Construction and transplantation of an engineered hepatic tissue using a polyaminourethane-coated nonwoven polytetrafluoroethylene fabric.

Soto-Gutierrez A, Navarro-Alvarez N, Rivas-Carrillo JD, Tanaka K, Chen Y, Misawa H, Okitsu T, Noguchi H, Tanaka N, Kobayashi N.

Transplantation. 2007 Jan 27;83(2):129-37.

Acute liver failure (ALF) is a serious disease with a high mortality rate. In severe cases, treatment of the disease, even when it is transient, requires transplantation and lifelong immune suppression. However, due to the shortage of donors, high costs, and the associated risks, treatments that involve temporary liver support are preferred over transplantation. Current bioartificial livers deal with issues of platelet consumption and hemodynamic instability. The researchers here have developed a new bioartificial device in which cells are grown on a polyaminourethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric. The PAU-coated PTFE fabric is used as a scaffold for human, porcine, and mouse hepatocytes. The researchers ran three separate sets of experiments, one for each type of liver cell.

The experiments that the researchers ran can fall into two sections: before transplantation and after transplantion into mice (transplantation occurred after 2 weeks). Several visual experiments were done to confirm the morphology and viability of cells that were grown on the PAU-coated PTFE fabric vs. cells grown on collagen IV. After 72 hours, cells on the fabric were uniformly distributed and showed spherical aggregates. Transmission electron microscopy revealed that these cells showed gap junctions and bile canaliculi. Mitochondrial viability, determined using an absorption assay, was better on the fabric than on collagen IV. The addition of a deleted-variant hepatocyte growth factor (dHGF) improved the viability of all three types of cells on both surfaces after 14 days.

Metabolic rate of ammonia and diazepam were also measured by introducing these into the cultures and measuring how much remained after 4 hours. Significantly higher rates of metabolism were measured in cells grown on fabric than cells on collagen. The addition of dHGF increased metabolic rates for all cells on both substrates. Albumin production was measured daily using ELISA. Cells on fabric maintained the level of albumin production, while cells on collagen showed no production on day 14. Addition of dHGF improved production for both sets.

The next step was to transplant the PAU-coated PTFE fabrics onto the surface of the spleens of mice suffering from ALF. Mice treated with various control transplants showed hypoglycemia, hyperammonemia, and encephalopathy, and died quickly of ALF. However, mice treated with the engineered hepatic tissue (EHT) device, which incorporated hepatocytes grown on PAU-coated PTFE fabric, showed improved survival rate. After 30 days, survival rate of mice with the EHT transplant was 60%, whereas survival rate for control mice was 0%. EHT also improved blood glucose and ammonia levels and reversed encephalopathy. The surviving mice were then sacrificed and histological examination of their livers showed normal structure, appropriate attachment, and strong expression of albumin.

The researchers have developed a device that can act as a functional artificial liver, at least in the short term. I think this article is interesting because of how thorough the research is. The researchers make several measurements, both pre- and post-transplantation, to fully determine the functionality of their device. It was also interesting because of the fact that the device showed such big improvements over growth on collagen substrates or over other control devices. Mainly, though, I chose it because liver tissue engineering interests me.

IBTWYPDCF (Itsy Bitsy Teeny Weeny Yellow Polka-Dot Collagen Fibers)

- To determine the efficacy of electropatterned collagen/PEO to promote cell attachment.
- To estimate the “minimal” pattern required for attachment.
- To demonstrate collagen I gene expression in 3T3 cells attached for 1 week.
- To determine viability of 3T3 cells on these surfaces after 2 weeks.

Team Blue Genes

In an 3D agarose culture designed to replace damaged or diseased cartilage, determine the effect of chondroitin sulfate and glucosamine on:
- Rex cell collagen II gene expression after 1 week of culture.
- amount of collagen II in the culture after two weeks.
- mechanical properties of the gel after two weeks.
- Rex cell viability after two weeks of culture.

Team Black Polo

Using smooth muscle cells in a 3D collagen culture:
- determine the mechanical properties of the gel after two weeks.
Using 3T3 fibroblasts in a 3D collagen culture:
- demonstrate collagen I gene expression after one week.
- determine for various collagen gel thicknesses the viability of cells after one week.

Team ‘Toga

In an 3D agarose culture designed to replace damaged or diseased skin, determine the effect of UV exposure on:
- 3T3 collagen I gene expression after 1 week of culture.
- amount of collagen I in the culture after two weeks.
- mechanical properties of the gel after two weeks.
- 3T3 viability after two weeks of culture.

Team T75

Determine the effect of “stiffness” (collagen gel concentration, possibly in an agarose mixture) on:
- HepG2 albumin gene expression after one week of culture.
- HepG2 albumin secretion rate after one week of culture.

Mango Tapioca

In a 3D agarose culture, determine the effect of ethanol on:

- HepG2 albumin gene expression after one week of culture.
- HepG2 albumin secretion rate after one week of culture.
- HepG2 viability after two weeks of culture.

The Boners

In a 3D agarose culture, determine the effect of vitamin C on:
- hFOB alkaline phosphatase gene expression after one week of culture.
- hFOB alkaline phosphatase activity after two week of culture.
- hFOB doubling time.

Team ACEE

For 3T3 fibroblasts in 2D culture:
- determine the effect of different collagen concentrations on collagen I gene expression after one week.
- determine the effect of different collagen concentrations on doubling time.
- determine the effect of different collagen concentrations on cell migration.

Epithelial contact guidance on well-defined micro- and nanostructured substrates

Ana I. Teixeira, George A. Abrams, Paul J. Bertics, Christopher J. Murphy and Paul F. Nealey

Journal of Cell Science 116, 1881-1892. 2003

http://jcs.biologists.org/cgi/reprint/116/10/1881

Teixeira et al. were interested in the effects of topographical nanoscale patterning of a substrate on the cell behavior of a epithelial cell culture. Previous studies had shown tendencies for cells to align along groove and ridge topographical features on the order of 1 micron in width as opposed to cell behavior on smooth surfaces which did not demonstrate alignment but rather remained round. This alignment of cells along topographical features is known as contact guidance. While previous studies had gone as small as 500 nm ridges and grooves, no one had yet demonstrated this phenomenon on substrates with 70 nm features, which is the biomimetic size for the felt-like surfaces of the corneal basement membrane. A better understanding of topographical signals to cell morphology would lead to clearer design requirements for cell cultures and tissue engineering.

This article is of particular interest to our team, the “Itsy Bitsy Tiny Weeny Yellow Polka Dot Collagen Fibers Group (IBTWYPDCFG)”, because it focuses on two key elements that we plan to further explore and develop in our project. First of all it focuses on micropatterning very thin topographical features and characterizes the effect on a specific cell functionality (alignment). Secondly it demonstrates successful cell adhesion to a Silicon Oxide substrate which we were concerned might be a problem. The difference between their micropatterning methods and ours is that they used a photoresist mask and Silicon etching procedure to achieve 70 nm groves with 600 nm depths consisting of pure silicon whereas we plan to use an electrospining technique of a polymer-collagen mixture to deposit 100 nm fibers onto a silicon wafer.

Other notable features in their experiment that we plan to deviate from include:

• They focused only on single cell attachment and alignment of corneal epithelial cells. We plan to culture many fibroblast cells in contact with each other and measure their proliferation and production of collagen as a function of the substrate pattern that they grow on.
• They measured their cell functionality based on cell alignment using both light microscopy as well as SEM and possessing the data in ImageJ. Our cell functionality measurements will be based more on techniques that we’ve practiced in lab that allow us to measure cell viability and the production of collagen by our seeded cells (Live/Dead assays, Western Blotting and RT-PCR).
• They were able to perform time lapse microscopy to measure the mobility of a seeded cell over a 12 hour period. We will not do that.

