Vitreous cryopreservation maintains the function of vascular grafts

Although successful human cryopreservation and revival likely won’t be available in the near future, it’s amazing to think that nowadays, living cells can be frozen for over a year before being thawed and re-cultured. As engineered vascular grafts increase in demand, a method is needed to store tissue constructs for indefinite periods of time, they will be of limited practical use. This need is the basis for work on cryopreservation.

Cryopreservation so far has mainly involved simply placing cells and solvent in low-temperature freezers in vials, cryoprotectant optional. Ice crystals formed through this method, however, cause damage to the tissue, and blood vessels do not maintain the same mechanical properties as when they are first obtained. To avoid this problem, scientists have developed an alternative technique for cryopreservation called vitrification. Rather than relying on the slow freezer, this process involves an extremely fast temperature decrease (e.g. plunging the sample into liquid nitrogen). This reduces the formation of ice crystals and instead causes the solution to become increasingly viscous and glass-like.

In this paper’s study, researchers tested the effects of freezing on the mechanical properties of rabbit blood vessels. Blood vessels were placed into one of three categories: fresh (control), slow-frozen (the original method for cryopreservation), or vitrification. After being cut into cross-sectional vein rings and undergoing their respective procedures, the vessels were treated with various drugs such as histamine or acetylcholine. Contractile tensions were measured to indicate response to these drugs.

The results show that slow-frozen blood vessels show a marked decrease in contractile response to the drugs compared to fresh tissue, while vitrified vessels mostly maintain this contractile response.

The graphs to the left show the differences in contractile response to various drugs among control (C), slow-frozen (F), and vitrified (V) vessels.

Microscope images of these tissues also reveal the extensive damage in slow-frozen tissue due to ice crystal formations, a feature largely absent in vitrified blood vessels.

Microscope images of slow-frozen (A) and vitrified (B-D) tissues are shown on the right. Slow-frozen tissue shows extensive ice crystal formation, while vitrified tissue remains mostly free of this damage.

The ultimate goal for cryopreservation is, as previously mentioned, storage and revival of complete organisms. The possibilities of such a technique are so varied that I won’t attempt to go into the details, lest I lose sight of the line dividing the real world with science fiction (Is cryopreservation of astronauts as in the movie 2001: A Space Odyssey possible?). Even nowadays, cryopreservation is used in such areas as sperm banks, where sperm can be frozen for long periods of time until someone wants them. There is a large need for effective storage of tissue for future use, and I predict that cryopreservation will become increasingly important in the medical and in the bioengineering fields.

Reconstruction of Functional Tissues with Cell Sheet Engineering

Joseph Yang, Masayuki Yamato, Tatsuya Shimizu, Hidekazu Sekine, Kazuo Ohashi, Masato Kanzaki, Takeshi Ohki, Kohji Nishida and Teruo Okano

The approach of seeding cells into biodegradable scaffolds has become a hallmark of modern tissue engineering. Rather than using biomaterials as scaffolding materials for tissue reconstruction, the investigators have created an alternative approach using polymer-coated culture surfaces to facilitate the non-invasive harvest of cultured cells as intact tissue sheets. The culture surface is grafted with poly(N-isoproplyacrylamide) (PIPAAm), a temperature responsive polymer that allows controlled attachment and detachment of living cells via temperature changes.

With this new method, cell types that secrete significant amounts of extracellular matrix (ECM) proteins, are cultured for prolonged periods to create sheet-like structures for tissue reconstruction. By covalently immobilizing PIPAAm onto conventional culture surfaces, changes in the surface properties can be controlled by varying the incubation temperature. At temperatures above 32 °C (lower critical solution temperature), culture surfaces are slightly hydrophobic and enables various cell types to attach, spread and proliferate as if on normal tissue culture polystyrene. When the temperature is below 32 °C, the PIPAAm-grafted surfaces spontaneously become hydrophilic. A hydration layer forms between the culture surface and the attached cells, allowing the harvest of confluent cells as intact sheets. Since the grafted surfaces facilitate spontaneous cell detachment, scientists can avoid using proteolytic enzymes such as typsin and collagenase. With non-invasive cell harvest, cell-to-cell junction and ECM proteins can be maintained. Cell sheet engineering had been used to create functional tissue sheets to treat a wide range of diseases including corneal dysfunction, esophageal cancer, tracheal resection, and cardiac failure. The researchers have also developed methods to create thick vascular tissues as well as, organ-like systems for the heart and liver by generating 3D tissues of cultured cells and deposited ECM proteins with the cell sheets. The article goes in more detail about each of these applications.

Using cell sheet engineering, numerous cell types have shown the maintenance of differentiated functions after low-temperature cell sheet harvest due to the preservation of cell surface proteins, such as growth factor receptors, ion channels, and cell-to-cell junction proteins. In addition, cell sheets can be easily transferred and attached to other surfaces (culture dishes, host tissues). A disadvantage is that engineered tissues created using this cell sheet method contain relatively little ECM. Thus, the method may not be ideal for the creation of cell-sparse tissues like bone and cartilage. Regardless, cell sheet engineering seems to be able to provide new possibilities in regenerative medicine and tissue engineering.

I chose this article because cell sheet engineering seemed interesting and it could be useful for future projects. This method is like an extension of the basic cell culture techniques taught in class - some day, I would like to try it out and see the results. Cell sheet engineering is a method that could be implemented with regeneration of specific tissues without the risks of using traditional scaffold-based methods. In addition, cell sheet engineering is novel alternative for research projects that require the re-creation of functional tissue structures.

Designing synthetic materials to control stem cell phenotype

This review discusses how stem cell behavior is manipulated using synthetic materials. Ligands have been designed to emulate the natural extracellular matrix, cell-cell contacts, and growth factors. Fate determination of the stem cells is regulated by material architecture and mechanical properties. Synthetic material systems are specifically designed to interact with cells on different length scales (e.g. macro vs cellular). Thus, they replicate the elements of natural stem cell niches (micro-environment needed to sustain self-renewal and control differentiation). However, synthetic materials differ from natural because they have the potential for improved control, repeatability, safety and scalability. Several classes of synthetic materials have been created to control stem cell phenotype: natural polymers (like mammalian ECM), synthetic polymers (like polyacrylamides), inorganic materials, and self-assembling peptides. Whichever class is used, the material must be processed and functionalized for specific clinical applications. Important material properties include ligand identity, presentation (conformity/orientation), and density. Optimizing these parameters creates materials that resemble the natural environment of the stem cells and allows for controlled mechanical properties.

Self-renewal and differentiation mechanisms are sensitive to many ligands/combinations of ligands: adhesion ligands from ECM, ligands from neighboring cells, and immobilized growth factors. Once a set of ligands is selected, it must be conjugated to the material for proper orientation. The way a material is organized and structured on the nanoscale (aka material architecture) is known to control cell signaling and organization. The geometry of the cellular interface with the material is a major factor in determining ligand engagement, molecular diffusion, and force transmission. Material architecture also determines bulk mechanical properties at larger scales. Both 2D and 3D architectures have been used for stem cell cultures. For 3D scaffolds, there are 3 predominant types: porous solids, hydrogels, and nanofibers. Material mechanics is largely determined by material's composition, water content, and structure. These affect intermolecular forces and stress distributions. Common techniques to change mechanical properties include altering molecular composition/connectivity, thermal processing, and creating porous composites. Mechanical properties affect cell migration and proliferation. Tensional homeostasis with the micro-environment (via integrins) causes cytoskeletal rearrangements and changes gene regulation pathways. The elastic modulus of culture material can alter or maintain human stem cell phenotype. Viscoelasticity is another mechanical property that may affect stem cell phenotype, but its effect is not fully known yet.

I chose this paper because I believe that studying how to manipulate stem cells is crucial, particularly if we want to successfully utilize stem cells in therapeutic applications. This paper discusses several properties that play significant roles in directing stem cell phenotype, but concludes that much work needs to be done in the future to realize the full clinical potential of stem cells.

-Rustin

Collective migration of an epithelial monolayer in response to a model wound

M. Poujade*, E. Grasland-Mongrain*, A. Hertzog†, J. Jouanneau†, P. Chavrier†, B. Ladoux‡, A. Buguin*, and P. Silberzan*§

The study of cell migration is important in wound healing. Usual methods of observing migratory behaviors of cells in vitro is the classical “wound healing” scratch assay, in which a razor blade or pipette tip is used to mechanically remove some cells in a confluent epithelium monolayer. However, researchers have been trying to develop new ways in proving how cells acquire their motility. In the scratch assay, the removed cells release intracellular content into the medium, sending chemical signals to neighboring cells to trigger migration. Another theory in why cells migrate is something called a “free surface” in which a removal of a barrier, which initially inhibits the growth of the epithelium, causes the cell to migrate. However, previous studies have been inconclusive and remain to be controversial.

