Reduced contraction of skin equivalent engineered using cell sheets cultured in 3D matrices

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The cell sheets that have been tissue-engineered in the past few years have had poor mechanical properties. Because of the fragility of cell sheets, external supports such as stainless steel rings and non-degradable polymeric membranes have to be used. In this experiment, they used two different types of 3D matrices with cultured fibroblast sheets to form a possible tissue engineered skin replacement that have the mechanical properties more similar to native skin. These would maintain their regenerative capacity while also showing reduced wound contraction when transplanted in vivo. The two 3D matrices they used were a weft-knitted poly (lacted-co-glycolic acid) mesh (PLGA), which has mechanical properties similar to native skin while also supporting dermal fibroblast attachment and proliferation, and a collagen sponge crosslinked with hyaluronic acid (CHA), which has greater resistance to enzymatic degredation along with reduced swelling.

The drawback in using cell sheets is that the poor mechanical properties results in difficulty in handling the sheets along with extensive contraction . The results of the expirement showed that the weft-knitted PLGA mesh was better than the CHA sponge because it provided high flexibility that was mechanically stable as a graft. The PLGA-cell sheet showed contraction comparable to autografts. These PLGA-cell sheets contracted less compared to using PLGA alone, showing a synergistic effect between the PLGA mesh and the cell sheets in preventing contraction in a wound, which would lead to less scar formation. The CHA matrix didn’t show great results in vivo because of its rigidity, which prevented it from adapting to the movement of the animals that resulted in wound healing under the matrices instead of over the matricies.

I chose this paper because our project is on TE skin. Although we won’t have a chance to experiment much using different types of 3D matrices like in this experiment, I thought it was a good reminder for us to think of design principles for our devices that can be used clinically in the real world. Even if we were to eventually create a skin sheet that showed nearly all the biological and chemical properties of natural skin tissue, putting the device into a patient for them to actually use is another huge consideration. If the device can’t be placed in a practical and viable way, then it is pretty much useless. It was also interesting to see the thought process of the entire experiment because they used a wide range of laboratory techniques, most of which we learned throughout the semester (cell culture, mechanical properties, microscopy, antibodies, etc.).

Cell-cell Interactions Are Essential for Maintenance of Hepatocyte Function in Collagen Gel But Not on Matrigel

Prabhas V. Moghe, Robin N. Coger, Mehmet Toner, Martin L. Yarmush

Full text at: http://www3.interscience.wiley.com/cgi-bin/fulltext/71004078/PDFSTART?CRETRY=1&SRETRY=0

Given the potential pharmacological, toxicological, and therapeutic potential, hepatocytes are often cultured on collagen as a monolayer and or induced three-dimensional aggregates. The study compared the importance of cellular aggregation and seeding density in two different hepatocyte culture systems, the two-dimensional monolayer in a collagen sandwiches and three-dimensional aggregates induced by the mouse sarcoma derived Matrigels. In order to determine the effectiveness of each of these systems in retaining hepatocyte function, the culture medium was measured for rat serum albumin content by enzyme-linked immunosorbant assay (ELISA) with purified rat albumin and peroxide-conjugated antibody and DNA content analysis.

Upon culturing, the hepatocytes cultured in the collagen sandwich after 10 days appeared more flattened and exhibited bright cell-cell contacts compared to the hepatocytes cultured in the Matrigel, where they appeared round and formed comparatively large three-dimensional aggregates. Albumin secretion were obtained each day for five different seeding densities (1.75, 3.5, 7.0, 17.5, and 70.0 x103 cells/cm2) and normalized to the amount of DNA present within each culture as obtained by the DNA content analysis. With high cell seeding densities the, both cultures exhibit similar levels in elevated function. However, as the cell seeding densities decreases, the collagen sandwich cultures exhibited a rapid deterioration in albumin secretion while the Matrigel cultures maintained similar levels of albumin secretion.

The decreasing secretion levels in the collagen sandwich and the maintenance of secretion levels in the Matrigel may be associated with the differences between the cell-cell and cell-matrix contact levels in the two cultures. As a result of the different morphologies that the collagen sandwich and the Matrigel induce, cell-cell contact and cell-matrix contact varies differently between the two systems. In both systems, the cell-cell interaction parameter decreases with cell decreasing cell density. The cell-matrix interaction parameter, however, remain relatively unchanged in the collagen sandwich as cell density decreases whereas the cell-matrix interaction parameter increases in the Matrigel as the cell density decreases. Thus, the paper concludes that in collagen sandwich systems, the maintenance of hepatocyte function corresponds to the amount of cell-cell contact whereas hepatocyte function is maintained from matrix derived cues.

