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MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells
Matrix Biology 20 Ž2001. 347᎐355
MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells Akemichi Ueno a,U , Yukiko Kitase b, Keiji Moriyamab, Hideo Inoue a a b
Department of Biochemistry, School of Dentistry, The Uni¨ ersity of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Department of Orthodontics, School of Dentistry, The Uni¨ ersity of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Received 20 October 2000; received in revised form 5 April 2001; accepted 17 May 2001
Abstract Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line ŽRPC-C2A. derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium ŽCM. from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3᎐6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor ŽTGF.- antibody and a soluble form of receptor for bone morphogenetic protein ŽBMP.-2r-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factorŽs. other than TGF- or BMP-2r-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells. 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Dental pulp cells; Mineralization; Conditioned medium; MC3T3-E1 preosteoblasts
1. Introduction Dental pulp is a unique soft connective tissue surrounded by dentin. Although both tissues have a common ancestry in the mesenchymal dental papilla ŽLinde, 1985., the former is unmineralized while the latter is highly mineralized. Dental pulp cells maintain the homeostasis of dental mineralized tissues. In response to appropriate stimuli Že.g. caries, chemicals or trauma., reparative dentin is formed by defensive pulp reactions ŽYamamura, 1985; Tziafas, 1995;
Abbre¨ iations: CM, conditioned medium; ECM, extracellular matrix; ALPase, alkaline phosphatase; TGF-, transforming growth factor-beta; BMP, bone morphogenetic protein; DSPP, dentin sialophosphoprotein; OC, osteocalcin; OPN, osteopontin; ON, osteonectin; TSP, thrombospondin U Corresponding author. Tel.: q81-88-633-7326; fax: q81-88633-7326. E-mail address: [email protected] ŽA. Ueno..
Holan, 1998.. With advancing age, the size of the dental pulp cavity is reduced as a result of peritubular dentin deposition ŽKvaal et al., 1994., whereas the incidence of pulp stones is not age-dependent ŽAlHadi Hamasha and Darwazeh, 1998.. The molecular mechanisms of these responses remain to be defined. To study these processes, it is useful to establish an in vitro mineralization system by a clonal cell line derived from dental pulp. Among several reports that showed mineralization by dental pulp-derived cells, Hayashi et al. Ž1993. observed mineralized tissues within multilayers of clonal RPC-K cells after a long period of culture Ž28 days.. They observed a vesicular structure around degenerate and necrotic cells. Some of these vesicles contained needle-like crystals. Other studies were done with non-clonal primary culture cells isolated from an enzymatic digest of dental pulp ŽNakashima, 1991; Kasugai et al., 1993; Yokose et al., 2000. or cells
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 1 . 0 0 1 4 1 - X
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outgrowing from cultured pulp explants ŽTsukamoto et al., 1992; Couble et al., 2000.. These primary cells are heterogeneous and are limited for obtaining a sufficient cell population. In these studies with RPC-K cells and primary cells, mineralized tissue formation has been observed only after a long period of culture and no further analyses have been performed. Extracellular matrix ŽECM. components in dental pulp consist of collagens and non-collagenous proteins as in other connective tissues. Non-collagenous proteins including osteocalcin ŽOC., osteopontin ŽOPN., osteonectin ŽON. and thrombospondins ŽTSPs., and glycosaminoglycans ŽGAGs. are also thought to be involved in the regulation of biomineralization ŽLinde, 1985.. Our previous studies showed that a clonal cell line, RPC-C2A established from rat incisor dental pulp by Kasugai et al. Ž1988., synthesizes chondroitin 4-sulfate and dermatan sulfate, but not chondroitin 6-sulfate among GAGs ŽNishikawa et al., 2000. in a similar composition with that of dentin, and that osteogenic transforming growth factor 1 ŽTGF-1. stimulates not only the incorporation of w 35 Sx sulfate into GAG but also the synthesis of ECM proteins including OPN and TSP1 ŽNishikawa et al., 2000; Islam et al., 1996.. Although this cell line exhibits high alkaline phosphatase ŽALPase. activity, no mineralized tissues in the matrix of RPC-C2A were observed ŽKasugai et al., 1988., even in the presence of TGF-1 ŽNishikawa et al., unpublished observation.. On the other hand, the mouse calvaria-derived osteogenic cell line MC3T3-E1 forms mineralized nodules in vitro ŽSudo et al., 1983.. This cell line secretes a number of cytokines including insulin-like growth factor ŽIGF. type I, TGF-, IGF-II, bone morphogenetic protein Ž BMP . -2 and BMP-4 ŽAmarnani et al., 1993; Suzawa et al., 1999.. In this study, we co-cultured RPC-C2A cells with MC3T3-E1 cells separated by a protein-permeable collagencoated membrane and established a new experimental system in which mineralized nodules were formed in the matrix of RPC-C2A cells to investigate the molecular mechanisms of supplementary mineralization of dental pulp.
by collagen-coated filters, which inhibit direct contact of the two cell lines but were permeable to cytokines, marked mineralization was observed in ECM of RPCC2A cells after 3 weeks. In contrast, co-culture with the osteoblastic osteosarcoma cell line ROS17r2.8 ŽLi et al., 1998. did not induce matrix mineralization of RPC-C2A ŽFig. 1a.. CM from MC3T3-E1 cells showed a similar stimulation of mineralized nodule formation when added to culture medium of RPC-C2A cells. This stimulation was proportional to the CM concentration with a maximum at 50% ŽFig. 1b.. Thus, 50% of CM was used in subsequent experiments. No mineralized nodule was observed with RDP4-1 cells ŽFig. 1c.. For quantification of the mineralized nodules, we measured the acid-soluble calcium content in ECM by o-cresolphthalein complexone method ŽFig. 1d,e.. Acid-soluble calcium contents in ECM of CM-treated RPC-C2A cells ŽFig. 1d. corresponded well with von Kossa-stained mineralized nodules shown in Fig. 1b. The maximal mineralized nodule formation observed with 50% CM was threefold that found with 25% CM. However, very few mineralized nodules were detected in 50% CM-treated RDP4-1 cells, another line of clonal pulp cells ŽFig. 1e.. Next, we compared the effects of CMs collected after different culture periods. Of the CMs collected at preconfluence Žy3᎐0 days. and the first CM Ž0᎐3 days., ᎏ eighth CM Ž19᎐21 days., the first CM Ž0᎐3 days., second Ž3᎐6 days. and third Ž6᎐9 days. were effective but others were ineffective ŽFig. 2a.. Because the second CM Ž3᎐6 days. was most effective, it was used in subsequent experiments, unless otherwise specified. Similar experiments were carried out with CM from ROS17r2.8 cells. However, the formation of mineralized nodules by RPC᎐C2A cells was not induced irrespective of the collection time of CM ŽFig. 2a. or percentage of its addition Ždata not shown.. The active factorŽs. in the CM was heat-labile because CM lost its activity completely on heating at 56⬚C for 30 min or 85⬚C for 10 min ŽFig. 2b.. 2.2. Effects of CM from MC3T3-E1 cells on ALPase acti¨ ity and mRNA le¨ els of non-collagenous ECM proteins in RPC-C2A cells
2. Results 2.1. Mineralized nodule formation by dental pulp cells The confluent RPC-C2A cells did not form mineralized nodules even on culture in medium C for more than 60 days. On the other hand, osteoblastic MC3T3-E1 cells formed mineralized nodules within 10 days after confluency Ždata not shown.. When these cells were co-cultured in compartments divided
The ALPase activity in RPC-C2A cells increased rapidly after confluency in the absence or presence of the CM. However, the activity did not increase any further after 3 days and decreased from 12 days after confluency in the absence of the CM. The addition of the CM resulted in a further increase from 3 days up to 6 days after confluency and 25᎐60% higher activities during days 6᎐15 than those in the absence of the CM ŽFig. 3a..
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Fig. 1. Effects of co-culture with MC3T3-E1 cells or ROS17r2.8 cells and CM from MC3T3-E1 cells on mineralized nodule formation by dental pulp cells. Ža. Confluent RPC-C2A cells and either MC3T3-E1 cells Ž1, 2. or ROS17r2.8 cells Ž3, 4. were co-cultured on Transwell-COL filters as described in Section 4. After culture for 21 days, pulp cell layers were stained with alizarin red S Ž1, 3. and von Kossa Ž2, 4.. Žb. and Žc.. Confluent RPC-C2A cells Žb. and RDP4-1 cells Žc. in six-well plates were cultured for 10 days in medium C containing 0% Ž1.; 25% Ž2.; 33% Ž3.; 50% Ž4.; 66% Ž5.; or 75% Ž6. of CM Ž3᎐6 days. from MC3T3-E1 cells. Mineralized nodules were examined by von Kossa staining. Žd,e.. Confluent RPC-C2A cells Žd. or RDP4-1 cells Že. were cultured in medium C in the presence of indicated concentrations of CM Ž3᎐6 days. from MC3T3-E1 cells. The acid-soluble calcium contents were measured by the o-cresolphthalein complexone method. Columns and bars show means and S.D.s, respectively, of results in three separate experiments. U Significant difference from control Ž0% CM..
