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Andres Castro
Nano‐Structured Selenium Coatings for Anti‐ Cancer Bone Implants
HOPE HIGH SCHOOL
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http://invsee.asu.edu/Moduhttp://invsee.asu.edu/Modules/spm/vispipe.jpgles/spm/vispipe.jpg 2
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http://video.google.com/videoplay?docid=3756607682964690734&hl=en
Introduction about bone implants and some problems
BONE IMPLANT: the use of implanted materials to repair bone.
MATERIALS: Titanium (popular implant materials) carbon nano‐tubes on anodized titanium
Plastics (such as UHMWPE –ultra high molecular weight polyethylene)
Ceramics and Glass‐Ceramic
Cr(chromium) ; Co(cobalt) ; Ni(nickel)
Stainless steel
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A hip implant
Human skeleton
A video showing a surgery for rib …
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Background: problems with current orthopedic applications
There were about 345,000 hospitalizations for hip fractures in 2003.1 Only one in four patients completely recovered.2 Bone fractures occurred 1,039,000 times in 2004.1The rate of hip and knee replacement failures increased 11% and 8%, respectively, in 2003 in the U. S.2Insufficient bonding between implant materials and surrounding bone tissues (osseointegration) may lead to implant loosening, and finally implant failure.
1 National Center for Health Statistics (NCHS). Website: www.cdc.gov/nchs2 American Academy of Orthopaedic Surgeons (AAOS). Website: www.aaos.orgLijie Zhang Division of engineering, Brown University.
www.helsinki.fi/research/news/2005/week12.htm
www.cdc.gov/nchs/ppt/icd9/att_TJR_oct04.ppt
www.lab.anhb.uwa.edu.au/.../Bone/Bon
e.htm
It is estimated 2380 individuals (1270 men and 1110 women) willbe diagnosed with bone and joint cancer and 1470 individuals will diefrom primary bone and joint cancer in 2008 in the U.S.[1]
Wear debris generated at articulating surfaces of orthopedic implants may lead to death of cells or create mitosis without organization
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1. American Cancer Society statistic data 2008. Website: http://www.cancer.org/docroot/STT/stt_0.asp4
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Introduction to selenium
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1. Selenium is a metalloid2. Selenium is trace element in our bodies3. Deficiency of selenium results in some diseases:
Kashin‐Beck disease, mental slowing, extreme fatigue,etc. Especially link between low selenium intake and high cancer incidence
4. Selenium is toxic at high levelssuch as 400 microgram per day
5. Dietary selenium is found in: Meats, cereals, fish, etc.
Electrons per shellAtomic number
glycinecysteineglutamate
CH2 CH2 CO
CHNH3
CO
OCCNHO
CH2
H
SH
NH CH2 CO
O
Glutathione is a tri‐peptide
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Antioxidants: Where are they and how they work
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Fruits and vegetable are good source ofanti‐oxidants
Anti‐oxidants protect cells from damagescaused by free radicals such as peroxyl radical, hydroxyl radical,superoxide radical
Anti‐oxidants neutralize free radicals
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A rougher terrain engineered on the nanoscale (top)promoted bone adhesion and inhibited bacterial growth much better than the smoother surface engineered on the microscale (bottom).Units are in microns. (Image: T. J. Webster)
Role of nanostructure toward improving bone implants
Smoother is not better. The faster endothelial cells(green) form a single smooth layer, the less chanceexposed vascular metal stents will provoke an immune response.Samples examined after 1, 3 and 5 days (left to right)show better coverage on nanotextured titanium (bottom row)than on conventional microstructured titanium.( Credit: Thomas Webster)
7
microns microns
microns
microns
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Approach and methods
• Coat nanostructured selenium on titanium, glass, and polyethylene
• Chemical steps:– Mixing sodium selenite with glutathione (GSH)
– Add sodium hydroxide to bring pH above 7
GSH + Na2SeO3 GSSeSG
Ti, glass, polyethylene
Se0
GSSG
pH > 7
(Diglutathione selenite)
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Procedures for making selenium coatings on Ti, Glass, and Polyethylene
Step 1: Clean the substrates: Soak and sonicate them in acetone and ethanol