Features that worked for them that we plan to emulate if possible include:

• The use of serum in the culture medium enhanced the cell functionality (specifically alignment). It would be useful to compare the results of two substrates one with serum, one without.
• The processing and cleaning of the silicon wafer followed a standard protocol similar to that used in Bekeley’s EE143 microprocessing lab. In particular close attention was paid to the removal of native oxide using Phiranha solution and the sterilization of the wafer prior to being used for tissue culture.
• The production of focal adhesions by the cell was measured using a mouse anti human vinculin primary antibody followed by a donkey anti-mouse immunoglobulin secondary antibody.

In the end Teixeira et al. were able to determine that the most dominant feature for cell alignment in their substrate was the groove depth, specifically that the highest percentage of alignment occurred when the groove depth was 600 nm. In addition they were able to show that cells had much higher rates of elongation on all patterned substrates (specifically the 70 nm biomimetic sized grooves and ridges) when compared to the blank Silicon Oxide substrates. This experiment was pretty cool and is hopefully useful in helping us prepare for ours. Yeah Science!

Quick Layer-by-Layer Assembly of Aligned Multilayers of Vascular Smooth Muscle Cells in Deep Microchannels

JIE FENG, Ph.D., MARY B. CHAN-PARK, Ph.D., JINYE SHEN, B.E., and VINCENT CHAN, Ph.D.

http://www.liebertonline.com/doi/abs/10.1089/ten.2006.0223

Bypass grafting is a common procedure used in cases of arterial disease. However, the availability of suitable native arteries/veins is limited, particularly when the patient has undergone the procedure before. The use of synthetic prostheses has not been met with much success for small diameter grafts, and so it is desirable for tissue engineering to produce blood vessels. The major challenge in this case is to reproduce the heterogeneous but well-organized 3D microstructure in human tissue. Native blood vessels usually consist of many layers of vascular smooth muscle cells (SMCs) that are aligned circumferentially. Reproducing this in vivo is a major challenge of tissue engineering.

Physical restrictions on SMCs during their culture has been shown to achieve aligned cellular microstructure. Thus, Feng et al. attempted to use microchanneled scaffolds, essentially wells with thin wall widths in contrast to a fairly wide and deep well on which to culture SMCs on. First, it was shown that monolayers of SMCs grew well in the microchannels and aligned uniformly when the layer was confluent. This required manipulating the channel widths to one where the cells grew well in. After this, a layer-by-layer (LBL) process was attempted. In the past, SMCs has been shown to grow when encapsulated in a collagen type I hydrogel, but these SMCs had a much lower proliferation and alpha-actin expression. Thus, the idea here was to use the hydrogel in thin layers to separate confluent layers of SMCs. When the first layer of SMCs were confluent, a layer of hydrogel was applied, upon which more SMCs were seeded. In this paper, four layers were attempted, which survived even after six days. In an extension to this, the hydrogel step was skipped, and five layers of SMCs survived for also the required six days.

Different initial cell seeding densities of SMCs for each layer was also explored. When the cell seeding density reached 200000 cells/cm², the time until the layer reached confluence was about half a day. This allows a thick layer of SMCs to be produced quickly, and the implication for clinical applications is apparent. This paper showed that a simple technique, such as the LBL, may go a long way toward creating blood vessel grafts. The authors showed that cells between the channels were connected by some cells that would grow over the walls, ensuring that cell-cell interaction was still present. The authors also proposed further work on creating the scaffold with different degradable materials. When the perfect material is achieved, hopefully timed to the growth of the SMCs, a good multilayer of SMCs will be easily producible, all perfectly aligned. This paper was also very interesting, in that many techniques used were similar to those done in the 115 labs. Simple live/dead assays with Trypan Blue was used, as well as staining of F-actin and alpha-actin to determine alignment of the expressed proteins.

Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization
John J. Stankus, Lorenzo Soletti, Kazuro Fujimoto, Yi Hong, David A. Vorp, William R. Wagner

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4N3GFKS-5&_user=4420&_coverDate=02%2F20%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=e39b34356796ae903bcac1108e628096

I know there have been a couple articles done on electrospun scaffolds, but I found an interesting paper on overcoming cellular seeding problems using an electrospray method. This paper uses electrospun biodegradable poly(ester urethane) urea (PEUU) to mimic the mechanical and architectural environment of native ECV found in blood vessels. The study uses microintegration (or electrohydrodynamic atomization), where cells are electrosprayed while the tubular conduits are being electrospun simultaneously around the cells. This insures that cells are numerous and of equal density throughout the whole construct. Compared to methods that require cell seeding and migration, this method offers full control of cell density. Tubular conduits and vascular SMCs were used (more specific to blood vessel TE). The construct was then cultured and the cellular viability, morphology, count etc. along with mechanical properties were measured.

The paper goes on to describe the synthesis of PEUU as it is a novel polymer that was developed by this lab for the purposes of the electrohydrodynamic atomization. Both the PEUU and SCMs are charged and sprayed through separate nozzles that sit perpendicular to each other to form 8cm conduits which were then cultured in serum for a full day before being removed from the apparatus and sectioned for further culturing (3 days) on TCPS plates or in spinner flasks.

SCM growth and viability was characterized using a MTT mitochondrial activity assay. Both the TCPS plates and spinner flasks showed SMC growth, with spinner flasks showing higher proliferation. The samples were then stained with H&E and analyzed using our friend ImageJ. After four days, cells had normal morphology and began to show signs of cellular spreading.

After 4 days, the biomechanical properties were also characterized. These included: static/dynamic compliances, burst pressure, stiffness index, and suture retention. They were able to model these properties, and these equations can be found in the paper if you’re really interested. In short, the constructs were able to take and average of 1750 mm Hg of pressure (compare to normal blood pressure in hundreds of mm Hg). Stress-stretch responses were all strong, and suture retention forces also seemed to increase with days in culture.

This paper was able to develop a process that has potential for future tissue engineered blood vessel production. Highly cellular tubular conduits were produced using a new microintegration technique that showed high cellularity and strong mechanical properties. SMCs were able to microintegrate into the conduits and proliferate very well. The construct itself was mechanically sound, was suturable, and retained their lumens after compression. The paper also found that constructs cultured on spinner flasks were more promising for TE of blood vessels, but that a non-thrombogenic luminal surface was needed first (for example: luminal seeding).

Scaled-Up Production of Mammalian Neural Precursor Cell Aggregates in Computer-Controlled Suspension Bioreactors
by Jane A. Gilbertson, Arindom Sen, Leo A. Behie, Michael S. Kallos

http://www3.interscience.wiley.com/cgi-bin/abstract/112446824/ABSTRACT?CRETRY=1&SRETRY=0

As a significant percentage of the population continues to grow older, the need for neurodegenerative therapies will likewise continue to grow. A very promising avenue of research is the use of neural precursor cells, or central nervous system stem cells, to repair and ideally cure central nervous system diseases such as Parkinson's disease. However, before cell-based therapies can be developed, there needs to be an substantial source of neural precursor cells available. So far, protocols to culture neural precursor cells exist only for small-scale bioreactors. In anticipation of the demand for neural precursor cells in the future, this group has decided to explore how to modify these existing protocols to scale-up production from a small-scale bioreactor to a large-scale computer-controlled bioreactor.

In scaling up the bioreactor vessel, adjustments must be made for changes in environment. Oxygen level is a primary concern for cell growth. Shear stress must also be maintained to prevent the cell aggregates from being damaged or from growing too large. Through equations and calculations from other studies, they concluded that these changes can be accounted for by adjusting the bioreactor's impeller to an appropriate rpm.

However, it was discovered that the fluid behavior of the system is altered by adding measuring probes into the system. These probes, otherwise unavailable for small-scale bioreactors, provide control over the bioreactor's pH, oxygen levels, and other environmental parameters. But with these probes, the hydrodynamic environment of the system was found to be drastically altered. Therefore, they adjusted the rpm of the impeller to create a similar hydrodynamic environment found in small-scale bioreactors, while at the same time maintain sufficient oxygen and shear stress levels.

