Differentiation of vascular smooth muscle cell sheet into contractile phenotype for vascular tissue engineering application

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Differentiation of vascular smooth muscle cell sheet into contractile phenotype for vascular tissue engineering application

Category: Dissertation

Subcategory: Biology

Level: Masters

Pages: 8

Words: 2200

Research Design

Background

In tissue engineering approaches, co-culture systems are routinely used in in-vivo settings. Co-culture systems consist of two or more different cell types that are closely cultured together. This helps in mimicking and replicating the in-vivo settings. This helps in increased viability of such cells compared to single or targeted cell (which needs to be reared).
Endothelial cells (ECs) are found in the luminal surface of blood vessels. In previous studies, it has been shown that damage or intervention to endothelial changes the phenotype of the associated smooth muscle cells in the lumen f the blood vessels. Angioplasty and radiotherapy cause various signaling cascades in ECs which leads to cross-talk in the SMCs. It is noted that ECs can significantly induce the phosphatidyl inositol pathway in SMCs, which changes their morphology. The SMCs become more proliferative and migrate in the lumen leading to atherosclerosis ADDIN EN.CITE ADDIN EN.CITE.DATA [7, 8]. These changes within the lumen impact its tonicity and increase the peripheral resistance leading to an increase in blood pressure. During vascular injury, a primary feature of atherosclerotic plaques is the synthetic state transited SMCs. They change their morphology from contractile state and become proliferative, which secretes excess ECM, leading to the narrowing of arteries ADDIN EN.CITE ADDIN EN.CITE.DATA [9, 10].
For instance, in angioplasty removal of diseased plaques from the blood vessels cause damage to ECs. Such action fails to proliferation of smooth muscle cells, which causes stenosis of blood vessels ADDIN EN.CITE ADDIN EN.CITE.DATA [9, 11]. Therefore, ECs should be grafted and engineered in such a way that it does not influence the morphological changes in the SMCs in vivo. Further tissue engineering should aim to maintain the contractile state of the SMCs and prevent their proliferation within the lumen. The tunica media (medial layer of the vessel wall) is an elastic and contractile layer, composed of highly ordered VSMCs and ECM, such as collagen and elastin. The contractile nature of these cells is required to maintain the tone of the blood vessels. Maintenance of vascular smooth muscle tone through contraction and relaxation is important for regulating peripheral resistance and blood pressure. The phenotypic changes of the VSMCs may be reflected from their mechanical properties. The mechanical properties of VSMCs are determined by the integrity of elastin and collagen in these cells. Elastin provides elasticity to the blood vessels while type-1 collagen resists changes in deformation of the blood vessels and makes them rigid ADDIN EN.CITE ADDIN EN.CITE.DATA [12].
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Principle of the Research Design
The goal of this project is to develop and characterize a vascular smooth muscle cell sheet that would preserve its contraction/relaxation phenotype. This development will be made available as a tissue engineered vascular patch. The tissue engineering vascular patch would be prepared through the combination of substrate micro-patterning and cell sheet harvesting techniques. The hypothesis contends that such tissue engineered grafts would be mechanically stable and align with the endothelial microenvironment. Finally, immuno-fluorescence staining of collagen and elastin would be performed to assess their organization, within the cell sheets grown on the substrate. Further, the tensile property the harvested cell sheet would be analyzed for mechanical properties. The research would be carried out in a step wise manner with defined aims as presented below.
Aim 1: Investigate the effect of substrate patterning on the cellular morphology and differentiation of VMSCs cell sheet.
Previous studies have implicated that VSMCs can be aligned on a patterned surface. These aligned cells exhibited an up-regulation of contractile phenotype markers, which helped in maintaining their contractile/relaxation phenotype. However, various studies have indicated that such changes are only noted with cells that were grown on patterned groves, but not as a confluent cell sheet. Therefore, the present study would be very important for understanding the effects of cellular alignment on cell phenotypes, as a confluent sheet. The study will confirm the feasibility of such VSMC cell sheet manufactured through fabrication of degradable hydro-gel substrate. The study will also investigate the effect of substrate patterning on the contractile phenotype of VSMCs, as a total unit and not as patterned groove.
Preparation of micro-patterned stamp to generate patterned substrate
In vitro techniques have been applied with cardiac cell cultures that mimicked ventricular myocardial phenotype in vivo. Such techniques were based on the principle of controlling the location and mechanical environment of the cultured cells. Microlithography technique was used to manufacture micro-structured silicon wafers. These are used to fabricate the micro-fluidic action of ECM proteins on the elastic membranes on the ventricular muscles in a patterned groove. Such applications helped in alignment of ventricular muscle cells (in vitro) which allowed for stretching and other phenotypic integrity [1].
