Therapy with Bortezomib plus Dexamethasone Induces Osteoblast Activation in Responsive Patients with Multiple Myeloma

Bortezomib is a novel proteasome inhibitor that has shown marked antitumor effects in patients with multiple myeloma (MM). We evaluated the feasibility and efficacy of bortezomib plus dexamethasone (BD) therapy and assessed bone metabolism in relapsed or refractory MM. Fourteen patients received 1.3 mg/m2 bortezomib on days 1, 4, 8, and 11 along with 20 mg/dose of dexamethasone on days 1, 2, 4, 5, 8, 9, 11, and 12 in a 21-day cycle. After 1 to 3 cycles of BD therapy, 9 patients (64%) achieved an objective response (5 very good partial responses and 4 partial responses). Notably, a rapid increase in the serum concentration of alkaline phosphatase (ALP) was observed in 6 of the treatment-responsive patients. Moreover, serum levels of bone-formation markers (bone-specific ALP and osteocalcin) significantly increased in 5 and 2 responsive patients, respectively. Radiographic examination showed improvement in bone lesions, suggesting that BD therapy induces osteoblast activation in responders. Adverse events included thrombocytopenia of grades 1 to 3, peripheral neuropathy of grades 1 to 2, and grade 3 ileus and were transient and manageable. Although severe lung injury has been reported among Japanese patients treated with bortezomib, no pulmonary complications were observed during BD therapy. Our results suggest that BD therapy is a safe and promising therapeutic approach for Japanese patients with MM.     

Polyelectrolyte multilayer films functionalized with peptides for promoting osteoblast functions

Layer-by-layer deposition of polyelectrolyte multilayer (PEM) thin films has recently been applied to biomaterial applications. This simple and versatile technique provides a wide variety of potential utilization by insertion of biomolecules such as cell adhesion peptides. In this work dual peptides containing RGD (a cell-binding domain) and LHRRVKI (a heparin-binding domain) were immobilized onto polystyrene by the PEM technique and the effects on osteoblast cell culture were investigated. These peptides were conjugated to the amino groups of poly(allylamine hydrochloride) and then adsorbed onto the top of a 10 layer poly(allylamine hydrochloride)/poly(acrylic acid) film assembled at either pH 2.0 or pH 6.5. Osteoblasts, isolated from neonatal rat calvariae, were then seeded and cultured on the peptide-conjugated surfaces. We found that the cells adhered and grew better on the RGD-conjugated PEM films. The osteoblasts exhibited a better differentiated phenotype on the pH 2.0 films than the pH 6.5 films with respect to calcium deposition. The incorporation of LHRRVKI did not support cell adhesion, growth and matrix mineral deposition. Our results showed that the efficacy of RGD conjugation on osteoblast behavior was affected by the base PEM film.     

脂肪来源干细胞体外成骨和成脂及成神经的诱导分化研究

目的 探讨脂肪组织来源干细胞 (adipose stem cell,ADSC) 的表面表型及向成骨、成脂和成神经多向分化的生物学特性.方法 取SD大鼠的腹股沟、附睾垫和腹膜后脂肪,Ⅰ型胶原酶消化、分离培养ADSC,检测第3代ADSC的CD29、CD44和CD45的表达情况.将ADSC加入成骨诱导剂,通过茜素红S染色鉴定成骨细胞;成脂诱导剂利用油红O染色鉴定脂肪细胞;神经干培养基诱导,免疫荧光染色鉴定诱导的神经元.结果 ADSC表达CD29(99.07±0.12)%、CD44(95.82±0.68)%和CD45(0.11±0.12)%.其经成骨诱导4周,茜素红S染色显示聚集的细胞团中央形成红色钙化结节,碱性磷酸酶染色可见到细胞的胞质内有紫红色颗粒;成脂诱导1周,油红O染色可见细胞质内有橙红色脂滴;经过神经干培养基诱导6 d后,免疫荧光染色显示诱导的细胞Nestin、NF-200及GFAP阳性表达.结论 ADSC表达间质干细胞的表面表型,体外经诱导培养后可向成骨细胞、脂肪细胞和神经元多向分化,表明 ADSC具有干细胞的多向分化潜能.     

RGD肽修饰的纯钛种植材料表面成骨细胞黏附增殖能力

目的:评价精氨酸-甘氨酸-天冬氨酸(RGD)肽修饰纯钛表面对细胞早期黏附增殖影响. 方法:利用分子自组装技术氨基化纯钛表面,化学接枝GYRGDS肽,计算不同时点材料表面黏附细胞数;不同浓度RGD肽液预处理接种前细胞,计算材料表面细胞黏附率;MTT法和扫描电镜分别检测材料表面细胞的增殖、生长.结果:RGD肽修饰的纯钛表面细胞早期黏附率、增殖活性和生长情况在各时点均优于未修饰组,高浓度肽液预处理对RGD肽修饰组表面细胞有较强黏附抑制作用.结论:采用分子自组装技术可将活性RGD肽稳定的化学接枝于纯钛表面,修饰后材料的细胞早期黏附增殖等生物学行为有明显改善.     

