Difference between revisions of "Team:TecCEM/Description"

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     <nav class="sidebar-index">
 
     <nav class="sidebar-index">
 
         <div class="sidebar-header">
 
         <div class="sidebar-header">
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             <h3>Project/ Experiments</h3>
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             <h3>Project/ Description</h3>
 
         </div>
 
         </div>
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         <a href="#experimentsSubmenu" data-toggle="collapse" aria-expanded="false" data-offset="100" data-change="sidemenu">
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         <a href="#descriptionSubmenu" data-toggle="collapse" aria-expanded="false" data-change="sidemenu">
 
             <span data-change="el" class="d-inline-block open"></span>
 
             <span data-change="el" class="d-inline-block open"></span>
 
             Index
 
             Index
 
         </a>
 
         </a>
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         <ul class="collapse list-unstyled" id="experimentsSubmenu">
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         <ul class="collapse list-unstyled" id="descriptionSubmenu">
 
             <li class="active">
 
             <li class="active">
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                 <a data-target="#description">Description</a>
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                 <a data-target="#background">Background</a>
 
             </li>
 
             </li>
 
             <li>
 
             <li>
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                 <a href="#proteinSubmenu" data-toggle="collapse" aria-expanded="false" data-offset="100" data-change="sidemenu">
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                <a data-target="#solution">The solution</a>
 +
            </li>
 +
            <li>
 +
                <a data-target="#extracellular">Extracellular matrix</a>
 +
            </li>
 +
            <li>
 +
                 <a href="#componentsSubmenu" data-toggle="collapse" aria-expanded="false" data-change="sidemenu">
 
                     <span class="d-inline-bock open" data-change="el"></span>
 
                     <span class="d-inline-bock open" data-change="el"></span>
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                     Protein
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                     Components
 
                 </a>
 
                 </a>
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                 <ul class="collapse list-unstyled" id="proteinSubmenu">
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                 <ul class="collapse list-unstyled" id="componentsSubmenu">
 +
                    <li>
 +
                        <a data-target="#collagen">Collagen</a>
 +
                    </li>
 +
                    <li>
 +
                        <a data-target="#tenascin">Tenascin</a>
 +
                    </li>
 +
                    <li>
 +
                        <a data-target="#heparin">Heparin</a>
 +
                    </li>
 +
                    <li>
 +
                        <a data-target="#chitosan">Chitosan</a>
 +
                    </li>
 
                     <li>
 
                     <li>
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                         <a href="#chitosan" data-toggle="collapse" aria-expanded="false" data-offset="100" data-change="sidemenu">
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                         <a data-target="#growth">Growth factor</a>
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                            <span class="d-inline-bock open" data-change="el"></span>
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                            Chitosan
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                        </a>
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                        <ul class="collapse list-unstyled" id="chitosan">
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                            <li>
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                                <a data-target="#encapsulation">Protein encapsulation protocol</a>
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                            </li>
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                            <li>
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                                <a data-target="#encapsulation-efficiency">Protein encapsulation efficiency
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                                    protocol</a>
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                            </li>
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                            <li>
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                                <a data-target="#liberation-and-stability">Protein liberation and stability
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                                    protocol</a>
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                            </li>
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                        </ul>
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                     </li>
 
                     </li>
 
                 </ul>
 
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     </nav>
 
     </nav>
 
     <div class="content">
 
     <div class="content">
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         <div class="container mt-3" id="description">
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         <div class="container" id="description">
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             <div class="row">
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             <div class="row" id="background">
 
                 <div class="col">
 
                 <div class="col">
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                     <p>This is our experiment section. Here we compile important protocols for the development of
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                    <h1>Background</h1>
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                         TecTissue, ranging from our bacterial transformation procedures to our cell proliferation
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                     <p>All over the globe, burns are a huge problem in the health sector, only in Mexico is the third
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                         assays.
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                        cause of infant mortality and around 120 thousand people per year suffer a burn accident, based
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                         We also address cell culture maintenance and protein loaded chitosan nanoparticles.
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                         in the public health sector. However, there is not the required infrastructure needed to treat
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                         Here you may find the protocol for our growth factor delivery to damaged cells and how much
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                         third-degree burns, which causes a high mortality rate (NTX, 2017).
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                         harm can be inflicted in vitro.
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                    </p>
 +
                    <p>
 +
                         Furthermore, the rate of child mortality from burns is 7 times higher in locations with low
 +
                        socioeconomic indicators, where each burn case could hit a cost of 141,750 USD due to the lack
 +
                        of specialized equipment and staff required to attend these delicate wounds.
 +
                         This problem has been ignored because of what the healing process implies, high costs and
 +
                         difficulties providing the specialized treatment. Then, we concluded that accelerating this
 +
                        process would have many advantages, not only in a faster skin regeneration but also it would
 +
                        have a psychological impact and reduce the economic cost.
 
                     </p>
 
                     </p>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
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        </div>
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            <div class="row" id="solution">
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        <div class="container mt-3">
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            <h1>Protocols</h1>
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            <h2>Chitosan nanoparticles</h2>
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            <div class="row">
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                 <div class="col">
 