Micro-patterned stamps were made by replica casting of poly-dimethylsiloxane (PDMS) against a silicon master, made by photolithography ADDIN EN.CITE <EndNote><Cite><Author>Camelliti</Author><Year>2006</Year><RecNum>32</RecNum><DisplayText>[1]</DisplayText><record><rec-number>32</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1442435915″>32</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Camelliti, P.</author><author>Gallagher, J. O.</author><author>Kohl, P.</author><author>McCulloch, A. D.</author></authors></contributors><auth-address>Kohl, P&#xD;Dept Physiol Anat &amp; Genet, Parks Rd, Oxford OX1 3PT, England&#xD;Dept Physiol Anat &amp; Genet, Parks Rd, Oxford OX1 3PT, England&#xD;Dept Physiol Anat &amp; Genet, Oxford OX1 3PT, England&#xD;Univ Calif San Diego, Dept Bioengn, La Jolla, CA 92093 USA</auth-address><titles><title>Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium</title><secondary-title>Nature Protocols</secondary-title><alt-title>Nat Protoc</alt-title></titles><periodical><full-title>Nature Protocols</full-title><abbr-1>Nat Protoc</abbr-1></periodical><alt-periodical><full-title>Nature Protocols</full-title><abbr-1>Nat Protoc</abbr-1></alt-periodical><pages>1379-1391</pages><volume>1</volume><number>3</number><keywords><keyword>human sinoatrial node</keyword><keyword>cardiac fibroblasts</keyword><keyword>mechanical stretch</keyword><keyword>ventricular myocytes</keyword><keyword>gene-expression</keyword><keyword>biaxial strain</keyword><keyword>hypertrophy</keyword><keyword>stress</keyword><keyword>tissue</keyword><keyword>heart</keyword></keywords><dates><year>2006</year></dates><isbn>1754-2189</isbn><accession-num>ISI:000251155400039</accession-num><urls><related-urls><url>&lt;Go to ISI&gt;://000251155400039</url></related-urls></urls><electronic-resource-num>10.1038/nprot.2006.203</electronic-resource-num><language>English</language></record></Cite></EndNote>[1](Figure 4.a).To generate micro-patterned topographical cues on the substrate, the PDMS was used to make a gelatin mold that provided a well for hydrogel substrate (Figure 4.b). A negative photo-resist layer (SU8-5, Microchem Corp.) was spin-coated on the silicon wafer that was soft baked 65’C, 95’C for a brief period. UV exposure selectively patterned the resist through the patterned mask.
After exposure, the silicon wafer was developed to remove the photo-resists that were not cross-linked with the substrates. This procedure created a master, which was used to generate patterned PDMS. A mixture of PDMS (monomer: curing agent = 10: 1) was poured against the master and cured in an oven. The patterned PDMS was peeled off from the master and was cut into proper size. Non-patterned PDMS was created by curing PDMS in a normal culture plate. Sterile PDMS was put on a culture plate, and warm gelatin solution (10wt % in PBS) was poured on top of it. The gelatin was then cooled by storing the plate in the refrigerator. Then the solidified gelatin was taken out and used as a mold for the hydrogel substrate.
1.5 % Al-ty or CMC-ty was mixed with HRP, before filling the gelatin mold. This was further cross-linked by adding H2O2on top. Once the hydrogel polymerized, warm PBS was poured onto the plate to melt out the gelatin mold. This was followed by several rinses with fresh PBS, to wash completely out excess gelatin. To confirm the stamping effect on the pattern, microscopic images were taken for PDMS, gelatin mold and hydro gel substrate (Figure 4.c)Most cell types are known to orient and can move along fibers with a range of 5–50 um in diameter ADDIN EN.CITE ADDIN EN.CITE.DATA [2, 3].Therefore, several different designs of master were fabricated with several pattern dimensions, within 50 um. However, the experiment was started with 30RG5H, to confirm the effect of a micro-patterned surface on the cytoskeleton morphology and contractile phenotype expression of VSMCs.
The experimentations were further extended to more diverse pattern designs. For example, 10RH5H, 30RG5H and 50RG5H, to compare the effect of pattern width on the cellular alignment behavior (unit: um, RG: width of ridge and groove, H: height). Figure 6 shows the representative microscopic images of the cross-sectional view of PDMS, with diverse width of pattern.
Figure 4. A scheme of photolithography procedure and strategy to fabricate micro-patterned substrate. a) The micropattern was developed on the silicon ‘master’, and the ‘master’ is used as a template to cast many reusable PDMS replication molds. b) Gelatin was used as a temporal mold to generate a patterned hydrogel substrate. C) Microscopic image showed that the pattern of PDMS stamp was stamped through gelatin mold to hydrogel substrate
Figure 6. Microscopic images of patterned PDMS. The cross-sectional view of patterned PDMS stamps generated from micropatterned wafers.
Cell culture tool development on hydrogel substrate
VSMCs (vascular smooth muscle cell) were chosen as a model cell since it is the main cell type that supports the strength, contractility and elasticity of the arteries. VMSCs were cultured in low glucose Dulbecco’s Modified Eagle Medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS), 1% ABAM (Antibiotic-Antimycotic) and 1% L-glutamine. The cells were maintained in a humidified incubator, with 5% CO2 at 37oC.