As a result, the authors of this article orchestrated a very simple but elegant design to study the response of the monolayer as a result of a free surface. The experiment involved culturing a monolayer of cells constrained within a microstencil, which is fabricated from polydimethylsiloxane (PDMS) that precisely defined the geometrical conditions of the monolayer. The stencil was removed and the cells were free to migrate as shown in Figure 1. There are some advantages with this assay in observing cell migration. First, it eliminates the need to “scratch” the monolayer, which might depend on the user and the tool used to scratch the surface. Next, the wounds are well defined and perfectly controlled, allowing the authors to observe the cells progress from its initial position. The authors concluded that their injury-free wound healing assay on MDCK epithelial cells showed that the free surface was sufficient in inducing cell migration. Moreover, they note that the cells migrate in a leader-follower pattern which a certain cell will take “lead” in migration.

I chose this paper because of the simplicity of the experiment. For my project, I plan to use a similar design in determining cell migratory patterns within various defined regions.

UTILITY AND CONTROL OF PROTEOGLYCANS IN TISSUE ENGINEERING

Proteoglycans (PGs) and their glycosaminoglycan (GAG) have been used commonly in tissue engineering (TE) to produce scaffolds. However, their specific biophysical and biological functions in TE have not been fully studied. Proteinglycan is an important part of the extracellular matrix (ECM). PG molecule consists of a core protein and the GAG chains. The core protein doesn’t have serine residues. GAG chains are attached to core protein by tetrasaccharide link. The variety of PGs are the result of the variety of GAG chains attach to core protein. GAGs are long chains of disaccharide units that are sulfated differently. PGs in the native tissues have roles in tissue remodeling, intracellular signaling, cell migration, cell compressibility and transparency, and other functions. PGs in the extracellular space are the following: basement membrane PGs, hyalectines, and small leucine-rich PGs (SLRPs). In this paper, we will discuss the functions of perlycan in the TE scaffold. Another category of PGs is the cell surgace PGs, which include syndecans, glypicans. Thrombomodulin, and CD44. The more we learn about functions of specific PG and its GAGs, the closer we are to reconstruct native environment of tissues.
This paper review two kinds of PGs and GAGs grafted in scaffolds. The first one is collagen-GAG scaffold. The collagen-GAG suspension is solidified, which results in the co-precipitation of collagen-GAG and the growing ice crystals. The GAG chains are then grafted to the scaffold through exposure to high temperature. This process also sterilizes the scaffold. Studies have proved that the present of GAGs in scaffold improve tissue growth and regeneration over the use of collagen alone. This type of scaffold help inducing cells to retain more PG aggregates in scaffold and retain more newly synthesized PGs within the cell layers. Collagen-GAG scaffolds recently have been used in nerve regeneration, artificial skin, osteogenic and chondrogenic tissue development. Studies have suggested that osteoblasts seeded in collagen-GAG scaffolds show greater adhesion, proliferation and make more markers. Silicone tube-collagen-GAG combination has shown more success in inducing greater number of axons per nerve. There are also many applications of this type of scaffold discussed in this paper.
Matrigel scaffolds have also been studied for TE applications. Matrigel is a soluble extract of Engelbreth-Holm-Swarm tumor cells’ basement membrane. Its components include laminin, fibril, nidogen, entactin, collagen IV, perlecan, and growth factors. This scaffold has been used to study tumor cell migration and invasion of the basement membrane. HSPG perlycan is one of the major component of matrigel which has not been studied widely. It is suggested to play key roles in blood vessel growth. The C-terminus of perlycan has been reported to inhibit endothelial cell migration, collagen-induced endothelial tube formation, and blood vessel growth in vivo. Therefore, it is suggested to contribute in the control of tumor cell growth.
There are still many type of PGs and GAG that are commonly used in TE. Scientists have done many studies on the total amount of PGs synthesized in engineered tissues. However, not many studies have been done to fully explore the potentials of PGs in creating the native environment of tissues.

Cells on chips

Info. of Article
Jamil El-Ali, Peter K. Sorger & Klavs F. Jensen, "Cells on chips", Nature, v. 442, 2006, pp. 403~411.
Full text: http://www.nature.com/nature/journal/v442/n7101/pdf/nature05063.pdf

Main idea of article
In the first section, the authors explain why the "cells on chip" is important to us. ("cells on chip" means to do cell-related biological research or analysis on chips. Some people also call them "lab-on-a-chip" or "micro-total-analysis-system"(uTAS)). The authors believe the significance of "cells on chip" will be similar to the role that miniaturization has played for microelectronics. Our intuition thought would be that "cells on chip" simply represent miniaturized versions of conventional laboratory techniques, but the authors tell you it is far beyond that. In micro world, there are many wonderful advantages that are not true in macro scale. For example, you can easily have favorable scaling of electrical fields due to small length and well-controlled laminar flows due to the low Reynolds numbers in microchannels. Moreover, instead of fabricating individual small-scale equipments piece by piece, the researchers have been making the microsystems incorporating several steps of an assay into a single system. By doing this, you can do experiments on a single chip quickly and effectively. You name the benefits: small sample request, far less labour intensive (just think about what people expect typical biology-majored students often do in their labs), and avoiding of potential error-prone laboratory manipulations. With developed micromaching technology, it is also easy to batch produce such devices cheaply. In a word, “cells on chip” will be a wonderful breakthrough, no matter you consider from the perspective of pure research, cost, or effectiveness.

After the introduction, the authors go into the details, extensively but logically introducing up-to-date progress in each step of a cell experiment ranging from cell culture, selection, lysis, separation to final analysis. The article is well organized. Roughly speaking, in each step, the authors will tell you the major biological mechanisms in this step, then how they can be realized in micro scale, and finish it with some vivid practical examples and beautiful pictures. I would like to introduce as detail as above, however, the topics of this article are so wide that you bet my summary would be far less attractive than those used by the authors. Since you know what they are going to talk about, why don't you explore it by your self?

Why it is worth your reading?

——Chosen from a possibly different view
Slightly different from most of you, I am a graduate student spending years in Electrical and Mechanical Engineering but having never learnt any biology (BioE115 is my first-ever bio-related class in college.) So my choice may be more from an engineer view rather than a biologist view. Read it to see whether your view is different from mine.

——Read it like watching movies
Instead of focusing on a specific phenomenon or application, this article is like an excellent scientific documentary. You won't have to face any never-heard-of nomenclatures or massive arguments and calculations.

——Open yourself to a new world
By using a short period of time (if you exclude pictures and references, this article has only about 5 pages of text content.), you may quite probably open yourself to an unknown world which is actually not far away from you. No matter how well you are familiar with biology or engineerings, you will find something new and worthy. Trust me on this.

——Be an early bird
With the well-known example of IC technology, device miniaturization is a long-term tendency in many fields. You had better know a little about it from now on.

——Trustable and strong
It is a REVIEW in NATURE written by several MIT professors. You can imagine how often you could see such a combination.

You are welcome to raise any questions. With spending years in microfabrication cleaning room, I believe I may be helpful in practical issues such as device design and fabrication (but weak in those too theorital and too biological).

Now, welcome to micro world, a both familiar and unknown world!

Titin expression in human articular cartilage and cultured chondrocytes: A novel component in articular cartilage biomechanical sensing?

(Antibody Recognition and RT-PCR applications...)

