This work was supported by the Swedish governmental strategic research program Trees and Crops for the Future (TC4F), VINNOVA, and the EC FP7 project ICON. Hannah Rasel is gratefully acknowledged for performing most of the experiments in the previous paper (Reference 5).

NOTE: Crambe seeds (cultivar Galactica) were supplied by the Plant Research International, Wageningen, Netherlands. Oil was extracted from the seeds by the method of Appelqvist13. Both the crambe meal and the wheat gluten were stored at -18 &#176;C until further use.
1. Dough Preparation
<ol>
	<li>Sieving crambe
	<ol>
		<li>Sieve the crambe meal with a round, fine-mesh stainless steel kitchen sieve (pore size: ~1.5 mm, 14 mesh), to remove large fiber fractions and uncrushed seeds. Store the sieved meal at -18 &#176;C to prevent material ageing.</li>
	</ol>
	</li>
	<li>Milling crambe
	<ol>
		<li>To reduce the particle size and make the material more homogeneous, mill the sieved crambe meal in a rotary ball mill.</li>
		<li>Mill 250 g of crambe meal each time in a 7 L jar with 21-25 mm diameter ceramic balls using a 53 rpm jar revolution rate and a milling time of 24 hr.</li>
	</ol>
	</li>
	<li>Conditioning
	<ol>
		<li>Before further processing, condition all the ball-milled crambe meal and wheat gluten powder in open jars for a minimum of 48 hr at 23 &#176;C and a relative humidity of 50% in a climate-controlled room.</li>
	</ol>
	</li>
	<li>Mixing the components
	<ol>
		<li>Grind the urea powder (stored in a closed beaker at ambient conditions) to fine particles with a mortar and pestle.</li>
		<li>Blend urea and glycerol (25.5 g of glycerol and 15 g of urea per 100 g of the final mixture).
		<ol>
			<li>Heat the glycerol to 65 &#176;C in a glass flask in an oil bath and add the urea powder slowly.</li>
			<li>Stir the mixture with a magnetic stirrer at 65 &#176;C until the urea powder is completely dissolved.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Mixing the crambe and wheat gluten
	<ol>
		<li>Blend the crambe meal powder and wheat gluten powder in a kitchen mixing machine for 5 min. For example, for a 60/40 (w/w) crambe/wheat gluten ratio, use 35.7 g of crambe meal and 23.8 g of wheat gluten per 100 g of the final mixture.</li>
	</ol>
	</li>
	<li>Mixing glycerol/urea with crambe/wheat gluten
	<ol>
		<li>Slowly add the glycerol/urea mixture to the crambe/wheat gluten blend in the kitchen mixing machine while stirring the mixture. Continue mixing for approximately 2 min, until a homogeneous dough is obtained. Prepare 500 g of the mixture each time.</li>
		<li>For the material with 60/40 (w/w) crambe and wheat gluten, use the following relative contents of the respective constituents: 35.7 g of crambe meal, 23.8 g of wheat gluten, 25.5 g of glycerol, and 15 g of urea (per 100 g). For the other two material combinations (i.e., 70/30 and 80/20), change only the crambe and wheat gluten contents. Keep the glycerol and urea contents the same as in the 60/40 combination.</li>
	</ol>
	</li>
</ol>
2. Film Extrusion
<ol>
	<li>Low-temperature profile
	<ol>
		<li>Perform the film extrusion in a twin-screw extruder. Set zones 1-10 (each 80 mm-long) along the extruder barrel at a low temperature profile (subsequently called &#34;low-T profile&#34;), as follows: 75-75-75-80-80-80-80-85-85-85 &#176;C. This prevents the wheat gluten from crosslinking in the barrel.</li>
		<li>Use a flat sheet die (45 mm x 0.7 mm) to extrude the films.</li>
		<li>Choose a screw speed between 30 rpm and 200 rpm and record the die pressure.</li>
		<li>Feed the dough manually through the hopper with the help of a wooden pusher to support the material flow towards the screws.</li>
		<li>At the die, pick up the extrudate with a conveyor belt operating at a speed of 2.0 m/min. Place air ventilation cooling (fans) along the belt.</li>
		<li>Run different die temperatures (105 &#176;C-(105 &#176;C), 110 &#176;C-(110 &#176;C), 125 &#176;C-(115 &#176;C), 130 &#176;C-(120 &#176;C), and 140 &#176;C-(125 &#176;C)) to select the conditions that give the smoothest extrudate with a minimum amount of voids. 
		NOTE: The values in the brackets correspond to the temperature in zone 11, next to the die. It is adjusted to reach the target temperature in the die.</li>
		<li>After extrusion, store the extrudates in sealed polyethylene bags until further processing or analysis in order to prevent ageing and atmospheric water absorption.</li>
