Articular cartilage chondrocyte apoptosis following impact injury in experimental study
Authors: Shao-kun, Fu-Xing Pei, Di Dong-hua, Zhao Jianzhong
【Abstract】 Objective] with the TUNEL labeling method and transmission electron
microscopy to observe the cartilage after impact injury in the evolution of chondrocyte apoptosis. [Method] 56 healthy adult New Zealand white rabbits were randomly divided into high and low energy groups, each of 28 randomly selected side of the hind
limbs of the experimental group, on the other side of the control group; different weights of the hammer hit the inside of the femur condylar cartilage damage caused by the different. After 4 d, 1,2,4,8,16,32 weeks to collect specimens, each time group 4
rabbits, carried out TUNEL labeling method and transmission electron microscopy to observe the apoptosis of chondrocytes. [Results] In the control group, specimens from the middle layer and calcified cartilage layer, apoptotic cells. Low-energy group, after
injury 4 d, 1,2-week specimens, cartilage intermediate layer of the transitional layer and apoptotic cells, the apoptotic rate between the control group had no statistical significance; 4 weeks after the specimen significantly reduced in apoptotic cells,
apoptosis rate were not statistically significant between groups. High-energy group, 4 d
after injury cartilage surface and transitional layer of light appears in apoptotic cells, and the injury the longer the greater the percentage of apoptotic cells, apoptosis rate
were statistically significant between groups . [Conclusion] cartilage high, low-energy
impact injuries suffered from an early chondrocyte apoptosis, apoptotic rate was higher; but low-energy injury 4 weeks after chondrocyte apoptosis returned to control
levels; high-energy injury cartilage apoptosis rate increased gradually over time.
Key words cartilage; cartilage cells; apoptosis; traumatic osteoarthritis
Recent studies have shown that osteoarthritis and cartilage injury in vitro model,
can be observed in chondrocyte apoptosis. With the increasing severity of osteoarthritis, cartilage cells, also increases the rate of apoptosis, mechanical damage can also induce chondrocyte apoptosis, which may be cartilage cells to mechanical damage, one of the earliest reactions [1,2] . Articular cartilage in vivo after injury by impact, cartilage cell apoptosis rate in the evolution of a lack of sufficient knowledge of the people. This experiment Newberry, Vrahas model based on the rabbit medial
femoral condylar cartilage of different models, with the TUNEL labeling method and transmission electron microscopy observation of cartilage injury in the evolution of chondrocyte apoptosis.
1 Materials and methods
1.1 Animals and Grouping
56 healthy adult rabbits, of either sex, weighing 3.0 ~ 3.5 kg, were randomly divided into light and heavy two groups of 28, randomly selected side of the hind limbs of the experimental group, on the other side of the control group. And then randomly divided
into two after 4 d, 1,2,4,8,16,32 weeks 7 hours groups, each with four animals.
1.2 The establishment of articular cartilage injury model
1.2.1 5% chloral hydrate 1.25 ml / kg intraperitoneal injection of anesthesia, supine position to take, hair removal, routine disinfection shop towel.
1.2.2 medial longitudinal incision length 3 cm, followed by skin incision, subcutaneous tissue, deep fascia, joint capsule will patella eversion, knee flexion 120 ?, so that femoral shaft with the horizontal plane perpendicular to a fixed pelvis with fixed pelvis, thigh, with the thigh fixed to prevent collisions with fixed unstable.
1.2.3 use the hammer 1.33,0.43 kg, respectively 46,20 cm height of free fall through the guide rod, the resulting energy of 6.3 J (can cause collagen lattice fault) and 0.9 J (not caused by collagen rack fault) [2,3], and through the body caused by collision medial femoral condyle cartilage damage. No control side impact, I step in the same test group. Gentamicin saline flush operation completed joint cavity, layer by layer suture the wound. Anesthesia conscious cage freely. After intramuscular injection of penicillin 200,000 units, 2 times a day qd for 3 d.
(4) was after 4 d, 1,2,4,8,16,32 weeks to collect samples for TUNEL labeling method
and transmission electron microscopy to observe the apoptosis of chondrocytes.
1.3 Morphological observation
1.3.1 drawn, fixed
After the expiration of each group to observe, in the same location with a sharp
double-sided blade Department subchondral bone were cut 0.5 cm × 0.4 cm × 0.2 cm of cartilage, subchondral bone and did not take to avoid the damage due to the process of specimen decalcified cells, causing false positive. Specimens cut two equal parts, one
were immediately placed in 10% neutral formalin solution at room temperature for a fixed 24h, paraffin-embedded tissue blocks for serial sections, sheet thickness of 51 μm, made of pathology standby. The other one were prepared to do transmission electron
1.3.2 TUNEL-end labeling
Biopsy conventional dewaxing, the gradient of alcohol, after replenishment, sliced dropping antigens proteinase K repair. Dropping 20% fetal calf serum and 3% bovine
serum albumin closure of non-specific reactions. In the DNA fragment labeled with 0.3% H2O2 blocking endogenous peroxidase, room temperature, 15 min. Slice dropping 20% goat serum, 3% bovine serum albumin and 1% blocking agent, 37 ?, 15
min, once again closed to non-specific reactions. Slice dropping 1:2 diluted POD 20 μl, home wet box, 37 ?, 30 min to the immune response to expand the signal. Mirror tap water reaction. Hematoxylin. Gradient alcohol dehydration, xylene and
transparent, gums Fengpian.
1.3.3 Transmission electron microscopy
Specimens were placed in 5% glutaraldehyde preservation fluid infiltration protection, double-sided blade with a sharp coronal cut into 1 mm3 pieces will be
cartilage, immediately put into 4 ? 5% glutaraldehyde solution, fixed 24 h later, 0.1 mol / L dimethyl arsenite buffer (pH7.35) rinse 24 h. 1% osmium tetroxide post-fixed 2
h, ethanol, progressively dehydrated resin-embedded, ultra-thin slicing machine 70 nm
transmission electron microscope observation filming.
1.4 Image Analysis
Each one TUNEL marker sectioning microscopy camera microscope (magnification 10 × 40), randomly selected four vision to capture the image into the computer, and the analysis system of doing image analysis, using target count, determination of chondrocyte apoptosis rate, obtained after the arithmetic mean apoptosis rate of each slice. Reposted elsewhere in the paper for free download http://
1.5 Statistical analysis
With the SPSS 12.0 statistical software package, according to high and low energy group and the corresponding control group were compared to 22, using t test, take 95% confidence interval were observed in all experimental groups and control group
the rate of chondrocyte apoptosis in the availability and Statistics learning differences.
Postoperative survival of all experimental animals were drawn to the scheduled time, incision healed well and no infection in 1 case.
2.1 TUNEL labeling observed rate of chondrocyte apoptosis
Cartilage cell nucleus orange for apoptotic cells, blue for the normal cells. Specimens in the control group in the middle layer and the calcified cartilage layer, apoptotic cells.
Low-energy group, after injury 4 d, 1,2-week specimens, cartilage intermediate layer of
the transitional layer and apoptotic cells, the apoptotic rate between the control group had no statistical significance; 4 weeks after the specimen in apoptotic cells significantly
reduced apoptosis rate were not statistically significant between groups (P