Ectopic study of calcium phosphate cement seeded with pBMP-2 modified canine bMSCs mediated by a non-viral PEI derivative

We have evaluated the ectopic new bone formation effects of CPC (calcium phosphate cement) seeded with pBMP‐2 (plasmids containing bone morphogenetic protein‐2 gene) transfected canine bMSCs (bone marrow stromal cells) mediated by a non‐viral PEI (polyethylenimine) derivative (GenEscort? II) in nude mice. Canine bMSCs were transfected with pBMP‐2 or pEGFP (plasmids containing enhanced green fluorescent protein gene) mediated by GenEscort? II in vitro, and the osteoblastic differentiation was explored by ALP (alkaline phosphatase) staining, ARS (alizarin red S) staining and RT—qPCR (real‐time quantitative PCR) analysis. Ectopic bone formation effects of CPC/pBMP‐2 transfected bMSCs were evaluated and compared with CPC/pEGFP transfected bMSCs or CPC/untransfected bMSCs through histological, histomorphological and immunohistochemical analysis 8 and 12 weeks post‐operation in nude mice. Transfection efficiency was up ～35% as demonstrated by EGFP (enhanced green fluorescent protein) expression. ALP and ARS staining were stronger with pBMP‐2 gene transfection, and mRNA expression of BMP‐2 (bone morphogenetic protein‐2), Col 1 (collagen 1) and OCN (osteocalcin) in pBMP‐2 group was significantly up‐regulated at 6 and 9 days. Significantly higher NBV (new bone volume) was achieved in pBMP‐2 group than in the control groups at 8 and 12 weeks (P<0.05). In addition, immunohistochemical analysis indicated higher OCN expression in pBMP‐2 group (P<0.01). We conclude that CPC seeded with pBMP‐2 transfected bMSCs mediated by GenEscort? II could enhance ectopic new bone formation in nude mice, suggesting that GenEscort? II mediated pBMP‐2 gene transfer is an effective non‐viral method and CPC is a suitable scaffold for gene enhanced bone tissue engineering.     

Obesity regulates miR-467/HoxA10 axis on osteogenic differentiation and fracture healing by BMSC-derived exosome LncRNA H19

This study explored the therapeutic effect of bone marrow mesenchymal stem cell-derived exosomes on the treatment of obesity-induced fracture healing. Quantitative real-time PCR was used to detect the expression of lncRNA H19, miR-467 and Hoxa10 and combined with WB detection to detect osteogenic markers (RUNX2, OPN, OCN). Determine whether exosomes have entered BMSCs by immunofluorescence staining. Alkaline phosphatase (ALP) and alizarin red staining (ARS) staining were used to detect ALP activity and calcium deposition. We found that high-fat treatment can inhibit the secretion of BMSCs-derived exosomes and affect the expression of H19 carried by them. In vivo and in vitro experiments show that high-fat or obesity factors can inhibit the expression of osteogenic markers and reduce the staining activity of ALP and ARS. The treatment of exosomes from normal sources can reverse the phenomenon of osteogenic differentiation and abnormal fracture healing. Further bioinformatics analysis found that miR-467 as a regulatory molecule of lncRNA H19 and Hoxa10, and we verified the targeting relationship of the three through dual luciferase report experiments. Further, we found similar phenomena in ALP and ARS staining. Bone marrow mesenchymal stem cell-derived exosomes improve fracture healing caused by obesity.     

饲料钙磷含量和钙磷比对生长期大鼠体重增长与骨骼发育的影响

目的研究饲料中不同的钙磷含量和钙磷比对生长期实验大鼠体重及骨骼发育的影响。方法采用完全随机化设计方案,取1月龄（100±10）g清洁级Wistar雄性大鼠70只,随机分成7组,每组10只,饲喂7种不同钙磷含量的饲料。自由采食,饮用去离子水,试验期42 d。各组于实验开始和结束时称重。试验结束后,处死动物并分离一侧胫骨和股骨用于实验指标的检测。结果当保持饲料中钙或者磷中的一种含量不变的情况下,调整另一种的含量即改变钙磷比例时,1.2∶1的正常钙磷比例组生长期实验大鼠增重及骨骼发育要明显好于0.4∶1的低钙磷比例组和4∶1的高钙磷比例组（P〈0.05）,并且当保持饲料中钙磷比例不变时,即使在低钙低磷和高钙高磷进食水平下,正常钙磷比例组大鼠增重及骨骼发育都较好,但低钙磷比例组和高钙磷比例组大鼠增重及骨骼发育则由于饲料中钙磷含量的变化波动较大（P〈0.05）。结论饲料中钙和磷需按比例添加,如果两者绝对含量相差过大就会影响到动物的增重及骨骼发育,本实验证明饲料钙磷比为1.2∶1时生长期实验大鼠增重及骨骼发育较好。     

