ASPN Synergizes with HAPLN1 to Inhibit the Osteogenic Differentiation of Bone Marrow Mesenchymal Stromal Cells and Extracellular Matrix Mineralization of Osteoblasts

Objective

Bone marrow mesenchymal stromal cells (BMSCs) are major sources of osteogenic precursor cells in bone remodeling, which directly participate in osteoporosis (OP) progression. However, the involved specific mechanisms of BMSCs in OP warrant mass investigations. Initially, our bioinformatics analysis uncovered the prominent up-regulation of Asporin (ASPN) and proteoglycan link protein 1 (HAPLN1) in osteoblasts (OBs) of OP patients and their possible protein interaction. Hence, this study aimed to explore the effects of ASPN and HAPLN1 on osteogenic differentiation of BMSCs, extracellular matrix (ECM) mineralization of OBs, and osteoclastogenesis, hoping to offer research basis for OP treatment.

Methods

GSE156508 dataset was used for analysis and screening to acquire the differentially expressed genes in OBs of OP patients, followed by the predicative analysis via STRING. OP mouse models were induced by ovariectomy (OVX), and ASPN and HAPLN1 expression was determined. BMSCs and bone marrow macrophages (BMMs) were isolated from OVX mice and induced for osteogenic differentiation and osteoclastogenesis, respectively. After knockdown experiments, we assessed adipogenic differentiation and osteogenic differentiation in BMSCs. Osteogenic (OPN, OCN, and COL1A1) and osteoclast (Nfatc1 and c-Fos) marker protein expression was determined. The binding of ASPN to HAPLN1 was analyzed.

Results

High expression of ASPN and HAPLN1 and their protein interaction were observed in OBs of OP patients via bioinformatics and in bone tissues of OVX mice. ASPN interacted with HAPLN1 in BMSCs of OVX mice. ASPN/HAPLN1 knockdown increased ALP, OPN, OCN, and COL1A1 protein expression and ECM mineralization in BMSCs while decreasing Nfatc1 and c-Fos expression in BMMs. These effects were aggravated by the simultaneous knockdown of ASPN and HAPLN1.

Conclusion

Our results indicate that ASPN synergises with HAPLN1 to suppress the osteogenic differentiation of BMSCs and ECM mineralization of OBs and promote the osteoclastogenesis in OP.     

Ⅰ型神经纤维瘤病成骨细胞生物学特性的研究

目的检测神经纤维瘤蛋白在Ⅰ型神经纤维瘤病(type1neurofibromatosis,NF1)脊柱侧凸患者成骨细胞中的表达,观察NF1脊柱侧凸患者成骨细胞的生物学特性。方法先天性脊柱侧凸患者10例,NF1脊柱侧凸患者8例,两组年龄和Cobb角相近。脊柱后路手术取髂骨松质骨,采用植块法进行成骨细胞培养。取第二代成骨细胞分别检测其增殖活性、碱性磷酸酶、Ⅰ型胶原和骨钙素等成骨细胞特异性分化指标,并采用免疫沉淀和Westernblot法检测神经纤维瘤蛋白在两组成骨细胞中的表达。结果NF1脊柱侧凸患者成骨细胞中神经纤维瘤蛋白表达水平明显低于先天性脊柱侧凸患者(灰度积分比值分别为1.05±0.06和2.59±1.40,P=0.002);碱性磷酸酶、Ⅰ型胶原和骨钙素水平均明显低于先天性脊柱侧凸患者(44.69IU/mg和51.38IU/mg,P=0.019;226.34ng/mg和249.93ng/mg,P=0.014;7.41ng/mg和8.87ng/mg,P=0.049)。NF1脊柱侧凸患者成骨细胞显示相对活跃的增殖活性(增殖倍数分别为3.34和2.70,P=0.049)。结论NF1脊柱侧凸患者成骨细胞神经纤维瘤蛋白表达降低的同时,其功能存在明显缺陷。成骨细胞功能缺陷可能是骨骼营养不良性改变和骨密度降低等各种骨骼异常共同的基础。     

