Control Substrata Research Articles

Electricity Production Using Plant–Microbial Fuel Cell (P-MFC)

The current climate change threat by green house gas emissions from the combustion of fossil fuels has necessitated a search for alternative non-polluting, reliable, renewable and sustainable sources of energy such as solar energy and it’s derivatives. The present work focuses on power generation by Plant-Microbial Fuel Cell using Phragmitesaustralis (Reed plant). The plants were grown in fuel-cell, graphite as anode and carbon felt as cathode, separated by proton-exchange-membrane. During anaerobic microbial metabolism of carbohydrates in the roots, protons and electrons are released, the electrons are donated to the anode by the microbes. These electrons can be channeled through a circuit bearing a load to the cathode. In this work, carbon granules as substratum (control), red soil and carbon granules mixture (30:70) as substratum in varied condition was considered. For control substratum, the max.voltage measured was 0.327 V and power density of 2.06x10-3 mW m-2 was obtained. When red soil mixed with carbon granules in the ratio 30:70, the voltage measured was 0.6 V and the power density was found to be 3.78x10-3 mW m-2. When graded red soil (0.0018 m) mixed with carbon granules in the ratio 30:70, the voltage measured was 0.623 V and the power density was found to be 3.98x10-3 mW m-2. The result proves that the plant microbial fuel cell can be used for generating electricity and is a promising renewable energy technology.

Factors regulating lake periphyton biomass and nutrient limitation status across a large trophic gradient

Abstract Because of the historical focus of limnology on pelagic processes, the factors controlling lake periphyton growth and nutrient limitation are understudied compared to the phytoplankton. We deployed nutrient‐diffusing substrata at 28 sites spanning a wide trophic status gradient in Lakes Superior and Michigan to assess periphyton biomass accrual on control substrata and the response of periphyton to single and combined phosphorus (P) and nitrogen (N) additions. Periphyton growth was unimodally related to a composite metric of site trophic status, with highest biomass at mesotrophic sites and lower growth at oligotrophic and highly eutrophic sites. Contrary to expectations, P limitation was rare. Instead, several lines of evidence pointed to primary N or N + P co‐limitation of periphyton. Limitation extent was negatively related to site trophic status, with stronger nutrient limitation at oligotrophic sites. Our results support the hypothesis that phytoplankton and periphyton biomass respond differently to nutrient enrichment and suggest that different nutrients may limit pelagic and benthic primary production, even in the same system. Our findings also support the use of periphyton as an early warning indicator of nutrient pollution and help explain why large, oligotrophic lakes may be especially susceptible to localised benthic algal blooms.

The Presence of Marine Filamentous Fungi on a Copper-Based Antifouling Paint

Marine biofouling is undesirable growth on submerged substances, which causes a major problem for maritime industries. Antifouling paints containing toxic compounds such as copper are used to prevent marine biofouling. However, bacteria and diatoms are usually found in biofilms developed on such paints. In this study, plastic panels painted with a copper-based self-polishing antifouling paint were exposed to biofouling for 6 months in the Marina Bandar Rowdha, Sea of Oman. Clean panels were used as a control substratum. Marine filamentous fungi from protected and unprotected substrate were isolated on a potato dextrose agar. Pure isolates were identified using sequences of the ITS region of rDNA. Six fungal isolates (Alternaria sp., Aspergillus niger, A. terreus, A. tubingensis, Cladosporium halotolerans, and C. omanense) were obtained from the antifouling paint. Four isolates (Aspergillus pseudodeflectus, C. omanense, and Parengyodontium album) were isolated from clean panels and nylon ropes. This is the first evidence of the presence of marine fungi on antifouling paints. In comparison with isolates from the unprotected substrate, fungi from the antifouling paint were highly resistant to copper, which suggests that filamentous fungi can grow on marine antifouling paints.

Laminin-dependent integrin β1 signaling regulates milk protein synthesis via prolactin/STAT5 pathway in bovine mammary epithelial cells

Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and β1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of β-casein. However, the mechanism by which β1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with β1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of β1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that β1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of β1 integrin was not sufficient to increase β-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of β1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of β-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, β1 integrin, and β-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking β1 integrin function, resulting in attenuated STAT5 activity and decreased β-casein synthesis. These results indicated that β1 integrin was a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for β-casein expression in BMECs.