Before seeding the cells, the substrates were coated with collagen type 1 (10ug/cm2). The cells were harvested with trypsin and seeded with a density of 5 x 104cells/cm2. During the initial stage of the study, a square ring-shaped PDMS with 5 mm thickness was fabricated. Plasma was added to attach the PDMS on the 100 mm round plate. PDMS formed a wall of the 100mm round plate.
Next, the smaller square shaped e1.5% hydrogel substrate (with 1 mm thickness) was fabricated to fit in the PDMS wall. The edges of the substrate were glued down with 1% hydrogel to fix the substrate on the bottom (once the 1% hydrogel solution was cross-linked).
Excess media was needed to fill out the whole 100 mm round plate, even though cells were only placed on the square sized hydrogel substrate at the center. The problem could have been solved by building a much higher PDMS wall. However, the occasional failure of the glue between the hydrogel substrate and the plate caused hydrogel to float in the media. This process did not guarantee the same cell seeding density. Therefore, to guide cells only on top of the surface and control the initial seeding density accurately, stainless steel rings were fabricated (Figure 5). For the O-ring, an outer diameter was matched with the good diameter of 12 well culture plates, and the inner diameter was big enough to cover the circular substrate. The rectangular ring was fabricated for further use in preparing the samples for mechanical characterization.
Figure 5. Cell culture set up to guide cells attached on the surface of the substrate. a) Schematic figure showing the function of the stainless steel O-ring. b) Image of O-ring with outer diameter matched to 12 well culture plate well diameter, c) Image of stainless steel rectangular ring in rectangular culture well plate.
Microscopic image and staining for cellular morphology and orientation characterization
During cell culture, the images of the cells on the hydrogel substrate were taken by a microscope. The images were further processed to analyze orientation of the cells within the sheets and the aspect ratio of the cells. Hence, they were used to observe the elongated and aligned cell morphology on the patterned substrate, as well as the randomly oriented cell morphology on the non-patterned substrate. 2D FFT method (two-dimensional fast Fourier transform) was used to measure cell alignment ADDIN EN.CITE <EndNote><Cite><Author>Ayres</Author><Year>2008</Year><RecNum>37</RecNum><DisplayText>[4]</DisplayText><record><rec-number>37</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1442435918″>37</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Ayres, C. E.</author><author>Jha, B. S.</author><author>Meredith, H.</author><author>Bowman, J. R.</author><author>Bowlin, G. L.</author><author>Henderson, S. C.</author><author>Simpson, D. G.</author></authors></contributors><auth-address>Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA 23116, USA.</auth-address><titles><title>Measuring fiber alignment in electrospun scaffolds: a user&apos;s guide to the 2D fast Fourier transform approach</title><secondary-title>Journal of biomaterials science. Polymer edition</secondary-title><alt-title>J Biomater Sci Polym Ed</alt-title></titles><periodical><full-title>Journal of biomaterials science. Polymer edition</full-title><abbr-1>J Biomater Sci Polym Ed</abbr-1></periodical><alt-periodical><full-title>Journal of biomaterials science. Polymer edition</full-title><abbr-1>J Biomater Sci Polym Ed</abbr-1></alt-periodical><pages>603-21</pages><volume>19</volume><number>5</number><edition>2008/04/19</edition><keywords><keyword>Anisotropy</keyword><keyword>Biocompatible Materials/*chemistry</keyword><keyword>*Fourier Analysis</keyword><keyword>Microscopy, Electron, Scanning</keyword><keyword>Tissue Engineering/*methods</keyword></keywords><dates><year>2008</year></dates><isbn>0920-5063 (Print)&#xD;0920-5063 (Linking)</isbn><accession-num>18419940</accession-num><work-type>Research Support, N.I.H., Extramural&#xD;Review</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/18419940</url></related-urls></urls><electronic-resource-num>10.1163/156856208784089643</electronic-resource-num><language>eng</language></record></Cite></EndNote>[4]. 2D FFT analysis, evaluated the distribution of the angular difference between the aligned cells within the cell sheet. This was done to compare the alignments across diverse patterns of the substrate. The aspect ratio of the cells on the substrate is an indicator of the cell morphology, as well its phenotype. Thakar et al. reported that SMCs that were grown on the micro-patterned PDMS exhibited lower cell shape index (CSI) than cells that were grown on non-patterned PDMS (CSI: 0.42) ADDIN EN.CITE ADDIN EN.CITE.DATA [5]. The proliferation rate of patterned SMCs (lower CSI) was higher than non-patterned SMCs (higher CSI). This feature implicated the proliferative property of synthetic phenotype.
To analyze the cytoskeleton structure of the cell sheet, the cells were fixed for staining the actin filaments (F-actin). Actin is one of the major cytoskeleton proteins in most cells. The staining of actin was carried out through various steps. After seven days of culture, the cells were fixed with 4% para-formaldehyde in PBS for 10 min at room temperature, followed by serial rinses with PBS. The cells were the made permeable with 0.5% Triton X-100 in PBS for 15 minutes, followed by several washes with fresh PBS. After several washes, the cells were incubated in blocking buffer for 1 hr. 1% BSA in PBS was used as the blocking buffer. F-actin was stained using rhodamine-phalloidin (1:200) and while the nuclei of the SMCs were stained with Hoechst (1:5,000).