Experimental study of the synergistic effect and network regulation mechanisms of an applied combination of BMP-2, VEGF,and TGF-β1 on osteogenic differentiation

The study aimed to explore the osteogenic effect induced by the combined use of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1), attain the best combination for osteogenic quality and efficiency, and explore the network regulation mechanisms of induced osteogenesis. MC3T3-E1 cells were cultured in vitro, and BMP-2, VEGF, and TGF β1 were added to osteogenic induction mediums in different combinations to conduct experiments. At 7 and 14 days, the alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining of the applied BMP-2 and VEGF combination were deeper and the quantitative analysis were higher than those of the other groups. After optimizing the time–effect relationship of the combined application, with BMP-2, VEGF, and TGF-β1 adding in the early stage and BMP-2 and VEGF adding in the late, the ALP and ARS staining of these groups were deeper and the quantitative analyses were meaningfully higher than the BMP-2 and VEGF combination group at 7 and 14 days. The expression of the RUNX2 gene and the Smad1 signaling pathway in the optimized combination group was also significantly higher. The results demonstrate that the combination of BMP-2, VEGF, and TGF-β1 applied according to the time–effect relationship can significantly promote osteogenic differentiation mainly through the classical BMP-receptor-Smad signal pathway.     

BMP9诱导人脐带间充质干细胞体内外成骨分化的作用研究

目的:探讨人骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)在体内外诱导人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)成骨分化的作用研究。方法:设立Ad-BMP9处理组和Ad-GFP对照组感染hUC-MSCs,两组细胞分别于3天、5天、7天进行ALP活性检测,14天后采用免疫组织化学染色检测骨钙素(Osteocalcin,OCN)、骨桥蛋白(Osteopotin,OPN)的表达情况,21天后茜素红染色检测矿化结节的形成;然后收集不同分组hUC-MSC用于裸鼠皮下注射成骨模型的建立,4周后取出离体骨进行Micro-CT扫描和分析,并进行H&E、Masson Trichrome、Alcain Blue染色。结果:BMP9处理组的ALP活性和矿化结节形成明显高于对照组,免疫组化染色结果显示BMP9诱导组的OCN、OPG的阳性表达明显高于对照组;裸鼠皮下注射成骨模型的观察结果显示,空白对照组没有形成肉眼可见的皮下包块,仅感染Ad-BMP9的hUC-MSCs能生成异位骨,且形成的异位骨骨量明显,骨密度平均值为396.05±0.60;H&E染色结果显示BMP9诱导生成的异位骨中形成部分成熟的骨基质和骨小梁,Masson Trichrome染色结果显示BMP9明显诱导hUC-MSCs的基质矿化作用,Alcain Blue染色结果显示BMP9明显诱导hUC-MSCs的软骨内成骨作用。结论:BMP9成功诱导人脐带间充质干细胞的体内外成骨作用,为临床骨组织工程的细胞疗法提供了明确的可行性。     

The chromosome aberrations in human peripheral blood lymphocytes 43-46 years after the acute radiation disease

Cytogenetic study of workers, who had an acute radiation syndrome of the medium (ARS II), severe (ARS III) and extremely severe (ARS III-IV) degrees in 1953-1957, was performed. Lymphocytes from peripheral blood were cultured and analyzed with using the routine chromosome staining (4 individuals) and FISH (2 individuals) methods. In each case 4000-1000 metaphase slides were analyzed with the group chromosome kariotyping. A high frequency of chromosome aberrations (CA) was revealed, i.e.: 9.33-9.8 CA per 100 cells for ARS II patients, 28.6 and 36.6 CA per 100 cells for patients with the severe ARS. The main type of rearrangement is stable CA (up to 90%). The CA frequency exceeds the level of spontaneous CA (control--20 individuals) and CA of the patients, who had Chronic Radiation Disease (CrRD) 45 years ago (20 individuals). By 43-46 years of the control. No cancer diseases or hematopoietic pathology were revealed by 43-46 years of follow-up.     

Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity

By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes.     

大鼠骨髓间充质干细胞的原代培养及鉴定

摘要 目的：研究大鼠BMSCs（骨髓间充质干细胞）原代培养与纯度鉴定的方法。方法：无菌环境中,从SD大鼠股骨与胫骨端采集骨髓,先行酶消化,利用全骨髓细胞悬液贴壁法对提取BMSCs实施传代培养,选取生长良好的第3代细胞进行鉴定;对BMSCs实施成脂与成骨诱导分化,同时经由油红O（ORO）与茜素红（ARS）染色法对诱导分化效果加以鉴定;借助流式细胞术（FCM）对CD34、CD44与CD90这3类BMSCs表面标志物的表达展开分析。结果：BMSCs是长梭状贴壁细胞,生长状态为纤维细胞样漩涡状;在第3代BMSCs传代期间,其第1-3 d发展至生长潜伏期,呈较慢速的生长;第3-5 d发展至对数生长期,呈高速生长;待至第7 d长速增殖最大,速度停止上升进入平缓期;BMSCs成骨、成脂诱导结束后,对其诱导分化鉴定发现：细胞出现明显形态学变化,通过ORO对脂肪染色,细胞显示橘红色;待成骨诱导培养结束,通过ARS对钙盐染色,显示红色,且出现矿化结节沉积,说明BMSCs具有良好的成骨、成脂分化能力;FCM测定发现：CD34表达呈阴性（1.09 %）,CD90（96.8 %）与CD44（92.4 %）皆呈阳性,与BMSCs表型相符。结论：经由全骨髓黏附培养技术有效分离BMSCs,且完成培养。     

氨基修饰的纳米纤维对人和大鼠 MSCs增殖分化的影响

目的:探讨氨基修饰后的静电纺丝纳米纤维对大鼠和人骨髓来源的间充质干细胞(Rat and human bone marrow mesenchymal stem cells, r MSCs and hMSCs)增殖及成骨分化的影响。方法:采用静电纺丝法制备聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)纳米纤维,用氨气等离子体处理其表面来接枝氨基;通过测量PLGA纳米纤维(NF)及氨基修饰后的纳米纤维(NF-NH_2)接触角来证明修饰效果;将r MSCs和hMSCs分别接种于NF和NF-NH_2,用CCK-8试剂盒检测接种后1, 3 (4), 7天的细胞增殖;接种后的21天,用茜素红S染色(ARS)法检测细胞成骨分化情况。结果:氨气等离子体处理后纳米纤维接触角从81.28±0.33降低至53.99±0.79,说明氨基修饰后的PLGA NF亲水性增加;CCK-8结果显示氨基修饰增加了r MSCs的黏附,接种24 h后r MSCs在NF和NF-NH_2上的检测吸光值分别为0.096±0.011和0.175±0.014(P0.001),而对hMSCs黏附和增殖没有影响,接种24 h后hMSCs在NF和NF-NH_2上的检测吸光值分别为0.237±0.004和0.238±0.006(P0.05);ARS染色结果显示氨基修饰后r MSCs成骨分化增多(在NF和NF-NH_2表面ARS染色区域比例分别13.147±3.223%和36.677±5.230%),而hMSCs在修饰前后的纳米纤维上均有表达(修饰前后ARS染色比例分别为50.283±2.942%和38.254±3.272%)。结论:氨基修饰的NF可以促进大鼠来源的MSCs黏附增殖以及成骨分化,而对人骨髓来源的MSCs没有显著影响,这提示我们MSCs的增殖分化行为可能具有种属依赖性。     

Moderate/subclinical calcium deficiency attenuates trabecular mass,microarchitecture and bone growth in growing rats