I chose this paper as, in the liver project, we are also testing the maintenance of hepatocyte function in a new matrix, agerose. However, we will be measuring albumin secretion rates using western blots and determining the number of viable cells after one week of culture using hemocytometer counts. This paper might provide insight into how cellular aggregation, seeding density, and the resulting cell morphology might affect rates of albumin secretion and hepatocyte functional maintenance in different culturing systems.

The application of new biosynthetic artificial skin for long-term temporary wound coverage

Full text article:
visit: http;//www.sciencediret.com
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Skin acts as a barrier to keep antigens out of the immune system, and to keep bodily fluids in. Open wounds where the epidermal and dermal layers are absent are prone to infection and need to have the skin repaired immediately to restore homeostasis. The most ideal way to do this is for an autologous skin graft because immune rejection would not be a factor. However this is not always ideal if the wound is large. The authors of the paper investigate a new method of healing skin wounds with engineered artificial skin that will either 1) cover the wound long enough for the wound to heal naturally or 2) cover the wound long enough until an autologous skin culture can be grown to an amount that will suffice in a permanent skin graft.

The artificial skin consists of a thin layer of silicone fused with acellular porcine dermis (APD), and therefore sufficiently named silicone acellular porcine dermis (SAPD).
A 4cm x 5cm skin wound was delivered onto each back of 36 rats; half of them received SAPD treatment over the entire wound and the other half received Biobrane (a manufactured biosynthetic skin substitute that also contains silicone fused to dermal porcine substance).

The wounds were analyzed weekly over a 6-week period using pathological tests. The Biobrane group exhibited wound contraction and digestion of the dermal layer after the 3rd week of grafting. Before the dermal layer was digested, its porcine dermis component faced immunorejection and caused wound inflammation. On the contrary the SAPD group exhibited no wound contraction and after the 6th week, SAPD maintained its well-organized porcine dermis, thereby creating a dermal template that allowed the containing procine collagen to incorporate into the recipient’s wound. No immunorejection was present with minimal inflammation.

Although SAPD proves to be the better artificial skin graft in the observations, one of the major drawbacks is its time dependency. After the 6th week of grafting, the silicone layer separates from the APD, causing dermal layer exposure and infection. As long as the wound can heal or a skin culture can be grown within 6 weeks, SAPD is a sufficient skin graft.

This article was my pick of the liter because its subject matter was somewhat relevant to my project of tissue-engineered skin. The paper provided little insight on methods since our materials vary from those found in the paper, but it was helpful to learn that the dermal layer was the important factor in a successful skin graft – the epidermal layer (in this case silicone) was to provide protection and was not crucial in the material-recipient incorporation. Also it was interesting to know that there is still much headway that can be made with artificial skin despite it being the first tissue organ ever to be engineered.

Physiologicallly relevant increase in temperature causes an increase in intestinal epithelial tight junction permeability

Karol Dokladny, Pope L. Moseley and Thomas Y. Ma
Am J Physiol Gastrointest Liver Physiol 290: 204-212,2006

http://ajpgi.physiology.org/cgi/content/full/290/2/G204

This paper discusses the impact of physiologically relevant increase in temperature (37 -41 degrees in Celsius) on epithelial tight junctions as well as the role of heat-shock proteins (HSPs) in regulating this heat stress. The study was done using a cell line known as caco-2 cells. Non-leaky epithelial tight junction barrier is "crucial in providing barrier function against epithelial penetration of pathogenic bacteria and toxic luminal antigens like endotoxins." As indicated by the study, the disruption of the tight junction barrier results in a "leaky" barrier that allows paracellular permeation of toxic luminal substances. The experimental data presented in the paper show that HSPs play an important protective role in preventing heat-induced disruption of the tight junction.

Western blot analysis was used to assess the HSP, occludin, ZO-1, and beta-actin protein that were expressed just as we did in lab. The researchers mixed the clear lysate derived from a centrifuged cell lysate with a Laemmli gel buffer, boiled for 7 minutes, and lated separated using SDS-PAGE gel. The protein from the gel was transfered to Nitrocellulose membrane overnight.

This paper was very interesting to me because it serves as a cautions to me as prospective tissue engineer to be mindful of little things that might end up ruining my engineered tissue. I was also intrigued by its mention as well as usage of certain techniques that I have learnt in class.

