Effect of crystallographic phases of TiO2 on hepatocyte attachment, proliferation and morphology

In this study, the effect of the crystallographic phases of titanium dioxide (TiO(2)) on hepatocyte response was investigated by culturing cells in a medium containing a dissolution of rutile TiO(2) powders, and on anatase and rutile ceramic discs. When the concentration of Ti ions exceeded 0.16 microg/mL, TiO(2) exhibited cytotoxicity. However, in a range of concentrations between 10(-6) and 10(-3)microg/mL, the Ti ion leached out from the powder stimulated cell proliferation. Cytocompatibility was also examined by the cell attachment and proliferation on the TiO(2) discs. The results show that the surface characteristic of the TiO(2) discs was the main factor influencing cell attachment, and the cells were better attached on the dense surface of the discs with high crystallinity than on the loose surface with low crystallinity. Both the surface characteristic and the crystallographic phase affected cell proliferation. In addition to the dense surface with high crystallinity, the anatase ceramics was in favor of cell proliferation as compared to the rutile ceramics sintered at the same temperature. In conclusion, the results suggest that the dense rutile ceramic with high crystallinity is a good substrate for hepatocytes.     

Effects of hypoxia on proliferation and differentiation of myoblasts

Oxygen is an environmental and developmental signal regulator, and its role is involved in energy homeostasis, development and process of differentiation. Myoblasts persist in skeletal muscle as satellite cells, which possess capability of self-renewing and differentiation into mature myofiber. Myoblasts play a critical role in postnatal muscle regeneration after injury as well as maintaining myofibers' function. Though oxygen is vital to nearly all forms of life, studies focused on investigating the effects of oxygen level on proliferation and differentiation of myoblasts are few. Lower oxygen concentration is more close to the level of oxygen in physiological and pathological environment in vivo. So physiological environment is actually optimum condition for myogenesis. It is significant for understanding repair and regeneration of skeletal muscle to study on effects of hypoxia on myogenesis. HIF-1 signaling pathway was involved in these processes as well as other signaling pathways would be, and accordingly, deep studying and further revealing the signaling pathways involved in mechanism will provide evidences or references for looking for novel targets for stem cells therapy and drug treatment.     

Isolation, growth and differentiation of adult rabbit skeletal myoblasts in vitro

A simple and reliable method is described for the growth and differentiation of myoblasts isolated from adult New Zealand White rabbit fast-twitch (tibialis anterior) and slow-twitch (soleus) skeletal muscle. Cells were dissociated mechanically, and expanded in DMEM supplemented with 20% horse serum. The myoblasts were differentiated by switching to DMEM supplemented with 10% horse serum when the myoblasts were 80–90% confluent. The myoblasts fused into multinucleated myotubes that spontaneously contracted. At the light microscopic level, the myotubes exhibited a striated pattern as revealed by positive immunostaining for sarcomeric proteins such as -actinin, myosin and titin.Electron microscopy demonstrated well ordered sarcomeres with localized mitochondria and sarcoplasmic reticulum. The only notable difference between the fiber types was in the initial response to isolation wherein the myoblast yield was four-fold greater from the soleus than the tibialis anterior, in agreement with satellite cell abundance in predominately slow-twitch vs fast-twitch muscles. These techniques repeatedly (n = 12) produced a population of healthy myoblasts isolated from either fast- or slow-twitch skeletal muscle which can be utilized for studies of skeletal muscle differentiation, assembly and signaling.     

Polyaniline, an electroactive polymer, supports adhesion and proliferation of cardiac myoblasts

Conductive polymers, such as polypyrrole, have recently been studied as potential surfaces/matrices for cell- and tissue-culture applications. We have investigated the adhesion and proliferation properties of H9c2 cardiac myoblasts on a conductive polyaniline substrate. Both the non-conductive emeraldine base (PANi) and its conductive salt (E-PANi) forms of polyaniline were found to be biocompatible, viz., allowing for cell attachment and proliferation and, in the case of E-PANi, maintaining electrical conductivity. By comparison to tissue-culture-treated polystyrene (TCP), the initial adhesion of H9c2 cells to both PANi and E-PANi was slightly reduced by 7% (P < 0.05, n = 18). By contrast, the overall rate of cell proliferation on the conductive surfaces, although initially decreased, was similar to control TCP surfaces. After 6 days in culture on the different surfaces, the cells formed confluent monolayers which were morphologically indistinguishable. Furthermore, we observed that E-PANi, when maintained in an aqueous physiologic environment, retained a significant level of electrical conductivity for at least 100 h, even though this conductivity gradually decreased by about 3 orders of magnitude over time. These results demonstrate the potential for using polyaniline as an electroactive polymer in the culture of excitable cells and open the possibility of using this material as an electroactive scaffold for cardiac and/or neuronal tissue engineering applications that require biocompatibility of conductive polymers.     