Adequate dietary calcium (Ca) intake is essential for bone accretion, peak bone mass (PBM) attainment, bone quality and strength during the mammalian growth period. Severe Ca deficiency during growing age results in secondary hyperparathyroidism (SHPT) and poor bone quality and strength. However, the impact of moderate Ca deficiency during rats early growth period on bone health and the reversibility with supplementing calcium later in adult life remains unclear. Female Sprague-Dawley (SD) rats (postnatal 28th day, P28) were initiated either with a moderate calcium-deficient diet (MCD, 0.25% w/w Ca) or a control diet (0.8% w/w Ca, control group) till P70. Thereafter, MCD rats were continued either with MCD diet or supplemented with calcium diet (0.8% w/w Ca, calcium supplemented group, CaS) till P150. Another group (control rats) were fed 0.8% w/w Ca containing diet from P28 till P150.MCD group, as compared to the control group, had significantly reduced serum ionized Ca and procollagen type 1 N-terminal propeptide (P1NP) at P70 while no significant change was observed in serum corrected Ca, inorganic phosphate (P), alkaline phosphatase (ALP), 25-hydroxy vitamin D [25(OH)D], intact parathyroid hormone (iPTH), and urinary C-terminal telopeptide of collagen 1 (CTX-1), Ca, and P. Femoral and tibial metaphysis in MCD rats had significantly reduced linear growth, cortical and trabecular volumetric BMD (vBMD), trabecular microarchitecture (BV/TV%, trabecular thickness, separation and number, structural model index and connectivity density), cortical thickness, and bone stiffness despite the absence of secondary hyperparathyroidism (SHPT). Continued MCD at P70–P150 results in persistence of compromised bone strength while calcium supplementation (CaS group) improved all the parameters related to bone strength and microarchitecture. Our results indicate that uncorrected moderate/subclinical calcium deficiency in growing rats can result in poor bone quality and strength despite the absence of SHPT. This finding could have relevance in children with poor calcium intake in childhood and adolescence.     

Light microscopic localization of labile calcium in hypertrophied chondrocytes of long bone with alizarin red S.

A method which localizes labile 5% ethylene glycol-bis-(beta-amino-ethyl ether)N-N'-tetraacetic acid-removable calcium in spherules within hypertrophied chondrocytes and in pericellular matrix using alizarin red S (ARS) is described. Fresh blocks of epiphyseal cartilage approximately 1 mm thick were immersed into 0.5-2% ARS solution containing 7% mounted on glass slides in 7% sucrose or in glycerol-gelatin. The stained tissue blocks were also dehydrated in acetone, cleared in xylene and mounted in Preservaslide. The ARS precipitated ionic calcium as red Ca-ARS salt which was birefringent in polarizing microscope, stable in water at pH 4-9 and in nonpolar organic solvent but soluble in polar solvents, especially in dimethyl sulfoxide. In contrast, ARS-stained insoluble calcium phosphate was stable even in dimethyl sulfoxide. Calcium in the hypertrophied chondrocytes, therefore, was thought to be present in a readily ionizable state instead of as insoluble calcium phosphate. Since addition of 7% sucrose retained as well as improved ARS localization of cellular calcium, the calcium was believed to be present in an osmotically sensitive, membrane-bound cytoplasmic compartment. The ARS-positive labile calcium in spherules which develop in the hypertrophied chondrocytes as well as in the pericellular matrix at the zone of provisional calcification suggested a preparatory stage in the process of cartilage calcification.     

Comparative study of clinical characteristics of amniotic rupture sequence with and without body wall defect: Further evidence for separation

BACKGROUND: Amniotic rupture sequence (ARS) is a disruption sequence presenting with fibrous bands, possibly emerging as a result of amniotic tear in the first trimester of gestation. Our comparative study aims to assess whether there is a difference in the clinical pattern of congenital limb and internal organ anomalies between ARS with body wall defect (ARS‐BWD) and ARS without BWD (ARS‐L). METHODS: Among 1,706,639 births recorded between 1998 and 2006, 50 infants with a diagnosis of ARS were reported to the Polish Registry of Congenital Malformations. The information on 3 infants was incomplete, thus only 47 cases were analyzed. These infants were classified into groups of ARS‐L (38 infants) and ARS‐BWD (9 infants). RESULTS: The ARS‐BWD cases were more frequently affected by various congenital defects (overall p < 0.0001), and in particular by urogenital malformations (p = 0.003). In both groups, limb reduction defects occurred in approximately 80% of cases; however, minor and distal limb defects (phalangeal or digital amputation, pseudosyndactyly, constriction rings) predominated in the ARS‐L group (p = 0.0008). The ARS‐L group also had a higher frequency of hand and upper limb involvement. CONCLUSIONS: This observation suggests that amniotic band adhesion in ARS‐L takes place at a later development stage. Although limited by a small sample size, our study contributes to the growing evidence that both ARS entities represent two nosologically distinct conditions. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Response of cortical and cancellous bones to mild calcium deficiency in young growing female rats: a bone histomorphometry study.

