ATP citrate lyase inhibition can suppress tumor cell growth

Submitted by Daniel Rosen

Georgia Hatzivassiliou1, Fangping Zhao1, Daniel E. Bauer1, Charalambos Andreadis2, Anthony N. Shaw3, Dashyant Dhanak4, Sunil R. Hingorani1, 2, David A. Tuveson1, 2 and Craig B. Thompson1, Corresponding Author Contact Information, E-mail The Corresponding Author

1Department of Cancer Biology, Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104 2Department of Medicine, Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104 3Department of Medicinal Chemistry, Metabolic and Viral Diseases Center of Excellence for Drug Discovery, GlaxoSmith-Kline, Collegeville, Pennsylvania 19426 4Department of Medicinal Chemistry, Musculoskeletal, Microbial and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmith-Kline, Collegeville, Pennsylvania 19426


Received 11 April 2005;

revised 9 September 2005;

accepted 28 September 2005.

Published: October 17, 2005.

Available online 17 October 2005.



Summary:

Human cancers are often detected by 18-F-2-deoxyglucose positron emission tomography (PET) because the cancers tend to exhibit high levels of aerobic glycolyis which can be detected by the PET scan. Although the increased levels of aerobic metabolism fulfill a need for increased ATP production, the majority of the ATP derives from glycolytic activity. The pyruvate end-product enters a modified Citric Acid/Krebs cycle and is shunted to producing citrate and then acetyl-CoA. The acetyl-CoA is a necessary building block for de-novo synthesis of fatty acids, which cancers cells do almost exclusively regardless of external supplies. Cancer cells engage in upregulated levels of fatty acid synthesis to (1) support membrane production and (2) permit post-translational modification of proteins (i.e. acetylation) specifically lipid modified signaling molecules. Both functions are essential for cancer growth. ATP Citrate Lyase (ACL) is an enzyme which converts Citrate to cytosolic acetyl-coA and it is also coordinately regulated with other lipgenic enzymes. For this potential to link glucose and lipid metabolism, ACL was studied and its inhibition was determined to suppress proliferation and promote differentiation in glucose dependent cancers.

Endogenous ACL levels in human lung adenocarcinoma cell line A549 were knocked down with siRNA oligonucleotides. As a control the siACL knockdowns were compared to siLUC (luciferase) knockdowns. In the first experiment a Western Blot was run comparing the two siRNA transfections production of ACL, which was compared to Actin (Figure 1). The results show decreased levels of ACL in the knockdowns which increases with time and there is no change in the controls. This is also quantified in figure 1B. Data in Figure 1 also shows a difference in lipid synthesis when tagged with D-[6-14C] glucose (for the glucose dependent lipid synthesis) for the siACL vs. siLUC lines and no change for acetate tagging (glucose independent lipid synthesis). Viability only decreased for the siACL line at extended time periods.

As shown in Figure 4. the in vivo effect of short hairpin knockdown strains of A549 cells resulted in reduced tumor weight and differentiation into glandular structures when injected in vivo into nude mice. The differentiated phenotype, being unexpected, was confirmed with a replicated experiment using K562 chronic myelogenous leukemia cell line. The pharmoacologic inhibitor of ACL, SB-204990 (not stated but presumably a Glaxo-smith-kline (GSK) propriety compound) was tested on a IL-3 dependent cell lines. The IL-3 stimulates glycolysis and cell proliferation which replicates a tumor cell profile. The SB-204990 compound induced delayed cell cycle entry (arrest in G1 phase at 15 microMolar and complete inhibition at higher doses). The compound was then tested against three tumor forming cell lines in vitro and in vivo via xenographs in nude mice. Two of the cell lines (A549 and PC3) exhibited sensitivity to SB-204990 treatment while the third line SKOV3 did not. The difference is indicated to be a result of the SKOV3 lower dependence and uptake of glucose.

Significance:

Because many human cancers display a higher than average glucose dependency, fueling growth and malignancy, an inhibitor of the utilization of that glucose makes an attractive target in anti-cancer therapy. Cancer cells require the extra glucose in order to generate de novo lipids specifically signaling molecules and membrane. One relatively upstream enzyme involved in this lipogenesis is ACL which when downregulated was shown in vitro and in vivo to have negative effects on cancer growth and also promoted differentiation—another indication of cancer retreat. Although several methods of downregulation including siRNA and pharmaceutical compounds produced the desired effect, it was only significant in cancer lines representative of cells which require an abnormally high amount of exogenous glucose and had a minor effect on cancer lines with lower glucose to lipid production ratios. Additionally, a separate glucose-independent pathway can rescue lipogenesis function in several cell lines. Despite these setbacks, ACL inhibitors remain attractive anti-cancer therapeutics due to upstream regulation of other lipogenic factors, selective targeting of cancer tissues and anti-neoplastic properties.

From the perspective of BioE 115 this paper employs three of the available cell lines, Western Blotting, and BrdU staining.

Hyaline Cartilage Regeneration Using Mixed Human Chondrocytes and Transforming Growth Factor-β1-Producing Chondrocytes

SUN U. SONG, Ph.D.,1 YOUNG-DEOG CHA, M.D.,1 JEOUNG-UK HAN, M.D.,1
IN-SUK OH, M.D.,2 KYOUNG BAEK CHOI, M.S.,3 YOUNGSUK YI, Ph.D.,3
JONG-PIL HYUN, B.S.,3 HYEON-YOUL LEE, B.S.,3 GUANG FAN CHI, Ph.D.,3
CHAE-LYUL LIM, M.S.,3 J. KELLY GANJEI, B.S.,4 MOON-JONG NOH, Ph.D.,4
SEONG-JIN KIM, Ph.D.,5 DUG KEUN LEE, Ph.D.,4 and KWAN HEE LEE, M.D.1,3,4

1Clinical Research Center, College of Medicine, Inha University, Inchon, South Korea.
2Department of Orthopedic Surgery, College of Medicine, Inha University, Inchon, South Korea.
3TissueGene Asia, Inchon, South Korea.
4TissueGene, Gaithersburg, Maryland.
5Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Address:
http://www.liebertonline.com/doi/abs/10.1089/ten.2005.11.1516

Summary
This goal of this study was to compare a mixture of transforming growth factor-β1 (TGF- β1)-producing human chondrocytes and primary human chondrocytes (hChon) in terms of cartilage regeneration with either hChon-TGF- β1 or hChon cells alone. This was to be studied in the context of hyaline articular cartilage, which has a very limited regenerative capacity and does not heal well after traumatic injury or other defects. Several other proposed methods for regenerating articular cartilage including use of mesenchymal stem cells have shown some promise but also several disadvantages.

Human chondrocyte cells transduced with retroviral vectors for human TGF- β1 were cultured and TGF- β1 production was measured with ELISA. RT-PCR on RNA extracted from cultured cells was conducted with primers for Type I and Type II collagen. A mixture of hChon-TGF- β1 and hChon cells or hChon-TGF- β1 cells alone were injected subcutaneously into the backs of nude mice with untransduced hChon cells injected as control. Human chondrocyte cells were also injected into rabbit knees with induced defects in the articular cartilage.

hChon-TGF- β1 cells were found by ELISA to produce about 20 ng/105 cells per 24 hours. RT-PCR checked that the transduced cells produced type II collagen continuously, while the untransduced did not. Both cell lines produced type I collagen. The tissue formed from injected cells were analyzed by hematoxylin-eosin (H&E), toluidine blue (TB) and safranin-O (S-O) staining, and found to be similar to normal cartilage tissue. Immunohistochemical staining found that TGF- β1 and human type II collagen was expressed in the new tissue, with greater amounts in tissue with greater amounts of transduced cells.

