Spinal Implant Material Testing, Let’s Take a “PEEK” at Si3N4
By Kristine Peterson
Abstract:
When it comes to implants and foreign materials being placed into patient’s
bodies, a surgeon wants to be sure to pick the very best. That’s why we asked
the question, what is best? PEEK, a thermoplastic material has been used as
the industry standard for many years, is technically biocompatible, but has
tendencies to form biofilms. On the other hand silicon nitride, Si 3N4, , is a
ceramic, which had also been FDA approved for use in spinal implants, is shown
to be resistant to biofilms, and promotes bone growth.(1)
In order to find the more effective substance an experiment was created to
compare the proliferation of MG-63 osteoblast-like cell line on the substances.
Cells would be grown on silicon nitride and Peek discs as well in culture treated
and non-culture treated 24-well plates. The amount of cells would be
measured according to the amount of crystal violet dye absorbed by the cell,
measured with a spectrophotometer. The amounts of cells would be compared
to our negative and positive controls, the culture treated and non-culture
treated plates.
It is hoped that the data will further prove that silicon nitride is superior to
Peek. This way those that need this type of treatment will be able to receive
the best quality and recovery.
Purpose: To perform experimentation that allows a comparison of implant
materials PEEK and Silicon Nitride. Specifically the rate of proliferation on
the surfaces.
Figure 1 Figure 2
Figure 3
Figure 1: Non-compressed Silicon NItride powder. Figure 2: compressed,
shaped and sintered Silicon Nitride implant. Figure 3: Peek Spinal implant.
Background:
Silicon nitride is a ceramic formed out of compressed powder, which is shaped
and then fired to harden. There are several unique qualities that silicon nitride
has as an implant: enhanced osteogenic response, has shown a better protein
absorption and increased bone growth.(1) Also antibacterial characteristics,
inhibiting bacterial infection and biofilm formation.(2) Finally, semi-radiolucent
with visible boundaries, giving it excellent imaging compatibility.
Peek, our comparison material, has been used for implants longer than
silicon nitride because it has a stiffness similar to bone.(3)
Crystal Violet is a triarylmethane dye. Once cells are fixed to wells and
dyed, acetic acid is used to resuspend the color. This is scanned on a
spectrophotometer at 570 nm and absorbances are recorded.
Methods: Phase 1: Determining the optimal seeding density and
harvesting time.
Establishment of cell cryopreservation of MG-63 cell in
DMSO.
Form a standard curve and growth curve in 24 well culture
treated plates.
Find cell counts by taking absorbance measurements of
crystal violet dye.
Analyze and graph data to find the optimal seeding density
and harvesting time of MG-63 cells to be used in phase two
as optimal growing conditions.
Results:
Figure 6
Figure 6
Figure 4: 24 well plate layout for material testing. The discs
are transferred from the left side to the right before staining.
Figure 5: example of crystal violet stained wells.
Figure 6:A comparative bar graph of absorbances of Si3N4,
Peek, positive and negative controls.
Figure 7:Standard Curve for the average absorbance graph.
(Figure 6)
Phase 2: Material Testing with PEEK, and Silicon Nitride.
Set up cultures of MG-63 cells on testing material discs,
positive and negative controls in 24 well plates.
Fix cells with methanol to wells and discs followed by
staining with crystal violet.
Find cell counts by taking absorbance measurements of
crystal violet dye.
Analyze data, comparing test groups to control groups and
determine the substance with higher performance.
Figure 4
Figure 5
Figure 7
Conclusion:
In the first phase of this project we found that the optimal seeding
density was between 20,000 and 60,000 cells. Along with that the
doubling time was between the 24-48 hour range. This allowed us to find
the optimal circumstances to grow our cells in.
Results from phase two showed that the average absorbances of the
silicon nitride material was considerably higher than the rest. At the
60,000 seeding density it produced a .5507 absorbance, which is higher
than the positive control and Peek materials which had .457 and .2827
absorbance respectively. When the standard curve equation is used to
calculate the number of cells, silicon nitride shows a 664,904.76 cell
yield, compared to the 186,333.33 cell yield of peek. Both of the positive
and negative controls used were effective in their function as
benchmarks.
Because of the nature of our data more testing will be needed to not
only show that our results are repeatable. As seen in the data, the
margin of error seems to be larger for the silicon nitride than the other
surfaces. This indicates that more testing is necessary. More attempts
along with becoming more experienced with the protocol should help get
more consistent results.
Silicon nitride proved to promote the growth of our MG-63 cells
more than Peek did, and even more than our positive control. This leads
us to believe that in an implant setting it would promote growth and a
speedy recovery for patients.
References:
(1) Kue, R., Sohrabi, A., Nagle, D., Frondoza, C., & Hungerford, D.
(1999). Enhanced proliferation and osteocalcin production by human
osteoblast-like MG63 cells on silicon nitride ceramic discs.
Biomaterials, 20(13), 1195-1201.
(2) Bal, S., Gorth, D., Puckett, S., Webster, T. J., R., & Ercan, B.
(2012). Decreased bacteria activity on Si3N4 surfaces compared with
PEEK or titanium. International Journal of Nanomedicine IJN, 4829.
(3) Skinner HB. Composite technology for total hip arthroplasty.
Clin Orthop Relat Res. 1988 Oct;(235):224–236.
Acknowledgments: My team at InnovaBio: Miranda
Wilson, Cody Jones, Marianne Longoria and Jordan Clawson.
Along with a special thanks to Kate Slessor, Mary Nelson