氯化鐠對原代培養的小鼠成骨細胞增殖、分化和礦化功能的影響

張金超李亞平 張 群 王書香

(河北大學化學與環境科學學院，河北省化學生物學重點實驗室，保定 071002)

氯化鐠對原代培養的小鼠成骨細胞增殖、分化和礦化功能的影響

張金超＊李亞平 張 群 王書香＊

(河北大學化學與環境科學學院，河北省化學生物學重點實驗室，保定 071002)

采用噻唑藍(MTT)法、堿性磷酸酶(ALP)比活性測定、油紅O染色、I型膠原測定以及礦化結節染色及定量分析等方法，研究了不同濃度的氯化鐠對原代培養的成骨細胞增殖、分化、礦化功能以及橫向分化為脂肪細胞的影響。結果表明:氯化鐠對成骨細胞增殖、分化、礦化功能以及橫向分化為脂肪細胞的影響與作用濃度和時間密切相關，但沒有呈現出時間和劑量依賴性。結果提示，氯化鐠對骨代謝的影響是復雜的,其具有保護還是損害作用取決于作用濃度和時間。作用濃度和時間是影響氯化鐠生物效應轉變的關鍵因素。

氯化鐠；成骨細胞；增殖；分化；礦化

0 Introduction

Praseodymium,one of the Ln (lanthanides),has been widely used in various fields due to its special physical and chemical natures.For example,salts of praseodymium may be used to color glasses and enamels,when mixed with some other materials,praseodymium produces an intense and unusually clean yellow color in glass.Along with other rare earths,praseodymium iswidely used asa core material for carbon arcs used in the motion picture industry for studio lighting and projection.However,these extensive applications increase the chance of human exposure to praseodymium and its compounds,thus raising deep concerns regarding their risks.Lnincorporated apatite has a series ofattractivepropertiesasapotentialbioactivebone implant material.However,Ln released may induce favorable/noxious reactions locally or systemically during corrosion of implanted materials.Severe hepatotoxicity has been detected after the administration of praseodymium[1].Jha et al.[2]reported that praseodymium oxide (Pr6O11)administered intraperitoneally to Swiss albino mice induced significant increase in the frequency of CAs (chromosomal aberrations)in bone marrow cells.Feyerabend etal.[3]reported that praseodymium showed cytotoxic effects atlower concentrations on MG63 cells.

It was reported that praseodymium was rapidly cleared from the blood and redistributed to tissues,primarily bone after administration[4].The similarity between Ln3+and Ca2+has been suggested to be responsible for some of their biochemical behaviors.Thus it is likely that the praseodymium intervenes in bone-remodeling process and affects bone cell function.Jha et al.[5]reported that Pr6O11promoted bone resorption in vivo.However,to the best of our knowledge the potential effects of praseodymium on bone metabolism have not been reported at the cell level.In this study,we investigated the effects of PrCl3on the proliferation, differentiation and mineralization of primary mouse OBs (osteoblasts)in vitro.

1 Experimental

1.1 Materials and Reagents

KM (Kunming)mice were purchased from the Laboratory Animal Center of Hebei Province.DMEM(Dulbeccos modified eagles medium)and trypsin were purchased from Gibco.NBS (neonatal bovine serum)was from Hangzhou Sijiqing Organism Engineering Institute.MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), penicillin,streptomycin, β-glycerophosphate, ascorbic acid,dexamethasone,collagenase type II,insulin,oil red O,ARS (alizarin red stain), sirius red, and cetylpyridinium chloride were purchased from Sigma(St.Louis,MO,USA).An ALP(alkaline phosphatase)activity kit was purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing,China)and a micro-protein assay kit was from Beyotime Biotechnology (Haimen, China).Praseodymium chloride (PrCl3,Purity ＞99.9%)was from Beijing Institute of Rare Earth Sci.&Tech.Co.,Ltd.Other chemicals used were of analytical grade.

1.2 Methods

1.2.1 Isolation and culture of primary OBs

Primary OBs were isolated mechanically from skulls of newborn KM mouse by a modification of the method previously reported[6].Briefly,skulls were dissected,endosteum and periosteum were stripped off,and bone slices were cut into approximately 1～2 mm2pieces and digested with 2.5 g·L-1trypsin for 30 min.Then bone slices were collected and digested twice with 1.0 g·L-1collagenase II with 1 h for each time.After being incubated overnight in a 5%CO2humidified incubator at 37℃,following which the used medium was changed.The medium was changed every 3 d in all the experiments.