Chondrocyte cells injected into cartilage defects in rabbits were found to refill the defect with new tissue. A mixture of cells filled the defect in 6 weeks while untransduced cells alone did not completely fill the defect. The 5:1 mixture of hChon:hChon-TGF- β1 cells were found to fill the gap better than the 1:1 ratio.

Histological and immunohistochemical staining of new tissue in both mice and rabbits show that the mixture of transduced and untransduced chondrocytes has higher efficacy in regenerating hyaline cartilage than only transduced cells. This may be because the untransduced chondrocytes serve as additional bulk material to fill the defect and can be targeted by TGF- β1 produced by transduced cells. While this study shows the effects of TGF- β1 on chondrocyte differentiation the exact molecular mechanism that controls cartilage repair is still unknown.

Significance
Osteoarthritis is caused by erosion of the articular cartilage around a joint, which is primarily composed of hyaline cartilage. It is often treated by total joint replacements, which have short lifespans and can cause biomechanical and immune responses in the host. This paper suggests a method that could lead to autografts that would enable patients to quickly regenerate lost hyaline cartilage, which would provide an alternative method of treatment.

Submitted by Chris Han

Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes

Feng Cao1., Roger A. Wagner2., Kitchener D. Wilson1,3., Xiaoyan Xie1, Ji-Dong Fu6, Micha Drukker4, Andrew Lee1, Ronald A. Li6, Sanjiv S. Gambhir1,3, Irving L. Weissman4, Robert C. Robbins5, Joseph C. Wu1,2*

Citation: Cao F, Wagner RA, Wilson KD, Xie X, Fu J-D, et al. (2008) Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes. PLoS ONE 3(10): e3474. doi:10.1371/journal.pone.0003474

Summary: Many applications of human embryonic stem cells (hESCs) have been explored over the past 48 years; one focus has been the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting repair/recovery after myocardial infarction. There are hopes of not only preserving cardiac function, but actual regeneration of diseased muscle because stem cells provide a potentially limitless source of cells. However, the pluripotency of stem cells also presents the serious risk of teratoma formation.

Feng et al. sought to characterize the molecular networks governing the differentiation of cardiomyocytes with an eye toward the transplantation of hESC-CMs to myocardial ischemia in vivo. To do so, both genomic and noninvasive imaging tools were utilized to understand the biological processes that could form the foundation of future stem cell therapy.

Feng et al. differentiated hESCs into cardiomyocytes utilizing the methods outlined in the Figure 1, below. They utilized RT-PCR analysis to track expression in the hESC-derived EBs as they differentiated into beating clusters. The expected stem cell markers (Oct4, NANOG, Rex1) were all observed early on, while early stage cardiac transcriptional factors (such as Nkx2.5 and MEF2C) appeared later between 14 and 28 days.


Figure 1: Schematic outlining the cardiomyocyte differentiation experimental design. The cells were maintained in an undifferentiated state on a feeder layer of mouse embryonic fibroblasts (MEFs). After the appearance of beating clusters in the embryoid bodies (EBs), the cells were separated by Percoll density gradient purification, which allowed for cardiomyocyte-enriched populations ranging from 40-45% beating EBs expressing the cardiac marker troponin-T as determined by FACS analysis.

Primary ventricular cardiomyocytes were used in microarray analysis. The significance analysis of microarrays (SAM) algorithm was then used to identify those genes which had changed expression significantly during differentiation from pluripotent hESCs to fetal heart cells.

In addition to performing transcriptional profiling of the cells, the authors also sought to compare their cultured cardiomyocyte population with what would likely be an optimal cell population for transplantation. Specifically comparisons of electrophysiological readings and energy metabolism revealed several points. First, these in vitro differentiated cells, while capable of contraction, had not yet faced the biomechanical stresses of in vivo cardiac development. Additionally, of the hESC-CMs, the ventricular-like derivatives were most like the ideal primary fetal heart cardiomyocytes, in terms of resting membrane potential.

Finally, the authors examined the effects of transplantation of hESC-CM into ischemic regions of the left ventricle of mice. These mice showed improvement, as measured by increased angiogenesis as well as left ventricular fractional shortening (LVFS) in comparison with controls. However, based upon a histologic evaluation of the transplanted hESC-CMs, there was very minimal integration of fluorescently tagged cells in the infracted areas as has been noted in previous studies. The authors hypothesize that the improvement seen in week 8 may relate to paracrine factors.

Significance: Through the research conducted, it was shown that hESC-CMs express cardiomyocyte genes at levels similar to those in 20-week fetal heart cells, which makes them promising candidates for use in vivo. Observations also indicated the importance of environment in the development of the hESC-CMs that fit the necessary parameters for potential stem cell therapeutics.

SPTLC1 Binds ABCA1 to Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

SPTLC1 Binds ABCA1 to Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

Authors: Norimasa Tamehiro, Suiping Zhou, Keiichiro Okuhira, Yair Benita, Cari E. Brown, Debbie Z. Zhuang, Eicke Latz, Thorsten Hornemann, Arnold von Eckardstein, Ramnik J. Xavier, Mason W. Freeman, and Michael L. Fitzgerald

Source: Biochemistry, Volume: 47, Issue: 23, Pages: 6138-6147, Published: 2008

Address: http://apps.isiknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=1&SID=3EdcFJ@AmofLj7loEAi&page=1&doc=1&colname=WOS

Summary:
This paper presents continuing research on the protein-protein interactions between a cholesterol and lipid tranporter, ATP-binding cassette transporter ABCA1, and a subunit of the serine palmitoyltransferase (SPT) holoenzyme, SPTLC1. The authors found that SPTLC1 blocks exit of ABCA1 from the endoplasmic reticulum and down-regulates ABCA1 efflux activity. Furthermore, this regulatory activity is independent of the enzymatic activity of the SPT holoenzyme.

Prior research from the same group showed that ABCA1 contained a C-terminus motif that conformed to the PDZ class I binding motif. By comparing the proteins that bound to wild-type ABCA1 against those that bound to ABCA1 lacking a functional PDZ binding motif, the group were able to identify several proteins that utilize the PDZ motif to interact with ABCA1. SPTLC1 was one of the proteins identified.

In this paper, the authors studied the interactions of SPTLC1 with ABCA1 in more detail.

First, co-immunoprecipitations were conducted with ABCA1. These experiments assume that if a target protein is captured via antibodies, then proteins associated with that target protein will also be pulled down. This paper found that SPTLC1 and ABCA1 form a complex in human macrophages and liver cells in physiologic settings.

Second, the authors studied the functional significance of this complex by inhibiting SPTLC1. This inhibition was enforced either at the translational level via siRNA knockdown of SPTLC1 mRNA or at the post-translational level via the chemical myriocin. In both cases of reduced SPTLC1 activity, ABCA1 cholesterol efflux to apoA-I was increased. Further testing using myriocin suggested that it was specifically the SPTLC1-ABCA1 complex that down-regulated ABCA1 activity.

