骨组织H亚型血管在骨质疏松与非骨质疏松骨折患者中的表现

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

目的 证实人骨组织中存在特殊亚型（H亚型和L亚型）血管,探讨H亚型血管在骨质疏松与非骨质疏松骨折患者中的表现.方法 设计前瞻性临床试验,收集2015年5月至2016年3月期间股骨近端转子间骨折行近端髓内钉固定患者的骨标本,研究对象均为绝经后女性,年龄60～70岁,平均（64.30±4.67）岁.按照术前双能X线骨密度T值将患者分为骨质疏松组（T值≤-2.5）和非骨质疏松组（T值＞-2.5）,每组10例.在保证不增加医源性骨损失的前提下,在髓内钉植入前使用特制器械取出骨块,约6 mm×10 mm,生理盐水冲洗骨块表面残余的血液,立即置入质量分数4％多聚甲醛溶液固定24 h,0.5 mol/L EDTA脱钙4周,质量分数20％蔗糖溶液脱水2周,OTC包埋、切片,应用一抗4℃孵育过夜,二抗常温孵育1h,激光共聚焦显微镜观察两组骨切片中特殊亚型血管面积的变化.结果 两组患者身高、体重相当,血常规及肝、肾功能血清指标正常,骨密度及血清骨代谢指标存在差异.两组患者骨标本中均存在特殊亚型血管,但与非骨质疏松组比较,骨质疏松组骨标本中特殊亚型血管的面积减少,两组的差异有统计学意义（t=26.66,P＜ 0.0001）.结论 人骨组织中存在特殊亚型血管,其面积大小在骨质疏松与非骨质疏松性骨折患者间存在差异,为骨质疏松性骨折的防治提供新的靶点.

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℃ 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℃ 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 yessel 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.