Autologous cartilage implantation (ACI) is a new medical alternative that especially benefits for patients who have a damaged cartilage. The goal of ACI is to provide the patient with a durable, load bearing functional joint surface by the repair of articular carticular with both biomechanical and histologic characteristics comparable to normal articular cartilage. There are two stages involved in this ACI procedure. First, some health cartilage cells are collected from the patient’s knee. Then, these biopsytissue are sent to a special laboratory to grow for weeks and transplant back to the patient’s damaged region for new cartilage regeneration. One of the advantages of ACI is the usage of the patient’s own cells could reduce the chances of infection. Besides, ACI has the ability to restore hyaline-like articular cartilage.

ACI is an advanced, cell-based orthobiological technology; however the quality of this treatment is still not very well determined. In this article, “Autologous Chondrocyte Implantation: Superior Biologic Properties of Hyaline Cartilage Repairs,” by Henderson I, Lavigne P, Valenzuela H, and Oakes B. (could be downloaded from https://webfiles.berkeley.edu/wincheung/shared/ ), the properties and qualities of ACI were tested and analyzed. The authors focused on three points. First, the different effects resulted from cartilage repairs of different tissue types in terms of stiffness and outcome. Second, the correlation between the anatomic locations and the properties of the repairs. Third, the correlation between the presence of an abnormal macroscopic appearance at arthroscopy and its biomechanical or histologic properties. The results were based on two standard scoring systems. The International Cartilage Repair Society (ICRS) visual scoring system and the International Knee Documentation Committee (IKDC) score, which evaluate the symptoms, activity level, and health-related quality of life measures.

There were two limitations in this study. First, among the symptomatic population, which the data were derived from, they had additional abnormalities unrelated to the ACI repair. This made the results obtained from them couldn’t easily be extrapolated to the asymptomatic population with ACI. Second, the limitation to study the potential relation between the repair’s biologic properties and the clinical outcome due to the indentometry had to base on proper instrument positioning and also the repair thickness, which could possibly cause inaccurate measurements. Although the histologic evaluation was based on a 2mm thick tissue sample, this couldn’t represent the total composition of the repair tissue, it is more adequate to test for reliable, reproducible, and repeatable results.

It was found at the average of 26.5 months after implantation, the repair was stiffer than normal cartilage. The greatest stiffness was recorded in the following orders: the hyaline articular cartilage, hyaline-like, fibrocartilage, hyaline-like/fibrocartilage group. Besides, there were no difference in the histologic quality resulted from the difference anatomic location of the repair. Regarding of the third focus of the study, the relationship between the presence of an abnormal macroscopic appearance at arthroscopy and its biomechanical or histologic properties, the ICRS and IKDC scores were inconsistent. Based on the ICRS scores, the patients who were macroscopically normal had greater maximum and normalized stiffness than patients who were macroscopically abnormal. On the other hand, the patients with or without abnormal macroscopic appearance scored no difference in IKDC scores. The IKDC scores increased with time in patients in both cases, hyaline cartilage and fibrocartilage. These scores indicated patients did well regardless of the quality of the repair at 3 years postoperatively because these scores were comparable to scores of asymptomatic patients in the ACI cohort. 24 months after ACI, the overall stiffness and macroscopic appearance of hyaline articular cartilage and hyaline-like repairs were similar to normal articular cartilage.

My grandma has inspired me to have a special interest in the field of tissue engineering, and be more specific – cartilage, because of her knee pain. Her cartilage is wearing out as a result of aging, and by the time I heard this ACI repair treatment, I thought it might bring hope to save my grandma from pain. Treating ACI as a possible treatment for my grandma, I want to know more about ACI, and that’s why this article was especially attractive to me because it fulfilled my curiosity about the quality of ACI repair. Besides, by knowing these mechanical and histologic properties of current ACI, we can make improvement and hence one step closer to the durable cartilage repair.

Yee-Wan (Win) Cheung

Viscoelastic Testing Methodologies for Tissue Engineered Blood Vessels
Joseph Berglund, Robert Nerem and Athanassios Sambanis
Journal of Biomech Engineering 2005 Vol 27

http://scitation.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBENDY000127000007001176000001&idtype=cvips&gifs=yes


Matching the complex mechanical behavior of native blood vessels is one of several obstacles in creating a suitable tissue engineered blood vessel. Many of the TE vessel mechanical testing methodologies have used single-point burst pressures measurements and uniaxial tensile testing as an indication of mechanical suitability. However,since blood flow is a pulsatile behavior, and native blood vessels exhibit a time-varying behavior, other methods are needed to correctly characterize the mechanical properties of TE vessels. In addition to performing uniaxial tensile testing, this paper reports the use of viscoelastic characterization methods to model the time-dependent behavior of TE and native vessels.