The mRNA of odontoblast-specific DSPP was undetectable in RPC-C2A cells under all experimental conditions employed wFig. 3bŽi.x. The mRNA levels of OC, OPN and ON began to increase just after confluency even in the absence of CM. OC mRNA increased linearly until 12 days without CM and the level on day 12 was more than six times that on day 0. The mRNA levels of OPN and ON reached maxima 6᎐9 days after confluency in the absence of CM. Unlike ALPase activity, CM depressed the elevations of both mRNAs after day 3 and the levels during days 9᎐15 were almost equal to those on day 0. Then the mRNA levels of OPN and ON on days 6᎐15 in the
absence of CM were higher than those in the presence of CM wFig. 3bŽii.x. 2.3. Effects of cytokines of MC3T3-E1 cells and their antagonists on mineralized nodule formation by RPCC2A cells Co-culture experiments suggested that an active factorŽs. in CM could be a cytokineŽs. of MC3T3-E1 cells. To search for an active factorŽs. in CM, we examined the effects of TGF- and BMPs because these growth factors are reported to be differentiation-related cytokines of MC3T3-E1 cells ŽAmarnani
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Fig. 2. Effects of MC3T3-E1 cell CM collected after different culture periods and heat treatment of CM on formation of mineralized nodules by pulp cells. Ža.. Confluent RPC-C2A cells Ž1᎐9. or RDP4-1 cells Ž10᎐12. were cultured for 10 days in medium C containing 0% Ž1. or 50% Ž2᎐12. CM collected after different culture periods Žsee Section 4 for details.. CM Žy3᎐0 days preconfluence. Ž2.; CM Ž0᎐3 days. Ž3 and 10.; CM Ž3᎐6 days. Ž4 and 11.; CM Ž6᎐9 days. Ž5 and 12.; CM Ž9᎐12 days. Ž6.; CM Ž12᎐15 days. Ž7.; CM Ž15᎐18 days. Ž8.; CM Ž18᎐21 days. Ž9.. Žb. Confluent RPC-C2A cells in 12-well plates were cultured for 10 days in medium C containing 0% Ž1. or 50% Ž2᎐4. CM before Ž2. and after heating at 56⬚C for 30 min Ž3. or at 85⬚C for 10 min Ž4. and stained by the von Kossa technique as described in Section 4.
et al., 1993; Suzawa et al., 1999.. TGF-1 and BMPs alone or in combination did not stimulate mineralized nodule formation ŽFig. 4a. and calcium deposition ŽFig. 4c.. Antagonists against these cytokines did not depress the effectiveness of CM from MC3T3-E1 cells ŽFig. 4b.. Increases Ž50%. in cytokines and their antagonists gave similar results Ždata not shown.. Although CM-induced calcium deposition was slightly inhibited by the addition of pan-specific TGF- neutralizing antibody or a soluble form of recombinant human BMP receptor IA, it was not statistically significant ŽFig. 4c. under the experimental conditions employed.
3. Discussion Confluent RPC-C2A pulp cells show high activity of ALPase ŽKasugai et al., 1988. and high levels of mRNA of OC and OPN, but not DSPP ŽFig. 3b.. The
syntheses of ECM proteins ŽOPN, TSP1, and matrix metalloproteinases. were stimulated by a variety of hormones and cytokines such as calcitriol ŽNagata et al., 1994., phorbol ester ŽIslam et al., 1996; Yokota et al., 1992., retinoic acid ŽOhishi et al., 1999., TGF-1 ŽIslam et al., 1996., and interleukin ŽIL.-1 ŽPanagakos et al., 1996.. These responses in RPC-C2A cells were quite similar to those of preosteoblasts and osteoblasts ŽAmarnani et al., 1993; Thrailkill et al., 1995; Kusano et al., 1998.. However, mineralized nodule formation by RPC-C2A cells has not been reported to date. There are reports that mineralization of ECM in vitro is influenced by co-cultured cells or their CM. For example, CM from the human prostatic carcinoma cell line PC-3 inhibited osteoblastic cell differentiation of primary fetal rat calvaria cells ŽSantibanez et al., 1996; Martinez et al., 1996; Kido et al., 1997.. On the contrary, Kimura et al. Ž1992. reported that CM from PC-3 stimulated the calcification of a human osteoblastic cell line, Tak-10, in medium supplemented with prostatic acid phosphatase and ␣glycerophosphate. Thus, the activity of CM from PC-3 cells is still controversial. Zimmermann et al. Ž1990. reported that endochondral mineralization in cultured cartilage was enhanced in the presence of calvarial cells or their CM, but inhibited by co-culture with skin fibroblast-like cells. Similarly, rat skin fibroblasts and human ligament fibroblasts inhibited mineralized bone nodule formation by rat bone marrow stromal cells ŽOgiso et al., 1991.. These results suggest that endochondral mineralization was stimulated by osteoblastic cells but inhibited by fibroblastic cells, and that these effects were mediated by soluble factors in CM. Osteoblastic MC3T3-E1 cells show marked formation of mineralized nodules after confluency ŽSudo et al., 1983. and secrete a variety of soluble factors into the CM ŽAmarnani et al., 1993; Suzawa et al., 1999.. Therefore, we examined the effect of CM from MC3T3-E1 cells on mineralized nodule formation by RPC-C2A cells. We developed a simple and rapid experimental system by which matrix mineralization of pulp-derived clonal cells could be induced. The von Kossa-positive mineralized nodules can be recognized within 9 days from the start of RPC-C2A culture, which is a much shorter time than approximately 28 days of culture required for RPC-K cells described by Hayashi et al. Ž1993. and 30 days of culture required in the case of PC-3 CM ŽKimura et al., 1992.. The CM-treated RPC-C2A cells formed mineralized nodules with increased activity of ALPase ŽFig. 3a., a marker enzyme of osteoblasts and significant decrease of mRNA levels of OPN and ON ŽFig. 3b., which are rich in ECM of osteoblasts ŽRoach, 1994., although the roles of these proteins in osteoblasts were not
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well defined. In the mineralizing cells, the production of OPN is one of the earliest Žseveral days before the onset of mineralization., and latest, secretory activities of the osteoblast lineage ŽDenhardt and Guo, 1993; McKee and Nanci, 1996.. The reason why the latter part of the OPN mRNA level behind its earlier peak is diminished by adding CM ŽFig. 3b. remains to be determined. Similar results have been reported.
Fig. 3.
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Malaval et al. Ž1994. demonstrated using rat bone marrow stromal cell cultures by mRNA levels, protein production andror enzymatic activity, that expressions of OC and ALPase increased concomitantly with the development of bone nodules, while OPN mRNA levels decreased and those of ON did not change significantly. In addition, they found, using rat calvaria cell cultures, that leukemia inhibitory factor ŽLIF. decreased ALPase activity and bone nodule production, but Northern blotting analysis showed early stimulation of OPN and no effect on ON ŽMalaval et al., 1995.. RPC-C2A cells expressed high mRNA of OC ŽFig. 3b., a marker protein of osteoblasts ŽGundberg et al., 1984., but an undetectable mRNA level of DSPP, a marker protein of odontoblasts ŽLinde, 1985., during CM-stimulated formation of mineralized nodules. These results suggested that the CM from MC3T3-E1 cells enhanced the differentiation of RPC-C2A cells to osteoblasts. An active factorŽs. in CM of MC3T3-E1 cells was secreted during a restricted period of culture Ž3᎐6 days after confluency. and was not detectable in replicating preosteoblasts or beyond day 9 of confluence ŽFig. 2a.. Moreover, it showed a strict cell-specificity to RPC-C2A cells ŽFig. 1.. The clonal RDP4-1 cells also exhibited high ALPase activity which increased with the duration of culture and produced abundant type I collagen as ECM ŽKawase et al., 1990., but matrix mineralization could not be achieved. Furthermore, CM from ROS17r2.8 cells ŽLi et al., 1998. did not induce matrix mineralization of RPC-C2A cells. This may be due to the fact that ROS17r2.8 cells do not form mineralized nodules by themselves on a conventional plastic culture dish ŽShteyer et al., 1986., unlike MC3T3-E1 cells. From these facts, we assumed that the active factorŽs. could be a cytokineŽs. of MC3T3-E1 cells, and examined the effects of TGF-1 and BMP-2r-4 ŽFig. 4., because these are representative osteogenic cytokines of MC3T3-E1 cells ŽAmarnani et al., 1993; Suzawa et al., 1999. and
Fig. 3. Effect of CM from MC3T3-E1 cells on ALPase activity and mRNA levels of non-collagenous ECM proteins in RPC-C2A cells. a. Confluent RPC-C2A cells were cultured in 35-mm dishes for the indicated periods in the absence Ž`. or presence Ž䢇. of 50% CM Ž3᎐6 days. from MC3T3-E1 cells. ALPase activity was assayed as described in Section 4. Values are means " S.D. for 3᎐4 separate experiments. U Significantly different from the control value Ž0% CM.. Žb. RPC-C2A cells were cultured for the indicated periods in medium C containing 0% Žy, `. or 50% Žq, 䢇. CM Ž3᎐6 days. from MC3T3-E1 cells. Total RNA extraction and Northern blot analysis were done as described in Section 4. The levels of unconfluent cells Žy3 day. are also shown. Results are shown by autoradiography Ži. and its quantitative analysis by a bioimaging analyzer Fujix BAS2000 II Žii.. Lane ²i: in Ži. shows mRNA of DSPP in 0.2 g of rat incisor odontoblast poly Aq RNA. Values in Žii. are averages of two separate experiments. kb, kilobases.
