Alkaline phosphatase activity from human osteosarcoma cell line SaOS-2: an isoenzyme standard for quantifying skeletal alkaline phosphatase activity in serum

Earlier we described a kinetic assay for quantifying skeletal alkaline phosphatase (ALP) isoenzyme activity in serum. The precision of the assay depends on including ALP standards for the skeletal, hepatic, intestinal, and placental isoenzymes. We wondered whether human osteosarcoma cells could provide an efficient alternative to human bone or Pagetic serum as a source of the skeletal ALP standard. ALP activities prepared from five human osteosarcoma cell lines were compared with a bone-derived ALP standard with respect to heat stability and sensitivity to chemical effectors. Two of the cell lines (SaOS-2 and TE-85) contained ALP activities that resembled the bone-derived standard. We selected SaOS-2 cells for additional evaluation (as a potential source of isoenzyme standard), because they contained 40-50 times more ALP activity than did the TE-85 cells. To include the SaOS-2 cell-derived ALP activity in the quantitative isoenzyme assay, we diluted the enzyme in a solution containing heat-inactivated (i.e., ALP-negative) human serum. Surprisingly, this dilution caused a 60-125% increase in maximum enzyme activity. In the quantitative assay of ALP isoenzyme in serum, the SaOS-2 derived ALP was indistinguishable from the serum skeletal ALP standard, with respect to the above criteria and assay variations. Evidently ALP from SaOS-2 cells is suited as a standard for measuring skeletal ALP activity in this assay.     

腺病毒介导的BMP-2基因转染骨髓基质干细胞及种植异种骨支架的体外观察

目的　用人骨形态发生蛋白 2腺病毒表达载体 (Ad BMP 2 )转染的兔骨髓基质干细胞 (BMSC) ,种植BCB(去抗原牛松质骨 )支架体外构建组织工程骨。方法　蛋白印迹法检测转染后细胞BMP 2的表达 ,ALP活性检测分析基因转染对细胞分化的影响。然后将转染后细胞接种到BCB支架上 ,扫描电镜观察细胞贴附、生长状况。结果　转染后 ,BMSC表达BMP 2 ,ALP活性明显增高。扫描电镜见转染细胞分布均匀 ,伸展良好。结论　Ad BMP 2可高效转染BM SC ,且促进细胞分化。转染后细胞在BCB上生长良好 ,BMP 2基因治疗的组织工程骨构建成功     

BMP6对小鼠骨髓基质细胞定向成骨分化的影响

目的初步研究BMP6对小鼠骨髓基质细胞定向成骨分化的影响,探讨其作为定向诱导细胞成骨分化细胞因子的可能性。方法BMP6腺病毒感染骨髓基质细胞（BMSC）。染色和定量检测碱性磷酸酶（ALP）的表达;实时荧光定量聚合酶链反应检测骨桥素（OPN）和骨钙素（OC）的表达;荧光素酶报告基因实验检测BMP6对于转录因子Smad和成骨关键基因Runx2的活化作用。结果BMP6能诱导BMSC细胞ALP的表达,且具有剂量依赖性;BMP6刺激后,BMSC细胞晚期成骨基因OPN、OC的表达均有明显的增加;BMP6可以活化Smad和成骨关键基因Runx2。结论BMP6可以促进小鼠骨髓基质细胞定向成骨分化。     

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein-induced bone formation

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)-induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2-induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.     

Oxy133, a novel osteogenic agent,promotes bone regeneration in an intramembranous bone‐healing model

