBLEM: Information about the long-term evaluation of 2-piece zirconia abutments (TPZAs) with bonded titanium inserts for implant-supported restorations is sparse. PURPOSE: The purpose of this clinical study was to evaluate the clinical outcome of TPZAs with bonded titanium inserts for implant-supported restorations. MATERIAL AND METHODS: This study retrospectively evaluated the records of patients who received implant restorations supported by TPZAs with bonded titanium inserts. The patients were recalled between January 2017 and December 2017 so that the restorations could be examined clinically and radiographically for technical and biological complications. RESULTS: A total of 27 patients with 30 restorations and 42 zirconia abutments (ZAs) were included in the study. The patient records revealed that all implants remained integrated. However, 5 restorations supported by seven ZAs in 4 patients had to be refabricated because of abutment failure before the recall. Six of the failed abutments were in the molar area, with a combination of zirconia and titanium as the connection platform. Additional complications included screw loosening (1 patient), ceramic chipping (1 patient), and peri-implantitis (1 patient). The mean observation period was 6.6 ±1.5 years (range: 3.6 to 9.2 years). CONCLUSIONS: After a mean observation period of 6 years after insertion, TPZAs with bonded titanium inserts appear to be a suitable treatment option in the anterior and premolar regions. However, in the molar area, the use of the same type of ZA without a complete metal-to-metal connection platform to support the restoration led to a high incidence of fracture.

Injectable, Antioxidative, and Tissue-Adhesive Nanocomposite Hydrogel as a Potential Treatment for Inner Retina Injuries.

Reactive oxygen species (ROS) have been recognized as prevalent contributors to the development of inner retinal injuries including optic neuropathies such as glaucoma, non-arteritic anterior ischemic optic neuropathy, traumatic optic neuropathy, and Leber hereditary optic neuropathy, among others. This underscores the pivotal significance of oxidative stress in the damage inflicted upon retinal tissue. To combat ROS-related challenges, this study focuses on creating an injectable and tissue-adhesive hydrogel with tailored antioxidant properties for retinal applications. GelCA, a gelatin-modified hydrogel with photo-crosslinkable and injectable properties, is developed. To enhance its antioxidant capabilities, curcumin-loaded polydopamine nanoparticles (Cur@PDA NPs) are incorporated into the GelCA matrix, resulting in a multifunctional nanocomposite hydrogel referred to as Cur@PDA@GelCA. This hydrogel exhibits excellent biocompatibility in both in vitro and in vivo assessments, along with enhanced tissue adhesion facilitated by NPs in an in vivo model. Importantly, Cur@PDA@GelCA demonstrates the potential to mitigate oxidative stress when administered via intravitreal injection in retinal injury models such as the optic nerve crush model. These findings underscore its promise in advancing retinal tissue engineering and providing an innovative strategy for acute neuroprotection in the context of inner retinal injuries.

Transplantation of embryonic stem cells improves the regeneration of periodontal furcation defects in a porcine model.

OBJECTIVES: Stem cell-based therapy promises to regenerate lost tissue. Embryonic stem (ES) cells are pluripotent and may provide a virtually unlimited source for transplantation. We investigated whether ES cell transplantation improved the regeneration of furcation defects in a porcine model. MATERIAL AND METHODS: Experimental periodontitis was induced in the buccal furcations of the bilateral mandibular 2nd premolars of six minipigs. After 4 weeks, the lesions were surgically debrided and implanted with collagen matrix alone (control site) or collagen matrix overlaid with porcine ES cells expressing green fluorescent protein (pES/GFP(+) ) (test site). After 3 months of healing, the clinical parameters were measured again. The treated teeth with adjacent tissue, and part of the major organs, were processed for GFP immunohistochemistry. RESULTS: We found no obvious teratoma or rejection. The test group had significantly better clinical parameters. Immunohistochemistry (IHC) showed that transplanted pES/GFP(+) cells had differentiated to new periodontal ligament and cementum in the test sites. Surprisingly, GFP(+) cells were also detectable in the repaired control cementum and remote organs. CONCLUSIONS: We conclude that using ES cells to improve the regeneration of periodontal furcation defects is feasible. More studies are required to assess this potential treatment's efficacy and safety.