不同浓度地塞米松诱导脂肪干细胞成骨分化的实验研究

目的地塞米松是MSCs成骨诱导分化的基础试剂,探讨诱导脂肪干细胞(adipose-derived stem cells,ADSCs)成骨分化过程中地塞米松的优选浓度,为进一步骨组织工程研究提供理论依据。方法 3月龄清洁级健康新西兰大白兔5只,雌雄不限,体重2～3 kg。取腹股沟区皮下脂肪4～6 mL,采用胶原酶消化离心贴壁法分离培养ADSCs,取第3代细胞进行实验。倒置相差显微镜观察细胞形态变化;联合CD44、CD106免疫荧光染色和成脂诱导分化鉴定ADSCs。调整细胞密度为1×105个/mL,分别用普通培养液(A组)及含0(B组)、1×10-9(C组)、1×10-8(D组)、1×10-7(E组)、1×10-6(F组)、1×10-5 mol/L(G组)地塞米松的成骨诱导培养液对ADSCs进行培养。MTT法检测细胞增殖情况;RT-PCR检测诱导细胞骨钙素(osteocalcin,OC)和核心结合因子α1(core binding factorα1,Cbfα1)的表达;测定ALP活性及矿化面积百分率;对矿化结节行茜素红染色。结果 ADSCs形态多为梭形、多角形,呈"漩涡状"排列;表面抗原分子CD44呈阳性,CD106呈阴性,成脂诱导后可观察到细胞内有脂滴形成,油红O染色呈阳性。MTT检测显示随地塞米松浓度升高,吸光度(A)值呈下降趋势;其中成骨诱导5、7 d时,D、E组A值比较差异有统计学意义(P<0.05)。RT-PCR检测示,成骨诱导7 d OC和Cbfα1 mRNA的表达分别在E组和D组达高峰;成骨诱导14 d ALP活性和矿化面积百分率均在D组达高峰,随后逐渐下降。D、E组间OC和Cbfα1 mRNA的表达量、ALP活性及矿化面积百分率比较差异均无统计学意义(P>0.05),与其余各组比较差异均有统计学意义(P<0.05)。成骨诱导14 d,G组细胞均死亡;茜素红染色除A、G组外均呈阳性。结论成骨培养液中地塞米松浓度为1×10-8 mol/L时,能在减少对细胞增殖抑制的同时,更有效地诱导ADSCs成骨分化。     

神经纤维瘤蛋白在先天性脊柱侧凸患者成骨细胞和软骨细胞中的表达

目的：检测神经纤维瘤蛋白在先天性脊柱侧凸患者成骨细胞和软骨细胞中的表达。方法：6例先天性脊柱侧凸患者，在后路手术时取髂骨及髂骨生长板，分离、培养成骨细胞和软骨细胞，分别行碱性磷酸酶染色和甲苯胺蓝染色。逆转录-多聚酶链反应（RT—PCR）检测神经纤维瘤蛋白mRNA．间接免疫荧光和Westemblot检测神经纤维瘤蛋白在成骨细胞和软骨细胞中的表达。结果：先天性脊柱侧凸患者成骨细胞和软骨细胞中存在Ⅱ型神经纤维瘤蛋白表达，该蛋白主要分布在细胞浆，所表达蛋白为三磷酸鸟苷酶活化蛋白（GAP）活性较弱的Ⅱ型异构体。结论：先天性脊柱侧凸患者成骨细胞和软骨细胞中存在神经纤维瘤蛋白表达，但该蛋白是否通过对成骨细胞和软骨细胞的影响导致骨骼系统异常还有待于进一步研究。     