Interaction of primary human trabecular meshwork cells with metal alloy candidates for microinvasive glaucoma surgery.

Microinvasive glaucoma surgery (MIGS) is a relatively new addition to the glaucoma treatment paradigm. Small metallic stents are inserted into the trabecular meshwork in order to increase aqueous humour drainage. MIGS procedures are rapidly being adopted owing to a more favourable side effect profile when compared with traditional surgery. Remarkably, this rapid rate of utilization has occurred without any published studies on the effect of metal alloys used in these stents on human trabecular meshwork cells (HTMCs). Therefore, this study aimed to determine the effect of candidate metal alloys for MIGS on HTMC morphology, viability and function. Human trabecular meshwork cells were cultured on the surfaces of titanium (polished and sandblasted), a titanium-nickel (nitinol) alloy and glass (as control substratum). Fluorescence imaging was used to assess cell morphology and spreading. A lactate dehydrogenase cytotoxicity assay, cell death detection ELISA, MTT cell viability assay, BrdU cell proliferation assay and fibronectin ELISA were also conducted. Cells cultured on sandblasted titanium exhibited significantly greater spreading than cells cultured on other substrata. In comparison, HTMCs cultured on nitinol displayed poor spreading. Significantly more cell death, by both necrosis and apoptosis, occurred on nitinol than on titanium and glass. Also, cell viability and proliferation were suppressed on nitinol compared with titanium or glass. Finally, HTMCs on both titanium and nitinol produced greater amounts of fibronectin than cells grown on glass. Substratum topography and metal alloy composition were found to impact morphology, viability and function of primary HTMC cultures.

Self-decontaminating photocatalytic zinc oxide nanorod coatings for prevention of marine microfouling: a mesocosm study

The antifouling (AF) properties of zinc oxide (ZnO) nanorod coated glass substrata were investigated in an out-door mesocosm experiment under natural sunlight (14:10 light: dark photoperiod) over a period of five days. The total bacterial density (a six-fold reduction) and viability (a three-fold reduction) was significantly reduced by nanocoatings in the presence of sunlight. In the absence of sunlight, coated and control substrata were colonized equally by bacteria. MiSeq Illumina sequencing of 16S rRNA genes revealed distinct bacterial communities on the nanocoated and control substrata in the presence and absence of light. Diatom communities also varied on nanocoated substrata in the presence and the absence of light. The observed AF activity of the ZnO nanocoatings is attributed to the formation of reactive oxygen species (ROS) through photocatalysis in the presence of sunlight. These nanocoatings are a significant step towards the production of an environmentally friendly AF coating that utilizes a sustainable supply of sunlight.

Abstract 232: Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols

Abstract Some cancer-preventive agents such as selenium and green tea polyphenols effectively prevent cancer in some cases, but fail in other cases. It could be possible that the cancer-preventive actions of these agents may be altered by the interaction of cancer cells with the extracellular matrix (ECM) components, thus making this an important issue to study. Others have previously cloned a family of human prostate cancer cell lines with a common lineage that show the progression of characteristics from low to high degrees of malignancy and mimic different stages of tumor progression seen in human prostate cancer. For this study, we used these prostate cancer cell lines with increasing tumorigenicity and invasive ability: WPE1-NA22 showing the lowest, WPE1-NB14 and WPE1-NB11 showing intermediate, and WPE1-NB26 showing the greatest. The low tumorigenic cell line, WPE1-NA22 resembles noninvasive prostatic intra-epithelial neoplasia and represents a good target for chemoprevention in humans. Initially, we grew these cells on the surface of ECM components, laminin, collagen I, and fibronectin and determined the tumor cell sensitivity to methylseleninic acid (MSA) and epigallocatechin-3-gallate (EGCG). Poly-L-lysine was used as a control substratum. Cell growth was determined by sulforhodamine B staining assay. Then we determined whether these ECM proteins could enhance tumor cell resistance to MSA and EGCG. ECM components, but not polylysine, decreased tumor cell sensitivity to these agents. The effect of ECM components was prevented by β1 integrin-blocking antibodies suggesting that β1 integrin plays a role in mediating intracellular signaling conferring resistance against MSA and EGCG. Previously, we have shown an increase in expression of protein kinase Cε (PKCε) in advanced prostate tumor cells WPE1-NB26. A decrease in expression of PKCε by siRNA approach decreased the tumor cell resistance caused by ECM components. Furthermore, an inhibition of cell survival enzymes phosphoinositide-3-kinase (PI3K) with its specific inhibitor LY294002 (50 μM) as well as Akt with its specific inhibitor MK-2206 (1 μM) decreased the effect of ECM components on tumor cell survival against MSA and EGCG. WPE1-NB14, a low tumorigenic cell line having low PKCε and Akt activities, responded less to ECM components and showed higher sensitivity to MSA and EGCG. Conceivably, ECM components, laminin, collagen, and fibronectin induced β1 integrin signaling to activate cell survival pathways involving PKCε and PI3K-Akt, thereby decreasing prostate tumor cell sensitivity to MSA and EGCG. Thus, ECM may be one of the limiting factors for the action of cancer-preventive agents. Citation Format: Rayudu Gopalakrishna, Tiffany Fan, Ronald Deng, David Rayudu, Zachary W. Chen, William S. Tzeng, Usha Gundimeda. Extracellular matrix components influence prostate tumor cell sensitivity to cancer-preventive agents selenium and green tea polyphenols. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 232. doi:10.1158/1538-7445.AM2014-232

