骨痂中转化生长因子-β1基因表达及利用骨痂植骨对术后功能的影响

背景:在骨折愈合过程中,若将骨折端周围的骨痂组织去除,骨折端比较难完全吻合,可以推测这些骨痂组织是人体在修复过程中的必然产物,应该是有益的,拟将这些骨痂做组织学分析和冰冻切片的原位杂交,观察其组织学内容和转化生长因子β1的含量,并做为供骨植于骨折端,了解骨痂植骨在骨折愈合中的作用。目的:通过测定骨痂中的组织学内容和转化生长因子β1的含量来了解延迟手术中骨折端周围骨痂组织的再利用价值。设计:采用骨痂组织原位杂交及随机分组对照的实验。单位:西安交通大学第二医院骨科、台州市中心医院骨科。材料:病例分别选择西安交通大学第二医院骨科和台州市中心医院骨科1994-07/2003-10骨折延迟手术的住院患者51例,在手术中取骨折延迟愈合的骨痂做标本(均已征得患者同意),Dig标记试剂盒购自BoehringerMannheim公司,转化生长因子β1寡核苷酸探针的序列:CTTGCTGTACTGTGTGTCCAA,由DNA合成仪合成,X线计算机系统(CR)购自日本富士公司。方法:在骨折后的2,4,6和8周,在手术中取出少量骨痂做标本,采用不脱钙的骨痂组织做冰冻切片,采用非同位素Dig标记原位杂交,观察转化生长因子β1在骨痂中的基因表达。术中将骨断端清理后,在骨折牢固固定的同时,再将原骨痂植于骨缺损处及骨折周围。对延迟手术的患者分别采用单纯骨痂植骨、髂骨植骨,骨痂加髂骨植骨,经1～4年患者复诊随访,平均1年9个月。主要观察指标:①骨折不同时期骨痂中的组织学内容和转化生长因子β1的含量。②不同植骨方式对骨折愈合时间的影响。结果:参加治疗51例,有7例因死亡和到外地生活而未能进行随访,评定44例,纳入结果分析。①骨痂中转化生长因子β1的基因表达:骨折后第2周,骨折端有少量纤维骨痂,软骨骨痂较多,转化生长因子β1在软骨小岛内的软骨细胞中高表达。骨折后4周左右转化生长因子β1在成骨细胞内表达显著。8周左右大量骨痂生长,并以软骨化骨为主,成纤维细胞、骨痂、成骨细胞、骨小梁越来越多,术后6周转化生长因子β1在各种细胞内的表达消失,但随着骨重建过程增强,8周时骨基质中转化生长因子β1表达又有增高趋势。②不同植骨方式的愈合时间:不同的植骨方式在愈合时间上没有显著性差异。结论:转化生长因子β1在骨愈合的平衡中发挥重要作用,不同阶段骨痂的组织变化和转化生长因子β1量的变化有着必然的联系,骨痂是机体修复和重建的产物,骨痂植骨再利用是一种可行的方法,它对需要植骨的患者可减少其痛苦和损伤。

Transforming growth factor-beta 1 expression in callus and the effect of callus bone graft on the post-operative function

BACKGROUND: In the healing process of fracture, if the callus tissue around the location of fracture is cleared, the end of fracture is difficult to be integrated completely, and more or less there would be some bone de fect. We deduce that these callus tissues are the necessary product during the process of bone reparation, which ought to be useful. Concerning this,we observed histological content and the volume of transforming growth factor-β1 (TGF-β1) with histological analysis and hybridization in situ of the frozen section of these calluses to investigate the effects of callus graft in the healing process of fracture.OBJECTIVE: To understand the salvaging value of bony callus organization around fracture in delayed operation by means of determining histological substances and contents of TGF-β1.DESIGN: Hybridization in situ of the callus tissue and randomized-grouping and controlled experiment.SETTING: Department of Orthopaedics, the Second Hospital of Xi'an Jiaotong University, and Department of Orthopaedics, Central Hospital of Taizhou City PARTICHANTS: Totally 51 inpatients who received delayed fracture operations in the Department of Orthopaedics of the Second Hospital of Xi 'an Jiaotong University and Central Hospital of Taizhou City from July 1994 and October 2003 were recruited. In some cases of delayed fracture operation, callus was obtained with the consent of patients, Dig labeled kit was bought from Boehringer Mannheim Corp. CTT GCT GTA CTG TGT GTC CAA, TGF-β1 oligonucleotide probe sequence, was synthe sized by DNA synthesizer. Computer X-ray system was bought from Japan Fuji Corp. METHODS: 2,4,6,8 weeks after fracture, a small amounts of callus was obtained. Frozen section was made from undecalcified callus tissue, and noneisotope Dig labeled hybridization in situ, observe gene expression of TGF-β1 in callus. In the operation, after clearing the bone stump and fas tening the fracture, original bony callus was grafted to the bone defect and around the fracture. Simple callus graft, flank bone graft or both were adopted respectively on patients in delayed operation with follow-up obser vations were given for 1-4 years, with the average of 1 year and 9 months,at outpatient clinic for recheck.MAIN OUTCOME MEASURES: ① Histological substance and TGF-β1 content of bony callus at different phases of the fracture. ② Effect of different mode of bone graft on the time of fracture healing. RESULTS: Totally 51 cases were treated, and 7 cases were not followed up for death or moving to other areas. Data of the followed-up 44 patients were collected into the stage of result analysis. ① Gene expression of TGF-β1 in bone callus: In the second week after fracture, there were a small amount of fibrous bone callus and comparatively much cartilage bone callus,and TGF-β1 was hypso-expressed in cartilage cells inside the cartilage islet; about four weeks later, TGF-β1 was notably expressed in os teoblasts; about eight weeks later, a host of calluses grew, mainly carti laginea, and the numbers of formed fibroblast, callus, osteoblasts and tra becular bone were increasing. TGF-β1 expression in all kinds of cells dis appeared after six weeks but had a tendency to rise again in bone matrix in the eighth week. ② There were no significant difference of healing time in different modes of bone graft .CONCLUSION: TGF-β1 plays an important role in balancing bone healing, and necessary connections exist between tissue change in bone callus and change in expression of TGF-β1. As callus is a product of recovery and reconstitution of organism, we regard callus graft salvage as a feasible method in reducing pain and damage to patients who need graft.