环氧乙烷处理同种异体皮质骨板移植组织学研究

目的研究环氧乙烷处理同种异体皮质骨板移植组织学表现.方法分别移植经过零下70℃深低温冷冻4周、零下70℃深低温冷冻4周+48℃环氧乙烷灭菌处理的山羊同种异体皮质骨板,对移植骨板以及周围软组织进行组织学检查.结果术后3～6周深低温冷冻组移植骨板周围软组织少量炎性细胞浸润,环氧乙烷+深低温冷冻组移植骨板周围软组织较多炎性细胞浸润,但炎细胞浸润评分差别无统计学意义(P＞0.05);术后6～24周两组移植骨板周围软组织炎性细胞浸润进一步减轻.深低温冷冻组和环氧乙烷+深低温冷冻组术后3周部分移植骨板原哈佛式管扩大,其内有少量间充质组织和新生血管;6周间充质细胞分化为破骨细胞和成骨细胞,破骨细胞使移植骨板哈佛式管进一步扩大以及出现骨吸收隙窝,在哈佛式管和骨吸收隙窝边缘出现成骨细胞贴附和新骨形成;12周移植骨板骨吸收隙窝缩小,成骨细胞和新骨形成增多,部分新骨改建形成板层骨;24周移植骨板基本完成吸收替代和改建塑形.两组移植骨板内无炎细胞浸润,组织学表现相似.结论环氧乙烷灭菌同种异体皮质骨板移植周围软组织出现炎细胞浸润,不影响移植骨板爬行替代和内部改建塑形.     

环氧乙烷处理同种异体皮质骨板移植组织学研究

目的 研究环氧乙烷处理同种异体皮质骨板移植组织学表现。方法 分别移植经过零下70℃深低温冷冻4周、零下70℃深低温冷冻4周+48℃环氧乙烷灭菌处理的山羊同种异体皮质骨板,对移植骨板以及周围软组织进行组织学检查。结果 术后3～6周深低温冷冻组移植骨板周围软组织少量炎性细胞浸润,环氧乙烷+深低温冷冻组移植骨板周围软组织较多炎性细胞浸润,但炎细胞浸润评分差别无统计学意义(P>0.05);术后6～24周两组移植骨板周围软组织炎性细胞浸润进一步减轻。深低温冷冻组和环氧乙烷+深低温冷冻组术后3周部分移植骨板原哈佛式管扩大,其内有少量间充质组织和新生血管;6周间充质细胞分化为破骨细胞和成骨细胞,破骨细胞使移植骨板哈佛式管进一步扩大以及出现骨吸收隙窝,在哈佛式管和骨吸收隙窝边缘出现成骨细胞贴附和新骨形成;12周移植骨板骨吸收隙窝缩小,成骨细胞和新骨形成增多,部分新骨改建形成板层骨:24周移植骨板基本完成吸收替代和改建塑形。两组移植骨板内无炎细胞浸润,组织学表现相似。结论 环氧乙烷灭菌同种异体皮质骨板移植周围软组织出现炎细胞浸润,不影响移植骨板爬行替代和内部改建塑形。     

Histochemical changes of rat skeletal muscles induced by cold acclimation

After cold acclimation of rats the augmentation of succinic dehydrogenase activity in the fast-twitch oxidative glycolytic (FOG) and the slow-twitch oxidative (SO) fibers was observed in entire regions of the soleus, the extensor digitorum longus, the plantaris, the longissimus and the gastrocnemius muscles. Furthermore, a tendency to increased proportion of the FOG and the SO fibers was observed more prominently in superficial regions than in deep regions of a large muscle such as the gastrocnemius muscle.     