                 <div class="col">
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                     <a class="btn-link text-notebook" data-toggle="collapse" href="#encapsulation" role="button"
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                     <h1>The solution</h1>
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                        aria-expanded="false" aria-controls="encapsulation">
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                     <p>Thus, we created TecTissue, a novel treatment that is capable of reducing wound healing time,
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                        <h3>Protein encapsulation protocol</h3>
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                         preventing infections and consequently, decreasing mortality rate. We achieved this with
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                     </a>
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                         synthetic biology, incorporating a growth factor and a scaffold.
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                    <div class="collapse" id="encapsulation">
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                    </p>
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                        <div class="mb-3">
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                    <p>
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                            <h4>Reactants</h4>
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                        TecTissue’s main objective is to provide families with a simple way to help in the reduction of
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                            <ul>
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                        medical bills and hospitalization time, make the recovery process easier and also create a
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                                <li>Chitosan low molecular weight from Sigma-Aldrich</li>
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                        chance of better life quality for the people that suffered from burns.
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                                <li>TPP from Sigma-Aldrich</li>
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                        We used recombinant proteins like collagen, composed by non-canonical amino acids. We also
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                                <li>NaOH 1M</li>
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                        implemented a growth factor produced by humans called Leptin. This growth factor has been
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                                <li>Acetic acid 1M</li>
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                         reported to be essential in different skin regeneration processes. There were some other
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                                <li>Distilled water</li>
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                         molecules used in our project such as Tenascin and heparin. Both of them, with the company of
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                                <li>Protein of interest (10 mg/mL)</li>
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                        collagen, compose our scaffold which is encapsulated with leptin in nanoparticles of chitosan,
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                            </ul>
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                         in order to have an efficient delivery in the skin.
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                         </div>
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                    </p>
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                         <div class="mb-3">
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                    <p>We tested the development of TecTissue in fibroblast cell line L-929 and human mesenchymal
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                            <h4>Procedure</h4>
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                         cells. To demonstrate our project it was necessary to elaborate a Mini Skin Simulator were the
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                            <h5><i>Stock solutions</i></h5>
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                        cell lines were provided with the conditions they required in order to get a positive
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                            <ol>
+
                        proliferation. This Mini Skin Simulator was automatized to create a friendly device for the
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                                <li>In a 15 mL Falcon tube add 30 mg of chitosan and 10 mL of distilled water (to get a
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                        manipulation of cells in a culture and to reduce the possible contamination.
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                                    solution with a concentration of 3 mg/mL).</li>
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                     </p>
−
                                <li>Add 10 microliters of acetic acid for each mL of chitosan solution to solubilize
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                                    the
+
−
                                    chitosan.
+
−
                                    To adjust the pH acetic acid and NaOH should be used.</li>
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                                NOTE: the pH should be adjusted depending on your protein of interest, taking into
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                                account
+
−
                                the isoelectric point, always maintaining the chitosan solution positively charged (pH
+
−
                                &lt;
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−
                                6.5)
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−
                                and the protein of interest negatively charged (preferred).
+
−
                                <li> In another 15 mL falcon
+
−
                                    tube add 10 mg of TPP and 10 mL of distilled water (to get a concentration of 1
+
−
                                    mg/mL).</li>
+
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                            </ol>
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−
                         </div>
+
−
                         <div class="mb-3">
+
−
                            <h5><i>
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                                    Nanoparticle preparation
+
−
                                </i></h5>
+
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                            <ol>
+
−
                                <li>In a 20 mL beaker add 1 mL of chitosan solution and 100 uL of your protein, stir
+
−
                                    the
+
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                                    mix at 1100 rpm with a magnetic stirrer (the size of nanoparticles is affected by
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                                    rpm
+
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                                    value; for smaller nanoparticles use higher rpm).</li>
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−
                                <li>Take 1 mL of the TPP solution and add it to the mix dropwise.</li>
+
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                                <li>Continue stirring for 1 hour.</li>
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                            </ol>
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                         </div>
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                        <div class="mb-3">
+
−
                            <h5><i>
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                                    Particle collection
+
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                                </i></h5>
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                            <ol>
+
−
                                <li>Transfer the mix to 2 1.5 mL Eppendorf tubes.</li>
+
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                                <i>NOTE: If nanoparticles are to be extracted centrifuge the tubes at 20,000 rpm for
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                                    30
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                                    minutes at 4°C.</i>
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                                <li>Eliminate the supernatant.</li>
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                                <li>The pellet will contain your protein of interest.</li>
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                                <li>If nanoparticles are to be used for liberation measurients or suspended in a
+
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                                    controlled pH solution, resuspend well and store at 4 °C.</li>
+
−
                            </ol>
+
−
                         </div>
+
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                        <div class="mb-3">
+
−
                            <h5> <i>
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−
                                    To preparation
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−
                                </i></h5>
+
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                            <p><i>To visualize chitosan nanoparticles some previous preparation steps must be carried
+
−
                                    out
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                                    (this preparation protocol may vary).</i></p>
+
−
                            <ol>
+
−
                                <li>A film of Formvar has to be previously prepared and used to coat a glass slide for
+
−
                                    the
+
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                                    creation of an 80-120 μm thick mibrane.</li>
+
−
                                <li>Place a copper grid on the Formvar mibrane for it to be absorbed and later rioved
+
−
                                    with a needle.</li>
+
−
                                <li>Add 20 μL of your solution of interest into the grid and let it be absorbed. Add a
+
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                                    solution of 1% (w/v) phosphotungstic acid until the sample dries.</li>
+
−
                                <li>View in a transmission electron microscope.</li>
+
−
                                <i>NOTE: Samples were observed at 150,000x.</i>
+
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                            </ol>
+
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                        </div>
+
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                     </div>
+
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
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             <div class="row">
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             <div class="row" id="extracellular">
 
