Extent Of Cell Proliferation Research Articles

An in vitro study into three different PRF preparations for osteogenesis potential.

To investigate the effect of Advanced Platelet-Rich Fibrin (A-PRF+), Leukocyte Platelet-Rich Fibrin (L-PRF), and injectable Platelet-Rich Fibrin (i-PRF) on osteogenesis of a human osteoblast-like cell line in vitro. Different PRF protocols are used in clinical dentistry in the last years. Recent literature documented the positive impact of PRF derivatives in vivo and in vitro, on different types of cells. However, hardly any literature comparing the new protocols for PRF (the A-PRF+ and i-PRF) with the original protocol of PRF (L-PRF) is present for osteoblast-like cells. A-PRF+, L-PRF, and i-PRF were prepared from six male donors and pre-cultured with 10mL culture medium for 6 days. 5 x103 cells/ml osteoblasts from the osteoblast cell line (U2OS) were seeded and cultured either with conditioned medium derived from the different PRF conditions or with regular culture medium. At five different time points (0, 7, 14, 21, 28 days), the osteogenic capacity of the cells was assessed with Alizarin Red S to visualize mineralization. Also in these cells, the calcium concentration and alkaline phosphatase activity were investigated. Using qPCR, the expression of alkaline phosphatase, osteocalcin, osteonectin, ICAM-1, RUNX-2, and collagen 1a was assessed. In osteoblast-like cells cultured with conditioned medium, the A-PRF+ conditioned medium induced more mineralization and calcium production after 28 days of culturing compared with the control (p< .05). No significant differences were found in the extent of cell proliferation between the different conditions. RUNX-2 and osteonectin mRNA expression in the cells were lower in all PRF-stimulated cultures compared with control at different time points. The i-PRF-conditioned medium induced more ALP activity (p< .05) compared with control and osteoblasts-like cells differentiated more compared with osteoblasts cultured with L-PRF. The three PRF preparations seem to have the capacity to increase the osteogenic potential of osteoblast-like cells. A-PRF+ seems to have the highest potential for mineralization, while i-PRF seems to have the potential to enhance early cell differentiation.

GABAergic regulation of cell proliferation within the adult mouse spinal cord

Manipulation of neural stem cell proliferation and differentiation in the postnatal CNS is receiving significant attention due to therapeutic potential. In the spinal cord, such manipulations may promote repair in conditions such as multiple sclerosis or spinal cord injury, but may also limit excessive cell proliferation contributing to tumours such as ependymomas. We show that when ambient γ-aminobutyric acid (GABA) is increased in vigabatrin-treated or decreased by GAD67 allele haplodeficiency in glutamic acid decarboxylase67-green fluorescent protein (GAD67-GFP) mice of either sex, the numbers of proliferating cells respectively decreased or increased. Thus, intrinsic spinal cord GABA levels are correlated with the extent of cell proliferation, providing important evidence for manipulating these levels. Diazepam binding inhibitor, an endogenous protein that interacts with GABA receptors and its breakdown product, octadecaneuropeptide, which preferentially activates central benzodiazepine (CBR) sites, were highly expressed in spinal cord, especially in ependymal cells surrounding the central canal. Furthermore, animals with reduced CBR activation via treatment with flumazenil or Ro15-4513, or with a G2F77I mutation in the CBR binding site had greater numbers of Ethynyl-2′-deoxyuridine positive cells compared to control, which maintained their stem cell status since the proportion of newly proliferated cells becoming oligodendrocytes or astrocytes was significantly lower. Altering endogenous GABA levels or modulating GABAergic signalling through specific sites on GABA receptors therefore influences NSC proliferation in the adult spinal cord. These findings provide a basis for further study into how GABAergic signalling could be manipulated to enable spinal cord self-regeneration and recovery or limit pathological proliferative activity.

Modeling, simulations, and optimization of smooth muscle cell tissue engineering for the production of vascular grafts.

