Effects of rinsing with arginine bicarbonate and urea solutions on initial enamel lesions in situ.

OBJECTIVE: The aim of this study was to investigate the effects of rinsing with arginine or urea solution on initial enamel lesions in situ. METHODS: Fourteen subjects who wore mandibular removable partial dentures embedded with bovine enamel blocks with artificial enamel lesions were included. The experiment included four 4-week rinsing periods with a 10-day washout period between each rinsing period. In each rinsing period, the subjects rinsed after meal or snack using water, or 2% arginine bicarbonate, or 1% urea, or 0.05% NaF solution, five times daily. The mineralization changes of the enamel lesions were assessed using quantitative light-induced fluorescence. RESULTS: All groups except the water group showed a statistically significant decrease in the fluorescence loss after treatment, compared with their respective baseline. Although both the arginine group and urea group showed more decrease in fluorescence loss than that of the water group, the decrease was not statistically significantly different from that of the water group. The decrease in fluorescence loss of the NaF group was statistically significant than that of the water group, arginine group, and urea group. CONCLUSION: Rinsing with arginine or urea solution offers limited remineralizing benefit to enamel lesions over a period of 4-week time.

Antibiofilm activity of three irrigation protocols activated by ultrasonic, diode laser or Er:YAG laser in vitro.

AIM: To investigate the impact of three irrigation protocols, activated by three different methods, on mature biofilms of Enterococcus faecalis in vitro. METHODOLOGY: Root canals in 280 single-rooted teeth were instrumented using a rotary Ni-Ti system. Biofilms of E. faecalis were generated based on a previously established protocol. Samples were randomly divided into three experimental (n = 80) and one control (n = 40) group based on the irrigation protocol employed: group 1 (NaOCl + Etidronic acid), 1 : 1 mixture of 6% NaOCl and 18% etidronic acid; group 2 (NaOCl-EDTA), 3% NaOCl followed by 17% EDTA; group 3 (NaOCl-EDTA-NaOCl), 3% NaOCl followed by 17% EDTA and a final flush of 3% NaOCl. Saline served as the control. Samples were further divided into four subgroups (n = 20) based on the activation method: subgroup A, no activation; subgroup B, ultrasonic activation; group C, diode laser; group D, Er:YAG laser. Confocal laser scanning microscopy was used to assess bacterial viability in situ. Root dentine powder was obtained for determining the colony-forming units (CFU mL(-1) ). Data were analysed by appropriate statistical analyses with P = 0.05. RESULTS: All experimental irrigation protocols caused complete destruction of the biofilm in the root canal lumen. Within the dentinal tubules, all groups had a significantly higher percentage of dead bacteria than the saline control (P < 0.05). There was no significant difference between NaOCl + etidronic acid and NaOCl-EDTA-NaOCl (P > 0.05), whereas both groups brought about more bacterial reduction than NaOCl-EDTA (P < 0.05). There was no significant difference between diode laser and Er:YAG laser in any of the groups (P > 0.05). Both diode and Er:YAG laser were more effective than ultrasonic activation and conventional syringe irrigation in reducing E. fecalis biofilms (P < 0.05). CONCLUSIONS: The use of NaOCl after or in combination with a chelator caused the greatest reduction of E. faecalis. Diode laser and Er:YAG laser activation were superior to ultrasonics in dentinal tubule disinfection.

Thermal response of a dental tissue induced by femtosecond laser pulses.

This paper reports a theoretical and experimental study for thermal transport in a thin slice of human tooth induced by a 120 fs, 800 nm pulse laser at a repetition rate of 1 kHz. The surface reflectivity of enamel and the convection heat transfer coefficient were determined using an inverse heat transfer analysis. Instead of a fully three-dimensional modeling, two simplified two-dimensional (2D) planar and axisymmetric heat conduction models were proposed to simulate the temperature fields. The temperature responses obtained from the 2D planar and axisymmetric model agree well with the experimental measurements. On the other hand, the one-dimensional (1D) result significantly differs from the 2D axisymmetric one, suggesting that care should be taken when a 1D thermal model is considered for estimating temperature response.