Through the passages in the bioreactors that were performed, the group concluded that by adjusting the protocol to account for oxygen and shear stress level, as well as the hydrodynamic environment of the bioreactor vessels, adequate cell viability and cell density can be achieved in large-scale bioreactors. They however note that the cells require some time to adapt to the new environment. With this study, sufficient quantities of neural precursor cells can be generated to further fuel further studies to apply these cells for clinical uses.

Overexpression of Telomerase Confers Growth Advantage,
Stress Resistance, and Enhanced Differentiation of ESCs
Toward the Hematopoietic Lineage
L. Armstrong, G. Saretzki, H. Peters, I. Wappler, J. Evans, N. Hole, T. von Zglinicki, M. Lako
Stem Cells 2005; 23: 516—529

Article:

http://stemcells.alphamedpress.org/cgi/reprint/23/4/516.pdf

Pubmed Link
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?holding=npg&cmd=Retrieve&db=PubMed&list_uids=15790773&dopt=Abstract

Telomeres are repeating DNA segments found at the ends of chromosomes. Most somatic cells in humans have very little telomerase activity and thus, their telomeres shorten with each replication. When the telomeres shorten, the cells begin to age and die. On the other hand, immortalized cells have noticeable amounts of telomerase activity and stable telomere lengths throughout each propagation. For example, embryonic stem cells (ESC) are pluripotent and can reproduce indefinitely in culture. Human ESC initially expresses a high level of telomerase activity due to the expression of the TERT gene (a reverse transcriptase responsible for adding on segments to the telomeres); but when ESC differentiate, TERT and telomerase activity decrease and telomeres shorten in the differentiated cells. Thus, enhancing or lengthening telomeres may lead to a revolutionary breakthrough in cell renewal technology.

In this study, the scientists over-expressed the TERT gene in mouse ESC and compared their performance and survival to a wild-type ESC (a control group). This is because in mice, ESCs also require telomerase RNA and its reverse transcriptase (TERT) to function. Also, mouse ESC decreases telomerase and TERT activity upon differentiation. However, scientists do not know if this down-regulation of telomerase is necessary for normal cell growth. Thus, the scientists wanted to look into the effects of continuous telomerase activity on cell proliferation, differentiation, death, and resistance to stresses for mouse ESC. They accomplished this through a various number of assays such as Flow Cytometry Analysis, Microarray Analysis, RT-PCR, Apoptosis Assay, and many others.

The results of the various assays showed that over-expressing TERT in mouse ESC increased the cell’s proliferation, resistance to apoptosis, enhanced the efficiency of differentiation (due to higher proliferation and better survival of progenitor cells in culture), and improved the cell’s resistance to oxidative stress. The paper also notes that results from other studies showed that telomerase had beneficial effects other than just extending telomere length—for example, it helped increase wound healing in mice. Overall, it was determined that telomerase is a “survival enzyme” for ESC and the cells it differentiates into. However, an important note the authors point out is that over-expression of TERT in differentiated cells have led to increased tumor growth—thus more work needs to be done to control TERT over-expression in differentiated cells.

I chose this paper because I was always interested in the fables of the fountain of youth. It seems that lengthening telomeres is the closest scientists have come to drinking from that fountain. This study is very important in that the results of increased ESC proliferation and enhancement of ESC differentiation due to TERT over-expression can have a great impact on tissue engineering treatments or cell-replacement therapies. For example, ESCs from bone, liver, or skin cells can be cultured to over-express TERT so that they can grow a workable number of cells for use in tissue engineered scaffolds. Or, one day, scientists may even be able to just grow the cells needed to directly replace the wounded or damaged organ. Much more work will need to be done, but this study was a promising step forward.

Human hepatoblast phenotype maintained by hyaluronan hydrogels

William S. Turner , Eva Schmelzer, Randall McClelland, Eliane Wauthier, Weiliam Chen, Lola M. Reid

J Biomed Mater Res B Appl Biomater. 2006 Dec 20

The matrix of the liver in fetal and embryonic tissues contain significant amounts of hyaluronan (HA), a glycosaminoglycan that aids in matrix stabilization, facilitation of cell migration, transport regulation, acting as a hormonal reservoir, and water/protein homeostasis. In adult liver tissue it is found in the presumed stem cell compartment called the Canals of Hering, often present when adult tissues are undergoing cellular expansion, wound repair, and regeneration. Therefore, because they are in association with hepatic stem cells and their immediate descendants heptoblasts, HAs are hypothesized to be candidates for matrix components in 3-D scaffolds for creating ex-vivo cultures of hepatic progenitors.

In this study, parenchymal cells (hepatoblasts) were isolated from liver tissue from human fetuses. HA matrices were created by crosslinking a pre-made HA solution, which became highly porous HA spongy hydrogels that absorbed water readily. The cultured progenitor cells, rich in hepatoblasts, were then seeded into the HA hydrogels and also in collagen gels and on culture plastic, which were both used as controls. The cultures were then fixed with paraformaldehyde and micro-sectioned for observation; analyzed for albumin and urea production; RNA, DNA, and protein were isolated and quantified; and gene expression was analyzed via quantitative real time RT-PCR. They were also stained with immunofluorescence outside HA hydrogels (using primary and secondary antibody coupling for HA receptors).

Results showed that HA hydrogels were able to maintain early staged hepatic progenitors in viable, proliferative, and phenotypically stable state over long culture periods. Immunostaining showed that human hepatic stem cells and hepatoblasts were positive for the specific HA receptor, CD44, and uptook the conjugates at higher rates than the other cells in the culture, such as stroma and endothelial cells. In the HA hydrogels, cells showed considerable aggregation and expansion and an increase in DNA production over time. Furthermore, hepatoblasts in HA hydrogels also produced less albumin and survived in culture longer than hepatoblasts in plastic culture. Urea production decayed slower in HA hydrogels than in the other two culture conditions. Both results showed that ability of HA hydrogels to maintain hepatoblast phenotype in culture as high levels of albumin and high decay rates of urea are characteristics of mature cells, while the opposite holds true for progenitor cells.

This research was significant as it explored and affirmed the possibility of creating ex-vivo constructs of liver tissue. So far, HAs have been the only culture condition discovered to allow the viability, proliferability, and maintenance of hepatoblasts. Because they will allow for the maintenance of a larger variety of specific cell lineage states (since hepatoblasts are pluripotent), HA hydrogels will be a prime reservoir for replenishing liver tissue/cells in medical research and cell therapy. Furthermore, this article was also significant as it contained many of the assays and imaging techniques that were discussed in lecture and performed in lab.

Human hepatoblast phenotype maintained by hyaluronan hydrogels

William S. Turner 1 *, Eva Schmelzer 2, Randall McClelland 2, Eliane Wauthier 2, Weiliam Chen 3, Lola M. Reid 1 2 4 *

1Department of Biomedical Engineering, UNC School of Medicine, Chapel Hill, North Carolina 27599 2Department of Cell and Molecular Physiology, UNC School of Medicine, Chapel Hill, North Carolina 27599 3Department of Biomedical Engineering, State University of New York, Stony Brook, New York 11794-2580 4Program in Molecular Biology and Biotechnology, Lineberger Cancer Center, UNC School of Medicine, Chapel Hill, North Carolina 27599

J Biomed Mater Res B Appl Biomater. 2006 Dec 20

The matrix of the liver in fetal and embryonic tissues contain significant amounts of hyaluronan (HA), a glycosaminoglycan that aids in matrix stabilization, facilitation of cell migration, transport regulation, acting as a hormonal reservoir, and water/protein homeostasis. In adult liver tissue it is found in the presumed stem cell compartment called the Canals of Hering, often present when adult tissues are undergoing cellular expansion, wound repair, and regeneration. Therefore, because they are in association with hepatic stem cells and their immediate descendants heptoblasts, HAs are hypothesized to be candidates for matrix components in 3-D scaffolds for creating ex-vivo cultures of hepatic progenitors.

