Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

A collagen network phase improves cell seeding of open-pore structure scaffolds under perfusion

Scaffolds with open-pore morphologies offer several advantages in cell-based tissue engineering, but their use is limited by a low cell seeding efficiency. We hypothesized that inclusion of a collagen network as filling material within the open-pore architecture of polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds increases human bone marrow stromal cells (hBMSC) seeding efficiency under perfusion and in vivo osteogenic capacity of the resulting constructs. PCL-TCP scaffolds, rapid prototyped with a honeycomb-like architecture, were filled with a collagen gel and subsequently lyophilized, with or without final crosslinking. Collagen-free scaffolds were used as controls. The seeding efficiency was assessed after overnight perfusion of expanded hBMSC directly through the scaffold pores using a bioreactor system. By seeding and culturing freshly harvested hBMSC under perfusion for 3 weeks, the osteogenic capacity of generated constructs was tested by ectopic implantation in nude mice. The presence of the collagen network, independently of the crosslinking process, significantly increased the cell seeding efficiency (2.5-fold), and reduced the loss of clonogenic cells in the supernatant. Although no implant generated frank bone tissue, possibly due to the mineral distribution within the scaffold polymer phase, the presence of a non crosslinked collagen phase led to in vivo formation of scattered structures of dense osteoids. Our findings verify that the inclusion of a collagen network within open morphology porous scaffolds improves cell retention under perfusion seeding. In the context of cell-based therapies, collagen-filled porous scaffolds are expected to yield superior cell utilization, and could be combined with perfusion-based bioreactor devices to streamline graft manufacture.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Clusterin facilitates COMMD1 and I-kB degradation to enhance NF-kB activity in prostate cancer cells

Secretory clusterin (sCLU) is a stress-activated, cytoprotective chaperone that confers broad-spectrum cancer treatment resistance, and its targeted inhibitor (OGX-011) is currently in phase II trials for prostate, lung, and breast cancer. However, the molecular mechanisms by which sCLU inhibits treatment-induced apoptosis in prostate cancer remain incompletely defined. We report that sCLU increases NF-κB nuclear translocation and transcriptional activity by serving as a ubiquitin-binding protein that enhances COMMD1 and I-κB proteasomal degradation by interacting with members of the SCF-βTrCP E3 ligase family. Knockdown of sCLU in prostate cancer cells stabilizes COMMD1 and I-κB, thereby sequestrating NF-κB in the cytoplasm and decreasing NF-κB transcriptional activity. Comparative microarray profiling of sCLU-overexpressing and sCLU-knockdown prostate cancer cells confirmed that the expression of many NF-κB–regulated genes positively correlates with sCLU levels. We propose that elevated levels of sCLU promote prostate cancer cell survival by facilitating degradation of COMMD1 and I-κB, thereby activating the canonical NF-κB pathway.