	</ol>
	</li>
	<li>Film extrusion using the high-temperature profile
	<ol>
		<li>Extrude films as described in section 2.1, but use a high-temperature profile (subsequently called &#34;high-T profile&#34;), as follows: 85-85-85-100-100-100-110-110-120-120-120 &#176;C for zones 1 to 11 of the extruder.</li>
		<li>Use die temperatures of 125 &#176;C and 130 &#176;C.</li>
	</ol>
	</li>
	<li>Film extrusion after pelletization
	<ol>
		<li>In order to get pellets, extrude the material as continuous strands in the extruder using a two-strand die.
		<ol>
			<li>Use the low-T profile for the extruder barrel, as described above, and a 60-rpm screw rotational speed.</li>
			<li>Use different die-(zone 11) temperatures (130 &#176;C-(125&#176;C), 125 &#176;C-(115 &#176;C), 105 &#176;C-(100 &#176;C), and 85 &#176;C-(85 &#176;C)) to obtain the strands with the smoothest surfaces.</li>
		</ol>
		</li>
		<li>Pelletization
		<ol>
			<li>After passing the conveyor belt (the belt situated after the extruder that helps to feed material out of the extruder), feed the strands into a pelletizer operated with a cutting speed of 7 m/min.</li>
		</ol>
		</li>
		<li>Film extrusion from the pellets
		<ol>
			<li>Feed the pellets manually into the extruder and extrude films with the low-T profile inside the barrel and with a 125 &#176;C-(115 &#176;C) flat-sheet die temperature. Use a screw rotational speed of 30 rpm.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Film extrusion using volumetric feeding
	<ol>
		<li>In order to simulate automatic feeding (commonly used in industry), use the pellets previously extruded at 85 &#176;C (steps 2.3.1-2.3.2.1).</li>
		<li>Connect the feeder to the extruder and choose the volumetric feeder mode of the hopper.</li>
		<li>Use a feeding volume of 35 kg/h and hopper and extruder screw speeds of 16 and 120 rpm, respectively.</li>
		<li>Extrude with the low temperature profile of the barrel and use a die-(zone 11) temperature of 125 &#176;C-(115 &#176;C).</li>
	</ol>
	</li>
</ol>
3. Post-extrusion Process (Compression Molding)
<ol>
	<li>Pressing with the frame
	<ol>
		<li>For the first setup, cut two extrudates into pieces of 4.4 cm x 7.0 cm and 2.6 cm x 7.0 cm. 
		NOTE: This is needed because the frame is broader than the extrudates.</li>
		<li>Place them next to each other in an aluminum rectangular frame (70 x 70 x 0.5 mm3).</li>
		<li>Sandwich the frame between two aluminum plates using poly(ethylene terephthalate) (PET) films on both sides to prevent adhesion, and then place them into the press.</li>
		<li>Set the pressure gauge on the press to 200 or 400 bar.</li>
		<li>For each molding pressure, press the films with a plate temperature of 110, 120, and 130 &#176;C for 10 and 20 min.</li>
		<li>As a reference to the pre-extruded samples, press films from unextruded material. Center 7.2 g of fresh material (from section 1.6) in the aluminum frame.</li>
		<li>Press with the same parameter settings as for the pre-extruded films above (steps 3.1.4- 3.1.5).</li>
	</ol>
	</li>
	<li>Pressing without a frame
	<ol>
		<li>Cut out and sandwich rectangular samples (4.4 cm x 4.4 cm) between two aluminum plates using PET films on both sides to prevent adhesion.</li>
		<li>Place them into the press. Set the pressure gauge to 50 bar, 75 bar, or 100 bar.</li>
		<li>For each molding pressure, press films for 5 or 10 min using 110 &#176;C, 120 &#176;C and 130 &#176;C plate temperatures.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Aeschelmann, F., Carus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bio-based building blocks and polymers in the world. Capacities, production and applications: Status quo and trends towards 2020. , (2015).</li><li>T&#252;re, H., G&#228;llstedt, M., Kuktaite, R., Johansson, E., Hedenqvist, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+network+structure+and+properties+of+wheat+gluten+extrudates+using+a+novel+solvent-free+approach+with+urea+as+a+combined+denaturant+and+plasticizer.">Protein network structure and properties of wheat gluten extrudates using a novel solvent-free approach with urea as a combined denaturant and plasticizer.</a> Soft Matter. 7, 9416-9423 (2011).