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BMP-2 induces heterotopic bone development in vivo and in vitro ŽKubler and Urist, 1993; Riley et al., 1996; Volek-Smith and Urist, 1996.. However, the addition of these factors to the culture medium ŽFig. 4a. and inhibition of these cytokines in the CM by neutralizing antibodies or soluble receptors had no significant effect ŽFig. 4b,c., indicating little participation of TGF-s or BMP-2r-4 in the CM-induced mineralization by RPC-C2A cells. The active factorŽs. is heat-labile ŽFig. 2b. and impermeable to an ultrafiltration membrane Žmol. wt. cutoff: 30 000. ŽUeno et al., unpublished observation., suggesting a proteinous moleculeŽs.. Identification of an active factorŽs. is now underway. Recently, Couble et al. Ž2000. and Gronthos et al.
Ž2000. reported that human dental pulp-derived cells can differentiate in vivo or in vitro to odontoblast-like cells which show biochemical and morphological characteristics of mature odontoblasts, an expression of DSPP mRNA and an appearance of long cellular processes, respectively. These and our results suggest the presence of progenitors of both odontoblasts and osteoblasts in dental pulp and that these cells participate in supplementary mineralization in dental pulp. Further studies, such as transplantation experiments, are essential to assess in vivo formation of mineralized ECM by CM-treated RPC-C2A cells.
4. Experimental procedures 4.1. Materials Tissue culture plasticware was obtained from Becton Dickinson Labware ŽFranklin Lakes, NJ, USA.. Transwell-COL Ž24 mm in diameter, 0.4-m pore size. membrane culture inserts were purchased from Corning-Costar ŽCambridge, MA, USA.. ␣-MEM medium was purchased from GibcoBRL Life Technologies ŽRockville MD, USA.. Eagle’s MEM was purchased from Nissui Pharmaceutical ŽTokyo, Japan.. Fetal bovine serum was purchased from Hyclone Laboratories ŽLogan, UT, USA. or JRH Biosciences ŽLenexa, KS, USA.. A BCA protein assay kit was obtained from Pierce ŽRockford, IL, USA.. Recombinant human TGF-1 was purchased from Roche Diagnostics ŽMannheim, Germany.. Recombinant human BMP-2 was provided by Yamanouchi Pharmaceutical ŽTokyo, Japan.. Recombinant human
Fig. 4.
Fig. 4. Effects of cytokines of MC3T3-E1 cells and their antagonists on mineralized nodule formation by RPC-C2A cells. Ža. Confluent RPC-C2A cells were cultured in 12-well plates for 10 days in the absence Ž1. or presence of 50% CM Ža mixture of the first to third collections. Ž2.; 10 ngrml recombinant human TGF-1 Ž3.; 50 ngrml human recombinant BMP-2 Ž4.; BMP-4 Ž5.; or TGF-1 q BMP-2 q BMP-4 Ž6. and then stained by the von Kossa method. Žb. A volume of 0.4 ml CM Ž3᎐6 days. from MC3T3-E1 cells was incubated at 37⬚C for 60 min without Ž1. or with either 8 g Ž4 l. of pan-specific TGF- neutralizing antibody Ž ␣TGF-. Ž2. or 320 ng Ž1.6 l. of a soluble form of recombinant human BMP receptor IA ŽsBMPrIA. Ž3.. The antagonist-treated CM was diluted with medium C to 50% CM and used for culture of confluent RPC-C2A cells in 12-well dishes for 10 days. Mineralized nodules in 12-well dishes were stained by the von Kossa technique as described in Section 4. Figures in Ža. and Žb. are typical results of two separate experiments. Žc. Mineralized nodules in well Nos. 1 and 6 in Ža. and well Nos. 1, 2 and 3 in Žb. were quantified by assay of acid-soluble calcium in ECM as described in Fig. 1d,e Žmean " S.D., n s 3.. Neither the differences between antagonist-treated samples Žcorresponding to b-2 and -3. and mineralized control Ždesignated as CM, b-1. nor the difference between cytokine-treated sample Ža-6. and unmineralized control Ža-1. were significant.
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BMP-4 and a pan-specific TGF- neutralizing antibody were purchased from GenzymerTechne ŽCambridge, MA, USA.. Soluble BMP receptor IA ŽNatsume et al., 1997; Otsuka et al., 1999. was kindly provided by Dr T. Hatta ŽNational Institute of Agrobiological Resources, Tsukuba, Japan.. w ␣- 32 Px dCTP Ž111 TBqrmmol. and the Hybond-Nq nylon membrane were from Amersham Pharmacia Biotech ŽBuckinghamshire, UK.. Restriction enzymes and a Random Primer DNA Labeling Kit Ver.2 were from T a k a ra S h u zo Ž K u sa tsu , Ja p a n . .  -G lycerophosphate, Hoechst No. 33258, calf thymus DNA and p-nitrophenyl phosphate were from Sigma ŽSt. Louis, MO, USA.. A RNeasy Mini Kit was from Qiagen ŽHilden, Germany.. L-Ascorbic acid, dexamethasone, silver nitrate, alizarin red S and Calcium C-test Wako were obtained from Wako Pure Chemical ŽOsaka, Japan.. 4.2. Cell culture The clonal cell lines RPC-C2A and RDP4-1, derived from incisor dental pulp of Wistar strain rats, were provided by Drs S. Kasugai ŽKasugai et al., 1988., and T. Kawase ŽKawase et al., 1990., respectively. For subculture, both lines were cultured in medium A wEagle’s MEM supplemented with 2.2 mgrml NaHCO3 , 0.292 mgrml L-glutamine, 60 grml kanamycin and 10% fetal bovine serum ŽFBS.x under a humidified atmosphere of 5% CO 2 in air at 37⬚C. MC3T3-E1 cells ŽSudo et al., 1983. and ROS17r2.8 cells ŽLi et al., 1998. were cultured in medium B Ž ␣-MEM supplemented with 0.22% NaHCO3 , 100 Urml penicillin G, 100 grml streptomycin, 10 grml gentamycin and 10% FBS.. For mineralization experiments, confluent cells were cultured in medium C Žmedium B further supplemented with 2 mM glycerophosphate, 50 grml L-ascorbic acid and 10y8 M dexamethasone.. In co-culture experiments, RPCC2A cells in medium A were seeded at a density of 14 000 cellsrcm 2 on Transwell-COL 0.45 m collagen-coated filters in six-well plates and MC3T3-E1 cells in medium B were plated at a density of 15 000 cellsrcm 2 in the bottom wells of six-well plates. When both cell lines became confluent, co-culture was started with 3 ml of medium C. In all cell cultures of this study, the medium was changed every 3 days, the medium volumes being 0.8, 1.8, 1.8, 3.5 and 8 ml for 12-well plates, six-well plates, 35-mm dishes, 60-mm dishes and 100-mm dishes, respectively.