The paper presents a transient, continuum, two-phase model of the tissue engineering in fibrous scaffolds, including transport equations for the flowing culture medium, nutrient and cell concentration with transverse and in-plane diffusion and cell migration, a novel feature of local in-plane transport across a phenomenological pore and innovative layer-by-layer cell filling approach. The model is successfully validated for the smooth muscle cell tissue engineering of a vascular graft using crosslinked, electrospun gelatin fiber scaffolds for both static and dynamic cell culture, the latter in a dynamic bioreactor with a rotating shaft on which the tubular scaffold is attached. Parametric studies evaluate the impact of the scaffold microstructure, cell dynamics, oxygen transport, and static or dynamic conditions on the rate and extent of cell proliferation and depth of oxygen accessibility. An optimized scaffold of 75% dry porosity is proposed that can be tissue engineered into a viable and still fully oxygenated graft of the tunica media of the coronary artery within 2 days in the dynamic bioreactor. Such scaffold also matches the mechanical properties of the tunica media of the human coronary artery and the suture retention strength of a saphenous vein, often used as a coronary artery graft.

Amifostine Suppresses the Side Effects of Radiation on BMSCs by Promoting Cell Proliferation and Reducing ROS Production.

This study is aimed at investigating the effect of amifostine (AMI) on rat bone marrow stromal stem cells (BMSCs) exposed to 2 Gy radiation. The BMSCs were divided into four groups, namely, group A that received 0 Gy radiation, group B that received 0 Gy radiation and AMI, group C that received 2 Gy radiation, and group D that received 2 Gy radiation and AMI. The proliferation, apoptosis, and distribution of BMSCs in the cell cycle, along with their osteogenesis ability, adipogenesis ability, and ROS production, were subsequently examined. The levels of ALP, PPARγ, P53, and TNFα were determined by Western blotting. The results demonstrated that the proliferation of BMSCs and the levels of ALP in group C were much lower than those in group A. The production of ROS and levels of PPARγ, P53, and TNFα in the group that received 2 Gy radiation were much higher than those in group A. Furthermore, the production of ROS and the levels of PPARγ, P53, and TNFα were much lower in group D than in group C. Additionally, the levels of ALP and extent of cell proliferation were much higher in group D than in group C. The results demonstrated the potential of AMI in reducing the side effects of radiation in BMSCs and in treatment of bone diseases caused by radiation.

Development of functionalized multi-walled carbon-nanotube-based alginate hydrogels for enabling biomimetic technologies.

Alginate is a hydrogel commonly used for cell culture by ionically crosslinking in the presence of divalent Ca2+ ions. However these alginate gels are mechanically unstable, not permitting their use as scaffolds to engineer robust biological bone, breast, cardiac or tumor tissues. This issue can be addressed via encapsulation of multi-walled carbon nanotubes (MWCNT) serving as a reinforcing phase while being dispersed in a continuous phase of alginate. We hypothesized that adding functionalized MWCNT to alginate, would yield composite gels with distinctively different mechanical, physical and biological characteristics in comparison to alginate alone. Resultant MWCNT-alginate gels were porous, and showed significantly less degradation after 14 days compared to alginate alone. In vitro cell-studies showed enhanced HeLa cell adhesion and proliferation on the MWCNT-alginate compared to alginate. The extent of cell proliferation was greater when cultured atop 1 and 3 mg/ml MWCNT-alginate; although all MWCNT-alginates lead to enhanced cell cluster formation compared to alginate alone. Among all the MWCNT-alginates, the 1 mg/ml gels showed significantly greater stiffness compared to all other cases. These results provide an important basis for the development of the MWCNT-alginates as novel substrates for cell culture applications, cell therapy and tissue engineering.

Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum.

