Summer 2013
Abstract
Reliable techniques to meet the needs of patients with peripheral nerve repair injuries need to be refined. One of the strategies often used is to create a nerve guidance channel or build a nerve conduit Silicone is has been found to work well as a conduit from an injury for regenerated axon cells if it does not contain foreign components. The cycle of the cleaning treatment was repeated until the Pentane-Acetone-Heat Procedure was successful and the plated cells survived for several weeks. The duration of the plating and cell count using the cleaned silicone lasted from May 3, 2013 to July 19, 2013. The laboratory resulted in the successful development of a Pentane-Acetone-Heat Procedure. The results were shown to be successful because of the large number of cells grown and the plated axon cells were able to grow well over almost six weeks.
List of Figures
Figure 1 Dilution of plated cells due to ml of suspension 4
Figure 2 Number of cells versus media amount 4
Figure 3 Number of cells versus time 5
Figure 4 Images of the cells on the plates over time 6
List of Tables
BackgroundInjuries from accidents or daily repetitive tasks are peripheral nerve injuries. Peripheral nervous system (PNS) injuries can occur after surgeries. Axon nerve regeneration is an important treatment for healing these types of injuries. Problems arise when trying to repair this type of injury because not enough nerve cells may remain from the damaged cells to make a repair. When not enough nerve ending is left to complete a connection an autograft may be needed. The autograft is not always a satisfactory choice if there is limited “availability of donor nerve and comorbidity associated with additional surgery” (Pfstir, Gordon, Loverde, Kochar (et al. 81).Therefore a biomedical engineering alternative is to prepare a synthetic matrix. In other words a substrate or matrix needs to be developed which is suitable for cells to regenerate rapidly and to thrive. Axon cells demonstrate targeted synaptic connection specificity; they do not develop connections with random cells. The target for the connection is the cell which the axons project towards or connect with. The event axon connection or axon projecting is called ‘the specificity of connections’ or ‘neuronal specificity.’ Researchers are trying to solve the problems that occur when relying solely on autografting. A survey of clinicians showed that repair is done in one of three ways; 78 percent of the repairs are made by surgery directly to the nerve, 15 percent of the time autografts are used, 4 percent of the time alternative methods are used while 3 percent of the time a repair is not attempted. (Pfister et al. 2) Although only a few centimeters of nerve are needed for the autografting procedure this amount may not be available. The Axon cells are the type of cells that need to be regenerated and grown to a healthy stable state so nerves can repair. An Axon cell is the portion of a neuron that carries the nerve impulses away from a cell body. Biomedical engineers have an opportunity to solve the problems associated with autografting. The basic problem is that healthy nerves are taken from other parts of the body to repair the damaged nerves in order to autograft an injury. It also takes a long time to be able to understand if the autograft was or was not a success. Months or years may pass before a doctor can be sure whether the recovery was satisfactorily accomplished, by that time the best time to intervene with more surgery has long passes. (Pfister et al. 22) Pfitser (et al. 32) have called for bioengineers to meet the challenge of developing “tissue engineered nerve graft alternatives” for peripheral nerve repair.
Introduction
In order to develop a reliable method to meet the needs of patients with peripheral nerve repair injuries many details of the axon regenerative techniques need to be refined. Yi-Cheng Huang and Yi-You Huang (2006) have discussed the biological complexities in terms of nerve regeneration. The difficulties a nerve injury cause are not only to the immediate area of the injury. Nerves may need to be taken from another part of the body to make the repair. The injury may negatively impact other parts of the body because “recovery is difficult and malfunctions in other parts of the body may occur because mature neurons don’t undergo cell division” (Huang and Huang 1). Therefore techniques to enhance the regeneration of the nerves are being researched to make sure the time necessary for the cells to grow the proper length are reached and that the cells remain healthy. One strategy is called creating a nerve guidance channel or building a nerve conduit. (Huang and Huang 1) According Huang and Huang (1) the channel building or conduit type of strategy requires a stable substrate for the growth of Axon cells, supporting cells, factors to enhance growth and an organic or synthetic extracellular matrix. The commercial PDMS formulas, Sylgard 527 and Sylgard 184 have been found to “support cell attachment and growth” (Palchesko, Zhang, Sun and Feinberg 2002). The usefulness of enhancing these capabilities for medical uses cannot be overstated. Mechanobiological applications have great potential for improving the “regulation of cell shape, proliferation, migration and differentiation” for injured or diseased cell tissues (Palchesko et al. 2002). Therefore research for ensuring the silicone is a clean substrate has important implications.