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cells were further cultured for a maximum of 24 days in medium C. CMs were collected every 3 days. CMs of the first and second collections were defined as CM Ž0᎐3 days. and CM Ž3᎐6 days., respectively. The CMs collected were centrifuged at 1000 = g for 20 min at 4⬚C to remove floating cells and cell debris and frozen at y80⬚C until use. For neutralization of cytokines in CMs, 0.4-ml volumes of CMs were pre-incubated at 37⬚C for 1 h with either a pan-specific TGF- neutralizing antibody or a soluble form of human recombinant BMP receptor IA ŽNatsume et al., 1997; Otsuka et al., 1999. at concentrations of 10 grml and 400 ngrml, respectively. 4.4. Staining of mineralized nodules After the dental pulp-derived cells were cultured to confluence in culture medium A in 12- or six-well plates or 35 mm dishes, the medium was replaced by medium C containing 0᎐75% CM. For the detection of mineralized nodules, the cells were fixed with 10% neutral buffered formalin and stained in situ by the von Kossa technique with 3% silver nitrate as described by Otsuka et al. Ž1999.. Mineralized nodules were also stained with 1% alizarin red S solution by the method of Park and Kim Ž1984.. All figures for mineralized nodules are typical results of three separate experiments, unless otherwise specified. 4.5. Quantification of mineralized nodules Mineralized nodules were quantified by the measurement of acid-soluble calcium deposition in ECM. Cells were washed three times with calcium, magnesium-free phosphate-buffered saline wPBSŽy.x and decalcified with 0.6 M HCl for 24 h. The concentration of calcium in HCl supernatants was determined colorimetrically with a commercially available kit which contains o-cresolphthalein complexone ŽCalcium C-test Wako. as described by Jono et al. Ž2000.. The absorbance at 570 nm was determined 5 min after the addition of a color reagent with a Beckman DU 8 spectrophotometer ŽFullerton, CA, USA.. After decalcification, the cells were solubilized with 0.1 M NaOH, 0.1% sodium dodecyl sulfate ŽSDS.. The protein content was measured with a BCA protein assay kit with bovine serum albumin as a standard. 4.6. Assays of ALPase acti¨ ity and DNA content
4.3. Preparation of CM MC3T3-E1 cells Ž9 = 10 5 cells. and ROS17r2.8 cells Ž6 = 10 5 cells. were subcultured for 3 days until they reached confluence in 100 mm dishes. The confluent
The pulp cells cultured in the absence or presence of CM were scraped into cold phosphate-buffered saline, sonicated in an ice-cold bath with a sonicator cell disruptor ŽHeat systems-Ultrasonics, Plainview,
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NY, USA., and centrifuged at 1800 = g for 20 min. ALPase activity in the supernatant was determined by the method of Lowry et al. Ž1954. with p-nitrophenyl phosphate as a substrate. The amount of pnitrophenol released on 30-min incubation at 37⬚C was measured as absorbance at 405 nm. The cellular DNA content was determined by the method of Labarca and Paigen Ž1980. with Hoechst Dye 33258 and calf thymus DNA as a standard. 4.7. Northern blot analysis Total RNA was isolated by guanidinium thiocyanate᎐cesium chloride ultracentrifugation and Northern blot analysis was performed as described previously ŽKawano et al., 1992.. Denatured RNA samples Ž5 g RNA per lane. were loaded on 1% agarose gels containing 6.5% formaldehyde and transferred to Hybond-Nq nylon filters. The filters were then hybridized with the probe labeled by random priming with w ␣- 32 PxdCTP and a Random Primer DNA Labeling Kit. After autoradiography and signal quantification with a Fujix Bioimaging Analyzer BAS2000 II ŽFuji film, Tokyo, Japan., the filters were stripped and reprobed with another probe. For the analysis of DSPP, an additional lane for 0.2 g of rat incisor odontoblast poly Aq RNA was included as a positive control. The cDNA probes Ž1 ; 5 = 10 9 cpmrg. used were 0.47 kb of rat OC cDNA ŽHirakawa et al., 1994., 1.2 kb of mouse OPN cDNA ŽIslam et al., 1996., 1.0 kb of mouse ON cDNA ŽNomura et al., 1988., and 0.47 kb of rat DSPP PCR product ŽRitchie and Wang, 1996., and 0.9 kb of human glyceraldehyde-3-phosphate dehydrogenase ŽGAPDH. cDNA was used as a control of RNA loading. PCR amplification was conducted on a portion of the reverse transcription reaction of poly Aq RNA from rat incisor odontoblasts with 5⬘-CAAGCCCCAAGGGCTGCCGA-3⬘ Žpositions q1 to q20. as 5 ⬘-p rim e r a n d 5 ⬘-C G T G T C G C T A C T G T CACTGCTG-3⬘ Žpositions q474 to q453. as 3⬘primer to generate a 474-bp PCR product for DSPP cDNA ŽRitchie and Wang, 1996.. 4.8. Statistical analysis Unless otherwise specified, results were compared by Student’s t-test and a P value of less than 0.05 was regarded as significant.
Acknowledgements The authors would like to thank Dr Keiko Miyoshi for her helpful comments and assistance in performing the experiments described in this report.
References Al-Hadi Hamasha, A., Darwazeh, A., 1998. Prevalence of pulp stones in Jordanian adults. Oral Surg. Oral Med. Oal Pathol. Oral Radiol. Endod. 86, 730᎐732. Amarnani, S., Merriman, H.L., Linkhart, T.A., Baylink, D.J., Mohan, S., 1993. Autocrine regulators of MC3T3-E1 cell proliferation. J. Bone Miner. Res. 8, 157᎐165. Couble, M.L., Farges, J.C., Bleicher, F., Perrat-Mabillon, B., Boudeulle, M., Magloire, H., 2000. Odontoblast differentiation of human dental pulp cells in explant cultures. Calcif. Tissue Int. 66, 129᎐138. Denhardt, D.T., Guo, X., 1993. Osteopontin: a protein with diverse functions. FASEB J. 7, 1475᎐1482. Gronthos, S., Mankani, N., Brahim, J., Robey, P.G., Shi, S., 2000. Postnatal human dental pulp stem cells ŽDPSCs. in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 97, 13625᎐13630. Gundberg, C.M., Hauschka, P.V., Lian, J.B., Gallop, P.M., 1984. Osteocalcin: isolation, characterization, and detection. Methods Enzymol. 107, 516᎐544. Hayashi, Y., Imai, M., Goto, Y., Murakami, N., 1993. Pathological mineralization in a serially passaged cell line from rat pulp. J. Oral Pathol. Med. 22, 175᎐179. Hirakawa, K., Hirota, S., Ikeda, T. et al., 1994. Localization of the mRNA for bone matrix proteins during fracture healing as determined by in situ hybridization. J. Bone Miner. Res. 9, 1551᎐1557. Holan, G., 1998. Tube-like mineralization in the dental pulp of traumatized primary incisors. Endod. Dent. Traumatol. 14, 279᎐284. Islam, M.R., Ueno, A., Nishikawa, H., Nagata, T., Inoue, H., 1996. Osteotropic factor-stimulated synthesis of thrombospondin in rat dental pulp cells. FEBS Lett. 393, 193᎐196. Jono, S., Peinado, C., Giachelli, C.M., 2000. Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification. J. Biol. Chem. 275, 20197᎐20203. Kasugai, S., Adachi, M., Ogura, H., 1988. Establishment and characterization of a clonal cell line ŽRPC-C2A. from dental pulp of the rat incisor. Arch. Oral Biol. 33, 887᎐891. Kasugai, S., Shibata, S., Suzuki, S., Susami, T., Ogura, H., 1993. Characterization of a system of mineralized-tissue formation by rat dental pulp cells in culture. Arch. Oral Biol. 38, 769᎐777. Kawano, M., Ueno, A., Ashida, Y., Matsumoto, N., Inoue, H., 1992. Effects of sialagogues on ornithine decarboxylase induction and proto-oncogene expression in murine parotid gland. J. Dent. Res. 71, 1885᎐1890. Kawase, T., Orikasa, M., Suzuki, A., 1990. A clonal prostaglandinresponsive cell line ŽRDP4-1. derived from rat dental pulp. Bone Miner. 11, 163᎐175. Kido, J., Yamauchi, N., Ohishi, K. et al., 1997. Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro. J. Cell. Biochem. 67, 248᎐256. Kimura, G., Sugisaki, Y., Masugi, Y., Nakazawa, N., 1992. Calcification in human osteoblasts cultured in medium conditioned by the prostatic cancer cell line PC-3 and prostatic acid phosphatase. Urol. Int. 48, 25᎐30. Kusano, K., Miyaura, C., Inada, M. et al., 1998. Regulation of matrix metalloproteinases ŽMMP-2, -3, -9, and -13. by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption. Endocrinology 139, 1338᎐1345. Kubler, N., Urist, M.R., 1993. Cell differentiation in response to partially purified osteosarcoma-derived bone morphogenetic protein in vivo and in vitro. Clin. Orthop. 292, 321᎐328.