Alginate/polycaprolactone composite fibers as multifunctional wound dressings.

We developed a composite wound dressing that provides multifunctional wound care. Alginate and polycaprolactone (PCL) were coelectrospun as composite fibers. Highly absorbent alginate provided a moist environment for wounds and PCL increased cell adhesion. Silver nanoparticles embedded in PCL fibers for long-term release inhibited the growth of microorganisms. In addition, plasmid DNA encoding platelet-derived growth factor-B (PDGF-B) were complexed with polyethylenimine (PEI) to form cationic nanoparticles which were then adsorbed on anionic alginate fibers through electrostatic interaction. As wound cells adhered to composite fibers, they were in situ transfected to express PDGF-B continuously. Moreover, calcium ions in alginate fibers were released into the wound site through ion exchange to accelerate hemostasis. Wound healing experiments demonstrated that PDGF-B gene-loaded composite fibers accelerated wound closure and promoted collagen formation. We expect this comprehensive study offers an ideal multifunctional solution to facilitate wound healing.

Click-Coupling to Electrostatically Grafted Polymers Greatly Improves the Stability of a Continuous Monitoring Sensor with Single-Molecule Resolution.

Sensing technologies for the real-time monitoring of biomolecules will allow studies of dynamic changes in biological systems and the development of control strategies based on measured responses. Here, we describe a molecular architecture and coupling process that allow continuous measurements of low-concentration biomolecules over long durations in a sensing technology with single-molecule resolution. The sensor is based on measuring temporal changes of the motion of particles upon binding and unbinding of analyte molecules. The biofunctionalization involves covalent coupling by click chemistry to PLL-g-PEG bottlebrush polymers. The polymer is grafted to a surface by multivalent electrostatic interactions, while the poly(ethylene glycol) suppresses nonspecific binding of biomolecules. With this biofunctionalization strategy, we demonstrate the continuous monitoring of single-stranded DNA and a medically relevant small-molecule analyte (creatinine), in sandwich and competitive assays, in buffer and in filtered blood plasma, with picomolar, nanomolar, and micromolar analyte concentrations, and with continuous sensor operation over 10 h.

Assessment of Antimicrobial Agents, Analgesics, and Epidermal Growth Factors-Embedded Anti-Adhesive Poly(Lactic-Co-Glycolic Acid) Nanofibrous Membranes: In vitro and in vivo Studies.

BACKGROUND: Postoperative tissue adhesion is a major concern for most surgeons and is a nearly unpreventable complication after abdominal or pelvic surgeries. This study explored the use of sandwich-structured antimicrobial agents, analgesics, and human epidermal growth factor (hEGF)-incorporated anti-adhesive poly(lactic-co-glycolic acid) nanofibrous membranes for surgical wounds. MATERIALS AND METHODS: Electrospinning and co-axial electrospinning techniques were utilized in fabricating the membranes. After spinning, the properties of the prepared membranes were assessed. Additionally, high-performance liquid chromatography and enzyme-linked immunosorbent assays were utilized in assessing the in vitro and in vivo liberation profiles of the pharmaceuticals and the hEGF from the membranes. RESULTS: The measured data suggest that the degradable anti-adhesive membranes discharged high levels of vancomycin/ceftazidime, ketorolac, and hEGF in vitro for more than 30, 24, and 27 days, respectively. The in vivo assessment in a rat laparotomy model indicated no adhesion in the peritoneal cavity at 14 days post-operation, demonstrating the anti-adhesive capability of the sandwich-structured nanofibrous membranes. The nanofibers also released effective levels of vancomycin, ceftazidime, and ketorolac for more than 28 days in vivo. Histological examination revealed no adverse effects. CONCLUSION: The outcomes of this study implied that the anti-adhesive nanofibers with sustained release of antimicrobial agents, analgesics, and growth factors might offer postoperative pain relief and infection control, as well as promote postoperative healing of surgical wounds.

Biodegradable Antimicrobial Agent/Analgesic/Bone Morphogenetic Protein-Loaded Nanofibrous Fixators for Bone Fracture Repair.