The objective of this experiment was to test for the presence of titin on human articular cartilage tissue (e.g. joints). This large protein has previously been detected on heart and skeletal muscle tissue, where a biomechanical strain-bearing role has been attributed to it. "...Titin accounts for most of the passive elastic response retention that prevents over stretching... acts as an adjustable spring element..." Being this the case, it might be reasonable to look for the expression of this protein on other stress/strain-bearing tissues where elasticity is also required (e.g. joints).
Cartilage samples were taken from three groups of donors: Adults with osteoarthritis (OA), adults without OA, and infants (disgusting sampling). Immunolabelling was done with specific antibodies for "four" different domains of Titin; the N-terminal Z1-Z2 domain, the Novex III exon, the PEVK region, and N2A region (Fig.1). [I have to point out that even when only "three"antibodies were mentioned in the abstract, the paper showed results for "four" antibodies. Even section 2.3 (immunohystochemistry) doesn't make it clear (maybe it is me... I'm open to suggestions)].
However, it seems interesting to see positive results for titin presence in cartilage tissue (previously detected on muscle only). More than just existing on cartilage tissue, titin differences between Osteoarthritis and normal tissues were not detectable (Fig. 2,3). This suggests a crucial structural role of titin in cartilage.
Another result that called my attention was the detection of titin in extracellular matrix (ECM), when it was claimed to be intracellular (Fig. 2-4). This has two possible explanations:1) "a cross-reaction with other member of the Ig-domain gene superfamily, to which titin belongs" (detection of something else that has a domain in common with titin), and 2) titin has both intra- and extracellular strain-bearing functions.
Third interesting outcome: Some of the infant samples showed negative results for N2A region of titin (table 1, fourth column). This was associated to possible expression of a different titin isoform during maturation of the cartilage. This changes can be related to skeletal growth or mechanical load needs. This is a good point in favor of the specificity of antibody recognition (optimum trial and error, huh?). There were also discrepancies on the OA patients results (table 1, columns 2 & 3). This was similar to findings of aberrant forms of titin on patients with OA or Charcot Marie Tooth disease (Banes et. al. 2004). This is open to argument, though. Banes' aberrations where on the N2A region while aberrations here might be on Z1-Z2 (X112 113 X) and PEVK (9D10 ) regions.
Presence of titin was also shown by RT-PCR done from cells cultured from the samples. However, some domain fragments could not be amplified (see table2, Fig. 5). Again, might be related to the titin isoforms during cartilage maturation. Howeer, PEVK region was detected in all tissues by antibody recognition but not by RT-PCR. This case remains not clear. Further research will be required to clarify this.













Secondary Reference Sites:
1- http://www.ks.uiuc.edu/~ericlee/Telethonin/
2- http://www.psc.edu/science/2000/schulten/rude_mechanicals.html

Tissue Engineering of Skeletal Muscle

http://www.liebertonline.com/doi/abs/10.1089/ten.2006.0408

Tissue Engineering

Tissue Engineering of Skeletal Muscle.

Wentao Yan, Sheela George, Upinder Fotadar, Natalia Tyhovych, Angela Kamer, Michael J. Yost, Robert L. Price, Charles R. Haggart, Jeffrey W. Holmes, Louis Terracio. Tissue Engineering. ahead of print. doi:10.1089/ten.2006.0408.

Although a considerable amount of research has been done in skeletal muscle tissue engineering, there has yet to be a method for replacing lost tissue. In their study, Yan et al. have suggested the possibility of reconstruction through an in vitro construction of skeletal muscle with satellite cells to be reconstituted in vivo. Their 3D skeletal construct, engineered by seeding satellite cells on an aligned matrix of type I collagen fibrils, has been shown to closely resemble adult skeletal muscle function and structure and brings us only one step closer to a striated muscle implant in the future.

Experimental results prove the validity of their construct. Immunohistochemical staining of actin filaments and MyoD, the regulatory protein in muscle differentiation, revealed the differentiation of the satellite cells to myotubes and the parallel orientation of the multilayer muscle cells that are characteristic of skeletal muscle. Real-time RT-PCR tracked the muscle cell maturation process by assessing gene expression for various proteins. mRNA expressions for embryonic and adult myosin heavy chain, dystrophin, and alpha- and beta-enolases were consistent with patterns of in vivo mRNA expression reported in previous studies. Cell viability, measured using Trypan blue, was determined to filter out the possibility of death with aberrations in gene expression. Twitch and titanic stresses were implemented on the cells to test for their function. Measurements of the construct showed that the amount of developed force increases with the amount of stretching until the force plateaus. This force-length relationship observed parallels with that of known skeletal muscle. It is clear from their analyses that their construct mimics the basic phenotypes of adult skeletal muscle; however, their research is still incomplete. Further examinations on thickening the culture, contributions of extracellular matrix components, and hypertrophy of muscle stretching are needed until we can perform the first muscle transplant.

As the paper indicates, muscle loss has no answer, so I believe this study has a lot of potential in solving the problem. What I found most interesting is how simple the muscle construct was—a plate painted with stripes of collagen, seeded with isolated satellite cells. This comes to show that a project as a complex as skeletal muscle tissue engineering need not necessarily be carried out with an intricate protocol, but rather one that is well thoroughly thought-out. In addition, I realized how universal and widely-applicable the techniques we learned in class are. In fact, most of the techniques Yan et al. used to characterize their construct were introduced to us in class—immunochemistry, RT-PCR, Trypan blue counting. For my project, I hope to research on a topic related to skeletal muscle engineering, and I feel that this paper will serve as a good guideline.

Bioreactor-based bone tissue engineering: The influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization

Currently, osteoblast cells have been successfully seeded and cultured into 3D ceramic and biodegradable polymer scaffolds for bone substitute replacement, but the cells’ ingrowth into the scaffolds have been limited, possibly due to limited nutrient diffusion (200-800μm in PLAGA foam) in static culture conditions. Some have tried bypassing this diffusion limitation by utilizing a rotating bioreactor, which has low shear, is 3D, and most importantly, has a high mass transfer rate, imparting dynamic culture conditions. However, the densities of conventional scaffolds are generally greater than the density of the surrounding medium, causing the scaffold to collide with the bioreactor walls due to centrifugal force, inducing cell damage and disrupting cell growth and mineral deposition.

To address these issues, the investigators developed a system that utilized 3D degradable microcarrier scaffolds and a rotating bioreactor. The 3D scaffolds were composed of different mixtures of heavier-than-water (HTW) and lighter-than-water (LTW) PLAGA microspheres that were sintered together. These scaffolds, because of their varying densities, were proposed to have different interactions with the bioreactor walls than conventional scaffolds. The scaffolds were seeded with rat calvarial osteoblastic cells, which had been isolated from 2-day-old Sprague Dawley rats via the enzymatic digestive method at a density of 2E4 cells/ mL.

The cell cultures were characterized for cell proliferation, differentiation, mineralized matrix synthesis, bone marker protein expression, and morphological analysis. After 7 days of culture, cells were shown to grow mainly on the surface of the static control scaffolds unlike the scaffolds cultured in the rotating bioreactor, where the cells seemed to proliferate preferentially on the interior, as seen by SEM (scanning electron microscopy) and MTT assays. The osteoblasts also retained their characteristics and activity, as determined by alkaline phosphatase activity, osteocalcin, osteopontin, mineralized matrix formation, and calcium quantification. These results, in conjunction with video visualization, demonstrate that the mixed scaffold does indeed decrease the amount of collisions with the reactor walls, allowing the cells to proliferate and grow normally. Furthermore, by controlling the level of fluid flow, enhanced differentiation and mineral deposition could be achieved.

I chose this paper because bioreactor tissue culture has both economical and research implications: not only does it allow for better cell growth penetration into a scaffold, but the method also allows for faster and possibly more affordable methods of producing tissue engineered replacements for not only bone, but other tissues as well.

Functional Living Trileaflet Heart Valves Grown In Vitro

I chose this paper because it was relevant to two of the classes I am taking right now. We have been talking about heart valves and heart-related mechanics in bioe 102, and the tissue engineering part is definitely related to our class. It might be relatively an old paper (2000) but I thought some of the techniques they used, like the pulse duplicator, were pretty interesting and overall an informative read.

In this study this group fabricated tri-leaflet heart valves by seeding autologous myofibroblasts and endothelial cells from sheep on a bioabsorbable polymer graft. A pulse duplicator was used to gradually increase the pressure and flow in order to mimic the function of heart so that the cells adjust themselves to in vivo conditions.

The major advancement of this approach over other tissue engineering attempts was that it does not have the shortcomings of mechanical valve replacements. Mechanical valve replacements have the risk of thromboembolism due to structural dysfunction and other mechanical shortcomings. Also these replacements often require anticoagulation therapy. Some valves may calcify prematurely and have limited durability. This approach eliminates these shortcomings by using autologous transplantation and mimicking in-vivo conditions using the pulse replicator.