A. Ueno et al. r Matrix Biology 20 (2001) 347᎐355 Kvaal, S.I., Koppang, H.S., Solheim, T., 1994. Relationship between age and deposit of peritubular dentine. Gerodontology 11, 93᎐98. Labarca, C., Paigen, K., 1980. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102, 344᎐352. Li, J., Tsuji, K., Komori, T. et al., 1998. Smad2 overexpression enhances Smad4 gene expression and suppresses CBFA1 gene expression in osteoblastic osteosarcoma ROS17r2.8 cells and primary rat calvaria cells. J. Biol. Chem. 273, 31009᎐31015. Linde, A., 1985. The extracellular matrix of the dental pulp and dentin. J. Dent. Res. 64 ŽSpec. Iss.., 523᎐529. Lowry, O.H., Roberts, N.R., Wu, M.L., Hixon, W.S., Crawford, E.J., 1954. The quantitative histochemistry of brain. J. Biol. Chem. 207, 19᎐37. Malaval, L., Modrowski, D., Gupta, A.K., Aubin, J.E., 1994. Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J. Cell. Physiol. 158, 555᎐572. Malaval, L., Gupta, A.K., Aubin, J.E., 1995. Leukemia inhibitory factor inhibits osteogenic differentiation in rat calvaria cell cultures. Endocrinology 136, 1411᎐1418. Martinez, J., Silva, S., Santibanez, J.F., 1996. Prostate-derived soluble factors block osteoblast differentiation in culture. J. Cell. Biochem. 61, 18᎐25. McKee, M.D., Nanci, A., 1996. Osteopontin: an interfacial extracellular matrix protein in mineralized tissues. Connect. Tissue Res. 35, 197᎐205. Nagata, T., Yokota, M., Ohishi, K. et al., 1994. 1␣ , 25-dihydroxyvitamin D 3 stimulation of osteopontin expression in rat clonal dental pulp cells. Arch. Oral Biol. 39, 775᎐782. Nakashima, M., 1991. Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch. Oral Biol. 36, 655᎐663. Natsume, T., Tomita, S., Iemura, S., Kinto, N., Yamaguchi, A., Ueno, N., 1997. Interaction between soluble type I receptor for bone morphogenetic protein and bone morphogenetic protein-4. J. Biol. Chem. 272, 11535᎐11540. Nishikawa, H., Ueno, A., Nishikawa, S. et al., 2000. Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor- in rat clonal dental pulp cells. J. Endod. 26, 169᎐171. Nomura, S., Wills, A.J., Edwards, D.R., Heath, J.K., Hogan, B.L.M., 1988. Developmental expression of 2ar Žosteopontin. and SPARC Žosteonectin. RNA as revealed by in situ hybridization. J. Cell Biol. 106, 441᎐450. Ogiso, B., Hughes, F.J., Melcher, A.H., McCulloch, C.A.G., 1991. Fibroblasts inhibit mineralized bone nodule formation by rat bone marrow stromal cells in vitro. J. Cell. Physiol. 146, 442᎐450. Ohishi, M., Horibe, M., Ikedo, D. et al., 1999. Effect of retinoic acid on osteopontin expression in rat clonal dental pulp cells. J. Endod. 25, 683᎐685. Otsuka, E., Yamaguchi, A., Hirose, S., Hagiwara, H., 1999. Characterization of osteoblastic differentiation of stromal cell line ST2 that is induced by ascorbic acid. Am. J. Physiol. 277, C132᎐C138. Panagakos, F.S., O’Boskey Jr., J.F., Rodriguez, E., 1996. Regulation of pulp cell matrix metalloproteinase production by cytokines and lipopolysaccharides. J. Endod. 22, 358᎐361.
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Park, E.H., Kim, D.S., 1984. A procedure for staining cartilage and bone of whole vertebrate larvae while rendering all other tissues transparent. Stain Technol. 59, 269᎐272. Riley, E.H., Lane, J.M., Urist, M.R., Lyons, K.M., Lieberman, J.L., 1996. Bone morphogenetic protein-2: biology and applications. Clin. Orthop. 324, 39᎐46. Ritchie, H.H., Wang, L.H., 1996. Sequence determination of an extremely acidic rat dentin phosphoprotein. J. Biol. Chem. 271, 21695᎐21698. Roach, H.I., 1994. Why does bone matrix contain non-collagenous protein? The possible roles of osteocalcin, osteonectin, osteopontin and bone sialoprotein in bone metabolism and resorption. Cell Biol. Int. 18, 617᎐628. Santibanez, J.F., Silva, S., Martinez, J., 1996. Soluble factors produced by PC-3 prostate cells decrease collagen content and mineralisation rate in fetal rat osteoblasts in culture. Br. J. Cancer 74, 418᎐422. Shteyer, A., Gazit, D., Passi-Even, L. et al., 1986. Formation of calcifying matrix by osteosarcoma cells in diffusion chambers in vivo. Calcif. Tissue Int. 39, 49᎐54. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S., Kasai, S., 1983. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96, 191᎐198. Suzawa, M., Takeuchi, Y., Fukumoto, S. et al., 1999. Extracellular matrix-associated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro. Endocrinology 140, 2125᎐2133. Thrailkill, K.M., Quarles, L.D., Nagase, H., Suzuki, K., Serra, D.M., Fowlkes, J.L., 1995. Characterization of insulin-like growth factor-binding protein 5-degrading proteases produced throughout murine osteoblast differentiation. Endocrinology 136, 3527᎐3533. Tsukamoto, Y., Fukutani, S., Shin-ike, T. et al., 1992. Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts. Arch. Oral Biol. 37, 1045᎐1055. Tziafas, D., 1995. Basic mechanisms of cytodifferentiation and dentinogenesis during dental pulp repair. Int. J. Dev. Biol. 39, 281᎐290. Volek-Smith, H., Urist, M.R., 1996. Recombinant human bone morphogenetic protein ŽrhBMP. induced heterotopic bone development in vivo and in vitro. Proc. Soc. Exp. Biol. Med. 211, 265᎐272. Yamamura, T., 1985. Differentiation of pulpal cells and inductive influence of various matrices with reference to pulpal wound healing. J. Dent. Res. 64 ŽSpec. Iss.., 530᎐540. Yokose, S., Kadokura, H., Tajima, Y. et al., 2000. Establishment and characterization of a culture system for enzymatically released rat dental pulp cells. Calcif. Tissue Int. 66, 139᎐144. Yokota, M., Nagata, T., Ishida, H., Wakano, Y., 1992. Clonal dental pulp cells ŽRDP4-1, RPC- C2A. synthesize and secrete osteopontin ŽSPP1, 2ar.. Biochem. Biophys. Res. Commun. 189, 892᎐898. Zimmermann, B., Wachtel, H.C., Somogyi, H., 1990. Endochondral mineralization in cartilage organoid culture. Cell Differ. Dev. 31, 11᎐22.
MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells Akemichi Ueno a,U , Yukiko Kitase b, Keiji Moriyamab, Hideo Inoue a a b
Department of Biochemistry, School of Dentistry, The Uni¨ ersity of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Department of Orthodontics, School of Dentistry, The Uni¨ ersity of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Received 20 October 2000; received in revised form 5 April 2001; accepted 17 May 2001
Abstract Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line ŽRPC-C2A. derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium ŽCM. from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3᎐6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor ŽTGF.- antibody and a soluble form of receptor for bone morphogenetic protein ŽBMP.-2r-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factorŽs. other than TGF- or BMP-2r-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells. 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Dental pulp cells; Mineralization; Conditioned medium; MC3T3-E1 preosteoblasts
1. Introduction Dental pulp is a unique soft connective tissue surrounded by dentin. Although both tissues have a common ancestry in the mesenchymal dental papilla ŽLinde, 1985., the former is unmineralized while the latter is highly mineralized. Dental pulp cells maintain the homeostasis of dental mineralized tissues. In response to appropriate stimuli Že.g. caries, chemicals or trauma., reparative dentin is formed by defensive pulp reactions ŽYamamura, 1985; Tziafas, 1995;
Abbre¨ iations: CM, conditioned medium; ECM, extracellular matrix; ALPase, alkaline phosphatase; TGF-, transforming growth factor-beta; BMP, bone morphogenetic protein; DSPP, dentin sialophosphoprotein; OC, osteocalcin; OPN, osteopontin; ON, osteonectin; TSP, thrombospondin U Corresponding author. Tel.: q81-88-633-7326; fax: q81-88633-7326. E-mail address: [email protected] ŽA. Ueno..