Phenotype gene expression profile
To determine the effect of regulated cell alignment on the substrate, quantitative TaqMan RT-PCR (real-time polymerase chain reaction analysis) was performed. The VSMC markers that were commonly used to define phenotypes are shown at Figure 7 ADDIN EN.CITE <EndNote><Cite><Author>Rensen</Author><Year>2007</Year><RecNum>34</RecNum><DisplayText>[6]</DisplayText><record><rec-number>34</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1442435916″>34</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Rensen, S. S.</author><author>Doevendans, P. A.</author><author>van Eys, G. J.</author></authors></contributors><auth-address>Department of Genetics and Cell Biology, Cardiovascular Research Institute Maastricht, University of Maastricht, the Netherlands.</auth-address><titles><title>Regulation and characteristics of vascular smooth muscle cell phenotypic diversity</title><secondary-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</secondary-title><alt-title>Neth Heart J</alt-title></titles><periodical><full-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</full-title><abbr-1>Neth Heart J</abbr-1></periodical><alt-periodical><full-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</full-title><abbr-1>Neth Heart J</abbr-1></alt-periodical><pages>100-8</pages><volume>15</volume><number>3</number><edition>2007/07/07</edition><dates><year>2007</year></dates><isbn>1568-5888 (Print)&#xD;1568-5888 (Linking)</isbn><accession-num>17612668</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/17612668</url></related-urls></urls><custom2>1847757</custom2><language>eng</language></record></Cite></EndNote>[6]. After 10 days of culture, cells were washed twice with PBS and mRNA was isolated. The mRNA was isolated from the cell/substrate construct, by using the RNeasy Plus Mini kit (Qiagen). cDNA synthesis was carried out using High Capacity RNA-to-cDNA Kit (Applied Biosystems). Quantitative PCR (qPCR) amplification was carried out using TaqMan Gene Expression Master Mix (Applied Biosystems). Relative gene expression of the cell/substrate construct was compared with non-patterned cell sheet(which was considered as controls) by the delta Ct (ddCt) method.
ACTB (beta actin, gene ID: 280979) was used as a housekeeping gene to normalize the gene expression level of 4 chosen contractile phenotype markers and 3 chosen synthetic phenotype markers. The chosen contractile phenotype markers were ACTA2 (Smooth Muscle Alpha-Actin, gene ID: 515610), MYH11 (smooth muscle myosin heavy chain 11, gene ID: 530050), TAGLN (Transgelin, Smooth muscle protein 22-alpha, gene ID: 513463) and CNN1 (smooth muscle calponin, gene ID: 534583). VIM (vimentin, gene ID: 280955), MYH10 (non-muscle myosin heavy chain 10, non-muscle, gene ID: 317655) and TPM4 (tropomyosin 4, gene ID: 535277) were chosen as the synthetic phenotype markers.
Figure 7. A schematic representation of expression levels of genes associated with a particular SMC phenotype ADDIN EN.CITE <EndNote><Cite><Author>Rensen</Author><Year>2007</Year><RecNum>34</RecNum><DisplayText>[6]</DisplayText><record><rec-number>34</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1442435916″>34</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Rensen, S. S.</author><author>Doevendans, P. A.</author><author>van Eys, G. J.</author></authors></contributors><auth-address>Department of Genetics and Cell Biology, Cardiovascular Research Institute Maastricht, University of Maastricht, the Netherlands.</auth-address><titles><title>Regulation and characteristics of vascular smooth muscle cell phenotypic diversity</title><secondary-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</secondary-title><alt-title>Neth Heart J</alt-title></titles><periodical><full-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</full-title><abbr-1>Neth Heart J</abbr-1></periodical><alt-periodical><full-title>Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation</full-title><abbr-1>Neth Heart J</abbr-1></alt-periodical><pages>100-8</pages><volume>15</volume><number>3</number><edition>2007/07/07</edition><dates><year>2007</year></dates><isbn>1568-5888 (Print)&#xD;1568-5888 (Linking)</isbn><accession-num>17612668</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/17612668</url></related-urls></urls><custom2>1847757</custom2><language>eng</language></record></Cite></EndNote>[6]
Aim 2: Investigate the cellular structure and viability of the harvested cell sheet and the effect of the differentiation on the mechanical property of VSMCs sheet.
