Frontal sinus obliteration after trauma: analysis of bone regeneration for two selected methods

We present a prospective randomized experimental study comparing bone regeneration obtained in 60 post-traumatic frontal sinuses obliterated with either calvarial bone dust (n = 30, group I) or calvarial bone and demineralized bone matrix (DBM; n = 30, group II). Radiological follow-up included high-resolution computed tomography with quantitative micro-density analysis in Hounsfield units (HU), together with a volumetric evaluation of the ossification at 6 and 24 months after surgical treatment. Epidemiological information and potential drawbacks were analysed. Bone volume and density data (HU) for the regenerated areas were subjected to statistical analysis at 6 and 24 months for both groups. Results were compared with reference values obtained from frontal and temporal bone in every patient. Complications developed for 10% of operated sinuses. The resulting bone formation (HU) in group I patients was significantly better than that obtained in group II. Ossification progressed in a statistically significant manner in both groups when compared at 6 and 24 months postoperatively. The use of DBM as a biomaterial associated with calvarial bone dust for sinus obliteration shows long-term safe results, similar to autogenous bone, but with a lower final bone density.     

腺病毒介导BMP-2和EGFP基因转染兔骨髓基质干细胞及种植脱钙骨的体外观察

目的用腺病毒载体介导人骨形态发生蛋白-2及EGFP基因转染兔骨髓基质干细胞(rBMSC),种植DBM(脱钙骨)支架体外构建组织工程骨。方法兔髓基质干细胞(rBMSC)的分离、培养;流式细胞仪检测细胞表面标记;转染后,荧光显微镜观察细胞最适感染复数(MOI)及蛋白印迹检测外源基因的表达情况;采用Urist提供的方法制备脱钙骨(DBM),用细胞计量法测定BMSCs与DBM复合培养的黏附率。然后将转染后细胞接种到DBM支架上,用荧光显微镜观察细胞是否成功粘附于支架材料上,扫描电镜观察细胞贴附、生长状况。结果 BMSCs细胞表型鉴定:CD44表达阳性,CD45表达阴性;转染后,蛋白印迹试验检测到BMP-2表达增高;骨髓间充质干细胞与脱钙骨基质的平均粘附率为(72.74±1.99)%;扫描电镜见转染细胞生长良好,伸出丝状突起,相互连接。结论成功培养及鉴定兔BMSCs,Ad-BMP-2/EGFP可高效转染兔BMSCs,转染后的细胞种植于DBM支架材料后生长状况良好,组织工程骨构建成功。     

In vitro proliferation and differentiation of human mesenchymal stem cells on hydroxyapatite versus human demineralised bone matrix with and without osteogenic protein-1

Background: Allografting is currently used in lower limb reconstruction surgery. Demineralised bone matrix (DBM) is more osteoinductive compared with allografts but lacks mechanical strength. Osteogenic protein-1 (OP-1) can improve the osteoinductivity of the allograft, however recent reports indicate significant allograft resorption when it is combined with OP-1. Objectives: Our hypothesis was that hydroxyapatite (HA) with human-mesenchymal stem cells (h-MSCs) and OP-1 (HA+h-MSCs+OP-1) has similar osteoinductive properties to human-DBM+h-MSCs. The objective was to evaluate h-MSC proliferation (by tritiated thymidine incorporation, total DNA Hoechst 33258 and scanning electron microscopy) and osteogenic differentiation (from alkaline phosphatase activity) in human demineralised bone matrix (h-DBM) and HA, with or without OP-1. Results: H-MSC proliferation on HA+OP-1 was significantly higher compared with that on HA at all time points (p < 0.05) and compared with DBM alone [day 1, (198.4 vs 95.4) p = 0.042; day 14 (286.1 vs 119.9), p < 0.001]. H-MSC proliferation was higher in DBM+OP-1, at all time points compared with HA+OP-1 but the difference was not statistically significant (p > 0.05). H-MSC differentiation was significantly higher in HA+OP-1 compared with HA (p < 0.05) but not significantly different from diffferentiation on DBM alone (p > 0.05). Differentiation was significantly higher on DBM+OP-1 at all time points compared with HA (p < 0.05) and with HA+OP-1 [day 1, (21.1 vs 10.1) (p = 0.03); day 7 (39.4 vs 7.1) (p < 0.01); day 14 (40.2 vs 14.4) (p < 0.001)]. Conclusions: When HA+h-MSCs is combined with OP-1 in vitro its osteogenic potential is similar to that of DBM+h-MSCs alone which may be adequate for non-weight-bearing applications. Mechanical testing however is of great importance for weight-bearing applications and the in vivo testing of the composite graft HA+h-MSCs+OP-1 vs DBM+h-MSCs is recommended.     