Holan, 1998.. With advancing age, the size of the dental pulp cavity is reduced as a result of peritubular dentin deposition ŽKvaal et al., 1994., whereas the incidence of pulp stones is not age-dependent ŽAlHadi Hamasha and Darwazeh, 1998.. The molecular mechanisms of these responses remain to be defined. To study these processes, it is useful to establish an in vitro mineralization system by a clonal cell line derived from dental pulp. Among several reports that showed mineralization by dental pulp-derived cells, Hayashi et al. Ž1993. observed mineralized tissues within multilayers of clonal RPC-K cells after a long period of culture Ž28 days.. They observed a vesicular structure around degenerate and necrotic cells. Some of these vesicles contained needle-like crystals. Other studies were done with non-clonal primary culture cells isolated from an enzymatic digest of dental pulp ŽNakashima, 1991; Kasugai et al., 1993; Yokose et al., 2000. or cells
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 1 . 0 0 1 4 1 - X
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outgrowing from cultured pulp explants ŽTsukamoto et al., 1992; Couble et al., 2000.. These primary cells are heterogeneous and are limited for obtaining a sufficient cell population. In these studies with RPC-K cells and primary cells, mineralized tissue formation has been observed only after a long period of culture and no further analyses have been performed. Extracellular matrix ŽECM. components in dental pulp consist of collagens and non-collagenous proteins as in other connective tissues. Non-collagenous proteins including osteocalcin ŽOC., osteopontin ŽOPN., osteonectin ŽON. and thrombospondins ŽTSPs., and glycosaminoglycans ŽGAGs. are also thought to be involved in the regulation of biomineralization ŽLinde, 1985.. Our previous studies showed that a clonal cell line, RPC-C2A established from rat incisor dental pulp by Kasugai et al. Ž1988., synthesizes chondroitin 4-sulfate and dermatan sulfate, but not chondroitin 6-sulfate among GAGs ŽNishikawa et al., 2000. in a similar composition with that of dentin, and that osteogenic transforming growth factor 1 ŽTGF-1. stimulates not only the incorporation of w 35 Sx sulfate into GAG but also the synthesis of ECM proteins including OPN and TSP1 ŽNishikawa et al., 2000; Islam et al., 1996.. Although this cell line exhibits high alkaline phosphatase ŽALPase. activity, no mineralized tissues in the matrix of RPC-C2A were observed ŽKasugai et al., 1988., even in the presence of TGF-1 ŽNishikawa et al., unpublished observation.. On the other hand, the mouse calvaria-derived osteogenic cell line MC3T3-E1 forms mineralized nodules in vitro ŽSudo et al., 1983.. This cell line secretes a number of cytokines including insulin-like growth factor ŽIGF. type I, TGF-, IGF-II, bone morphogenetic protein Ž BMP . -2 and BMP-4 ŽAmarnani et al., 1993; Suzawa et al., 1999.. In this study, we co-cultured RPC-C2A cells with MC3T3-E1 cells separated by a protein-permeable collagencoated membrane and established a new experimental system in which mineralized nodules were formed in the matrix of RPC-C2A cells to investigate the molecular mechanisms of supplementary mineralization of dental pulp.
by collagen-coated filters, which inhibit direct contact of the two cell lines but were permeable to cytokines, marked mineralization was observed in ECM of RPCC2A cells after 3 weeks. In contrast, co-culture with the osteoblastic osteosarcoma cell line ROS17r2.8 ŽLi et al., 1998. did not induce matrix mineralization of RPC-C2A ŽFig. 1a.. CM from MC3T3-E1 cells showed a similar stimulation of mineralized nodule formation when added to culture medium of RPC-C2A cells. This stimulation was proportional to the CM concentration with a maximum at 50% ŽFig. 1b.. Thus, 50% of CM was used in subsequent experiments. No mineralized nodule was observed with RDP4-1 cells ŽFig. 1c.. For quantification of the mineralized nodules, we measured the acid-soluble calcium content in ECM by o-cresolphthalein complexone method ŽFig. 1d,e.. Acid-soluble calcium contents in ECM of CM-treated RPC-C2A cells ŽFig. 1d. corresponded well with von Kossa-stained mineralized nodules shown in Fig. 1b. The maximal mineralized nodule formation observed with 50% CM was threefold that found with 25% CM. However, very few mineralized nodules were detected in 50% CM-treated RDP4-1 cells, another line of clonal pulp cells ŽFig. 1e.. Next, we compared the effects of CMs collected after different culture periods. Of the CMs collected at preconfluence Žy3᎐0 days. and the first CM Ž0᎐3 days., ᎏ eighth CM Ž19᎐21 days., the first CM Ž0᎐3 days., second Ž3᎐6 days. and third Ž6᎐9 days. were effective but others were ineffective ŽFig. 2a.. Because the second CM Ž3᎐6 days. was most effective, it was used in subsequent experiments, unless otherwise specified. Similar experiments were carried out with CM from ROS17r2.8 cells. However, the formation of mineralized nodules by RPC᎐C2A cells was not induced irrespective of the collection time of CM ŽFig. 2a. or percentage of its addition Ždata not shown.. The active factorŽs. in the CM was heat-labile because CM lost its activity completely on heating at 56⬚C for 30 min or 85⬚C for 10 min ŽFig. 2b.. 2.2. Effects of CM from MC3T3-E1 cells on ALPase acti¨ ity and mRNA le¨ els of non-collagenous ECM proteins in RPC-C2A cells
2. Results 2.1. Mineralized nodule formation by dental pulp cells The confluent RPC-C2A cells did not form mineralized nodules even on culture in medium C for more than 60 days. On the other hand, osteoblastic MC3T3-E1 cells formed mineralized nodules within 10 days after confluency Ždata not shown.. When these cells were co-cultured in compartments divided
The ALPase activity in RPC-C2A cells increased rapidly after confluency in the absence or presence of the CM. However, the activity did not increase any further after 3 days and decreased from 12 days after confluency in the absence of the CM. The addition of the CM resulted in a further increase from 3 days up to 6 days after confluency and 25᎐60% higher activities during days 6᎐15 than those in the absence of the CM ŽFig. 3a..
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Fig. 1. Effects of co-culture with MC3T3-E1 cells or ROS17r2.8 cells and CM from MC3T3-E1 cells on mineralized nodule formation by dental pulp cells. Ža. Confluent RPC-C2A cells and either MC3T3-E1 cells Ž1, 2. or ROS17r2.8 cells Ž3, 4. were co-cultured on Transwell-COL filters as described in Section 4. After culture for 21 days, pulp cell layers were stained with alizarin red S Ž1, 3. and von Kossa Ž2, 4.. Žb. and Žc.. Confluent RPC-C2A cells Žb. and RDP4-1 cells Žc. in six-well plates were cultured for 10 days in medium C containing 0% Ž1.; 25% Ž2.; 33% Ž3.; 50% Ž4.; 66% Ž5.; or 75% Ž6. of CM Ž3᎐6 days. from MC3T3-E1 cells. Mineralized nodules were examined by von Kossa staining. Žd,e.. Confluent RPC-C2A cells Žd. or RDP4-1 cells Že. were cultured in medium C in the presence of indicated concentrations of CM Ž3᎐6 days. from MC3T3-E1 cells. The acid-soluble calcium contents were measured by the o-cresolphthalein complexone method. Columns and bars show means and S.D.s, respectively, of results in three separate experiments. U Significant difference from control Ž0% CM..
The mRNA of odontoblast-specific DSPP was undetectable in RPC-C2A cells under all experimental conditions employed wFig. 3bŽi.x. The mRNA levels of OC, OPN and ON began to increase just after confluency even in the absence of CM. OC mRNA increased linearly until 12 days without CM and the level on day 12 was more than six times that on day 0. The mRNA levels of OPN and ON reached maxima 6᎐9 days after confluency in the absence of CM. Unlike ALPase activity, CM depressed the elevations of both mRNAs after day 3 and the levels during days 9᎐15 were almost equal to those on day 0. Then the mRNA levels of OPN and ON on days 6᎐15 in the
absence of CM were higher than those in the presence of CM wFig. 3bŽii.x. 2.3. Effects of cytokines of MC3T3-E1 cells and their antagonists on mineralized nodule formation by RPCC2A cells Co-culture experiments suggested that an active factorŽs. in CM could be a cytokineŽs. of MC3T3-E1 cells. To search for an active factorŽs. in CM, we examined the effects of TGF- and BMPs because these growth factors are reported to be differentiation-related cytokines of MC3T3-E1 cells ŽAmarnani
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Fig. 2. Effects of MC3T3-E1 cell CM collected after different culture periods and heat treatment of CM on formation of mineralized nodules by pulp cells. Ža.. Confluent RPC-C2A cells Ž1᎐9. or RDP4-1 cells Ž10᎐12. were cultured for 10 days in medium C containing 0% Ž1. or 50% Ž2᎐12. CM collected after different culture periods Žsee Section 4 for details.. CM Žy3᎐0 days preconfluence. Ž2.; CM Ž0᎐3 days. Ž3 and 10.; CM Ž3᎐6 days. Ž4 and 11.; CM Ž6᎐9 days. Ž5 and 12.; CM Ž9᎐12 days. Ž6.; CM Ž12᎐15 days. Ž7.; CM Ž15᎐18 days. Ž8.; CM Ž18᎐21 days. Ž9.. Žb. Confluent RPC-C2A cells in 12-well plates were cultured for 10 days in medium C containing 0% Ž1. or 50% Ž2᎐4. CM before Ž2. and after heating at 56⬚C for 30 min Ž3. or at 85⬚C for 10 min Ž4. and stained by the von Kossa technique as described in Section 4.
et al., 1993; Suzawa et al., 1999.. TGF-1 and BMPs alone or in combination did not stimulate mineralized nodule formation ŽFig. 4a. and calcium deposition ŽFig. 4c.. Antagonists against these cytokines did not depress the effectiveness of CM from MC3T3-E1 cells ŽFig. 4b.. Increases Ž50%. in cytokines and their antagonists gave similar results Ždata not shown.. Although CM-induced calcium deposition was slightly inhibited by the addition of pan-specific TGF- neutralizing antibody or a soluble form of recombinant human BMP receptor IA, it was not statistically significant ŽFig. 4c. under the experimental conditions employed.