Polyaniline,an electroactive polymer,supports adhesion and proliferation of cardiac myoblasts

Conductive polymers, such as polypyrrole, have recently been studied as potential surfaces/matrices for cell- and tissue-culture applications. We have investigated the adhesion and proliferation properties of H9c2 cardiac myoblasts on a conductive polyaniline substrate. Both the non-conductive emeraldine base (PANi) and its conductive salt (E-PANi) forms of polyaniline were found to be biocompatible, viz., allowing for cell attachment and proliferation and, in the case of E-PANi, maintaining electrical conductivity. By comparison to tissue-culture-treated polystyrene (TCP), the initial adhesion of H9c2 cells to both PANi and E-PANi was slightly reduced by 7% (P < 0.05, n = 18). By contrast, the overall rate of cell proliferation on the conductive surfaces, although initially decreased, was similar to control TCP surfaces. After 6 days in culture on the different surfaces, the cells formed confluent monolayers which were morphologically indistinguishable. Furthermore, we observed that E-PANi, when maintained in an aqueous physiologic environment, retained a significant level of electrical conductivity for at least 100 h, even though this conductivity gradually decreased by about 3 orders of magnitude over time. These results demonstrate the potential for using polyaniline as an electroactive polymer in the culture of excitable cells and open the possibility of using this material as an electroactive scaffold for cardiac and/or neuronal tissue engineering applications that require biocompatibility of conductive polymers.     

CMF1-Rb interaction promotes myogenesis in avian skeletal myoblasts.

CMF1 protein is expressed in developing striated muscle before the expression of contractile proteins, and depletion of CMF1 in myoblasts results in inability to express muscle-specific proteins. Previous studies of CMF1 identify a functional Rb-binding domain, which is conserved in the murine and human homologues. Here, we show that CMF1 binds Rb family members, while a CMF1 protein with deletion of the Rb-binding domain (Rb-del CMF1) does not. Myogenic cell lines over-expressing Rb-del CMF1 proliferate normally, but exhibit markedly impaired differentiation, including dramatically reduced contractile proteins gene expression and failure to fuse into myotubes. Furthermore, by quantitative real-time polymerase chain reaction, MyoD and Myf5 mRNA levels are comparable to wild-type, while myogenin and contractile protein mRNA levels are significantly attenuated. These data demonstrate that CMF1 regulates myocyte differentiation by interaction with Rb family members to induce expression of myogenic regulatory factors.     

Myotubularin-deficient myoblasts display increased apoptosis, delayed proliferation, and poor cell engraftment

X-linked myotubular myopathy is a severe congenital myopathy caused by deficiency of the lipid phosphatase, myotubularin. Recent studies of human tissue and animal models have discovered structural and physiological abnormalities in myotubularin-deficient muscle, but the impact of myotubularin deficiency on myogenic stem cells within muscles is unclear. In the present study, we evaluated the viability, proliferative capacity, and in vivo engraftment of myogenic cells obtained from severely symptomatic (Mtm1δ4) myotubularin-deficient mice. Mtm1δ4 muscle contains fewer myogenic cells than wild-type (WT) littermates, and the number of myogenic cells decreases with age. The behavior of Mtm1δ4 myoblasts is also abnormal, because they engraft poorly into C57BL/6/Rag1null/mdx5cv mice and display decreased proliferation and increased apoptosis compared with WT myoblasts. Evaluation of Mtm1δ4 animals at 21 and 42 days of life detected fewer satellite cells in Mtm1δ4 muscle compared with WT littermates, and the decrease in satellite cells correlated with progression of disease. In addition, analysis of WT and Mtm1δ4 regeneration after injury detected similar abnormalities of satellite cell function, with fewer satellite cells, fewer dividing cells, and increased apoptotic cells in Mtm1δ4 muscle. These studies demonstrate specific abnormalities in myogenic cell number and behavior that may relate to the progression of disease in myotubularin deficiency, and may also be used to develop in vitro assays by which novel treatment strategies can be assessed.     

