The overall goal of this procedure is to engineer freestanding nano fibers, nanostructures, and 2D matrices from single or multiple extracellular matrix proteins using surface initiated assembly. This is accomplished by first preparing poly dimethyl soane or PDMS stamps by pouring the PDMS pre polymer onto a topographically patterned master mold and allowing it to cure. The second step is to cut out the cured PDMS stamps and coat them with a solution containing the desired extracellular matrix proteins.
Next, the stamps are washed, dried, and micro contact printed onto a poly N iso propyl acrylamide, or pipe PAM coated glass cover slip. The final step is to hydrate the cover slip with warm 40 degrees Celsius deionized water and allow it to gradually cool reduction of the solution. Temperature below about 32 degrees Celsius triggers the dissolution of the thermally sensitive pipe PAM layer and the release of assembled protein nano fibers or nano structures.
Ultimately, the assembled nano fibers and nano structures are freestanding in solution as confirmed by phase contrast and or fluorescence microscopy, and can be used in further applications such as tissue engineering scaffolds. The main advantage of this technique over existing methods such as face separation or electro spinning, is the ability to engineer ECM protein nano fibers using a technique that really mimics the way cells normally build ECM fibers in vivo. The surface initiated assembly technique has a number of unique advantages, including the capability to control protein composition, fiber morphology, and scaffold architecture.
Further, we can engineer ECM protein nano fibers from extracellular matrix proteins such as fiber nin laminate, and collagen type four, which has proved difficult using other types of methods. This method can help answer key questions in the field of tissue engineering by enabling researchers to develop constructs with specific and well-defined physical and chemical cues to direct a variety of cell behaviors such as adhesion and differentiation. If you're new to this technique, you should remember to continuously inspect the quality of your stems as well as the fidelity of the micro contact printed patterns.
This is essential to obtain proper assembly of the ECM nano fibers and nanostructures. To begin this procedure, prepare the poly dimethyl soane or PDMS pre polymer by combining the elastomer base and curing agent in a 10 to one weight per weight ratio, typically 80 grams of base and eight grams of curing agent are used to ensure there is sufficient PDMS to cover the master mold in a one centimeter thick layer mix and DGAs the PDMS using a centripetal mixer set to the following mix at 2000 RPM for two minutes, DGAs at 2000 RPM for two minutes. Poor enough.
PDMS pre polymer over the master mold to form a one centimeter thick layer here, the PDMS by baking at 65 degrees Celsius for four hours. Once cured, use a scalpel to cut out the region containing the pattern to form the PDMS stamp. To distinguish the feature side from the backside of the PDMS stamp.
Cut a notch out of one of the corners on the backside of the stamp. Begin this procedure by cleaning the 25 millimeter diameter glass cover slips, sonicate the cover slips in 95%ethanol for one hour, and then dry them in a 65 degrees celsius oven. Next, prepare the poly and isopropyl acrylamide or piam solution dissolve piam in one butanol at a concentration of 10%Center a clean glass cover slip on the vacuum chuck of the spin coer and pipette 200 microliters of the pipe Pam solution onto the glass surface to cover the surface entirely.
Spin coat the cover slip at 6, 000 RPM for one minute. Clean the PDMS stamps by sonication in 50%ethanol for 30 minutes, and then drying under a stream of nitrogen drying and all subsequent steps should be performed in a biosafety cabinet to maintain sterility for applications where the extracellular matrix or ECM nano structures will be used with cells. The micro contact printing step is the most difficult aspect of this procedure.
It's important to inspect the PDMS stamps for any visible defects prior to use. Also, do not use protein solution that is older than two weeks Place. The PIAM coated cover slips inside a closed Petri dish and sterilize using UV exposure.
45 minutes under the UV light in a biosafety cabinet is sufficient. Coat the pattern surface of each PDMS stamp with 200 microliters of the protein solution. Fibronectin or FN is used here at a concentration of 50 micrograms per milliliter in sterile distilled water.
Incubate for one hour at room temperature after one hour wash the PDMS stamps and distilled water to remove excess protein and dry thoroughly under a stream of nitrogen. It is important to remove the water completely to avoid premature dissolution of the pipe. Pam coating on the cover slip and improper protein transfer.
Perform micro contact printing by placing the feature side of the PDMS stamp in contact with the pipe PAM coated cover slip if required. Use forceps to tap lightly on the back of the stamps to remove any air bubbles and ensure uniform contact after five minutes. Peel off the PDMS stamp from the cover slip.
At this stage. Depending on the study, additional ECM proteins can be patterned to create more complex and multi-component structures. Once the ECM pattern has been printed, place the patterned pipe PAM coated cover slip in a 35 millimeter Petri dish and inspect the pattern fidelity using phase contrast microscopy.