Current reconstructive techniques for complex craniofacial osseous defects are challenging and are associated with significant morbidity. Oxysterols are naturally occurring cholesterol oxidation products with osteogenic potential. In this study, we investigated the effects of a novel semi‐synthetic oxysterol, Oxy133, on in vitro osteogenesis and an in vivo intramembranous bone‐healing model. Rabbit bone marrow stromal cells (BMSCs) were treated with either Oxy133 or BMP‐2. Alkaline phosphatase (ALP) activity, expression of osteogenic gene markers and in vitro mineralization were all examined. Next, collagen sponges carrying either Oxy133 or BMP‐2 were used to reconstruct critical‐sized cranial defects in mature rabbits and bone regeneration was assessed. To determine the mechanism of action of Oxy133 both in vitro and in vivo, rabbit BMSCs cultures and collagen sponge/Oxy133 implants were treated with the Hedgehog signalling pathway inhibitor, cyclopamine, and similar outcomes were measured. ALP activity in rabbit BMSCs treated with 1 μm Oxy133 was induced and was significantly higher than in control cells. These results were mitigated in cultures treated with cyclopamine. Expression of osteogenic gene markers and mineralization in BMSCs treated with 1 μm Oxy133 was significantly higher than in control groups. Complete bone regeneration was noted in vivo when cranial defects were treated with Oxy133; healing was incomplete, however, when cyclopamine was added. Collectively, these results demonstrate that Oxy133 has the ability to induce osteogenic differentiation in vitro in rabbit BMSCs and to promote robust bone regeneration in vivo in an animal model of intramembranous bone healing. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of basic fibroblast growth factor in mouse embryonic stem cell culture and osteogenic differentiation

Embryonic stem cells are actively explored as a cell source in tissue engineering and regenerative medicine involving bone repair. Basic fibroblast growth factor (bFGF) has been a valuable growth factor to support the culture of human stem cells as well as their osteogenic differentiation, but the influence of bFGF on mouse embryonic stem (mES) cells is not known. Towards this goal, D3 cells were treated with bFGF during maintenance conditions and during spontaneous and osteogenic differentiation. In feeder‐free monolayers, up to 40 ng/ml of exogenous bFGF did not support self‐renewal of mES without LIF during cell expansion. During spontaneous differentiation in high‐density cultures, bFGF stimulated cell proliferation under certain conditions but did not influence differentiation, as judged by stage‐specific embryonic antigen‐1 expression. The addition of bFGF reduced the alkaline phosphatase (ALP) activity associated with osteoblast activity during differentiation induced by osteogenic supplements, although the extent of mineralization was unaffected by bFGF. The bFGF increased the mesenchymal stem cell marker Sca‐1 in an mES cell population and led to an enhanced increase in osteocalcin and runx2 expression in combination with BMP‐2. These results suggest that bFGF could be utilized to expand the cell population in high‐density cultures in addition to enriching the BMP‐2 responsiveness of mES cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Tumor necrosis factor-α accelerates the calcification of human aortic valve interstitial cells obtained from patients with calcific aortic valve stenosis via the BMP2-Dlx5 pathway

Calcific aortic valve stenosis (CAS) is the most frequent heart valve disease in the elderly, accompanied by valve calcification. Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine secreted mainly from macrophages, has been detected in human calcified valves. However, the role of TNF-α in valve calcification remains unclear. To clarify whether TNF-α accelerates the calcification of aortic valves, we investigated the effect of TNF-α on human aortic valve interstitial cells (HAVICs) obtained from patients with CAS (CAS group) and with aortic regurgitation or aortic dissection having a noncalcified aortic valve (control group). HAVICs (2 × 10(4)) were cultured in a 12-well dish in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The medium containing TNF-α (30 ng/ml) was replenished every 3 days after the cells reached confluence. TNF-α significantly accelerated the calcification and alkaline phosphatase (ALP) activity of HAVICs from CAS but not the control group after 12 days of culture. Furthermore, gene expression of calcigenic markers, ALP, bone morphogenetic protein 2 (BMP2), and distal-less homeobox 5 (Dlx5) were significantly increased after 6 days of TNF-α treatment in the CAS group but not the control group. Dorsomorphin, an inhibitor of mothers against decapentaplegic homologs (Smads) 1/5/8 phosphorylation, significantly inhibited the enhancement of TNF-α-induced calcification, ALP activity, Smad phosphorylation, and Dlx5 gene expression of HAVICs from the CAS group. These results suggest that HAVICs from the CAS group have greater sensitivity to TNF-α, which accelerates the calcification of aortic valves via the BMP2-Dlx5 pathway.     