Third, expression of two different dominant-negative SPTLC1 mutants (that do not have functional enzymatic activity) still resulted in ABCA1 down-regulation, suggesting that enzymatic activity was not required for negative regulation. Furthermore, expressing SPTLC1 (wild-type and dominant negative) with SPTLC2 to form the SPT holoenzyme still resulted in down-regulation of ABCA1. Lastly, to study the physical distribution of ABCA1 and SPTLC1, confocal microscopy was used. Cells containing ABCA1-GFP and SPTLC1 were compared to cells transfected with only ABCA1-GFP. This experiment revealed that SPTLC1 reduced cell-surface distribution of ABCA1, and ABCA1 tended to localize in the ER. Addition of myriocin disrupted the SPTLC1-ABCA1 complex and resulted in greater surface concentrations of ABCA1. Further experimentation suggested that SPTLC1 negatively regulated cell surface ABCA1 levels but did not affect total ABCA1 protein levels.

Lastly, to study the physical distribution of ABCA1 and SPTLC1, confocal microscopy was used. Cells containing ABCA1-GFP and SPTLC1 were compared to cells transfected with only ABCA1-GFP. This experiment revealed that SPTLC1 reduced cell-surface distribution of ABCA1, and ABCA1 tended to localize in the ER. Addition of myriocin disrupted the SPTLC1-ABCA1 complex and resulted in greater surface concentrations of ABCA1. Further experimentation suggested that SPTLC1 negatively regulated cell surface ABCA1 levels but did not affect total ABCA1 protein levels.

Significance:
ABCA1 is an important transporter of cholesterol to the extracellular apolipoprotein A-I (apoA-I), resulting in the formation of high density lipoprotein (HDL). In patients with Tangier’s disease, a defect in ABCA1 results in a severe reduction of HDL; this leads to atherosclerosis. On the other hand, the SPT holoenzyme is involved in the de novo synthesis of sphingolipids, which become incorporated into specialized lipid rafts and membrane bilayers. Consequently, SPT has been shown to have a positive correlation with atherosclerosis. Studying the effects of interactions between these two proteins could give us more insight into methods of suppressing atherosclerosis.

Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes

It is believed that the function and structure of cartilage is directed and maintined by the mechanical forces placed upon it, helping in part to create the stratification shown in fig. (a). This figure can be found in their original context in the paper linked to above.

Summary:
This article examines the growth and cultivation of chondrocyte cells for therapeutic uses in humans, with a focus on the design of bioreactors that can (at least partially) recreate the physical forces experienced by such cells in vivo, which are believed to affect their functionality. Chondrocytes are the type of cells that make the different kinds of cartilage in the human body. Cartilage itself is primarily composed of a complex ECM designed to resist different kinds of forces depending on its type and location. Articular cartilage in particular has the lowest volumetric cell density in the human body (99% ECM by volume), and is practically avascular, relying on diffusion and the movement of synovial fluid. Additionally, it must be able to withstand and absorb forces equal to several times a person’s body weight (particularly true of the articular cartilage in the hip and knee). These factors make it very difficult and slow for cartilage to heal itself, and sufficient damage can make self-repair impossible, emphasizing the desire for engineered cartilage tissue that can replace the damaged regions. Articular cartilage is thin and can survive reasonably hypoxic environments, side-stepping the vascularization problem of most engineered tissues; however, merely growing chondrocytes in collagen gels is not enough – the tissues structure and function are highly influenced by the dynamic processes it is subject to in the body, including hydrostatic pressure, compression, and other mechanical stimuli. The second half of the paper focuses almost entirely on different types of bioreactors that have been created to explore solutions to these issues by subjecting chondrocytes to these forces in a multitude of different manners, both statically (applying a constant force) and dynamically (varying the force, more closely approximating the natural environment). It ends with a discussion of using something like a ‘bedside bioreactor’ to grow cartilage implants from a patient’s own stem cells, but makes the point that before such devices are viable, more research must be done on the process of chondrocyte differentiation and the role mechanical forces play in it.

Significance:
One line from the paper underlines the societal importance of chondrocyte tissue engineering: “Currently, more than 40 million US American citizens (approximately 15% of the overall population of the USA) suffer from arthritis. It is estimated that nearly 60 million US American citizens will be affected by the year 2020.” That is a considerable portion of the population whose lives could be improved through the use of engineered cartilage tissue. The importance of mechanical and biological components seems to make this a task well suited to bioengineering. In addition, articular cartilage benefits from not being complicated by the need for a method of vascularization that beleaguers thicker tissues, increasing the chances that a method for the effective culturing of implantable cartilage tissue could be found in the near future.
(And on a merely personal note, having had surgery that involved removing a chunk of cartilage from my knee, I think it would be really great if we could develop something that could repair it before the bones in my knee wear out!)

17 β-Estradiol responsiveness of MCF-7 laboratory strains is dependent on a n autocrine signal activating the IGF type I receptor

17β-Estradiol responsiveness of MCF-7 laboratory strains is
dependent on an autocrine signal activating the IGF type I receptor
Irene HL Hamelers, Richard FMA van Schaik, John S Sussenbach and
Paul H Steenbergh*

Address: Utrecht Graduate School of Developmental Biology, Department of Physiological Chemistry, University Medical Center Utrecht, P. O.
Box 85060, 3508 AB Utrecht, The Netherlands
Email: Irene HL Hamelers - I.Hamelers@nki.nl; Richard FMA van Schaik - F.M.A.vanSchaik@med.uu.nl;
John S Sussenbach - j.sussenbach@hccnet.nl; Paul H Steenbergh* - P.H.Steenbergh@med.uu.nl
* Corresponding author

Published: 11 July 2003
Cancer Cell International 2003, 3:10
Received: 28 January 2003
Accepted: 11 July 2003
This article is available from: http://www.cancerci.com/content/3/1/10
© 2003 Hamelers et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.

Summary:

Human MCF-7 cells have been used as a model for breast cancer cell growth. Literature states that the serum-starved MCF-7 cells can be induced to proliferate by only adding 17 β-Estradiol (E2). However, the level of the mitogenic response to E2 fluctuates in different MCF-7 laboratory strains. In this paper, the E2-sensitivity of three different MCF-7 laboratory strains was studied.

With about the same levels and activities of the estrogen receptor (ER), the MCF-7 NKI is most E2-sensitive, the MCF-7 ATCC is intermediate E2-sensitive, while the MCF-7S is non-responsive to E2. Yet, the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells can be inhibited by both suramin and IGF type I receptor blocking antibodies. Therefore, E2-induced proliferation of all three MCF-7 strains depends on IGF type I receptor activation.

To examine whether E2-induced cell cycle in all three MCF-7 strains is dependent on activation of the IGF type I receptor, two DNA synthesis assays were performed; one with suramin addition and the other with αIR3 antibody addition. As results, IGF-induced incorporation of 3H-TdR is completely inhibited with suramin (Figure 3 in the article) and with αIR3 (Figure 4 in the article).