A mutation of the reln gene gives rise to the Reeler mouse (reln−∕−) displaying an ataxic phenotype and cerebellar hypoplasia. We have characterized the neurochemistry of postnatal (P0–P60) reln−∕− mouse cerebella with specific attention to the intervention of cell proliferation and apoptosis in the P0–P25 interval. Homozygous reln−∕− mice and age-matched controls were analyzed by immunofluorescence using primary antibodies against NeuN, calbindin, GFAP, vimentin, SMI32, and GAD67. Proliferation and apoptosis were detected after a single intraperitoneal BrdU injection and by the TUNEL assay with anti-digoxigenin rhodamine-conjugated antibodies. Quantitative analysis with descriptive and predictive statistics was used to calculate cell densities (number/mm2) after fluorescent nuclear stain (TCD, total cell density), labeling with BrdU (PrCD, proliferating cell density), or TUNEL (ApoCD, apoptotic cell density). By this approach we first have shown that the temporal pattern of expression of neuronal/glial markers in postnatal cerebellum is not affected by the Reeler mutation. Then, we have demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to reduction of cortical size and cellularity (TCD), and that TCD is, in turn, linked to quantitative differences in the extent of cell proliferation and apoptosis, as well as derangements in their temporal trends during postnatal maturation. Finally, we have calculated that PrCD is the most important predictive factor to determine TCD in the cerebellar cortex of the mutants. These results support the notion that, beside the well-known consequences onto the migration of the cerebellar neurons, the lack of Reelin results in a measurable deficit in neural proliferation.

Therapeutic Efficacy of Orally Delivered Doxorubicin Nanoparticles in Rat Tongue Cancer Induced by 4-Nitroquinoline 1-Oxide.

Oral cancer is one of the most significant cancers in the world, and squamous cell carcinoma makes up about 94% of oral malignancies. The aim of the present study was to compare the efficacy of doxorubicin plus methotrexate - loaded nanoparticles on tongue squamous cell carcinoma induced by 4NQO and compare it with the commercial doxorubicin and methotrexate delivered orally on seventy SD male rats. 70 rats were divided into five groups. During the study, the animals were weighed by a digital scale once a week. Number of mortalities was recorded in the data collection forms. At the end of the treatment, biopsy samples were taken from rat tongues in order to evaluate the severity of dysplasia and the extent of cell proliferation. The results were analyzed using ANOVA, descriptive statistics and chi-square test. No statistically significant difference was found in the mean weight of five groups (p>0.05). No significant relationship was found between groups and mortality rate (P = 0. 39). In addition, there was a significant relationship between groups and the degree of dysplasia (P <0.001). The statistical analysis showed a significant relationship between groups and the rate of cell proliferation (p <0.001). The results of the present study showed that the use of doxorubicin plus methotrexate - loaded nanoparticles orally had more therapeutic effects than commercial doxorubicin plus methotrexate.

Wound healing potential of scaffolds prepared from porcine jejunum and urinary bladder by a non-detergent/enzymatic method.

Scaffolds prepared using extracellular matrices of mammalian organs/tissues, when used as grafts, have wound healing potential. This paper evaluated the physical properties and invivo wound healing potential of jejunum-derived scaffold (JDS) and urinary bladder-derived scaffold (UDS) of porcine origin prepared by a non-detergent/enzymatic method. The former had higher flexural rigidity and suture retention strength compared to the latter, but both of them had the essential flexural rigidity and suture retention strength required for skin grafts. Full thickness skin-wounds on rabbit dorsum were treated with these scaffolds and the wound healing ability was compared by studying histomorphology parameters such as re-epithelialisation, collagen deposition, angiogenesis, proliferation of cells, mesenchymal cell infiltration and myofibroblast response. The extent of these reactions was assessed using histomorphometry. The results indicated that both grafts initiated healing faster than those wounds without any graft, as evidenced by the extent of cell proliferation and mesenchymal cell infiltration. The myofibroblast response persisted longer in the non-graft assisted wound healing reaction compared to the healing in the graft assisted wounds. Moreover, the JDS induced higher cell proliferation and greater angiogenesis than UDS probably indicating better healing by the former. The results suggested that JDS and UDS prepared by non-detergent/enzymatic method have potential clinical applications.