Nitrogen limitation of the phytobenthos in Alpine lakes: results from nutrient‐diffusing substrata

Summary In the Alps, as in other mountainous regions across the world, nitrogen (N) enrichment of ecosystems via atmospheric deposition is a major environmental concern. Nitrogen enrichment can cause acidification in poorly buffered lakes and streams, but here we assessed the less well‐known effects of eutrophication on the phytobenthos of lakes and its consequences for grazing. To simulate the effects of N deposition, we conducted experimental nutrient additions (N and N+P) in two lakes in the French Alps, in summer 2012. In each lake, we deployed nutrient‐diffusing substrata for 22 days. The substrata were placed within cages that either allowed or prevented access by macrograzers. We tested the hypotheses that N enrichment: (i) increases phytobenthic biomass, (ii) alters phytobenthic taxonomic composition and (iii) reduces benthic grazing. Supporting the first hypothesis, at the end of the experiments N‐enriched substrata had a greater phytobenthic biomass than unenriched substrata, indicating N limitation. Supporting the second hypothesis, the taxonomic composition of the phytobenthos differed between N‐enriched and control substrata. Green algae, including filamentous forms, were favoured by N enrichment to a greater extent than diatoms and cyanobacteria. By contrast, the third hypothesis was not supported. Grazing had the same effect on the biomass of the phytobenthos regardless of nutrient treatment. Our results highlight the sensitivity of Alpine lakes to deposition of atmospheric N. Its ecological effects are not limited to acidification, but also include increased biomass (eutrophication) and altered taxonomic composition of the phytobenthos. Because filamentous green algae favoured by N enrichment are usually unpalatable to macrograzers, inputs of atmospheric N could reduce the efficiency of transfer of energy from the phytobenthos up through the food web.

Chitosan–gelatin biopolymers as carrier substrata for limbal epithelial stem cells

The aim of this work was to evaluate semi-synthetic biopolymers based on chitosan (CH) and gelatin (G) as potential in vitro carrier substrata for human limbal epithelial cells (hLECs). To that end, human corneal epithelial cells (HCE) were cultured onto different CH-G membranes. None of the polymers were cytotoxic and cell proliferation was higher when CH was functionalized with G. Expression levels of corneal epithelial markers (K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) were better maintained in HCE cells grown on CH-G 20:80 membranes than other proportions. Consequently, CH-G 20:80 was chosen for the subsequent expansion of hLECs. Cells derived from limbal explants were successfully expanded on CH-G 20:80 membranes using a culture medium lacking components of non-human animal origin. The expression levels found for corneal (K3 and K12) and limbal epithelial stem cells (K15) specific markers were similar to or higher than those found in limbal cells grown onto the control substratum. Our results demonstrate that CH-G 20:80 membranes are suitable for the expansion and maintenance of stem cells derived from the limbal niche. These results strongly support the use of polymers as alternative substrata for the transplantation of cultivated limbal cells onto the ocular surface.