3. Discussion Confluent RPC-C2A pulp cells show high activity of ALPase ŽKasugai et al., 1988. and high levels of mRNA of OC and OPN, but not DSPP ŽFig. 3b.. The
syntheses of ECM proteins ŽOPN, TSP1, and matrix metalloproteinases. were stimulated by a variety of hormones and cytokines such as calcitriol ŽNagata et al., 1994., phorbol ester ŽIslam et al., 1996; Yokota et al., 1992., retinoic acid ŽOhishi et al., 1999., TGF-1 ŽIslam et al., 1996., and interleukin ŽIL.-1 ŽPanagakos et al., 1996.. These responses in RPC-C2A cells were quite similar to those of preosteoblasts and osteoblasts ŽAmarnani et al., 1993; Thrailkill et al., 1995; Kusano et al., 1998.. However, mineralized nodule formation by RPC-C2A cells has not been reported to date. There are reports that mineralization of ECM in vitro is influenced by co-cultured cells or their CM. For example, CM from the human prostatic carcinoma cell line PC-3 inhibited osteoblastic cell differentiation of primary fetal rat calvaria cells ŽSantibanez et al., 1996; Martinez et al., 1996; Kido et al., 1997.. On the contrary, Kimura et al. Ž1992. reported that CM from PC-3 stimulated the calcification of a human osteoblastic cell line, Tak-10, in medium supplemented with prostatic acid phosphatase and ␣glycerophosphate. Thus, the activity of CM from PC-3 cells is still controversial. Zimmermann et al. Ž1990. reported that endochondral mineralization in cultured cartilage was enhanced in the presence of calvarial cells or their CM, but inhibited by co-culture with skin fibroblast-like cells. Similarly, rat skin fibroblasts and human ligament fibroblasts inhibited mineralized bone nodule formation by rat bone marrow stromal cells ŽOgiso et al., 1991.. These results suggest that endochondral mineralization was stimulated by osteoblastic cells but inhibited by fibroblastic cells, and that these effects were mediated by soluble factors in CM. Osteoblastic MC3T3-E1 cells show marked formation of mineralized nodules after confluency ŽSudo et al., 1983. and secrete a variety of soluble factors into the CM ŽAmarnani et al., 1993; Suzawa et al., 1999.. Therefore, we examined the effect of CM from MC3T3-E1 cells on mineralized nodule formation by RPC-C2A cells. We developed a simple and rapid experimental system by which matrix mineralization of pulp-derived clonal cells could be induced. The von Kossa-positive mineralized nodules can be recognized within 9 days from the start of RPC-C2A culture, which is a much shorter time than approximately 28 days of culture required for RPC-K cells described by Hayashi et al. Ž1993. and 30 days of culture required in the case of PC-3 CM ŽKimura et al., 1992.. The CM-treated RPC-C2A cells formed mineralized nodules with increased activity of ALPase ŽFig. 3a., a marker enzyme of osteoblasts and significant decrease of mRNA levels of OPN and ON ŽFig. 3b., which are rich in ECM of osteoblasts ŽRoach, 1994., although the roles of these proteins in osteoblasts were not
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well defined. In the mineralizing cells, the production of OPN is one of the earliest Žseveral days before the onset of mineralization., and latest, secretory activities of the osteoblast lineage ŽDenhardt and Guo, 1993; McKee and Nanci, 1996.. The reason why the latter part of the OPN mRNA level behind its earlier peak is diminished by adding CM ŽFig. 3b. remains to be determined. Similar results have been reported.
Fig. 3.
351
Malaval et al. Ž1994. demonstrated using rat bone marrow stromal cell cultures by mRNA levels, protein production andror enzymatic activity, that expressions of OC and ALPase increased concomitantly with the development of bone nodules, while OPN mRNA levels decreased and those of ON did not change significantly. In addition, they found, using rat calvaria cell cultures, that leukemia inhibitory factor ŽLIF. decreased ALPase activity and bone nodule production, but Northern blotting analysis showed early stimulation of OPN and no effect on ON ŽMalaval et al., 1995.. RPC-C2A cells expressed high mRNA of OC ŽFig. 3b., a marker protein of osteoblasts ŽGundberg et al., 1984., but an undetectable mRNA level of DSPP, a marker protein of odontoblasts ŽLinde, 1985., during CM-stimulated formation of mineralized nodules. These results suggested that the CM from MC3T3-E1 cells enhanced the differentiation of RPC-C2A cells to osteoblasts. An active factorŽs. in CM of MC3T3-E1 cells was secreted during a restricted period of culture Ž3᎐6 days after confluency. and was not detectable in replicating preosteoblasts or beyond day 9 of confluence ŽFig. 2a.. Moreover, it showed a strict cell-specificity to RPC-C2A cells ŽFig. 1.. The clonal RDP4-1 cells also exhibited high ALPase activity which increased with the duration of culture and produced abundant type I collagen as ECM ŽKawase et al., 1990., but matrix mineralization could not be achieved. Furthermore, CM from ROS17r2.8 cells ŽLi et al., 1998. did not induce matrix mineralization of RPC-C2A cells. This may be due to the fact that ROS17r2.8 cells do not form mineralized nodules by themselves on a conventional plastic culture dish ŽShteyer et al., 1986., unlike MC3T3-E1 cells. From these facts, we assumed that the active factorŽs. could be a cytokineŽs. of MC3T3-E1 cells, and examined the effects of TGF-1 and BMP-2r-4 ŽFig. 4., because these are representative osteogenic cytokines of MC3T3-E1 cells ŽAmarnani et al., 1993; Suzawa et al., 1999. and
Fig. 3. Effect of CM from MC3T3-E1 cells on ALPase activity and mRNA levels of non-collagenous ECM proteins in RPC-C2A cells. a. Confluent RPC-C2A cells were cultured in 35-mm dishes for the indicated periods in the absence Ž`. or presence Ž䢇. of 50% CM Ž3᎐6 days. from MC3T3-E1 cells. ALPase activity was assayed as described in Section 4. Values are means " S.D. for 3᎐4 separate experiments. U Significantly different from the control value Ž0% CM.. Žb. RPC-C2A cells were cultured for the indicated periods in medium C containing 0% Žy, `. or 50% Žq, 䢇. CM Ž3᎐6 days. from MC3T3-E1 cells. Total RNA extraction and Northern blot analysis were done as described in Section 4. The levels of unconfluent cells Žy3 day. are also shown. Results are shown by autoradiography Ži. and its quantitative analysis by a bioimaging analyzer Fujix BAS2000 II Žii.. Lane ²i: in Ži. shows mRNA of DSPP in 0.2 g of rat incisor odontoblast poly Aq RNA. Values in Žii. are averages of two separate experiments. kb, kilobases.
A. Ueno et al. r Matrix Biology 20 (2001) 347᎐355
352
BMP-2 induces heterotopic bone development in vivo and in vitro ŽKubler and Urist, 1993; Riley et al., 1996; Volek-Smith and Urist, 1996.. However, the addition of these factors to the culture medium ŽFig. 4a. and inhibition of these cytokines in the CM by neutralizing antibodies or soluble receptors had no significant effect ŽFig. 4b,c., indicating little participation of TGF-s or BMP-2r-4 in the CM-induced mineralization by RPC-C2A cells. The active factorŽs. is heat-labile ŽFig. 2b. and impermeable to an ultrafiltration membrane Žmol. wt. cutoff: 30 000. ŽUeno et al., unpublished observation., suggesting a proteinous moleculeŽs.. Identification of an active factorŽs. is now underway. Recently, Couble et al. Ž2000. and Gronthos et al.
Ž2000. reported that human dental pulp-derived cells can differentiate in vivo or in vitro to odontoblast-like cells which show biochemical and morphological characteristics of mature odontoblasts, an expression of DSPP mRNA and an appearance of long cellular processes, respectively. These and our results suggest the presence of progenitors of both odontoblasts and osteoblasts in dental pulp and that these cells participate in supplementary mineralization in dental pulp. Further studies, such as transplantation experiments, are essential to assess in vivo formation of mineralized ECM by CM-treated RPC-C2A cells.
