Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro

Background and Objective: A number of bone filling materials containing calcium (Ca++) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effect that local release of Ca++ and P ions have on biological reactions is not fully understood. In this study, we investigated the effects of various levels of Ca++ and P ions on the proliferation, osteogenic differentiation, and mineralization of human periodontal ligament cells (hPDLCs). Materials and Methods: hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca++ to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated by FITC-Annexin V/PI double staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase (ALP) activity, and Alizarin red S/von Kossa staining. The mRNA expression of osteogenic related markers was analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Results: Within the ranges of Ca++ and P ions concentrations tested, we observed that increased concentrations of Ca++ and P ions enhanced cell proliferation and formation of mineralized matrix nodules; whereas ALP activity was reduced. The RT-PCR results showed that elevated concentrations of Ca++ and P ions led to a general increase of Runx2 mRNA expression and decreased ALP mRNA expression, but gave no clear trend on OCN mRNA levels. Conclusion: The concentrations and ratios of Ca++ and P ions could significantly influence proliferation, differentiation, and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 mM Ca++ ions and 4.5 mM P ions were the optimum concentrations for proliferation, differentiation, and mineralization in hPDLCs.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Human neural cell interactions with orientated electrospun nanofibers in vitro

Aim: Electrospun nanofibers represent potent guidance substrates for nervous tissue repair. Development of nanofiber-based scaffolds for CNS repair requires, as a first step, an understanding of appropriate neural cell type-substrate interactions. Materials & methods: Astrocyte–nanofiber interactions (e.g., adhesion, proliferation, process extension and migration) were studied by comparing human neural progenitor-derived astrocytes (hNP-ACs) and a human astrocytoma cell line (U373) with aligned polycaprolactone (PCL) nanofibers or blended (25% type I collagen/75% PCL) nanofibers. Neuron–nanofiber interactions were assessed using a differentiated human neuroblastoma cell line (SH-SY5Y). Results & discussion: U373 cells and hNP-AC showed similar process alignment and length when associated with PCL or Type I collagen/PCL nanofibers. Cell adhesion and migration by hNP-AC were clearly improved by functionalization of nanofiber surfaces with type I collagen. Functionalized nanofibers had no such effect on U373 cells. Another clear difference between the U373 cells and hNP-AC interactions with the nanofiber substrate was proliferation; the cell line demonstrating strong proliferation, whereas the hNP-AC line showed no proliferation on either type of nanofiber. Long axonal growth (up to 600 µm in length) of SH-SY5Y neurons followed the orientation of both types of nanofibers even though adhesion of the processes to the fibers was poor. Conclusion: The use of cell lines is of only limited predictive value when studying cell–substrate interactions but both morphology and alignment of human astrocytes were affected profoundly by nanofibers. Nanofiber surface functionalization with collagen significantly improved hNP-AC adhesion and migration. Alternative forms of functionalization may be required for optimal axon–nanofiber interactions.

Evaluation of silk sericin as a biomaterial: In vitro growth of human corneal limbal epithelial cells on Bombyx mori sericin membranes

Sericin and fibroin are the two major proteins in the silk fibre produced by the domesticated silkworm, Bombyx mori. Fibroin has been extensively investigated as a biomaterial. We have previously shown that fibroin can function successfully as a substratum for growing cells of the eye. Sericin has been so far neglected as a biomaterial because of suspected allergenic activity. However, this misconception has now been dispelled, and sericin’s biocompatibility is currently indisputable. Aiming at promoting sericin as a possible substratum for the growth of corneal cells in order to make tissue-engineered constructs for the restoration of the ocular surface, in this study we investigated the attachment and growth in vitro of human corneal limbal epithelial cells (HLECs) on sericin-based membranes. Sericin was isolated and regenerated from the silkworm cocoons by an aqueous procedure, manufactured into membranes, and characterized (mechanical properties, structural analysis, contact angles). Primary cell cultures from two donors were established in serum-supplemented media in the presence of murine feeder cells. Membranes made of sericin and fibroin-sericin blends were assessed in vitro as substrata for HLECs in a serum-free medium, in a cell attachment assay and in a 3-day cell growth experiment. While the mechanical characteristics of sericin were found to be inferior to those of fibroin, its ability to enhance the attachment of HLECs was significantly superior to fibroin, as revealed by the PicoGreen® assay. Evidence was also obtained that cells can grow and differentiate on these substrata.

The influence of architecture on degradation and tissue ingrowth into three-dimensional poly(lactic-co-glycolic acid) scaffolds in vitro and in vivo

The in vitro and in vivo degradation properties of poly(lactic-co-glycolic acid) (PLGA) scaffolds produced by two different technologies - thermally induced phase separation (TIPS), and solvent casting and particulate leaching (SCPL) were compared. Over 6 weeks, in vitro degradation produced changes in SCPL scaffold dimension, mass, internal architecture and mechanical properties. TIPS scaffolds produced far less changes in these parameters providing significant advantages over SCPL. In vivo results were based on a microsurgically created arteriovenous (AV) loop sandwiched between two TIPS scaffolds placed in a polycarbonate chamber under rat groin skin. Histologically, a predominant foreign body giant cell response and reduced vascularity was evident in tissue ingrowth between 2 and 8 weeks in TIPS scaffolds. Tissue death occurred at 8 weeks in the smallest pores. Morphometric comparison of TIPS and SCPL scaffolds indicated slightly better tissue ingrowth but greater loss of scaffold structure in SCPL scaffolds. Although advantageous in vitro, large surface area:volume ratios and varying pore sizes in PLGA TIPS scaffolds mean that effective in vivo (AV loop) utilization will only be achieved if the foreign body response can be significantly reduced so as to allow successful vascularisation, and hence sustained tissue growth, in pores less than 300 μm. © 2005 Elsevier Ltd. All rights reserved.

Microbial colonisation of human follicular fluid and adverse in vitro fertilisation outcomes

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

In vitro estimation of foetal liver volume using ultrasound, x-ray computed tomography and magnetic resonance imaging

Sixteen formalin-fixed foetal livers were scanned in vitro using a new system for estimating volume from a sequence of multiplanar 2D ultrasound images. Three different scan techniques were used (radial, parallel and slanted) and four volume estimation algorithms (ellipsoid, planimetry, tetrahedral and ray tracing). Actual liver volumes were measured by water displacement. Twelve of the sixteen livers also received x-ray computed tomography (CT) and magnetic resonance (MR) scans and the volumes were calculated using voxel counting and planimetry. The percentage accuracy (mean ± SD) was 5.3 ± 4.7%, −3.1 ± 9.6% and −0.03 ± 9.7% for ultrasound (radial scans, ray volumes), MR and CT (voxel counting) respectively. The new system may be useful for accurately estimating foetal liver volume in utero.

A tan in a test tube -in vitro models for investigating ultraviolet radiation-induced damage in skin

Presently, global rates of skin cancers induced by ultraviolet radiation (UVR) exposure are on the rise. In view of this, current knowledge gaps in the biology of photocarcinogenesis and skin cancer progression urgently need to be addressed. One factor that has limited skin cancer research has been the need for a reproducible and physiologically-relevant model able to represent the complexity of human skin. This review outlines the main currently-used in vitro models of UVR-induced skin damage. This includes the use of conventional two-dimensional cell culture techniques and the major animal models that have been employed in photobiology and photocarcinogenesis research. Additionally, the progression towards the use of cultured skin explants and tissue-engineered skin constructs, and their utility as models of native skin's responses to UVR are described. The inherent advantages and disadvantages of these in vitro systems are also discussed.