Once the differentiation of VSMCs cell sheet was confirmed via patterning on a hydrogel substrate, further characterizations of the cell sheet are necessary. Various studies pointed out regarding the poor viability of harvested cell types in vivo. The following evaluations were implicated:
viability and cellular structure of the harvested cell sheet
mechanical characterization of contractile phenotype of the VSMCs cell sheet
the effect of endothelial cell sheet on VSMCs cell sheet differentiation
co-culture system will also be developed
Enzymatic release of VSMCs sheet
The cell sheet was detached from the hydrogel substrate by adding enzyme into the medium. To harvested cell sheet was transferred to different substrates. These were cover glass, tissue culture plate or other hydrogel substrate. The cell sheet and different substrates were coated with collagen and then, the cell sheet/substrate construct were flipped over to the different substrate and incubated for 1 hr with fresh medium. This was done so that cell sheets would completely adhere to the new substrate. Cellulase or alginate lyase containing medium was used for 1 or 2 hrs at 37oC to degrade the hydrogel. After the substrate had been degraded, fresh medium was added to stabilize the cell sheet for further analysis or culture.
Viability of harvested cell sheet
To evaluate the survival efficacy of harvested cell sheet, live and dead assay kit were used. The LIVE/DEAD® Viability/Cytotoxicity Kit (Life Technologies) discriminated live cells from dead cells. This was done by simultaneously staining with green-fluorescent calcein-AM to indicate intracellular esterase activity and red-fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity. After aspirating the medium, the cell sheet was washed with 1x PBS and then incubated with the staining solution (5 µL calcein AMand 20 µL ethidium homodimer-1 in 10 mL PBS) for 30 minutes at room temperature. Then the cells were observed for survival efficacy from the fluorescence microscopic images. The viability study was not only done after degradation of the hydrogel but even after 24 hrs and 48 hrs when the cell sheets were placed in normal culture condition.
Contractile phenotype of harvested VSMCs sheet
The study confirmed that the cells proliferated on the hydrogel substrate, to form a confluent cell sheet. Further, the micro-pattern on the substrate guided the differentiation of VSMCs resulting in a contractile phenotype. To investigate whether the cell sheet supported phenotype maintenance compare to cell suspension, the contractile/synthetic phenotype gene expression will be analyzed following the same procedure we performed in previous PCR experiment. For one group we will harvest the cell sheet by degrading the hydrogel substrate, and the other group we will collect cell suspension by traditional trypsin-EDTA treatment.
‘Homo-, Hetero type’ cell sheet
In this study, co-culture system will be developed using stainless rings to separate the hydrogel substrates with different cell types. However, they will be kept in the same well to share co-culture medium. VSMCs phenotype expression profile will be analyzed with or without ECs in the same well. This will be done to compare the effect of ECs on differentiation of VSMCs in the hydrogel substrate system.
Extracellular matrix characterization and mechanical property analysis
. Characterization of mechanical property of VSMCs sheet is important for understanding the physiology of VSMCs and mechanics of arterial wall ADDIN EN.CITE <EndNote><Cite><Author>Wanjare</Author><Year>2013</Year><RecNum>39</RecNum><DisplayText>[13]</DisplayText><record><rec-number>39</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1442435919″>39</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Wanjare, M.</author><author>Kusuma, S.</author><author>Gerecht, S.</author></authors></contributors><auth-address>Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences-Oncology Center and Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, MD 21218, USA.</auth-address><titles><title>Perivascular cells in blood vessel regeneration</title><secondary-title>Biotechnology journal</secondary-title><alt-title>Biotechnol J</alt-title></titles><periodical><full-title>Biotechnology journal</full-title><abbr-1>Biotechnol J</abbr-1></periodical><alt-periodical><full-title>Biotechnology journal</full-title><abbr-1>Biotechnol J</abbr-1></alt-periodical><pages>434-47</pages><volume>8</volume><number>4</number><edition>2013/04/05</edition><keywords><keyword>Animals</keyword><keyword>Humans</keyword><keyword>Muscle, Smooth, Vascular/cytology/*physiology</keyword><keyword>Pericytes/cytology/*physiology</keyword><keyword>Regeneration/*physiology</keyword><keyword>Tissue Engineering/*methods</keyword></keywords><dates><year>2013</year><pub-dates><date>Apr</date></pub-dates></dates><isbn>1860-7314 (Electronic)&#xD;1860-6768 (Linking)</isbn><accession-num>23554249</accession-num><work-type>Research Support, N.I.H., Extramural&#xD;Review</work-type><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/23554249</url></related-urls></urls><custom2>3743428</custom2><electronic-resource-num>10.1002/biot.201200199</electronic-resource-num><language>eng</language></record></Cite></EndNote>[13]. We used a customized uni-axial tensile tester, for measuring the mechanical property of cell sheet. This was previously developed by Daniel Beckman ADDIN EN.CITE <EndNote><Cite><Author>Isenberg</Author><Year>2012</Year><RecNum>207</RecNum><DisplayText>[14]</DisplayText><record><rec-number>207</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1449300848″>207</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Isenberg, B. C.