凝胶包埋骨髓基质干细胞复合脱钙骨基质修复关节软骨缺损简

目的探索Ⅱ型胶原凝胶包埋的自体骨髓基质干细胞（BMSCs）接于同种异体脱钙骨基质（DBM）材料修复兔关节软骨缺损的效果。方法15只健康成年新西兰大白兔,雌雄不限,体质量约3．0kg,兔龄6。9个月;以Urist方法制作同种异体DBM材料。以Ⅱ型胶原蛋白配制水凝胶．以水凝胶包埋兔BMSCs并接种于同种异体DBM材料,构建组织工程复合物。在新西兰大白兔股骨髁关节面制造软骨缺损。分组进行修复。将健康成年新西兰大白兔27只（雌雄不限,体质量约2．5kg．兔龄3～4个月）共54侧膝关节随机分为Ⅱ型胶原／DBM／BMSCs修复组（实验组）、Ⅱ型胶原／DBM修复组（实验对照组）及空白对照组。于术后4周、8周及12周各处死9只动物,取材对修复组织进行大体及组织学观察,根据Wakitani法对修复组织进行评分．数据输入SPSS11．5软件进行统计学分析,比较各组的评分差异是否具有统计学意义。结果实验组Ⅱ型胶原／DBM／BMSCs植入后形成透明软骨样修复．表面光滑平坦,与周围软骨及软骨下骨结合良好;实验对照组Ⅱ型胶原／DBM植入后有部分软骨样修复：而空白对照组仅有少量纤维性修复。根据组织学评分标准,实验组组织学评分为（20．25±1．64）分,高于实验对照组[（7．46±1．29）分]及空白对照组[（6．00±2．09）分]。结论Ⅱ型胶原自体BMSCs复合同种异体DBM支架材料修复全层关节软骨缺损的效果良好,是一种修复软骨缺损的行之有效的方法。     

过氧乙酸-乙醇联合辐照灭菌对脱钙骨基质成骨诱导活性的影响

目的探讨过氧乙酸-乙醇(PES)联合辐照灭菌对脱钙骨基质(DBM)成骨诱导活性的影响。方法取SD大鼠长管骨制作成骨粉装入胶囊后分为两组进行消毒灭活处理:PES+20k Gy的钴-60射线消毒组(实验组)和20k Gy钴-60射线消毒组(对照组)。将DBM骨胶囊植入40只SD大鼠体内,分别在2、4、6、8周处死SD大鼠,将之前植入的DBM骨胶囊取出,对DBM的骨诱导活性进行检测。结果从大体观察看,实验组所植DBM骨块呈散在颗粒状、形态不完整,对照组所植DBM骨块呈卵圆形、质地较硬、形态完整;从新生微血管数(MVQ)看,实验组MVQ值少于对照组,差异有统计学意义(0.0001);从新生骨生长速率看,对照组的生长速率为1.59 m/d,而实验组生长速率过低,无法测量;从钙、磷、碱性磷酸酶含量看,实验组钙含量小于对照组,差异有统计学意义(0.05),实验组磷含量小于对照组,差异有统计学意义(0.001),实验组碱性磷酸酶含量小于对照组,差异有统计学意义(0.05)。结论使用PES联合辐照灭菌以后会降低DBM的成骨诱导活性。     

Studies in the diabetic mutant mouse: IV. DBM,a modified diabetic mutant produced by outcrossing of the original strain

Summary Body weight, blood glucose, serum insulin and islet morphology were studied in DBM mice, modified diabetic mutants produced by crossing mice from the original black diabetic strain (C 57 BL/Ks-db) with normal misty colored mice (C 57 BL/6J-m). DBM mutants appeared to live longer than most mice from the original strain. Only 1 of 6 animals, 7 months of age or older, showed the fall in body weight observed in the majority of the original mutants during the terminal stage of the disease. Blood glucose levels in both types of mutants were similar, generally reaching levels of 400 to 500 mg glucose/100 ml blood. The mean insulin level in older (18 weeks or greater) DBM mutants was significantly greater than in the original mutants. These differences could be explained by the failure of DBM mutants to develop the marked decrease in numbers of beta cells seen in older mutants from the original strain.U.S.P.H.S. Research Career Development Awardee, Grant 1 K4-AM-7394.     

Western and dot immunoblotting analysis of viral antigens and antibodies: application to murine hepatitis virus

Viral proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred quantitatively to nitrocellulose by electroblotting in SDS-containing buffer. Monoclonal antibodies directed against previously defined epitopes on the viral proteins were used as probes to detect viral protein synthesis and processing, as well as expression in animal tissues. Circulating polyclonal antibodies were also probed and characterized for their polypeptide specificities. Under appropriate conditions, this Western immunoblotting technique was quantitative. Finally, a highly sensitive dot immunoblotting assay was used to analyze the sensitivity to denaturation of various epitopes on the viral proteins. This assay detected picogram quantities of viral antigens and antibodies.     