PURPOSE: Postoperative infection and pain management are of great concern to orthopedic surgeons. Although there are several protocols available to deal with these aspects, they are fraught with complications, such as cartilage damage, cardiovascular and neurological intoxication, and systemic adverse responses. Therefore, it is necessary to develop safe and effective perioperative protocols. In the current study, antimicrobial agents/analgesics/growth factor-embedded biodegradable hybrid fixators (polycaprolactone fixator + poly[lactide-co-glycolide] sheath-core structured nanofibers) for bone fracture repair were designed. METHODS: The biodegradable hybrid fixators were fabricated using solution-extrusion three-dimensional printing and electrospinning. In vitro, the characteristics of the hybrid fixators were examined. Additionally, the release of the incorporated vancomycin, ceftazidime, lidocaine, and bone morphogenetic protein-2 (BMP-2) was evaluated. The in vivo efficacy including drug-eluting properties, fracture repair, and pain management of the biomolecule-loaded nanofibrous fixators was investigated in rabbit rib-fracture models. RESULTS: The nanofibrous fixators released vancomycin, ceftazidime, and lidocaine in a sustained manner under both in vitro and in vivo conditions and protected BMP-2 from burst release. The implantation of these hybrid fixators around the fractured rib significantly improved animal activities and bone union, indicating that the inclusion of analgesic in the fixator effectively reduced postsurgical pain and thereby helped in recovery. CONCLUSION: The novel biomolecule-loaded nanofibrous hybrid fixators resulted in excellent therapeutic outcomes. These fixators may be effective in the repair of rib fractures in clinical settings and may help mitigate surgical complications, such as infection, nonunion, and intolerable postoperative pain.

Tissue concentrations of Fe, Zn, Cu and Mn of four Taiwanese toothed cetaceans.

Concentrations of Fe, Mn, Zn, and Cu in the muscle, lung, liver, and kidney tissues of 49 cetaceans, including 11 Kogia sima (Ks), 10 Lagenodelphis hosei (Lh), 14 Grampus griseus (Gg), and 14 Stenella attenuata (Sa) from 1994 to 2012 in Taiwan were measured. Ks exhibited the highest Fe, and the lowest Zn, Cu and Mn tissue concentrations. The Kogiid and Delphinid groups were significantly categorized by nMDS analysis. Fe muscle concentrations were found to be positively correlated with Ks, Lh, and Gg but not Sa body length. The different levels of Zn-, Cu-, and Mn- tissue concentrations of the two families were due to their different SOD systems. Their calves contained higher Cu liver concentrations. The four essential elements in cetaceans regulate homeostasis to meet their eco-physiological demand. The baseline levels for these four elements in these four tissues in the Delphinid group are defined.

Highly sensitive analysis of cholesterol and sitosterol in foods and human biosamples by liquid chromatography with fluorescence detection.

A simple and sensitive method is described for the quantitative analysis of important animal and plant sterols (cholesterol and sitosterol) by liquid chromatography with fluorimetric detection. The method is based on the derivatization of cholesterol and sitosterol with a fluorescent reagent (naproxen acyl chloride) in toluene. The resulting derivatives were isocratically separated on a C(8) column with a mixed solvent of methanol-isopropanol-water (90:5:5, v/v) as a mobile phase and monitored with a fluorimetric detector (excitation 231 nm and emission 352 nm). The linear range for the quantitation of cholesterol or sitosterol was 0.1-2.0 microM with a detection limit (S/N=3 with 10 microl injected) of about 25 nM. Recoveries of cholesterol spiked in milk (n=5) ranged over 99-104% with relative standard deviations (RSD) less than 6.0%. Application of the method to the analysis of cholesterol or sitosterol in milk, saliva and urine proved simple and feasible.

Antimicrobial activity of Antrodia camphorata extracts against oral bacteria.