</li><li>Belyea, R. L., Steevens, B. J., Restrepo, R. J., Clubb, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+in+Composition+of+By-Product+Feeds.">Variation in Composition of By-Product Feeds.</a> J. Dairy. Sci. 72 (9), 2339-2345 (1989).</li><li>G&#243;mez-Estaca, J., Gavara, R., Catal&#225;, R., Hern&#225;ndez-Mu&#241;oz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+proteins+for+producing+food+packaging+materials:+A+review.">The potential of proteins for producing food packaging materials: A review.</a> Packag. Technol. Sci. , (2016).</li><li>Lazzeri, L., Leoni, O., Conte, L. S., Palmieri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+technological+characteristics+and+potential+uses+of+Crambe+abyssinica+products.">Some technological characteristics and potential uses of Crambe abyssinica products.</a> Ind. Crops and Prod. 3, 103-112 (1994).</li><li>Lalas, S., Gortzi, O., Athanasiadis, V., Dourtoglou, E., Dourtoglou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+Characterisation+of+Crambe+abyssinica+Hochst+Seed+Oil.">Full Characterisation of Crambe abyssinica Hochst Seed Oil.</a> J. Am. Oil Chem. Soc. 89, 2253-2258 (2012).</li><li>Carlson, K. D., Tookey, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crambe+Meal+as+a+Protein+Source+for+Feeds.">Crambe Meal as a Protein Source for Feeds.</a> J. Am. Oil Chem.Soc. 60, 1979-1985 (1983).</li><li>Massoura, E., Vereijken, J. M., Kolster, P., Derksen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteins+from+Crambe+abyssinica+oilseed.+II.+Biochemical+and+functional+properties.">Proteins from Crambe abyssinica oilseed. II. Biochemical and functional properties.</a> J. Am. Oil Chem. Soc. 75, 323-335 (1988).</li><li>Quispea, C. A. G., Coronadoc, C. J. R., Carvalho, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycerol:+Production,+consumption,+prices,+characterization+and+new+trends+in+combustion.">Glycerol: Production, consumption, prices, characterization and new trends in combustion.</a> Renew. Sust. Energ. Rev. 27, 475-493 (2013).</li><li>Kuktaite, R., Plivelic, T. S., T&#252;re, H., Hedenqvist, M. S., G&#228;llstedt, M., Marttila, S., Johansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+hierarchical+protein+polymer+structure:+urea+and+temperature+effects+on+wheat+gluten+films.">Changes in the hierarchical protein polymer structure: urea and temperature effects on wheat gluten films.</a> RSC Advances. 2, 11908-11914 (2012).</li><li>Bennion, B. J., Daggett, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+for+the+chemical+denaturation+of+proteins+by+urea.">The molecular basis for the chemical denaturation of proteins by urea.</a> Proc.Natl.Acad.Sci. 100, 5142-5147 (2003).</li><li>Rasel, H., Johansson, T., G&#228;llstedt, M., Newson, W., Johansson, E., Hedenqvist, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioplastics+based+on+agricultural+side-stream+products:+Film+extrusion+of+Crambe+abyssinica/wheat+gluten+blends+for+packaging+purposes.">Development of bioplastics based on agricultural side-stream products: Film extrusion of Crambe abyssinica/wheat gluten blends for packaging purposes.</a> J. Appl. Polym. Sci. 133, 42442 (2016).</li><li>Appelqvist, L. -. &. #. 1. 9. 7. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+studies+on+a+multisequential+method+for+determination+of+oil+content+in+oilseeds.">Further studies on a multisequential method for determination of oil content in oilseeds.</a> J. Am. Oil Chem. Soc. 44, 209-214 (1967).</li><li>Verbeek, C. J. R., van den Berg, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+Processing+and+Properties+of+Protein-Based+Thermoplastics.">Extrusion Processing and Properties of Protein-Based Thermoplastics.</a> Macromol. Mater. Eng. 295, 10-21 (2010).</li><li>Ralston, B. E., Osswald, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscosity+of+Soy+Protein+Plastics+Determined+by+Screw-Driven+Capillary+Rheometry.">Viscosity of Soy Protein Plastics Determined by Screw-Driven Capillary Rheometry.</a> J Polym. Environ. 16, 169-176 (2008).</li><li>Nur Hanani, Z. A., Beatty, E., Roos, Y. H., Morris, M. A., Kerry, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacture+and+characterization+of+gelatin+films+derived+from+beef,+pork+and+fish+sources+using+twin+screw+extrusion.">Manufacture and characterization of gelatin films derived from beef, pork and fish sources using twin screw extrusion.</a> J. Food Eng. 113, 606-614 (2012).</li></ol>

