組織工学のためのエレクトロ繊維のポスト処理

The aim of this procedure is to produce electro spun biodegradable scaffolds that can be sterilized and made thick enough and of appropriate mechanical strength for clinical tissue engineering. This is accomplished by first electro spinning biodegradable polymers into primary architectures, either random or reliant and orientated or sequentially layered. The second step is to apply post spinning modifications to the scaffolds by putting several layers together and sterilizing them.

Next cells are introduced to the scaffolds and their performance during and after culture is assessed. The final step is to subject the cells to an exercise regime to mimic the physiological stress the cells will encounter in the body in order to prepare them for transplantation. Ultimately, we can assess the response of cells to exercise by measuring the extracellular matrix they produce in response to dynamic tension.

This technique is versatile and flexible, allowing a large variety of scaffolds to be made with properties specifically modified for the required application. For example, scaffolds can be designed to treat difficult conditions where tissue repairs needed, such as replacement of extensive skin loss in badly burned patients or repair of damaged oral mucosa following cancer or trauma. Though this video shows a lasting production of cells as a marker of dynamic stress, you could also consider a range of extracellular matrix proteins as well as assess many other aspects of cell biology using exercised or non-exercise cells.

Generally, individuals new to this method will struggle to obtain reproducible scaffolds cause the electro spinning process is affected by many variables. Visual demonstration of the processing of electro spun scaffolds post spinning is, is important. It's rarely discussed.

A knowledge of the process is necessary for the technique to be successfully applied in the clinic. Begin by coating a rotating mandrel collector with aluminum foil with the shiny side facing outwards. Then place four five milliliter syringes loaded with five milliliters of the polymer of interest.

Here polylactic acid is used into a syringe pump. Set the flow rate to 40 microliters per minute per syringe, and the working distance to 17 centimeters from needle tip to mandrel. Next, charge the syringe needles to plus 17, 000 volts and electro spin from the appropriate distance onto the aluminum foil coated mandrel for aligned fibers.

Rotate the manl at 1000 RPM for random fibers. Rotate the manl at 200 RPM. Electro bun scaffolds can be stored on the aluminum foil in a seal container at four degrees Celsius in the presence of a desiccant for at least four months after electro spinning PHBV Lotus second polymer.

Again here. PLA has shown the layering on top of the PHBV then using the parameters and normal conditions for that polymer spin. Again, this additive process builds up a double layer of scaffold producing a bilayer to produce a multilayered scaffold by heater.

Kneeling place four sheets of PLGA on top of each other and then heat a kneel the sheets at 60 degrees Celsius for three hours to produce a multilayered scaffold by vapor kneeling. Pour 10 milliliters of DILU methane or DCM in an appropriate sized container. Then place four sheets of PLGA on top of each other and suspend them two centimeters above the DCM.

Finally, seal the container for one hour at room temperature to achieve aseptic scaffold production by electro spinning, dissolve medical grade polymers sterilized by incubation in DCM and then electro. Spin the polymer solution onto sterile foil wrapped around a sterilized mandrel. Then in a sterile lanar flow hood, incubate the scaffold in antibiotic-free growth media for the appropriate period to verify the sterility of the samples per experimental ethanol disinfection.

Place the scaffolds into an ethanol and distilled water solution for 15 minutes. For peracetic acid sterilization. Immerse the scaffolds in paracetic acid and PBS solution for three hours.

At room temperature for gamma sterilization, irradiate the scaffolds with a dose of three kilo gray using a caesium source first, cut the scaffolds into five millimeter by 20 millimeter rectangles. Then use a micrometer to measure the scaffold thickness. Then place the rectangles into a Bose electro force 3, 100 instrument.

Calculate the young's modulus and elasticity from the load versus displacement as a stress strain curve plotted by the instrument. Begin by labeling the cells with a fluorescent dye before seeding the cells. Then incubate confluence, human dermal fibroblasts in trypsin and EDTA for five minutes at 37 degrees Celsius.

Then spin down the detached cells for 10 minutes at 150 times G at room temperature and re suspend the pellet in five milliliters of DMEM. After counting, adjust the cell concentration for seeding and seed the cells onto the scaffolds. Now image the surface of the labeled scaffolds with a fluorescent microscope to investigate the penetration of cells Deeper into scaffolds, a multiphoton confocal microscope can be used.

This can achieve around a 200 micron penetration into most scaffolds with or without cells. Then after culturing the seeded scaffolds, fix the samples in one milliliter formaldehyde in PBS for 20 minutes at 37 degrees Celsius. After fixing, wash the samples three times with one milliliter of PBS, now incubate each of the samples in 200 microliters of elastin primary antibodies for 30 minutes at 37 degrees Celsius.

Wash the samples three more times in one milliliter of PBS and then incubate the samples in a solution of secondary antibody in PBS containing DPI for 30 minutes. Finally, after washing the samples one last time in PBS image, the samples on the fluorescence microscope using 365 nanometer excitation and 460 nanometer remission for the DPI and 480 nanometer excitation and 533 nanometer remission for the secondary antibody. First autoclave the flow regulation apparatus and the balloon then in a clean room, unpack the apparatus in a laminar flow hood in a position for electro spinning.