                 <div class="col">
 
                 <div class="col">
−
                     <a class="btn-link text-notebook" data-toggle="collapse" href="#encapsulation-efficiency" role="button"
+
                     <h1>Extracellular matrix</h1>
−
                        aria-expanded="false" aria-controls="encapsulation-efficiency">
+
                     <p>The extracellular matrix (ECM) is a non-cellular structure found on the outside of the cell.
−
                        <h3>Protein encapsulation efficiency protocol</h3>
+
                         Among other features, the organization of the cells, tissue, organs and protection are the most
−
                     </a>
+
                         important. It can be used as a biological material for damaged organs or tissue regeneration.</p>
−
                    <div class="collapse" id="encapsulation-efficiency">
+
                    <p>In order to generate an extracellular matrix, four molecules were used: a collagenous peptide,
−
                        <p>
+
                         Tenascin type III-like, heparin and chitosan.</p>
−
                            This protocol will evaluate and standardize encapsulation efficiency when working with a
+
                    <figure class="figure text-center">
−
                            specific
+
                        <img src="https://static.igem.org/mediawiki/2018/d/de/T--TecCEM--ScaffoldDes1.jpg " class="figure-img img-fluid rounded"
−
                            protein. It is highly recommended to work with a highly purified protein sample, so as to
+
                             alt="Scaffolddes1">
−
                            get the
+
                         <figcaption class="figure-caption"><strong>Figure 1. Scaffold diagram.</strong></figcaption>
−
                            most reliable quantification. Measurients are performed according to the Bradford assay.
+
                    </figure>
−
                         </p>
+
                    <p>Collagen V was shown to bind heparan sulfate proteoglycans through its heparin binding site
−
                         <p>
+
                         (HepV) with higher affinity than other collagens. A recombinant fragment of the α1(V) chain
−
                            <i>
+
                        (Ile824 to Pro950) binding site binds heparin and heparan sulfate by electrostatic interactions
−
                                Detection range: 0.1-1.4 mg/mL
+
                        [4]. The purpose of including this domain was to promote the binding of heparin, which then
−
                            </i>
+
                         interacts with tenascin C fibronectin type III domain V (TNCIII5), forming a matrix that boosts
−
                        </p>
+
                        cell adhesion.</p>
−
                         <p>NOTE: Bradford reactant must be at room tiperature and shaken gently before starting
+
                    <p>We supplemented the scaffold with a recombinant human leptin β (LepB) to accelerate cell
−
                            the
+
                        proliferation, as we validated that it could achieve a controlled drug delivery when
−
                            protocol.
+
                        encapsulated. Due to its wide use as a biomaterial, chitosan nanoencapsulation was employed.
−
                        </p>
+
                        The efficacy of the system was evaluated with MTT proliferation assay performed in 96-well
−
                        <div class="mb-3">
+
                        plates.</p>
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                            <h4>
+
                    <div class="text-center">
−
                                <i>
+
                        <iframe width="560" height="315" src="https://static.igem.org/mediawiki/2018/5/54/T--TecCEM--Scaffold.mp4 "
−
                                    Calibration curve of BSA
+
                            frameborder="0" allow="autoplay; encrypted-media" allowfullscreen></iframe>
−
                                </i>
+
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                            </h4>
+
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                        </div>
+
−
                        <div class="mb-3">
+
−
                             <h5> BSA Stock solution</h5>
+
−
                            <ol>
+
−
                                <li>Prepare 10 mg/mL BSA solution</li>
+
−
                                <li>Store in ice for further use</li>
+
−
                            </ol>
+
−
                         </div>
+
−
                        <div class="mb-3">
+
−
                            <h5>Dilutions</h5>
+
−
                            <p>
+
−
                                Loaded and ipty nanoparticles are prepared under the same conditions (agitation,
+
−
                                tiperature, pH,
+
−
                                and reactant concentrations). Thus, we used a sample of ipty encapsulation supernatant
+
−
                                as
+
−
                                a blank
+
−
                                to construct a standard curve to estimate protein encapsulation efficiency. Volumes of
+
−
                                supernatant
+
−
                                were mixed with volumes of BSA stock solution to obtain dilutions of known protein
+
−
                                concentrations.
+
−
                            </p>
+
−
                            <ol>
+
−
                                <li>Refer to encapsulation protocol to prepare ipty chitosan nanoparticles.</li>
+
−
                                <li>Prepare encapsulation solution aliquots.</li>
+
−
                                <li>Centrifuge samples after splitting the initial encapsulation volume at 13,400 rpm
+
−
                                    for 30
+
−
                                    min.</li>
+
−
                                <li>Supernatant will be used to derive the curve. Do not discard.</li>
+
−
                            </ol>
+
−
                            <p><i>Prepare dilutions according to the following table and label each tube. A small-scale
+
−
                                    procedure
+
−
                                    was adapted from Sigma Aldrich to perform Bradford assay on the prepared dilutions.</i></p>
+
−
                            <p><i>
+
−
                                    <b>Table 1.</b> Dilutions to derive a standard curve for encapsulation efficiency
+
−
                                    quantification
+
−
                                </i></p>
+
−
                         </div>
+
−
                        <table>
+
−
                            <thead>
+
−
                                <tr>
+
−
                                    <th>
+
−
                                        Dilution
+
−
                                    </th>
+
−
                                    <th>
+
−
                                        BSA concentration (mg/mL)
+
−
                                    </th>
+
−
                                    <th>
+
−
                                        Volume of BSA stock solution 10 mg/mL (uL)
+
−
                                    </th>
+
−
                                    <th>
+
−
                                        Volume of ipty nanoparticle encapsulation supernatant (uL)
+
−
                                    </th>
+
−
                                    <th>
+
−
                                        Final volume (uL)
+
−
                                    </th>
+
−
                                </tr>
+
−
                            </thead>
+
−
                            <tbody>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        0
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        1
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0.26
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        2.6
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        97.4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0.52
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        5.2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        94.8
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        3
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0.78
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        7.8
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        92.2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        1.04
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        10.4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        89.6
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        5
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        1.4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        14
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        86
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                            </tbody>
+
−
                         </table>
+
−
                        <div class="mb-3">
+
−
                            <h5>
+
−
                                Experimental procedure</h5>
+
−
                            <ol>
+
−
                                <li>Place 6.5 μL of each pattern of BSA (0 mg/mL to 1.4 mg/mL) in sterile 0.6 mL tubes.</li>
+
−
                                <li>Add 193.5 μL of Bradford reagent to each tube. The final volume is 200 μL.</li>
+
−
                                <li>Vortex gently.</li>
+
−
                                <li>Incubate 5-45 min at room tiperature (until a change in color is noticeable).</li>
+
−
                                <li>Transfer 50 μL to a spectrophotometer cell.</li>
+
−
                                <li>Blank with the tube of null BSA concentration + Bradford reagent.</li>
+
−
                                <li>Take absorbance at 595 nm for the samples and record it.</li>
+
−
                                <li>Derive a standard curve for protein concentration in encapsulation supernatant.</li>
+
−
                            </ol>
+
−
                            <p><i>Note: There shouldn’t be a time difference higher than 10 minutes between each read.</i></p>
+
−
                        </div>
+
−
                        <div class="mb-3">
+
−
                            <h5><i>
+
−
                                    Efficiency quantification
+
−
                                </i></h5>
+
−
                            <p>
+
−
                                Refer to protein encapsulation protocol here using BSA.
+
−
                                NOTE: Calculate initial protein concentration before stirring and record it.
+
−
                            </p>
+
−
                            <p>
+
−
                                Experimental procedure</p>
+
−
                            <ol>
+
−
                                <li>Prepare eight 1 mL aliquots of loaded chitosan nanoparticles: four ipty, four
+
−
                                    containing
+
−
                                    the protein of interest.</li>
+
−
                                <li>Centrifuge aliquots at 13,400 rpm for 30 min.</li>
+
−
                                <li>Take 6.5 μL of the supernatant and measure absorbance at 595 nm with 193.5 μL of
+
−
                                    Bradford
+
−
                                    reagent. Riiber to incubate this mix at room tiperature 5-45 minutes (until a
+
−
                                    change of
+
−
                                    color is noticeable).</li>
+
−
                                <li>Record reads and estimate protein concentration in the supernatant using the
+
−
                                    previously
+
−
                                    derived standard curve.</li>
+
−
                                <li>Calculate encapsulation efficiency at this initial time as follows.</li>
+
−
                            </ol>
+
−
                        </div>
+
−
                        <div class="text-center">
+
−
                            <figure class="figure text-left">
+
−
                                <img src="https://static.igem.org/mediawiki/2018/9/99/T--TecCEM--Figure5Improvement.png"
+
−
                                    class="figure-img img-fluid rounded" alt="IMP-1">
+
−
                            </figure>
+
−
                        </div>
+
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
−
             <div class="row">
+
             <div class="row" id="components">
 
