Scale‐up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low‐density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro‐CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time‐point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low‐density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1213–1220, 2012     

Assessment of adhesion formation to intra-abdominal polypropylene mesh and polytetrafluoroethylene mesh

BACKGROUND: The development of intra-abdominal adhesions, bowel obstruction, and enterocutaneous fistulas are potentially severe complications related to the intraperitoneal placement of prosthetic biomaterials. The purpose of this study was to determine the natural history of adhesion formation to polypropylene mesh and two types of polytetrafluoroethylene (ePTFE) mesh when placed intraperitoneally in a rabbit model that simulates laparoscopic ventral hernia repair. MATERIALS AND METHODS: Thirty New Zealand white rabbits were used for this study. A 10-cm midline incision was performed for intra-abdominal access and a 2 cm x 2 cm piece of mesh (n = 60) was sewn to an intact peritoneum on each side of the midline. Two types of ePTFE mesh (Dual Mesh and modified Dual Mesh, W.L. Gore & Assoc., Flagstaff, AZ) and polypropylene mesh were compared. The rate of adhesion formation was evaluated by direct visualization using microlaparoscopy (2-mm endoscope/trocar) at 7 days, 3 weeks, 9 weeks, and 16 weeks after mesh implantation. Adhesions to the prosthetic mesh were scored for extent (%) using the Modified Diamond Scale (0 = 0%, 1 50%). At necropsy the mesh was excised en bloc with the anterior abdominal wall for histological evaluation of mesothelial layer growth. RESULTS: The mean adhesion score for the polypropylene mesh was significantly greater (P < 0.05) than Dual Mesh at 9 weeks and 16 weeks and modified Dual Mesh at 7 days, 9 weeks, and 16 weeks. Fifty-five percent (n = 11) of the polypropylene mesh had adhesions to small intestine or omentum at necropsy compared to 30% (n = 6) of the Dual Mesh and 20% (n = 4) of the modified Dual Mesh. There was a significantly greater percentage (P < 0.003) of ePTFE mesh mesothelialized at explant (modified Dual Mesh 44.2%; Dual Mesh 55.8%) compared to the polypropylene mesh (12.9%). CONCLUSIONS: Serial microlaparoscopic evaluation of intraperitoneally implanted polypropylene mesh and ePTFE mesh in a rabbit model revealed a progression of adhesions to polypropylene mesh over a 16 week period. The pore size of mesh is critical in the development and maintenance of abdominal adhesions and tissue ingrowth. The macroporous polypropylene mesh promoted adhesion formation, while the microporous nature of the visceral side of the ePTFE served as a barrier to adhesions.     

种植性兔VX2肝肿瘤血供的研究

目的探讨种植性兔VX2肝肿瘤生长的自然血供特点。方法VX2瘤株接种于新西兰兔肝左叶,制成模型瘤兔15只,将其按瘤体直径分为3组:A组直径<2.0cm、B组直径2.0～3.0cm、C组直径>3.0cm,分别应用彩色多普勒超声仪检测各组VX2瘤兔的门静脉和肝动脉的血流动力学指标,以及肝动脉血管造影显示瘤体供血情况,分析不同直径大小的瘤体血流分布及构成。结果A组的VX2兔肝肿瘤供血不丰富,肝动脉部分参与供血;B组的VX2兔肝肿瘤瘤内血流丰富,供血以肝动脉为主;C组的VX2兔肝肿瘤中心部出现坏死,瘤周血流丰富,供血以肝动脉为主。C组的VX2兔肿瘤肝动脉最大血流速度(Vmax)(60.3±10.9cm/s)与A组(50.4±12.2cm/s)相比有差异(P<0.01),阻力指数(RI)(0.9±0.12)与A组(0.5±0.02)相比有差异(P<0.01);门静脉平均血流速度(V)及门静脉内径(D)各组差异不大(P>0.05)。结论种植性VX2肝肿瘤主要供血血管为肝动脉,肝动脉最大血流速度和阻力指数随肿块增大明显增加,多普勒流速曲线呈高速高阻型;门静脉平均血流速度及门静脉内径随肿块增大变化不明显。     