The methods and results were previously presented as an article by Rasel et al.5.

The reason the die pressure was of such high importance was probably due to the fact that the material needed a certain pressure to avoid bubble formation. However, the different components could phase separate if the pressure was too high. When extruding at too low a temperature, the cohesion was poor, possibly due to a low degree of crosslinking, while too high a temperature resulted in the release of gas (probably moisture together with urea and protein degradation products).
The two step extrusion (i.e., where strands were first extruded, pelletized, and then extruded again) resulted in a more homogeneous extrudate because of the more extensive blending that the first extrusion step provided.
The poor dough cohesion when decreasing the urea concentration from 15 to 10 wt% was probably due to a lower crosslink density. In analogy to this, a lower glycerol concentration, and thus a lower ability to dissolve the urea, resulted in poorer films unless a higher die pressure was applied.
Increasing the crambe meal concentration, and thus decreasing the wheat gluten concentration, resulted in a lower degree of aggregation/network formation. This lowered the viscosity of the material in the extrudate, resulting in the need to increase the die temperature to 130 &#176;C to raise the viscosity and generate homogeneous films.
It is difficult, if not impossible, to extrude plasticized crambe into films of sufficient quality for any use. We show here that this can be overcome by blending crambe with a more readily extrudable protein like wheat gluten. For the best quality, the extrudates need to be compression-molded in a separate step after extrusion.
It is shown here that extrusion works on a smaller scale, and upscaling is likely to be more demanding. Extrusion, along with injection molding, are the most important commercial methods for producing plastics. In order to replace existing conventional plastics, it is necessary that the protein material can be produced with the same techniques14-16. We show here that it is possible to extrude crambe oilseed meal with the help of wheat gluten.
Possible applications include packaging and applications for various extruded profiles (e.g., rods and cylinders). We consider the most critical step during the preparation of the samples to be the extrusion step. The final quality of the films depended strongly upon the extrusion parameters and the properties of the material prior to extrusion.