Application of a new bioresorbable film to guided bone regeneration in tibia defect model of the rabbits

Aim is to study the effect of calcium alginate film (CAF) on guided bone regeneration (GBR). Circular bone defects with 5 mm diameter were created in both tibias in 60 rabbits. The defects covered with CAF served as the experimental site, and with collagen membrane (CM) or with no membrane both served as the control site. Healing was analyzed by gross, X-ray, electromicroscopy, histology, immuno-histochemical studies, and an image pattern analysis system after 1, 2, 4, 6, and 8 weeks. The CM control sites showed more macrophages, and CM were absorbed more slowly while collecting fewer osteoinductive factors (p < 0.01) in the early weeks. CAF induced dense bone formation, whereas CM induced less new bone, and the blank control sites effected the worst. In conclusion, the effect of CAF group gave better results than blank control group and CM group on GBR in this animal model.     

The effect on bone regeneration of a liposomal vector to deliver BMP-2 gene to bone grafts in peri-implant bone defects

Successful bone-implant osseointegration in large peri-implant bone defects is often difficult, even through autologous bone grafting. Recently, cell-mediated regional gene therapy was introduced to deliver potent morphogens or growth factors in regenerative medicine. We applied liposomal vectors carrying bone morphogenetic protein (BMP)-2 cDNA directly into freshly created peri-implant bone defects on pig calvariae, with or without autologous bone graft. The BMP-2 gene was efficiently introduced into immigrating cells and trabecular cells lining the marginal bone surrounding the bony defect. After 1 week, abundant BMP-2 protein was detected throughout the peri-implant bone defect by immunohistochemistry. At 4 weeks, BMP-producing cells were still present in the defect and peri-implant area, which significantly enhanced new bone formation, compared with the control groups. Interestingly within a week of BMP-2 gene delivery with bone grafts, most osteoblastic cells lining the grafted bone chips also produced BMP-2. Particulated bone was immediately reorganized into newly formed trabecular bone. Grafted bone without BMP-2 gene delivery was still scattered and new bone matrix formation was not detected until 4 weeks after bone grafting. In conclusion, direct application of the BMP-2 gene using a liposomal vector enhanced bone regeneration in a bony defect and gene delivery combined with bone graft could induce a rapid osseointegration of the bone-implant interface at earlier stage.     

Histochemical studies of hepatocellular carcinomas in the rat induced by aflatoxin

A histochemical study has been done on a group of 18 hepatocarcinomas induced by aflatoxin. One hundred per cent, incidence of hepatocarcinomas is induced by feeding 5 p.p.m. aflatoxin for 6 weeks. The carcinomas were trabecular hepatocarcinomas with a mixed adenomatous pattern and showed considerable variation in histochemical reactions throughout the lesions. There was a patchy distribution of glycogen and glucose-6-phosphate and the membrane ATPase was present along much of the canalicular border of the cells but with an abnormal and tortuous pattern. Aniline hydroxylase was present in varying amounts in both trabecular and adenomatous carcinomas. It is concluded that the histological variants of hepatocarcinoma are all derived from hepatocytes, but no unique changes were observed related to the progress involved in malignant neoplasia. The observations form a basis for comparison with early lesions seen prior to the recognition of carcinoma.     

Umbilical cord and bone marrow mesenchymal stem cell seeding on macroporous calcium phosphate for bone regeneration in rat cranial defects

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be harvested at a low cost without an invasive procedure. However, there has been no report on comparing hUCMSCs with human bone marrow MSCs (hBMSCs) for bone regeneration in vivo. The aim of this study was to investigate hUCMSC and hBMSC seeding on macroporous calcium phosphate cement (CPC), and to compare their bone regeneration in critical-sized cranial defects in rats. Cell attachment, osteogenic differentiation and mineral synthesis on RGD-modified macroporous CPC were investigated in vitro. Scaffolds with cells were implanted in 8-mm defects of athymic rats. Bone regeneration was investigated via micro-CT and histological analysis at 4, 12, and 24 weeks. Three groups were tested: CPC with hUCMSCs, CPC with hBMSCs, and CPC control without cells. Percentage of live cells and cell density on CPC in vitro were similarly good for hUCMSCs and hBMSCs. Both cells had high osteogenic expressions of alkaline phosphatase, osteocalcin, collagen I, and Runx2. Bone mineral density and trabecular thickness in hUCMSC and hBMSC groups in vivo were greater than those of CPC control group. New bone amount for hUCMSC-CPC and hBMSC-CPC constructs was increased by 57% and 88%, respectively, while blood vessel density was increased by 15% and 20%, than CPC control group at 24 weeks. hUCMSC-CPC and hBMSC-CPC groups generally had statistically similar bone mineral density, new bone amount and vessel density. In conclusion, hUCMSCs seeded on CPC were shown to match the bone regeneration efficacy of hBMSCs in vivo for the first time. Both hUCMSC-CPC and hBMSC-CPC constructs generated much more new bone and blood vessels than CPC without cells. Macroporous RGD-grafted CPC with stem cell seeding is promising for craniofacial and orthopedic repairs.     