放射性~(125)I粒子组织间植入对家兔尿道损伤的实验研究

目的:研究不同剂量的放射性125I粒子对家兔尿道的放射性损伤。方法:麻醉下将放射性125I粒子植入雄性家兔尿道旁1.0cm处。125I粒子的放射性粒子活度分别为14.8MBq(A组)、29.6MBq(B组)和44.4MBq(C组),对照组(D组)仅尿道旁种植相当于粒子大小无放射性的无菌铅管1粒。植入后4周,摄尿道片,观察粒子位置等情况;原手术切口切开,取放射粒子周围2.0cm范围内的家兔尿道组织作肉眼、光学显微镜和电子显微镜观察,进行放射性损伤的评价。结果:术后4周,肉眼及光学显微镜观察,实验组与对照组粒子周围的尿道粘膜、粘膜下及肌层所见基本一致;C组少部分电镜视野中观察到尿道上皮胞质出现较多空泡变性、空化、嵴稀疏等超微结构的损伤。光镜下尿道入射性损伤评分,A、B、C、D组分别为(2.20±0.18)、(2.23±0.15)、(2.27±0.10)、(2.10±0.17)分,A、B、C组与D组相比,差异无显著性(P>0.05)。对线粒体作FlaMeng半定量分析,A、B、C、D各组评分分别为(1.23±0.13)、(1.34±0.25)、(1.41±0.30)、(1.12±0.13)分,A、B、C各组与D组(对照组)相比,差异无显著性(P>0.05)。结论:放射性125I粒子对尿道放射性损伤随粒子的放射性活度的增加而逐渐加重,呈明显的放射性活度效应关系;正常剂量的放射性粒子对尿道的损伤是很轻微的,是安全可行的。     

Free radical damage as a biomarker of bladder dysfunction after partial outlet obstruction and reversal

OBJECTIVE

To investigate the use of free‐radical generation as a result of protein carbonylation and nitrotyrosination to characterize the level of bladder dysfunction after partial bladder outlet obstruction (PBOO) and reversal.

MATERIALS AND METHODS

We surgically created PBOO in male New Zealand White rabbits; after 4 weeks of PBOO, one group of six rabbits was assessed, while the PBOO was relieved in two additional groups of six rabbits each that were assessed at 4 and 8 weeks after relieving the PBOO. Six sham‐operated rabbits served as controls. Sedated rabbits were assessed by cystometry and the bladders were then removed for contractile, histological and molecular studies. Western blotting was used to determine the level of carbonylation and nitrotyrosination at the protein level.

RESULTS

The PBOO group had significant decreases in the contractile responses to field stimulation, ATP, carbachol and KCl. The responses to all forms of stimulation increased significantly at 4 weeks after reversal, and further increased to near normal levels by 8 weeks. Similarly, compliance and cystometric values also returned to near normal values after reversal. The hypertrophied smooth muscle of the obstructed bladders regressed to near‐normal size. There was a significant increase in the level of carbonylation and nitrotyrosination after PBOO, and a progressive decrease in the 4‐week reversal groups, nearing control values by 8 weeks.

CONCLUSIONS

Significantly increased carbonylation and nitrotyrosination levels after PBOO correlated with the severe dysfunction in the obstructed rabbits. Similarly, decreased levels of oxidation and nitration correlated with the functional recovery after reversal.     

营养性高脂血症对雄性新西兰兔睾丸和阴茎发育的影响

目的:探讨营养性高脂血症对雄性新西兰兔睾丸和阴茎发育的影响。方法:雄性新西兰兔36只,随机分为实验组(n=20)和对照组(n=16),高脂饲料建立营养性高脂血症动物模型。全自动生化分析仪检测外周血总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)和低密度脂蛋白胆固醇(LDL-C);放射免疫法检测外周血睾酮(T)、黄体生成素(LH)和卵泡刺激素(FSH);HE染色观察睾丸和阴茎发育的形态学改变。结果:与对照组比较,实验组TC、TG、LDL-C明显升高,T、LH和FSH水平明显降低,差异有极显著性(P<0.01);与对照组比较,实验组阴茎长度缩短(P<0.05),睾丸系数下降(P<0.01)。光镜下睾丸生精上皮破坏明显,阴茎组织有明显的脂肪细胞沉积。结论:幼年起摄入大量高脂食物极易诱发营养性高脂血症,并可造成下丘脑-垂体-性腺轴功能紊乱,导致阴茎发育短小,睾丸生精上皮破坏。     

人参多糖对家兔肝缺血/再灌注损伤时血小板聚集功能的影响

目的探讨人参多糖对家免肝缺血/再灌注损伤时血小板聚集功能的影响。方法30只家免,随机均分为对照组（C组）、缺血/再灌注组（IR组）和人参多糖组（GP组）。观察血小板最大聚集率（Ptmax）、最大聚集时间（PtT）、聚集坡斜率（PtS）及谷丙转氨酶（ALT）的变化,电镜观察肝组织形态学改变。结果IR组与C组比较,各时间点Ptmax、PtS、ALT明显升高,PtT显著缩短,肝组织超微结构发生异常改变;人参多糖可逆转上述指标的异常变化。结论人参多糖可通过抑制血小板聚集功能,改善肝脏微循环,从而防治肝脏缺血/再灌注损伤。     