1.2.2 Cell viability assay

OB viability was determined by MTT assay as described previously[7].In brief,OBs were seeded in 96-well tissue culture plates at the density of 2×104cells per well and incubated at 37℃,in a 5%CO2humidified incubator.After the addition of PrCl3at different concentrations (final concentration 1×10-9,1×10-8,1×10-7,1×10-6,1×10-5and 1×10-4mol·L-1),18,24,36 and 48 h furtherincubationswere performed.Control wells were prepared by addition of NaCl.Wells containing DMEM without cells were used as blanks.Wells containing NaF(1×10-6mol·L-1)were used as positive control.Upon completion of the incubation,20 μL of MTT (5.0 mg·mL-1in 1×PBS(phosphate buffered saline))was added and incubated for another 4 h at 37℃.The supernatant was removed,and dimethyl sulfoxide (DMSO)was added to each well.The OD (optical density)was measured on a microplate spectrophotometer (MD VersaMax,USA)at a wavelength of 570 nm.The cell viability(%)was calculated according to the formula:(ODtreated-ODblank)/(ODcontrol-ODblank)×100.

1.2.3 ALP activity

The OBs (2×104cells per well)were plated in 48-well culture plates,and treated with PrCl3at final concentrations of 1×10-9,1×10-8,1×10-7,1×10-6,1×10-5and 1×10-4mol·L-1.Control,blank and positive wells were prepared as above.Upon completion of the incubation,the plates were washed thrice with icecold PBS and lysed by two cycles of freezing and thawing.Aliquots of supernatants were subjected to ALP activity and protein measurement by an ALP kit and a micro-protein assay kit,respectively.All results were normalized by protein content. The differentiation promotion rate (%)was calculated according to the formula: (ALP activitytreated-ALP activityblank)/(ALP activitycontrol-ALP activityblank)×100.

1.2.4 Collagen production analysis

Collagen production analysis was performed as previously described with some modifications[8].Briefly,OBs(2×104cells per well)were plated in 48-well culture plates and cultured overnight at 37℃,in a 5%CO2humidified incubator.PrCl3was added at final concentrations of 1×10-9,1×10-8,1×10-7,1×10-6,1×10-5and 1×10-4mol·L-1.Control,blank and positive wells were prepared as above.Upon completion of the incubation,the plates were washed thrice with icecold PBS and fixed for 1 h with 500 μL fixation solution of 10%formaldehyde and then washed with distilled water.200 μL of dye solution (30 mg sirius red in 30 mL saturated solution of picric acid)per well was added with mild agitation for 1 h.After that,they were washed with 0.01 mol·L-1HCl to remove the excess of sirius red.The dye fixed collagen was observed and extracted with 200 μL 0.1 mol·L-1NaOH solution per well.The OD was measured at a wavelength of 550 nm.All results were normalized by protein content.Synthesis of type collagen promotion rate (%)was calculated according to the formula:(typeⅠcollagentreated-typeⅠ collagenblank)/(typeⅠcollagencontrol-type Ⅰ collagenblank)×100.

1.2.5 Mineralized matrix formation assay

The OBs (2×104cells per well)were plated in 24-well culture plates and cultured overnight at 37℃,in a 5%CO2humidified incubator.The medium was then changed to differentiation medium containing 10 mmol·L-1β-glycerophosphate and 50 μg·mL-1ascorbic acid,PrCl3was added at final concentrations of 1×10-9,1×10-8,1×10-7,1×10-6,1×10-5and 1×10-4mol·L-1.Control,blank and positive wells were prepared as above.Upon completion of the incubation,the formation of mineralized matrix noduleswas determined by ARS.Briefly,Cell monolayers were fixed in 95%ethanol for 10 min at room temperature,then washed by PBS twice and stained with 0.1%ARS for 30 min at room temperature.Quantitation of ARS was performed by elution with 10% (W/V)cetylpyridium chloride for 10 min at room temperature and theOD wasmeasured at570 nm[9].The mineralized function promotion rate (%)was calculated according to the formula: (ODtreated-ODblank)/(ODcontrol-ODblank)×100.