Effect of surface processing on the attachment, orientation, and proliferation of human gingival fibroblasts on titanium.

The adhesion, orientation, and proliferation of human gingival fibroblasts was studied on electropolished (elpTi), etched (etchTi), and sandblasted (sblTi) titanium surfaces. The texture, chemical state, and composition of the titanium surfaces were analyzed using a surface tracing instrument and electron spectroscopy for chemical analysis. Considerable differences were evident in the surface texture and chemical composition of the differently treated titanium plates. Electropolishing produced the smoothest and cleanest surface. Human gingival fibroblasts attached, spread, and proliferated on all titanium surfaces. However, cells on elpTi exhibited an extremely flat morphology and seemed to form cellular bridges with adjacent cells, whereas the etchTi and sblTi surfaces harbored both round and flat cells with many long processes. Cells on elpTi appeared to grow in thick layers with no specific orientation, whereas on etchTi surfaces they were migrating along the parallel, irregular minor grooves caused by mechanical polishing, and on sblTi surfaces they seemed to grow in clusters. Stress-fiber type actin bundles and vinculin-containing focal adhesions were present in cells spreading on elpTi and etchTi surfaces but not in cells spreading on sblTi surfaces. Cell shape, orientation, and proliferation appear to depend on the texture of the titanium surface and probably also on the properties of the oxide layer and adjacent bulk material. Our findings suggest that smooth or finely grooved titanium surfaces could be optimal in implants adjacent to soft tissues as they support the attachment and growth of human gingival fibroblasts.     

Effects of hydroxylapatite coating crystallinity on biosolubility, cell attachment efficiency and proliferation in vitro.

A hydroxylapatite (HA) coating with approximately 97% crystalline HA content (MP-1 treated HA coating, MP-HA) was tested in vitro for its biosolubility and cellular biocompatibility. The MP-HA coating was compared with a standard HA coating with approximately 63% crystallinity (SHA) and an amorphous HA coating with approximately 25% crystallinity (AHA), as well as a titanium (Ti) surface without HA coating as a control. The topographic study with scanning electron microscopy indicated that MP-HA appeared more coarse, with projected nodules which altered the shape of cells attached to the substrate. Biosolubility study indicated that MP-HA had the least effect on the culture medium pH, while AHA (P < 0.01) and SHA (P < 0.05) significantly raised the medium pH up to 8.2 and 7.75, respectively. X-ray diffraction (XRD) analysis showed essentially unchanged levels of the total soluble phases of all coatings after incubation with culture medium, except that the CaO phase was rapidly dissolved from AHA coatings and completely eliminated from SHA coatings. Cultures of human gingival fibroblasts on these HA coatings showed that MP-HA and SHA had about the same cell attachment efficiency which was relatively lower than that of AHA coatings. MP-HA generated significant higher cell proliferation rate relative to AHA (P < 0.01) and SHA (P < 0.05). This study indicated that surface chemistry and topography of lower crystallinity might be favorable to cell attachment, but that elevated medium pH might result in a cytotoxic effect that inhibits the proliferation of attached cells on coating surfaces.     

Effect of procyanidolic oligomers on cultured mesenchymal cells. I. Effect on attachment, proliferation and detachment of cells

The effect of procyanidolic oligomers (OPC) was studied on mesenchymal cells in culture: human skin fibroblasts (FB) and porcine aorta smooth muscle cells (CML). In presence of OPC part of the freshly seeded FB did not attach. There was no significant effect on the attachment of CML. Proliferation of FB was also slowed down in a dose-dependent manner. Proliferation of CML-s was also decreased, but much less than for FB-s. The detachment of the cells was also studied by adding trypsin to previously attached cells. Detachment of FB-s was strongly inhibited in presence of OPC in a dose-dependent manner. Much less effect was seen on CML. It appears therefore that OPC may interact with some components of the FB cell membrane and modify the attachment, proliferation and detachment of these cells. The only cell kinetic parameter significantly influenced by OPC for CML was their rate of proliferation. This may be due to the different constitution of the CML cell surface as compared to the FB cell surface.     

Pitx2c overexpression promotes cell proliferation and arrests differentiation in myoblasts.