When upgrading a material from a low value to a higher value, two main issues need to be considered: the type of potential end-product(s) and the required properties. This study focuses on the extrusion of protein-based plastics for potential use in packaging for two reasons. The present variety of packages is extensive, but the request for renewable and biodegradable low-cost packaging material has increased rapidly in the last decade. This trend seems to be continuing, as most brand owners and legislators are searching for options to create plastics from petroleum1. The required material properties for packaging are, in many cases, more demanding than for other plastic products. However, if a successful material is obtained, the potential market is very large.
Packaging material needs to fulfil a number of criteria to be suitable. The exact criteria differ depending on the type of package, filling/sealing systems, transport, storage, content, appearance, product design, etc. All these parameters should be considered by a packaging developer, but all cannot be of the highest priority at once when initiating the development of a new and unexplored material. The properties in focus for this study were the mechanical and barrier performances.
Extrusion is the processing method of choice for two reasons: extrusion is a common and efficient method for making packaging plastics, and it generally does not involve a solvent, as in solution casting. Thus, no drying step is needed at the end of the process2.
Wheat gluten is also a side stream material coming from a starch product3. It has shown potential as a packaging plastic in a number of studies. Despite this, some challenges remain4. Crambe abyssinica is an interesting oilseed plant in that it is not a food resource and can be grown in many different agronomic conditions5,6. As with wheat gluten, crambe protein is a byproduct, in this case, from oil production. It is obtained as a defatted crambe meal, with protein as the largest component. It also contains a sizeable amount of nitrogen-free extracts, such as carbohydrates and fiber7,8. The meal has relatively poor cohesive properties and needs to be blended with a material of higher cohesion. In this study, wheat gluten is used as a supportive additive to the crambe meal. To improve the toughness/extensibility of the protein material, a plasticizer is commonly used as an additive as well. In this study, glycerol is used, which is a side product of the plant oil industry (e.g., rape seed methyl ester fuels) and is readily available at a low cost9. Urea, also renewable, is used as a denaturant in order to give the extrudate the proper cohesion2,10,11. It may also work as a plasticizer.
Renewable materials, especially those that are used directly from nature, without purification, modification, or chemical synthesis, are, in most cases, not suitable for high-temperature treatment. The challenge is to find suitable processing parameters and compositions that result in an extrudate with properties that allow it to compete with products from petroleum.
This study focuses on the characterization of the mechanical and barrier properties of a new bio-based material produced from crambe meal processed with different additives and at different conditions12. The full details of the mechanical and oxygen barrier features are found in Rasel et al.12.