1.2.6 Oil red O stain and measurement

The OBs (2×104cells per well)were plated in 48-wellculture plates,afterbeing induced by adipogenic supplement(10 μg·mL-1insulin,10-7mol·L-1dexthamethone)and treated with PrCl3at final concentrations of 1×10-9,1×10-8,1×10-7,1×10-6,1×10-5and 1×10-4mol·L-1.Control,blank and positive wells were prepared as above.Upon completion of the incubation, fat droplets within differentiated adipocytes from OBs were stained according to the literature[10].Cells were fixed in 4%formaldehyde,washed in water and stained with a 0.6%(w/v)oil red O solution (60%isopropanol,40%water)for 15 min at room temperature.For quantification,cells were washed extensively with water to remove unbound dye,then isopropyl alcohol was added to the culture plates.After 5 min,the OD of the extract was measured at a wavelength 510 nm.The adipocytic transdifferentiation promoting rate (%)was calculated according to the formula:(ODtreated-ODblank)/(ODcontrol-ODblank)×100.

1.2.7 Statistical analysis

Data were collected from at least four separate experiments.Results were expressed as meanshstandard deviation (SD).Statistical differences were analyzed using SPSS t-test.P values less than 0.05 were considered to indicate statistical differences.

2 Results and discussion

2.1 Results

2.1.1 Effect of PrCl3on the viability of OBs

As shown in Fig.1,the results reveal that PrCl3decreases the viability of OBs at all tested concentrations for 18 h.After 24 and 48 h of PrCl3treatment,Prdecreasesthe viability ofOBsat concentrations of 1×10-9,1×10-8,1×10-7,1×10-6and 1×10-5mol·L-1,but turns to increase the viability of OBs at the highest concentration of 1 ×10-4mol·L-1.After 36 h of PrCl3treatment,it shows no significant effect on the viability of OBs at concentrations of 1×10-9,1×10-8,1×10-7,1×10-6and 1×10-5mol·L-1,but also turns to increase the viability of OBs at the highest concentration of 1×10-4mol·L-1.

2.1.2 Effect of PrCl3on ALP activity of OBs

As shown in Fig.2,after 18 h of PrCl3treatment,it promotes the differentiation of OBs at the highest concentration of 1 ×10-4mol·L-1,and inhibits the differentiation of OBs at other tested concentrations.PrCl3has no significant effect on the differentiation of OBs at the lowest concentration of 1×10-9mol·L-1,and significantly inhibits the differentiation of OBs at other tested concentrations for 36 h.

2.1.3 Effect of PrCl3on synthesis of type I collagen of OBs

As shown in Fig.3,on day 3,6 and 9,PrCl3has no significant effect on synthesis of type I collagen of OBs at concentrations of 1×10-9,1×10-8,1×10-7,and 1×10-6mol·L-1.On day 3,PrCl3has no significant effect on synthesis of type I collagen of OBs,but it turns to promote synthesis of type I collagen of OBs at a concentration of 1×10-5mol·L-1on day 6 and 9.On day 3,6 and 9,it promotes synthesis of type I collagen of OBs at the highest concentration of 1×10-4mol·L-1in a time-dependent manner.

2.1.4 Effect of PrCl3on the formation of mineralized matrix nodules

As shown in Fig.4,after 17 d of PrCl3treatment,it significantly promotes the formation of mineralized matrix nodules at all tested concentrations without evidentdose-dependence.On 21 d,ithas no significant effect on the formation of mineralizedmatrix nodules at concentrations of 1×10-9,1×10-8,1×10-7,and 1 ×10-6mol·L-1,but turns to promote the formation of mineralized matrix nodules at concentrations of 1×10-5and 1×10-4mol·L-1.

2.1.5 Effect of PrCl3on the adipocytic transdifferentiation of OBs

As shown in Fig.5,after 12 and 15 d of PrCl3treatment,ithas no significant effect on the adipocytic transdifferentiation of OBs at concentrations of 1×10-9,1×10-8,and 1×10-7mol·L-1,but turns to promote the adipocytic transdifferentiation of OBs at concentrations of 1×10-6,1×10-5,and 1×10-4mol·L-1.