6种中药提取物对离体新西兰白兔阴茎海绵体的松弛作用

目的:比较6种中药提取物(甲基莲心碱、粉防己碱、葛根素、灯盏花乙素、人参皂苷Rg1和人参皂苷Rb1)松弛离体新西兰白兔阴茎海绵体的作用。方法:采用离体新西兰白兔阴茎海绵体肌条张力记录法,观察6种中药提取物对去氧肾上腺素(PE)诱导收缩的阴茎海绵体肌条的松弛作用。结果:在(10-8~10-3)mol/L,甲基莲心碱、粉防己碱、葛根素和灯盏花乙素对PE诱导收缩的肌条具有浓度依赖性松弛作用,其IC50分别为4.60×10-6、3.73×10-5、8.03×10-4和3.33×10-3mol/L。而人参皂苷Rg1和人参皂苷Rb1对PE诱导收缩的肌条松弛作用较弱,终浓度为10-3mol/L时,仅分别松弛(16.32±13.36)%和(11.21±7.59)%。结论:6种中药提取物对离体新西兰白兔阴茎海绵体的松弛作用,甲基莲心碱最强。     

实验性睾丸缺血超声表现与再灌注后组织变化相关性研究

目的:探讨不同程度急性单侧睾丸缺血的二维、彩色多普勒(CDU)及超声造影表现与再灌注后睾丸病理变化的相关性。方法:32只大白兔随机分成对照组、缺血组(A、B、C组),每组8只。缺血组在超声监测下制成不同程度的单侧睾丸缺血模型,A组,睾丸回声均匀、血流信号轻度减少;B组,睾丸回声不均匀、血流信号明显减少;C组,睾丸出现放射状或小片状低回声、血流信号消失。各组在出现上述声像图变化后予以再灌注。缺血前及再灌注前分别行双侧睾丸超声造影。1个月后观察各组缺血睾丸病理变化。比对分析各组超声表现与生精功能之间的关系。结果:超声造影:与对照组比较,A、B组缺血睾丸造影参数到达时间、速度、达峰时间、峰值减半时间有显著差异(P<0.05),峰值基础强度差A组无明显差异(P>0.05)、B组有显著差异(P<0.05);C组无造影剂充盈。1个月后缺血睾丸Johnsen评分:与对照组(9.10±0.11)比较,A组(8.70±0.39)无显著差异(P>0.05),B组(6.01±0.88)有显著差异(P<0.05),C组(3.16±1.05)有极显著差异(P<0.01)。结论:与CDU相比,超声造影能较好地反映缺血睾丸组织的血流灌注情况,睾丸缺血的不同超声表现与再灌注后生精功能明显相关,超声检查能对再灌注后睾丸生精功能的预测提供帮助。     

Regional differences in responses of rabbit detrusor to electrical and adrenergic stimulation: influence of outlet obstruction

OBJECTIVE: To examine regional responses of control and obstructed rabbit detrusor strips to electrical and adrenergic stimulation, and determine whether outlet obstruction causes regional variations in blood flow throughout the detrusor, as the detrusor smooth muscle of the bladder body has previously been considered homogeneous in its pharmacological properties. MATERIALS AND METHODS: Fourteen male rabbits had the bladder outlet surgically obstructed for 2 weeks and were compared with 10 unoperated control rabbits. Blood flow was measured with the bladder empty and at capacity, using fluorescent microspheres. Paired dorsal and ventral strips were harvested from the midline equatorial detrusor and electrically and adrenergically stimulated. RESULTS: Obstructed rabbits had significantly higher bladder capacities and bladder weights than control rabbits. Dorsal strips from both control and obstructed rabbits contracted in response to noradrenaline, whereas ventral strips relaxed. The addition of prazosin, a nonselective alpha1-adrenergic-receptor blocker, completely blocked the contraction in dorsal strips, but had no effect on responses of ventral strips. There was also a regional difference in response to electrical stimulation, with ventral strips generating significantly more tension than dorsal strips in both control and obstructed rabbits. There were no regional differences in detrusor blood flow. Obstruction resulted in significantly lower responses to all forms of stimulation, and significantly less blood flow throughout the detrusor. CONCLUSION: There are regional differences in adrenergic receptor function and response to electrical-field stimulation throughout control and obstructed rabbit detrusor, a region that was previously thought to be functionally homogeneous. These differences must be recognized and acknowledged to obtain accurate and reproducible data from in vitro studies of the bladder.