TE vessels were made from rat collagen (2 mg/ml) and human dermal fibroblast (1 x 106 cells/ml). TE vessels were made to form a tubular structure with a 3mm inside diameter. Some TE vessels were combined with acellular support sleeves made from untreated and glutaraldehyde crosslinked collagen. Common carotid arteries were taken from 1 to 2 year old pigs.

Uniaxial tensile testing was used to measure overall strength (ultimate tensile strength). Stepwise stress relaxation and creep testing was performed (for more details see paper link). Mathematical modeling, using 3 and 4 parameter constitutive, models was used to characterize stress relaxation and creep data.

This paper also reports geometrical changes for all samples after 8 and 23 days in culture. Interestingly, this study found that the addition of acellular support sleeves had minimal effect on gel compaction. However, a difference in mechanical properties was observed amoung the samples. The Glutaraldehyde treated vessel constructs exhibited the highest burst strength but were still inferior to the native arteries. The untreated vessel constructs seemed to match the overall behavior of the native vessels the best.

I chose this article because it provides a framework for characterizing the mechanical suitability of TE vessels. It also illustrates the use of mathematical models which is something of interest to me.

Chondrocyte transplantation for osteochondral defects with the use of suspension culture

Toshihiro Izumi, Toshiyuki Tominaga, Junichi Shida1, Futoshi Onishi & Moritoshi Itoman Department of Orthopaedic Surgery, Kitasato University School of Medicine,
1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan

http://www.springerlink.com/content/g523121861680452/fulltext.pdf

Cartilage graft is a common technique applied in repairing chondral or osteochondral defects. One way of performing cartilage graft is by autologous chondrocytes transplantation following cell culture. However, one of the potential problems of this method is that chondrocytes may change their phenotype during monolayer culture. To solve this problem, the experiment presented in this research paper hypothesized that chondrocytes cultured over agarose (suspension culture) as a source of graft material can retain the expression of type II collagen and glycosaminoglycans, and the expression of these two products for chondrocytes cultured in a plastic glass (2D culture) is also measured as a control.

105 cells/cm2 of rat chondrocytes are isolated from coast cartilage and are dispersed in Ham’s F-12 medium supplemented with 0.2 mg/ml bovine serum albumin, 50 mg/ml ascorbic acid, antibiotics–antimycotic, 10% fetal bovine serum ,and 25mM HEPES, pH 7.4. The isolated cells were collected, and seeded on six well plate either uncoated or precoated with 1.5% agarose. Chondrocytes cultured in uncoated dishes represented the monolayer culture, while chondrocytes in coated dishes represented suspension culture. Cultures were then maintained in a humidified atmosphere consisting of 95% air and 5% CO2 at 37 oC. Cells were cultured for two weeks, and medium was changed twice a week. After 14 days in culture, the expression of glycosaminoglycans is examined by a histochemical test in which the chondrocytes are stained with safranin O (a biological stain used in histology). On the other hand, the expression of type II collagen is examined first by an acid guanidinehiocyanate–phenol–chloroform method for RNA extraction and following by northern analysis. The cultured chondrocytes are then transplanted to a rat with osteochondral defects.

The result of this study shows that chondrocyte phenotype was maintained in suspension culture over agarose, as assessed by northern analysis and histochemistry compared with chondrocyte monolayer culture. Moreover, this study shows that suspension culture chondrocyte grafts in femoral osteochondral defect also resulted in hyaline cartilage formation compared with controls in this study. Surface of the condylar defect was smooth, and the matrix produced in the defect contained glycosaminoglycans.

The reasons I choose this paper are because it is related to our project and may provide some clues to accomplish the objects in our experiment. Instead of concerning the mechanical properties of the chondrocytes / agrose cell culture, measuring the expression of collagen II of our chondrocytes after one week of culture is also the other big challenge in this project. This problem can get complicated as we do not know which portion of the collagen is from the calf serum and which portion is from the cells. Therefore, solely running the sample and a negative control in western blot cannot tell us whether the cells are expressing collagen II or not. The application of northern blot used in this paper suggests a possible way to deal with this problem since the mRNA of collagen II would only come from the chondrocytes. Nevertheless, we still have to come up with some ways to quantify the protein.

Engineering Vascularized Skeletal Muscle Tissue
Nature Biotechnology 23, 879 - 884 (2005)
Shulamit Levenberg, Jeroen Rouwkema, Mara Macdonald, Evan S Garfein, Daniel S Kohane, Diane C Darland, Robert Marini, Clemens A van Blitterswijk, Richard C Mulligan, Patricia A D'Amore & Robert Langer
Full Text: http://www.nature.com/nbt/journal/v23/n7/full/nbt1109.html

Vascularization is a critical factor in the viability of bioenegineered tissue. In relatively thin tissues with low metabolic activity such as skin, little vascularization is required. Yet in thicker tissues that require a large supply of nutrients and removal of waste, vascularization has been a critical road block in achieving clinical viability.