                 <div class="col">
 
                 <div class="col">
−
                     <a class="btn-link text-notebook" data-toggle="collapse" href="#liberation-and-stability" role="button"
+
                     <h1>Components</h1>
−
                         aria-expanded="false" aria-controls="liberation-and-stability">
+
                    <h2 id="collagen">Collagen</h2>
−
                        <h3>Protein liberation and stability protocol</h3>
+
                    <p>Collagen is the most abundant type of ECM used in experimental procedures to provide the cells
−
                     </a>
+
                        with a scaffold for the reconstruction of multiple tissues.</p>
−
                     <div class="collapse" id="liberation-and-stability">
+
                    <p>A typical molecule of collagen has a fibrillar structure that consists of a helix made of three
−
                        <p>
+
                        α chains. Most of the known types of collagen have a similar primary structure: Gly-X-Y, where
−
                            This protocol will assess the protein release and nanoparticle stability in aqueous
+
                        X and Y can represent any amino acid, which generally are proline and hydroxyproline (Hyp),
−
                            solutions.
+
                        respectively. The amino acids occupying X and Y sites have been shown to change the collagen
−
                            We quantified protein liberation by Bradford assay.
+
                        function. Furthermore, proline hydroxylation plays a crucial role by providing the triple helix
−
                         </p>
+
                        with improved thermostability and structural integrity. Despite, with a specific set of
−
                        <div class="mb-3">
+
                        conditions, exogenous Hyp has been shown to be activated by Pro-tRNA synthetase when supplied
−
                             <h4> <i>
+
                        in the culture medium. So, Hyp is successfully incorporated in protein synthesis.</p>
−
                                    Materials
+
                    <figure class="figure text-center">
−
                                </i>
+
                        <img src="https://static.igem.org/mediawiki/2018/7/7f/T--TecCEM--ColDes7.jpg " class="figure-img img-fluid rounded"
−
                            </h4>
+
                            alt="Scaffolddes1">
−
                             <ul>
+
                         <figcaption class="figure-caption"><strong>Figure 2. Collagen production.</strong></figcaption>
−
                                 <li>PBS pH 7.4</li>
+
                     </figure>
−
                                 <li>Bradford reagent</li>
+
 