In this study, parenchymal cells (hepatoblasts) were isolated from liver tissue from human fetuses. HA matrices were created by crosslinking a pre-made HA solution, which became highly porous HA spongy hydrogels that absorbed water readily. The cultured progenitor cells, rich in hepatoblasts, were then seeded into the HA hydrogels and also in collagen gels and on culture plastic, which were both used as controls. The cultures were then fixed with paraformaldehyde and micro-sectioned for observation; analyzed for albumin and urea production; RNA, DNA, and protein were isolated and quantified; and gene expression was analyzed via quantitative real time RT-PCR. They were also stained with immunofluorescence outside HA hydrogels (using primary and secondary antibody coupling for HA receptors).

Results showed that HA hydrogels were able to maintain early staged hepatic progenitors in viable, proliferative, and phenotypically stable state over long culture periods. Immunostaining showed that human hepatic stem cells and hepatoblasts were positive for the specific HA receptor, CD44, and uptook the conjugates at higher rates than the other cells in the culture, such as stroma and endothelial cells. In the HA hydrogels, cells showed considerable aggregation and expansion and an increase in DNA production over time. Furthermore, hepatoblasts in HA hydrogels also produced less albumin and survived in culture longer than hepatoblasts in plastic culture. Urea production decayed slower in HA hydrogels than in the other two culture conditions. Both results showed that ability of HA hydrogels to maintain hepatoblast phenotype in culture as high levels of albumin and high decay rates of urea are characteristics of mature cells, while the opposite holds true for progenitor cells.

This research was significant as it explored and affirmed the possibility of creating ex-vivo constructs of liver tissue. So far, HAs have been the only culture condition discovered to allow the viability, proliferability, and maintenance of hepatoblasts. Because they will allow for the maintenance of a larger variety of specific cell lineage states (since hepatoblasts are pluripotent), HA hydrogels will be a prime reservoir for replenishing liver tissue/cells in medical research and cell therapy. Furthermore, this article was also significant as it contained many of the assays and imaging techniques that were discussed in lecture and performed in lab.

In vivo engineering of organs: The bone bioreactor

Molly M. Stevens, Robert P. Marini, Dirk Schaefer, Joshua Aronson, Robert Langer, and V. Prasad Shastril
PNAS (2005) 102: 11450-114555.

Currently, the repair of major bone defects, such as those associated with tumor resections and spinal fusions, requires bone grafts. Typically, autologous bone grafts are harvested from the iliac crest. However, the harvesting procedure is frequently accompanied by pain and morbidity. To address this issue, the authors tested an in vivo bioreactor designed to produce autologous bone without requiring the administration of growth factors or cell transplantation.

The bone bioreactor “space” was created in the tibia of rabbits by injecting a biocompatible gel composed of calcium and alginate between the periosteum and long bone. The maturation of bone within the bioreactor was observed over 12 weeks. The engineered bone was harvested from the reactor and then transplanted into tibial defects to determine how well it would integrate with existing bone.

The authors observed that after 6 weeks, the engineered bone demonstrated all the histological and structural characteristics of native lamellar bone. Also, they determined that both the compressive strength and ultimate strength of the engineered bone was within the range for compact bone. Moreover, histological and radiographical tests revealed that after 6 weeks, transplanted neo-bone integrated with native bone within a tibial defect.

The authors also demonstrated that by adding the angiogenic factor, Suramin, to the gel matrix, cartilage formation instead of bone formation occurred within the bioreactor. However, no quantitative analysis on the cartilage was performed.

The authors have established that in vivo engineering of autologous bone without the need for growth factors or transplanted cells is possible. Their approach may potentially lead to the generation of large quantities of autologous bone at an easily accessible surgical site. The study may have important implications for bone banking and bone transplantations.

http://www.liebertonline.com/doi/pdfplus/10.1089/ten.2006.12.3395


Quantitative Analysis of Radiation-Induced DNA Break Repair
in a Cultured Oral Mucosal Model
HINNE A. RAKHORST, M.D.,1 WENDY M.W. TRA, B.Sc.,1 SANDRA T.
POSTHUMUS-VAN SLUIJS, B.Sc.,1 STEVEN E.R. HOVIUS, M.D., Ph.D.,1 PETER C.
LEVENDAG, M.D., Ph.D.,2 ROLAND KANAAR, Ph.D.,2,3 and STEFAN O.P. HOFER, M.D., Ph.D.1

This study uses tissue engineering to study the effects of radiation on skin tissue from the mucosal membrane. This work is extremely relevant to the condition of oral mucositis, a side effect experienced by many patients receiving chemotherapy. This condition can consist of ulcers in the mouth and even malnutrition. Usually this condition is treated by stopping the chemotherapy until the skin heals, which unfortunately allows for the spread of cancer. Using oral keratinocytes and fibroblasts and, experiments were conducted in vitro to assess the effects of radiation on the tissue, especially its effect on proteins involved with the DNA double-strand break repair mechanism. I feel that this paper is both interesting and important because it showcases how tissue engineering can be used to create a very important in vitro model to test conditions that can be dangerous or unfeasible in vivo.

The first step to the experiment involved constructing a mucosal substitute. This was done by obtaining fibroblasts from human buccal mucosa. Fibroblasts and keratinocytes were seeded into an acellular dermal carrier and were then gamma-irradiated. Next, the tissue cultures were examined to quantify P53 binding protein 1, MRE11 and RAD51—all proteins involved in double strand break (DSB) repair. These proteins were quantified using antibodies; furthermore, live-dead assays were performed to measure cell proliferation and apoptosis due to radiation.

The results indicated clear changes in the cell and tissue morphology as a result of the radiation. First the nuclei became much smaller and showed chromatin condensation (pyknosis). Further staining showed increased apoptosis most likely due to extreme damage to the DNA. The amount of the DSB-repair proteins was quantified through immunofluorescence visualization. These DSB-repair proteins would conglomerate around the site of the DSB forming ionizing radiation-induced foci (IRIF) in the nucleus. These IRIFs could be visualized, and thus the number of IRIFs per nucleus could be calculated and measured as an indication of the amount of double strand breaks that were occurring due to radiation. Using the protein P53, the expected trend of increased IRIFs with increased radiation occurred. However, in the case of other proteins such as MRE11, the pattern was less clear; thus the MRE11 protein is not as good of an indicator of double strand break levels. Cell proliferation staining also did show some peaks despite radiation at the low 2 Gy level indicating the potential for the cells to recuperate and grow. Overall, this study was a good model to display the effect of radiation on DNA double strand breakage; further study of the radiation effects with full understanding of cell cycle and immunological responses (i.e. cytokines), could lead to an even better understanding of radiation on skin tissue and how this may affect oral mucositis.

A Novel Culture System Shows that Stem Cells Can be Grown in 3D and Under Physiologic Pulsatile Conditions for Tissue Engineering of Vascular Grafts
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WM6-4JHMSYW-4-M&_cdi=6926&_user=4420&_orig=search&_coverDate=05%2F15%2F2006&_sk=998679997&view=c&wchp=dGLbVzW-zSkWA&md5=23cf603dfae4de45db6ff27649bdc890&ie=/sdarticle.pdf

Oscar Abilez, M.D.,*,†,1 Peyman Benharash, M.D.,*,† Mahncy Mehrotra,*,† Emiko Miyamoto, B.S.,*,‡ Adrian Gale, B.S.,*,§ Jean Picquet, M.D.,*,† Chengpei Xu, M.D., Ph.D.,*,† and Christopher Zarins, M.D.,*,†

As cardiovascular disease has become more prominent, various methods to develop better vascular grafts came into light in tissue engineering. This study specifically tests for a culture system that would successfully grow and differentiate embryonic stem cells into endothelial, smooth muscle, and fibroblast cells that can be assembled into vascular grafts. Because the nature of vascular system requires pulsatile condition due to the pumping of heart and blood pressure, the authors have designed a new bioreactor system that incorporates flow with regular pulses above a 3D culture of stem cells.

