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Chapter 3
Charatherization
To ensure that our scaffold was suitable for our purposes we managed a series of tests. It’s really important in this type of research made this tests because our scaffold were thought to put in a human body and they have to respect a specific characteristic.
3.1 SEM
In the Scanning Electron Microscope (SEM) an electronic gun produce an electron beam which is focused whit great intensity (producing one “spot”) in a point of the surface thanks to a system of lenses. This spot is moving rapidly back and forth on the sample ad the scanning movement and the scanning movement is effected by the deflector of beam, a system of electron loads placed between the condenser and the sample.
The intense spot of electron that hits the sample excite the molecules that are found on the surface and makes their reach extremly high energy states. These molecules re-emit this energy in different forms, including high-energy electrons called
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secondary electrons (SE).
In a SEM, the image is built by the secondary electrons rather than the illuminating beam.
The secondary electrons that move away from a point of surface are captured by a detector placed on the side of the surface and converted in an electrical pulses and sent in a real time to a monitor where there is a another similar scanning at same time. The result is a black and white image with a really high resolution and large depth of field totally similar to a normal picture.
For this reason the SEM image are immediately intelligible and intuitive to understand.
The normal resolution of SEM is around 5 nm. The sample is analyzed under high vacuum because the air could stop the electron beam and the sample has to be metalized to avoid that the secondary electrostatic charges may disturb the scanning.
3.2 DSC
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the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature.
The characterization of thermal properties of scaffold was made by DSC 7. The samples was put in an aluminum mold and a second mold was a control. Both the mold was put in calorimetric cell with a continue flow of nitrogen to maintain inert the atmosphere. A software make a regulation of the temperature in the furnace and add or remove heat to the cell with a predetermined velocity. The samples’s temperature was been registered by thermocouples and the instrument calculate the difference between the two samples and transformed it in energy with the obligation that ΔT=0.
If there are no transition the value of ΔT remain constant otherwise the instrument registered a positive or negative value depending on whether the event is esothermic or endothermic.
The final result is an experimental curve that show the dependence of heat flow from temperature.
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The spectroscopy techniques allow to measure the absorbance, the emission or dispersion of electromagnetic radiation.
The absorption spectroscopy techniques, like IR or UV-vis, measure the energy absorbed form the sample as a difference from the intensity of incoming and transmitted radiation. The radiation make possible tha passage of electron from a low to high energetic level and just a determined light energy can do this because the nergy level are quantized.
The type of excitation depends on wavelenght. The wavelenght of he IR radiation is from 2.5 to 25 µm but, generally, the information to detect the organic compounds are between 2.5 to 15 µm.
In a IR-scanning a photon beam with different energy are sent on a sample. The beam cover all the IR spectrum and a detector measure the intensity of exiting energy to determinate which radiation are absorbed and how much.
An absorbance spectrum report the quantity of absorbed light in function of wavelenght and, because it depends on the energy level of the sample, the spectrum will be different for any substance. In other hands it is possible to obtain a quantity
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analysis because the size of the spectrum will depend on how much substance is in. In a spectrum is it possible to see just the absorbance of wavelenght that correspond to a transition between roto-vibrational levels.
The molecular vibrations are classified acording to the movement: Stretching: the movement is along the bond axis
Bending: the vibration will change the angle of bond
Every vibration will determinate a specific spectrum in a specific wavelength and the form and the position of the spectrum make possible to understand how the sample is made.
3.4 Chemical imaging
The last generation instrument of IR-spectroscopy have a device of micro pointing with a software control. That allow the perfect reconstruction of the IR-image and of the spectral map.
The analytical spectrum used is between 5000 and 300 cm-1. The high number of vibration make possible that also a simple molecules can have a high number of absorbance bands a really difficult interpretative spectrum. The chemical imaging
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allows to obtain information about chemical composition, structure and distribuition of materials.
3.5 Degradation test
To understand the possible behavior of our scaffold in human body we performed a degradation test in saline solution, particularly we used PBS (Phosphate buffered saline,is a water-based salt solution containing sodium phosphate, and, in some formulations, potassium chloride and potassium phosphate. The osmolarity and ion concentrations of the solutions match those of the human body which has the task to emulate the human body fluid).The test was performed using the samples and putting their in a 10 mL of olution. Then the samples was put into a shaker at 37°C for 1 – 3 – 7 – 14 – 21 – 28 days. We measured the weight of the samples before and after the test to be certain that there was a degradation. As a control we used a porous scaffold made with just pure PGS.
3.6 Porosity
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this calculation we can get a first idea about the goodness of our scaffold. The calculation is (1,138 −1,138𝑊
𝑉 ) ∗ 100 where W is the weight of the sample and V is
the volume of the sample.
3.7 Ink test
To correlate the theoretical porosity with the effective porosity we did a test with colored ink to evaluate the velocity that the sample employs to absorb a solution. More porosity mean more velocity.
3.8 Permeability test
Permeability measures the ability of fluids to flow through a porous material. The permeability is a constant in Darcy’s formula which binds the pressure gradient to the flow rate through two constant:
The viscosity of the fluid
The porous characteristic of the material Permeability is described by Darcy’s formula: 𝑘 = µ𝑙𝑉
𝑆∆𝑝 where k is the permeability in
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meters), µ is the viscosity of the fluid, V is the volumetric flow rate and Δp is the pressure difference at the ends of the sample.
To perform a permeability test we used a machine with a peristaltic pump to generate a water flow, a cell where we put a sample, a manometer, a cylinder and a chronometer.
The pump generate a pressure measurable with the manometer, the water flows in the sample and the operator take the time that the water employs to through the sample and fill the cylinder. So we have all the date to calculate the permeability by Darcy’s formula.
3.9 Cell colture test
To be sure that our scaffold were suitable to a future transplant we performed a cell culture test. The tests were done with C2C12 murine cell. These cells are a mouse myoblast cell line. C2C12 cells were originally obtained by Yaffe and Saxel through serial passage of myoblasts cultured from the thigh muscle of C3H mice after a crush injury. These cells are capable of differentiation. C2C12 cells are a useful tool to study the differentiation of myoblast and osteoblast, to express various proteins, and to
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explore mechanistic pathways.
The tests were carried out following a standard procedure. After the tests the samples were shown with fluorescence microscope to see the attachment and the proliferation of the cell on the surface of the scaffold.
To see the cells we used a fluorescent colorant as DAPI for nucleus and Alexa Fluor® 594 Goat Anti-Mouse IgG for α-tubulin so for the cytoskeleton.