−
                             </ul>
+
                     <h2 id="tenascin">Tenascin</h2>
 +
                    <p>Tenascin C or TNC is a protein that is located in the extracellular matrix forming a
 +
                        disulfide-bonded hexabrachion. This protein has a positive regulation in the regeneration and
 +
                        tissue remodeling, playing an important role in the regulation of this process. The TNC
 +
                        presence stimulates the migration of fibroblasts, furthermore inhibits the contraction of the
 +
                        fibrin-fibronectin matrix in order to prevent the premature contraction of the matrix before
 +
                        the adequate deposition of collagen. The third domain of TNC consists of a series of up to 15
 +
                        fibronectin type III-like repeats (TNCIII). The subdomain TNCIII5 has the responsibility of the
 +
                         binding with heparin. The binding between heparin and tenascin is involved in affinity with
 +
                        many growth factors, specifically with Fibroblast Growth Factor (FGF) as well as TGF-β and
 +
                        IGF-BP. The high affinity of TNC for an extensive range of growth factors is mediated mainly by
 +
                        the TNCIII5 domain and that, despite being a high-end affinity, a promiscuous binding occurs.</p>
 +
                    <figure class="figure text-center">
 +
                        <img src="https://static.igem.org/mediawiki/2018/a/a4/T--TecCEM--TenDes6.jpg " class="figure-img img-fluid rounded"
 +
                             alt="Scaffolddes1">
 +
                        <figcaption class="figure-caption"><strong>Figure 3. Tenascin production.</strong></figcaption>
 +
                    </figure>
 +
                    <div class="row my-3">
 +
                        <div class="col-6">
 +
                             <figure class="figure text-left">
 +
                                 <img src="https://static.igem.org/mediawiki/2018/3/31/T--TecCEM--TenDes5.jpg " class="figure-img img-fluid rounded"
 +
                                    alt="TEN-1">
 +
                                 <figcaption class="figure-caption">Figure 4. Heparin-Tenascin binding</figcaption>
 +
                             </figure>
 
                         </div>
 
                         </div>
−
                         <div class="mb-3">
+
                         <div class="col-6">
−
                             <h4>
+
                             <div class="text-center">
−
                                 <i>
+
                                 <video width="100%" height="240" controls>
−
                                     Standard curve derivation
+
                                     <source src="https://static.igem.org/mediawiki/2018/4/45/T--TecCEM--Tenani.mp4" type="video/mp4">
−
                                </i>
+
                                 </video>
−
                            </h4>
+
                             </div>
−
                            <ol>
+
−
                                <li>Use Bradford assay to derive a standard curve for a standard protein (BSA, for
+
−
                                    instance).
+
−
                                </li>
+
−
                                <li>Prepare 6 dilutions from a 10 mg/mL stock solution of the standard protein
+
−
                                    according to
+
−
                                    the
+
−
                                    table. A small-scale procedure was adapted from Sigma Aldrich to perform Bradford
+
−
                                    assay
+
−
                                    on
+
−
                                    the prepared dilutions.
+
−
                                </li>
+
−
                            </ol>
+
−
                            <p>
+
−
                                <i>
+
−
                                    <b>Table 1.</b> Dilutions for standard curve derivation using PBS
+
−
                                 </i>
+
−
                             </p>
+
 
                         </div>
 
                         </div>
−
                        <table>
+
                    </div>
−
                            <thead>
+
                    <h2 id="heparin">Heparin</h2>
−
                                <tr>
+
                    <p>Heparin is a highly sulfated glycosaminoglycan (GAG) which is known to be able to interact with
−
                                    <th>
+
                        multiple growth factors, improving its efficiency of function. Due to its capacity to increase
−
                                        Dilution
+
                        the efficacy of growth factors: vascular endothelial growth factor (VEGF) and fibroblast growth
−
                                    </th>
+
                        factors 1 and 2 (FGF-1 and FGF-2). In addition, heparin has the main characteristic of being an
−
                                    <th>
+
                        anti-aging factor.</p>
−
                                        Standard protein concentration (mg/mL)
+
                    <h2 id="chitosan">Chitosan</h2>
−
                                    </th>
+
                    <p>Chitosan (CS) is a biocompatible, biodegradable, and antimicrobial natural polymer. It is
−
                                    <th>
+
                        degraded into N-acetyl glucosamine by lysozymes and into carbon dioxide via glycoprotein
−
                                        Volume of stock solution 10 mg/mL (uL)
+
                        pathway.</p>
−
                                    </th>
+
 