黏附肽改性纯钛表面成骨细胞的早期黏附能力研究

目的 对纯钛表面钝化态氧化膜进行生物化学活化改性,探讨生物化学改性对钛表面成骨细胞黏附的影响.方法 24个纯钛试件打磨抛光后均分为4组,每组6个;纯钛组不处理,碱-热处理组行碱-热处理,溶胶涂层组行碱-热处理+溶胶涂层处理,黏附肽组行碱-热处理+溶胶涂层+黏附精氨酸-甘氨酸-天冬氨酸-丝氨酸肽(Glycine-Tyrosine-Arginine-Glycine-Asparticacid-Serine,GYRGDS)处理.使用X射线光电子能谱(X-ray photoelectron spectroscopy,XPS)、傅里叶变换红外光谱计(Fourier transform infrared spectrometer,FTIR)分析各组钛表面化学元素及功能基团组成;在各组钛试件表面接种成骨细胞,经2.05 Pa流体剪切力作用30 min、1 h、2 h,计算细胞残留率.结果 碱-热处理组钛表面富含大量活性羟基,溶胶涂层组钛表面富含大量羟氧基团.黏附肽组钛表面经XPS及FTIR分析检测到酰胺峰,显示GYRGDS肽成功固定于钛表面.流体剪切力作用30 min、1 h和2 h后黏附肽组钛表面的成骨细胞残留率(分别为93.0%、54.4%、34.4%)均高于相应时间点其他组.结论 GYRGDS肽修饰后的钛表面成骨细胞黏附稳定性均优于非GYRGDS肽修饰组.     

唑来膦酸对兔成骨细胞分化及骨保护素分泌的影响

目的 探讨不同浓度的唑来膦酸对成骨细胞分化的影响及其对骨保护素(osteoprotegerin,OPG)分泌的促进作用,以期为唑来膦酸的进一步局部应用提供理论依据.方法 利用组织块培养法进行成骨细胞原代及传代培养.原代培养3d后对细胞爬片行碱性磷酸酶(alkaline phosphatase,ALP)染色,原代培养18 d后将细胞爬片用钙结节染色并鉴定;将唑来膦酸加入含传代第3代成骨细胞悬液的培养液中使其终浓度为0(对照组)、10-5、10-6、10-7、10-8及10-9 mol/L(5个实验组),收集细胞上清液进行ALP检测,用放射免疫测定法测定骨钙素的含量;培养3 d后收集细胞,结合蛋白质印迹法测定细胞分泌的OPG.结果 成骨细胞ALP染色阳性,钙结节茜素红染色呈橘红色钙化结节,唑来膦酸浓度为10-6、10-7、10-8及10-9 mol/L的实验组可促进骨钙素含量增加[分别为(2.80±0.51)、(3.20±0.33)、(4.70±0.35)、(3.40±0.36)μg/L]、ALP活性增强[分别为(5.91±0.35)、(7.62±0.33)、(10.00±0.38)、(8.91±0.29)U/L]、OPG表达升高[分别为(526 955.7±64 068.9)、(661 447.9±75 223.4)、(753 083.3±70 300.0)、(655 184.1±63 401.1)],与对照组[骨钙素含量为(2.40±0.35)μg/L、ALP活性为(4.28±0.36)U/L、OPG表达为(64 713.7±28 715.6)]相比差异均有统计学意义(P＜0.01),并在10-8mol/L组达峰值.结论 唑来膦酸可以提高成骨细胞活性,促进OPG分泌.     

四种纳米粒径钛片生物相容性检测

目的研究4种纳米粒径钛片的生物相容性。方法分别在常温、100℃、250℃和380℃,采用直流磁控溅射法,形成4种纳米粒径表面的钛片。在24孔培养板上,将SD（Sprague-Dawley）乳鼠第3代成骨细胞接种于4种纳米粒径的钛片和未处理的钛片表面,分别为常温组、100℃组、250℃组、380℃组、非处理组;另设1个空白对照组,细胞培养板内不放入钛片。四甲基偶氮唑盐比色法测定6组细胞的增殖情况。结果培养后第1、4、7天,250℃组和380℃组材料表面细胞持续增殖,其中250℃组最高;培养后第7天,4个温度处理组细胞光密度值与非处理组的差异均有统计学意义（P〈0.05）。结论纳米化纯钛材料表面可促进成骨细胞的增殖。     