Effects of maltitol and xylitol chewing-gums on parameters involved in dental caries development.

AIM: The effects on plaque parameters of sugar free chewing-gums (CG) sweetened with either maltitol or xylitol were assessed to better understand the role polyols can play in dental caries prevention. MATERIALS AND METHODS: A double-blind, parallel, randomised, controlled study was conducted in China. Subjects (N = 258, age = 13 to 15 years-old) were divided into 4 groups: 2 receiving polyols CG, containing respectively maltitol or xylitol, a group receiving gum base (placebo) and a negative control group not receiving any gum. CG were chewed for 30 days. This corresponds to a 10 g consumption of polyol per day. Plaque parameters (growth, pH, bacteria and insoluble glucans) were evaluated throughout the experimental period. RESULTS: All parameters studied were significantly modified with gum base compared to no-gum: plaque pH increased; plaque growth, bacteria (S. mutans, S. sobrinus, A. viscosus and Lactobacillus) and insoluble glucans decreased. Maltitol and xylitol CG led similarly to a higher plaque pH (AUC, p⋜0.05) on short (at baseline after the first CG consumption) and long term (after 4 weeks of daily CG consumption), with or without saliva stimulation compared to both control and placebo groups. They led to a decrease in plaque growth (p=0.02) over the experimental period compared to controls. Moreover, they significantly reduced the concentration of 4 cariogenic bacteria species (p⋜0.05) in dental plaque compared to gum base. CONCLUSION: Sugar free CG sweetened with either maltitol or xylitol can similarly reduce plaque acidogenicity compared to gum base through a decrease in oral bacteria presence. The use of a gum base placebo allowed to isolate effects on parameters involved in dental caries development specific to maltitol and xylitol, and to show these effects were similar.

A Cuff Lead for Delivering Ionic Direct Current (iDC) to Block Neural Activities of Sciatic Nerve.

Direct current (DC) applied extracellularly can block action potential (AP) propagation in a neuron. This suppression paradigm has been proposed as a possible treatment for blocking nociceptive pain. However, the application of DC is limited in duration due to the charge injection constraint imposed by the evolution of electrochemical reactions at the metal electrode. To prolong the application of DC, a microfluidic lead filled with conductive electrolyte can be used to separate the metal electrode from the target nerve. Here, we describe a tripolar nerve cuff lead fabricated with biocompatible silicone to block the APs in the rat sciatic nerve. This lead has a self-curling silicone membrane to wrap around sciatic nerve for secured mechanical attachment and electrical isolation between the nerve and the surrounding muscle. In-vivo testing showed that delivering 1.4mA DC via the cuff lead blocked the nerve activity and reduced the evoked compound action potential (eCAP) to 30% of its unblocked response.

X-ray microscopy and tomography detect the accumulation of bare and PEG-coated gold nanoparticles in normal and tumor mouse tissues.

We demonstrate that, with appropriate staining, high-resolution X-ray microscopy can image complicated tissue structures--cerebellum and liver--and resolve large or small amounts of Au nanoparticles in these tissues. Specifically, images of tumor tissue reveal high concentrations of accumulated Au nanoparticles. PEG (poly(ethylene glycol)) coating is quite effective in enhancing this accumulation and significantly modifies the mechanism of uptake by reticuloendothelial system (RES) organs.

The myotubularin family of lipid phosphatases in disease and in spermatogenesis.