Microarray analysis reveals that high mobility group A1 is involved in colorectal cancer metastasis

Tumor size indicates the extent of cell proliferation in most cases of colorectal cancer (CRC), although there are some advanced small tumors with metastases. Lymph node metastasis is a significant factor that greatly impacts disease prognosis in CRC cases. The underlying factors that cause lymph node metastasis in CRC cells are not fully understood. We investigated the mechanism that might induce CRC metastasis by focusing on smaller sized (<2 cm) invasive tumors. We carried out gene expression array analysis for CRC cases; group 1 consisted of 6 cases with tumors <2 cm with metastases, and group 2 consisted of 65 cases with tumors >2 cm without metastases. Results were validated using gene expression array data from an additional 77 cases and another bulk case set of 172 cases. Gene ontology and pathway analysis using microarray data revealed that anti-apoptotic activity had a crucial role in CRC metastasis. High mobility group A1 (HMGA1) was identified as a biomarker for poor prognosis and metastasis formation. HMGA1 expression levels were higher in lymph node-positive cases than in lymph node-negative cases, even in subgroup analysis of submucosal invasive cases. The present study strongly supports the clinical significance of HMGA1 expression as a predictive indicator of lymph node metastasis in CRC cases, even in submucosal invasive cases which could be cured by local resection.

THE EFFECTS OF MORINDA CITRIFOLIA, MOMORDICA CHARANTIA AND CENTELLA ASIATICA EXTRACTS ON LIPOPROTEIN LIPASE AND 3T3-L1 PREADIPOCYTES

ABSTRACT The effect of Morinda citrifolia fruit (MCFE), Momordica charantia (MCE) and Centella asiatica (CAE) extracts on lipoprotein lipase (LPL) activity, proliferation and differentiation of 3T3-L1 preadipocytes was investigated. The inhibitory effect of the extracts on LPL activity was determined in vitro. The extent of cell proliferation was determined utilizing 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the degree of differentiation was measured by Oil Red O staining assay. Extracts were analyzed for total phenolic compounds and flavonoids utilizing Folin–Ciocalteu assay and high-performance liquid chromatography, respectively. Results showed that, at 1 mg/mL, MCFE demonstrated the highest inhibitory effect (21.4 ± 2.3%), compared to the other extracts tested. On the other hand, MCE demonstrated the highest inhibitory effect on both proliferation and differentiation of preadipocytes (LC50of 1.6 mg/L). LC50 refers to the concentration of extracts (mg/mL) to cause a 50% reduction in viable cells. Results also revealed that all extracts consisted of high concentration of phenolic compounds, with catechin and epicatechin predominantly present in all three extracts, that maybe responsible for the activities measured. PRACTICAL APPLICATION Obesity has become an important public health issue, being a risk factor for various types of cancer, cardiovascular diseases, diabetes and metabolic disorders. Classical weight loss programs, such as low-fat diets, often fail to achieve a long-term maintenance of weight loss. As a consequence of that, alternative such as the use of natural products in prevention and treatment of obesity is studied. Results from the study indicate that both Momordica charantia and Morinda citrifolia fruit extracts exert antiobesity effects by inhibiting proliferation and differentiation of preadipocytes and lipoprotein lipase activity in vitro, respectively. Both fruits have great potential to be used as the main ingredient in developing functional foods for antiobesity purposes.

Regulation of hippocampal progenitor cell survival, proliferation and dendritic development by BDNF

BackgroundEnvironmental enrichment (EE) is known to enhance BDNF levels and neurogenesis in the adult hippocampus. To examine the role of BDNF in modulating EE-mediated adult hippocampal neurogenesis, we conditionally ablated BDNF expression in the hippocampus (cKO mice) and have assessed proliferation, survival, differentiation and dendritic development of hippocampal progenitors.ResultsWe show that while the extent of cell proliferation and neuronal fate differentiation in the hippocampus of cKO mice is not different from wild-type (WT) littermates maintained in either standard or enriched conditions, reduced BDNF levels significantly impaired the survival of newborn cells in both housing conditions. In addition, while highly active enriched WT mice exhibited a robust increase in progenitor cell proliferation, highly active cKO mice showed a modest increase in cell proliferation compared to standard housed or underactive cKO mice.ConclusionsThere results argue that while BDNF plays a role in exercise-induced cell proliferation, other factors must contribute to this phenomenon. We also show that dendritic development was impaired in cKO mice maintained in standard housing conditions, and that EE rescued this phenotype.