The purpose of the present study was to clarify the differences in the alterations of cellular activities of osteoblasts and osteoclasts, mineralization, and bone mass in cortical and cancellous bones of young growing rats with mild calcium deficiency. Twenty female Sprague-Dawley rats, 6 weeks of age, were randomized by the stratified method into two groups with 10 rats in each group: 0.5% (normal) calcium diet group and 0.1% (low) calcium diet group. After 10 weeks of feeding, bone histomorphometric analysis was performed on cancellous bone of the proximal tibia as well as cortical bone of the tibial shaft. Calcium deficiency increased eroded surface (ES/bone surface [BS]) and the number of osteoclast (N.Oc/BS) with an increase in osteoblast surface (ObS/BS), but decreased bone formation rate (BFR/BS) in cancellous bone. However, cancellous bone volume was preserved, while cortical bone area was decreased as a result of decreased periosteal bone gain and enlargement of the marrow cavity. These results suggest that short-term mild calcium deficiency in young growing female rats increased bone resorption by increasing osteoclastic recruitment, and suppressed mineralization followed by increased osteoblastic recruitment in cancellous bone, but cancellous bone loss was counteracted through redistribution of calcium from cortical bone to cancellous bone.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.     

Activation of cannabinoid receptor 2 alleviates glucocorticoid-induced osteonecrosis of femoral head with osteogenesis and maintenance of blood supply

In glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH), downregulated osteogenic ability and damaged blood supply are two key pathogenic mechanisms. Studies suggested that cannabinoid receptor 2 (CB2) is expressed in bone tissue and it plays a positive role in osteogenesis. However, whether CB2 could enhance bone formation and blood supply in GC-induced ONFH remains unknown. In this study, we focused on the effect of CB2 in GC-induced ONFH and possible mechanisms in vitro and in vivo. By using GC-induced ONFH rat model, rat-bone mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) to address the interaction of CB2 in vitro and in vivo, we evaluate the osteogenic and angiogenic effect variation and possible mechanisms. Micro-CT, histological staining, angiography, calcein labeling, Alizarin red staining (ARS), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) staining, TUNEL staining, migration assay, scratch assay, and tube formation were applied in this study. Our results showed that selective activation of CB2 alleviates GC-induced ONFH. The activation of CB2 strengthened the osteogenic activity of BMSCs under the influence of GCs by promotion of GSK-3β/β-catenin signaling pathway. Furthermore, CB2 promoted HUVECs migration and tube-forming capacities. Our findings indicated that CB2 may serve as a rational new treatment strategy against GC-induced ONFH by osteogenesis activation and maintenance of blood supply. Subject terms: Prognostic markers, Calcium and phosphate metabolic disorders     

Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis

Rat mesenchymal stem cells (rMSCs) represent a small portion of the cells in the stromal compartment of bone marrow and have the potential to differentiate into bone, cartilage, fat, and fibrous tissue. These mesenchymal progenitor cells were maintained as primary isolates and as subcultured cells in separate closed modular incubator chambers purged with either 95% air and 5% CO(2) (20% or control oxygen) or 5% oxygen, 5% CO(2), and 90% nitrogen (5% or low oxygen). At first passage, some cells from each oxygen condition were loaded into porous ceramic vehicles and implanted into syngeneic host animals in an in vivo assay for osteochondrogenesis. The remaining cells were continued in vitro in the same oxygen tension as for primary culture or were switched to the alternate condition. The first passage cells were examined for in vitro osteogenesis with assays involving the quantification of alkaline phosphatase activity and calcium and DNA content as well as by von Kossa staining to detect mineralization. Cultures maintained in low oxygen had a greater number of colonies as primary isolates and proliferated more rapidly throughout their time in vitro, as indicated by hemacytometer counts at the end of primary culture and increased DNA values for first passage cells. Moreover, rMSCs cultivated in 5% oxygen produced more bone than cells cultured in 20% oxygen when harvested and loaded into porous ceramic cubes and implanted into syngeneic host animals. Finally, markers for osteogenesis, including alkaline phosphatase activity, calcium content, and von Kossa staining, were elevated in cultures which had been in low oxygen throughout their cultivation time. Expression of these markers was usually increased above basal levels when cells were switched from control to low oxygen at first passage and decreased for cells switched from low to control oxygen. We conclude that rMSCs in culture function optimally in an atmosphere of reduced oxygen that more closely approximates documented in vivo oxygen tension.     