Antrodia camphorata (A. camphorata) is a unique, endemic and extremely rare mushroom species native to Taiwan, and both crude extracts of and purified chemical compounds from A. camphorata have been reported to have a variety of significant beneficial effects, such as anti-tumor and anti-inflammatory activity. However, reports on the effects of A. camphorata against dental pathogens have been limited. Oral health is now recognized as important for overall general health, including conditions such as dental caries, periodontal disease and rheumatoid arthritis. Streptococcus mutans (S. mutans) and Porphyromonas gingivalis (P. gingivalis) are the most common bacteria associated with dental plaque and periodontopathic diseases, respectively. Thus, our study examined the ability of five various crude extracts of A. camphorata to inhibit the growth of dental bacteria and anti-adherence in vitro. Among the extracts, the ethanol, ethyl acetate and chloroform extracts exhibited the lowest MICs against P. gingivalis and S. mutans (MIC = 4∼16 µg/mL). The MIC of the aqueous extract was greater than 2048 µg/mL against both P. gingivalis and S. mutans. In vitro adherence of S. mutans was significantly inhibited by the addition of either the ethyl acetate extract or chloroform extract (MIC = 16∼24 µg/mL), while the ethanol extract (MIC = 32∼64 µg/mL) exhibited moderate inhibitory activity. Based on the result of this study, the ethyl acetate and chloroform extracts of A. camphorata may be good candidates for oral hygiene agents to control dental caries and periodontopathic conditions.

Molecular epidemiology of enterovirus 71 infection in the central region of Taiwan from 2002 to 2012.

Enterovirus 71 (EV71), a causative agent of hand, foot, and mouth disease can be classified into three genotypes and many subtypes. The objectives of this study were to conduct a molecular epidemiological study of EV71 in the central region of Taiwan from 2002-2012 and to test the hypothesis that whether the alternative appearance of different EV71 subtypes in Taiwan is due to transmission from neighboring countries or from re-emergence of pre-existing local strains. We selected 174 EV71 isolates and used reverse transcription-polymerase chain reaction to amplify their VP1 region for DNA sequencing. Phylogenetic analyses were conducted using Neighbor-Joining, Maximum Likelihood and Bayesian methods. We found that the major subtypes of EV71 in Taiwan were B4 for 2002 epidemic, C4 for 2004-2005 epidemic, B5 for 2008-2009 epidemic, C4 for 2010 epidemic and B5 for 2011-2012 epidemic. Phylogenetic analysis demonstrated that the 2002 and 2008 epidemics were associated with EV71 from Malaysia and Singapore; while both 2010 and 2011-2012 epidemics originated from different regions of mainland China including Shanghai, Henan, Xiamen and Gong-Dong. Furthermore, minor strains have been identified in each epidemic and some of them were correlated with the subsequent outbreaks. Therefore, the EV71 infection in Taiwan may originate from pre-existing minor strains or from other regions in Asia including mainland China. In addition, 101 EV71 isolates were selected for the detection of new recombinant strains using the nucleotide sequences spanning the VP1-2A-2B region. No new recombinant strain was found. Analysis of clinical manifestations showed that patients infected with C4 had significantly higher rates of pharyngeal vesicles or ulcers than patients infected with B5. This is the first study demonstrating that different EV 71 genotypes may have different clinical manifestations and the association of EV71 infections between Taiwan and mainland China.

Gradient conducting polymer surfaces with netrin-1-conjugation promote axon guidance and neuron transmission of human iPSC-derived retinal ganglion cells.

Major advances have been made in utilizing human-induced pluripotent stem cells (hiPSCs) for regenerative medicine. Nevertheless, the delivery and integration of hiPSCs into target tissues remain significant challenges, particularly in the context of retinal ganglion cell (RGC) restoration. In this study, we introduce a promising avenue for providing directional guidance to regenerated cells in the retina. First, we developed a technique for construction of gradient interfaces based on functionalized conductive polymers, which could be applied with various functionalized ehthylenedioxythiophene (EDOT) monomers. Using a tree-shaped channel encapsulated with a thin PDMS and a specially designed electrochemical chamber, gradient flow generation could be converted into a functionalized-PEDOT gradient film by cyclic voltammetry. The characteristics of the successfully fabricated gradient flow and surface were analyzed using fluorescent labels, time of flight secondary ion mass spectrometry (TOF-SIMS), and X-ray photoelectron spectroscopy (XPS). Remarkably, hiPSC-RGCs seeded on PEDOT exhibited improvements in neurite outgrowth, axon guidance and neuronal electrophysiology measurements. These results suggest that our novel gradient PEDOT may be used with hiPSC-based technologies as a potential biomedical engineering scaffold for functional restoration of RGCs in retinal degenerative diseases and optic neuropathies.