This paper was actually a follow-up on another study. In the previous study the group had tried a transplantation of a single pulmonary valve. In this study they improved the original polymer graft to decrease the absorbability and strength of the graft. After cells were harvested for 14 days on the heart valve constructs they were implanted into the same lambs. The polymer graft would temporarily provide mechanical support and would gradually get absorbed as the cells produce their own ECM, which replaces the polymer. After transplantation different analysis were done on the TE valve periodically. These test compared different properties of the TE heart valve with normal heart valves and a control experiment. Control experiment was done using the same material, except that the cells and polymer graft were not put on the pulse duplicator. Some of these test included Polymer degradation analysis, tissue microconstructure, mechanical properties, and tissue analysis. Histology results showed that the cells were organized in a layered fashion, denser in the outer layer compared to inner layers. Mechanical tensile testing revealed that the strength of the implants were originally higher than normal tissue, but gradually leveled off to normal. Tissue analysis also showed collagen content 140% of normal tissue in 4 week, which increased to 180% (plateau level). DNA content was initially 65%, but rose up to 150% in 20 weeks.

Overall the valves showed higher formation of matrix proteins, a more organized structure, and favorable mechanical properties over the control experiment (static cell culture) The study only reported follow up until week 20 over which no evidence of thrombus, stenosis, or aneurysm, however at 16-20 weeks, central pulmonary regurgitation was reported.

Functional Living Trileaflet Heart Valves Grown In Vitro

Cell growth: stress and proliferation are related!!!

Recently, I have spent countless sleepless nights thinking to myself, how can I understand epithelial monolayer mechanics better so I can help the sick and the hungry of the world? Well, thanks to Terry and BE115, I think I've found an article that'll help me end my Pursuit of Sleepyness.

Title: Emergent patterns of growth controlled by multicellular form and mechanics
By: Celeste M. Nelson, and others. (U. Penn)
Link to the article

So in BE115, we've been culturing lots of Fibroblast cells. We do bunch of stuff to them and we finally seed them to a piece of plastic, and a week later, BOOM, There they are, gloriously filling up the whole dish. But our observation stops there. We kind of wave our hands in the air and convince ourselves that because of contact-inhibition, there are no more proliferation that'll interest us. And rightly so! With sunny beach few miles away and snowy slopes and fine wine country only a few hours away, who has time to think about these things? Well, Apparently, people in Pennsylvania do. It's winter, it's snowed up to your hip and it's so cold. If you spit, it'll freeze and shatter by the time it got to the ground. So what do these good Pennsylvanians do? They sit in their toasty labs and do research on multicellular form and mechanics. Heaven bless Pennsylvania!

Here's a summary of their work.

Tissue structures can influence patterns of proliferation, and in return, it can influence tissue structures. I.e. there's a kind of feedback mechanism. Traditionally, scientists have thought that most important aspects of tissue structural fates lie with soluble growth factors call morphogens. They are distributed asymmetrically in our body, and these in turn can influence various important bodily changes such as tissue segregation, gastrulation, epithelial folding, tubulogenesis, and differential cell growths.

Apparently, though, physical stuffs like shape and stress of the tissue can influence how these changes come about as well.

To test this, they made several microfabricated wells of different shapes and sizes:

1) A square well
2) A rectangular well
3) A circular well
4) A pyramidal mesh

They seeded these with bovine pulmonary artery endothelial cells (which is apparently contact-inhibited), and they tried to measure proliferative rates. (They have markers that fluoresce during DNA synthesis.) And they found that proliferation is greatest at the (see figure 1):

1) Corners. (for square and rectangular wells)
2) Short edges. (for rectangular wells)
3) Perimeter (for square, rectangular, and circular wells)
4) Valleys (for pyramidal mesh)

These are all regions of high tractional stress. But just for completeness sake, they made some FEA models to predict that levels of local tractional stresses and proliferation match. And apparently they do, very well! (See figure 2)

They tried to test their theory that stress is related to (or even causes) proliferation with several other non-computational methods. First, they used elastomeric force sensor arrays (those microfabricated devices with small poles everywhere) and found that, indeed, locations of high stress coincided with regions of high proliferation. (figure 3 B, D)

They also found that when you increase or decrease the internal stress by varying the composition of cytoskeletal components with pharmacological factors, proliferation increased and decreased as expected. (Figure 3, F, H)

Overall, I think this was an excellent paper demonstrating how external factors such as stress and shape can influence the growth of a colony of cells. This is of course very important, since being able to understand and replicate tissue genesis for organ replacement can significantly better everyone's standard of living.

And off to sleep I go.

See you everyone.

David.

Cell organization in soft media due to active mechanosensing

I. B. Bischofs and U. S. Schwarz*
Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany
Communicated by Tom C. Lubensky, University of Pennsylvania, Philadelphia, PA, June 10, 2003 (received for review January 21, 2003)

Until recently, biologists have considered an adherent cell’s physiological function to be the main influence on its mechanical activity. Scientists suggest a new theory: adherent cells orient themselves in their environments to maximize effective stiffness. This theory relies on fairly complex mathematical equations whose derivations were far from trivial. Before constructing any formulas, scientists had to find a way to calculate the stress and strain on a cell due to its surroundings. They then tried to measure how much work a cell must do on its surroundings to build up a certain force at its point of contact. They came up with a complicated integral calculating the work, where minimum work corresponded to the cell sensing maximum stiffness. Note that they are not implying that cells actually execute this minimal work; rather, the effectiveness of the measurement lies in allowing them to compare different values of work in different elastic environments. Thus, they can see the varying degrees of effectiveness in extracting information through mechanosensing.
The results showed that cells interacting with homogeneous external strains orient themselves with the direction of stretch. Specifically, experiments with fibroblasts on elastic substrates and collagen gels support this orientation corresponding to minimal work. The scientist’s model also agrees with preexisting results regarding the mechanical activity of cells on boundaries. They also investigated cooperative effects of cells. Their work-minimizing model suggested that two neighboring cells would completely align their dipoles to maximize stiffness. This agrees with experimental results where strings of cells align themselves parallel to the external strain. Thus, this paper proposed various mathematical formulas for calculating the work done by various types of cells. Then, with the equations, found the orientations of the cells that minimized the work. Then, the paper compared these predictions with previously found experimental results. The consistent agreement between the predictions and experimental results provides a strong case supporting the validity of the optimization principle of cells in soft media.
I chose to read and summarize this article because of its substantial mathematical and physical content. I think work is a fascinating physical phenomenon. When I glossed over the articles, all the integrals and equations for work in this paper sparked my interest. I wanted to understand what the formulas represented biologically. Explaining biological phenomena with the tools in math and physics is, in my opinion, a very precise and beautiful way of understanding why cells behave the ways they do. This paper, in particular, is important because it presents a theory to describe previously observed experimental results on cell orientation. Understanding why cells orient themselves in a certain way is so much more exciting, to me, than just learning how they orient themselves. With math and physics, we have found ways to deepen our understandings of observed phenomena.

Progress and opportunities for tissue-engineered skin

MacNeil, Sheila. “Progress and opportunities for tissue-engineered skin.” Nature. Vol 445
pg.874-880. 22 Feb 2007.


Individuals who suffer a loss of both layers of skin (epidermis and dermis) more than a diameter of 4 cm usually cannot heal without a graft. In these cases surgeons will remove from healthy areas of the body a split-thickness graft that comprises of a full epidermis and partial dermis layer. The graft integrates with the wounded area and protects the area like a skin band-aid. The area the graft was taken from will be able to regenerate an epidermis layer as long as there are enough residual epidermal cells left in the remaining dermis layer. Although this method works well, sometimes it is not always best to create another wound to heal another depending on the person’s health—this is where skin tissue engineering is necessary.
Skin grafts must restore barrier function or promote healing, therefore engineered skin must do the same. Engineered skin is composed of the same cells naturally found in skin in a 3D structure: keratinocytes (for barrier) and fibroblasts (for strength and resilience). One of its major advantage is that skin cells can be cultured and grown at a much higher rate than in the human body, however, a major fallback is immune rejection to the cultured cells or support mediums. Some grafts use fetal bovine serum to sustain the cells that can cause an unwanted allergic response. Also a successful engineered skin graft is one where the individual’s own skin cells take over and replace the cells in the graft so near seamless integration of foreign and host tissue is necessary.
Skin grafts must also be a proper scaffold structure that allows angiogenesis to occur because without a blood source the graft will die and obviously not integrate with the surrounding skin. Cell migration needs to take place to aid in integration and replacement of cells also, so that is why artificial skin is so complex. There are substitutes that consist of just the epidermis layer, just the dermis layer, and few with both the epidermis layer and dermis layer (the most needed one).
Besides the clinical application, skin grafts can also aid researchers in learning about cell-to-cell extra-cellular interaction, regulation of pigmentation, disease penetration/invasion (i.e. melanoma), skin contraction, and many more specific mechanisms. This added bonus furthers motivation in finding a good skin graft. Currently the main goals to improve upon the already established research include ease of use, clinical efficacy and practicality, introducing TGFβ-family factors to promote wound healing and reduce scar formation, reducing graft contraction while increasing graft attachment

I chose this paper because it shows me how much the field of bioengineering has advanced, and yet, how much more it can broaden—it is amazing that skin was one of the first engineered tissue and yet there is still no perfect skin substitute. Skin is the largest human organ and the primary barrier that block harmful antigens from entering the body. The immune system is largely dependant on skin and so there is high motivation to find suitable skin substitutes for individuals with large surface wounds. Thus, for these reasons I believe major advances should continue to be made in this aspect of bioengineering.

Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Kazyo Ohashi, Takashi Yokoyama, Masayuki Yamato, Hiroyouki Kuge, Hiromichi Kanehiro, Masahiro Tsutsumi, Toshihiro Amanuma, Hiroo Iwata, Joseph Yang, Teruo Okano, and Yoshiyuki Nakajima

link: http://www.nature.com/nm/journal/v13/n7/pdf/nm1576.pdf

There are currently two major methods for implanting hepatic tissue in patients who do not require whole liver organ transplants. In one treatment, hepatocytes, suspended in extracellular matrix components, are injected into the body via the portal vein. In the second method, hepatocytes are implanted with a biodegradable scaffold that serves as a structural foundation for cell attachment. Unfortunately, both treatments have many risks and disadvantages. For example, the first treatment may result in embolization – the injected cells obstruct portal circulation. The second method may lead to fibrosis, inflammatory reactions, or immunogenic reactions, as a consequence of introducing a foreign object into the body, the. Furthermore, hepatic cell viability is short (on the scale of days) in both treatments because the body does not well vascularize the engineered tissue. As a result, multiple transplantations are required.

To address these issues, the investigators developed protocols to produce long living and functional 2D and 3D hepatic tissues, to optimize vascularization of the engineered tissues, and to implant them without biodegradable scaffolds. To create the tissues, the investigators isolated primary hepatocytes, expressing human alpha 1-antitrypsin (hA1AT), from transgenic mice. They grew the cells on culture dishes coated with temperature-sensitive polymer poly(N-isopropylacrylamide) or (PIPAAm). At 37°C, the cells grew and remained attached to the slightly hydrophobic polymer. After the cells reached confluency, the investigators lowered the temperature. Below 32°C, the polymer became hydrated and caused the cells to detach uniformly as a 2D sheet. To make the 3D tissues, 2D sheets were layered on top of one other. In the control, the scientists used the proteolytic enzyme collagenase to separate the tissue from the polymer. The scientists found that the tissue prepared by varying temperature had better structural and functional integrity than the control. The engineered tissues produced more hA1AT, had better drug metabolism, and retained intercellular connectivity or extracellular matrix components. In the control, the enzymes destroyed fibronectin which was important for cell adhesion. However, in the cells detached by lowering temperature, fibronectin was still present.

Vascularization of transplanted tissue is essential for prolonged tissue viability. In the study, the investigators inserted the engineered hepatocyte tissue into the subcutaneous area of mice. In order to vascularize the region, the scientists implanted a device that secreted fibroblast growth factor. After a vascular network had formed, the scientists removed the device and transplanted the tissue. In the mouse, these cells had longer viability than those implanted using conventional methods. The engineered cells also maintained hepatocyte-specific functions. For example, using BrdU and immunofluorescence staining, the scientists found that the cells were able to profilerate in the mouse. They were also able to detect plasma exchange between the engineered tissue and the body.

The advantages of the new protocol are that it is simple, minimally invasive, and free of materials that can elicit a detrimental immune response. There are, however, some disadvantages to the technique. These tissues were uniform and did not have structural polarity. The formation of sinusoidal and biliary surfaces is necessary to create tissue that can replace whole organs. Thus, this technique can not yet be used to replace whole organ transplantation. Nevertheless, I chose this paper because the technology has potential to benefit patients with liver diseases that do not require an organ transplants, including those with hemophilia and congenital metabolic liver diseases, such glycogen storage disease and congenital deficiency of coagulation factor VII. Since the cells must be incubated on the plates for a few days to reach confluency, scientists can genetically modify the cells to optimize treatment for particular liver diseases. In the future, this technology can be extended to develop larger sections of liver that would lower the demand for organ transplantation.

Transferable anticancer innate immunity in spontaneous regression/complete resistance mice

Transferable anticancer innate immunity in spontaneous regression/complete resistance mice

Amy M. Hicks, Gregory Riedlinger, Mark C. Willingham, Martha A. Alexander-Miller, C. Von Kap-Herr, Mark J. Pettenati, Anne M. Sanders, Holly M. Weir, Wei Du, Joseph Kim, Andrew J. G. Simpson, Lloyd J. Old, and Zheng Cui

Full Text Article: http://www.pnas.org/cgi/content/full/103/20/7753

This paper explored the properties and mechanisms behind the cancer immunity of spontaneous regression/complete resistance (SR/CR) mice, and its implications for the treatment of cancer. Spontaneous regression/complete resistance (SR/CR) mice have the ability to resist very high doses of cancer cells that are lethal to wild type (WT) mice even at low doses. The researchers found that this remarkable ability is mediated by the rapid infiltration of leukocytes, involving natural killer cells, macrophages, and neutrophils, which then triggers rapid cytolysis. These cell types from SR/CR mice also have the ability to independently kill cancerous cells in vitro. Remarkably, this resistance was also completely transferable to wild type mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against future cancer or eradication of established malignancy.

The researchers found that the primary immune response was innate, (i.e. present without prior cancer exposure), but also found signs of adaptive immune responses, as the elimination of cancer cells was faster upon further exposures. Mice started out as spontaneous regression phenotypes, but after further exposure were entirely immune. In addition, they found the method involved the formation of rosettes between leukocytes and cancer cells that enable cytolysis of the cancerous cells. Interestingly, each individual type of leukocyte was able to kill cancer cells alone, without the other two types present. The researchers also developed an in vitro cancer-killing assay to identify SR/CR mice without needing to challenge them directly with cancer cells.

Next, the ability of transferred leukocytes to kill established tumors in wild-type mice was established. With injections of SR/CR leukocytes (4*10^8) from spleens and bone marrow, many solid tumors regressed completely or partially regressed and stabilized. In addition, the fully regressed mice were cancer-free at least until publication, more than 10 months after the experiment. This conferred immunity also worked in the absence of the adaptive immune system both in transferred leukocytes (no T and B cells) and in the recipient mice. Finally, Wild Type mice, when injected with the SR/CR leukocytes, stayed immune to large doses of cancerous cells even up to 12 months after transfer, and to multiple doses.

The most pressing need for future research is in determining the exact mechanism of the innate cancer immune response, as well as characterizing its behavior, if present, in humans. Determining the nature of the genetic change in SR/CR mice would open the door for finding other similar mutations, as well as improve the chances of finding such a mutation in human immune cells. Also, finding such immunity in human cells would open the possibility for using such immune cells as a cancer treatment.

These results could have a tremendous impact upon cancer treatment in the near future. If these properties and abilities are present in Human cells, then it could be possible to cure and prevent many varied types of cancer through simple immune cell transfusions. However, as the exact mechanism is not known, and the frequency of such natural immunity is rare in mice, it may be difficult at best to begin such treatments in the near future. Despite these shortcomings, this research shows great promise toward finding a cure for cancer.

Lung Tissue Engineering Technique with Adipose Stromal Cells Improves Surgical Outcome for Pulmonary Emphysema

Lung Tissue Engineering Technique with Adipose Stromal Cells Improves Surgical Outcome for Pulmonary Emphysema
Norihisa Shigemura, Meinoshin Okumura, Shinya Mizuno, Yukiko Imanishi, Akifumi Matsuyama, Hiroyuki Shiono, Toshikazu Nakamura and Yoshiki Sawa
Link: http://ajrccm.atsjournals.org/cgi/content/abstract/174/11/1199

Lung Tissue Engineering Technique with Adipose Stromal Cells Improves Surgical Outcome for Pulmonary Emphysema is a paper studying and exploring new avenues of tissue engineering of the lung. Problems relating to lung dysfunction include the disease Emphysema. Emphysema, which can be caused by smoking, is a disease characterized by the loss of tissue elasticity which inhibits the function of alveoli, trapping air within the lung, preventing gas exchange with the environment. Symptoms include a shortness of breadth under small amounts of stress. Lung volume reduction surgery (LVRG), which is in essence removal of inefficient tissue, is the main method to correct this disease, but problems incurs in the mending of the remaining lung tissue.