The particular experiment consisted of PBS solution flowing on top of a culture system at a pre-assigned rate and pressure controlled through a computer. A CCD camera gathers pictures and video files that observe the displacement of cells within the 3D scaffold. The displacement and the unison in movement signify the ability of mESC (mouse embryonic stem cell) to successfully differentiate into vascular cells. Two controls were tested along with stem cells in 3D Matrigel matrix: a positive control with beads in 3D Matrigel and a negative control with stem cells in 2D culture.

The results somewhat showed similarity with a regular fluid flow mechanism. Due to the shear stress on only the top of the culture, a gradient of maximum displacement was observed; the cells / beads that were closest (the top-most layer of culture) to the PBS flow moved the furthest along with the flow, while those on the bottom barely changed its position. Cells in 2D system were completely washed away, assuring that in pulsatile condition, only 3D scaffold enables stem cells to maintain its unison and hence function to differentiate.

The drawback of this experiment was in the approximation of the applied shear stress. The fluid used for the flow and the geometrical dimension of the culture system in the experiment did not accurately represent the actual condition in vascular system of a mouse, in terms of shear stress calculation. However, this problem can be easily fixed in later experiments by substituting a solution that better describes the system and changing the dimension of 3D scaffold when designing one.

The significance of this study is that it is one of the first and successful researches to attempt tissue engineering using stem cells, and not already differentiated cells. The autologous stem cells would significantly reduce the problems faced with the ongoing vascular grafts (i.e. material availability and immunological rejection). The combination of stem cell research with tissue engineering, such as this one, seems to be promising.

Mechanoregulation of gene expression in fibroblasts

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T39-4MY6G9W-1-7&_cdi=4941&_user=4420&_orig=search&_coverDate=04%2F15%2F2007&_sk=996089998&view=c&wchp=dGLbVtb-zSkzk&md5=62474072cb124b85f7f4b650feeedb2d&ie=/sdarticle.pdf

James H.-C. Wanga, , , Bhavani P. Thampattya, Jeen-Shang Linb and Hee-Jeong Imc aMechanoBiology Laboratory, Departments of Orthopaedic Surgery, Bioengineering, Mechanical Engineering, and Physical Medicine and Rehabilitation, University of Pittsburgh, 210 Lothrop St., BST, E1640, Pittsburgh, PA 15213, United StatesbDepartment of Civil and Environmental Engineering, School of Engineering, University of Pittsburgh, United StatescDepartments of Biochemistry and Internal Medicine, Rush University Medical Center, Cohn Research BD 558, 1735 W. Harrison, Chicago, IL 60612, United States

Intro: Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This paper reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested.

used: Among many mechanoresponsive cells in our body, especially fibroblasts (collagen producing ECM cells) are studies for mechanotransduction resulting in gene regulation in this paper. Fibroblasts are also important for maintenance of most of the ECM components such as proteoglycans, growth factors, and cytokines.

experimental method: To mimic in vivo environment, tensile mechanical loading on fibroblasts in vitro is introduced using a substrate stretching method. This method is versatile; the loading parameters, including magnitude, frequency, and duration, are easy to control; and the mechanical properties of substrates as well as surface chemistry for cell attachment can be readily altered. The substrate deformation induced by substrate stretching is characterized by substrate surface strains.
To determine the effects of mechanical loading on fibroblasts, static or cyclic stretching is applied to a group of cells cultured on smooth, flexible elastic membranes (e.g.,silicone elastomers). And the mechanical force can be calculated by observating fluorescent beads attached on the sillicone substrate (for those in 116, we learned about this!:) ). In fact, 3D simulation of ECM by using collagen gels is more effective and accurate in terms of mimicking what's happening in vivo. Gene expression can be observed using PCR or RT-PCR.

Results: Induced collagen production, change in ECM components, growth factor, MMP secretion, TIMP (MMP inhibitor) expression, focal adhesion change, etc. (refer to Table 1.).

Discussion:
Gene regulation in fibroblasts depends on mechanical loading conditions: type (e.g., tension vs.compression), magnitude, frequency, and duration. The mechanoregulation of gene expression in fibroblasts also depends on the tissue location from which fibroblasts are derived, as well as ECM protein with which cells interact. To better understand how tissue homeostasis is maintained and how pathological conditions initiate and develop, it is necessary to study the effects of various mechanical loading conditions on fibroblasts. One challenging task in future research is to understand how a cell “decides” its response from “crosstalks” of many mechanotransduction signals, since mechanotransduction mechanisms do not function in isolation, but rather by an integrated network of various signaling pathways. Another particularly challenging task is to identify specific “force receptors,” for which specific proteins at the membrane–cytoskeletal interface(e.g., integrins and G proteins) are good candidates. Ultimately, additional research in mechanoregulation of gene expression in fibroblasts will aid in developing new therapeutic strategies and new approaches to engineering tissue constructs for improved repair and regeneration of connective tissues.

Proliferation and differentiation of transplantable rabbit epithelial sheets engineered with or without an amniotic membrane carrier

http://www.iovs.org/cgi/reprint/48/2/597

It reports a novel method of engineering transplantable, carrier-free corneal epithelial sheets by using a biodegradable fibrin sealant and compares its characteristics with epithelial sheets cultivated on denuded amniotic membrane carriers.

Stratified corneal epithelial sheets were prepared in culture dishes coated with biodegradable fibrin glue. Amniotic membrane (AM) carriers served as the control. The quality of cultivated sheets was compared by immunohistochemistry for cytokeratin (K)3, K12, K14, p63, occludin, and integrin beta1; electron microscopy; and colony-forming assays. K3 protein expression was compared by Western blot analysis. In a limbal-deficient rabbit transplantation model, postoperative adaptation and proliferation of BrdU-labeled cell sheets were examined by histology and anti-Ki67 staining.

Epithelial sheets were successfully engineered by using a biodegradable fibrin sealant. Cell sheets in both groups were multilayered, expressed K3, K12, and K14, and had functioning occludin(+) apical tight junctions as well as p63 and integrin beta1 staining in basal cells. The carrier-free sheets appeared to be more differentiated than the AM sheets, which was also demonstrated by the higher levels of K3 in the Western blots. The colony-forming efficiency of dissociated cells was similar in both groups, although larger colonies were observed on the AM sheets. AM sheets retained higher levels of BrdU-labeled cells and fewer Ki67(+) cells compared with carrier-free sheets after transplantation.

Tissue engineering with a commercially available fibrin sealant was an effective means of creating a carrier-free, transplantable corneal epithelial sheet. Carrier-free sheets were more differentiated compared with AM sheets, while retaining similar levels of colony-forming progenitor cells.

Cell Movement Is Guided by the Rigidity of the Substrate
Chun-Min Lo, Hong-Bei Wang, Micah Dembo, and Yu-li Wang
Biophys J. 2000 Jul;79(1):144-52.

In creating any tissue construct, it is important to recognize that any artificially engineered tissue or synthetic biomaterial must not only provide function and immunogenic compatibility, but must often encourage proliferation and cell migration from outside of the construct as well. To that end, it is critical for tissue engineers to understand the basic mechanisms which underlie cellular migration. In this paper, a group from the University of Massachusetts Medical School looked at how changing the rigidity of the substrate might affect the movement of the cells cultured on top of it. NIH 3T3 fibroblasts were cultured on sheets of flexible polyacrylamide that were coated with collagen 1. By varying the concentration of a bis-acrylamide crosslinker in the collagen, the researchers were able to create a substrate with two varying zones of stiffness (a softer side and a stiffer side), and examine how cells behaved as they moved across the boundary.