In most tissue engineering transplantations, vascularization occurs post implant via the host and through the addition of growth factors. Unfortunately this technique has not been successful for large, thick tissues. The other option for tissuevascularization is to begin the process prior to implantation. In the paper, "Engineering vascularized skeletal muscle tissue," Levenberg etal. were able to produce prevascularization prior to implantation and viability post implantation in an in-vivo model.

The first step in the experiment by Leavenberg was creating a co-culture of muscle and endothelial cells. When co-cultured and analyzed over time endothelial tubes formed around the elongated muscle cells showing the beginings of vascularization. This concept was then extended by culturing three different cells together; skeletal muscles, endothelial cells, and fibroblasts. The fibroblasts were added due to their ability to differentiate into smooth muscle cells, which would act to stabilize the endothelial tubes. This tri-culture showed even greater stabilization then the co-culture with the presence of smooth muscle cells and also the increased production of angiogenic growth factors such as VEGF. Having shown proof of concept in-vitro the tri-cultured tissues were then implanted into mice.

In the mice three different sites received tissue, one of which was abdominal muscle. The implant showed evidence of viability, and very interestingly, it appeared that the new muscle appeared toalign with the fibers of the host tissue. This revealed the possibility of both functionality and viability in the implant.

The study by Levenberg etal. was one of the first to show the ability to create prevascularization in thick complex tissues. The results of this research creates great possibilities for advancement of tissue engineering in such complex metabolically active tissues as liver or brain. In addition, the techniques developed could be used for studyingmulticellular processes, such as angiogenesis in-vitro.

Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision
Kwok-Fai So, and Gerald E. Schneider
Rutledge G. Ellis-Behnke, Yu-Xiang Liang, Si-Wei You, David K. C. Tay, Shuguang Zhang,
doi:10.1073/pnas.0600559103
PNAS 2006;103;5054-5059; originally published online Mar 20, 2006;
http://www.pnas.org/cgi/content/full/103/13/5054

This paper discusses the regeneration of the optic nerve by aiding neuron reconnection on a nanometer scale scaffold created from amino acids. The experiment consisted of severing optic nerves in hamsters and then patching them with a self assembling peptide nanofiber scaffold (SAPNS). The reason for choosing such a scaffold is motivated by the inert properties of amino acids in the cellular environment. This scaffold also provides nutrients for growing cells and aids in the healing of the wound.

This article suggests also that nerves can be reattached using cell grafts from nerves in the legs of the animal. This is not as desirable as the SAPNS method as legs are often damaged by such a process. Tests were done after 30, 45 and 90 days and healing evidence was found. Imaging with an SEM and optical microscopes of the wound area also showed the SAPNS aiding in the healing of the nerve cells.

I chose this article because I think it is interesting. Scaffolds for engineered cells are almost as important as the growth of the cells. A biologically and chemically inert scaffold that functions on the nanometer scale is something that could benefit many tissue engineered devices at a variety of size scales.

I am skeptical about the results of the paper. The movies that show the hamster reactions are hard to quantify, and the possibility of another means of neuron regeneration was largely disregarded or not even mentioned to be as likely as the SAPNS aiding growth method. However, the paper does detail the experiment well and discusses the insertion of the scaffold into the wound and the various controls they used to eliminate random hamster visual artifacts. There is also visual evidence of the growth.

Compressive Strains at Physiological Frequencies Influence the Metabolism of Chondrocytes Seeded in Agarose

David A. Lee and Dan L. Bader

Full text link: http://www3.interscience.wiley.com/cgi-bin/fulltext/109929044/PDFSTART

The experiment presented in the article studies the universality of mechanotransduction pathways for chondrocyte metabolism under the application of static and dynamic mechanical strains. Specifically, the experiment aims to characterize whether chondrocyte deformation under mechanical strain has the capability of stimulating three major markers of chondrocyte metabolism: proteoglycan synthesis, cell division, and protein synthesis.