The effect of ex vivo dynamic loading on the osteogenic differentiation of genetically engineered mesenchymal stem cell model

Mechanical loading has been described as a highly important stimulus for improvements in the quality and strength of bone. It has also been shown that mechanical stimuli can induce the differentiation of mesenchymal stem cells (MSCs) along the osteogenic lineage. We have previously demonstrated the potent osteogenic effect of MSCs engineered to overexpress the BMP2 gene. In this study we investigated the effect of mechanical loading on BMP2‐expressing MSC‐like cells, using a special bioreactor designed to apply dynamic forces on cell‐seeded hydrogels. Cell viability, alkaline phosphatase (ALP) activity, BMP2 secretion and mineralized substance formation in the hydrogels were quantified. We found that cell metabolism increased as high as 6.8‐fold, ALP activity by 12.5‐fold, BMP2 secretion by 182‐fold and mineralized tissue formation by 1.72‐fold in hydrogels containing MSC‐like cells expressing BMP2, which were cultured in the presence of mechanical loading. We have shown that ex vivo mechanical loading had an additive effect on BMP2‐induced osteogenesis in genetically engineered MSC‐like cells. These data could be valuable for bone tissue‐engineering strategies of the future. Copyright © 2010 John Wiley & Sons, Ltd.     

人脐带间充质干细胞分离培养鉴定及诱导分化实验

目的 建立人脐带间充质干细胞(MSCs)分离培养方法,并进行生物学鉴定及定向诱导分化研究。方法 采用酶消化Wharton胶法体外分离培养人脐带干细胞,通过流式细胞仪分析其表面标记分子,并向成骨、成脂方向诱导分化,钙钴法染色检测ALP,茜素红染色检测矿化结节,油红“O”染色检测脂肪滴,进行干细胞的成骨成脂活性鉴定。结果 分离培养的脐带间充质干细胞CD73,CD90和CD105均表达阳性,阳性率为92.45%,95.45%和96.45%,CD34表达阴性,阳性率仅为1.07%。成骨诱导3周后ALP染色胞质内可见黑色颗粒,4周后可见大量矿化结节。成脂诱导2周后细胞内出现大量脂肪小滴,多数细胞形态变圆,油红“O”染色阳性。结论 脐带来源的间充质干细胞具有成骨、成脂多向分化潜能,为临床干细胞治疗研究的种子细胞来源奠定了基础。     

全髋关节置换者骨髓间充质干细胞中成骨细胞相关因子的基因表达

背景：全髋关节置换后假体的使用寿命与似体周幽有效的骨生成密切相关，这包括骨髓问充质干细胞的募集。碱性磷酸酶、Runx2、MSX2、骨形态发生蛋白2及坩形态发生蛋白受体1a，b的基因表达町反映出骨髓质充质干细胞的成骨潜能。目的：分析个髋关节置换患者骨髓间充质干细胞相关成骨基因的表达水平与性别、年龄及体质摄指数的关系。方法：选取12例接受全髋关节置换的患肯，术在取患者股骨近端的骨髓组织于体外在a-MEM与体积分数20％胎牛血清培养基中扩增至子一代（Passage1），取出细胞进行流式细胞学或RNA检测，应用反转录一聚合酶链反应用检测成骨相关信号凶jrSOST、碱性磷酸酶、Runx2．MSX2、骨形态发生蛋白2及骨形态发生蛋白受体1a，b的RNA表达水平。骨形态发生蛋白2与骨形态发生蛋白1a，b的关系应用线性回归分析；成骨相关因子bSOST的关联性应用Pearson分析。结果与结论：骨髓间充质干细胞成骨相关信号因子与患者的性别、年龄及体质最指数无明显相关性。线性回归分析表明，骨形态发‘雠臣白2与骨形态发生蛋白受体1a存存娃著相关性（P〈001），但与骨形念发生蛋白受体1b无市H关性。SOST与碱性磷酸酶、MSX2及骨形态发生蛋白受体1a有相关性（尸〈0.05）。提示全髋哭节簧换后假体的稳定性町能与患者的成骨能力相关。尽管性别、年龄及体质量指数等与成骨潜能无明显联系，但这些凶索仍然町能影响假体的稳定性。     