Pitx2 is a paired-related homeobox gene that has been shown to play a central role during development. In the mouse, there are three isoforms, Pitx2a, b, and c, which differ only in their amino terminal regions. Pitx2 is expressed in myotomes, myoblasts, and myofibers and may be involved in muscle patterning. However, the mechanism by which Pitx2 acts in muscle cell lineages as well as the distinct functions of the individual isoforms have not been investigated. In this study, we used Sol8 myoblasts to investigate the function of Pitx2 in skeletal myogenesis. We found that Pitx2c is the main Pitx2 isoform present in Sol8 myoblasts. Overexpression of Pitx2c in Sol8 myoblasts inhibited myocyte differentiation and myotube formation. Furthermore, Sol8 cells overexpressing Pitx2c maintained high proliferative capacity and a significant up-regulation of the cell cycle genes cyclin D1, cyclin D2, and c-myc. Gene expression analysis for Pax3 and the s MyoD and myogenin showed that Pitx2c-overexpression caused Sol8 cells to remain as myoblasts, in an undifferentiated myogenic state. Furthermore, down-regulation of the muscle-specific genes sTnI and MyHC3 demonstrated that Sol8-overexpressing Pitx2c myoblasts failed to reach terminal differentiation. This study sheds light on previously unknown functions of the Pitx2c isoform in balancing proliferation vs. differentiation in a myogenic cell line.     

Membranes for biohybrid liver support systems--investigations on hepatocyte attachment,morphology and growth

The biological properties of four different membranes were studied regarding their possible application in biohybrid liver support systems. Two of them, one made of polyetherimide (PEI), and a second based on polyacrylonitrile-N-vinylpyrollidone co-polymer (P(AN-NVP)), were recently developed in our lab and studied for the first time. Together with pure polyacrylonitrile (PAN) membranes, the three preparations were characterised as ultra-filtration membranes. Their ability to support cell attachment, morphology, proliferation and function of human hepatoblastoma C3A cells was studied. The role of surface morphology for the interaction with hepatocytes was highlighted using a commercial, moderately wettable polyvinylidendifluoride (PVDF) membrane with micro-filtration properties. Comparative investigations showed strongest interaction of C3A cells with PAN membranes, as the focal adhesion contacts were more expressed and cell growth was also high. However, the functional activity in terms of albumin synthesis was reduced. Very similar results were obtained with the most hydrophobic PEI membrane. In contrast, the most hydrophilic membrane P(AN-NVP) was found to provoke stronger homotypic adhesion (E-cadherin expression) of C3A cells and less substratum attachment (focal adhesions), but enhanced albumin secretion. However, proliferation of C3A cells was lowered. Micro-porous PVDF membrane showed very good initial attachment, but the resulting cell material and cell-cell interaction were relatively poor developed. Among four membranes tested, PEI seems to be the most attractive membrane for biohybrid liver devices, as it provides good surface properties for hepatocytes interaction, but in addition it is highly thermostable, which would permit steam sterilisation. No simple relationship, however, between the wettability of the membranes and their ability to support hepatocyte adhesion and function was found in this study.     

Fibronectin silanized titanium alloy: a bioinductive and durable coating to enhance fibroblast attachment in vitro

Long term success of percutaneous implants is dependent on soft tissue attachment to prevent infection and epithelial downgrowth, which leads to failure of the implant. Fibronectin coatings are known to enhance fibroblast attachment in vitro, but are subject to desorption from serum protein competition in vivo. This paper quantifies the binding of fibronectin to titanium alloy by silanization and the durability of this attachment when soaked in protein-rich fluid compared with adsorbed fibronectin. The biological activity of fibronectin bound to silanized titanium alloy was confirmed by analyzing cell area, morphology, immunolocalization of focal contacts, and metabolism of dermal fibroblasts. This was compared with both adsorbed fibronectin and uncoated titanium alloy. Silanized titanium alloy bound over twice the amount of fibronectin compared to untreated titanium alloy. On soaking in fetal calf serum there was no significant loss of fibronectin (p = 0.589) from the silanized surface but a significant 44% loss (p = 0.002) from untreated surfaces. Fibroblasts on silanized fibronectin had significantly larger cell areas and more vinculin focal contact markers when compared to untreated surfaces (p < 0.005). The results confirm the durability of silanized fibronectin from protein competition and bioactive effect on fibroblasts.     

大鼠骨骼肌成肌细胞与乳鼠心肌细胞共培养对缝隙连接蛋白43表达的影响

目的 观察体外共培养的乳鼠心肌细胞与大鼠骨骼肌成肌细胞(简称L6细胞)以及乳鼠心肌细胞与转染外源性缝隙连接蛋白43(Cx43)基因的L6细胞(简称L6-Cx43细胞)间Cx43蛋白的表达情况,从而探讨共培养时心肌环境对成肌细胞的作用.方法 将乳鼠心肌细胞与4′,6-二脒基-2-苯基吲哚标记的大鼠骨骼肌成肌细胞进行共培养...     