肉苁蓉含药血清诱导骨髓间充质干细胞向成骨细胞分化的实验研究

目的:研究补肾中药肉苁蓉含药血清对骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)定向分化的影响。方法:全骨髓培养法获得原代骨髓间充质干细胞,经胰酶消化传代培养至第4代,将第4代BMSCs以5×106接种于2块6孔板,每板3组孔,分为空白组、地塞米松组、肉苁蓉组,2 d后换液,加入诱导液。空白组加入含10%胎牛血清(FBS)的L-DMEM培养基继续培养,地塞米松组在基础培养基中加入诱导剂(含β甘油磷酸钠10 mmol/L,地塞米松0.1μmol/L,维生素C 50 mg/L),肉苁蓉组为含10%肉苁蓉含药血清的L-DMEM培养基。第10天取出1块6孔板做ALKP染色,另一6孔板于第20天做6孔板茜素红染色。结果:第10天肉苁蓉组及地塞米松组ALKP染色阳性,第12天可见白色钙结节,第20天茜素红染色阳性。结论:BMSCs在肉苁蓉含药血清诱导下可向成骨细胞分化,分别作为组织工程的种子细胞和诱导因子对治疗骨质疏松、骨折不愈合将有良好的前景。     

Isolation, characterisation and osteogenic potential of human bone marrow stromal cells derived from the medullary cavity of the femur

Marrow stromal cells (MSC) are increasingly being introduced in orthopaedic practice as potentially powerful effectors of bone regeneration. Since cell recovery of MSC is affected by a high degree of individual variability, sources for collecting adequate amounts of safe and effective MSC under routine conditions are needed. We analysed if femoral bone marrow, which is usually discarded during total hip arthroplasty procedures, is a reliable source of MSC to enhance bone healing and regeneration. Mononuclear cells were isolated, assayed for typical MSC markers, harvested under appropriate culture conditions and evaluated for their ability to differentiate into osteoblasts. Cell recovery and osteogenic potential were independent from donor gender or age, suggesting that elderly individuals are eligible for autologous cell therapy. Although heterogeneous, the pool of MSC recoveredfrom femoral marrow without further in vitro selection or manipulation proved highly effective in proliferating and differentiating along the osteogenic lineage. In conclusion, this source of MSC offers a valuable tool to be used to promote osteogenesis and implant fixation.     

Down-regulation of the Wnt, estrogen receptor, insulin-like growth factor-I, and bone morphogenetic protein pathways in osteoblasts from rats with chronic spinal cord injury

Objectives

To investigate the anabolic response of osteoblasts to chronic spinal cord injury and to identify potential signaling pathways that are associated with the osteogenic response after spinal cord injury by using in-house microarray analyses in osteoblasts.

Methods

Ten young male Sprague-Dawley rats were randomized into spinal cord injury (SCI) and SHAM groups. The tibiae were assessed for DXA and bone histomorphometry, and osteoblasts from femora were used for microarray analysis.

Results

SCI rats showed lower BMD and deteriorated microstructure in the proximal tibiae as compared with SHAM rats. The Wnt, BMP/TGF, estrogen receptor (ER), and IGF-I pathways were down-regulated in osteoblasts from spinal cord-injured rats.

Conclusion

Down-regulation of the Wnt, BMP/TGF, ER, and growth hormone/IGF-I pathways is associated with decreased bone formation after spinal cord injury.     

大鼠雪旺细胞对两种来源的成骨细胞增殖分化影响的研究

目的 研究应用雪旺细胞(SCs)对两种来源的成骨细胞(OB)的增殖与分化的影响,为构建神经化组织工程骨提供体外实验理论依据.方法 通过采用股骨、胫骨髓腔冲洗法获得SD大鼠骨髓基质干细胞(BMSCs),采用胰蛋白酶消化新生鼠颅骨获得OB及采用消化后组织块法获得SCs.将BMSCs在成骨诱导液作用3周,鉴定BMSCs向OB分化.采用96孔共培养板将第2代SCs种植于上室,颅骨来源OB种植下室,共培养为实验组,无SCs干预共培养为对照组.将诱导分化的OB种植于下室,第2代SCs种植于上室,共培养为实验组,无SCs干预共培养为对照组,分别于共培养后1、3、5、7、9d采用甲基噻唑基四唑(MTT)进行增殖比较.采用6孔共培养板,实验组上室种植SCs,下室分别种植颅骨来源及BMSCs来源的OB,不加SCs的两种来源的OB作为对照组,分别共培养后于3、7 d检测两种来源OB的碱性磷酸酶、骨钙素、骨桥蛋白、骨形态发生蛋白-2 mRNA表达.结果 SCs对颅骨来源的OB在共培养3、5、7、9 d 4个时间点均有明显促增殖作用,在碱性磷酸酶、骨桥蛋白、骨形态发生蛋白-2mRNA在各个时间段均起到抑制作用.SCs对大鼠BMSCs来源的OB在共培养1、3 d无明显促增殖作用,在5、7、9 d有明显促增殖作用,在成骨培养基的环境里,SCs可促进BMSCs诱导的OB分化.结论 选择BMSCs来源的OB 与 SCs共培养更适合用于构建神经化组织工程骨.     