This research group pursed methods of increasing the rate of tissue repair after undergoing LVRG in medical applications. This study has tracked the effects of hepatocyte growth factor (HGF), which induces lung repair and regrowth. Such growth factors are secreted in adipose tissue, specifically by adipose tissue-derived stromal cells (ASCs), which can be found in epidermal tissues. The main question under discernment is whether external cells that are introduced into the wounded area can aid in the reconstruction of the tissue. Addition of the HGF in tissue undergoing surgery can increase efficiency of the lung by increasing the rate of cell growth and stabilize regular function.

The paper described a plethora of experiments to make essential measurements of tissue repair and regrowth. Green florescent protein (GFP) was introduced to mark and label ASCs to see if cells would adhere to a scaffolding. Effects of ASCs attached to a PGAF (polyglycolic acid felt sheet) scaffold were monitored. Rats were used and induced with Emphysema by the introduction of Elastase. As a control, scaffolds without ASCs were used in one set of rats while another set contained ASCs in the scaffold. PCNA antigen introduction was utilized to count cell number. ELISA was used to monitor angiogenesis growth factors (VEGF) and HGF. Laser Doppler was also used to quantify blood flow and capillary density. Blood gas analyzers aided in the measuring of oxygen in the blood at rest. Measurements of oxygen levels were also taken from rats on a treadmill, to monitor performance of the lungs under external stress.

Results of introducing such modified were positive and as expected. ELISA produced results that showed a massive increase in the concentrations of HGF in the ASCs rich scaffolds. Furthermore, cell number also increased as a result of the introduction of stem cells as well as an increase in vascularization and blood flow from the secretion of VEGF and other angiogenic growth factors. On the treadmill experiments, oxygen levels in rats with ASCs were about double than in controlled rats. All in all, these experiments illustrated the success of this lab. Surely the hypothesis was correct and proven.

Future directions include clinical applications. While experiments were done on rats in the laboratory, human experimentations should also be conducted to determine the usefulness in humans and in the medicinal field. More so, investigations concerning age are areas of great consideration. Since lung disease can affect those of all ages, exploration in age dependence must also be traversed. Perhaps using the same techniques and fundamental concepts on other systems in the body can also be explored, and will also lead to favorable results.

This topic is relevant is important because of its implications. With high numbers of smokers world wide, it can be hypothesized that emphysema is a condition that has the potential to make a large impact on human populations. This paper validates the tissue engineering of a new solution by methods beyond just considering the localized tissue engineered zone. While similar techniques in class were used to characterize the performance, other tests concerning the entire specimen in physical endurance tests were also administerd. Thus, one should consider the overall implication of a tissue engineered device. Moreover, this article also validates the implication of tissue engineering in society. For research, it is sometimes difficult to infer the bigger picture of a work. This paper clearly illustrates the identification of a societal issue while posing a novel design for the improvement of post surgical recovery. The evidence of tissue engineering and its success is solidified.

Biocompatible Inkjet Printing Technique for Designed Seeding of Individual Living Cells

http://www.liebertonline.com/doi/pdfplus/10.1089/ten.2005.11.1658

MAKOTO NAKAMURA, M.D., Ph.D.,1 AKIKO KOBAYASHI, M.D.,
FUMIO TAKAGI, M.S., AKIHIKO WATANABE, Ph.D., YUKO HIRUMA, D.D.S., Ph.D.,
KATSUHIRO OHUCHI, M.S., YASUHIKO IWASAKI, Ph.D.,
MIKIO HORIE, Dr.Eng., IKUO MORITA, Ph.D., SETSUO TAKATANI, Dr.Eng.

Organ transplantation is the current solution to end-stage organ failures. However, the availability of suitable donors is severely limited and many patients cannot afford to wait too long for an organ transplant. While making tissue-engineered organs using biological materials is a promising solution to the shortage of organ donors, it is very difficult to recapitulate the precise spatial arrangements of different cell types and extracellular components of an actual organ. This paper investigated the feasibility of a novel TE technique called “bioprinting”. Inspired by inkjet printing technology in commercial printers, this technique can potentially allow for high-resolution seeding of living cells in an arrangement similar to biological tissues.

First, a cell printing system was constructed using an electrostatically driven inkjet head made of glass and silicon. This inkjet head is biocompatible and does not generate too much heat, which is important in keeping the cells alive as they are being ejected. Bovine endothelial cell suspensions were then used as the “ink” and printed onto PET culture disks as one-dot-thick lines. After printing, the culture disks were incubated and checked later for cell viability.

Comparing with floating and adhered endothelial cells, the authors found that the dots were printed at nearly the same size and resolution. Phase-contrast microscopy indicated that the 0-4 cells were situated inside the printing dots, depending on the concentration of the original cell suspension. Time-lapse recordings showed some of the cells could adhere onto the culture disks for proliferation, a strong indication that the cells were still alive. Finally, SEM images showed no changes in cell morphology before and after printing, indicating that no significant mechanical damages were inflicted on the cells during ink ejection.

The results led the authors to believe that inkjet printing of living cells is a feasible method for making TE organs. Besides the ability to precisely arrange cells in high resolution, there are also other potential advantages in this inkjet system. For example, the concept of color printing can easily be applied. By using growth factors, polymers, ECMs etc as “color ink”, we can simultaneously position these essential components along with the living cells. Also, inkjet printing can be operated at high speed, thus reducing the time to make an organ. The printer can also transmit digital data to a computer, allowing bioengineers to design organs virtually to fit clinical and individual needs.

There are a few issues to consider in future developments of this technique. Since living cells and other biological molecules are used, the inkjet system must be biocompatible. Heat generation during the printing process must also be minimized because most biological materials are heat-sensitive. The authors believe an electrostatically driven system used in their experiments is the most suitable for this purpose. In conclusion, biocompatible inkjet printing seems to be a great alternative to organ transplantation, even though many essential improvements such as “color printing” discussed above still need to be developed.

Antithrombogenic Property of Bone Marrow Mesenchymal Stem Cells in Nanofibrous Vascular Grafts

http://www.pnas.org/cgi/content/full/104/29/11915?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&fulltext=craig+hashi&searchid=1&FIRSTINDEX=0&resourcetype=HWCIT

Craig K. Hashi†, Yiqian Zhu‡, Guo-Yuan Yang‡, William L. Young‡, Benjamin S. Hsiao§, Karin Wang§, Benjamin Chu§,
and Song Li†¶

Nanostructured biomaterial is the main focus not only in this article but also in the field of tissue engineering. First, electrospinning is used to produce such nanofibrous scaffolds made of synthetic polymers, such as poly-L-lactic acid, which are biocompatible, biodegradable, and have configurable mechanical properties. In fact, the use of electrospinning allows the structure and morphology of electrospun scaffolds to be manipulated to resemble ECM, including porosity that allows homing and penetration of cells into the membranes for tissue regeneration. Afterwards, mesenchymal stem cells (MSC) were seeded on the nanofibrous membrane, and the graft was implanted into the common carotid artery of rats via bypass procedure. Sixty days after implantation, the remodeling of vascular grafts was examined in vivo, showing that acellular grafts had significant intimal thickening and inward remodeling in the lumen compared to the MSC-seeded grafts that showed little intimal thickening. This suggests that nanofibrous scaffold alone could not remain patent in the long run, but with MSCs, inflammatory responses induced by the scaffolds could be blocked. Furthermore, cross-sections of the vascular grafts were stained for CD31 and MHC to locate endothelial cells (EC) and smooth muscle cells (SMC), respectively, as well as for collagen and elastin. The well-organized EC and SMC distribution in and the formation of an elastic lamina layer adjacent to the lumen of MSC-seeded grafts represented that in native arteries, insinuating that cellular nanofibrous scaffolds allowed self-assembly and remodeling in vivo and that long-term patency of such vascular grafts could be achieved.