</author><author>Backman, D. E.</author><author>Kinahan, M. E.</author><author>Jesudason, R.</author><author>Suki, B.</author><author>Stone, P. J.</author><author>Davis, E. C.</author><author>Wong, J. Y.</author></authors></contributors><auth-address>Department of Biomedical Engineering, Boston University, College of Engineering, Boston, Massachusetts 02215, USA.</auth-address><titles><title>Micropatterned cell sheets with defined cell and extracellular matrix orientation exhibit anisotropic mechanical properties</title><secondary-title>J Biomech</secondary-title></titles><periodical><full-title>J Biomech</full-title></periodical><pages>756-61</pages><volume>45</volume><number>5</number><keywords><keyword>Animals</keyword><keyword>Anisotropy</keyword><keyword>Arteries/*cytology</keyword><keyword>Ascorbic Acid/pharmacology</keyword><keyword>Biomechanical Phenomena</keyword><keyword>Cattle</keyword><keyword>Cell Culture Techniques/*methods</keyword><keyword>Cells, Cultured</keyword><keyword>Dimethylpolysiloxanes/pharmacology</keyword><keyword>Extracellular Matrix/*physiology</keyword><keyword>Hemodynamics/physiology</keyword><keyword>Muscle, Smooth, Vascular/*cytology</keyword><keyword>Myocytes, Smooth Muscle/*cytology</keyword><keyword>Tissue Engineering/*methods</keyword></keywords><dates><year>2012</year><pub-dates><date>Mar 15</date></pub-dates></dates><isbn>1873-2380 (Electronic)&#xD;0021-9290 (Linking)</isbn><accession-num>22177672</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/22177672</url></related-urls></urls><electronic-resource-num>10.1016/j.jbiomech.2011.11.015</electronic-resource-num></record></Cite></EndNote>[14]. This tensile stretcher has a motor, which provides sufficient translational resolution to stretch materials with slow elongation rates. The force can be transmitted directly to the force sensor that is placed outside of the tissue bath, where the cell sheet sample can be loaded. Once the cell sheets are harvested from the substrate, they can be tested in different stretching direction. Absolute values of the failure stress, failure strain, and stiffness for non-patterned and patterned cell sheet will be investigated in different stretching condition. These conditions would be parallel stretching or perpendicular stretching. The mechanical data will be compared with contractile phenotypic expression, cell alignment, and ECM orientation. The analysis would be based on the correlation co-efficient of such parameters.
Aim 3: Develop a thick and strong multi-layered cell sheet for in vivo tissue engineered vascular patch study.
To develop a biomaterial from the findings of two aims, a three-dimensional structure would be assembled. This would be done to validate the applicability of the cell sheet as a tissue engineered vascular patch. The cell sheet stacking strategy will be implemented to fabricate a 3D multi-layered cell sheet, followed by three-dimensional visualization and assessment of mechanical properties of VSMCs. These results will support further design criteria of in vivo vascular patches. Such design will portray the number of layers and thickness of the cell sheet, which may be implicated as tissue engineered vascular patches.
Strategy to obtain multi-layer cell sheet.
Cell sheet engineering approach has the potential to develop a tissue engineered vascular patch. They can improve the remodeling response of the VSMCs with direct interaction with the surrounding host tissue. To be applicable as in vivo patch, the strength and thickness should be investigated. The strategy to obtain multiple layer of cell sheet, which can make thicker and stronger cell sheet about monolayer, will be evaluated next.
Figure 8 reflects the strategy to fabricate the multiple layers of cell sheets. Similar to the cell sheet transfer procedure, the cell sheet cultured on one type of hydrogel (Al-ty or CMC-ty) are transferred to cell sheet cultures, on a different type of hydrogel. After degrading the top layer hydrogel substrate, it results in bilayer cell sheets on the other type of hydrogel substrate. By repeating this procedure, multiple layers of thick cell sheets can be generated. Moreover, each aligned cell sheet can be organized in the desired direction as layer by layer.
Figure 8. A scheme of cell-sheet stacking strategy. a) Single cell sheet transfer procedure to other substrates. b) Cell sheet stacking strategy using opposite type of hydrogel substrate.
To visualize the bilayer cell sheet, each cell sheet will be pre-labeled with different cell tracker before stacking. Perpendicular direction stacking and parallel direction stacking can be compared to observe the direction of alignment in bilayer cell sheet. Such directional alignment is important for ensuring the mechanical property of the vascular patches. It is known that the medial layer consists of multiple layers of VSMCs and ECM, which are arranged in distinct spiral configurations in natural blood vessel ADDIN EN.CITE ADDIN EN.CITE.DATA [2]. Fiber orientation for each layer differs slightly from adjacent layers, which is responsible for variation of diameter of the blood vessels ADDIN EN.CITE ADDIN EN.CITE.DATA [15]. The spatial arrangement of VSMCs and the ECM could play a critical role in the function of the medial layer. Therefore, as a bio-mimetic approach, the patterned cell sheet can be stacked in slightly different angle compared to non-pattern cell sheet. The non-patterned cell sheet may be oriented as parallel stacked cell sheet or perpendicularly stacked cell sheet. After studying the alignments of such patches, they would be analyzed for their mechanical properties.