脂肪基质干细胞培养及其定向分化为成骨细胞、软骨细胞特性的研究

目的研究脂肪基质干细胞（adipose—derived stem cells，ADSCs）分离培养的方法，探讨大鼠ADSCs在体外向成骨细胞、软骨细胞分化的能力。方法从成年SD大鼠腹股沟处无菌获取脂肪组织，胶原酶消化分离，培养出ADSCs。基础培养基传至第二代时改换诱导培养基，分别诱导向成骨、软骨细胞分化，培养2～4周，碱性磷酸酶（alkaline phosphatase，ALP）、Vonkossa染色鉴定向成骨细胞分化能力；阿新蓝染色鉴定向软骨细胞分化能力。RT—PCR检测成骨细胞、软骨细胞标志基因。结果大鼠脂肪能够分离培养出生长旺盛的ADSCs；向成骨细胞诱导，ALP、Vonkossa染色阳性，RT—PCR检测有ALP、Osteocalcin及Osteopontin表达；向软骨细胞诱导，阿新蓝染色阳性，RT—PCR检测有Ⅱ型胶原、X型胶原和Aggreean表达。结论大鼠脂肪组织可以分离培养出ADSCs，生物学特性与骨髓基质干细胞（mesenchymal stemcells，MSCs）相似，能够向成骨细胞、软骨细胞分化，有希望成为组织工程理想的种子细胞来源。     

The incidence of bilateral cryptorchidism is increased and the fertility potential is reduced in sons born to mothers who have smoked during pregnancy

PURPOSE: Recent studies have demonstrated a high prevalence of cryptorchidism, decreasing semen quality and increasing incidence of testicular cancer. These changes seem to be interrelated, and may be symptoms of a common underlying entity with foundations in fetal life. We investigated the influence of maternal smoking on fertility status in offspring cryptorchidism. MATERIALS AND METHODS: We prospectively studied consecutive patients presenting to the pediatric surgery department between 1996 and 2005. A total of 157 boys 1 to 5.9 years old underwent surgery for cryptorchidism with simultaneous testicular biopsy, and exhibited well preserved testicular parenchyma. Only white patients with Danish-speaking mothers who had reported pregnancy history including smoking habits during pregnancy and history of the offspring were included. The patients had cryptorchidism only and none received hormonal treatment before surgery. The number of spermatogonia and gonocytes per tubule cross-section was assessed and compared to normal values from autopsy material. RESULTS: The group of boys with cryptorchidism whose mothers had smoked heavily during pregnancy (ie more than 10 cigarettes daily throughout the pregnancy) had a significantly increased risk of bilateral cryptorchidism (52%, or 11 of 21 patients), and a decreased number of spermatogonia and gonocytes per tubule cross-section, which was absolute (0.097 [0 to 0.75]) and age related (14% [0% to 198%] of normal for age) compared to boys whose mothers did not smoke (20%, or 22 of 112 patients, 0.140 [0 to 2.14] and 37% [0% to 563%] of normal for age, p <0.01, p <0.05 and p <0.05, respectively). CONCLUSIONS: A close relationship between maternal smoking during pregnancy and adverse trends in offspring reproductive health in relation to cryptorchidism was observed.     