Figure 3 (left column): Effect of suramin on the induction of DNA-synthesis by IGF-I and E2 in the three MCF-7 strains
Figure 4 (right column): Effect of an IGF-RI blocking antibody on the induction of DNA-synthesis by IGF-I and E2 in the three MCF-7 strains

Significance:

The results of this article indicate that E2-responsiveness of MCF-7 cells is affected by the secretion of an autocrine factor activating the IGF-IR. The blockage of the IGF-RI-pathway prevents all three MCF-7 strains from responding to E2. Breast cancer therapy is generally targeted at inhibiting estrogen action. This study suggests that the inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way to treat or even prevent breast cancer.

I personally chose this paper because my group decided to examine MCF-7 cells’ VEGF secretion and estradiol as its stimulus. This article provides information of MCF-7 cell culture and Western blotting, which should be useful for our project.

MCF-7 breast cancer cell line grown in agarose culture for study of COX-2 inhibitors in three-dimensional growth system

MCF-7 breast cancer cell line grown in agarose culture for study of COX-2 inhibitors in three-dimensional growth system

David H. Kinder ^a and Amy L. Aulthouse ^b

^a Department of Pharmacological and Biomedical Sciences, College of Pharmacy, Ohio Northern University, Ada, OH 45810, USA ^b Department of Biological Sciences, College of Arts and Sciences, Ohio Northern University, Ada, OH 45810, USA

Received 21 March 2003;

accepted 13 October 2003.

Available online 18 February 2004.

Summary: This paper reports a method for studying pharmaceutical COX-2 inhibitors in the MCF-7 breast cancer cell line where cells are grown in a three-dimensional agarose matrix. Cyclo-oxygenase (COX) has been shown to be elevated in several cancers and clinical studies have shown that certain cancer types can be prevented or at least minimized by patients who take cyclo-oxygenase inhibitors. In order to overcome the drawback of the insensitivity of cancer cell lines grown in monolayer format to the COX-inhibitors, this paper utilizes a three-dimensional growth pattern in which cells were grown in 3-D agarose culture in the same method used for human chondrocytes, allowing the system to be free of cellular attachment to plastic and allow in vivo tumor-like growth patterns.

The cell line chosen was the estrogen receptor positive breast adenocarinoma MCF-7, and the treatment was the COX-2 selective drug etodolac (Lodine). MCF-7 cancer cells were suspended in agarose and plated in a cell culture dish where they form small multicellular ‘tumors’ in the agarose. Then, they were treated with COX-2 inhibitors for 2.5 weeks to induce cell death (determined by trypan blue exclusion), and this was compared to cells grown in monolayer given the same drug concentration.

In 3-D culture, it was observed that there was increasing amount of dead MCF-7 cells (either single cells or colonies) with treatment of etodolac, supporting the concept that etodolac, and presumably other COX-2 inhibitors, were eliminating polyps or decreasing tumor formation by inducing a form of cell death, presumably apoptosis. Moreover, etodolac did not show any cytotoxicity or cell growth inhibitory effects in monolayer culture at 250ug/ml drug concentrations, yet it had a clear effect on the growth of cells grown in agarose cultures where 3-D colonies were formed at the same drug concentration.

Ultimately, the paper introduced a method for growing cancer cells that circumvents the growth characteristics of monolayer cultures by allowing 3-D growth habits.

Significance: Breast cancer is one of the most common type of cancer (10.4% of all cancer incidence) and occurs in both women and men worldwide. Although breast cancer in men is about 100 times less common than women, men with breast cancer have the same statistical survival rates as women (American Cancer Society). Being able to mimic the growth of the breast cancer cells in vitro through methods like a three-dimensional agarose matrix allows for a more progressive study on matters like the mechanism of cell death and growth in presence of certain kinds of drugs, ultimately to discover biochemical or physio-chemical factors that will improve or replace biological functions.

For my project in Bioe115, I hope to evaluate the affect of chemical stimuli or inhibitor at varying concentrations on MCF-7 breast cancer cells cultured in well-plates to ultimately measure the secretion of VEGF (vascular endothelial growth factors), which is believed to be the key mediator of angiogenesis in breast cancer. Hopefully, with the aid of this paper, I can perform my study of VEGF production with a 3-D agarose matrix that mimics the environment of a cancer growing in vivo.

Ranieri Cancedda, Beatrice Dozin, Paolo Giannoni, and Rodolfo Quarto: Tissue engineering and cell therapy of cartilage and bone

Canceda et al. provides a comprehensive practical and biochemical analysis and evaluation of different treatments for bone and cartilage lesions, as well as providing sufficient background information about the biology of appropriate cells and tissues relevant to the discussion. The apparent motive for this paper is the widespread cartilage and bone damage and their increase vulnerability with age, and the challenges current treatment methods pose to clinicians.

The field or regenerative medicine aims at using innate regenerative biochemical pathways to heal damaged tissue. Different tissues have different regenerative potentials. Bone tissue is much more prone to regeneration than cartilage because of its continuously changing morphology through the lifetime of an organism. These differences lead to different types of treatment for the lesions, depending on their area of infliction.

Common surgical treatment for joint damage are microfracturing, autologous osteochondral transfer (mosaicplasty), and fresh osteochondral allograft. However, these procedures are limited by donor characteristics like number, compatibility, and by skill level the procedure require. Similarly, bone function is restored using bone transport (Ilizarov method), bone graft transplant, and a combination of osteotomy and bone transport. These techniques are restricted by a variety of factors: complication rate, recovery time, and extent of damage. Finally, a more small-scale approach has been autologous chondrocyte implantation, which has shown very high success rate, has not been evaluated comparatively yet.

Engineering bone and cartilage requires several central considerations: use of suitable cell types, use of suitable scaffold, and the engineering process itself. Two cell types have been targeted for use for cartilage and bone tissue engineering: mature chondrocytes and mesenchymal stem or progenitor cells; bone marrow stromal cells are a specific type of cell that is considered an adult stem cell. The main issue with mature chondrocytes is their limited and age-dependent proliferation potential and fragile differentiation potential. This issue is bypassed when using progenitor or stem cells due to their intrinsic high proliferation and differential potentials over long period of time. Another noteworthy advantage is their ability to promote vascularization, which allows nutrients to be more easily delivered to more areas of the engineered tissue. A specific subset of these cells, bone marrow stromal cells (BMSCs), is limited by the cells’ sensitivity to their microenvironment, loss of lineage potential with doubling iterations, and their lack of telomerase. Nevertheless, it is widely use for treatment because it is easy and safe to obtain. Some of the aforementioned issues can be surmounted by addition of growth factors like fibroblast growth factor-2 (FGF-2).

Scaffolds are designed to provide physical support for the extracurricular matrix (ECM), permit diffusion of nutrients and cell waste, and integrate with the surrounding tissue. Ideally, the scaffold would degrade at a rate corresponding to the rate of production of ECM, allow vascularization, and be biocompatible and resorbable. The former has not been achieved yet. Additional major drawbacks of common scaffolds are much less-than-ideal control of cell-substrate interactions and release of active molecules such as minerals, pore size, and geometry. Currently several types of natural scaffolds are used: collagen I, fibrin, alginates, and hyaluronic acid. Synthetic scaffolds are favorable to natural ones because there is better control of substance delivery and there is less of a biohazard potential; however, this type of scaffold usually induces an immune response from the host.