In the experiment, static and dynamic mechanical strains were applied on agarose-chondrocyte cylinders using a specially designed cell straining apparatus. The level of gross compressive strain used was 15%, which lies in the middle of the 0-30% physiological range for cell strain in intact cartilage subjected to physiological loads. For dynamic loading, the frequencies studied were 0.3 Hz, 1 Hz, and 3 Hz – all three were within the physiological range. The agarose-chondrocyte cylinders were prepared from chondrocytes removed from the metacarpophalangeal joints of 18-month-old steers, embedded in 3% agarose, and cultured under strain for 48 hours in DMEM with 20% fetal calf serum. Unstrained agarose-chondrocyte cylinders were used for control.

Chondrocyte viability was determined for each of the experimental group using trypan blue assay and the results showed that viability ranged from 97.8 to 99.1% of the time-zero viability. Glycosaminoglycan (GAG) synthesis by chondrocytes embedded in agarose was determined using a rapid spectrophotometric procedure that involved papain digestion of tissue or culture medium that provides glycosaminoglycans for assay. The results, normalized to the control cultures, indicated a significant reduction in glycosaminoglycan synthesis for static strain and 0.3 Hz dynamic strain, but a stimulation of glycosaminoglycan synthesis to 40% more than the unstrained control for dynamic strain at 1 Hz. Dynamic strain at 3 Hz did not significantly alter glycosaminoglycan synthesis. Incorporation of [³H]thymidine was analyzed as a marker for cell division and the results showed that its uptake was inhibited in cylinders subjected to static strain as compared to static strain, but was stimulated in cylinders subjected to dynamic strain at all three frequencies. Finally, incorporation of [³H]proline was analyzed as a marker for protein synthesis. The results showed that when compared to unstrained control, [³H]proline uptake was inhibited in cylinders subjected to both static and dynamic strain at all frequencies investigated. Because the three parameters investigated in the study were each influenced by the strain regimens in a distinct manner, it was concluded that that the mechanotransduction pathways involved were likely to be uncoupled.

I chose this article because I feel that this study is particularly important for the development and design of cell-seeded cartilage repair systems for cartilage diseases such as arthritis. As a matter of fact, a balance between cartilage matrix synthesis and degradation is critical both for the maintenance of articular cartilage function and skeletal development and growth. Because the focus of this study is on the mechanical aspects that can alter this balance, it is thus able to provide a general understanding of chondrocyte regulation in cartilage when subjected to various types of compressive strains. Additionally, the results from the study can give insights on how a tissue engineer can design an experiment that closely mimics human physiological conditions. The study presented made this attempt by subjecting the chondrocyte cell culture to dynamic loading at various frequencies so that it is more similar to normal walking. This way, more applicable results can be drawn from the experiment that can be used to generalize the effects of mechanotransduction on articular cartilage under physiological loading in human joints.

Reduced Contraction of Skin Equivalent Engineered using Cell Sheets Cultured in 3D Matrices

Full Text available at following link:
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TWB-4K18VTF-2&_coverDate=09%2F30%2F2006&_alid=481170456&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5558&_sort=d&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=36f4a4e1bea9fa127e704ef716f0eb78

The main objective of the experiment described by this paper was to improve the mechanical properties of cell sheets used to generate tissue engineered dermal equivalents. Cell sheets have been used for a variety of tissue engineering purposes that range from artificial blood vessels to myocardial patches and artificial skin. While they have the advantage that the cells on the sheet can be used to engineer a natural neo-tissue, a major shortcoming of cell sheets is that they contract when removed from culture surfaces, causing graft sizes to be reduced. Cell sheets are also very fragile and difficult to handle. This paper describes a technique for resolving these shortcomings in which human fibroblasts sheets are cultured in combination with three-dimensional matrices to form a contiguous dermal construct that does not contract over time.

The group in this study hypothesized that fibroblast sheets grown in conjunction with three-dimensional matrices will maintain their regenerative capacity and result in reduced wound contraction. To test their hypothesis they used their technique with two kinds of 3D matrices: a PLGA scaffold, and a collagen sponge cross linked with hyaluronic acid (CHA). I won’t go into to much of the details of their materials and methods, but I will note that the 3D matrices and cell sheets were fabricated separately, at which point cell sheets were peeled off the Petri dishes on which they were grown and then folded over the matrices to from the 3D dermal equivalent. Human keratinocytes were then seeded on top of each dermal equivalent and allowed to culture for seven days. The constructs were then either raised in an air-water interface for four weeks to induce cell stratification, or they were transplanted onto full-thickness wounds in nude rats for four weeks.