流体剪切力对RGD肽修饰纯钛表面细胞粘附稳定性影响的研究

目的研究精氨酸-甘氨酸-天冬氨酸（Arg-Gly-Asp,RGD）肽修饰的纯钛表面是否有助于提高成骨细胞的抗剪切能力,比较剪切力大小对细胞粘附的影响。方法实验分为空白处理纯钛组（CpTi组）、对照处理碱-热陈化组（AWTi组）、对照处理溶胶涂层组（ATTi组）和实验处理粘附肽修饰组（RGDTi组）。利用流体应力加力系统,定常流下作用于4组纯钛表面的成骨细胞,计算平均剪切力分别为2.05、3.12、4.20Pa时4组纯钛表面细胞残留率。结果细胞脱落量与剪切力的大小成正比,RGDTi组在2.05、3.12、4.20Pa的流体剪切力作用下,表面细胞残留率分别为92.8%、70.1%、66.8%,多于其他3组。结论RGD有利于提高成骨细胞在纯钛表面粘附稳定性,增加成骨细胞的抗剪切力。     

多肽P-15对大鼠成骨细胞附着增殖分化的影响

目的研究多肽P-15对小牛骨表面生长的大鼠成骨细胞附着、增殖、分化的影响。方法对照组和实验组分别采用单纯无机小牛骨粉（anorganic bone mineral,ABM）和复合P-15的无机小牛骨粉接种大鼠成骨细胞进行体外培养。四甲基偶氮唑蓝[3-（4,5-dimehyl-2-thiazolyl）-2,5-diphenyl-2H-tetrazoliumbromide,MTT]比色法检测大鼠成骨细胞在2组骨粉表面附着、增殖的速度,倒置相差显微镜观察成骨细胞在2组骨粉表面及周围的增殖情况,用对硝基苯磷酸二钠盐（p-nitrophenyl phosphate,PNPP）法检测2组成骨细胞碱性磷酸酶（alkaline phosphatase,ALP）活性。结果成骨细胞体外培养24h,实验组与对照组MTT比色法检测的光密度值分别为1.597±0.035、1.239±0.063,差异有统计学意义（P〈0.01）。培养72h,实验组和对照组PNPP法检测的ALP活性分别为（35.533±1.051）金氏单位/100mL和（34.645±1.187）金氏单位/100mL,实验组略高于对照组,但差异无统计学意义（P〉0.05）。结论多肽P-15加快了大鼠成骨细胞在骨粉表面粘附、增殖的速度,但对大鼠成骨细胞的分化无明显促进作用。     

IL-17A stimulates the expression of inflammatory cytokines via celecoxib-blocked prostaglandin in MC3T3-E1 cells

Objective

The prostaglandins (PGs) released from osteoblasts can alter the process of bone remodelling. Recently, we showed that compressive force induced the expression of pro-inflammatory cytokine interleukin (IL)-17s and their receptors in osteoblastic MC3T3-E1 cells and that IL-17A was expressed most highly. Consequently, in the current study we examined the effect of IL-17A and/or celecoxib on PGE₂ production and the expression of cyclooxygenases (COXs) and inflammatory cytokines in MC3T3-E1 cells. We also examined the effects of PGE₂ and cyclohexamide on the expression of inflammatory cytokines.

Methods

Cells were cultured with or without IL-17A (0.1, 1.0, or 10 ng/ml) in the presence or absence of 10 μM celecoxib, a specific inhibitor of COX-2, for up to 72 h. Cells were pretreated with or without 10 μg/ml cycloheximide, protein synthesis inhibitor, for 30 min, and then cultured with 10 ng/ml IL-17A for 24 h. Cells were also cultured with or without 1.5 ng/ml PGE₂ for 24 h. PGE₂ production was determined by ELISA. The expression of COX-1, COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α mRNAs and proteins was determined by real-time PCR and ELISA, respectively.

Results

The expression of COX-2, IL-1α, IL-6, IL-8, IL-11, and TNF-α, as well as PGE₂ production increased in the presence of IL-17A, whereas COX-1 expression did not change. Celecoxib blocked the stimulatory effect of IL-17A on the expression of COX-2, IL-1α, IL-6, IL-8, and IL-11 as well as PGE₂ production, whereas it did not block TNF-α expression. Cycloheximide pretreatment suppressed the expression of IL-17-induced inflammatory cytokines. The expression of IL-1α, IL-6, IL-8, and IL-11 increased by the addition of PGE₂, whereas TNF-α expression was not affected.

Conclusion

These results suggest that IL-17A stimulates the expression of bone resorption-related inflammatory cytokines through an autocrine mechanism involving celecoxib-blocked PGs, mainly PGE₂, in osteoblasts.