Chondroitin sulfate proteoglycans in demyelinated lesions impair remyelination

Failure of remyelination is a critical impediment to recovery in multiple sclerosis (MS). Chondroitin sulfate proteoglycans (CSPGs) have been reported to accumulate in MS lesions, and we thus examined the functional roles of CSPGs on oligodendrocyte precursor cells (OPCs), oligodendrocytes, and remyelination. We evaluated the expression of CSPGs in lysolecithin-injected mouse spinal cord, an animal model of demyelination and spontaneous remyelination. The functional impact of CSPGs on OPCs and remyelination was investigated using cultured adult murine and human OPCs and by treating demyelinated mice with xyloside to reduce the CSPG deposition that occurred following injury. Early and robust upregulation of CSPGs following lysolecithin-induced demyelination was cleared during remyelination. In culture, CSPGs anchored onto the substratum reduced the adhesion of mouse and human OPCs and their subsequent morphological differentiation into process-bearing oligodendrocytes. Soluble CSPGs added to already adherent OPCs reduced the development of processes, whereas the acquisition of mature myelin proteins was unimpeded. Stripe assays of alternating CSPG and control substrata confirmed the nonpermissive nature of CSPGs for OPC adhesion and morphological differentiation. Enzymatic degradation of CSPGs with chondroitinase ABC was sufficient to overcome CSPG-dependent inhibition of human oligodendrocytes. Finally, in vivo xyloside treatment to reduce CSPG synthesis in lysolecithin-demyelinated mice increased numbers of OPCs and oligodendrocytes in lesions, and culminated in improved remyelination. These results identify CSPGs as a nonpermissive substrate for OPCs and oligodendrocytes, and as a prominent impediment to remyelination. The data suggest the requirement for the neutralization of CSPGs for repair after demyelination.

The effect of surface microgrooves and anodic oxidation on the surface characteristics of titanium and the osteogenic activity of human periodontal ligament cells

ObjectiveThe purpose of this study was to investigate the effect of titanium (Ti) surface microgrooves and anodic oxidation on the surface characteristics of titanium and the osteogenic activity of human periodontal ligament cells (PLCs) cultured on theses surfaces. DesignMechanically ground Ti was used as the control substratum (NE0). Truncated V-shaped microgrooves, 60μm-wide and 10μm-deep in cross-sections, were created on the Ti substrata by photolithography (NE60/10). Anodically oxidized Ti (NE0AO) and anodically oxidized microgrooved Ti (NE60/10AO) were also prepared. Scanning electron microscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were performed for surface characterization. Cell proliferation assay, osteoblast differentiation assay, and quantitative real-time PCR analysis were performed to compare the osteogenic activity of PLCs on NE0, NE60/10, NEAO, and NE60/10AO. ResultsA decrease in the microgroove-width of NE60/10AO compared to NE60/10 due to Ti oxide layer generation by anodic oxidation was detected with XRD and XPS. Cell proliferation, osteoblast differentiation, and osteo-related gene expression were enhanced on the NE60/10AO substrata compared with NE0, NE60/10, and NE0AO. ConclusionsThe combination of Ti surface microgrooves and subsequent anodic oxidation treatment synergistically upregulated osteo-related gene expression, despite showing limited ability to increase cell proliferation and osteoblast differentiation levels in PLCs.

Texture direction of combined microgrooves and submicroscale topographies of titanium substrata influence adhesion, proliferation, and differentiation in human primary cells

ObjectiveThis study aimed to identify the optimal micro- and submicroscale topographies of titanium (Ti) substrata that would most significantly influence adhesion, proliferation, and other activities of these cells. DesignTruncated V-shaped microgrooves in 60μm-wide and 10μm-deep cross-sections with 0°, 30°, 60°, or 90° angles between the microgrooves and ridge-top submicroscale texture were created on the Ti substrata (designated NE60/10-0°, NE60/10-30°, NE60/10-60° and NE60/10-90°, respectively). Ground titanium with submicroscale texture but with no microgrooves was used as the control substratum, NE0. Scanning electron microscopic observation and the assays determining the cell adhesion, cell proliferation and osteoblast differentiation were performed. ResultsCells more actively migrated into the microgrooves on NE60/10-30° than into the microgrooves on any other substrata tested, suggesting that the cells utilise the increased surface area of the substrata at the microscale level. NE60/10-0° and NE60/10-30° substrata generally enhanced adhesion, proliferation, alkaline phosphatase activity, and osteoblast differentiation of human primary cells when compared to other Ti substrata, and significant correlations were observed between these cellular activities. ConclusionsHere, we show that the contact guidance of human primary cells grown on Ti substrata can be controlled more by specific submicroscale textures on ridge tops than by the dimensions of surface microgrooves only. Also, the degree of angles created between the submicroscale textures and microgrooves on Ti substrata significantly affect cell adhesion, proliferation and differentiation in human primary cells.