The MTM (myotubularin)/MTMR (myotubularin-related) protein family is comprised of 15 lipid phosphatases, of which nine members are catalytically active. MTMs are known to play a fundamental role in human physiology as gene mutations can give rise to X-linked myotubular myopathy or Charcot-Marie-Tooth disease, which manifest in skeletal muscle or in peripheral neurons respectively. Interestingly, studies have shown MTMR2 and MTMR5, two MTM family members, to be highly expressed in the testis, particularly in Sertoli and germ cells, and knockout of either gene resulted in spermatogenic defects. Other studies have shown that MTMR2 functions in endocytosis and membrane trafficking. In the testis, MTMR2 interacts and co-localizes with c-Src/phospho-Src-(Tyr4¹6), a non-receptor protein tyrosine kinase that regulates the phosphorylation state of proteins at the apical ES (ectoplasmic specialization), a unique type of cell junction found between Sertoli cells and elongating/elongated spermatids. In the present review, we highlight recent findings that have made a significant impact on our understanding of this protein family in normal cell function and in disease, with the emphasis on the role of MTMs and MTMRs in spermatogenesis. We also describe a working model to explain how MTMR2 interacts with other proteins such as c-Src, dynamin 2, EPS8 (growth factor receptor pathway substrate 8) and ARP2/3 (actin-related protein 2/3) at the apical ES and the apical TBC (tubulobulbar complex; tubular-like invaginations that function in the disassembly of the apical ES and in the recycling of its components) to regulate spermiation at late stage VIII of the seminiferous epithelial cycle.

Assembly of the antifreeze glycoprotein/trypsinogen-like protease genomic locus in the Antarctic toothfish Dissostichus mawsoni (Norman).

To investigate the genomic architecture underlying the quintessential adaptive phenotype, antifreeze glycoprotein (AFGP) that enables Antarctic notothenioid survival in the frigid Southern Ocean, we isolated the AFGP genomic locus from a bacterial artificial chromosome library for Dissostichus mawsoni. Through extensive shotgun sequencing of pertinent clones and sequence assembly verifications, we reconstructed the highly repetitive AFGP genomic locus. The locus comprises two haplotypes of different lengths (363.6 kbp and 467.4 kbp) containing tandem AFGP, two TLP (trypsinogen-like protease), and surprisingly three chimeric AFGP/TLP, one of which was previously hypothesized to be a TLP-to-AFGP evolutionary intermediate. The ~100 kbp haplotype length variation results from different AFGP copy number, suggesting substantial dynamism existed in the evolutionary history of the AFGP gene family. This study provided the data for fine resolution sequence analyses that would yield insight into the molecular mechanisms of notothenioid AFGP gene family evolution driven by Southern Ocean glaciation.

Photodynamic therapy enhances liposomal doxorubicin distribution in tumors during isolated perfusion of rodent lungs.

BACKGROUND: Photodynamic therapy (PDT) at low drug-light conditions can enhance the transport of intravenously injected macromolecular therapeutics through the tumor vasculature. Here we determined the impact of PDT on the distribution of liposomal doxorubicin (Liporubicin™) administered by isolated lung perfusion (ILP) in sarcomas grown on rodent lungs. METHODS: A syngeneic methylcholanthrene-induced sarcoma cell line was implanted subpleurally in the left lung of Fischer rats. Treatment schemes consisted in ILP alone (400 µg of Liporubicin), low-dose (0.0625 mg/kg Visudyne®, 10 J/cm(2) and 35 mW/cm(2)) and high-dose left lung PDT (0.125 mg/kg Visudyne, 10 J/cm(2) and 35 mW/cm(2)) followed by ILP (400 µg of Liporubicin). The uptake and distribution of Liporubicin in tumor and lung tissues were determined by high-performance liquid chromatography and fluorescence microscopy in each group. RESULTS: Low-dose PDT significantly improved the distribution of Liporubicin in tumors compared to high-dose PDT (p < 0.05) and ILP alone (p < 0.05). However, both PDT pretreatments did not result in a higher overall drug uptake in tumors or a higher tumor-to-lung drug ratio compared to ILP alone. CONCLUSIONS: Intraoperative low-dose Visudyne-mediated PDT enhances liposomal doxorubicin distribution administered by ILP in sarcomas grown on rodent lungs which is predicted to improve tumor control by ILP.