Dose-dependent pharmacokinetics of a new reversible proton pump inhibitor, DBM-819, after intravenous and oral administration to rats: hepatic first-pass effect.

The dose-dependent pharmacokinetic parameters of DBM-819 were evaluated after intravenous (5, 10 and 20 mg/kg) and oral (10, 20 and 50 mg/kg) administrations of the drug to rats. The hepatic first-pass effect was also measured after intravenous and intraportal administrations of the drug, 10 mg/kg, to rats. After intravenous administration, the dose-normalized (based on 5 mg/kg) area under the plasma concentration-time curve from time zero to time infinity, AUC, at 20 mg/kg (27.0 and 45.8 microg min/ml) was significantly greater than that at 5 mg/kg due to saturable metabolism. After oral administration, the dose-normalized (based on 10 mg/kg) AUC(0-12 h) at 50 mg/kg (25.1, 18.3 and 49.2 microg min/ml) was significantly greater than those at 10 and 20 mg/kg again due to saturable metabolism. After oral administration of DBM-819, 10 mg/kg, 2.86% of oral dose was not absorbed and the extent of absolute oral bioavailability (F) was estimated to be 46.7%. After intraportal administration of DBM-819, 10 mg/kg, the AUC was 51.9% of intravenous administration, suggesting that approximately 48.1% was eliminated by liver (hepatic first-pass effect). The considerable hepatic first-pass effect of DBM-819 was also supported by significantly greater AUC of M3 (3.70 and 6.86 microg min/ml), a metabolite of DBM-819, after intraportal administration. The AUCs of DBM-819 were not significantly different (comparable) between intraportal and oral administrations of the drug, 10 mg/kg, suggesting that gastrointestinal first-pass effect of DBM-819 was almost negligible in rats. At 10 mg/kg oral dose of DBM-819, the hepatic first-pass effect was approximately 48.1%, F was approximately 46.7 and 2.86% was not absorbed from gastrointestinal tract in rats.     

应用脱钙骨基质和硫酸钙颗粒治疗骨缺损的初期结果

目的探讨使用骨移植替代物脱钙骨基质（DBM）和硫酸钙颗粒（OSTEOSET）治疗骨缺损的疗效。方法对同期手术治疗的52例骨缺损患者的缺损处,分别于术后第1、4、8周及3、6、12个月进行随访并摄X线片直至骨缺损愈合。统计分析患者的临床资料、X线片辅助分析骨缺损修复程度和并发症。结果随访8-12个月,植骨术后6、12个月分别有95%和99%患者的骨缺损显示有明显的新骨形成,修复水平为60%-100%。并发症为3例（5.77%）,经换药后治愈,未发生骨缺损不愈合。结论DBM和OSTEOSET在骨愈合早期可加速骨缺损的修复,并可作为新鲜自体髂骨的替代物。     

低温保存对人骨髓基质干细胞在脱钙骨上体外增殖及成骨能力的影响

目的研究低温保存对人骨髓基质干细胞(BMSCs)在脱钙骨(DBM)上生长特性及成骨能力的影响。方法取3例志愿者骨髓(3～5mL),密度梯度离心、差速贴壁法获得BMSCs。第3代BMSCs在-196℃下保存24h,37℃复苏,测定细胞成活率。低温保存前、后的BMSCs分别用成骨诱导液诱导培养,至90%融合时,收集细胞接种在DBM支架上,并测定细胞在DBM上的粘附率。DiI荧光染料标记BMSCs,例置相差显微镜、荧光显微镜和SEM观察低温保存前、后的BMSCs在DBM上的生长及基质分泌情况,MTT法测定细胞在DBM上的增殖活性。通过测定碱性磷酸酶(ALP)活性和骨钙素(OCN)含量观察细胞在DBM上的成骨能力。结果复苏细胞的存活率为(90．24±0．02)%。低温保存前、后的BMSCs在DBM上的粘附率分别为(97．25±1．17)%和(97．00±1．09)%。倒置相差显微镜及SEM观察显示低温保存前、后的BMSCs在DBM上粘附、生长良好,有大量细胞外基质分泌、沉积。低温保存前、后的BMSCs在DBM上MTT吸光度值和ALP活性的检测结果差异无显著性意义(P＞0．05)；体外培养12d时,MTT吸光度值及ALP活性同时达到峰值。低温保存前、后的BMSCs在DBM上分泌OCN的量差异无显著性意义(P＞0．05),OCN含量随着培养时间延长而不断上升,在观察期(16d)内未出现平台期。结论低温保存对人BMSCs在DBM上的体外增殖、粘附及成骨能力影响差异无显著性意义,低温保存的人BMSCs可作为组织工程骨的种子细胞。