Discussion & Conclusion of Research Design
Cell sheet engineering approach has the potential for developing a vascular tissue engineered patch, in vivo. It minimizes the immune response due to a scaffold degradation, as well as improved localization or survival efficacy. It is also expected that the cell-mediated vascular network can remodel, in response to direct interaction with surrounding host tissue. Several previous studies have showed the applicability of cell sheet-based technique as a potential tissue engineered vascular graft. Human fibroblast sheet-derived vascular grafts implanted in nude rats exhibited physiological mechanical strength and positive scaffold remodeling and maturation, as detected from histological analysis ADDIN EN.CITE <EndNote><Cite><Author>L&apos;Heureux</Author><Year>2006</Year><RecNum>132</RecNum><DisplayText>[16]</DisplayText><record><rec-number>132</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1449185888″>132</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>L&apos;Heureux, N.</author><author>Dusserre, N.</author><author>Konig, G.</author><author>Victor, B.</author><author>Keire, P.</author><author>Wight, T. N.</author><author>Chronos, N. A.</author><author>Kyles, A. E.</author><author>Gregory, C. R.</author><author>Hoyt, G.</author><author>Robbins, R. C.</author><author>McAllister, T. N.</author></authors></contributors><auth-address>Cytograft Tissue Engineering, Inc., 3 Hamilton Landing, Suite 220, Novato, California 94949, USA. [email protected]</auth-address><titles><title>Human tissue-engineered blood vessels for adult arterial revascularization</title><secondary-title>Nat Med</secondary-title></titles><periodical><full-title>Nat Med</full-title></periodical><pages>361-5</pages><volume>12</volume><number>3</number><keywords><keyword>Adult</keyword><keyword>Animals</keyword><keyword>Arteries/*growth &amp; development</keyword><keyword>*Blood Vessel Prosthesis</keyword><keyword>Blood Vessel Prosthesis Implantation</keyword><keyword>Blood Vessels/*cytology/*growth &amp; development/transplantation</keyword><keyword>Cells, Cultured</keyword><keyword>Dogs</keyword><keyword>Humans</keyword><keyword>Primates</keyword><keyword>Rats</keyword><keyword>Rats, Nude</keyword><keyword>Time Factors</keyword><keyword>*Tissue Engineering</keyword></keywords><dates><year>2006</year><pub-dates><date>Mar</date></pub-dates></dates><isbn>1078-8956 (Print)&#xD;1078-8956 (Linking)</isbn><accession-num>16491087</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/16491087</url></related-urls></urls><custom2>PMC1513140</custom2><electronic-resource-num>10.1038/nm1364</electronic-resource-num></record></Cite></EndNote>[16]. Zhao et al. reported that MSC-derived cell sheet were rolled to form tissue-engineered vascular graft (TEVG), and then implanted into rabbit common carotid artery ADDIN EN.CITE ADDIN EN.CITE.DATA [17]. The in vivo result showed patency of the graft, production of collagen, elastin fibers formation and EC monolayer formation.
In the previous experiments, it was observed that the contractile phenotype of VSMCs sheet can be regulated and maintained within degradable hydrogel-based system, by applying micro-patterns on the substrate. Future studies may be carried out to evaluate whether the contractile phenotype differentiated VSMCs can be maintained in vivo, as well. VSMCs in native blood vessel have contractile phenotype and play an important role for the contraction of blood vessel, regulation of tone of blood vessels, blood pressure and blood flow. Meanwhile synthetic phenotype VSMCs is shown during injury responses, such as plaque formation or restenosis, caused by proliferation and secreted excess ECM by VSMCs.