Roles of exogenously regulated bFGF expression in angiogenesis and bone regeneration in rat calvarial defects

Regulation of transgene expression and function is important for gene therapy because it allows complex biological processes to be controlled and monitored. Basic fibroblast growth factor (bFGF) is an effective angiogenic factor and bone regeneration factor; it can induce differentiation of mesenchymal stem cells (MSCs) in?vitro and bone regeneration in?vivo. Further, exogenous regulation of controllable bFGF expression in the bone regeneration area safely allows bone formation and regeneration. In our study, we constructed a recombinant adeno-associated virus type 2 (rAAV2)-based bFGF gene delivery system, which is regulated by tetracycline or doxycycline (Dox, an analogue of tetracycline). We evaluated the regulatory effects of this system on bFGF transgenic expression in?vitro and in?vivo. We found that bFGF could increase the mRNA expression levels of osteoblast differentiation factor and the activity of alkaline phosphatase (ALP). Dox could effectively regulate bFGF expression, thus controlling MSC differentiation. After in?vivo transplantation of genetically engineered MSCs, animals not treated with Dox showed significant bone formation and angiogenesis compared with the group treated with Dox. Dox may also effectively regulate angiogenesis and bone regeneration in?vivo. Therefore, the inducible bFGF system is an effective way of regulating bone regeneration and formation.     

Effects of Laddec on the formation of calcified bone matrix in rat calvariae cells culture.

Osteoblasts from 21 day-old fetal rats calvaria were isolated using a collagenase digestion procedure. Cells were cultured in the presence of Laddec (a highly purified bovine xenograft) and Bio-Oss (natural bone mineral). Optical microscopic observations showed that osteoblasts attached on the plastic culture dishes and formed close contact with biomaterial particles. By day 5, the osteoblasts formed a confluent monolayer. A cytozymatic method showed intense alkaline phosphatase (ALP) staining around and between the substrate granules of the two materials. By day 14, inverted phase microscopic observations showed that osteoblasts formed bone nodules around and between substrate particles. In addition, at this time, the Von Kossa staining was positive. Using a conventional assay, ALP specific activity was higher in the presence of Laddec than in the presence of Bio-Oss. A quantitative morphological method using image analysis showed that the proportion of mineralized bone formed around biomaterial particles in relation to total bone was increased with Laddec (15% more than with Bio-Oss). Ultrastructural observations by TEM showed the presence of an electron dense collagen-free layer at the biomaterial/bone interface and a collagenous matrix deposited at the periphery, indicating the bioactivity of the biomaterials. These results indicated that Laddec increased the expression of ALP in osteoblast cultures and facilitated the formation of multiple cell layers, providing a culture environment suitable for mineralization.     