Fibroblast growth factor-2-overexpressing myoblasts encapsulated in alginate spheres increase proliferation, reduce apoptosis, induce adipogenesis, and enhance regeneration following skeletal muscle injury in rats

The fibroblast growth factor 2 (FGF-2) is known as pleiotropic cytokine with myoblast proliferative properties. In the present study, we tested the hypothesis that gene transfer of human FGF-2 via transplantation of genetically modified L8-myoblast encapsulated in alginate modulates the skeletal muscle recovery after crush injury in Wistar rats. Therefore, we performed a crush injury to the soleus muscle and transplanted alginate spheres containing myoblasts genetically modified to overexpress human FGF-2 (FGF-2) or a luciferase (LUC) cDNA at the site of injury. Animals that underwent muscle injury without transplantation of alginate spheres served as control (control). At day 4 after trauma the FGF-2 group showed significant higher mean values of cell proliferation (bromodeoxyuridine immunohistochemistry) and significant lower values of cell apoptosis (terminal deoxynucleotidyl transferase nick end labeling histology) compared to animals receiving luciferase-overexpressing myoblasts. At the same time point adiponectin expression (ACRP30 immunohistochemistry) was increased in the FGF-2 group exclusively. The p75(NTR) expression (p75(NTR) immunohistochemistry) significantly improved in both the FGF-2 and LUC group compared to the control group. Functional analysis of the injured muscle did not reveal a significant increase of the muscle force in the FGF-2 group compared to the control and LUC group 14 days after injury. In vitro analysis for 14 days of the FGF-2-modified spheres demonstrated at day 7 and day 14 a significant increase of the relative cell count as well as of the relative viable cell count in the FGF-2 myoblast spheres compared to luciferase myoblast spheres. Additionally, the expression of FGF-2 (enzyme-linked immunosorbent assay analysis) and luciferase (chemiluminescence analysis) persisted in vitro for 4 and 14 days, respectively. These results demonstrate that FGF-2-overexpressing myoblasts cannot considerably improve muscle strength but are able to modulate the proliferation as well as the apoptosis of injured muscle tissue mainly by conducting adipogenesis.     

Effect of penicillin on the succession, attachment, and morphology of segmented, filamentous microbes in the murine small bowel.

Indigenous segmented filamentous microbes attach to murine ileal epithelial cells. These microbes can be seen on the epithelial surface with a scanning electron microscope. They colonize preferentially the distal ileum in mice. Penicillin, placed in the animal's drinking water, eliminates the microbes from the mouse ileum, but recolinization of the ileum is observed 4 to 5 weeks after the penicillin treatment is stopped. Within 3 to 5 h after rats are given penicillin, the morphology of the microbes is changed. Their external surfaces are wrinkled or broken. Vacated and partially vacated attachment sites are observed. Almost all of the organisms disappear from murine ilea after the animals are exposed to penicillin for 10 h. These observations are discussed in relation to the microbe itself and in its interaction with ileal epithelial cells.     

Effect of ProNectin F derivatives on cell attachment and proliferation

ProNectin F (PnF) was chemically modified by introducing some functional groups to prepare various derivatives of primary amino (PnF-N₁), tertiary amino (PnF-N₃), quaternary ammonium (PnF-N₄), carboxyl (PnF-COOH) and sulfonyl groups (PnF-SO₃H). When C3H10T1/2 cells were cultured on non-treated dishes coated with the derivatives, the number of mesenchymal cells attached to the culture dishes increased for the coating with PnF-COOH and PnF-SO₃H, even at their low adsorption amount. The cytotoxicity was high for the coating of PnF-N₁ and PnF-N₄ compared with that of the PnF-N₃, PnF-COOH and PnF-SO₃H. The treatment with integrin α5 and αV antibodies suppressed the cell attachment to the dishes coated with PnF-COOH and PnF-SO₃H. The phosphorylation of extracellular signal-regulated kinase (ERK) was upregulated for cells attached to the dishes coated with PnF-COOH and PnF-SO₃H, indicating their enhanced proliferation. It is concluded that the chemical derivatization of PnF enhanced the ability of cell attachment and proliferation.