Biomedical Engineering Reference
In-Depth Information
72 h. The supernatant fluid was withdrawn and centrifuged to prepare the extraction
medium, then refrigerated at 4
o
C before the cytotoxicity test. The control groups involved
the use of DMEM medium as negative controls. Cells were incubated in 96-well cell culture
plates at 5
×
10
4
cells/ml medium in each well and incubated for 24 h to allow attachment.
The medium was then replaced with 100μl of extracts. After incubating the cells in a
humidified atmosphere with 5% CO2 at 37
o
C for 2, 4 and 7 days, respectively, cell
morphology was observed by optical microscopy (Nikon ELWD 0.3 inverted
microscope).The neutral red viability assay was performed according to published
procedures. A stock solution of neutral red (Beyotime, China) was prepared in water (1%).
The stock solution was diluted to 50 μg/ml in complete culture medium and 200μl of the
staining solution were added to each well after removal of the exposure medium. The cells
were incubated for 3 h at 37°C, The cells were then fixed with 200μl for-
maldehyde/CaCl2(3.7%/l%) and destained with 200μl methanol/glacial acetic acid (50%/l%),
The plates were shaken for 60 min at room temperature using a plate shaker. Optical densities
were measured at 540 nm in a multiwell spectrophotometer (Bio-RAD 680).The cell relative
growth rate (RGR) was calculated according to the following formula:
RGR= OD
test
/ OD
negative
×
100%
2.6 Animal test
2.6.1 Surgery
Animal tests were approved by the Ethnics Committee of the First Affiliated Hospital of
Harbin Medical University. The in-vivo degradation experiments were performed in the
animal laboratory of the hospital. A total of 15 adult New Zealand rabbits (6 females),
2.0~2.5kg in weight, were used. In the experimental group, sodium pentobarbital (30mg kg-
1) was administered to perform anesthesia by intravenous injection. The sterile Mg-Zn-Ca
alloy rod sample was implanted into the femora of the rabbit.
After operation, all animals received a subcutaneous injection of penicillin to avoid a wound
contamination and were allowed to move freely in their cages without external support.
After operation, five rabbits were sacrificed randomly at 1, 2 and 3 months, respectively.
2.6.2 Degradation and histological analysis
The bone samples with magnesium implants were fixed in 2.5% glutaraldehyde solution
and then embedded in epoxy resin for microstructure analysis. The samples were sliced by
hard tissue slicer (ZJXL-ZY-200814-1). Samples were made perpendicular to the long axis of
the implant to get a cross-section of the implant and surrounding bone tissue. The cross-
section microstructure was observed by an optical microscope (Nikon ELWD 0.3 inverted
microscope) and a scanning electronic microscope (Hitachi S-5500). The residual implant
areas were measured on the cross-section images using analysis software. The ratio of the
residual cross-section area of implants to the original cross-section area (residual
area/implant area×100%) was used to assess the in vivo degradation rate of magnesium
alloys. The element distributions in the residual implants and the degradation layer after 3
months implantation were analyzed.
For histological analysis, the bone samples with magnesium implants were fixed in 4%
formaldehyde solution, dehydrated, and then decalcified in ethylene diamine tetra acetate.
Then, the specimens were embedded in paraffin and cut into films with 5μm in thickness.