The expression, regulation and function of kallikrein 4 in prostate cancer

Prostate cancer is a significant health problem faced by aging men. Currently, diagnostic strategies for the detection of prostate cancer are either unreliable, yielding high numbers of false positive results, or too invasive to be used widely as screening tests. Furthermore, the current therapeutic strategies for the treatment of the disease carry considerable side effects. Although organ confined prostate cancer can be curable, most detectable clinical symptoms occur in advanced disease when primary tumour cells have metastasised to distant sites - usually lymph nodes and bone. Many growth factors and steroids assist the continued growth and maintenance of prostatic tumour cells. Of these mitogens, androgens are important in the development of the normal prostate but are also required to sustain the growth of prostate cancer cells in the early stage of the disease. Not only are androgens required in the early stage of disease, but also many other growth factors and hormones interact to cause uncontrolled proliferation of malignant cells. The early, androgen sensitive phase of disease is followed by an androgen insensitive phase, whereby androgens are no longer required to stimulate the growth of the tumour cells. Growth factors such as transforming growth factor  and  (TGF/), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factors (IGFs), Vitamin D and thyroid hormone have been suggested to be important at this stage of disease. Interestingly, some of the kallikrein family of genes, including prostate specific antigen (PSA), the current serum diagnostic marker for prostate cancer, are regulated by androgens and many of the aforementioned growth factors. The kallikrein gene family is a group of serine proteases that are involved in a diverse range of physiological processes: regulation of local blood flow, angiogenesis, tissue invasion and mitogenesis. The earliest members of the kallikrein gene family (KLK1-KLK3) have been strongly associated with general disease states, such as hypertension, inflammation, pancreatitis and renal disease, but are also linked to various cancers. Recently, this family was extended to include 15 genes (KLK1-15). Several newer members of the kallikrein family have been implicated in the carcinogenesis and tumour metastasis of hormone-dependent cancers such as prostate, breast, endometrial and ovarian cancer. The aims of this project were to investigate the expression of the newly identified kallikrein, KLK4, in benign and malignant prostate tissues, and prostate cancer cell lines. This thesis has demonstrated the elevated expression of KLK4 mRNA transcripts in malignant prostate tissue compared to benign prostates. Additionally, expression of the full length KLK4 transcript was detected in the androgen dependent prostate cancer cell line, LNCaP. Based on the above finding, the LNCaP cell line was chosen to assess the potential regulation of full length KLK4 by androgen, thyroid hormone and epidermal growth factor. KLK4 mRNA and protein was found to be up-regulated by androgen and a combination of androgen and thyroid hormone. Thyroid hormone alone produced no significant change in KLK4 mRNA or protein over the control. Epidermal growth factor treatment also resulted in elevated expression levels of KLK4 mRNA and protein. To assess the potential functional role(s) of KLK4/hK4 in processes associated with tumour progression, full length KLK4 was transfected into PC-3 cells - a prostate cancer cell line originally derived from a secondary bone lesion. The KLK4/hK4 over-expressing cells were assessed for their proliferation, migration, invasion and attachment properties. The KLK4 over-expressing clones exhibited a marked change in morphology, indicative of a more aggressive phenotype. The KLK4 clones were irregularly shaped with compromised adhesion to the growth surface. In contrast, the control cell lines (parent PC-3 and empty vector clones) retained a rounded morphology with obvious cell to cell adhesion, as well as significant adhesion to their growth surface. The KLK4 clones exhibited significantly greater attachment to Collagen I and IV than native PC-3s and empty vector controls. Over a 12 hour period, in comparison to the control cells, the KLK4 clones displayed an increase in migration towards PC-3 native conditioned media, a 3 fold increase towards conditioned media from an osteoblastic cell line (Saos-2) and no change in migration towards conditioned media from neonatal foreskin fibroblast cells or 20% foetal bovine serum. Furthermore, the increase in migration exhibited by the KLK4 clones was partially blocked by the serine protease inhibitor, aprotinin. The data presented in this thesis suggests that KLK4/hK4 is important in prostate carcinogenesis due to its over-expression in malignant prostate tissues, its regulation by hormones and growth factors associated with prostate disease and the functional consequences of over-expression of KLK4/hK4 in the PC-3 cell line. These results indicate that KLK4/hK4 may play an important role in tumour invasion and bone metastasis via increased attachment to the bone matrix protein, Collagen I, and enhanced migration due to soluble factors produced by osteoblast cells. This suggestion is further supported by the morphological changes displayed by the KLK4 over-expressing cells. Overall, this data suggests that KLK4/hK4 should be further studied to more fully investigate the potential value of KLK4/hK4 as a diagnostic/prognostic biomarker or in therapeutic applications.

A continuum model of the growth of engineered epidermal skin substitutes

We present a porous medium model of the growth and deterioration of the viable sublayers of an epidermal skin substitute. It consists of five species: cells, intracellular and extracellular calcium, tight junctions, and a hypothesised signal chemical emanating from the stratum corneum. The model is solved numerically in Matlab using a finite difference scheme. Steady state calcium distributions are predicted that agree well with the experimental data. Our model also demonstrates epidermal skin substitute deterioration if the calcium diffusion coefficient is reduced compared to reported values in the literature.

Insulin increases De Novo Steroidogenesis in prostate cancer cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