In the end, both PLGA and HCA constructs to varying degrees came to resemble human skin with mechanical properties far more stable than those inherent to cell sheets alone. Each possessed a stratified keratinocyte layer that resembled native epidermis. In addition, cell migration of the fibroblasts from the sheet into the 3D scaffold was observed for both PLGA and HCA, creating a 3D matrix of fibroblasts resembling that of the dermis. However, following transplantation, it was found that the CHA sponges sloughed off the wound within two weeks. The PLGA scaffolds, in contrast, took after 1 week post-transplantation and registered a take rate of 100%. Wounds re-epithelialized 3 weeks after transplantation. This demonstrates both the successes and failures of the experiment. While both constructs exhibited properties that mimicked those of human skin, in the end it was found that the CHA matrix did not take due to its higher than normal rigidity, which prevented it from conforming to changing wound topography as the animals moved.

I chose to post this paper because of the similarity of its objectives with those of our class project as applied to tissue engineered skin, in addition to the fact that many of the methods that were used to characterize their 3D constructs were similar to or the same as the techniques that we have learned in this course. Like our project, the objective of the experiment described by this paper was to create a 3-dimmensional skin substitute that is easy to work with and has mechanical properties comparable to those of the dermis. To accomplish this, they had to characterize their construct by observing the cells using microscopy and ensure that the fibroblasts were correctly distributed in their 3D matrix. In addition, like we have done in class, they used immunostaining techniques to verify that their cells were continuing to express their proteins of interest and generating a matrix that resembled that of human skin.

Human Tissue Engineered Blood Vessel For Adult Arterial Revascularization

Full text available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1513140

Abstract available at (PubMed link):

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16491087&query_hl=9&itool=pubmed_docsum

I chose this article for two main reasons. First of all, I believe that it would be helpful to other Bioengineering 115 students whose projects focus on tissue-engineered blood vessels. Besides that I think this article is a very comprehensive one. It thoroughly describes the technique for preparing and testing one particular type of the TEBV (tissue-engineered blood vessel).

In the experiment described human skin fibroblast cells were isolated from bypass patients and then cultured into sheets. Complex media supplemented with bovine serum, glutamine, penicillin, streptomycin and sodium ascorbate was used to feed the culture. As in natural blood vessels, three distinct cell layers (living adventitia, decellularized internal membrane and endothelium) were formed in the TEBV. Temporary Teflon®-coated stainless steel tube was used as a mechanical support during the formation of the first two layers (adventitia and IM (internal membrane)) and was removed later. When the vessel was ready, the natural conditions were simulated by subjecting the TEBV to the pulsating stream with the flow rate varying from 3ml/min to 150ml/min.

Mechanical testing was conducted to estimate (1) the burst pressure and (2) the suture retention strength of the vessels. Compliance was calculated based on the change in vessel diameter in response to increasing pressure (high resolution digital imaging was used to measure the vessel diameter).

The final phase of the experiment involved canine, nude rats and primate studies. In all trials immunosuppressive drugs were administered prior and following the introduction of the TEBV into the body. Tissue fixation and histology at different time periods were used to examine the condition of the newly incorporated blood vessels. The analysis revealed successful tissue integration, vasa vasorum formation and cell accumulation on the luminal side of the IM. Furthermore, the shape and size of the TEBV was approximately unchanged and proteoglycan expression was suppressed. This is indicative of the relative success of the preclinical trials.

In the end, authors suggest that if clinical trials of the TEBV prove successful further research should focus on decreasing time for the vessel production and other applications of allogeneic TEBVs.

Biochemical and Functional Changes of Rat Liver Spheroids During Spheroid Formation and Maintenance in Culture: I. Morphological Maturation and Kinetic Changes of Energy Metabolism, Albumin Synthesis, and Activities of Some Enzymes

Mingwen Ma, Jinsheng Xu, and Wendy M. Purcell
http://www3.interscience.wiley.com/cgi-bin/abstract/106563224/ABSTRACT?CRETRY=1&SRETRY=0

There have been many studies in the past observing how liver cells function when cultured on monolayers. Cells that aggregate into spheroids function much longer and superior (liver-like) to those cultured as monolayers in a dish. Spheroids are gobular formation of cells that form a three-dimensional, multicellular aggregate. Such a formation is favored for the cells will maintain cell-cell contact and maintain more liver-specific functions, as in secretions. Nevertheless, does spheroids really act like liver cells? The question is still being studied. Although spheroids can secrete, form (aggregate), and act as do liver cells, the cells may not be functional because it still undergoes a major environmental change, cultured in vitro. This paper describes how liver cells respond to the different environmental change of being cultured outside the body. It evaluates the liver cells/spheroids morphological formation and functional and biochemical parameters for 21 days. Spheroids grown in culture were compared to in vivo liver cells by measuring spheroid consumption, secretion, and activity.