To determine the antithrombogenic property of MSCs in vivo and antiplatelet adhesion property in vitro, short-term experiments were performed to examine the lumen of acellular and cellular grafts. SEM and cross-sectional staining showed that acellular grafts had significant amounts of platelet aggregation and presence of thrombus formation on luminal surface, whereas MSC-seeded grafts exhibited very little platelet aggregation and very minimal thrombus formation on luminal surfaces. In addition, another experiment was done to reveal that heparin sulfate proteoglycans on the MSC surface plays an essential role in resisting platelet adhesion and aggregation. These results suggest that the long-term patency of MSC-seeded grafts was primarily due to the antithrombogenic property and antiplatelet adhesion property of MSCs.

The use of combining artificial nanofibrous vascular grafts and bone marrow mesenchymal stem cells is essential in the field of tissue engineering, especially in treating cardiovascular diseases since cardiovascular disease is the number one killer in the United States. By helping the body build itself a new blood vessel through tissue engineering, researchers are able to avoid problems involving replacement vein or artery harvested from another part of body, limited availability of such vessels, and most importantly, thrombosis of small synthetic vascular grafts. Through further research, it is possible to eventually develop and construct tissue-engineered vascular grafts via nanostructured biomaterials that are nonthrombogenic and have long-term patency, utilizing the potential of the antithrombogenic property of mesenchymal stem cells, and may be applied to clinical settings.

Living Autologous Heart Valves Engineered From Human Prenatally Harvested Progenitors

Do¨rthe Schmidt, MD; Anita Mol, PhD; Christian Breymann, MD; Josef Achermann, PhD;
Bernhard Odermatt, MD; Matthias Go¨ssi, PhD; Stefan Neuenschwander, PhD; Rene´ Preˆtre, MD;
Michele Genoni, MD; Gregor Zund, MD; Simon P. Hoerstrup, MD, PhD

Congenital heart valve defects is a problem that is currently only treated with surgery such as heart valve replacements, using mainly synthetic materials. Such treatment has many adverse effects such as lack of growth over the patient's lifetime, risks of thrombosis, and lifetime anticoagulation drugs needed to cope with the transplant. The authors of this paper developed a new tissue engineered heart valve using autologous cells obtained from a typical CVS (chorionic villus sampling) during preganancy.

In this study, the authors obtained the umbilical cord blood-derived EC progenitor cells from a CVS aspiration. The cells were isolated from the sampled tissue via digestion by collagenase and centrifugation/pelleting. These isolated EC progenitors were then cultured, and a separate batch were cryopreserved for later use. The scaffold for the heart valve leaflets were made from PGA (polyglycolic acid) coated with poly-4-hydroxybutyric acid. The authors fabricated the scaffold into the proper shape of the heart valve leaflets, then seeded the cells (chorionic villi) onto the scaffolds and placed the device in a perfusion bioreactor.

Two conditions were then imposed: one with straining to provide cyclic mechanical stimulation, and one without. The device was then endothelialized (to prevent platelet adhesion) by seeding with differentiated EPCs previously obtained. Characterization of the device were assays of two main types: stainng for tissue composition and mechanical testing. H&E staining showed tissue organization of the tissue engineered device to be very similar to native neonatal valve leaflets. Other types of stains have shown the expected organization of collagen with collagen fibers on the periphery layer and GAG (glucosaminoglycans) in the center of the leaflets. The authors also took SEM images of the device before and after cell seeding to confirm the endothelialization of the leaflet and look at the surface morphology inside the engineered device.

Mechanical testing of the device on a tensile tester produced stress-strain curves that show the leaflets grown in the mechanically loaded bioreactor to be stiffer (higher Young's modulus), but not as pliable as the one's that were not strained. However, the authors only made some preliminary statements and did not offer any suggestions on how to balance strength and pliabilityas to find the most appropriate way to mechanically stimulate these leaflets to best match native tissue.

This research holds much promise it that it offers a cell source for future work on pediatric tissue engineering, especially cardiovascular tissue engineering. If developed, this methods offers a minimally invasive way to harvest progenitor cells that can be used in other applications. This paper also demonstrates many of the tissue engineering challenges discussed in lecture (staining, tissue characterization) as well as some of the techniques we've been exposed to in lab (cell culture, microscopy).

CREATION OF VIABLE PULMONARY ARTERY AUTOGRAFTS THROUGH TISSUE ENGINEERING

CREATION OF VIABLE PULMONARY ARTERY AUTOGRAFTS THROUGH TISSUE ENGINEERING

Toshiharu Shinoka, MDa

Dominique Shum-Tim, MDa

Peter X. Ma, PhDb

Ronn E. Tanel, MDc

Noritaka Isogai, MDd

Robert Langer, PhDb

Joseph P. Vacanti, MD,

John E. Mayer, Jr., MDa

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMF-4HGNNXN-6&_user=4420&_coverDate=03%2F31%2F1998&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=2aaa034538142cf14bcd79da754170a5

Problems with the cardiac system are one of the most common medical problems in the United States. As a result, there is a need to find viable arterial replacements. However, current homografts and medical prophetesses can become clogged by calcification and tissue ingrowth. They also lack the ability to grow and are likely sites of infection. This paper describes a procedure this group used to pulmonary arteries by seeding cells onto biodegradable polymer scaffolds and the results they found after implanting these arteries into lambs.

They harvested cells from small sections of carotid artery or of the jugular vein in young lambs, homogenized these tissues and then used flow cytometry to sort out the epthilial cells from the fibroblasts. The epthilial cells were then seeded onto a tubular scaffold made of polyglactin woven mesh and sealed with a non-woven PGA mesh inside the lumen. This matrix was roughly 95% porous before seeding and was designed to biodegrade over 6 to 8 weeks. After the cells became confluent on the scaffolds, the TE arteries were implanted into the same lambs they had been harvested from and were allowed to grow for between 75 to 169 days before the lambs were killed and the TE arteries removed and examined.

They found that these artificial ateries show no signs of thrombus formation or calcification and that the original polymer scaffold had disappeared. All of the arteries showed an increase over their original diameter, although they still were not quite as large as the native arteries and they were somewhat thinner as well. Histological examination showed that the TE vessels resembled normal arteries in structure. Further testing revealed that they had about 70% of the normal collagen content and that they contained more calcium as well, although no overt calcification was visible.

The authors hope that this method could solve problems with immune rejection and donor scarcity for blood vessels. By employing a biodegradable scaffold, they can allow the TE vessels to grow and expand more than other implants are capable of, making them potentially useful in children with cardiac problems. These arteries can use the existing systems in the body to grow, repair and maintain themselves, giving them much better long term potential than inert implants. A major limitation right now with these blood vessels is their stiffness before implantation, when the polymer scaffold is still present.

I remember from previous BioE classes that tissue engineers have had a great deal of trouble creating artificial blood vessels that can take the strain put upon them by the pulsing pressure effects generated by the heart. The group in this article succeeded in this area and their technique offers considerable promise for clinical applications. I think they mostly need to investigate ways to reduce the amount of time it takes to prepare a device, since growing up an appropriate number of cells from the patients took several weeks in this case.

Review paper: Bioengineering Skin Using Mechanisms of Regeneration and Repair

By Anthony D. Metcalfe and Mark W. J. Ferguson
on Biomaterials doi:10.1016/j.biomaterials.2007.07.031

The paper talks about the development of bioengineered skin up to the present technology, which emphasizes on the potential skin substitutes using the mechanism of regeneration and repair. The major development of bioengineered skin started about three decades ago when scientists found out about culturing keratinocytes from skin in vitro. This finding later developed into autologous skin transplantation, such as skin grafts containing all of the epidermis and part of the dermis from undamaged area of the body to a large injured area. This method, nonetheless, is not practical when the patient has a severe large damaged skin (e.g. a burn injury). Then cadaver skin is sometimes used to cover the wound temporarily although it is limited, expensive, and questionable in safety (e.g. spreading virus and other diseases). Further, both methods are not commercial off-the-shelf products, which then lead to the researches of engineered skin substitutes.

Many companies have developed and commercialized skin replacement therapy. Their products have been improving significantly, which include collagen matrix seeded with dermal fibroblasts and keratinocytes, acellular dermal substitute from bovine collagen and shark chondroitin sulfate, and cell suspension that can be sprayed onto wound. Despite the progressive technology, many companies went bankrupt and currently have financial difficulties. One of the main reasons is the high process automation costs since the companies also need to change the current medical practice to accept the usage of tissue-engineered products. This lack of usage is thought to be related to the drawbacks of skin substitutes, which include the lack of cellular mechanisms in wound healing, the lack of normal skin composition, and scarring at the margins.