Proliferation, Osteogenic Differentiation, and Distribution of Rat Bone Marrow Stromal Cells in Nonwoven Fabrics by Different Culture Methods

The proliferation, osteogenic differentiation, and distribution patterns of stromal cells from rat bone marrow were investigated in a three-dimensional nonwoven fabric of polyethylene terephthalate fiber by the static, agitated, and stirred culture methods; stirring speeds were 10, 50, and 100 rpm in the stirred culture method. The culture method affected the time profile of proliferation and osteogenic differentiation of cells or their distribution in the fabric. The extent of cell proliferation and osteogenic differentiation became higher in order of the stirred at 100 rpm = the stirred at 50 rpm > the stirred at 10 rpm > the agitated > the static methods. In addition, the cells were more uniformly proliferated in the fabric by the stirred culture method with time than they were proliferated in the fabric by other methods. The alkaline phosphatase (ALP) activity and calcium content were higher for cells cultured by the stirred culture method than those cultured by other methods. The total ALP activity, calcium content, and bone mineral density were higher for every stirred method than those for other methods. However, the distribution uniformity of cells differentiated was low irrespective of the culture method. It is concluded that the extent of proliferation and differentiation of cells or their distribution uniformity in the nonwoven fabrics was influenced by the culture method.

International Union of Pharmacology. LXI. Peroxisome Proliferator-Activated Receptors

The three peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors of the nuclear hormone receptor superfamily. They share a high degree of structural homology with all members of the superfamily, particularly in the DNA-binding domain and ligand- and cofactor-binding domain. Many cellular and systemic roles have been attributed to these receptors, reaching far beyond the stimulation of peroxisome proliferation in rodents after which they were initially named. PPARs exhibit broad, isotype-specific tissue expression patterns. PPARalpha is expressed at high levels in organs with significant catabolism of fatty acids. PPARbeta/delta has the broadest expression pattern, and the levels of expression in certain tissues depend on the extent of cell proliferation and differentiation. PPARgamma is expressed as two isoforms, of which PPARgamma2 is found at high levels in the adipose tissues, whereas PPARgamma1 has a broader expression pattern. Transcriptional regulation by PPARs requires heterodimerization with the retinoid X receptor (RXR). When activated by a ligand, the dimer modulates transcription via binding to a specific DNA sequence element called a peroxisome proliferator response element (PPRE) in the promoter region of target genes. A wide variety of natural or synthetic compounds was identified as PPAR ligands. Among the synthetic ligands, the lipid-lowering drugs, fibrates, and the insulin sensitizers, thiazolidinediones, are PPARalpha and PPARgamma agonists, respectively, which underscores the important role of PPARs as therapeutic targets. Transcriptional control by PPAR/RXR heterodimers also requires interaction with coregulator complexes. Thus, selective action of PPARs in vivo results from the interplay at a given time point between expression levels of each of the three PPAR and RXR isotypes, affinity for a specific promoter PPRE, and ligand and cofactor availabilities.

Effects of baicalein on the proliferation and differentiation of porcine preadipoctyes

To investigate the effects of Baicalein (BAI) on the proliferation and differentiation of porcine preadipocytes and to elucidate its potential mechanism, porcine primary preadipocytes were cultured in vitro. The morphologic changes of preadipocytes brought about by differentiation were observed by Oil Red O staining. The extent of cell proliferation was detected by MTT assay. The degrees of adipogenesis and differentiation were measured by Oil Red O staining extraction assay. The activity of fatty acid synthase (FAS) was detected by spectrophotometry. The mRNA expression of special peroxisome proliferation activated receptor-γ2 genes (PPARγ2) was detected by reverse transcriptase polymerase chain reaction (RT-PCR). When preadipocytes differentiated into adipocytes, the shape of the preadipocytes changed from shuttle to oval and round, and the preadipocytes were filled with lipid droplets of different sizes. The proliferation of preadipocytes was inhibited by the treatment of 160–640 μmol/L BAI ( P<0.05). The mRNA expression of PPARγ2 and FAS activity and the differentiation of preadipocytes was repressed by 40–320 μmol/L BAI treatment ( P<0.05). It is concluded that the proliferation and differentiation of preadipocytes are inhibited by BAI to some degree. The effect of BAI on differentiation of preadipocytes may be due to the inhibition of the mRNA expression of PPARγ2 and the reduction of the FAS activity.