壳聚糖温敏凝胶并PRP对骨髓基质细胞增殖分化影响

目的观察壳聚糖温敏凝胶与富血小板血浆（PRP）混合物浸提液对犬骨髓基质细胞（BMSCs）增殖分化的影响。方法制备壳聚糖温敏凝胶及PRP,将二者按不同体积比混合后分为5组：A组,纯壳聚糖温敏凝胶;B组,壳聚糖温敏凝胶：PRP=2：1;C组,壳聚糖温敏凝胶：RP=1：1;D组,壳聚糖温敏凝胶：PRP=1：2;E组,纯PRP,单纯培养液作为对照组。骨髓穿刺法原代培养犬BMSCs,通过MTT法及ALP试剂盒观察以上各组浸提液及单纯培养液对第3代犬BMSCs增殖及分化的影响。结果与对照组比较,B、c、D、E组浸提液均可以促进BMSCs的增殖及ALP水平的表达（F=8．22～112．31,P〈0．01）。结论壳聚糖温敏凝胶混合PRP后可以缓释PRP,促进犬BMSCs增殖分化,有望作为支架材料用于牙周组织再生的研究。     

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.     

BMP9促进间充质干细胞C3H10T1/2成骨分化的研究

目的确认骨形态发生蛋白9(Bone Morphogenetic Proteins 9,BMP9)是否促进间充质干细胞C3H10T1/2定向成骨分化,并初步探讨其分子机制。方法用重组鼠BMP9蛋白处理C3H10T1/2细胞。化学发光法检测碱性磷酸酶(ALP)活性;免疫组化检测骨桥素(OPN)的表达;茜素红S染色检测细胞钙盐沉积;Western Blot检测转录因子Smad1/5/8的磷酸化情况。结果 BMP9可以诱导C3H10T1/2细胞早期成骨指标ALP的活性,也可促进晚期成骨指标OPN的表达和钙盐沉积;BMP9刺激后,C3H10T1/2细胞中转录因子Smad1/5/8的磷酸化水平增加。结论 BMP9可以促进间充质干细胞C3H10T1/2定向成骨分化。     

Bone marrow mesenchymal stem cells,platelet‐rich plasma and nanohydroxyapatite–type I collagen beads were integral parts of biomimetic bone substitutes for bone regeneration

Platelet rich plasma (PRP), which includes many growth factors, can activate osteoid production, collagen synthesis and cell proliferation. Nanohydroxyapatite‐type I collagen beads (CIB), which mimetic natural bone components, are not only flexible fillers for bone defect but also encourage osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) are often used as an abundant cell source for tissue engineering. We used a rabbit model to combine PRP, CIB and BMSCs (CIB+PRP+BMSC) into a bone‐like substitute to study its impact on bone regeneration, when compared to defect alone, PRP, CIB+PRP, and PRP+BMSC. CIB+PRP upregulated more alkaline phosphatase (ALP) activity in BMSCs than PRP alone at 4 weeks postoperation. CIB+PRP+BMSC and PRP+BMSC did not differ significantly in DNA content, total collagen content, and ALP activity at 8 weeks. In histological assay, both CIB+PRP+BMSC and PRP+BMSC showed more bone regeneration at 4 and 8 weeks. Higher trabecular bone volume in tissue volume (BV/TV) (31.15±2.67% and 36.93±1.01%), fractal dimension (FD) (2.30±0.18 and 2.65±0.02) and lower trabecular separation (Tb.Sp) (2.30±0.18 and 1.35±0.16) of CIB+PRP+BMSC than of other groups at 4 and 8 weeks, and approach to of bone tissue (BV/TV=24.35±2.13%; FD=2.65±0.06; Tb.Sp=4.19±0.95). CIB+PRP+BMSC significantly enhanced new bone formation at 4 week. Therefore, nanohydroxyapatite‐type I collagen beads combined with PRP and BMSCs produced a bone substitute with efficiently improved bone regeneration that shows promise to repair bone defects. Copyright © 2012 John Wiley & Sons, Ltd.     

