Correlations between type I and II collagen expressions and mechanical strength in osteoporotic fracture healing
Yuan Shao-hui, Liu Wei, Wu Bin-qi, Han Xi-guang, Bo Chao-gang
Abstract
BACKGROUND: Studies have demonstrated that osteoporosis is a complex pathophysiological process involving changes of type I collagen number and physical or chemical properties. However, reports reporting correlations between type I and II collagen expression are few.
OBJECTIVE: To disclose the change rules of type I and II collagen protein of osteoporotic fracture healing in rats and the correlation of them with mechanical strength.
METHODS: Rats were randomly divided into osteoporotic fracture and general fracture groups. Rats in the former group received ovariectomy. The expressions of type I, II collagen were observed by Western blot at 1, 2, 4, 5, 8 and 12 weeks. Bone mineral density was examined by dual-energy X-ray absorptiometry, and biomechanical testing were performed at 4, 8, 12 and 16 weeks postoperatively.
ESULTS AND CONCLUSION: There were significant differences between the osteoporotic fracture group and general fracture group in type I and II collagen expression and bone mineral density at different time points after injury (P < 0.05). With the injury time prolonged, the downward trend was The type I and II collagen were correlated with maximum torque, elastic modulus, and maximum torsion angle in the osteoporotic fracture group (P < 0.05). The results showed that abnormal secretion of type I and II collagen osteoporotic fracture healing Strength and reduce the quality of fracture healing, which is the main reason for re-fracture.
Yuan SH, Liu W, Wu BQ, Han XG, Bo CG. Correlations between type I and II collagen expressions and mechanical strength in osteoporotic fracture healing. Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu. 2011;15(2): 208-212.
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Summary
BACKGROUND: Studies have shown that osteoporosis is a complex pathophysiological process involving changes in the amount and physical and chemical properties of type I collagen, but the association between osteoporosis and type I and type II collagen has rarely been reported so far.
OBJECTIVE: To observe the changes of type I and type II collagen expression in osteoporotic fracture healing and its correlation with biomechanical properties.
Methods: Rats were randomly divided into osteoporotic fracture group and general fracture group. Osteoporosis model was established in the osteoporotic fracture group. The protein expression of type I and type II collagen in osteophytes was detected by Western blot at 1, 2, 4, 5, 8 and 12 weeks after fracture injury. Bone mineral density was measured at 4, 8, 12 and 16 weeks after injury. Biomechanical performance test.
RESULTS AND CONCLUSION: The expression of type I and type II collagen and bone mineral density in osteoporotic fracture group were significantly different from those in the general fracture group at each time point after injury (P < 0.05), and with the damage The extension of time is more obvious. There was a correlation between type I and type II collagen and maximal torque, elastic modulus and maximum torsion angle in the osteoporotic fracture group (P < 0.05). The results showed that the abnormal secretion of type I and type II collagen during the healing process of osteoporotic fracture resulted in the decrease of mechanical strength and fracture healing quality, which affected fracture healing and was the main cause of re-fracture.
Key words: collagen; osteoporosis; bone mineral density; biomechanics; bone tissue engineering
Doi:10.3969/j.issn.1673-8225.2011.02.005
Yuan Shaohui, Liu Wei, Wu Binqi, Han Xiguang, et al. Expression of type I and type II collagen and its mechanical properties during osteoporotic fracture healing[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2011,15(2):208 -212. [http://www.crter.org http://cn.zglckf.com]
0 Preface
Osteoporosis with low bone mass and bone tissue microstructural destruction
To be characterized, its most serious consequence is the occurrence of osteoporotic fractures. Currently commonly used osteoporosis evaluation indicators include bone mineral density, biochemical indicators of bone metabolism, bone biomechanical strength and trabecular bone microstructure. Based on the establishment of osteoporotic rat fracture model, the bone organic matter, namely bone I and type II collagen, was used as the main research object to observe the changes of collagen content during osteoporotic healing of osteoporotic fracture in rats. The correlation between changes and changes in biomechanical strength provides a theoretical basis for the diagnosis and treatment of osteoporotic fractures.
1 Materials and methods
Design: Randomized controlled animal experiments.
Time and place: Completed at the Central Laboratory of the First Affiliated Hospital of Harbin Medical University from January 2009 to June 2010.
material:
Experimental animals: 50 healthy female SPF SD rats of 8 months old, with a body mass of 300-350 g, provided by the Animal Center of the Central Laboratory of the First Affiliated Hospital of Harbin Medical University. The method of treatment of animals in accordance with the experiment is in line with the "Guiding Opinions on Treating Experimental Animals" issued by the Ministry of Science and Technology of the People's Republic of China [1].
Main instruments and reagents:
Reagents and instruments
Fax itron Sample Radiation System US Itron Corporation
AG-I Biomechanical Measurement System Japan Shimadzu Corporation
DPX2L Dual Energy X-Ray Bone Densitometer United States Lunar Corporation
WDS2100 Computer Electronic Capability Test System Japan Matsushita Corporation
SPECTRAMAX 190 Continuous Spectral Microplate Densitometer US MDC Company Beauty
Gel Doc2000 Image Analysis System National Bio-Rad Company
Mouse anti-human I, II collagen monoclonal antibody US Invitrogen
Rabbit anti-mouse IgG US Santa Cruz
experimental method
Grouping and Modeling: 50 rats were randomly divided into osteoporotic fracture group and general fracture group, with 25 rats in each group. Each group of animals was intraperitoneally injected with anesthesia
After (Ketamine 0.1 g/kg), the rat's respiratory heartbeat was stabilized and then fixed on the operating table, and disinfected after depilation. In the osteoporotic fracture group, the bilateral ovaries were sutured after laparotomy, and the general fracture group was sutured after excision of a small amount of adipose tissue.