Prooxidant activity and toxicity of nordihydroguaiaretic acid in clone-9 rat hepatocyte cultures

Nordihydroguaiaretic acid (NDGA) is a polyphenol. It is present at high concentrations in the leaves of the evergreen desert shrub, Larrea tridentate (Creosote bush), which has a long history of medicinal use traditionally by the native Americans and Mexicans. It is generally believed that the antioxidant properties of NDGA are responsible for the medicinal value of this desert shrub. The clone-9 rat hepatocyte cultures were used as an in vitro model to assess the hepatotoxic potential of NDGA and to determine whether it exhibits any prooxidant activity. The hepatocyte cultures were treated with NDGA for 2 h at 37 °C at concentrations of 0–100 μM. After the treatment period the cells, the culture supernatants and cell lysates were assayed for evaluation of prooxidant activity and toxicity of NDGA. Oxidative stress level and oxidative cell injury as measured by the peroxidation of membrane lipids and DNA double-strand breaks were used to index prooxidant activity. Cytotoxicity as measured by the leakage of the liver enzyme lactate dehydrogenase (LDH) into the culture medium, mitochondrial function and extent of cell proliferation were used as the endpoints of toxicity. Significant concentration-dependent differences were observed in these biomarkers over the concentration range examined demonstrating the prooxidant activity and toxicity of NDGA in clone-9 rat hepatocyte cultures.

Ontogeny of protein expression of CD208, surfactant protein A and D, thyroid transcription factor‐1 (TTF‐1), and cell proliferation antigen (Ki‐67) in lung epithelia of pre‐ and full‐term lambs

Children born preterm have an increased susceptibility to respiratory syncytial virus (RSV) compared to those born full-term. RSV initially infects and damages epithelial cells, which are important for adaptive and innate immune responses such as expression of surfactant proteins A (SP-A) and D (SP-D) which are known to protect against RSV infection. However, the extent of expression preterm, as well as the effect of cell proliferation and differentiation has on the production of these proteins have not been determined. SP-A and D protein production as well as the extent of cell proliferation (Ki67 expression) and differentiation (CD208 antigen cells) and Thyroid Transcription Factor-1 (TTF-1; a SP transcription factor) were determined by immunohistochemistry in lung from preterm (80% gestation), full-term, and post-term (15 days of age) lambs. Cell differentiation (CD208+ cells) increased significantly with age; however, no significant changes were seen with proliferation (Ki67 antigen), SP-AD, or TTF-1. SP-AD expression was present in bronchial and bronchiolar epithelial cells, and occasional alveolar epithelial cells in all age groups, TTF-1 was present in non-ciliated airway cells and type II cells as described previously. This work suggests that cell differentiation increases with gestation and that SP-A and D production is present in bronchioles and alveoli in late gestation and at birth. Supported NIH NIAID Awards 5R01AI062787-02 and 5K08AI055499-03, USDA/CSREES/NRI-CGP 2003-35204-13492, and the JG Salsbury Endowment.

Housekeeping gene expression during fetal brain development in the rat—validation by semi-quantitative RT-PCR

Mammalian gene expression is usually carried out at the level of mRNA where the amount of mRNA of interest is measured under different conditions such as growth and development. It is therefore important to use a “housekeeping gene”, that does not change in relative abundance during the experimental conditions, as a standard or internal control. However, recent data suggest that expression of some housekeeping genes may vary with the extent of cell proliferation, differentiation and under various experimental conditions. In this study, the expression of various housekeeping genes (18S rRNA [18S], glyceraldehydes-3-phosphate dehydrogenase [G3PDH], β-glucuronidase [BGLU], histone H4 [HH4], ribosomal protein L19 [RPL19] and cyclophilin [CY]) was investigated during fetal rat brain development using semi-quantitative RT-PCR at 16, 19 and 21 days gestation. It was found that all genes studied, with exception to G3PDH, did not show any change in their expression levels during development. G3PDH, on the other hand, showed increased expression with development. These results suggest that the choice of a housekeeping gene is critical to the interpretation of experimental results and should be modified according to the nature of the study.