Next, connect a 50 milliliter syringe filled with PBS to the branch pipe of a three-way tap and inflate the balloon with the PBS until it is turgid. Now electro spin the desired polymer onto the balloon. Using the normal spinning conditions and a working distance of 10 centimeters, the wet fibers are sticky enough to adhere to the surface of the balloon without subsequently detaching.

Once the scaffold has dried, place the balloon into a sterile vessel and then transport the vessel to a lamina flow hood Suitable for cell culture. Culturing cells on a scaffold on a distended balloon to subject the cells to biomechanical conditioning is the trickiest part of this procedure. It is essential to base the balloon repeatedly with the cell solution in order to attach as many cells to the scaffold as possible.

We find this takes about 20 minutes. In the hood, remove the balloon from the vessel and place it onto a sterile surface, such as the Petri dish used here and pipette cell suspension onto the coated balloon every 20 seconds for 20 minutes to attempt to distribute the cells evenly over the balloon surface. Then place the balloon back into the culture vessel and add prewarm media appropriate to the cell type.

Finally, connect the inflation apparatus to a syringe pump and inflate and deflate the balloon as required to give biaxial distension. A computer controlled syringe pump can be used to achieve a more complex distension regime. Electro spinning can be utilized to create scaffolds with random and ordered architectures.

This is repeatable and the fibers are uniform. For example, perpendicular fibers can be created by electro spinning a set of aligned fibers onto aluminum foil by rotating the foil 90 degrees, and then immediately electro spinning a second set of aligned fibers on top of them. Many types of polymers can be electros spun with characteristics that can vary considerably as shown here for P-L-A-P-H-B-V-P-C-L and PLGA note that P-L-A-P-C-L and PLGA are all micro fibrous uniform scaffolds.

PHBV is spun as a pearl necklace with nano fibers connecting five to 20 micron sized beads. Here the scaffolds were initially spun using PHBV and then the syringes were filled with PLA and was spun on top of the PHBV scaffolds. These images show representative single PHBV and PLA layers.

This image illustrates what a cross section of A-P-H-B-V-P-L-A bilayer with a dense nano fibrous par necklace, PHBV layer and a more open micro fibrous PLA layer may look like all three scaffold types facilitate cell attachment and proliferation. If thicker scaffolds are required, vapor and heater kneeling can be employed to fuse layers of scaffolds together as demonstrated in these next few images. A kneeled scaffold layers do not delaminate and it can be very difficult to find the junction between layers.

This image shows a section through a vapor and kneeled PLA scaffold where initial fibrous scaffolds of approximately 150 microns have been placed together. And then DCM vapor was used to make much thicker scaffolds of up to 500 microns. This image shows the same section of the vapor nil bilayer at approximately two times The previous magnification in this image observe how the scaffold consists of layers of much thicker fibers in dispersed with layers of thinner fibers created by heater, kneeling layers of thin and thick fibers together to produce scaffolds of complex mechanical properties.

Once again, this image shows the same heater kneeled scaffold at four times. The previous magnification by layer membranes can be made with different cell types, each being cultured on a separate membrane without intermingling as demonstrated in these images of human dermal fibroblasts colored with two different fluorescent cell tracker dyes. The first image is a fibroblasts on electros bun PLA.

The cells were fixed and stained with here the dappy stained fibroblasts were seeded onto electros bun PHBV. In this experiment, the fibroblasts were prestained with the vital dye cell tracker green. Note that the cells are found on the PLA side of the bilayer.

This image is of a representative section through a bilayer with red stained fibroblasts on the lower PHBV surface and green stain fibroblasts on the upper PLA surface. In this image, the fibroblasts prestained with cell tracker red while growing on A-P-H-B-V surface. Thus, the use of vital fluorescent dyes provides a convenient methodology for looking at the distribution of cells on a scaffold while the cells are still growing.

The method of sterilization impacts the scaffold and the subsequent cell culture. This line graph illustrates the effects of paracetic acid, gamma irradiation, and ethanol on the ultimate tensile strength and young's modulus of A PLG. A scaffold.

Each of the sterilization methods changes the ultimate tensile strength and the elasticity of the scaffolds. The culture of cells on these scaffolds further reduces their ultimate tensile stress, but increases the elasticity. This bar graph illustrates the effect that the sterilization method has on the fiber diameter of an individual scaffold.

Gamma irradiation has no significant effect on fiber diameter, whereas paracetic acid and ethanol reduce fiber diameter by approximately 50%Thus, the best way to achieve sterile scaffolds is to produce them under aseptic conditions. This last series of images demonstrates that cells remain viable during dynamic distension and also produce increased amounts of elastin. Note the development of the dark blue color produced by the growing cells as detected with a metabolic indicator.MTT.

Here, the cells in blue cultured on a balloon and subjected to biaxial distension developed elastin fibers as indicated by the green staining. This contrasts markedly to the lack of elastin when the same cells are maintained under static conditions on an identical balloon scaffold. Following this procedure, other methods like static culture and fluid flow of the cells in the scaffolds can be performed to investigate how the cells respond to different culture conditions.

The development of culturing cells on a scaffold on a balloon, which can be distended, has proved very useful because it's paved the way for researchers in the field of tissue engineering to now explore the effect of dynamic conditioning of cells in tissue engineered constructs Don't forget that working with high voltage power supplies, solvents, lasers, and human cells can be hazardous and proper training and precautions must always be taken while performing this procedure.