Changes of H type microvessel in patients with osteoporotic and nonosteoporotic fractures in bone

Abstract

Objective To confirm a special vessel(H type and L type) in human bone and explore the changes of the vessel in patients with osteoporotic and nonosteoporotic fractures in bone. Methods Design prospective clinical study: from May 2015 to March 2016, 20 postmenopausal women with intertrochanteric fractures of the femur aged from 60 to 70 years old, with an average age of (64.30±4.67) years, were divided into 2 groups that were osteoporosis (T≤-2.5) and nonosteoporosis (T >-2.5) according to bone mineral density (BMD). There are 10 cases in osteoporotic and nonosteoporotic groups, respectively. BMD was measured by dual energy X-ray absorptiometry (DEXA) at hip and lumbar vertebra from L1 to L4 before surgery. A set of special instruments was designed to remove the bone sample, about 6mm×10mm while maintaining little bone loss caused by iatrogenic injury. Bone specimens were washed by 0.9% normal saline, fixed immediately by 4% paraformaldehyde (PFA) for 24 hours, decalcified by 0.5 mol/L ethylene diamine tetraacetic acid (EDTA) for 4 weeks, dehydrated by 20% sugar and 2% polyvinylpyrrolidone (PVP) solution for two weeks at 4°C and then embedded in O.C.T. compound (Tissue-Tek) and made for slices for histologic observation by confocal microscopy. These slices are washed by 2.44 mmol/L PBST (Triton-100) three times and blocked by 5% bovine serum albumin (BSA) for one hour at room temperature. Sections were washed three times by 2.44 mmol/L PBST and incubated with primary antibodies at 4°C overnight. After three washes with 2.44 mmol/L PBST, the sections were then incubated with fluorescein-conjugated secondary antibodies together with nuclear counterstaining dye (DAPI) at room temperature. After another three washes, slides were mounted with 5.43 mol/L glycerol and visualized under a confocal microscope (Olympus). The blood vessel area of a special subtype was measured using Image-Pro Plus (Media Cybernetics) based on color recognition. Changes of a special vessel between two groups were observed and statistical analyses were performed by SAS software. Results The patients have similar weight and height. Blood routine examination and liver and kidney functions are normal. There are lots of differences in BMD and biochemical markers of bone metabolism. A special vessel subtype was found in human bone samples, but area of the vessel in patients with osteoporotic fractures was significantly less than with nonosteoporotic fractures. The result of two groups showed significant difference in area of a special vessel (t=26.66, P<0.0001). Conclusion This study preliminarily confirmed that the special vessel subtype actually exists in human bone. The area of the special vessel shows significantly different between two groups. That will provide new targets for prevention and treatment of osteoporotic fractures. Key words: Osteoporosis, postmenopausal; Microvessels; Bone density