可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损

背景：自体髂骨移植一直被认为是骨缺损修复的“金标准”,但其来源有限。 目的：验证应用可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损的效果。 方法：80只雄性SD大鼠建立双侧颅骨缺损模型,随机分为3组：骨修复材料+骨碎补总黄酮组采用可注射骨修复材料结合骨碎补总黄酮灌胃修复大鼠颅骨缺损;骨修复材料+去离子水组采用可注射骨修复材料结合去离子水灌胃修复大鼠颅骨缺损;羟基磷灰石+去离子水组采用羟基磷灰石结合去离子水灌胃修复大鼠颅骨缺损,1次/d,持续8周。于建模后2,4,8周取颅骨标本进行苏木精-伊红染色和Masson染色组织学观察。 结果与结论：羟基磷灰石组新骨形成和材料降解速度较慢;可注射骨修复材料组新骨形成和材料降解较羟基磷灰石组快,利于血管及纤维组织长入;骨碎补总黄酮灌胃可以促进血管及纤维组织长入材料,促进成骨。与羟基磷灰石相比,可注射骨修复材料结合骨碎补总黄酮修复大鼠颅骨缺损,可促进新骨形成,缩短骨缺损修复时间。     

Histochemical studies on the early proliferative lesion induced in the rat liver by aflatoxin

Male inbred Fischer rats were fed a diet containing 5 p.p.m. aflatoxin for 1, 3, 4½ and 6 weeks at which times groups were killed for histological and histochemical study. Aflatoxin produced a scattered individual cell necrosis of parenchymal cells by 1 week. At 3 weeks small basophilic proliferative foci were seen which increased in size and abundance to 6 weeks. These foci showed starvation-resistant glycogen, variable depletion of glucose-6-phosphatase, succinic dehydrogenase, aniline hydrogenase, membrane ATPase and acid phosphatase. At 6 weeks the foci showed the presence of gamma glutamyl transpeptidase and glucose-6-phosphate dehydrogenase. The basophilic foci were not preceded by other focal histological and histochemical change. The basophilic proliferative lesions are observed when an irreversible change has been induced in the liver. The role of such lesions in the histogenesis of hepatocellular carcinoma is discussed.     

The double cantilever beam test applied to mode I fracture characterization of cortical bone tissue

The primary objective of this work was to analyse the adequacy of the Double Cantilever Beam (DCB) test in determining fracture toughness under pure mode I loading of cortical bone tissue. A new data reduction scheme based on specimen compliance and the crack equivalent concept was used to overcome the difficulties inherent in crack monitoring during its growth. It provides a complete resistance curve, which is fundamental in estimating the fracture energy. A cohesive zone model was used to simulate damage initiation and propagation, thus assessing the efficacy of the proposed testing method and data reduction scheme. Subsequently, the DCB test was applied to evaluate the mode I fracture energy of hydrated and thermally dehydrated cortical bone tissue from young bovine femur, in the tangential–longitudinal propagation system. The results obtained demonstrate the efficacy of the DCB test and the proposed data reduction scheme on the bone fracture characterization under mode I loading.     

烟碱拮抗秋水仙碱诱导的乳大鼠大脑皮质神经元凋亡

目的：探讨烟碱拮抗秋水仙碱诱导乳大鼠大脑皮质神经元凋亡的作用机制。方法: 培养Sprague-Dawley（SD）乳大鼠大脑皮层神经元，Hoechst33258核荧光染色进行凋亡率计数检测神经元凋亡, Akt（ser473）Western blotting分析试剂盒做激酶分析。结果: 0.1 μmol/L秋水仙碱可降低皮层神经元的存活率，诱导大鼠皮层神经元凋亡。但是，当10 μmol/Ｌ烟碱预孵2 h后再加入0.1 μmol/L秋水仙碱作用24 h, 烟碱可拮抗秋水仙碱的此种凋亡作用,将凋亡率从（62±1）％ 降低到 (38±2) ％,P<0.05。Akt（ser473）磷酸化在0.5 h时开始下降，于6 h时最低，仅为对照组的1/3。且Akt（ser473）磷酸化水平随时间（0.5、1、6、12和18 h）呈钟型性改变,分别是对照组的1.3、3.7、2.4、2.1和1.9倍,P<0.05。结论: 烟碱对大鼠皮质神经元凋亡的拮抗作用可能与其激活Akt有关。     