Step 2: Put the cleaned substrates into beakers
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Ultrasonic cleanerTitanium
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Step 3: Add GSH and Sodium selenite into the beakers Step 4: Add sodium hydroxide
to each beaker to bring the pH to over 7
Step 5: Wait 10 minutes Step 6: Take the samples outRinse with distilled water
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Scanning electron microscope (SEM) to visualize the surface of the samples
For glass and polyethylene (non‐conductive)Coating them with gold to make the surfaceconductive for SEM (using the sputterer)
Put the samples in the SEM chamber
Mount the samples onto sample holder
Take the image using the computer
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Prepare the healthy and cancer bone cells for cell experiments
Image of cancer bone cells Image of healthy bone cells
Magnification: 25x
12Magnification: 25x
Magnification: 40x
Magnification: 40x
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Cell experiment method
1. Seed bone cells on the substrates (uncoated and coated with Se substrates) at a seeding density of 3500 cells/cm2
2. Incubate at 37oC, 5%CO2 for 4 hours
3. After 4 hours, remove media, rinse substrates with PBS to remove dead, unadhered cells
4. Fix the cells onto the substrate using Formaldehyde 2%
5. After fixing, stain the cells with DAPI which stains the nucleus of the cells making the nucleus fluorescent (visible under fluorescence microscope)
6. Count the number of cells under a fluorescence microscope to determine cell densities on each substrate
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Results
• Scanning electron microscope image results
• Cell experiment results
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Scanning electron microscopy imagesUncoated Ti
Ti with a low level of selenium coating (low dose)
Ti with a medium level of selenium coating (medium dose)
Ti with a high level of selenium coating (high dose)
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Seleniumnanoparticles
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Higher magnification SEM images
High DoseHigh Dose‐High Magnification
High Dose‐High Magnification
Size of selenium nanoparticles is around 50 nm
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Seleniumnanoparticles
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Previous results of selenium coating on PE (by Phong Tran)
Our trial on glass and polyethylene
Uncoated Glass
Coated glass but possibly not successful
Coated polyethylene but possibly not successful
Unsuccessfully coated glass
Unsuccessfully coated PE
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Seleniumnanoparticles
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Decreased cancer cell densities on Ti with more selenium nanoparticles
Uncoated Ti Ti with low Se dose Ti with medium Se dose
Ti with high Se dose
Uncoated Low Dose
MediumDose
HighDose
Cell de
nsity (num
ber of cells/cm
2 )
18Titanium
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Increased healthy bone cell densities on Ti with more selenium nanoparticles
Uncoated Low Dose
MediumDose
HighDose
Cell de
nsity (num
ber of cells/cm
2 )
Uncoated Ti Ti with low Se dose Ti with medium Se dose
Ti with high Se dose
19Titanium
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Conclusions
1. Learned about chemopreventive properties of selenium
2. Selenium is not an antioxidant: it is structural component of an antioxidant enzyme:Glutathione peroxidase
3. Excessive free radicals damage the cells and result in some cells mutating into cancerous cells
4. Selenium coatings on titanium improve healthy bone cell adhesion and inhibit cancerousbone cell adhesion
5. Teaching and learning experience:
1. Hands‐on experience is good to motivate and engage students
2. Reframe approach to teach difficult concepts to students
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Acknowledgments
Phong Tran, Ph.D Candidate,Physics Department, Brown University
Thomas J. Webster.Associate Professor of EngineeringDivision of Engineering and OrthopedicsBrown University
Karen HaberstrohBrown University
NSF Funded "GK‐12: Physical Processes in the Environment".
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The author also thanks Lijie Zhang, Division of Engineering, Brown University for her help withthe statistic data on bone implants