Toxic Effects of Single-Walled Carbon Nanotubes in the Development of E. coli Biofilm

The impact of single-walled carbon nanotubes (SWNTs) on the different developmental stages of biofilms has been investigated using E. coli K12 as a model organism. Specifically, we investigated (i) the impact of SWNT concentration on cell growth and biofilm formation, (ii) toxic effects of SWNTs on mature biofilms, and (iii) formation of biofilm on SWNT-coated surfaces. The results show that at the initial stage of biofilm formation, SWNTs come into contact with bacterial cells prior to biofilm maturation and inhibit their growth. Furthermore, the results suggest that bacteria in mature biofilms are less sensitive to the presence of SWNTs than cells in other biofilm stages, similar to previous observations of biofilm resistance to antimicrobials. In mature biofilms, the soluble exopolymeric substances (EPS) secreted by the biofilm play an important role in mitigating the toxic effects of SWNTs. Upon exposure to SWNTs, biofilms without soluble EPS in the supernatant had a much more significant loss of biomass because of cell detachment from the biofilm than biofilms containing soluble EPS. To observe similar cell loss, biofilms with soluble EPS needed SWNT concentrations that were 10 times higher compared to biofilms without soluble EPS. Finally, SWNTs deposited onto surfaces affected significantly the subsequent biofilm development. Analysis of the total biomass and the area occupied by cells indicates that a SWNT-coated substratum has 10 times less biofilm colonization and biomass production than a control substratum without SWNTs.

Axon growth-promoting properties of human bone marrow mesenchymal stromal cells

Mesenchymal stromal cells are promising candidate donor cells for promoting functional tissue repair following traumatic spinal cord injury (SCI), however, the mechanism(s) of action remain poorly defined. Here, we describe an in vitro study of the axon growth-promoting properties of highly enriched populations of adult human mesenchymal stromal cells (hMSC). A random, non-oriented pattern of neuritic outgrowth was observed from dissociated adult rat DRG neurons seeded onto confluent A431 cells and PLL/laminin positive control substrata. Confluent hMSC formed arrays of similarly orientated cell bodies and processes which supported the regeneration of significantly more primary neurites but a slightly lower overall neuritic length than was observed over the PLL/laminin control substrate. The hMSC exerted a strong influence on the direction of neuritic outgrowth, with many regenerating processes following the orientation of underlying hMSC. The production of extracellular matrix appeared to be responsible for neuritic directionality, but the release of growth factors was a significant promoter for DRG neuritic outgrowth. This suggests that further investigations into the properties of hMSC may be of particular interest in the development of transplant-mediated strategies intending to promote functional axonal regeneration after SCI.

Myelin-Associated Glycoprotein (MAG) Protects Neurons from Acute Toxicity Using a Ganglioside-Dependent Mechanism

Myelin-associated glycoprotein (MAG), a protein expressed on the innermost wrap of myelin, contributes to long-term axon stability as evidenced by progressive axon degeneration in Mag-null mice. Recently, MAG was also found to protect axons from acute toxic insults. In the current study, rat dorsal root ganglion neurons were cultured on control substrata and substrata adsorbed with myelin proteins. Neurons on myelin-adsorbed surfaces were resistant to acute degeneration of neurites induced by vincristine, a cancer chemotherapeutic agent with neuropathic side effects. Myelin-mediated protection was reversed by anti-MAG antibody and was absent when cells were cultured on extracts from Mag-null mouse myelin, confirming the protective role of MAG. Gangliosides (sialylated glycosphingolipids) are one functional class of axonal receptors for MAG. In the current studies, a direct role for gangliosides in mediating the acute protective effects of MAG was established. Treatment of neurons with sialidase, an enzyme that cleaves the terminal sialic acids required for MAG binding, reversed MAG's protective effect, as did treatment with (1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of glycosphingolipid biosynthesis. In contrast, treatment with phosphatidylinositol-specific phospholipase C, an enzyme that cleaves Nogo receptors (NgR, another class of MAG receptor), or with a peptide inhibitor of an NgR-associated signaling molecule p75(NTR), failed to diminish MAG-mediated protection. Inhibiting the Rho-associated protein kinase ROCK reversed protection. We conclude that MAG protects neurites from acute toxic insult via a ganglioside-mediated signaling pathway that involves activation of RhoA. Understanding MAG-mediated protection may provide opportunities to reduce axonal damage and loss.