Silicone polydimethylsiloxane (PDMS) is has been found to work well as a conduit from an injury for regenerated axon cells. Silicone is a synthetic nondegradable conduit material that has been used for many years in the technique. (Pfitser et al. 98-101) Silicone must be washed to make sure no foreign materials will stop the growth. The building of a bridge for the Axon nerves depends also on whether or not the mammalian cells being used are compatible on surfaces of poly(dimethylsiloxane) (PDMS). (Lee, Jiang, Ryan and Whitesides 2004) Axon cells need to be able to regenerate so foreign components in the silicone must be cleaned out of the silicone for success. The axon cells grow by fasciculation which means the cells grow in tracts and a main structural function is to use adhesion on other axon cells. In order to repair the injury the axon cells need to enter the target cell population. This lab report takes an important action by trying to refine the cleansing process of the silicone.
Materials
Pentane
Acetone
Axon non-proliferating cells
Poly(dimethylsiloxane) aka Silicone
Vacuum oven
Plating materials
Method
Learning the best methods for cleaning and assembling the silicone wells (as well as the proper disposal) was the first step in the methodology. The preparation of the silicone wells is a critical part of ensuring good data for this type of experiment. A problem arises in regeneration of cells if the particles or microorganisms present in the silicone solvent is not or cannot be removed prior to use. Reverse osmosis is used as a cleaning strategy but not all of the particles and microorganism are always removed. The reason is because the silicone needs to have an affinity for Axon cells so they would have a longer life expectancy and stay adhered well to the surface of the matrix. The cells needed to be examined under a microscope.
Therefore three methodologies were considered to reach the primary objective of a thin layer of silicone for the special wells and for observation. First of all curing the silicone was considered as a possibility but curing makes the silicone too thick. The second method considered was to spin the silicone on a disc so it could spread out into a thinner layer. The third option, and the option finally used, was to treat the silicone after reverse osmosis cleaning so any foreign particles or microorganisms would be removed. The reason the foreign components need to be removed because research indicates that if the foreign components are present cells die after a few days or weeks which is not enough time for the required regeneration.
Training for silicon well preparation was accomplished. And then trials were carried out in order to perfect the procedure for the silicone wash. (See table 1) It was observed that the
pentane made the PDMS shrink. The treatment of the silicone with pentane wash, acetone wash and heat treatment was completed after three days. The cells were plated and a control was prepared. Silicone that had not been treated for cleaning was used as a control.
Note
Axon cells are non-proliferating so that a comparison of cell growth is possible when made to the proliferating cells.
In order to avoid bubbles the PDMS (the silicone) was placed at the bottom of the each of the wells. (If bubbles form the substrate will detach from the well bottoms. Lee et al. 11691)
Other avenues, such as 'homemade' silicone, were not fully investigated.
Results and Discussion
The training for appropriate technique for cleaning the PDMS helped make the final part for the experiment successful. The PDMS had to be cleaned to be useful in growing new cells. Pentane was found to shrink the PDMS. The laboratory resulted in the successful development a Pentane-Acetone-Heat Procedure to clean the silicone.
Preliminary experiments were preformed to attempt to gain regeneration of cells in the silicone treated samples for several weeks. At the end of the first week it was observed that the treated silicone wells outlasted the control with the non-treated silicone wells. The control’s cells died at the expected rate for cells without treated silicone. Whereas the cells on the treated silicone remained fairly health for about 9 days before their health began to decline. Therefore two weeks were spent improving laboratory practice. The cycle of silicone treatments was repeated until the Pentane-Acetone-Heat Procedure was successful and plated cells survived for several weeks.