Transforming Growth Factor (TGF)- 1 Releasing Tricalcium Phosphate/Chitosan Microgranules as Bone Substitutes

Tricalcium phosphate (TCP)/chitosan composite microgranules were developed as bone substitutes and tissue engineering scaffolds with the aim of obtaining a high bone forming efficacy. The microgranules have the ability to fill various types of defect sites with closer packing. In addition, the transforming growth factor-beta 1 (TGF-beta1) was added to the microgranules in order to improve bone-healing efficacy. TCP/chitosan microgranules were fabricated by dropping a TCP suspended chitosan solution into a NaOH/ethanol solution. TGF-beta1 was incorporated into the TCP/chitosan microgranules by soaking the microgranules into the TGF-beta1 solution. Scanning electron microscopy (SEM) observations as well as experiments examining the release of TGF-beta1 from chitosan and TCP/chitosan microgranules were performed. SEM was used to examine the cell morphologies on the microgranules, and the extent of cell proliferation was evaluated using a dimethyl-thiazol tetrazolium bromide (MTT) assay. The differentiated cell function was assessed by measuring the alkaline phosphatase activity as well as performing an osteocalcin assay. The size of the prepared microgranules was 350-500 microm and TCP powders were observed on the surface of the microgranules. TGF-beta1 was released from the TCP/chitosan microgranules at a therapeutic concentration for 4 weeks. The proliferation of osteoblasts on the TGF-beta1 loaded microgranules was the highest among the microgranules. SEM indicated that the seeded osteoblastic cells were firmly attached to the microgranules and proliferated in a multilayer fashion. The ALPase activity and osteocalcin content of all the samples increased during the culture period. These results suggest that the TCP/chitosan microgranules are potential bone substitutes with a drug releasing capacity and a osteoblastic cells culture scaffold.

Fas stimulation activates NF-κB in SK-Hep1 hepatocellular carcinoma cells

The TNF-receptor family has a dual signaling pathway, including induction of apoptosis and NF-kappaB activation associated with cell survival. Hepatocellular carcinoma (HCC) cells express TNF-receptor family members and the signaling from these receptors induces NF-kappaB activation. However, the role of Fas in induction of NF-kappaB activation in HCC cells is not well understood. In this study, SK-Hep1, HepG2 or HLE cells were stimulated by anti-Fas agonistic antibody. Fas stimulation induced NF-kappaB activation in a dose-dependent manner in SK-Hep1 and HepG2 cell lines, but not in HLE cells. Anti-Fas agonistic antibody or the metabolic inhibitor, cyclo-heximide (CHX), failed to kill SK-Hep1 cells, but co-incubation with anti-Fas agonistic antibody and CHX was effective for induction of apoptosis. SK-Hep1 cell lines receiving Fas stimulation had increased viability, but the extent of cell proliferation was not dose-dependent. The observation suggests that Fas stimulation may contribute to HCC cell survival or proliferation.

Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures

The response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was examined in vitro. The PCA thin film was prepared on polystyrene culture dishes using highly metastable calcium phosphate ion solution at low temperatures. The PCA thin film was formed through fusion and transformation of granular calcium phosphate particles, which had initially formed on the surface, into a film of calcium phosphate apatite crystal. The PCA thin film was used for cell culture without additional surface treatment. The osteoblastic cell behaviors including adhesion, proliferation, expression of the marker genes, and calcified matrix formation were examined on the PCA thin film using primary osteoblasts or MC3T3-E1 cells. The cells were well attached and had spread in a slender shape over the PCA thin film. The extent of cell proliferation on the PCA thin film is as much as on the plain dishes. In addition, a much larger number of calcified nodules had formed on the PCA thin film than on the plain dish. The expression of the marker genes such as alkaline phosphatase, osteocalcin, osteopontin, osteonectin was apparent. These results demonstrate that the osteoblasts exhibit a full spectrum of cellular activity including the adequate differentiation on the PCA thin film . Therefore, a PCA thin film can be used as a coating material for biomaterials where the surface is not adequate for inducing the full activity of bone cells.