恒河猴体内组织工程骨血管化的监测

Monitoring the vascularization of tissue engineered bone in rhesus

目的:采用X线、放射性核素骨显像、磁共振灌注成像三种不同方法监测组织工程骨血管化的情况,对比分析各种方法的优缺点。方法:实验于2005-05/08在南方医科大学南方医院动物实验中心完成。①选用13只成年雄性恒河猴的26条下肢,取1条下肢用于骨髓基质干细胞的培养,其余25条下肢分为5组:β-磷酸三钙+骨髓基质干细胞+血管束组、β-磷酸三钙+血管束组、β-磷酸三钙+骨髓基质干细胞组、单纯β-磷酸三钙组、空白对照组,5条/组。β-磷酸三钙直径12mm,长度20mm,横截面呈C型。血管束来源于恒河猴胫骨内侧皮下隐动脉分支,其外径约0.8～1.0mm。②13只恒河猴麻醉后,取胫前直切口,沿胫前肌与胫骨间隙进入。于胫骨外侧放置7孔AO重建钛钢板,除第4孔外其余孔2.5mm钻头钻孔,3.5mm丝攻攻丝后钛螺钉固定。于钢板第3～5孔之间用线锯锯断胫骨,切除该段相应骨膜,造成2cm的段缺性骨与骨膜缺损。然后根据动物分组填入各自相应材料,制备动物模型。③各组术后4,8,12周行磁共振灌注成像检查并计算信号强度-时间曲线的最大线性斜率(SSmax)及基线值(SIbaseline),拍摄恒河猴胫骨X线片并计算其透光度,同时行放射性核素骨显像检查并计算摄取比值。结果:实验选用13只成年雄性恒河猴的25条下肢,全部进入结果分析。①各组术后骨缺损区X线检查结果:各组骨缺损区透光度随着术后周数的延长均呈现不同的下降趋势。与术后4周比较,术后8,12周β-磷酸三钙+骨髓基质干细胞+血管束组均明显下降(P=0.001);与术后8周比较,术后12周β-磷酸三钙+血管束组、β-磷酸三钙+骨髓基质干细胞组均明显下降(P=0.002,P=0.021)。②各组术后骨缺损区放射性核素骨显像结果:β-磷酸三钙+骨髓基质干细胞+血管束组、β-磷酸三钙+血管束组、β-磷酸三钙+骨髓基质干细胞组、单纯β-磷酸三钙组感兴趣区同位素计数比值均为术后8周最高,术后12周最低。③各组术后骨缺损区磁共振灌注成像情况:术后4,8,12周β-磷酸三钙+骨髓基质干细胞+血管束组的SSmax值最高,且术后8周与4周相比SSmax有较大幅度的提高(P=0.003)。术后12周β-磷酸三钙+骨髓基质干细胞+血管束组5个样本的SSmax与同期X线透光度呈负相关(r=-1.0,P=0.000)。结论:信号强度-时间曲线的SSmax能够准确反映组织工程骨的血管化情况,磁共振灌注成像检查具有无创、无辐射、高灵敏度和定量分析的优点。

AIM： To monitor the vascularization of tissue engineered bone with 3 different methods, X-rays, radionuclide bone imaging, and magnetic resonance perfusion imaging, and compare and analyze the merits and shortcomings of 3 methods. METHODS： The experiment was conducted in the Animal Experimental Center, Nanfang Hospital of Southern Medical University between May and August 2005,①26 lower limbs were selected from 13 adult male rhesus, one of which was adopted to culture the bone marrow stroma cells （BMSCs）, and the rest 25 lower limbs were divided into 5 groups： β-tricalcium phosphate （β-TCP） ＋ BMSCs ＋vascular bundle group, β-TCP ＋ vascular bundle group, β-TCP ＋ BMSCs group, β-TCP group and blank control group with 5 limbs in each group, The diameter of β-TCP was 12 mm, and the length was 20 mm. The transverse section was in C type. The vascular bundles were obtained from the branches of arteria saphena inside the tibia of rhesus, the external diameter of which was 0.8-1,0 mm,② Vertical incisions were made before the shin of 13 rhesus after anesthesia, and medical tools entered along the interspace of tibial muscle anterior and tibia. A 7-pore AO reconstituted titanium steel board was placed in the extra-lateral of tibia, and all pores except the 4^th pore were drilled with a 2.5-mm drill bit, and then fixed with titanium screw after being tightened with 3.5-mm taps, The tibia between the 3^rd pore and 5^th pore was sawed off with wire saw, and corresponding periosteum in the segment was cut off to make a 2-cm defeet of bone and periosteum, Corresponding materials were then filled in animals aecording to the group to establish animal model,③ At 4, 8 and 12 weeks after operation, the magnetic resonance perfusion imaging was used to examine, and the greatest linear slope rate of signal intensity-time curve （SSmax） and value of baseline （SIbaseline） were ealculated, The transmissivity was calculated according to the X-ray of tibia in rhesus, meanwhile, radionuclide bone imaging was adopted for inspection and the ratio was calculated, RESULTS： A total of 25 enrolled lower limbs of 13 adult male rhesus were involved in the analysis of results, ①X-ray of bone defect in all groups after operation： The transmissivity in bone-defect area of all groups were descended to different extend with the increased time. Compared with the 4^th week after operation, that in the β-TCP ＋ BMSCs ＋vascular bundle group at 8 and 12 weeks after operation were significantly decreased （P=0.001）. Compared with the 8^th week after operation, that in the β-TCP ＋vascular bundle and β-TCP ＋ BMSCs group at 12 weeks after operation were obviously decreased （P=0.002, P=0.021）.②Results of radionuclide bone imaging in bone-defect area of all groups： The count ratio of isotope in region of interest of β-TCP＋ BMSCs ＋ vascular bundle group, β-TCP ＋ vascular bundle group, β-TCP ＋ BMSCs group and β-TCP group were the highest on the 8^th week after operation, and were the lowest at the 12^th week.③The magnetic resonance perfusion imaging of bone-defect area in all groups： At 4, 8 and 12 weeks after operation, the SSmax of β-TCP＋ BMSCs ＋ vascular bundle group was the highest, and that on the 8^th week was significantly increased than that on the 4^th week （P=0.003）. At 12 weeks after operation, the SSmax of 5 samples in the β-TCP＋ BMSCs ＋ vascular bundle group were in megative correlation with the transmissivity of X-ray in synchronization （r=-1.0,P=0.000）. CONCLUSION： The SSmax of signal intensity-time curve can accurately reflect the vascularization of tissue engineered bone, while the magnetic resonance perfusion imaging has some advantages such as high-sensitive, quantitative, noninvasive and non-radiant.