Evaluation of methods for cultivating limbal mesenchymal stromal cells

Background: Mesenchymal stromal cells (MSC) with similar properties to bone marrow derived mesenchymal stromal cells (BM-MSC) have recently been grown from the limbus of the human cornea. We presently contribute to this novel area of research by evaluating methods for culturing human limbal MSC (L-MSC). Methods: Four basic strategies are compared: serum-supplemented medium (10% foetal bovine serum; FBS), standard serum-free medium supplemented with B-27, epidermal growth factor, and fibroblast growth factor 2, or one of two commercial serum-free media including Defined Keratinocyte Serum Free Medium (Invitrogen), and MesenCult-XF (Stem Cell Technologies). The phenotype of resulting cultures was examined using photography, flow cytometry (for CD34, CD45, CD73, CD90, CD105, CD141, CD271), immunocytochemistry (α-sma), differentiation assays (osteogenesis, adipogenesis, chrondrogenesis), and co-culture experiments with human limbal epithelial (HLE) cells. Results: While all techniques supported to varying degrees establishment of cultures, sustained growth and serial propagation was only achieved in 10% FBS medium or MesenCult-XF medium. Cultures established in 10% FBS medium were 70-80% CD34-/CD45-/CD90+/CD73+/CD105+, approximately 25% α-sma+, and displayed multi-potency. Cultures established in MesenCult-XF were >95% CD34-/CD45-/CD90+/CD73+/CD105+, 40% CD141+, rarely expressed α-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of MesenCult-XF-grown L-MSC. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker ∆Np63, along with the corneal differentiation marker cytokeratin 3. Conclusions: We conclude that MesenCult-XF® is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells.

The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration

Epidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/RAF/MEK/ERK pathway.

IGF2 increases de novo steroidogenesis in prostate cancer cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.

Non-aqueous synthesis of hexagonal ZnO nanopyramids : gas sensing properties

Zinc oxide (ZnO) nanopyramids were synthesized by a one-pot route in a non-aqueous and surfactantfree environment. The synthesized metal oxide was characterized using SEM, XRD, and TEM to investigate the surface morphology and crystallographic phase of the nanostructures. It was observed that the ZnO nanopyramids were of uniform size and symmetrical, with a hexagonal base and height of ∼100 nm. Gas sensing characterization of the ZnO nanopyramids when deposited as thin-film onto conductometric transducers were performed towards NOx and C2H5OH vapor of different concentrations over a temperature range of 22–350 ◦C. It was observed that the sensors responded towards NO2 (10 ppm) and C2H5OH(250 ppm) analytes best at temperatures of 200 and 260 ◦C with a sensor response of 14.5 and 5.72, respectively. The sensors showed satisfactory sensitivity, repeatability as well as fast response and recovery towards both the oxidizing and the reducing analyte. The good performance was attributed to the low amount of organic impurities, large surface-to-volume ratio and high crystallinity of the solvothermally synthesized ZnO nanopyramids.

Effect of bone morphogenetic protein-4 (BMP-4) on the expression of Sox2, Oct-4 and c-Myc in human periodontal ligament cells during long-term culture

Recent studies demonstrated endogenous expression level of Sox2, Oct-4 and c-Myc is correlated with the pluripotency and successful induction of induced pluripotent stem cells (iPSCs). Periondontal ligament cells (PDLCs)have multi-lineage diferentiation capability and ability to maintain undifferentiated stage, which makes PDLCs a suitable cell source for tissue repair and regeneration. To elucidate the effect of in vitro culture condition on the stemness potential of PDLCs, we explored the cell growth, proliferation, cell cycle, and the expression of Sox2, Oct-4 and c-Myc in PDLCs from passage 1 to 7 with or without the addition of recombinant human BMP4(rhBMP4). Our results revealed that BMP-4 promoted cell growth and proliferation, arrested PDLCs in S phase of cell cycle and upregulated PI value. It was revealed that without the addition of rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs only maintained nucleus location until passage 3, then lost nucleus location subsequently. The mRNA expression in PDLCs further confirmed that the level of Sox2 and Oct-4 peaked at passage 3, then decreased afterwards, whereas c-Myc maintained consistently upregulation along passages. after the treatment with rhBMP4, the expression of Sox2, Oct-4 and c-Myc in PDLCs maintained nucleus location even at passage 7 and the mRNA expression of Sox2 and Oct-4 significantly upregulated at passage 5 and 7. These results demonstrated that addition of rhBMP-4 in the culture media could improve the current culture condition for PDLCs to maintain in an undifferentiated stage.

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.

Harmonic quality of multilevel cascade inverters with random carrier phase pulse width modulation

The output harmonic quality of N series connected full-bridge dc-ac inverters is investigated. The inverters are pulse width modulated using a common reference signal but randomly phased carrier signals. Through analysis and simulation, probability distributions for inverter output harmonics and vector representations of N carrier phases are combined and assessed. It is concluded that a low total harmonic distortion is most likely to occur and will decrease further as N increases.