Sustained epidermal growth factor receptor levels and activation by tethered ligand binding enhances osteogenic differentiation of multi‐potent marrow stromal cells

Epidermal growth factor receptor (EGFR)-mediated signaling helps regulate bone development and healing through its effects on osteogenic cells. Here, we show how EGFR activity and osteogenic differentiation responses in primary human bone marrow-derived multipotent stromal cells (MSCs) are influenced by presenting covalently tethered epidermal growth factor (tEGF) on the culture substratum, a presentation mode that reduces EGFR internalization and restricts signaling to the cell surface. In both absence and presence of tEGF, MSCs increase expression levels of EGFR and its heterodimerization partner HER2 during the course of osteogenic differentiation. tEGF substrata increased levels of phosphorylated EGFR and phosphorylated extracellular regulated kinase (ERK) compared to control substrata, and these elevations were associated with a twofold enhancement of MSC alkaline phosphatase activity at day 7 and matrix mineralization at day 21. Surprisingly, addition of soluble EGF (sEGF) to cells cultured on tEGF substrata reduces osteogenic differentiation, even though EGFR signaling is more strongly activated in acute, short-term manner by sEGF treatment than by tEGF treatment. A striking concomitant result of the sEGF effects is near-complete downregulation of EGFR and HER2, demonstrating that the tEGF/EGFR interaction is dynamically reversible even though temporally sustained. Taken together, our results show that enhanced MSC osteogenic differentiation corresponds to a sustained combination of receptor expression and ligand presentation, both of which are maintained by tEGF.

Genetically Engineered Elastin-Like Polymer as a Substratum to Culture Cells from the Ocular Surface

Purpose: To investigate epithelial cell adhesion and proliferation on a newly developed elastin-like polymer (ELP) that mimics the functional characteristics of extracellular matrices. Materials and Methods: A genetically engineered ELP with cell attachment sequences was adsorbed onto glass coverslips as 1, 2, or 3 molecular films. Conjunctival epithelial cells from a human cell line and human skin fibroblast cells (as controls) were plated onto coverslips with three different substrata: plain glass, Thermanox®, and ELP-coated. Cells (104) were plated after EDTA- or trypsin-based detachment. To test adhesion, epithelial and fibroblast cells were incubated for 4 hr, stained with hematoxylin, and counted. To study proliferation, Ki-67-positive epithelial cells were counted after 1, 3, and 5 days in culture. Immunostaining for conjunctival and adhesion markers was performed. Results: Epithelial cell, but not fibroblast, adhesion on ELP was significantly enhanced compared to that of control substrata. Epithelial cells detached with EDTA alone adhered significantly better than those detached with trypsin. By day 5, epithelial cell proliferation on ELP was significantly greater than that on plain glass. Epithelial cells grown on ELP expressed conjunctival and adhesion markers. Conclusions: The recombinant ELP resembling the ocular surface extracellular matrix was a suitable substratum to sustain epithelial cell attachment and growth. This type of polymer may be suitable for tissue engineering to restore vision by reconstructing the ocular surface.