<table border="0" cellpadding="0" cellspacing="0" width="889">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td>Crambe meal</td>
			<td>Plant Research International</td>
			<td></td>
			<td>Defatted crambe meal, Residual from oil extraction of cultivar Galactica seeds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Wheat gluten</td>
			<td>Lantm&#228;nnen Reppe AB</td>
			<td style="width:119px;"></td>
			<td>It contains 77 % (w/w) gluten, 8.1 % (w/w) starch and 1.34 % (w/w) fat.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Glycerol</td>
			<td>Karlshamn Tefac AB</td>
			<td style="width:119px;">99.5 % purity</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Urea</td>
			<td style="width:169px;">Sigma Aldrich</td>
			<td style="width:119px;">purity &#8805; 99.5 %</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">The dough&#160;</td>
			<td style="width:169px;"></td>
			<td style="width:119px;"></td>
			<td>(per 100 g) prepared with 35.7 g crambe meal, 23.8 g wheat gluten, 25.5 g glycerol and 15 g urea, hence with a liquid (glycerol/urea) to solid (crambe/wheat gluten) ratio of 0.342.</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Round, fine meshed stainless steel kitchen sieve</td>
			<td style="width:119px;"></td>
			<td>Sieve the crambe meal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Rotary ball mill</td>
			<td>Pascal Engineering</td>
			<td style="width:119px;"></td>
			<td>Milling crambe/The volume of the mill house is 7 l and it contained 215 ceramic balls, each with a diameter of 25 mm.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mortar and pestle</td>
			<td style="width:169px;"></td>
			<td></td>
			<td>Grinding urea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Kitchen machine Cloer 660</td>
			<td>Cloer</td>
			<td style="width:119px;"></td>
			<td>Blending crambe and wheat gluten</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Twin-screw extruder Type LTE20-48</td>
			<td style="width:169px;">Labtech Engineering LTD</td>
			<td style="width:119px;"></td>
			<td>Compounding and film extrusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat sheet die</td>
			<td style="width:169px;"></td>
			<td style="width:119px;"></td>
			<td>Produce extruded flat films with a cross-section of 45 mm x 0,7 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Air Cooling Conveyor Unit type LAC-2.6</td>
			<td>Labtech Engineering LTD</td>
			<td style="width:119px;"></td>
			<td>Used in the extrusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pelletizer Type LZ-120</td>
			<td>Labtech Engineering LTD</td>
			<td style="width:119px;"></td>
			<td>Making pellets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polystat 200T Hot Press&#160;</td>
			<td>Servitec Machine GmbH</td>
			<td style="width:119px;"></td>
			<td>Hot press to press extrudates</td>
		</tr>
	</tbody>
</table>

film extrusion of crambe abyssinica/wheat gluten blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

The blended materials (60 wt% crambe meal and 40 wt% wheat gluten) resulted in a tough dough after the initial mixing procedure. The material was rested for a few minutes before the first extrusion. However, the dough had a too high a viscosity to be able to be fed into the extruder hopper in a regular manner. Therefore, it was fed piece-by-piece, directly into the screw. The screws had a constant speed, and the resulting film extrudate was continuous and had a visually smooth surface. An example of an extruded film is shown in Figure 1.
The die pressure and temperature were found to be the two most important processing parameters to control in order to obtain homogeneous and smooth film extrudates. Too low a die temperature, typically below 110 &#176;C, did not result in continuous film extrudates, whereas a temperature above 130 &#176;C resulted in the formation of bubbles in the material. The most suitable die temperature to obtain homogeneous and smooth films was found to be around 125 &#176;C.
To get the most homogeneous extrudates, a two-step process was found to be advantageous, where, in the first step, strands were extruded at a lower temperature (typically 85 &#176;C) and pelletized. The pellets were then fed to the hopper for the second extrusion step.
When the urea content was decreased from 15 to 10 wt%12, the cohesion of the dough decreased substantially, resulting in a powder-like material; no continuous film could be extruded12.
When the glycerol concentration was decreased (with a retained 15 wt% urea), the dough was found to be more brittle, and the urea did not fully dissolve in the glycerol. Also, a considerably higher die pressure was required to get homogeneous films. However, these films were found to be smoother and more homogeneous than those with a higher glycerol content.
When increasing the crambe meal powder concentration and decreasing the wheat gluten concentration, the extruded films appeared darker, but also smoother and more homogeneous5. The feeding rate could also be increased12. The drawback was that the films were only partially continuous, and film breakages appeared a few meters apart. However, when increasing the die temperature to approximately 130 &#176;C, continuous films could be prepared, although with some discolorations12.
Compression molding without a frame yielded thin (thickness: 0.1-0.2 mm) films that were very flexible and translucent (Figure 2).
Depending on how the extrudates were made and what they contained, the stiffness ranged from 4.9-5.6 MPa and the strength from 0.3-0.7 MPa, whereas the extensibility ranged from 7 to 16%12. The corresponding values for the extrudates after compression molding were 6.4-15.0 MPa, 0.3-1.1 MPa, and 8-19%5. Details of the mechanical measurements are given in reference12. 64 mm-long dumbbell specimens were tensile-tested according to ASTM D882-02 at 23 &#177; 1 &#176;C and 50 &#177; 1% RH, with a crosshead speed of 10 mm/min. Figure 3 shows the importance of extruding crambe with the addition of wheat gluten. The strength, and especially the extensibility, decreased with decreasing wheat gluten content. The oxygen permeability ranged from 17 to 39 cm3 mm/(day m2 atm), depending on the composition and whether a compression molding step (with a frame) was used or not.
<img alt="Figure 1" src="/files/ftp_upload/54770/54770fig1.jpg" /> 
Figure 1: Extruded material. Extruded film using a die temperature of 130 &#176;C. It contains 35.7 wt% crambe, 23.8 wt% wheat gluten, 25.5 wt% glycerol, and 15 wt% urea. The width of the film is 44 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54770/54770fig2.jpg" /> 
Figure 2: Compression-molded films. Extruded material compression-molded, without a frame, into thin, translucent films using a 130 &#176;C press temperature at 75 bar for 10 min. The flat and wrinkled films are from the same material. The width of the left film is ~17 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54770/54770fig3.jpg" /> 
Figure 3: Mechanical properties as a function of crambe content. Maximum stress (filled circles) and strain at maximum stress (open circles) as a function of crambe content in the crambe/wheat gluten mixture. The error bars represent the standard deviations. Maximum stress was obtained from the maximum force per initial sample cross-section of the narrow part of the dumbbell specimen (narrow section: 16 mm long and 4 mm wide). The low-temperature profile was used with an initial die temperature of 125 &#176;C and a zone 11 temperature of 115 &#176;C. The screw speed was 30 rpm, and the extrusion was performed without prior pelletization. Data obtained from reference12. <a href="http://cloudflare.jove.com/files/ftp_upload/54770/54770fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Film Extrusion of Crambe abyssinica/Wheat Gluten Blends at JoVE.com