More efficient, regenerative treatments of bone and cartilage lesions are being tested on animals models. Cancedda et al. makes mark of important consideration with regards to choosing an appropriate animal model for testing: the regenerative capacity, matrix layer thickness, and relative load carried by animals of different species vary greatly. Animal testing is meant to tests specific aspects of the cartilage repair ability, as opposed to how well it will work in humans; thus, a good animal model would be an adult, big animal, and its spontaneous healing would be limited by inflicting heavy damage on the tissue.

According to Cancedda et al., future work should focus on creating a matrix that is biodegradable yet resistant, permits cell filtration, survival, proliferation, differentiation, and integrates with surrounding tissue.

The encompassing scope of this article provides the reader and the scientific community with a thorough recapitulation of current bone and cartilage lesion treatment methods, with a focus on tissue engineering and the major design considerations behind this field. Moreover, critical animal model identification aspects are highlighted. This article can serve as the origin of concept generation towards better tissue engineering design by presenting what is on the table currently. It can also be used to stem more cell-control research.

I chose this article specifically because of its comprehensive scope and the crucial, prevalent clinical issue it discusses. I know that arthritis is an extremely common illness that has many sources; I was curious to know what treatments are currently on the market. These treatments can also be extrapolated to damage originating from tissue removal due to cancer, which is of great concern to me as a human being. I feel that I am much more well-informed about treatment options for such harms, when I will have to face them.

Differences in Interleukin-1 Response Between Engineered and Native Cartilage

Differences in Interleukin-1 Response Between Engineered and Native Cartilage

E. Lima et al. Tissue Engineering: Part A. Vol. 14(10):2008.

(link to pdf of summary)


The goal of cartilage tissue engineering is to replace degenerated cartilage with engineered cartilage tissue that has similar physical characteristics. Although fragile at formation, the engineered tissue matures over time and forms its own extracellular matrix that provides strength and protection. Clinicians choose whether to implant immature engineered tissue and allow it to mature in vivo or mature engineered tissue in vitro before implanting it.

The authors of this article hypothesized that in vitro maturation would provide better cartilage implants because the cartilage would have developed better protection against the harsh chemo-mechanical environment found in a diseased joint. Diseased joints usually have chronic inflammation, and so proinflammatory cytokines, such as Interleukin-1α (IL-1α) are present, which increase the catabolic rate of cartilage in vivo. To test their hypothesis, the authors explored the response of engineered cartilage versus native cartilage when exposed to inflammatory factor interleukin-1α in vitro.

The article consists of 3 studies. In all 3 studies, the authors compared the following physical characteristics to formulate their results: Young’s Modulus (E), Dynamic Modulus(G), GAG concentration, and Collagen concentration. The first 2 properties are mechanical properties, whereas the latter 2 are chemical properties.

In the first study, the authors compare native cartilage explants (bovine source) to engineered cartilage tissue. After 14 days of culture growth, IL-1a was introduced to some of the explants and engineered cartilage (considered immature at this time point). The samples that grew without IL-1a after day 14 served as controls. The properties of all the groups were analyzed at day 28. Compared to its control, the difference in all 4 properties of the explants was statistically insignificant, whereas the engineered tissue’s properties had degenerated significantly.

In the second study, the authors studied engineered cartilage at 3 different stages: at day 0 (immature), day 14 (immature) and day 28 (mature). These were the time points at which IL-1a was introduced to each culture. There was also a fourth control group that was cultured without IL-1a at any time point. After a total of 42 days, the properties of all groups were analyzed. The results showed that the two immature groups were significantly degenerated compared to the control group. The mature engineered tissue had no statistically significant differences in any of the 4 measured properties.

In the third study, the authors studied the growth of engineered cartilage and native explants in the presence of IL-1a as well as dexamethasone (dex), an anti-inflammatory chemical mediator that counters the effects of IL-1a. Engineered cartilage that had been grown in the presence of dex since the start had statistically insignificant properties compared to the control. Engineered cartilage that was grown in presence of both dex and IL-1a for 28 days didn’t different significantly either; even after removing dex (after day 28), the cartilage properties didn’t differ significantly. However, engineered cartilage that was grown in dex + IL-1a for only 14 days showed significant degradation once the dex was removed after day 14.

Results from all 3 studies signify that the response of engineered cartilage tissue to inflammatory factor IL-1a varies greatly depending on the level of maturity of the tissue culture and will significantly impact the clinical success of the engineered tissue after implantation. This study supports the idea that engineered cartilage constructs should be implanted when the tissue is functionally mature because the presence of an extracellular matrix will not only protect cells mechanically, but will also protect the cells from chemical degradation.

Significance:

Articular cartilage degeneration due to diseases such as osteoarthritis, rheumatoid arthritis, and other injuries lower the quality of life of a person significantly by making even the simplest of actions, such as walking or running, excruciatingly painful. These diseases are very common, especially in the older population. Currently, degenerated cartilage after repair surgeries is either replaced by a synthetic substitute (e.g. in total knee arthroscopy), or healthy cartilage (from external sources) is implanted in the hopes of promoting repair. Although both processes improve a patient’s quality of life, both of them have several flaws. Cartilage tissue engineering is a very important avenue of research as it provides a chance to improve the above mentioned methods of repairing or replacing degenerated cartilage. This paper in particular is quite useful as it provides a good insight into what kind of studies must be conducted to optimize the growth of cartilage tissue in vivo. The researchers reproduced some of the harsh in vivo chemical conditions that the cells would face in vitro and then analyzed different cultures to determine whether the maturity of the engineered tissue significantly alters its properties when placed in harsh chemical conditions. This paper has very practical conclusions that can improve the clinical success of engineered cartilage implants drastically.

Cell Adhesion Strength to Bioceramics and Morphology

Cell Adhesion Strength to Bioceramics and Morphology

Tetsuya TATEISHI, Takashi USHIDA

Biomechanics Division, Mechanical Engineering Laboratory, Agency of Industrial

Science & Technology, Namiki 1-2, Tsukuba, Ibaraki 305, Japan

The authors of this paper set out to find and improve the characteristics of cell/biomaterial interfacing. To do this they setup a control of fibroblast cells on a standard alumina cell culturing plate, and compared the adhesion properties to an alumnia cell culture plate coated with fibronectin. the two cultures were tested under conditions of vertical stress in hopes that the results will yield information as to better adhere cells to biomaterials.

The specimens were put under vertical load by attaching the culture upside down and attaching to a centrifuge to produce a vertical acceleration. The cultures were then imaged to see the number of lost cells and area. The results found that the fibronectin coated plates had a larger adhesion area( 1200 microns squared vs 500 microns squared). This is thought to correlate with higher strength because cells adhere to material surfaces with adhesion plaques where fibronectin receptors interact with fibronectin of the surfaces. So if adhesion area correlates to number of plaques this would mean a stronger bond. The results of the experiment supported this theory. The mesured rate of cell peeling was higher on the alumina plate( 80% as apposed to 50% of fibronectin coated).

A mathematical model was used to predict cell peeling by characterizing the rupture process of fibronecting receptor bonds. The results of the model gave a distribution that was very close to the results shown in the non coated cell plate.