Three months after surgery, all experimental animals under anesthesia established two fracture models: fracture model of osteoporotic fracture group and fracture model of general fracture group [2]. The lateral femoral incision of the two groups of rats was taken, and the anterior and lateral muscle groups were retrogradely fixed. All experimental mice were immediately taken with the Fax itron sample radiology system on the day after surgery to determine whether the model was successful or not [3].
Western blot was used to detect the expression of type I and type II collagen. The animals in both groups were sacrificed at 1, 2, 4, 5, 8 and 12 weeks after fracture injury [4-5], and each time phase was 3, and the callus was Store the specimens at -80 °C until the specimens are ready. Decalcification of fracture specimens in 200 g/L ethylenediaminetetraacetic acid at 4 °C
1 week. The decalcified bone tissue was extracted by tissue lysis method, and the protein concentration was determined by Bradford method [6].
Western blot immunoblotting analysis of type I and type II collagen in osteophytes: protein was first subjected to 10% polyacrylamide gel electrophoresis, transferred, and nitrocellulose membrane was taken out and placed in 20 g/L bovine serum albumin/PBST solution. In 37 capsules overnight, the type I collagen primary antibody was a mouse anti-human type I collagen monoclonal antibody (1:1500), and the secondary antibody was a rabbit anti-mouse IgG (1:2 000). Type II collagen primary antibody is type II collagen monoclonal antibody (1:200), the next day rinsing nitrocellulose membrane 3 times, 5 min / time, then add the appropriate diluted secondary antibody, room temperature 2 h, remove and rinse again 3 times , 5 min / time, ECL color development, X-ray film exposure development.
Five fracture end protein samples were taken at each time point for the detection of type I and type II collagen. Place X-ray film on the Gel Doc2 000 chart
Like an analysis system. The average absorbance value of the target band was measured, and the average value of the five experimental results was taken as the relative value of the protein content.
Bone mineral density measurement of osteophytes: 4 rats were sacrificed in each group at 4, 8, 12, and 16 weeks after the establishment of the fracture model [7], and femur specimens were taken.
The DPX2L dual-energy X-ray absorptiometry was performed with the femur callus as the center and scanned for 2 cm. After the test, it was coated with saline wet gauze, sealed in a plastic bag and placed in a refrigerator at -20 °C for mechanical testing.
Mechanical properties test: femur specimens from which bone mineral density has been measured
The -20 °C refrigerator was gradually thawed and rewarmed at room temperature. The thickness of the femur was measured with the thickness of the femur and the sagittal diameter and transverse diameter of the epiphysis. The fracture healing time of the two groups was calculated. Cross-sectional area of the iliac crest.
The bone surface of 3 mm × 4 mm was selected on the edge of the epiphysis. The surface of the bone was flat and rough with coarse sandpaper. The surface fraction of 100% acetone was deesterified and dried. The three-point bending test was carried out using the WDS2100 computer electronic versatile test system. The span of the bearing point is 30 mm, the midpoint of the humerus is taken at the loading point, and the loading speed is 2 mm/min. The ends of the tibia are fixed with square bone cement to prevent the humerus from rotating when loading, keeping the position of each specimen consistent. The specimen is kept wet during the test, and the femoral torsion strength test is performed to measure the maximum torque and elastic modulus of the two epiphyseal tissues (g× Cm2/deg) and maximum twist angle (deg/cm).
MAIN OUTCOME MEASURES: Expression of type I and type II collagen after injury in both groups
And its correlation with post-injury bone mineral density and biomechanical properties (maximum torque, elastic modulus, maximum torsion angle).
Statistical analysis: measurement data is expressed by x±s, using SPSS13.0 system
The software package analyzes the data for statistical analysis. The significance test between the expression levels of type I and type II collagen was performed by two-sample t test; the ratio of the mechanical parameters of each pair of samples was calculated, and the difference between groups was judged by the variance method, I,
The correlation between type II collagen and maximum torque, elastic modulus and maximum torsion angle was analyzed by linear regression. P < 0.05 was considered significant.
result
2.1 Analysis of the number of experimental animals 50 rats were included in the results analysis, no death and infection, no shedding.
2.2I, type II collagen expression See Figure 1 and Table 1.
Wuhan Hege Biotechnology Co., Ltd.
The expression of type I and type II collagen in osteoporotic fracture group was significantly different from that in the general fracture group at each time point after injury (P <0.05), and as the damage time increases, the downward trend is more obvious.
Bone mineral density changes in the epiphyseal tissue The bone mineral density values of the two groups were lower than the general fracture group at 4, 8, 12, and 16 weeks after the establishment of the fracture model (P < 0.05); both groups were at 8 weeks.