2.2 Discussion

Bone is a specialized and dynamic organ that undergoes a continuous remodeling in an adult life,which include bone formation of OBs and bone resorption of osteoclasts in a coupled manner.OBs progress through a three-stage process of differentiation: proliferation, differentiation and mineralization.OB differentiation is a crucial aspect of bone formation and remodeling.An increase in bone specific ALP activity in vitro reflectsthe maturation from an earlier to a more mature stage of OB differentiation[11-12].The formation of mineralized bone nodules is the sign for the final stage of OB differentiation.In addition,both OBs and adipocytes derive from bone marrow stromal cells.There is more and more evidence that suggests the existence of a great degree of plasticity between OBs and adipocytes,and this transdifferentiation is reciprocal[13].It was reported that adipocytes were secretory cells that might influence hematopoiesis and osteogenesis.Adipocytes synthesized and released a variety of peptide and nonpeptide compounds orsecreted cytokines,and the main effect of these cytokines was a stimulation of bone resorption[14].So there may have protective effect on bone by inhibiting OB transdifferentiation.

In the present study,the model of primary OBs wasused to study the effectofPrCl3on the proliferation,differentiation and mineralization of OBs.The results indicate that the effect of PrCl3on the proliferation,differentiation and mineralization of OBs is closely related to the concentration and culture time,but has no obvious dose-or time-dependent tendency.These results are accordantwith our previous report[15-16].In addition,our experimental results also suggest that the different species of rare earth ions may behave differently.These differences may relate to the physico-chemical characteristics of the respective cations depending upon features,such as their ionic radii or charge densities.

In addition,in cellular studies of the biological effect of multivalent metal cations,researchers are always concerned about the precipitation paradox.The cations,known as hard Lewis acids tend to precipitate as hydroxides and phosphates/carbonates in cellculture media and thus complicate the experimental conditions and cause confusion.In fact,numerous pieces of evidence show that the biological effect of lanthanide-based complexes observed is actually in the presence of phosphates and bicarbonates.The particles formed are able to exert the biological effect under physiological conditions[17].A multi-phase model has been developed and terbium (Tb)speciation in human blood plasma has been studied.Ata concentration of 10-5mol·L-1,Tb（Ⅱ） was mostly bound to phosphate to form precipitate of TbPO4.As the concentration ofTb（Ⅱ） increases,phosphate is exceeded and another kind of precipitate of Tb2(CO3)3appears.Among the tiny soluble Tb（Ⅱ） species,Tb（Ⅱ）mainly distributes in[Tb(Tf)]at low concentration and in[Tb(HSA)],[Tb2(Tf)],[Tb(IgG)],[Tb(Lactate)]2+,[Tb(CitArgH)]and free Tb（Ⅱ）at high concentration[18].Up to now,the working species of praseodymium to exert the effect on the proliferation,differentiation and mineralization of OBs are not well-understood and need to be solved.

In conclusion,the effect of PrCl3on the bone metabolism is very complicated.The concentration and culture time are key factors for switching its biological effects from damage to protection.The mechanism for the effect of PrCl3on the proliferation,differentiation, adipocytic transdifferentiation and mineralization function ofprimary OBsand the working species of praseodymium to exert these effects remain to be further studied.

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Effect of Praseodymium Chloride on Proliferation,Differentiation and Mineralization of Primary Mouse Osteoblasts in vitro

ZHANG Jin-Chao＊LI Ya-Ping ZHANG Qun WANG Shu-Xiang＊
(College of Chemistry&Environmental Science,Chemical Biology Key Laboratory of Hebei Province,Hebei University,Baoding,Hebei 071002,China)

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide),ALP (alkaline phosphatase)activity,oil red O assays,synthesis of typeⅠcollagen and ARS (alizarin red-S)stain were employed to evaluate the effect of PrCl3on proliferation,differentiation and mineralization function of primary OBs(osteoblasts)in vitro.The results indicate that the effect of PrCl3on the above functions and the adipocytic transdifferentiation of primary OBs is closely related to the concentration and incubation time,but is not time and dose-dependent.This suggests that the effect of PrCl3on bone metabolism is complicated;concentration and culture time are the key factors for switching the biological effect of PrCl3from damage to protection.

praseodymium chloride;osteoblasts;proliferation;differentiation;mineralization

O0614.33+4

A

1001-4861(2012)08-1749-06

2012-01-17。收修改稿日期：2012-03-17。

國家自然科學基金(No.20971034)，河北省自然科學基金重點(No.B2009000161)資助項目。

＊通訊聯系人。E-mail：jczhang6970@yahoo.com.cn;wsx@hbu.edu.cn