Induction of periodontal disease via retentive ligature, lipopolysaccharide injection, and their combination in a rat model.

Periodontitis is a highly prevalent, chronic immune-inflammatory disease of the periodontium that results in the periodontium and alveolar bone loss's progressive destruction. In this study, the induction of periodontal disease via retentive ligature, lipopolysaccharide, and their combination at three different times were compared in a rat model. Seventy-two Sprague Dawley rats were distributed into four treatment groups: 1) control group with no treatment; 2) application of 4/0 nylon ligature around second maxillary molars; 3) combination of ligature and LPS injection (ligature-LPS); 4) intragingival injection of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to the palatal mucosa of the second maxillary molars. Six rats were sacrificed from each group after 7, 14, and 30 days of periodontal disease induction. Alveolar bone loss, attachment loss, number of inflammatory cells, and blood vessels were evaluated histologically. A micro-CT scan was used as a parameter to know the rate of alveolar bone loss. Parametric data were analyzed using two-way ANOVA followed by Bonferroni correction with a significance set at 5%. Non-parametric data were analyzed using Kruskal-Wallis, followed by multiple comparisons with Bonferroni correction. The histological results revealed significant destructive changes in the periodontal tissues and alveolar bone following the ligature and ligature-LPS induction techniques. These changes were evident as early as seven days, maintained until 14 days post-treatment, and declined with time. The ligature technique was effective in inducing acute periodontal disease. The LPS injection technique did not induce alveolar bone loss, and its combination to ligature added insignificant effects.

Preparation of silver nanoparticles with antimicrobial activities and the researches of their biocompatibilities.

Silver nanoparticles were prepared by chemical reduction method using chitosan as stabilizer and ascorbic acid as reducing agent in this work. The silver/chitosan nanocomposites were characterized in terms of their particle sizes and morphology by using UV spectrophotometer, nano-grainsize analyzer, and transmission electron microscopy. Antibacterial activities of these nanocomposites were carried out for Staphylococcus aureus and Escherichia coli. The silver nanoparticles exhibited significantly inhibition capacity towards these bacteria. Detailed studies on the biocompatibility of the silver/chitosan nanocomposites were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell adhesion test. The results indicated that these silver/chitosan nanocomposites were benefit for the proliferation and adhesion of L-929 cells, and the biocompatibilities between the nanocomposites and the cells would become better with the culturing days. We anticipated that these silver/chitosan nanocomposites could be a promising candidate as coating material in biomedical engineering and food packing fields wherein antibacterial properties and biocompatibilities are crucial.

A microfluidic system integrated with shape memory alloy valves for a safe direct current delivery system.

Direct current (DC) has potential as a clinical and scientific tool to accelerate wound healing, increase the permeability of the skin to drug treatment and modulate neural activity. But long duration delivery of DC unavoidably causes hazardous electrolysis at the tissue-electrode interface. To be able to deliver long duration DC, we previously proposed a design for a safe direct current stimulator (SDCS). This device uses alternating current that does not cause chemical reactions at the metal electrodes within the device, but delivers ionic direct current output to the tissue via microfluidic valves. We previously developed and published designs of multiple SDCS components including microfluidic, electronic, data processing, and energy systems. In this paper we focus on the development of the integrated microfluidics needed by the SDCS system. We developed a fabrication method and characterized valve performance within the multi-valve microfluidic system. We used poly-dimethylsiloxane (PDMS) to fabricate three microfluidic chips that integrated valves actuated by 50-µm Nitinol (NiTi) shape memory alloy (SMA) wire. We tested system operation by driving SMA valves with a current pulse and recording the valve response with an electrical assay. The valve operation complied with the SDCS system requirements. The time for valves to open was rapid at 0.177 ± 0.04 seconds, and the time for the valves to close was 0.265 ± 0.05 seconds. Open microfluidic channel impedance for unrestricted ionic current flow was 15.90 ± 8.28 kΩ and it increased by a factor of 40 to restrict ionic current flow at 678 ± 102 kΩ for the closed valves.