Depending on the pattern, A CCD camera may be necessary to resolve the features of the pattern. Fluorescence microscopy can also be used to inspect the pattern provided the ECM proteins are fluorescently labeled. After verifying pattern fidelity add three milliliters of 40 degrees Celsius distilled water to the Petri dish and allow the water to gradually cool the dissolution of the pipe.
PAM layer and the release of the ECM protein patterns can be monitored using phase contrast microscopy. Typically to ensure the ECM protein nanostructures have been released, the water is cooled to room temperature well below the lower critical solution temperature of pipe, pam, which is 32 degrees Celsius. If the application does not permit the use of optical techniques, the release can be monitored by measuring the solution temperature.
Nano fibers and other nano structures will be floating in the water after the release of the pipe PAM layer. For further applications, the nano fabrics and other nano structures need to be manipulated, but the exact approach will depend on the experimental objective. Representative results are presented here to demonstrate that surface initiated assembly or SIA is capable of engineering ECM protein nano fibers with precise control over fiber dimensions.
An array of fibronectin rectangles, 50 micrometers in length and 20 micrometers in width were patterned onto a piam coated cover slip addition of 40 degrees Celsius DI water and subsequent cooling below the lower critical solution. Temperature of piam triggered the dissolution of piam and the release of the fibroin nano fibers upon release. The fibers contracted because they were under an inherent prestress.
When patterned onto the piam surface Alice analysis of the fibronectin nanofiber dimensions pre-release revealed they were mono disperse with an average length of 50.19 plus or minus 0.49 micrometers and average width of 19.98 plus or minus 0.17 micrometers. Upon release the nano fibers contracted appreciably but remained mono disperse with an average length of 14.15 plus or minus 0.92 micrometers and average width of 2.65 plus or minus 0.32 micrometers. Atomic force microscopy provided a higher resolution perspective of the fiber dimensional changes associated with the SIA release process.
Notably, pre-release fibers had a uniform thickness of about five nanometers, whereas post-release fibers had a thickness on the order of several hundred nanometers while the length and width decreased. Using the SIA process, it is possible to engineer a variety of ECM protein nanostructures with tunable size, shape, and composition. For example, fibronectin nano fibers initially 20 micrometers in width and one centimeter in length were patterned onto a pipe.
PAM coated cover slip upon cooling and pipe. Pam dissolution, the nano fibers were released forming long threads with a reduced width of about three micrometers. Furthermore, since the pattern is defined by the surface topography of the PDMS stamp used for micro contact printing, it is possible to engineer complex ECM protein nanostructures as proof of concept.
Multi-armed fibroin stars were created. Thermal release resulted in the contraction of the arms, but not the central region of the star where the arms joined together. SIA also works with other ECM proteins such as laminate or LN, and multiple ECM proteins can be incorporated into the same structure.
In this example, orthogonal interconnected, 20 micrometer wide lines of fibronectin shown in red and 50 micrometer wide lines of laminin shown in green integrated into A 2D nano fabric were patterned and then released upon release both types of nano fibers contracted, but the overall interconnectivity and square lattice structure was maintained. These results demonstrate that SIA can be used to engineer ECM materials with a variety of compositions and structures. Lastly, some instances of failed SIA of ECM nano fibers are shown.
One cause is improper release of an incomplete pattern due to poor transfer of ECM protein to the piam surface. During micro contact printing, the presence of holes, irregular edges and other defects will create nano fibers and nanostructures that are incomplete and prone to breakage and fragmentation upon release. Rapid dissolution of pipe PAM can also cause poor pattern fidelity after release.
For example, using 20 degrees Celsius water, a temperature already below the lower critical solution. Temperature of piam will cause the piam to rapidly swell and dissolve. This can cause the nano fibers to snap back and break as illustrated in the 32nd image and form random unorganized configurations as illustrated in the 52 second image.
Before starting this procedure, the condition of the PDMS stems for defects and also make sure that all surfaces are free of dust. Otherwise, it'll prevent proper protein transfer and lead to poor assembly of the A CM structures. So following this procedure, the release nano fibers or nanostructures can be used to do a number of things such as analyze mechanical properties of the protein fibers, or used as scaffolds for tissue engineering.
After watching this video, you should have a good understanding of how to engineer ECM protein nano fibers and nanostructures with tuneable composition, geometry and architecture. Specifically, you shall understand how to micro contact print patterns of ECM proteins onto python coated glass cover slips, and then thermally triggering the dissolution of the surface to assemble the ECM structures.