Significance:

Many applications of bioengineering involve in vitro implementation of devices. The common problem with these devices is their incompatibility with the cells around them. An ability to vary the inter-material properties would be valuable in implants such as an artificial hip joint. In a hip joint the stem that is implanted into the bone needs to be a place of high friction so the implant doesn’t slip. On the other end, the surface needs to have a minute friction coefficient so that there is no rubbing between the prosthesis and the pelvis. To be able to vary the friction on cell/material interfaces would allow for a more viable and longer lasting prognosis.

Long-term culture of muscle explants from Sparus aurata

Funkenstein, B., Balas, V., Skopal, T., Radaelli, G., Rowlerson, A.
Tissue and Cell 23 (2006) 399-415


Summary:

This paper is about the development of a fish myoblast cell line from Sparus aurata fry muscle explants. The mechanisms of somatic growth and development, regulated by hormones, growth factors, and cytokines, are relatively well conserved among vertebrates. Fish skeletal muscle, like all vertebrate muscle, develops from myoblasts in the somatic mesoderm, and they can continue to grow significantly into juvenile life through continuous hyperplasia and hypertrophy. Hyperplasia is the proliferation of cells within an organ or tissue beyond that which is normally seen, and can result in the gross enlargement of an organ/tissue. Hyperplasmic growth of muscle refers to an increase in muscle due to the formation of new fibers from myogenic precursor cells. Hypertrophy is the increase in the size of an organ or area of tissue due to an increase in the size of cells, not a change in the number of cells. Hypertropic growth requires existing fibers to acquire additional nuclei, which they can do by fusing with mononucleate myoblasts. It has not yet been fully established if the same or different myogenic precursors underlie these two growth mechanisms. Many labs have previously attempted to develop primary cultures of fish myoblasts to further investigate this, however, although the myoblasts can be isolated and grown in culture, they do not proliferate. The authors of this paper were successful in developing a culture system of proliferative fish muscle cells.
In order to create the cell cultures, they took white muscle explants from Sparus aurata fry. The explants were cultured in both sterile cell culture flasks and on glass cover slips. After various culture periods the explants were either dehydrated, embedded in paraffin and sliced using a microtome, fixed to the cover slips and stained using immunohistochemistry, or solublized in order to study proteins. Anti-PCNA was used to assess cellular proliferation, and the TUNEL assay was used to follow apoptosis in the original muscle fibers. Early differentiation was tracked by staining with anti-Myf5, and terminal differentiation by staining for desmin, myosin and parvalbumin. RT-PCR was also performed on some muscle explants to study gene expression.
The results of this study show that muscle explants from Sparus aurata can be grown in long-term culture. New fibers will appear as the original ones degenerate. The in vitro system mimics parts of the in vivo situation of damaged muscle and subsequent regeneration. The system should be useful for studying the interaction between growth inhibitors, like MSTN, and growth factors, like IGFs, as well as for studying fish muscle cell precursors.

Significance:
This article is important because it is declaring the development of a new cell line from primary tissue. Use of this new cell line in future studies could provide further insight into muscle regeneration and myogenic precursor proliferation. This may eventually lead to promising future research in human skeletal muscle/muscle regeneration. The development of new cell lines is important in and of itself because it could positively impact research involving in vitro cultured cells. New cell lines could be used to help validate previous experiments by providing new environments in which researchers can test their hypothesis. They can also enable studies that were previously difficult to conduct, for example the effect of IGFs on fish skeletal muscle cells should now be easier to follow.
Additionally, many of the techniques used in this study were covered in class. This is a great application of the tools we have been learning to use in lab! Yay!

In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs

Cullen, D.K., Stabenfeldt, S.E., Simon, C.M, and C. Tate. 2007. In Vitro Neural Injury Model for Optimization of Tissue-Engineered Constructs. Journal of Neuroscience Research 85: 3642-3651.

Summary:
Traumatic brain injury (TBI) is a common form of neurological damage caused by physical deformation of the brain. This particular type of injury can cause permanent damage to an individual’s cognitive, motor, or sensory functions. Current attempts to restore brain function via therapeutic means have shown only limited success, particularly because of our limited understanding of the intricate pathological mechanisms initiated by the body in response to TBI. It is known that a hostile environment is created as a result of trauma-related destruction of neural cells. One aspect of the body’s response can be reactive astrogliosis in which a glial scar is formed to effectively cut off injured tissue from the rest of the brain. This glial scar is composed of hypertrophic astrocytes that are characterized by an abundance of chondroitin-sulfate proteoglycans (CSPGs) and increased expression of intermediate filaments like glial fibrillary acidic protein (GFAP). Another main feature of post-injury environment is that cell death may be delayed for months after injury onset. Tissue engineering provides one potential approach for addressing such issues, as it could enable researchers to optimize cells to be transplanted into such a hostile environment as well as allow for prolonged treatment of this injury. As of now, however, the biggest obstacles to implementing effective methods in vivo have been the abundance of donor cell death and lack of integration of these cells with host brain tissue following transplantation.



Figure 1




Figure 2

To confront the complications associated with in vivo cell transplantation in treating TBI, the authors of this paper developed a 3D in vitro model to mimic specific aspects of the hostile post-injury environment consistent with TBI. Cell cocultures were generated using neurons from embryonic day 17-18 rat fetuses and astrocytes from postnatal day 0-1 rat pups. These neuronal-astrocytic cocultures were initially plated at a neuron:astrocyte ratio of 1:1 and allowed to culture in vitro for 21 days in order to allow for maturation of the cells and formation of neural networks (Figure 1). The cocultures were then separated into three groups and either treated with transforming growth factor-β1 (TGF-β1, a cytokine shown previously to induce astrogliosis), subjected to mechanical deformation, or left untreated (control conditions). Mechanical deformation of cocultures was performed using a 3D cell-shearing device. The resulting heterogenous distribution of strains on cells was characteristic of the sort of mechanical injury that occurs in vivo via TBI. The cocultures that received TGF-β1 served as experimental controls by mimicking TBI-induced astrogliosis but without the associated precursor of cell death. This set-up allowed researchers to isolate how astrogliosis alone affects transplanted neural stem cells (NSCs). 48 hours post-insult (i.e. after treatment with TGF-β1 or mechanical loading), NSCs were delivered in medium to each of the cocultures.

First, the viability of astrocytes and neurons in the cocultures was assessed using a live-dead assay (Figure 2). A significant (i.e. P<0.05) decrease in the percentage of viable cells accompanied by increase in spatial density of dead cells following mechanical loading was found (as revealed by fluorescent confocal microscopy), but no significant change in cell viability amongst cocultures treated with TGF-β1 was observed. Immunochemistry was also employed to detect reactive astrogliosis by tracking markers for GFAP and CSPG, two substances known to be in abundance when astrocytes are undergoing this process of growth due to local neuron death. In fact, a significant increase in CSPG expression in the matrix was found post-insult only in TGF-β1-treated cultures (relative to controls). GFAP expression was used to examine both astrocytic reactivity and hypertrophy. In both the TGF-β1-treated cocultures and mechanically-deformed cocultures, the authors found a significant increase in GFAP immunoreactivity accompanied by an increase in astrocyte process density compared to cell in the control cocultures. Additionally, the viability of donor cells post-transplantation was examined using TUNEL staining, an assay that interacts with fragmented DNA of dying cells. A significant increase in the proportion of TUNEL+ NSCs was observed upon transplantation into the mechanically injured cocultures compared the other two coculture types (Figure 3). Thus factors specific to the mechanically-damaged environment but not the strictly astrogliotic environment were found to negatively impact NSC survival; but this same environment yielded no abnormal NSC differentiation patterns relative to the other two types of cocultures (Figure 4).