Disruption of Mtmr2 produces CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis.

Mutations in MTMR2, the myotubularin-related 2 gene, cause autosomal recessive Charcot-Marie-Tooth (CMT) type 4B1, a demyelinating neuropathy with myelin outfolding and azoospermia. MTMR2 encodes a ubiquitously expressed phosphatase whose preferred substrate is phosphatidylinositol (3,5)-biphosphate, a regulator of membrane homeostasis and vesicle transport. We generated Mtmr2-null mice, which develop progressive neuropathy characterized by myelin outfolding and recurrent loops, predominantly at paranodal myelin, and depletion of spermatids and spermatocytes from the seminiferous epithelium, which leads to azoospermia. Disruption of Mtmr2 in Schwann cells reproduces the myelin abnormalities. We also identified a novel physical interaction in Schwann cells, between Mtmr2 and discs large 1 (Dlg1)/synapse-associated protein 97, a scaffolding molecule that is enriched at the node/paranode region. Dlg1 homologues have been located in several types of cellular junctions and play roles in cell polarity and membrane addition. We propose that Schwann cell-autonomous loss of Mtmr2-Dlg1 interaction dysregulates membrane homeostasis in the paranodal region, thereby producing outfolding and recurrent loops of myelin.

Controlling Liquid-Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish.

Liquid-liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein-protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.

Asparaginase pharmacodynamics differ by formulation among children with newly diagnosed acute lymphoblastic leukemia.

Polyethylene glycol-conjugated (PEG) asparaginase is approved for use in patients who develop allergy to other forms of asparaginase, although its ability to deplete asparagine systemically in patients with hypersensitivity has not been well elucidated. In 53 children with newly diagnosed acute lymphoblastic leukemia, we serially assessed asparagine concentrations in cerebrospinal fluid (CSF) and plasma as well as serum anti-asparaginase antibodies. All patients received native Escherichia coli (Elspar) asparaginase during induction therapy; patients received PEG asparaginase during reinductions when available, and those who developed allergy received Erwinia asparaginase. All eight patients who developed clinical evidence of allergy to asparaginase had anti-asparaginase antibodies. Among patients who had no antibodies, those who received E. coli had lower mean (+/-s.d.) CSF asparagine (0.29+/-0.63, n=9) than those who received PEG (0.77+/-0.82, n=4) (P=0.007). Results were similar for plasma asparagine. There was no situation where asparagine concentrations were more effectively depleted by PEG than by other preparations. None of the five patients who developed thrombosis had an allergy or antibodies to asparaginase at the time of the thrombosis. We conclude that asparagine concentrations were less effectively depleted by PEG than by E. coli asparaginase at the doses commonly used. The risk of thrombosis may be affected by the intensity of asparaginase exposure.

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

C(60) and water-soluble fullerene derivatives as antioxidants against radical-initiated lipid peroxidation.

C(60), vitamin E, and three C(60) derivatives (polar 1 and water-soluble C(3)/D(3)C(60)s) were examined for their antioxidant effects on prevention of lipid peroxidation induced by superoxide and hydroxyl radicals. The protection effect on lipid peroxidation was found to be in the sequence: C(60) >/= vitamin E > 1 > none, for liposoluble antioxidants, and C(3)C(60) >> D(3)C(60) > none, for water-soluble ones. Fluorescence quenching of PyCH(2)COOH (Py = pyrene) by both C(3)- and D(3)C(60)s shows that the Stern-Volmer constant, K(SV), is about the same for both quenchers in aqueous solution. Upon addition of liposomes, the fluorescence quenching becomes more efficient: 5-fold higher in K(SV) for C(3)C(60) than for D(3)C(60). When Py(CH(2))(n)()COOH (n = 1, 3, 5, 9, or 15) was incorporated in lipid membranes, the K(SV)s all were small and nearly equal for D(3)C(60) but were quite large and different for C(3)C(60) with the sequence: n = 1 < 3 < 5 < 9 < 15. The better protection effect of C(3)C(60) on lipid peroxidation than that of D(3)C(60) is attributed to its stronger interaction with membranes. Overall, the antioxidation abilities of the compounds examined were rationalized in terms of the number of reactive sites, the location of antioxidant in lipid membranes, and the strength of interactions between antioxidants and membranes.