Osteogenic and chondrogenic potential of biomembrane cells from the PMMA‐segmental defect rat model

A layer of cells (the “biomembrane”) has been identified in large segmental defects between bone and surgically placed methacrylate spacers or antibiotic‐impregnated cement beads. We hypothesize that this contains a pluripotent stem cell population with potential valuable applications in orthopedic tissue engineering. Objectives using biomembranes harvested from rat segmental defects were to: (1) Culture biomembrane cells in specialized media to direct progenitor cells along bone or cartilage cell differentiation lineages; (2) evaluate harvested biomembranes for mesenchymal stem cell markers, and (3) define relevant gene expression patterns in harvested biomembranes using microarray analysis. Culture in osteogenic media produced mineralized nodules; culture in chondrogenic media produced masses containing chondroitin sulfate/sulfated proteoglycans. Molecular analysis of biomembrane cells versus control periosteum showed significant upregulation of key genes functioning in mesenchymal stem cell differentiation, development, maintenance, and proliferation. Results identified significant upregulation of WNT receptor signaling pathway genes and significant upregulation of BMP signaling pathway genes. Findings confirm that the biomembrane has a pluripotent stem cell population. The ability to heal large bone defects is clinically challenging, and novel tissue engineering uses of the biomembrane hold great promise in treating non‐unions, open fractures with large bone loss and/or infections, and defects associated with tumor resection. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1198–1212, 2012     

The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.     

Osteogenic differentiation of adipose-derived stromal cells treated with GDF-5 cultured on a novel three-dimensional sintered microsphere matrix

BACKGROUND CONTEXT: It is well known that under the proper conditions multipotential bone marrow stromal cells are capable of osteogenic differentiation. Recently studies have demonstrated that an analogous subpopulation of cells exist within adipose tissue. Although early studies characterizing these adipose-derived stromal (ADS) cells in culture exist, investigations exploring the characteristics and viability of these cells cultured on a three-dimensional sintered microsphere matrix are absent. PURPOSE: To characterize and investigate the viability of ADS cells cultured on bioengineered three-dimensional sintered microsphere matrices (SMM). STUDY DESIGN: Basic science, laboratory study. PATIENT SAMPLE: Sixty SMM total. Six underwent examination by scanning electron microscopy, 18 for cellular viability, 18 for biochemical assay, and 18 for evaluation by gene expression. OUTCOME MEASURES: The SMM were examined under scanning electron microscopy to evaluate for adherence, migration, and proliferation at 7, 14, and 28 days. Cellular viability was assessed using colorimetric assay for mitochondrial dehydrogenases activity in viable cells (MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay) at each corresponding time point. Osteoblastic differentiation was determined using biochemical assays for alkaline phosphatase activity and gene expression for alkaline phosphatase (ALP), osteocalcin (OC), and core binding factor alpha-1 (Cbfa1). METHODS: Multipotential ADS cells from adult Sprague Dawley rats were isolated and maintained in media. Sintered microsphere matrices of poly(lactide-co-glycolide) [85:15] were prepared using solvent evaporation technique followed by mechanical sieving and fabricated by heating in metal molds. ADS cells were then seeded on the SMM and cultured in media with growth and differentiation factor-5 (GDF-5). Treated samples and controls were evaluated at 7, 14, and 28 days. Statistical significance was set at p<.05. RESULTS: Multipotential ADS cells were capable of being isolated from adipose tissue. Scanning electron microscopy evaluation revealed cells adherent to the scaffold surface in a monolayer by 7 days. Cytoplasmic extensions were seen linking the cells on adjacent microspheres. Migration and proliferation resulting in extension of the cellular elements into the scaffold was apparent by 14 days. MTS confirmed cell viability within the scaffold throughout the 28-day study. Osteoblastic differentiation was confirmed using biochemical assays for alkaline phosphatase activity and gene expression for ALP, OC, and Cbfa1. CONCLUSIONS: This is the first study to investigate the fate of ADS seeded on a three-dimensional sintered microsphere matrix. The results of this study confirm that ADS cells, when treated with GDF-5, are not only capable of adhering to the bioengineered scaffold, but also remain viable and demonstrated the ability to migrate, proliferate, and subsequently undergo osteogenic differentiation under the conditions described. These early findings support the concept that ADS cells cultured on a SMM may serve as a viable alternative to more traditional methods of bone graft materials.     