Based upon these findings, the researchers decided to further explore NSC viability upon transplantation into a mechanically-damaged environment. Specifically, they hypothesized that the method with which cells are delivered to the injury site could affect post-injury survival of donor cells. Furthermore, they suspected that tissue-engineered bioactive scaffolds could improve donor cell survival by adding structural and adhesive support for the cells. NSCs were delivered to both mechanically-damaged and control 3D cocultures via three different delivery methods (a media vehicle, a methylcellulose (MC) scaffold, or a methylcellulose-laminin (MC-LN) scaffold). Frequency of caspase activation was used to gauge the efficacy of delivery, since caspase activation signals the initiation of caspase-related apoptotic pathways. When NSCs were delivered in either the MC or MC-LN scaffolds to the mechanically-injured cocultures, a significant reduction in caspase activation was observed. The authors also simultaneously examined growth of donor cell processes and found that growth was significantly improved by the use of an MC-LN scaffold relative to use of a media vehicle. These results suggest that delivery of NSCs to mechanically-injured cocultures using MC-LN scaffolds decreases donor cell death attributable to caspase-apoptotic mechanisms and also promotes integration of these cells with the host environment.

Overall, the results of this paper showed that according to 3D in vitro models of brain tissue post-TBI, other factors affecting mechanical injury (beyond just astrogliosis) are responsible for donor cell death following delivery to injured brain tissue. Additionally, the use of bioactive scaffolds for donor cell delivery provide a promising option for improving donor cell survival and success post-transplantation.

Figure 3


Figure 4


Significance:
According to current figures published by the U.S. Department of Defense, 10 to 20 percent of returning soldiers present symptoms characteristic of mild TBI, so one can see that this is a pressing medical issue that demands the attention of today's scientific and medical communities. This paper demonstrates the promising development of a 3D in vitro model to serve as a surrogate environment that mimics the biological response of brain tissue damaged via TBI and its possible contribution to engineering more optimal conditions for improved NSC survival upon transplantation. Typically, finding optimal donor cell conditions prior to therapeutic attempts has required expensive and time-consuming in vivo studies. The model developed by these authors, however, could possibly serve as a better alternative in the early stages of testing. Additionally, this paper provides the framework for the development of a model that is flexible enough to allow for trialwise elucidation of other factors hypothesized to affect NSC survival in vivo. Another aspect of this research is that it reinforced the possible contribution of optimized tissue-engineered scaffolds for the transplantation of donor cells to areas affected by mechanical injury. Overall, this paper provides a more feasible method for further improvement of transplantation strategies, strategies which might someday results in proven methodology for the treatment of TBI and other neurological deficits.

I chose this paper, because it suits my interest in traumatic brain injury research while also expanding upon many of the cell culture techniques/concepts we’ve learned in this class. As an undergraduate researcher, I conduct brain imaging and rehabilitation research and work primarily with individuals who have TBIs. But prior to discovering this article, I was virtually unaware of efforts being made in the tissue engineering field to regenerate damaged tissue crucial to our most important neural networks. So I found this paper very stimulating and promising!

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells (Cynthia Chuang)

Effects of oestradiol and tamoxifen on VEGF, soluble VEGFR-1, and VEGFR-2 in breast cancer and endothelial cells

S. Garvin, U.W. Nilsson, and C. Dabrosin
Division of Gynecologic Oncology, University Hospital, SE-581 85 Linko¨ping, Sweden
British Journal of Cancer (2005) 93, 10005-1010. Published online 18 October 2005.

Summary

This paper sought to discover the relation between cancer and endothelial cells and the effect of oestradiol and tamoxifen on both types of cells. Vascular endothelial growth factor (VEGF) is a key mediator of tumor angiogenesis, which includes neovascularisation in human breast cancer, and acts via two tyrosine kinase receptors VEGFR-1 and VEGFR-2. Oestradiol has been proven to increase levels of VEGF while tamoxifen inhibits the secretion of VEGF in breast cancer in vivo. Ratio of sVEGFR-1 to VEGF is a strong indicator of disease-free and overall survival in breast cancer patients.

In order to investigate the effects of oestradiol and tamoxifen on sVEGFR-1 and VEGFR-2 in human cell lines in vitro and a mouse model of breast cancer in vivo. Human umbilical vein endolethial cells were isolated and grown in Dulbecco's modified Eagle's medium. Cell were used from passages 2-3. Mice were implanted with pellets that either either continuously released oestradiol or released a placebo. MCF-7 cells were cultured in Dulbecco's modified Eagle's medium, trypsinised, seeded into Petri dishes, and incubated. The cells were then treated with or without oestradiol, or a combination of oestradiol and tamoxifen. The medium was changed every day, and on day 7 secreted VEGF and sVEGFR-1 was quantified using Bio-Rad DC Protein Assay. Tumor growth was determined through volume calculation.



Figure 1: Tumor sections from nude mice with MCF-7 stained with anti-von Willebrand's factor. (A) oestradiol group (B) oestradiol+tamoxifen-treated groups. Arrows indicate examples of positively stained vessels.

Results indicated that oestradiol decreased secreted sVEGFR-1, increased secreted VEGF, and decreased the ratio of sVEGFR-1/VEGF in MCF-7 human breast cancer cells. Addition of tamoxifen significantly countered the effects of oestradiol. Additionally, tamoxifen and oestradiol exert dual effects on the angiogenic environment in breast cancer by regulating cancer cell-secreted angiogenic ligands (e.g. VEGF and sVEGFR-1) and by affecting VEGFR-2 expression of endothelial cells.

Significance

Breast cancer is one of the most common forms of cancer in women and one of the leading causes of cancer death. This study demonstrates the advances being made to better understand the linkage between breast cancer cells' individual molecular components, oestradiol, and tamoxifen. It is an example of the use of tissue engineering as it attempts to better understand the principles of breast cancer cell growth. Application of results can be used to develop therapeutic strategies aimed at replacement and repair of tumor cells. In this case, combination of tamoxifen and oestradiol delivered to breast tumor cells is a viable solution that should be further explored to eliminate or reduce breast cancer.

My lab group is interested in conducting an experiment based on the understanding of oestradiol's relationship with MCF-7 breast cancer cells and VEGF secretion to determine gene expression and optimal VEGF secretion rate. This paper is a valuable resource as it gives (1) a protocol and conditions to grow MCF-7 cells and (2) amount of oestradiol that can be added to physiologically mimic local production and accumulation of oestradiol in human breast tumors in vivo. Additionally, this paper's reference section will serve as additional resources for exploration.