A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.

Proteolysis of integrin alpha5 and beta1 subunits involved in retinoic acid-induced apoptosis in human hepatoma Hep3B cells.

Our previous report demonstrated that all-trans-retinoic acid (ATRA) induces detachment and death under serum starvation in several human tumor cell lines. In this study, we examined the influence of cell-extracellular matrix interaction on the ability of ATRA to induce apoptosis. Plating of human hepatoma Hep3B cells onto poly-hydroxyethylmethacrylate-coated plates in the absence of serum resulted in the acceleration of ATRA-induced apoptosis. In contrast, ATRA-induced apoptosis was significantly suppressed by plating cells onto Matrigel-coated plates but not suppressed by culturing onto collagen-, laminin-, vitronectin-, or fibronectin-coated plates. Exogenously added soluble collagen, laminin, fibronectin, vitronectin or Matrigel failed to suppress ATRA-induced apoptosis. Results from the adhesion assay indicated that the cell attachment to fibronectin was significantly inhibited by ATRA. Treatment with perturbing antibody against integrin alpha5 or beta1 subunits resulted in promotion of ATRA-induced apoptosis. Moreover, the proteolytic cleavage of alpha5beta1 integrin and focal adhesion kinase (FAK) proteins is linked to the early phase of the ATRA-induced apoptotic process. Furthermore, ATRA-induced detachment, death, and cleavage of alpha5beta1 integrin and FAK were drastically suppressed by plating cells onto Matrigel-coated plates. These findings provide evidence that abrogation of cell adhesion, through proteolysis of alpha5beta1 integrin and FAK, is closely linked to ATRA-induced apoptosis in Hep3B cells.

Optimizing the sterilization of PLGA scaffolds for use in tissue engineering.

There are few suitable techniques available to sterilize biodegradable polyester three-dimensional tissue engineering scaffolds because they are susceptible to degradation and/or morphological degeneration by high temperature and pressure. We used a novel polyllactide-co-glycolide) scaffold (Osteofoam) to determine the optimal sterilization procedure--i.e. a sterile product with minimal degradation and deformation. Initial studies, found that an argon plasma created at 100W for 4min was optimal for sterilizing Osteofoam scaffolds without affecting their morphology. The RFGD plasma sterilization method was compared to two well-established techniques--ethylene oxide (ETO) and gamma-irradiation (gamma)--which were in turn compared to disinfection in 70% ethanol. Disinfection in 70% ethanol serves as a useful control because it affects neither the morphology nor the molecular weight of the polymer: yet, ethanol is unsuitable as a sterilization method because it does not adequately eliminate hydrophilic viruses and bacterial spores. The three sterilization techniques, ETO, gamma and RFGD plasma, were compared in terms of their immediate and long-term effects on the dimensions, morphology, molecular weight and degradation profile of the scaffolds. Scaffolds shrank to 60% of their initial volume after ETO sterilization whereas their molecular weight (Mw) decreased by approximately 50% after gamma-irradiation. Thus, both ETO and gamma-irradiation posed immediate problems as sterilization techniques for 3-D biodegradable polyester scaffolds. During the in vitro degradation study, all sterilized samples showed advanced morphological and volume changes over time relative to ethanol (EtOH) disinfected samples, with the greatest changes observed for gamma-irradiated samples. ETO, RFGD plasma sterilized and EtOH disinfected samples showed similar changes in Mw and mass over the 8-week time frame. Overall, of the three sterilization techniques studied, RFGD plasma was the best.