Influence of microgroove dimension on cell behavior of human gingival fibroblasts cultured on titanium substrata

The purpose of this study was to determine the dimension of surface microgrooves on titanium (Ti) substrata that shows the greatest positive influence on characterizing specific cell behavior of cultured human gingival fibroblasts. Commercially pure Ti disks with surface microgrooves of monotonous 3.5 mm in depth and respective 15, 30, and 60 microm in width were fabricated using photolithography and used as the culture substrata in the three experimental groups in this study (TiD15, TiD30, and TiD60 groups), whereas the smooth Ti disk was used as the control substrata (smooth Ti group). Human gingival fibroblasts were cultured on the four groups of Ti substrata on successive timelines. Cell behaviors, such as adhesion, morphology,viability and proliferation, and gene expression were analyzed and compared between all groups using crystal violet stain, scanning electron microscopy (SEM), XTT assay, and reverse transcriptase-polymerase chain reaction, respectively. SEM demonstrated that cells were able to readily descend into the microgrooves of TiD30 at the early phase of culture. Cells on the ridge edges or in groove corners were spindle shaped with abundant filopodia formation toward the acid-etched surface inside the microgrooves, thus mimicked the cell shape in three-dimensional (3D) nanoenvironment. TiD15 significantly increased the cell viability and proliferation compared with the smooth Ti substrata after 72 h of culture. Up-regulation of fibronectin(FN) and alpha5 integrin genes was noted in cells cultured on TiD15 and TiD30. Gene expression pattern specific to the cells in 3D-matrix culture, such as down-regulation of alpha-smooth muscle actin gene along with up-regulation of FN and p21 genes, was pronounced in cells cultured on TiD30. This study indicates that surface microgrooves of both 15 and 30 microm in widt hand a monotonous 3.5 microm in depth on Ti substrata increase various cell behaviors of cultured human gingival fibroblasts.

COMPARISON OF PLGA, PCL, AND CHITOSAN IN SALIVARY GLAND BRANCHING MORPHOGENESIS

Branching morphogenesis is a fundamental morphogenetic process in generating glandular tissues. Although the mechanism of branching morphogenesis has been well-explored in the salivary gland development, its interaction with different biodegradable materials has never been investigated. For the purpose of salivary gland regeneration, recapitulation of morphogenetic processes on biodegradable materials might be requisite. Toward this aim, biodegradable biomaterials including poly-lactic-co-glycolic acid (PLGA), poly-epsilon-caprolactone (PCL), and chitosan were examined in the submandibular gland (SMG) culture systems to elucidate their possible impact on salivary morphogenesis. It was found that when SMG explants were cultured on PLGA and PCL, the explants failed to form well-developed branching phenotypes with limited cell migration (5.6 ± 8.8 μm; 10.0 ± 14.1 μm) and decreasing cell viability (56.9% ± 12.5%; 50.3% ± 8.1%). On the contrary, explants cultured on chitosan showed well-developed branches, which were superior in number to those on the control substrata, without any alteration of the morphogenetic phenotypes. Furthermore, the increased cell migration (267.8 ± 45.2 μm) and explants viability (146.8% ± 18.4%) along with the greater deposition of type III collagen, altogether account for better SMG morphogenesis on chitosan. According to the results, it was found that branching morphogenesis of SMG was affected by different biodegradable materials. Chitosan might be an appropriate biodegradable material for salivary morphogenesis, and has applicable potential in the regeneration of salivary tissue.

Bombyx moriSilk Fibroin Membranes as Potential Substrata for Epithelial Constructs Used in the Management of Ocular Surface Disorders

Membranes were prepared from fibroin, a protein isolated from the domesticated silkworm (Bombyx mori) silk, and evaluated as a potential substratum for corneal limbal epithelial cells. These membranes (i.e., B. mori silk fibroin [BMSF] membranes) were cast from dialyzed solutions of fibroin protein (4% w/v) dispensed into 35-mm-diameter culture dishes and dried at room temperature (23-24 degrees C). The resulting material was transparent, easy to handle, and supported levels of human limbal epithelial (HLE) cell growth comparable to that observed on tissue culture plastic. Remarkably, these results were obtained utilizing a commercial serum-free medium (CnT-20) designed for the ex vivo expansion of corneal epithelial progenitor cells. The potential benefits of serum proteins on this culture system were examined through addition of fetal bovine serum (FBS) either to fibroin stocks prior to membrane casting or by supplementation of the CnT-20 medium. Membranes cast in the presence of FBS displayed increasing opacity and induced little change in HLE growth. Supplementation of CnT-20 medium with FBS deterred cell growth on all substrata, including tissue culture plastic control substrata. The remarkable properties of BMSF membranes demonstrated under serum-free conditions warrant investigation of this material as a substratum in the creation of tissue-engineered constructs for the restoration of diseased or damaged ocular surface.