By placing the cell sheets under a Zeiss IM-35 microscope equipped with a 40x lens, the researchers were able to record both phase-contrast and fluorescence images at five minute increments. Traction forces were inferred by watching the movement of fluorescent beads which were embedded near the substrate surface. An image processing algorithm was used to record the movements of individual beads and convert those into a map of displacement vectors. By knowing the position of the cell boundary and the Young’s modulus, as well as the Poisson ratio of the gel, they were able to calculate the traction stresses generated by the cell. Similarly, migration speeds were calculated by looking at time-lapse phase images recorded over a period of one hour, and noting the change of the position of the center of the nucleus at 15 minute intervals.

They found that cells migrated preferentially from the softer side of the collagen gel towards the stiffer side, a tendency that they termed “durotaxis”. That is to say, cells that approached the boundary from the softer side had no problems migrating across it. When the cells moved across this boundary, they also showed an increase in the spreading area of the cell as well as the traction forces that they were able to generate. On the other hand, the cells that were moving in the opposite direction (from the stiff side to the softer one) did not want to cross the boundary. Those cells displayed a smaller spreading area or changed directions entirely. They suggest that by changing the strains that are experienced in varying regions of the substrate (i.e. in front of or behind a polarized cell), one may be able to redirect the movement of the cell. They also found that the 3T3 cells that were placed on the stiffer substrate were able to generate stronger traction forces than those placed on a softer one.

This information is useful because it shows that cell movement is directed not only by gradients of chemical signals, but by purely physical interactions at the cell-substrate interface as well. The authors propose that this may work through a sensory feedback mechanism involving the lamellipodia, in which expansion and retraction may affect some ion channel regulation or receptor-ligand interactions that lead to some downstream biochemical changes at the cellular level. This was one of the most awesome papers ever, and a fun thing to read if you have some free time on Friday night.

Angiogenic and inflammatory response to biodegradable scaffolds in
dorsal skinfold chambers of mice

Martin Ru¨ ckera,
, Matthias W. Laschkeb, Dominik Junkerb, Carlos Carvalhoc,
Alexander Schramma, Rolf Mu¨ lhauptc, Nils-Claudius Gellricha, Michael D. Mengerb
Biomaterials 27 (2006) 5027–5038

One of the most prominent issues yet to be resolved in tissue engineering is the move from 2D to 3D tissues. The major limiting factor, of course, is the limits of diffusion and the necessity of a vascular network to bring nutrients and remove wastes. When an engineering device is implanted, often a scaffold is involved. The scaffold needs certain mechanical properties to support the device, but it is also important that the scaffold encourages, or at the very least does not hinder, necessary biological processes such as angiogenesis. I chose this paper because of its rather direct approach to investigating this problem.

This study attacks this issue by testing some common tissue engineering scaffold materials and their effects on neovascularization/angiogenesis in vivo. They compared three types of implants --poly(L-lactide-co-glycolide) (PLGA), collagen–chitosan–hydroxyapatite hydrogel, and isogeneic calvarial bone blocks—as well as a sham control. The scaffolds were examined in vivo using a dorsal skin chamber in balb/c mice and intravital fluorescence microscopy that stained blood plasma and white blood cells. Computer technology assisted the researchers in making volumetric blood flow and cell counting analysis. At the end of the experiment, histology and immunohistochemistry experiments were used to study the scaffolds ex vivo.

The quick and dirty results showed that the PLGA scaffold was similar to the isogeneic bone implant immunological response and neovascularization. Although, the isogeneic implant has a larger and more complex vascular networks which may be due inherent growth factors in bone matrix. The hydragel, meanwhile, showed little vascularization and high levels of apoptotic cells near the border of the implant. The hydragel also incited a high inflammatory response. End of story—the researchers believe that the hydragel may be toxic.

One quality issue stressed by these researchers was the effect of pore size of the scaffold. Different pore sizes can effect angiogenesis (see paper for references) and the researchers were concerned that normal scaffold preparation techniques resulted in a large distribution of pore sizes. In this study the researchers used a special technique (rapid prototyping/3D printing) to make sure the scaffolds had a homogenous pore size.

Controlled Microchannelling in Dense Collagen Scaffolds by Soluble Phosphate Glass Fibers

Showan N. Nazhat, Ensanya A. Abou Neel, Asmeret Kidane, Ifty Ahmed, Chris Hope, Matt Kershaw, Peter D. Lee, Eleanor Stride, Nader Saffari, Jonathan C. Knowles, and Robert A. Brown

Biomacromolecules 2007, 8, 543-551

This technical paper discusses three issues regarding scaffolds: strength, cell viability and formation of microchannels. One of the main issues in tissue engineering design is diffusion of nutrients to cells in dense 3D scaffolds. Utilizing fiber formation technology, this group unidirectionally aligned phosphate glass fibers (PGF) within a 30-40 um diameter in a collagen I matrix, compressed the gel into a sheet, and then rolled the sheet into a spirally assembled scaffold. This technique was performed with or without human oral fibroblasts to determine cell viability under compression.

The phosphate glass fiber scaffold (without cells) underwent mechanical testing. Using a densely packed collagen spiral scaffold as a control, it was shown that a scaffold containing 30% PGF increased Young's Modulus 100x and deformed little prior to failure

In addition, cell viability was determined to be approximately 80% in 30% PGF compressed sheets. Live cells were visualized using calcein AM in the green channel while propidium iodide showed dead cells in the red channel. Confocal laser microscopy captured the images. More death was seen in the spiral scaffold as compared to the sheet. This may be due to improper nutrient diffusion due to the close packing of collagen and cells. No PGF had degraded to form microchannels at that point. In addition, the cells had no preference for PGF or collagen.

To measure degradation of the channels, ion chromatography was utilized. Note, in the figures, that there is mainly a linear trend in the first 6 hours for PGF degradation, and the scaffolds containing more PGFs had a steeper slope. Ultrasound imaging was then used to confirm channel formation. Both microbubbles and dH2O were forced through the scaffold. Ultrasound imaging confirmed microchannel formation on several planes for the PGF degraded scaffolds. However, for the spiral scaffold with only collagen, no microchannels were formed.

Thus, this group demonstrated the potential for possible vascularization of a densely packed scaffold and the use of PGFs as a way to increase mechanical strength. However, to ensure QA, the group needs to refine the technique so that all the fibers remain aligned as this scaffold will probably be utilized "off the shelf". In addition, a worthy future experiment would be to monitor degradation of PGFs within a cell-containing scaffold. It needs to be confirmed that cells remain viable while microchannels are formed. In addition, they could run this experiment for time points longer than 24 hours.

A micro-spherical heart pump powered by cultured cardiomyocytes

Yo Tanaka,ab Kae Sato,c Tatsuya Shimizu,bd Masayuki Yamato,bd Teruo Okanobd and

Takehiko Kitamori*abce

Received 22nd August 2006, Accepted 30th October 2006

First published as an Advance Article on the web 13th November 2006

http://www.rsc.org/ej/LC/2007/b612082b.pdf

There are many effective implanted device. However, majority of them require power source that is either provided through the wire to the device, or has a batter that needed to periodically replace. Therefore, A microfluidic device powered without the need for external energy sources or stimuli is needed. This paper discuss the creation of a micro-spherical heart-like pump powered by spontaneously contracting cardiomyocyte sheets driven without a need for external energy sources or coupled stimuli. This device was fabricated by wrapping a beating cardiomyocyte sheet exhibiting large contractile forces around a fabricated hollow elastomeric sphere (5 mm diameter, 250 mm polymer thickness) fixed with inlet and outlet ports. Fluid

oscillations in a capillary connected to the hollow sphere induced by the synchronously pulsating cardiomyocyte sheet were confirmed, and the device continually worked for at least 5 days in this system.

Cell sheets are obtained routinely utilizing a commercial cell culture system based on the temperatureresponsive polymer, poly(N-isopropylacrylamide) (PIPAAm). Importantly, these living cell sheets are harvested intact, handled, manipulated and transferred to various devices while maintaining their regular and robust pulsating phenotype.