−
                                    <th>
+
                    <p>CS is water-insoluble except in acidic mediums (< 6.5 pH), where amino groups act as weak bases,
−
                                        Volume of PBS pH 7.4 (uL)
+
                            being easily protonated. Furthermore, when positively charged, it’s able to interact with
−
                                    </th>
+
                            negative surfaces such as cell membranes, mucus lining, and anionic polymers. (Jose,
−
                                    <th>
+
                            Kunjanchan and Lammers, 2010). For CS to be employed as a delivery vehicle low isoelectric
−
                                        Final volume (uL)
+
                            value proteins are better encapsulated at a pH greater than the pI value (pH>pI), this is
−
                                    </th>
+
                            because of the electrostatic interactions. (Quan and Wang, 2007) Nanoparticle formation is
−
                                </tr>
+
                            favored by CS ability to bind to polyanions due to the formation of complexes between both
−
                            </thead>
+
                            oppositely charged entities. The polyanion we chose to work with was sodium
−
                            <tbody>
+
                            tripolyphosphate (TPP) under constant stirring.</p>
−
                                <tr>
+
                    <div class="text-center">
−
                                    <td>
+
                        <iframe width="560" height="315" src="https://static.igem.org/mediawiki/2018/9/97/T--TecCEM--Quitosan2.mp4"
−
                                        0
+
                            frameborder="0" allow="autoplay; encrypted-media" allowfullscreen></iframe>
−
                                    </td>
+
                    </div>
−
                                    <td>
+
                    <h2 id="growth">Growth factor</h2>
−
                                        0
+
                    <p>Leptin is a regulator weight hormone (16 kDa) that controls the velocity for reducing fat. This
−
                                    </td>
+
                        hormone is produced by adipocytes and secreted into the bloodstream. Leptin is mainly
−
                                    <td>
+
                        synthesized in adipocytes, including subcutaneous adipocytes. However, the synthesis of leptin
−
                                        0
+
                        and its receptors has been detected in human and mice fibroblasts and keratinocytes. First of
−
                                    </td>
+
                        all, leptin signalling goes like this:</p>
−
                                    <td>
+
                    <figure class="figure text-center">
−
                                        100
+
                        <img src="https://static.igem.org/mediawiki/2018/5/5f/T--TecCEM--MetabolicLep.jpg" class="figure-img img-fluid rounded"
−
                                    </td>
+
                            alt="Scaffolddes1">
−
                                    <td>
+
                        <figcaption class="figure-caption"><strong>Figure 5. Leptin pathway.</strong></figcaption>
−
                                        100
+
                    </figure>
−
                                    </td>
+
                    <p>Leptin and its receptor are also expressed by human hair follicles. Several studies have been
−
                                </tr>
+
                        shown that human follicles papilla cell lines (not neonatal human dermal fibroblast) express
−
                                <tr>
+
                        leptin mRNA and produce significant amount of leptin in vitro. Also leptin induces
−
                                    <td>
+
                        STAT3-dependent signalling in human keratinocytes. This have shown that mice whose follicular
−
                                        1
+
                        keratinocyte and epidermal lack functional STAT3 are viable and display seemingly normal skin
−
                                    </td>
+
                        and hair follicle morphology, both, hair follicle cycling and wound healing are severely
−
                                    <td>
+
                        compromised. So, if STAT3 is disrupted or in deficit, skin regeneration and keratinocyte
−
                                        0.26
+
                        migration is retarded, just like their hair cycle progression. </p>
−
                                    </td>
+
                    <div class="row my-3">
−
                                    <td>
+
                         <div class="col-6">
−
                                        2.6
+
                             <figure class="figure text-left">
−
                                    </td>
+
                                 <img src="https://static.igem.org/mediawiki/2018/a/a3/T--TecCEM--LeptinDes4.jpg" class="figure-img img-fluid rounded"
−
                                    <td>
+
                                     alt="BOB-6">
−
                                        97.4
+
                             </figure>
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0.52
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        5.2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        94.8
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        3
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        0.78
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        7.8
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        92.2
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        1.04
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        10.4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        89.6
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                                <tr>
+
−
                                    <td>
+
−
                                        5
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        1.4
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        14
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        86
+
−
                                    </td>
+
−
                                    <td>
+
−
                                        100
+
−
                                    </td>
+
−
                                </tr>
+
−
                            </tbody>
+
−
                        </table>
+
−
                         <div class="mb-3">
+
−
                             <ul>
+
−
                                 <li>For each dilution mix 6.5 uL of the sample and 193.5 uL of Bradford reactant for a
+
−
                                    final
+
−
                                    volume of 200 uL and leave it react for 20 minutes (Sigma Aldrich suggests 5-45
+
−
                                    minutes).</li>
+
−
                                <li>Read the absorbance of the riaining dilutions using dilution 0 as blank.</li>
+
−
                                <li>Graph absorbance reads vs concentration.</li>
+
−
                                <li>Use a linear trend to get the equation to compute protein concentration evaluating
+
−
                                     correlation coefficient.</li>
+
−
                             </ul>
+
 
                         </div>
 
                         </div>
−
                         <div class="mb-3">
+
                         <div class="col-6 text-center">
−
                             <h4><i>Protein release behavior</i></h4>
+
                             <video width="100%" controls>
−
                            <ol>
+
                                 <source src="https://static.igem.org/mediawiki/2018/4/45/T--TecCEM--Leptinanim.mp4" type="video/mp4">
−
                                 <li>Refer to protein encapsulation protocol to prepare enough loaded-nanoparticles for
+
                            </video>
−
                                    six
+
                        </div>
−
                                    1 mL
+
                        <div class="col-12 text-center">
−
                                    aliquots (some volume is lost in every transfer).</li>
+
                            <figcaption class="figure-caption"><strong>Figure 6. Leptin production</strong></figcaption>
−
                                <li>Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.</li>
+
−
                                <li>Discard supernatant.</li>
+
−
                                <li>Resuspend pellet in a volume of PBS pH 7.4 equal to the original volume.</li>
+
−
                                <i>NOTE: Given the low solubility of chitosan in neutral pH solutions, some protocols
+
−
                                    iploy
+
−
                                    mild to moderate sonication to disrupt possible non-dissolved pellet.</i>
+
−
                                <li>Prepare aliquots as previously stated.</li>
+
−
                                <li>Refer to protein encapsulation protocol to prepare enough ipty nanoparticles for
+
−
                                    six 1
+
−
                                    mL
+
−
                                    aliquots (some volume is lost in every transfer).</li>
+
−
                                <li>Centrifuge the total encapsulation volume at 20000 rpm for 20 minutes.</li>
+
−
                                <li>Discard supernatant.</li>
+
−
                                <li>Resuspend pellet in a volume of PBS pH 7.4 equal to the original volume.</li>
+
−
                                <li>Prepare aliquots as previously stated.</li>
+
−
                                <li>Label all aliquots to measure thi at time 0, 2, 4, 6, 12, 24, and 48 h. Store at 37
+
−
                                    °C
+
−
                                    and
+
−
                                    100 rpm.</li>
+
−
                                <li>At the right time, centrifuge the aliquots at 20000 rpm for 20 minutes.</li>
+
−
                                <li>Take 193.5 μL of Bradford reagent and mix with 6.5 μL of centrifugation
+
−
                                    supernatant.
+
−
                                    Vortex
+
−
                                    gently.</li>
+
−
                                <li>Incubate tube at room tiperature for 20 minutes.</li>
+
−
                                <li>Transfer 50 μL to a spectrophotometer cell.</li>
+
−
                                <li>Measure absorbance and calculate protein concentration in the supernatant using the
+
−
                                    previously derived standard curve. Blank should be PBS pH 7.4 + Bradford reagent as
+
−
                                    stated
+
−
                                    above.</li>
+
−
                            </ol>
+
 
                         </div>
 
                         </div>
−
                        <p><i>NOTE: to achieve a time-efficient protocol, a previous standardization of protein
 
−
                                encapsulation
 
−
                                efficiency is strongly suggested (refer to protein encapsulation efficiency
 
−
                                protocol).
 