The effects of immobilization on vascular canal orientation in rat cortical bone

It is well established that bone is capable of adapting to changes in loading; however, little is known regarding how loading specifically affects the internal 3D microarchitecture of cortical bone. The aim of this study was to experimentally test the hypothesis that loading is a determinant of the 3D orientation of primary vascular canals in the rat tibial diaphysis. Left tibiae from 10 rats (30 weeks old) that had been immobilized (sciatic neurectomy) for 27 weeks, right SHAM-operated tibiae from these same rats (internal control) and right tibiae from 10 normal age-matched rats (external control) were scanned by micro-CT. Mean canal orientation (for the whole bone segment and by region), percent porosity, canal diameter and canal separation were quantitatively assessed in 3D. Canal orientation in the immobilized tibiae was significantly (P < 0.001) more radial (by 9.9°) compared to the external controls but did not differ from the internal controls (P = 0.310). Comparing the external and internal controls, orientation was significantly (P < 0.05) more radial in the internal control group (by 6.8°). No differences were found for percent porosity and canal separation. Canal diameter was significantly greater in the immobilized vs. internal (P < 0.001) and external control (P < 0.001) tibiae. The differences in orientation relative to the external controls indicated that the organization of cortical bone in the rat is affected by loading. Although the predicted difference in canal orientation was not detected between immobilized and internal control groups, the distributions of individual canal orientations, from which the mean values were derived, revealed distinctive patterns for all three groups. The internal controls exhibited an intermediate position between the immobilized and external controls, suggesting that paralysis on the contralateral side resulted in altered loading relative to the normal state represented by the external control. This was also evident in a regional analysis by quadrant. The loaded bones had the same cross-sectional shape; however, their internal structure differed. These results provide novel insights into the impact of loading on the 3D organization of primary cortical bone and have implications for understanding the relation between cortical bone adaptation, disease and mechanical properties.     

Prosthetic devices shaped as tubular chambers for the treatment of large diaphyseal defects by guided bone regeneration

Guided tissue regeneration is based on the hypothesis that the different tissues have unequal abilities to penetrate a wounded area during the healing process. The use of a device acting as a chamber allows the growth of a particular tissue and prevents the ingrowth of other tissues which impair the healing process. At the same time the chamber protects and maintains in situ the intrinsic growth factors so that they may perform their specific activity. Guided tissue regeneration currently plays a well-recognized role mostly in dentistry and peripheral nerve surgery but interesting perspectives have also opened up in orthopedics. Considering the possibility of using guided bone regeneration in the repair of diaphyseal bone defects, this updated survey highlights some critical points and pathways related to the state-of-the-art of this promising procedure, focusing particularly on the properties of the material to make the tubular chamber, the use of osteopromotive factors and the most appropriate animal model to be used for the experimental evaluation.     

黄芪诱导骨髓间充质干细胞向神经干细胞分化

目的:观察黄芪注射液诱导大鼠骨髓间充质于细胞(BMSCs)分化的特点以及黄芪对BMSCs活性的影响。方法:分别使用浓度为20、50、100、200μl/ml的黄芪注射液诱导BMSCs,分别在1、5、24 h光镜观察细胞形态变化,免疫细胞化学方法观察巢蛋白、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP-2)的表达,MTT方法检测黄芪对BMSCs活性的影响。结果:诱导后细胞体积增大,胞核固缩,核质比减小,有细长突起形成,部分细胞间可见网络状连接。巢蛋白阳性细胞在100μl/ml浓度诱导24 h组染色最强,而NSE和GFAP阳性细胞在200μl/ml浓度诱导24 h组阳性细胞数量最多,染色最强,MAP-2抗体染色只在100μl/ml浓度诱导24 h组和200μl/ml浓度诱导24 h组出现少量阳性细胞。不同浓度的黄芪注射液以及作用不同时间均对BMSCs的活性产生影响。结论:在黄芪作用下,BMSCs表达神经干细胞特异标志物,随着时间的延长,进一步表达神经元特异标志物和胶质细胞特异标志物。