Laminin is an essential part of the DCM, the extracellular matrix, especially Laminin-111 in the context of some cell types. Evidence suggests that the microstructure of laminin informs signaling cells. Polymerized laminin-1 exhibit a fractal microstructure showing robust biological activity and, as compared to conventional aggregated laminin-1.
Poly laminin's fractal network indicates that an assembly structure conductive to the functional differentiation of epithelial cells as well as other cell types. This is why researchers can choose between three different methods for assembling this biologically robust laminin polymer depending on their study subject. Currently, acute local injections of poly laminin produced under pH acidification has been shown to be effective in regenerating human spinal cord injury in large animals and human clinical studies.
Basic studies of poly laminin in heart failure and breast cancer are ongoing. Future studies using these methods to polymerize laminin-1 into fractal networks could identify the receptors and signaling necessary for epithelial as other cell types differentiation and investigate cell matrix interaction in both normal and abnormal microenvironments Thaw laminin-111 on ice in a refrigerator overnight. Then, use low protein binding tips to aliquot it into low protein binding micro centrifuge tubes.
Freeze aliquots and store them at 80 degrees Celsius. To make the polyLM polymerization buffer, weigh out and mix one millimolar calcium chloride and 20 millimolar sodium acetate in ultrapure water. Adjust the pH to four with hydrochloric or acetic acid, then filter the buffer through a 0.2 micrometer filter and store it at four degrees Celsius.
Add the polyLM polymerization buffer to thawed laminin aliquots for a final concentration of 100 micrograms per milliliter and mix the solution by pipetting. Pipette the diluted laminin on glass cover slips and incubate them at 37 degrees Celsius for 30 minutes. Seed dystroglycan knock-out and dystroglycan knock-in cells for laminin stimulation at 10, 500 cells per centimeter squared in well plates or cover slip bottom dishes.
After removing the supernatant, resuspend the laminin in an appropriate volume of DMEM F12 supplemented with three microgram per milliliter prolactin, 2.8 micromolar hydrocortisone, and five microgram per milliliter insulin. Immediately, add the laminin to the seeded cells to stimulate them. Seed DG knock-in cells at 5, 000 cells per centimeter squared and DG knock-out cells at 8, 000 cells per centimeter squared and culture them at 37 degrees Celsius in a humidified incubator, changing the media every two to three days and passaging them every four to five days.
Count the cells with a hemocytometer or automated cell counter to ensure appropriate growth rates. Remove media from the cells and add the laminin. Culture the cells for two days before fixing them with 10%formalin.
Use a laser scanning microscope with immersion objectives to image the laminin structure. Then analyze for fractal properties using box counting dimension algorithms available in MATLAB Toolboxes or in Image J.These methods plot the number of boxes containing laminin-1 compared to the size of the boxes on a log log plot. The slope of the relationship between these parameters is the box counting dimension.
Nonfractal geometries will have a dimension of zero, one, or two while fractal geometries have a non integer dimension. Laminin-111 is a trimetric protein with three self assembling domains at the end of its short arms. When treated suitably, the short arms can polymerize into a hexagonal lattice, which displays diffusion limited aggregate fractal properties at larger spatial scales.
Laminin incubated in calcium containing neutral pH buffers form small aggregates, which have a box counting fractal dimension of approximately two. In contrast, laminin-1 incubated in calcium containing pH four buffer, or in neutral buffer with nidogen-1 form space filling lacey networks with a fractal dimension of 1.7. Laminin-1, cultured with dystroglycan expressing cells form similar lacey networks.
When pH seven laminin and polyLM are imaged with SEM, the differences in microstructure become more apparent. At low resolution, polyLM networks appear spread out compared to pH seven laminin. At high resolution, pH seven laminin aggregates are apparent, whereas a spread hexagonal lattice can be seen in polyLM.
Following this procedure, it's possible to evaluate the fractal properties of the polymers. It is important to guarantee the production of the biomemetic polymer of poly laminin. Poly laminin and pH seven laminin plus nidogen present fractional dimension.
However, pH seven laminin without nidogen does not. Since poly laminin is biomemetic, the use of this fractal polymer of laminin will allow researchers to reproduce in vivo environment in an in vitro setting.