−
                                Since
 
−
                                you already know your protein encapsulation efficiency, protein liberation
 
−
                                calculations
 
−
                                may
 
−
                                be
 
−
                                performed as follows.</i></p>
 
 
                     </div>
 
                     </div>
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                </div>
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                    <div class="row my-3">
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            </div>
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                        <div class="col-6">
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            <div class="row">
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                            <figure class="figure text-left">
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                <div class="col">
+
                                <img src="https://static.igem.org/mediawiki/2018/4/4d/T--TecCEM--LeptinDes3.jpg" class="figure-img img-fluid rounded"
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                    <a class="btn-link text-notebook" data-toggle="collapse" href="#nanoparticle" role="button"
+
                                    alt="BOB-8">
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                        aria-expanded="false" aria-controls="nanoparticle">
+
                            </figure>
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                        <h3>Nanoparticle stability</h3>
+
                        </div>
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                    </a>
+
                        <div class="col-6 text-center">
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                    <div class="collapse" id="nanoparticle">
+
                             <figure class="figure text-left">
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                        <p>
+
                                 <video width="100%" controls>
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                             When studying the nanoparticle behavior in a certain environment several studies are
+
                                     <source src="https://static.igem.org/mediawiki/2018/b/b1/T--TecCEM--Encapsulacion.mp4"
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                            carried out to assess particle stability throughout time. Such procedures comprise Z
+
                                        type="video/mp4">
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                            potential measurient and visual examination of size, shape, and particle physical
+
                                 </video>
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                            integrity. A stability monitoring is suggested as particles may change their shape,
+
                             </figure>
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                            degrade, and conglomerate when subjected to different stimuli. Such a study is helpful
+
−
                            to
+
−
                            predict the behavior of the created nanoparticles throughout time and greatly improves
+
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                            the
+
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                            design of drug release experiments. Here we include a suggested simple procedure to
+
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                            visually evaluate particle sizes and integrity. You can also use a Z potential
+
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                            measurient
+
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                            equipment, or NanoSight NS300, as we did.</p>
+
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                        <div class="mb-3">
+
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                            <h4><i>Transmission electron microscopy</i></h4>
+
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                            <ol>
+
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                                 <li>Refer to protein encapsulation protocol to prepare a final volume of 2 mL
+
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                                     chitosan
+
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                                    nanoparticles (loaded or ipty).</li>
+
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                                <li>Store at the desired conditions.</li>
+
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                                <li>Perform Ti preparation procedure on a 100 μL sample.</li>
+
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                                 <li>At relevant times observe to evaluate nanoparticle integrity (size,
+
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                                    conglomeration,
+
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                                    and shape).</li>
+
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                             </ol>
+
 
                         </div>
 
                         </div>
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                         <div class="mb-3">
+
                         <div class="col-12 text-center">
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                             <h4><i>NanoSight</i></h4>
+
                             <figcaption class="figure-caption"><strong>Figure 7. Leptin nanoencapsulation</strong></figcaption>
−
                            <ol>
+
−
                                <li>Refer to protein encapsulation protocol to prepare a final volume of 2 mL
+
−
                                    chitosan
+
−
                                    nanoparticles (loaded or ipty).</li>
+
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                                <li>Store at the desired conditions.</li>
+
−
                                <li>Dilute samples if required.</li>
+
−
                                <li>At relevant times evaluate nanoparticle size distribution (statistical data
+
−
                                    provided in the analysis sheet is useful to evaluate particle behavior).</li>
+
−
                            </ol>
+
 
                         </div>
 
                         </div>
−
                        <p><i>NOTE: Some devices like NanoSight NS500 are able to measure Z potential as well.</i></p>
 
 
                     </div>
 
                     </div>
 
                 </div>
 
                 </div>

Revision as of 20:37, 17 October 2018

Cell Gif

Description

Background

All over the globe, burns are a huge problem in the health sector, only in Mexico is the third cause of infant mortality and around 120 thousand people per year suffer a burn accident, based in the public health sector. However, there is not the required infrastructure needed to treat third-degree burns, which causes a high mortality rate (NTX, 2017).

Furthermore, the rate of child mortality from burns is 7 times higher in locations with low socioeconomic indicators, where each burn case could hit a cost of 141,750 USD due to the lack of specialized equipment and staff required to attend these delicate wounds. This problem has been ignored because of what the healing process implies, high costs and difficulties providing the specialized treatment. Then, we concluded that accelerating this process would have many advantages, not only in a faster skin regeneration but also it would have a psychological impact and reduce the economic cost.

The solution

Thus, we created TecTissue, a novel treatment that is capable of reducing wound healing time, preventing infections and consequently, decreasing mortality rate. We achieved this with synthetic biology, incorporating a growth factor and a scaffold.

TecTissue’s main objective is to provide families with a simple way to help in the reduction of medical bills and hospitalization time, make the recovery process easier and also create a chance of better life quality for the people that suffered from burns. We used recombinant proteins like collagen, composed by non-canonical amino acids. We also implemented a growth factor produced by humans called Leptin. This growth factor has been reported to be essential in different skin regeneration processes. There were some other molecules used in our project such as Tenascin and heparin. Both of them, with the company of collagen, compose our scaffold which is encapsulated with leptin in nanoparticles of chitosan, in order to have an efficient delivery in the skin.