芹菜素改善酒精性骨质疏松症大鼠肠道菌群、调节骨钙代谢——

探究芹菜素改善酒精性骨质疏松症大鼠肠道菌群、调节骨钙代谢的作用。

60只SD健康大鼠采用随机数字表法分为模型组、正常组、常规治疗组以及芹菜素低、中、高剂量组,每组10只。模型组大鼠每天上午用白酒（10 mL/kg）灌胃+生理盐水（10 mL/kg）灌胃;正常组大鼠每天上午用生理盐水（10 mL/kg）灌胃+生理盐水（10 mL/kg）灌胃;常规治疗组大鼠每天上午用白酒（10 mL/kg）灌胃+碳酸钙、阿法D3混悬液（分别以85 mg/kg、0.05 μg/kg剂量溶于10 mL/kg生理盐水）灌胃;芹菜素低、中、高剂量组每天上午分别用白酒（10 mL/kg）灌胃+芹菜素溶液（分别以20、40、80 mg/kg剂量溶于10 mL/kg生理盐水）灌胃,每天1次,持续16周。使用双能X线骨密度检测仪测定各组大鼠右侧股骨骨密度;苏木素–伊红（HE）染色观察各组股骨组织病理学改变;16S核糖体脱氧核糖核酸（16S rDNA）测序技术检测各组肠道菌群多样性及门、属水平相对丰度;Connectify法测定各组骨钙（Ca）含量,改良Neuman-Lorgan法测定各组骨羟脯氨酸（HOP）含量,并计算二者比值。

模型组大鼠骨松质结构改变,骨小梁稀疏松散、粗细不匀,大量断裂,骨髓腔扩大明显,有大量纤维组织。常规治疗组和芹菜素3个剂量组以上病变情况均有不同程度改善,且芹菜素作用呈剂量依赖性,芹菜素中剂量组与常规治疗组差异不显著。与正常组比,模型组、常规治疗组和芹菜素3个剂量组大鼠骨密度、骨Ca、骨HOP含量及二者比值、Chao1指数、Ace指数以及肠道菌群厚壁菌门、毛螺旋菌属、粪球菌属、乳杆菌属、普氏菌属、罗氏菌属和布劳特菌属水平相对丰度降低,拟杆菌门、变形菌门、拟杆菌属、肠杆菌属和别样棒菌属水平相对丰度升高,差异均有统计学意义（均P＜0.05）;与模型组比,常规治疗组和芹菜素3个剂量组大鼠骨密度、骨Ca、骨HOP含量及二者比值、Chao1指数、Ace指数以及肠道菌群厚壁菌门、毛螺旋菌属、粪球菌属、乳杆菌属、普氏菌属、罗氏菌属和布劳特菌属水平相对丰度均升高,拟杆菌门、变形菌门、拟杆菌属、肠杆菌属和别样棒菌属水平相对丰度降低,差异均有统计学意义（均P＜0.05）,芹菜素对骨密度、骨Ca、骨HOP含量及二者比值、Chao1指数、Ace指数以及肠道菌群门、属水平相对丰度作用呈剂量依赖性,且芹菜素中剂量组与常规治疗组骨密度、骨Ca、骨HOP含量及二者比值、Chao1指数、Ace指数以及肠道菌群门、属水平相对丰度比较差异均无统计学意义（均P＞0.05）。

芹菜素能够提高酒精性骨质疏松症大鼠骨密度和骨Ca、骨HOP含量,推测是通过调节肠道菌群相对丰度,促进有益菌群生长,抑制致病菌群生长实现的。

     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

A Simple Method Using Alizarin Red S For the Detection of Calcium in Epoxy Resin Embedded Tissue

This report presents a simple procedure for staining 1-2 μm epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue 0 solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralition. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.