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

film-extrusion-of-crambe-abyssinicawheat-gluten-blends

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Bioengineering

9.9K Views.  SIG Combibloc. The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Video: Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Rat Model of Adhesive Capsulitis of the Shoulder

This proposal aims to create an in vivo rat model of adhesive capsulitis for researching potential treatment options for this condition and other etiologies of comparable arthrofibrosis. The model includes extra-articular fixation of the shoulder in rats via scapular to humeral suturing, resulting in a secondary contracture without invading the intra-articular space and resulting in decreased rotational ROM and increased joint stiffness.
We used 10 Sprague-Dawley rats for the purpose of this study. Baseline ROM measurements were taken before glenohumeral immobilization. The rats were subjected to 8 weeks of immobilization before the fixation sutures were removed and changes in ROM and joint stiffness were evaluated. To evaluate whether immobilization resulted in a significant reduction in ROM, changes in kinematics were calculated. ROM was measured at each time point in the follow-up period and was compared to the baseline internal and external ROM measurements. In order to evaluate the stiffness, joint kinetics were calculated by determining the differences in torque (text and tint ) needed to reach the initial external rotation of 60&#176; and initial internal rotation of 80&#176;.
After the removal of the extra-articular suture fixation on follow-up day 0, we found a 63% decrease in total ROM compared to baseline. We observed continuous improvement until week 5 of follow-up, with the progress slowing down around a 19% restriction. On week 8 of follow-up, there was still an 18% restriction of ROM. Additionally, on follow-up day 0, we found the torque increased by 13.3 Nmm when compared to baseline. On week 8, the total torque was measured to be 1.4 &#177; 0.2 Nmm higher than initial measurements. This work introduces a rat model of shoulder adhesive capsulitis with lasting reduced ROM and increased stiffness.

This protocol presents an in vivo rat model of adhesive capsulitis. The model includes an internal fixation of the glenohumeral joint with extra-articular suture fixation for an extended time, resulting in a decreased rotational range of motion (ROM) and increased joint stiffness.