Neural tissue engineering: A self-organizing collagen guidance conduit

Neural Tissue Engineering: A Self-Organizing Collagen Guidance Conduit

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James B. Phillips, Stephen C.J. Bunting, Susan M. Hall, Robert A. Brown. Tissue Engineering. September 1, 2005, 11(9-10): 1611-1617. doi:10.1089/ten.2005.11.1611.


Nerve autografts are currently widely used to bridge gaps in transected peripheral nerves. However, this practice is not ideal as it involves harvesting donor tissue which might cause donor site morbidity. So far, numerous compounds have been used in order to produce tissue-engineered conduits but only empty tubes have been approves for human use. The guidance provided by empty tubes on the tissue level is, however, limited and a better device would effectively be multilayerd to provide guidance at the cellular level.

This paper reports on an implantable device which delivers aligned collagen guidance conduit into a peripheral nerve injury site. The conduit contains Schwann cells. These cells, which were in tethered rectangular gels were observed to result in uniaxial alignment. The practical use of this methodology was tested in 3-D culture where it demonstrated the ability to guide neurite extension from dissociated dorsal root ganglia. Rectangular tethered gels were seeded with Schwann cells and allowed to contract and axonal growth was detected after 3 days by fluorescence immunostaining. Neurons seeded extended neurites parallel to the axis of tension in the central region of the gel which was known to contain aligned Schwann cells. No cells growth was seen at the stress-shielded delta zones at the ends.

http://www.liebertonline.com/na101/home/literatum/publisher/mal/journals/production/ten/2005/11/9-10/ten.2005.11.1611/pdfimages_v02/master.img-005.jpg

For an implantable construct, a silicon tube was incorporated within the tethering site which aided the uniaxial cell generation to form a bridge of aligned collagen fibrils with Schwann cells. The device was tested for surgical nerve regeneration in a rat sciatic nerve model with a 5mm defect. The implanted aligned Schwann cell collagen constructs tethered in silicon tubes were implanted into short gaps in transected rat sciatic nerves where they supported axonal outgrowth. This outgrowth generation was greater than observed in controls: 1)nerves across empty conduits of plain silicon tubes with no self aligned collagen and 2) no device.

The significance of this paper is that it describes a protocol for producing a novel device to deliver a self-aligning cell-seeded conduit for use in neural tissue engineering. The relative simplicity and robustness of this new device compared to current procedures can make it an alternative to nerve autografts. Moreover, the application shown here can be applied to create 3-D engineered tissue models.

Engineering Light-Gated Ion Channels: A Review

Matthew R. Banghart, Matthew Volgraf, Dirk Trauner

Biochemistry [2006]

Summary:

Ion channels found in cellular membranes can be controlled by a variety of stimuli, including ligands, voltage, and in some cases, light.  These Ion channels in turn control the electro-chemical properties of the cell, by changing conductance based on the external stimulus.  By artificially fabricating or specific placement of these cells, engineered cultures can be better controlled by a predetermined and controlled stimulus.  A light-gated reversible ion channel has many practical applications in tissue engineering due to the quick response of a light source compared to delayed reaction times with ligand bonding, as well as the ease of stimulation compared to many of the aforementioned methods.

The switches that control the gates can function in a few specific ways:  through changing the local electric field, moving hindering protein subgroups, or by inducing a reversible conformational change within a gating protein.  There are a few basic criterions to engineering a gated ion channel to ensure functionality on the cellular level.  It is important to make sure the gate functions on a cellular time scale, with activation times on the order of microseconds.  Generally, when engineering gated ion channels, it is more important to focus on a complete “off” position than an “on” configuration because a constant partial flow of ions within a cell could be toxic to the cell.  The mechanism by which light gating works is by inducing a cis-trans conformational change when a certain wavelength of light is shone upon a protein linked to the ion gate.

When a light is shone upon the system in (A), a conformational change brings the two halves of the ion gate together, allowing ions to pass.  In (B), the natural configuration of the protein is such that there is a large steric group that blocks the ion channel, and when light is shone upon the system, the protein opens to a trans configuration, thereby removing the blocking steric group.

Current applications of light-gated ion channels include simple control of neurons by controlling dopamine cells, as well as muscular control.  A test with fruit flies showed a correlation between dopamine release and exploration of environment; light-gated ion channels regulated dopamine channels such that when a light was shone on the insects, dopamine release was triggered, resulting in the fruit fly exploring the container in which it was kept.  Muscular control using light-gated ion channels works based on the requirement of certain ions to trigger or relax muscles.  By using a light gated ion channel in conjunction with a light that causes sodium or potassium ion gates to transport, muscle contraction can be coordinated to a programmed light source.

Significance:

Light gated ion channels have promising applications to tissue engineered materials based on their ability to transport ions that could be used as signaling mechanisms to promote cellular metabolism.  If ions could be isolated that cause production of certain proteins or other potentially helpful molecules (like dopamine, for instance) then one could imagine having bunches of light-gated ion channels that respond to different frequencies of light, that release different cellular signals for production of different molecules within one tissue.

Exposure to carbon nanotube material: assessment of nanotube cytotoxicity using human keratinocyte cells.

Exposure to carbon nanotube material:

assessment of nanotube cytotoxicity using human keratinocyte cells.

Shvedova AA, Castranova V

Summary:

In this paper, the authors studied the adverse affects of single-walled carbon nanotubes (SWCNT) that are exposed to human epidermal keratinocytes. They performed these tests because carbon nanotubes, a relatively new particle, have been shown to have novel properties that have potential use in the medical world. These applications include drug delivery, cancer treatment, and a device strengthening. The paper was mostly interested in the oxidative stressed that was placed on the cells and the morphological changes that took place in the cell.

To measure the oxidative stress placed on the cells, there were many approaches taken. The first was to detect the free radicals present in the cell. They used ESR spin trapping to detect free radicals by recording the spectrum from the mixture containing the cells exposed to the SWCNTs. The results indicated a large free radical OH production that was SWCNT stimulated.

In order to determine if this stress caused a morphological change in the cells, scanning and transmission microscopy of the cells was used. Transmission microscopy showed that there were cell morphological changes in the nucleus, mitochondria, tonofiliments, and other cytoplasmic organelles.

Another attempt to measure oxidative stress came with measuring the total antioxidant reserve in the cells. This was measured through a chemiluminescence assay was used. Results showed that there was a “concentration-dependent decrease in antioxidant reserve level”

Conclusions:

The paper concluded that it is the catalytic iron that is used in the production of nanotubes that causes the oxidative stress that is present in the cells. This comes from nanotubes that are not refined. Unrefined nanotubes have been shown to contain 30% iron composition. The iron has the ability to catalyze one electron reactions leads to the extra oxidative stressed placed on the cells.

Relevance:

This paper is relevant to my project because of the many biological uses that nanotubes may have in the future. We are interested in the effects of nanotubes on cells because of the therapeutic potential that they hold in the medical world. After reading the conclusion, it is slightly off topic from the reasons I am pursuing the question about the effects of nanotubes on cells. Since they concluded that iron places this stress on the cells, simply refining the nanotubes can fix the problem. We are more interested in the actual carbon structure, and the effects it makes on the cells. It is still important to realize that the refinement of nanotubes is important in biocompatibility, and it is important to know that if we use unrefined nanotubes in our experiment, it can alter our results.