The cellular mechanisms in wound healing involve inflammation, cell proliferation and migration, cell recruitment, angiogenesis, and ECM deposition. The process involves several essential cellular components such as: (1) cytokines and growth factors which determines the cell function within the wound e.g. interleukin-1 (IL-1) that transitions inflammation to remodeling phase; (2) fibroblasts that produce ECM for tissue remodeling, scar formation and repair; (3) bone marrow-derived stem cells that produce new skin cells; and (4) fibrocytes (fibroblast-like cells) that induce angiogenesis, recruit lymphocytes, and promote cell migration into the wound. A control over these cellular components is important in skin replacement therapy. An example is the promising cell resource in recreating normal skin composition: hair follicle bulge. Researchers found that the hair follicle bulge contains multipotent skin stem cells that allow a differentiation into sebaceous glands, epidermis, and hair follicles in skin transplantation. Nonetheless, inhibition of the Wnt protein is found to inhibit this folliculogenesis, whereas the overexpression shows the opposite result. Therefore, the Wnt protein may be a key factor to manipulate the wound-induced folliculogenesis.

To resolve scarring issue, fetal wound healing is an ideal model since it engages in regenerative process that does not end in scarring. Two of major differences of fetal wound healing from adult wound healing are: (1) reduced inflammatory response and (2) more molecules involved in repair. Inflammatory response of accumulating fibrocytes uncontrollably is hypothesized to cause scarring. As to engineer skin substitutes for a scar-free healing, the manipulation can be done in depositing ECM molecules as seen in embryonic wound healing.

In addition to resolving the three issues in skin substitutes, the medical interest in skin repair is mostly focusing on repair and regeneration mechanism. The regeneration process can be derived from either undifferentiated stem cells or existing tissues. Researchers have modeled mammalian wound repair and regeneration by punching the ears of three strains of mice (a crude thumb punch and a clinical biopsy punch). The MRL/MpJ mice healed faster than the others with more blastema (undifferentiated cells) formation and thickened tip epithelium. However, the regeneration only occurs when the punch is on the ear because its small dimensions with epithelium covering on both sides allow the diffusion of growth factors in a gradient similar to fetal wound healing. Transforming growth factor-b (TGF-b) is the major growth factor in wound healing. The three isoforms in mammals are expressed differently in embryo and adult wound healing. Embryonic wounds express high TGF-b3 and low TGF-b1 and TGF-b2; where adult wounds express high TGF-b1 and TGF-b2. Further studies found that neutralization of TGF-b1 and TGF-b2 or addition of TGF-b3 results in improved scarring repair or scar-free healing. Other growth factors that could be incorporated into a skin substitute are vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1). VEGF promotes angiogenesis of short, narrow, and highly branched (sprouting) vessels with normal pericyte coverage, where Ang-1 induces broader, longer vessels without increase in branching or sprouting, but with higher pericyte coverage. Combination of VEGF and Ang-1 generates larger, less branched vessels and more developed microvessels, which have a better functionality.

Another big emphasis in skin engineering is placed on the biomatrices design. The artificial ECM has been varied from naturally occurring materials including glucosaminoglycans, fibronectin, collagen, hyaluronan, and alginates, and synthetic materials including PGA, PLA, PLG, PCL, and PET. Ideal biomatrices have the characteristics of (1) non-toxic and non-immunogenic upon implantation, (2) biodegradable by proteases, (3) adherence of cells, and (4) competent in mechanical properties with the ECM in normal skin. The design of biomatrices has developed up to the design of scaffolding that has defined shapes with microporous for directing tissue growth up to when the host cells can reoccupy and resynthesize ECM. The matrix structure can be modified by varying solid content or gelation conditions, or by adding cross-linkers. Specific pore size can also be controlled using the new techniques of 3D printing and electrospinning. Synthetic materials have been shown to have a major disadvantage: the lack of cell-recognition signals. An important signal that can be lost is mechanotransduction signal (controlling cell phenotype) which is transmitted via cell adhesion to the matrix. Adding RGD sequences (Arg-Gly-Asp) is found to promote cell adhesion and migration. Latest development of biomatrices has reached to the synthetic biopolymer technology: hydrogels.

Hydrogels has a homogeneous, nanoscale microstructure that restricts cell migration to proteolytic remodeling. When it is based on polyethylene glycol (PEG) under a specific reaction, the hydrogels can be formed from aqueous precursors in the presence of cells. Therefore, important ligands can be added, for example the RGD-containing peptides for cell adhesion and protease-sensitive oligopeptides for MMP degradation. Moreover, PEG acts as an inert due to its hydrophilicity and resistance to protein adsorption so that it presents biological signals only from the attached peptides. In an experiment using the hydrogel in cell mixing and implantation to regenerate hair follicles, the researchers found that PEG hydrogels are fluid enough for cell reorganization and regeneration. Hydrogels have also been developed on fibronectin functional domain (FNfd’s) coupled to a Hyaluronan (HA) backbone. It is shown to be compatible for growing human dermal fibroblasts. Overall, hydrogels with combinations of proteins such as TGF-b, MMP, and VEGF lead to a more regenerative and anti-scarring wound healing.

Lastly, the area in skin substitutes that needs to be more focused on is biomechanics. A skin substitute needs to have appropriate mechanical properties (strength, elasticity, etc.) and handling properties (can be manipulated). Techniques to analyze the mechanical properties include torsional analysis, cutometry (elasticity), tensiometry (tension), gas-bearing electrodynamomtery, and indentometry (biological agents). Past study has shown that excessive tension by cells in biomaterials inhibits angiogenesis in fibronectin fibrils, which may explain the failure of skin substitutes to vascularize. On the other hand, angiogenesis is shown to be promoted in human umbilical vein endothelial cells (HUVEC) in vitro on a fibrin-based matrix covalently bonded with ligand L1Ig6.

Overall, despite the difficulties in meeting the clinical and commercial expectations, skin replacement therapy has developed significantly and benefited to patients. Continued researches will overcome challenges and may lead to advanced regenerative tissue-engineered replacement therapies.

I choose this paper because skin substitutes is always an interesting subject and currently a growing area in bioengineering. Thus, this review paper may update our knowledge regarding bioengineered skin, and perhaps may interest some of you to specifically study more about this.

Force Mapping in Epithelial Cell Migration

Olivia du Roure, Alexandre Saez, Axel Buguin, Robert H. Austin, Philippe Chavrier, Pascal Silberzan, and Benoit Ladoux

Supporting Figure 7: http://www.pnas.org/cgi/data/0408482102/DC1/1

Supporting Figure 8: http://www.pnas.org/cgi/data/0408482102/DC1/2

Supporting Movie: http://www.pnas.org/content/vol0/issue2005/images/data/0408482102/DC1/08482Movie1.mpg

Mechanotransduction plays a role in a number of cell processes. As such, it is important to gain a better understanding of the forces and stresses that a cell exerts on its substrate. To quantify the magnitude of these forces, an array of PDMS micro-posts (1-2 μm in diameter) were fabricated and cells were cultured on the array. Each post acts as a linear spring where the deflection of the top of the post is directly related to the force acting on the top of the post. Thus, by monitoring the deflection of each post, the corresponding traction forces and stresses can be calculated.

Prior experiments using an array of microposts to detect cell traction forces limited some cell functions. To combat this issue, the posts were spaced far closer together (2 μm to 4 μm center to center). As a result of this increased post density, cell adhesion, locomotion and proliferation were similar to the behavior of cells on regular 2D substrates. Thus, cell forces generated during these processes could be studied.

For single cells, the greatest traction forces occur around the edges of cells with far less mechanical activity underneath the nucleus and middle of the cell. While the average traction stress transmitted by single cells was approximately .6 nN/μm², the maximal stress was much larger with a magnitude of 3.8 ± .1 nN/μm². For a cell monolayer, the largest forces were found at the edge of the monolayer, oriented normally to the edge in the direction opposite of advancement. Furthermore, larger stresses occurred at cell-cell interactions in the monolayer while lower stresses developed underneath cell nuclei.

A number of prior methods for quantifying cell traction forces are limited in their ability to accurately estimate the magnitude and location of these forces. Meanwhile, prior post-arrays limited and affected cell behavior, distorting the information extracted. The importance of this research is twofold: one, the ability of cells to behave normally on an array of posts is demonstrated and two, cell traction forces and stresses were quantified more accurately during cell processes due to the localization of forces on singular posts. Accurately quantifying the magnitude of traction stresses during cell processes is integral to many applications in tissue engineering.