强骨宝方含药血清对糖尿病模型鼠体外培养成骨细胞的影响

目的：探讨糖尿病模型鼠强骨宝方含药血清促进体外培养成骨细胞分化、矿化的最佳时相与量效。方法：采用胰蛋白酶-Ⅱ型胶原酶消化法从1-2日龄SD大鼠颅盖骨中分离出成骨细胞，鉴定细胞后，用不灭活的不同时效（灌胃3、5d，末次灌胃后1、3h）、不同浓度（5％、10％、20％）的强骨宝方糖尿病模型鼠含药血清加入成骨细胞培养体系，培养7、18d，分别观察各组ALP活性及矿化结节形成量。结果：各浓度的糖尿病模型鼠强骨宝方含药血清对成骨细胞分泌ALP及矿化结节形成的作用均优于模型对照组，并接近于正常对照组水平，其中以20％添加浓度的作用最明显。在时相方面，两指标均以灌药3d或5d、末次灌胃后1h取得血清作用最明显。结论：糖尿病模型大鼠强骨宝方含药血清对体外培养成骨细胞（OB）的分化及矿化功能有促进作用。考虑到实验的时间与成本，以灌胃3d、末次灌胃后1h、20％不灭活的强骨宝方糖尿病模型鼠含药血清对成骨细胞分化与矿化作用最适宜。     

成年脊髓损伤大鼠脊髓神经干细胞的体外培养及分化研究

目的 探讨大鼠脊髓损伤后脊髓神经干细胞的分离培养方法及分化情况.方法 采用Allen法制作大鼠脊髓损伤模型,利用无血清培养和单细胞克隆技术在成年脊髓损伤7 d大鼠脊髓中分离具有单细胞克隆能力的神经干细胞,并进行培养鉴定.结果 从成年脊髓损伤7 d大鼠脊髓中成功分离出神经干细胞,该细胞具有连续克隆能力,可传代培养,表达神经巢蛋白抗原.分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原.结论 致伤7 d的成年大鼠脊髓组织体外町培养出神经十细胞,并分化为神经无细胞、星形胶质细胞和少突胶质细胞,有可能参与脊髓损伤的修复过程.     

Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP-2 from calvariae of GFP transgenic mice

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.     

Risk of autism spectrum disorder in children born to mothers with systemic lupus erythematosus and rheumatoid arthritis in Taiwan

Objectives

To determine whether offspring of Taiwanese mothers with systemic lupus erythematosus or rheumatoid arthritis have a higher risk of autism spectrum disorder.

Osteogenic potential and responsiveness to leptin of mesenchymal stem cells between postmenopausal women with osteoarthritis and osteoporosis

The aim of this study was to compare the osteogenic potential and responsiveness to leptin of mesenchymal stem cells (MSCs) from bone marrow between postmenopausal women with osteoarthritis (OA) and osteoporosis (OP). MSCs of the proximal femur from OA and OP donors were cultured under control and different experimental mediums. After verifying the availability of primary cells, their osteogenic potential and responsiveness to leptin were compared between two groups. Similar patterns of cell growth were shown in both OA and OP groups. However, after the sixth passage, the viability of undifferentiated cells decreased more in OP than in OA donors. Under the same osteogenic supplements condition, the mRNA expression of osteogenesis‐specific genes, osteocalcin (OC) and alkaline phosphatase (ALP) were higher in OA group. Comparison of bone matrix mineralization was parallel to that of mRNA expression. The level of bone‐specific ALP (BAP) was higher in cells from donors with OA, whereas osteoprotegerin (OPG) was higher in OP group. This difference in BAP expression proved to be insignificant after the administration of leptin. Although leptin upregulated the expression of OPG, a significant difference still existed between OA and OP. In conclusion, differential osteogenic potential and responsiveness to leptin of MSCs were noted between postmenopausal women with OA and OP. Differential biological behavior of MSCs seems to be partly related to the different distribution of bone mass between OA and OP populations. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1067–1073, 2009