The duration of the plating and cell count lasted from May 3, 2013 to July 19, 2013. (See appendix for table of all data recorded per dates) This was a good timeframe which allowed time to observe, examine and study any changes in the plated cells.
Figure 3 is the most useful graph because it shows the growth, peak and decline of the cell population from May 3, 2013 to July 19, 2013. During weeks 6 and 7 the cell population peaked. The Axon cell population declined after Week 7 in a pattern approximately symmetrical to the growth of the population before Week 6. This graph demonstrates that the Pentane-Acetone-Heat Procedure developed in the experiment for washing the silicone worked well. The pentane shrunk the silicone.
Several weeks of cell growth are needed to make sure the Axon cells can bridge gaps for healing and then regenerate. Another method of demonstrating the success of the experiment was to study the cells on the plates. Figure 4 a-f and Figure 5 a-d provide photographs of the plated cells. The exposure, contrast and brightness were manipulated on the .tif images in order to better view the cells. The images depict a lacey, fragile looking design; and that design is composed of the Axon cells. The images in Figure 4 and Figure 5 do not allow the viewer to see much detail (due to the exposure manipulation). The confluence of the cells is considered to be the number of cells covering one layer of the plate. Then the confluence is equal to approximately 95%. So the confluence is another measure of the success of the procedure for cleaning the silicone. None of the Axon cells died, the cells grew on each plate.
First set Silicone Well 1
First set Silicone Well 2
First set Silicone Well 3
First set Silicone Well 4
First Set Silicone Well 5
First set Silicone Well 6
SET 2 Well 1
Set 2 Silicone well 2
Set 2 Silicone Well 3
Set 2 Silicone Well 4
Conclusion
Medical bioengineering have an opportunity to enhance the healing of patients with nerve injuries. An alternative to autografting is needed. A matrix on which axon cells can regenerate and thrive is essential. Axon cells are well suited to heal injuries that have caused a nerve gap in patients. The growth of the cells used in procedures for bridging the gap must be healthy and regenerate quickly for patient recovery. Autograft has been a common procedure but this has not shown itself to be satisfactory for two reasons (a) the nerve cells must be taken from another part of the body and (b) the timing to check if the cells regenerate and thrive does not allow for timely intervention by the surgeon if the cells die. Silicone has been used to provide a matrix for axon cells to grow on but there is a challenge to success. No foreign particles or components can be in the silicone or the axon cells will die. Therefore this experiment focused on the preparing a successful method for cleaning the silicone. The laboratory resulted in the successful development a Pentane-Acetone-Heat Procedure to clean the silicone. The pentane shrinks up the PDMS. The results were proved successful because the plates because plated axon cells were able to grow well during a period of almost six weeks. This was demonstrated by increased cell counts.
Works Cited
Huang, Yi-Cheng and Yi-You Huang. “Tissue engineering for nerve repair.” Biomedical Engineering: Applications, Basis and Communications 18.3 (2006): 1-11.
Lee, Jessamine Ng, Jiang, Xingyu, Ryan, Declan, and George M. Whitesides. “Compatibility of Mammalian Cells on Surfaces of Poly(dimethylsiloxane).” Langmuir 20 (2004): 11684-11691. Print.
Lee, Jessamine Ng, Park, Cheolmin and George M. Whitesides. “Solvent compatibility of Poly(dimethylsilocane)-based microfluidic devices.” Analytical Chemistry 75 (2003): 6544-6554. Print.
Palchesko, R.N., Zhang, R. N., Sun, Y. and Feinberg, A. W. “Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechnobiology in muscle and nerve. PLOS ONE, (2012) http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0051499
Pfister, Bryan J., Gordon, T., Loverde, Joseph R., Kochar, Arshneel S., Mackinnon, Susan E. and D. Kacy Cullen. “Biomedical engineering strategies for peripheral nerve repair: surgical applications, state of the art, and future challenges.” Critical Review™ in Biomedical Engineering, 39.2 (2011): 81-124. Print.
Appendix
Table A1.1 Data for Cell plating counts