We tested the development of TecTissue in fibroblast cell line L-929 and human mesenchymal cells. To demonstrate our project it was necessary to elaborate a Mini Skin Simulator were the cell lines were provided with the conditions they required in order to get a positive proliferation. This Mini Skin Simulator was automatized to create a friendly device for the manipulation of cells in a culture and to reduce the possible contamination.

Extracellular matrix

The extracellular matrix (ECM) is a non-cellular structure found on the outside of the cell. Among other features, the organization of the cells, tissue, organs and protection are the most important. It can be used as a biological material for damaged organs or tissue regeneration.

In order to generate an extracellular matrix, four molecules were used: a collagenous peptide, Tenascin type III-like, heparin and chitosan.

Scaffolddes1
Figure 1. Scaffold diagram.

Collagen V was shown to bind heparan sulfate proteoglycans through its heparin binding site (HepV) with higher affinity than other collagens. A recombinant fragment of the α1(V) chain (Ile824 to Pro950) binding site binds heparin and heparan sulfate by electrostatic interactions [4]. The purpose of including this domain was to promote the binding of heparin, which then interacts with tenascin C fibronectin type III domain V (TNCIII5), forming a matrix that boosts cell adhesion.

We supplemented the scaffold with a recombinant human leptin β (LepB) to accelerate cell proliferation, as we validated that it could achieve a controlled drug delivery when encapsulated. Due to its wide use as a biomaterial, chitosan nanoencapsulation was employed. The efficacy of the system was evaluated with MTT proliferation assay performed in 96-well plates.

Components

Collagen

Collagen is the most abundant type of ECM used in experimental procedures to provide the cells with a scaffold for the reconstruction of multiple tissues.

A typical molecule of collagen has a fibrillar structure that consists of a helix made of three α chains. Most of the known types of collagen have a similar primary structure: Gly-X-Y, where X and Y can represent any amino acid, which generally are proline and hydroxyproline (Hyp), respectively. The amino acids occupying X and Y sites have been shown to change the collagen function. Furthermore, proline hydroxylation plays a crucial role by providing the triple helix with improved thermostability and structural integrity. Despite, with a specific set of conditions, exogenous Hyp has been shown to be activated by Pro-tRNA synthetase when supplied in the culture medium. So, Hyp is successfully incorporated in protein synthesis.

Scaffolddes1
Figure 2. Collagen production.

Tenascin

Tenascin C or TNC is a protein that is located in the extracellular matrix forming a disulfide-bonded hexabrachion. This protein has a positive regulation in the regeneration and tissue remodeling, playing an important role in the regulation of this process. The TNC presence stimulates the migration of fibroblasts, furthermore inhibits the contraction of the fibrin-fibronectin matrix in order to prevent the premature contraction of the matrix before the adequate deposition of collagen. The third domain of TNC consists of a series of up to 15 fibronectin type III-like repeats (TNCIII). The subdomain TNCIII5 has the responsibility of the binding with heparin. The binding between heparin and tenascin is involved in affinity with many growth factors, specifically with Fibroblast Growth Factor (FGF) as well as TGF-β and IGF-BP. The high affinity of TNC for an extensive range of growth factors is mediated mainly by the TNCIII5 domain and that, despite being a high-end affinity, a promiscuous binding occurs.

Scaffolddes1
Figure 3. Tenascin production.
TEN-1
Figure 4. Heparin-Tenascin binding

Heparin

Heparin is a highly sulfated glycosaminoglycan (GAG) which is known to be able to interact with multiple growth factors, improving its efficiency of function. Due to its capacity to increase the efficacy of growth factors: vascular endothelial growth factor (VEGF) and fibroblast growth factors 1 and 2 (FGF-1 and FGF-2). In addition, heparin has the main characteristic of being an anti-aging factor.

Chitosan

Chitosan (CS) is a biocompatible, biodegradable, and antimicrobial natural polymer. It is degraded into N-acetyl glucosamine by lysozymes and into carbon dioxide via glycoprotein pathway.

CS is water-insoluble except in acidic mediums (< 6.5 pH), where amino groups act as weak bases, being easily protonated. Furthermore, when positively charged, it’s able to interact with negative surfaces such as cell membranes, mucus lining, and anionic polymers. (Jose, Kunjanchan and Lammers, 2010). For CS to be employed as a delivery vehicle low isoelectric value proteins are better encapsulated at a pH greater than the pI value (pH>pI), this is because of the electrostatic interactions. (Quan and Wang, 2007) Nanoparticle formation is favored by CS ability to bind to polyanions due to the formation of complexes between both oppositely charged entities. The polyanion we chose to work with was sodium tripolyphosphate (TPP) under constant stirring.

Growth factor

Leptin is a regulator weight hormone (16 kDa) that controls the velocity for reducing fat. This hormone is produced by adipocytes and secreted into the bloodstream. Leptin is mainly synthesized in adipocytes, including subcutaneous adipocytes. However, the synthesis of leptin and its receptors has been detected in human and mice fibroblasts and keratinocytes. First of all, leptin signalling goes like this:

Scaffolddes1
Figure 5. Leptin pathway.

Leptin and its receptor are also expressed by human hair follicles. Several studies have been shown that human follicles papilla cell lines (not neonatal human dermal fibroblast) express leptin mRNA and produce significant amount of leptin in vitro. Also leptin induces STAT3-dependent signalling in human keratinocytes. This have shown that mice whose follicular keratinocyte and epidermal lack functional STAT3 are viable and display seemingly normal skin and hair follicle morphology, both, hair follicle cycling and wound healing are severely compromised. So, if STAT3 is disrupted or in deficit, skin regeneration and keratinocyte migration is retarded, just like their hair cycle progression.

BOB-6
Figure 6. Leptin production
BOB-8
Figure 7. Leptin nanoencapsulation