In this study, based on the mechanical characterization results of tissue engineered vascular patch, the number of cell sheet layers need to be stacked may be extrapolated. Such extrapolations will indicate whether these vascular patches may be applicable in vivo also. The tissue engineered vascular patch will be implanted (or wrapped) in nude rats as abdominal aorta interposition grafts. A different number of layers and pattern or non-patterned cell sheet will be implanted. The control group will consist of non-pattern VSMCs sheet or cell pellets. The philosophy is that the blood flow will give a stretching stimuli to the wrapped cell sheet that has enhanced contractile property due to tissue engineering ADDIN EN.CITE <EndNote><Cite><Author>Huang</Author><Year>2014</Year><RecNum>122</RecNum><DisplayText>[18]</DisplayText><record><rec-number>122</rec-number><foreign-keys><key app=”EN” db-id=”9f9atarptaawxeex2x05frrqdwsp9xextdvf” timestamp=”1449180738″>122</key></foreign-keys><ref-type name=”Journal Article”>17</ref-type><contributors><authors><author>Huang, A. H.</author><author>Niklason, L. E.</author></authors></contributors><auth-address>Department of Biomedical Engineering, Yale University, New Haven, CT, 06511, USA, [email protected]</auth-address><titles><title>Engineering of arteries in vitro</title><secondary-title>Cell Mol Life Sci</secondary-title></titles><periodical><full-title>Cell Mol Life Sci</full-title></periodical><pages>2103-18</pages><volume>71</volume><number>11</number><keywords><keyword>Arteries/immunology/*pathology/surgery</keyword><keyword>Biocompatible Materials/metabolism</keyword><keyword>Biomechanical Phenomena</keyword><keyword>Bioreactors</keyword><keyword>*Blood Vessel Prosthesis</keyword><keyword>Collagen/metabolism</keyword><keyword>Endothelial Cells/*cytology/physiology</keyword><keyword>Endothelium, Vascular/cytology/physiology</keyword><keyword>Extracellular Matrix/metabolism/physiology</keyword><keyword>Fibrin/metabolism</keyword><keyword>Graft Survival/immunology</keyword><keyword>Humans</keyword><keyword>Mechanotransduction, Cellular</keyword><keyword>Tissue Culture Techniques</keyword><keyword>*Tissue Engineering</keyword><keyword>Tissue Scaffolds</keyword></keywords><dates><year>2014</year><pub-dates><date>Jun</date></pub-dates></dates><isbn>1420-9071 (Electronic)&#xD;1420-682X (Linking)</isbn><accession-num>24399290</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/24399290</url></related-urls></urls><custom2>PMC4024341</custom2><electronic-resource-num>10.1007/s00018-013-1546-3</electronic-resource-num></record></Cite></EndNote>[18].The synthesis rate of ECM proteins is also known to be significantly increased by cyclic stretching on VSMCs. The implanted engineered vessels will be harvested for gross observation, histological and immune-histochemical analysis to confirm the localization, cellular structure, and contractile phenotype expression. The cell sheet will also be subjected H&E staining; Masson’s trichrome staining for collagen, and immunostaining for contractile protein markers.

ADDIN EN.REFLIST 1.Camelliti, P., et al., Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium. Nature Protocols, 2006. 1(3): p. 1379-1391.2.Sarkar, S., et al., Vascular tissue engineering: microtextured scaffold templates to control organization of vascular smooth muscle cells and extracellular matrix. Acta biomaterialia, 2005. 1(1): p. 93-100.3.Curtis, A. and M. Riehle, Tissue engineering: the biophysical background. Physics in medicine and biology, 2001. 46(4): p. R47-65.4.Ayres, C.E., et al., Measuring fiber alignment in electrospun scaffolds: a user’s guide to the 2D fast Fourier transform approach. Journal of biomaterials science. Polymer edition, 2008. 19(5): p. 603-21.5.Thakar, R.G., et al., Cell-shape regulation of smooth muscle cell proliferation. Biophys J, 2009. 96(8): p. 3423-32.6.Rensen, S.S., P.A. Doevendans, and G.J. van Eys, Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation, 2007. 15(3): p. 100-8.7.Campbell, J.H. and G.R. Campbell, Endothelial cell influences on vascular smooth muscle phenotype. Annu Rev Physiol, 1986. 48: p. 295-306.8.Brown, D.J., et al., Endothelial cell activation of the smooth muscle cell phosphoinositide 3-kinase/Akt pathway promotes differentiation. J Vasc Surg, 2005. 41(3): p. 509-16.9.Lilly, B., We have contact: endothelial cell-smooth muscle cell interactions. Physiology (Bethesda), 2014. 29(4): p. 234-41.10.Doran, A.C., N. Meller, and C.A. McNamara, Role of smooth muscle cells in the initiation and early progression of atherosclerosis. Arterioscler Thromb Vasc Biol, 2008. 28(5): p. 812-9.11.Yu, P.J., et al., Vascular injury and modulation of MAPKs: a targeted approach to therapy of restenosis. Cell Signal, 2007. 19(7): p. 1359-71.12.Miyazaki, H., Y. Hasegawa, and K. Hayashi, Tensile properties of contractile and synthetic vascular smooth muscle cells. Jsme International Journal Series C-Mechanical Systems Machine Elements and Manufacturing, 2002. 45(4): p. 870-879.13.Wanjare, M., S. Kusuma, and S. Gerecht, Perivascular cells in blood vessel regeneration. Biotechnology journal, 2013. 8(4): p. 434-47.14.Isenberg, B.C., et al., Micropatterned cell sheets with defined cell and extracellular matrix orientation exhibit anisotropic mechanical properties. J Biomech, 2012. 45(5): p. 756-61.15.O’Connell, M.K., et al., The three-dimensional micro- and nanostructure of the aortic medial lamellar unit measured using 3D confocal and electron microscopy imaging. Matrix biology : journal of the International Society for Matrix Biology, 2008. 27(3): p. 171-81.16.L’Heureux, N., et al., Human tissue-engineered blood vessels for adult arterial revascularization. Nat Med, 2006. 12(3): p. 361-5.17.Zhao, J., et al., A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells. Artif Organs, 2012. 36(1): p. 93-101.18.Huang, A.H. and L.E. Niklason, Engineering of arteries in vitro. Cell Mol Life Sci, 2014. 71(11): p. 2103-18.