Primary liver cells were obtained from rats and cultured into spheroids onto plates. Various biochemical assays were ran to compare how spheroids relate to in vivo liver cells. Total protein, glucose, galactose, albumin, pyruvate secretion were determined as well as LDH, g-GT, GPT, and GOT activity. Results were as following: galactose and pyruvate consumption was maintained at a relatively stable level throughout the assay. Glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, then decreased up to maturity and remained stable after. g-GT and LDH activities were initially extremely low and increased as spheroids matured. Albumin secretion decreased rapidly before the formation of the spheroid and increased during maturity. Through the observations made throughout the 21 culture day, the group were able roughtly to divide liver spheroid formation into two days: immaturity (days 1-5) and maturity (> day 5). Throughout spheroid immaturity, the liver cells undergoes a period of biochemical and functional turbulence and after maturity, the spheroid tends to maintain relatively stable functions up to 21 days. As compared to liver cells cultured on monolayers, spheroids are much more related to in vivo liver cells. Nevertheless, it does not imply that spheriods grown in culture can be injected in vivo. Thus, in order to study how liver cells act in vitro, one should develop spheroids and observe them only after maturity, for when they mature, most of their functions have been recovered or maintained at relatively stable levels.

I chose this paper because it relates to the final project in this course. Since, the purpose of the final project can be to propose a method to create a bioartificial liver in a sense, it is crucial to understand the relationship between a liver grown in vitro to one in vivo. I have heard a lot about the importance of spheroids to mimic cells or organs in vivo. Nevertheless, one knows that it is noticeably different for the environment is completely different; the spheroids are grown in a dish. Nevertheless, despite the dramatic difference in environment, this study provides insight about how spheriods are similar and different than a liver. In addition, it gives insight as to parameters and techniques for comparing how well the spheroids mimic the a real liver. For instance, if one wants to observe or test spheroids, one must wait at least 5 days after plating the cells to allow the cells to aggregate and mature. After that, there are a variety of tests that display whether the cells are liver-like or still immature. As in the case for our final project, the amount of production of albumin indicates spheroid maturity and growing liver-like properties.

Functional Tissue Engineering of Articular Cartilage through Dynamic Loading of Chondrocyte-Seeded Agarose Gels

The article can be viewed at http://scitation.aip.org/getabs/servlet/GetabsServlet?prog=normal&id=JBENDY000122000003000252000001&idtype=cvips&gifs=yes

Articular cartilage is limited in its ability to regenerate and to repair as it is not supplied by blood vessels. In spite of the previous efforts in tissue engineering of articular cartilage, current cartilage replacements are only morphologically and biochemically similar to natural cartilage. Their mechanical functionality still needs to be ameliorated for better cartilage substitutes, which is the goal of the authors of this paper. The authors hypothesized that more mechanically functional cartilage can be engineered by subjecting the tissues to dynamic loading at physiological levels.

Three studies were used to test the hypothesis. In the first study, hydrogels made of agarose and alginate at various concentrations without cells were tested for their material properties by a loading device to assess which material is more suitable for use in scaffolds. In the second study, 2% agarose and 2% alginate hydrogels with chondrocytes and acellular controls were cultured without loading. Compositional analysis of sulfated glycosaminoglycan and confined compression testing were carried out for comparison. In the third study, chondrocyte-seeded 2% agarose gels were cultured in two conditions: unconfined compression loading and unloading. Sulfated glycosaminoglycan and hydroxyproline compositional analysis, as well as confined and unconfined stress relaxation testing, were performed at different times.

From the first study, subphysiological peak stresses of agarose gels were observed when they were subjected to physiological cartilage strain levels. The authors found it more reliable to evaluate the aggregate modulus from the equilibrium stress-strain response rather than the transient response. From the second study, the peak equilibrium aggregate modulus measured for the cell-seeded agarose gels was three-fold better than that for cell-seeded alginate gels; moreover, initial increase in sulfated glycosaminoglycan content lasts longer for cell-seeded agarose gels, suggesting that agarose be a better scaffold material. From the third study, the equilibrium modulus of cell-seeded agarose gels with dynamic loading measured at day 28 was 21-fold better than that measured at day 0; under unloading condition, agarose gels only showed a three-fold increase between the same time points. Lastly, the sulfated glycosaminoglycan content of loaded agarose gels remained accumulated at day 21 whereas that of unloaded hydrogels plateaued at day 14.

This paper was chosen because of its relevance to the class project on cartilage. I am amazed at the time period within which they were able to achieve an unprecedented 21-fold increase in the equilibrium modulus of the tissue. Moreover, their study provided a good example of how to design experiments with good controls.