兔骨髓间充质干细胞的分离、培养及鉴定

目的探讨兔骨髓间充质干细胞分离、培养和鉴定的方法。方法全骨髓贴壁法提取兔骨髓中的间充质干细胞,流式细胞术检测细胞表面分子CD14、CD29、CD34、CD44、CD45和CD90等标志的表达以及不同代次兔BM-SCsDNA含量。通过定向分化检测兔BMSCs的多向分化潜能。结果兔骨髓间充质干细胞呈形态较为均一的小梭形、克隆样生长。流式细胞术检测结果显示,传代至第12代和20代的兔骨髓间充质干细胞检测对数生长期细胞的DNA含量结果显示细胞为正常二倍体细胞,无明显变异。细胞表面表达CD29（61.71%）、CD44（60.2%）,不表达CD14（0.4%）、CD34（0.34%）、CD45（0.64%）和CD90（0.04%）。兔BMSCs的定向分化结果显示其可向成骨细胞、脂肪细胞及成软骨细胞分化。结论成功获得具有多向分化潜能的兔骨髓间充质干细胞,其具有特定的细胞表面抗原,具有容易获取、增殖能力强等特点,是优良的组织工程种子细胞。     

Bone morphogenetic protein expression in human atherosclerotic lesions.

Artery wall calcification associated with atherosclerosis frequently contains fully formed bone tissue including marrow. The cellular origin is not known. In this study, bone morphogenetic protein-2a, a potent factor for osteoblastic differentiation, was found to be expressed in calcified human atherosclerotic plaque. In addition, cells cultured from the aortic wall formed calcified nodules similar to those found in bone cell cultures and expressed bone morphogenetic protein-2a with prolonged culture. The predominant cells in these nodules had immunocytochemical features characteristic of microvascular pericytes that are capable of osteoblastic differentiation. Pericyte-like cells were also found by immunohistochemistry in the intima of bovine and human aorta. These findings suggest that arterial calcification is a regulated process similar to bone formation, possibly mediated by pericyte-like cells.     

人脱落乳牙干细胞骨向分化相关分子Runx2的动态表达

背景：Runx2被认为是成骨基因表达的主要调节因子,是一种向成骨细胞分化的不可或缺的转移因子,在成骨细胞发育、分化、调控、骨钙化形成及骨修复过程中起着极其重要的作用。 目的：观察人脱落乳牙干细胞生物学特征,探讨乳牙干细胞骨向分化潜能,以及不同时间点成骨相关转录因子Runx2的动态表达规律。 方法：体外分离、培养人脱落乳牙干细胞。流式细胞仪检测乳牙干细胞表面标志。体外脂向诱导分化4周后进行油红O染色,检测脂滴形成情况;矿化诱导21 d进行茜素红染色,检测矿化结节形成情况。于成骨诱导的不同时间点,用RT-PCR技术检测Runx2的动态表达。 结果与结论：通过酶消化法和有限稀释法获得了乳牙干细胞,流式细胞仪检测显示CD146和STRO-1均有不同程度的表达。成脂诱导油红O染色可见橙红色阳性颗粒。矿化诱导茜素红染色呈阳性。RT-PCR技术检测Runx2在成骨诱导第0天已有表达,0-6 d成明显增强趋势,6-12 d显著下降,12-18 d表达再次上升,以后相对平稳。结果表明乳牙干细胞可体外分离、培养,表达间充质干细胞表面标志,具有成脂和成骨向分化能力。Runx2在乳牙干细胞成骨分化各阶段均有表达,早期和晚期表达上升,中期表达有下降趋势。