Pumping action is driven with only chemical energy input from culture milieu without any requirement for coupled external energy sources, unlike conventional actuators. However, to mimic the heart of most animals, check valves and multiple chambers are required.

A micro-spherical heart has at least two directions for possible applications: (1) as an electric powerless bio-actuator to drive fluids in implanted micro-chemical or biochemical medical implant devices without using external power sources, and (2) as a component of a cardiovascular circulatory system micro-model to study mechanisms of circulatory physiology, pathology, and developmental biology, or for medical diagnoses of patients using cultured autologous cells.

I choose this article because powering for the any sort of device has always been a great problem. For example the pace maker in heart the battery has life limit, and every time the replace of battery creates a tremendous harm to patience. The ability to utilize biological environment to provide power to the device is indeed ingenious. I like this article also because its creativity and amazed by people’s ability to create cardiomyocyte sheet which can contract upon control and can survive with the nutrient from the environment.

Novel biodegradable electrospun membrane: scaffold
for tissue engineering

Shanta Raj Bhattarai, Narayan Bhattarai, Ho Keun Yi, Pyong Han Hwang,
Dong Il Cha, Hak Yong Kim

http://www.eng.uq.edu.au/files/course/files/CHEE4020/Choy.pdf

This paper presents the results of using electrospun membranes as the extra-cellular matrix in tissue engineering. The study uses the copolymer PPDO/PLLA-b-PEG to fabricate a nanofibrous matrix. The electrospinning consists of a syringe, a ground electrode, and a high voltage power supply.

The copolymer solution is charged and injected through a capillary tip. Because of its charge, the solution is drawn towards the collator as a whipping jet. While the jet travels, the solvent evaporates and leaves a continuous fiber that builds up on the collector target. The design of the fiber can be controlled by the rotation and translation of the collector drum while collecting the fiber. This produces a nonwoven fibrous mat. The morphology of the electrospun membrane is examined with SEM.

The NIH 3T3 cells were seeded onto the nanofibrous scaffold as a monolayer and the cultures were allowed to incubate. The number of cells in a nanofibrous scaffold was found at certain time intervals with a cell proliferation assay. The DNA content was also assayed and used to determine the cell number in the cell culture matrices using a standard DNA digestion procedure. After incubating for 7 days, the seeded 3T3 cells reached a plateau at six times more than the original population during the 10 days of incubation. Because the cell was able to proliferate, the electrospun membrane can be determined to promote cell growth and nontoxic for cell culture.

Cell migration occurs through the pores in the electrospun membrane. The paper states that pore properties such as porosity, pore dimension and pore volume, are parameters related to how the cells accommodate. The cells use amoeboid movement to migrate through the pores. The study also shows that cells seeded on the scaffold interacted with their environment. The cells maintained a normal phenotypic shape, which shows that the cells function biologically within this structure. Because the cells adhere onto the fibers, proliferate, and pack together on the structure surface, the cells must favor the electrospun membrane. The cells also crosslink the nanofiber and integrate with the surrounding fibers to form a three-dimensional cellular network.

The architecture of the structure of the nanofibrous membrane is similar to that of the natural extra-cellular matrix, so that the scaffold of the PPDO/PLLA-b-PEG copolymer may be suitable for tissue engineering applications. The studies in the paper have not optimized the physical properties of the nanofibrous membrane that would be best for cell attachment, growth, and proliferation, but the structure of the membrane could be suitable for soft tissue such as skin.

Demystifying SDS-PAGE:



Cute, no?

Potential of Nanofiber Matrix as Tissue-Engineering Scaffolds

ZUWEI MA, Ph.D. MASAYA KOTAKI, Ph.D. RYUJI INAI,
and SEERAM RAMAKRISHNA, Ph.D.

It's been well documented that cell behaviors depends greatly on the surrounding ECM properties. So in order for a TE device to be successful ECM properties need to be taken into account. This paper explores the use of nanofibers to fulfill that need and the various techniques commonly used to manufacture nanofibers.

Of the 3 common nanofiber production techniques electrospinning is the most used and oldest technique. Although the technique is fairly successful at contructing various nanofibers ranging from biodegradable polymers to collagen, the harsh 10-20 kV condition during spinning. Good luck trying to embedd proteins or cells during the spinning process.

Various research groups have shown PLGA(poly lactic co glycolic acid), PCL(polycaprolactone) or collagen nanofibers can be used to successfully culture fibroblast, cartilage, mesenchymal stem cells, chondrocytes and smooth muscle cells while maintaining their "selfness". The nanofiber structure is capable of supporting cell attachment and proliferation.
Nanofiber's physical properties like fiber diameter, alignment, can be manipulated to match the native physical conditions of various cell's ECM. In cases like TE blood vessel, the nanofiber can be aligned in the direction of the blood flow to help prevent atherosclerosis(hardening of the blood vessel) and the surrouding scaffold for smooth muscles cells can be aligned in concentric circles to help withstand pressure.

For a TE device to perform properly inside a patients body, one can't just slap in the device with no regard for the ECM. This field still have alot of potential for future improvements. One future technique is mimicking spider silk spinning. Such a technique can enalbe TE devices to have time released shells filled with growth factors or nanofibers embedded with living cells.

Contractile Three-Dimensional Bioengineered Heart Muscle for Myocardial Regeneration
Wiley Periodicas, Journal of Biomedical Materials Research (2006): 719-731
Yen-Chih Huang, Luda Khait, Ravi K. Birla
Department of Biomedical Engineering (Section of Cardiac Surgery), The University of Michigan, Ann Arbor, Michigan

Bioengineered heart muscle (BEHM) is being investigated as a method of treatment for early stage congestive heart failure. Various methods are currently under investigation including biodegradable gels/hydrogels and synthetic scaffolds. One of the biggest limitations of these methods is trying to provide a controllable mechanical environment for the engineered tissue that is a close match to the physiological environment of the native tissue; often the tissue constructs lack the necessary mechanical strength under physiological conditions.

This study investigated the use of a biodegradable fibrin gel as a novel method to engineer functional three-dimensional heart muscle. The fibrin gel functions as a support matrix to promote the formation of three-dimensional heart muscle, which forms from the self-organization of primary cardiac cells. The fibrin has the advantage that it has controllable degradation kinetics, so the rate of its degradation can be matched to the rate of tissue formation. This is important because it allows for the cardiac cells to organize into tissue and then allows for the gradual transfer of mechanical forces in the native environment from the matrix to the tissue.

Primary cardiac myocytes were isolated from newborn rats, and then two methods were used to form the BEHM: the layering method and the embedding method. With the layering approach, cells were plated on the surface of a tissue culture plate coated with PDMS, followed by thrombin and fibrin. In the second method, cells were suspended with thrombin and fibrin and then plated on PDMS tissue culture plates. Both approaches resulted in a three-dimensional tissue construct of cardiac tissue within seven days, and in both cases the monolayer of cardiac cells delaminated due to the spontaneous contraction of the cells.

Overall, the investigators found that the layering approach was better for myotube formation, which is necessary for the formation of cardiac tissue. The cardiac cells spread more, and the cell monolayer underwent spontaneous contractions with a consistent frequency that resembled the regular contractions of native myocardial tissue. The cells that were embedded in the fibrin gel didn’t spread as much, which is likely why only isolated patches of cells underwent contractions.

The investigators also looked at mechanical strength of the BEHMs generated via the two methods. The plating density didn’t affect the stimulated active force for the constructs produced via the embedded method, but the plating density did significantly affect the force for constructs that were produced via the layering method (the lower the density, the higher the forces). Additionally, the investigators found that by changing the media daily, they were able to maintain the stimulated active force for a period of two months. Unfortunately, these measured forces for the BEHMs was much lower compared to the specific force of native thin cardiac muscle.

Because my interests lie in tissue engineering, I found this paper interesting. In particular, it was exciting to me because it presents a rather new approach to generate engineered tissue; there has already been tremendous research investigating the use of synthetic scaffolds and hydrogels. Because this method is so novel there is a huge amount of research to be done, targeting some of the major problems (including the lower forces generated by BEHMs as compared to native cardiac tissue as mentioned earlier). Despite the limitations, this method is an exciting potential alternative to scaffolds and hydrogels in tissue engineering of cardiac muscle.

Bone modeling adaptation as a method for promoting development of bone tissue engineered construct in vitro

Zhang Chunqiu, Zhang Xizheng, Dong Xin and Zhu Weimin
School of Mechanical Engineering, Tianjin University of Technology, Tianjin 300191, China
Tianjin Institute of Medical Equipment, Tianjin 300161, China
Department of mechanics, College of Mechanical Science and Engineering, Nanling Campus, Jilin University, Changchun 130022, China

Received 11 September 2006; accepted 18 September 2006. Available online 22 January 2007.

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WN2-4MW90G9-1&_user=4420&_coverDate=01%2F22%2F2007&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=0846f3e0f7263064a1b3a422f48ea9ff

This paper focuses on using the bone modeling adaptation to promote the development of a bone tissue engineered construct in vitro. It is found that in the modeling process, in vivo bone formation increases bone formation in order to adapt to overload strains. So, this fact is taken advantage of in the lab as external mechanical forces are applied onto cell-seeded scaffolds to produce overload strains in the in vitro constructs. This produces bone cell differentiation and extracellular matrix deposition, which initiates bone formation on the interior of the scaffolds. The development of the construct is produced step by step as it accomodates itself to the new mechanical environment. Once the full bone construct is produced, these can be used as innovative off the shelf, bone-like substitutes for patients who suffer from long-term bone defects.

The cells that deposit themselves on the interior of the scaffold in the development of BTEC include osteocytes, bone lining cells, osteoblasts and osteoprogenitors. When the base of the template becomes filled with a large amount of osteocytes, the construct then matches native bone tissue very well and can be the BTEC desired by surgeons for use in replacing defected bone. Scaffold strains induced by the mechanical demands placed on the construct in vitro serve to produce a mechanical environment that is similiar to native tissue. The stains include everything from deformations of bone cells stuck on the interior surfaces of the scaffold to force induced stimuli such as flow shear, flow potential and even changes of signaling molecules, nutrients, and waste products. In the development of the BTEC, osteocytes direct osteoblasts to decide the location where it is mechanically neccessary for more bone tissue to be added. These osteocytes then become thicker within the construct and the bone-like construct can finally withstand strong forces after a long period of time.

This introduced model for creating bone-like constructs than can be used as substitues for native bone tissue holds great value for medicinal uses because present autologous bone grafting presents several drawbacks. Its limitations include the limited available amount for transplantation, the lack of structural integrity to withstand functional loads, and increased patient morbidity at the site of harvest. Production of off the shelf bone-like substitutes is still a major challenge even if the proper scaffolds, engineered cells and growth factors are given, but the bone modeling adaptation seems to be promote the development of BTEC in vitro rather well. Hopefully, in the future, this method can be used to promote the widespread use of tissue engineered bone-like substitutes to replace current autologous grafting methods.

In Vitro-Cultured Meat Production

P.D. Edelman, D.C. McFarland, V.A Mironov, J.G. Matheny. Commentary: In vitro-cultured meat production. Tissue Eng. 2005 May-Jun;11(5-6):659-62

http://www.hedweb.com/animimag/invitro-culturedmeat.pdf

Most of what we consider edible meat is formed of skeletal muscle tissue. The idea of creating cultured meat basically involves growing large numbers of myoblasts on a scaffold that are differentiated into myofibers which can be harvested, processed and cooked into a meat product (boneless, ground). More ambitious ideas would be producing highly structured meat, like steak, which has been shown to be more difficult to tissue engineer in vitro due to the lack of blood circulation.

Skeletal muscle is formed of various cell types. Fibers are formed from myoblasts and satellite cells that join to form large multinucleated syncytia. The most practical cell source for cultured meat is either embryonic myoblasts or skeletal muscle satellite cells. Satellite cells have been cultured from chickens, turkeys, pigs, lambs, and cattle. They have differentiated to form immature muscle fibers called myotubes. Replicating unprocessed meat would be difficult requiring fibroblasts for the connective tissue and possibly fat cells within the same culture organized into a 3D structure. Replicating a processed meat source would be much simpler and could use a single line of myogenic cells.

The quantity of cells that could be reproduced is not known and may be limited by a fixed number of doublings called the Hayflick limit. Satellite cells from turkey were shown to express telemorase giving them a higher Hayflick limit. A single satellite cell with a Hayflick limit of 75 could theoretically satisfy the world demand for meat. Species not expressing telemorase may require transfection with the telemorase gene or proliferation of stem cells in culture before differentiation into myoblasts. Differentiation of myoblasts into myotubes has been shown with the aid of mechanical or electromagnetic stress on the growing cells.

Myoblasts require a scaffold for attachment allowing proliferation and differentiation. The scaffold must be edible for production of meat and flexible to allow stressing of myoblasts to induce differentiation. One possible scaffold would be to use edible, porous microspheres that stretch in responses to changes in pH or temperature. These spheres could be made of collagen, cellulose, alginate or chitosan. Thin sheets of muscle fiber could be cultured and mechanically stretched to induce development of aligned myotubes. The thin scaffold membranes could be extracted from the meat or left on if made of edible material. Several sheets of tissue could be combined to get the required thickness or processed to make a meat product. A scaffold for non-processed meat would be much more difficult to produce because of vascularization requirements that would require a network of edible, elastic, and porous material to exchange nutrients with the cells.

The media used to grow these cells would likely have to be cheap yet contain high levels of nutrients. Some promising alternatives include serum free media made from shitake mushroom extract. Proper levels of growth factors are also necessary for the media. Large volume bioreactors that maintain low shear and uniform perfusion such as rotating bioreactors would enhance the growth process.

Cultured meat could provide many benefits including control of ratio of saturated and unsaturated fat, reduction of food borne disease, improvement of meat nutrient content, and reduction of resources and pollution associated with farm animals. Whether or not cultured meat will be cost efficient is debatable but the technological obstacles are fewer than those for tissue engineering in clinical applications

Biological Designer Self-Assembling Peptide Nanofiber Scaffolds Significantly Enhance Osteoblast Proliferation, Differentiation and 3-D Migration

Akihiro Horii, Xiumei Wang, Fabrizio Gelain, and Shuguang Zhang

Published online 2007 February 7. doi: 10.1371/journal.pone.0000190.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1784071

The authors have developed new biomimetic designer self-assembling peptide scaffolds to enhance pre-osteoblast proliferation, differentiation and migration. Self-assembling peptide nanofiber scaffolds have been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold. The authors designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The self-assembling peptide scaffolds showed to be effective at enhancing cellular proliferation and differentiation. Particularly, they selected three active motifs which included cell secreted signal peptide (osteogenic growth peptide), cell attachment domain of extracellular matrix (osteopontin) and designed RGD cell attachment sequence. RGD ( a short peptide sequence Arg–Gly–Asp) is present in fibronectin and many other extracellular matrix proteins.

The results of the study clearly demonstrated that the designer self-assembling peptide scaffolds significantly enhanced mouse pre-osteoblast cell proliferation and differentiation. It also stimulated cell migration into the 3D scaffold. These experiments showed that cell proliferation is enhanced using the mix of functional and pure RADA16 peptide scaffold. These results suggest that designer peptide scaffolds may be useful for bone regeneration and bone tissue engineering.

The study was interesting because of its future potential. The simple addition of short, biologically active peptide motifs have been shown here to significantly enhance particular cellular activities. This opens a new way to design new biologically active scaffolds for specific tissue repair and tissue engineering.