4. Experimental procedures 4.1. Materials Tissue culture plasticware was obtained from Becton Dickinson Labware ŽFranklin Lakes, NJ, USA.. Transwell-COL Ž24 mm in diameter, 0.4-m pore size. membrane culture inserts were purchased from Corning-Costar ŽCambridge, MA, USA.. ␣-MEM medium was purchased from GibcoBRL Life Technologies ŽRockville MD, USA.. Eagle’s MEM was purchased from Nissui Pharmaceutical ŽTokyo, Japan.. Fetal bovine serum was purchased from Hyclone Laboratories ŽLogan, UT, USA. or JRH Biosciences ŽLenexa, KS, USA.. A BCA protein assay kit was obtained from Pierce ŽRockford, IL, USA.. Recombinant human TGF-1 was purchased from Roche Diagnostics ŽMannheim, Germany.. Recombinant human BMP-2 was provided by Yamanouchi Pharmaceutical ŽTokyo, Japan.. Recombinant human
Fig. 4.
Fig. 4. Effects of cytokines of MC3T3-E1 cells and their antagonists on mineralized nodule formation by RPC-C2A cells. Ža. Confluent RPC-C2A cells were cultured in 12-well plates for 10 days in the absence Ž1. or presence of 50% CM Ža mixture of the first to third collections. Ž2.; 10 ngrml recombinant human TGF-1 Ž3.; 50 ngrml human recombinant BMP-2 Ž4.; BMP-4 Ž5.; or TGF-1 q BMP-2 q BMP-4 Ž6. and then stained by the von Kossa method. Žb. A volume of 0.4 ml CM Ž3᎐6 days. from MC3T3-E1 cells was incubated at 37⬚C for 60 min without Ž1. or with either 8 g Ž4 l. of pan-specific TGF- neutralizing antibody Ž ␣TGF-. Ž2. or 320 ng Ž1.6 l. of a soluble form of recombinant human BMP receptor IA ŽsBMPrIA. Ž3.. The antagonist-treated CM was diluted with medium C to 50% CM and used for culture of confluent RPC-C2A cells in 12-well dishes for 10 days. Mineralized nodules in 12-well dishes were stained by the von Kossa technique as described in Section 4. Figures in Ža. and Žb. are typical results of two separate experiments. Žc. Mineralized nodules in well Nos. 1 and 6 in Ža. and well Nos. 1, 2 and 3 in Žb. were quantified by assay of acid-soluble calcium in ECM as described in Fig. 1d,e Žmean " S.D., n s 3.. Neither the differences between antagonist-treated samples Žcorresponding to b-2 and -3. and mineralized control Ždesignated as CM, b-1. nor the difference between cytokine-treated sample Ža-6. and unmineralized control Ža-1. were significant.
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BMP-4 and a pan-specific TGF- neutralizing antibody were purchased from GenzymerTechne ŽCambridge, MA, USA.. Soluble BMP receptor IA ŽNatsume et al., 1997; Otsuka et al., 1999. was kindly provided by Dr T. Hatta ŽNational Institute of Agrobiological Resources, Tsukuba, Japan.. w ␣- 32 Px dCTP Ž111 TBqrmmol. and the Hybond-Nq nylon membrane were from Amersham Pharmacia Biotech ŽBuckinghamshire, UK.. Restriction enzymes and a Random Primer DNA Labeling Kit Ver.2 were from T a k a ra S h u zo Ž K u sa tsu , Ja p a n . .  -G lycerophosphate, Hoechst No. 33258, calf thymus DNA and p-nitrophenyl phosphate were from Sigma ŽSt. Louis, MO, USA.. A RNeasy Mini Kit was from Qiagen ŽHilden, Germany.. L-Ascorbic acid, dexamethasone, silver nitrate, alizarin red S and Calcium C-test Wako were obtained from Wako Pure Chemical ŽOsaka, Japan.. 4.2. Cell culture The clonal cell lines RPC-C2A and RDP4-1, derived from incisor dental pulp of Wistar strain rats, were provided by Drs S. Kasugai ŽKasugai et al., 1988., and T. Kawase ŽKawase et al., 1990., respectively. For subculture, both lines were cultured in medium A wEagle’s MEM supplemented with 2.2 mgrml NaHCO3 , 0.292 mgrml L-glutamine, 60 grml kanamycin and 10% fetal bovine serum ŽFBS.x under a humidified atmosphere of 5% CO 2 in air at 37⬚C. MC3T3-E1 cells ŽSudo et al., 1983. and ROS17r2.8 cells ŽLi et al., 1998. were cultured in medium B Ž ␣-MEM supplemented with 0.22% NaHCO3 , 100 Urml penicillin G, 100 grml streptomycin, 10 grml gentamycin and 10% FBS.. For mineralization experiments, confluent cells were cultured in medium C Žmedium B further supplemented with 2 mM glycerophosphate, 50 grml L-ascorbic acid and 10y8 M dexamethasone.. In co-culture experiments, RPCC2A cells in medium A were seeded at a density of 14 000 cellsrcm 2 on Transwell-COL 0.45 m collagen-coated filters in six-well plates and MC3T3-E1 cells in medium B were plated at a density of 15 000 cellsrcm 2 in the bottom wells of six-well plates. When both cell lines became confluent, co-culture was started with 3 ml of medium C. In all cell cultures of this study, the medium was changed every 3 days, the medium volumes being 0.8, 1.8, 1.8, 3.5 and 8 ml for 12-well plates, six-well plates, 35-mm dishes, 60-mm dishes and 100-mm dishes, respectively.
353
cells were further cultured for a maximum of 24 days in medium C. CMs were collected every 3 days. CMs of the first and second collections were defined as CM Ž0᎐3 days. and CM Ž3᎐6 days., respectively. The CMs collected were centrifuged at 1000 = g for 20 min at 4⬚C to remove floating cells and cell debris and frozen at y80⬚C until use. For neutralization of cytokines in CMs, 0.4-ml volumes of CMs were pre-incubated at 37⬚C for 1 h with either a pan-specific TGF- neutralizing antibody or a soluble form of human recombinant BMP receptor IA ŽNatsume et al., 1997; Otsuka et al., 1999. at concentrations of 10 grml and 400 ngrml, respectively. 4.4. Staining of mineralized nodules After the dental pulp-derived cells were cultured to confluence in culture medium A in 12- or six-well plates or 35 mm dishes, the medium was replaced by medium C containing 0᎐75% CM. For the detection of mineralized nodules, the cells were fixed with 10% neutral buffered formalin and stained in situ by the von Kossa technique with 3% silver nitrate as described by Otsuka et al. Ž1999.. Mineralized nodules were also stained with 1% alizarin red S solution by the method of Park and Kim Ž1984.. All figures for mineralized nodules are typical results of three separate experiments, unless otherwise specified. 4.5. Quantification of mineralized nodules Mineralized nodules were quantified by the measurement of acid-soluble calcium deposition in ECM. Cells were washed three times with calcium, magnesium-free phosphate-buffered saline wPBSŽy.x and decalcified with 0.6 M HCl for 24 h. The concentration of calcium in HCl supernatants was determined colorimetrically with a commercially available kit which contains o-cresolphthalein complexone ŽCalcium C-test Wako. as described by Jono et al. Ž2000.. The absorbance at 570 nm was determined 5 min after the addition of a color reagent with a Beckman DU 8 spectrophotometer ŽFullerton, CA, USA.. After decalcification, the cells were solubilized with 0.1 M NaOH, 0.1% sodium dodecyl sulfate ŽSDS.. The protein content was measured with a BCA protein assay kit with bovine serum albumin as a standard. 4.6. Assays of ALPase acti¨ ity and DNA content
4.3. Preparation of CM MC3T3-E1 cells Ž9 = 10 5 cells. and ROS17r2.8 cells Ž6 = 10 5 cells. were subcultured for 3 days until they reached confluence in 100 mm dishes. The confluent
The pulp cells cultured in the absence or presence of CM were scraped into cold phosphate-buffered saline, sonicated in an ice-cold bath with a sonicator cell disruptor ŽHeat systems-Ultrasonics, Plainview,
354
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NY, USA., and centrifuged at 1800 = g for 20 min. ALPase activity in the supernatant was determined by the method of Lowry et al. Ž1954. with p-nitrophenyl phosphate as a substrate. The amount of pnitrophenol released on 30-min incubation at 37⬚C was measured as absorbance at 405 nm. The cellular DNA content was determined by the method of Labarca and Paigen Ž1980. with Hoechst Dye 33258 and calf thymus DNA as a standard. 4.7. Northern blot analysis Total RNA was isolated by guanidinium thiocyanate᎐cesium chloride ultracentrifugation and Northern blot analysis was performed as described previously ŽKawano et al., 1992.. Denatured RNA samples Ž5 g RNA per lane. were loaded on 1% agarose gels containing 6.5% formaldehyde and transferred to Hybond-Nq nylon filters. The filters were then hybridized with the probe labeled by random priming with w ␣- 32 PxdCTP and a Random Primer DNA Labeling Kit. After autoradiography and signal quantification with a Fujix Bioimaging Analyzer BAS2000 II ŽFuji film, Tokyo, Japan., the filters were stripped and reprobed with another probe. For the analysis of DSPP, an additional lane for 0.2 g of rat incisor odontoblast poly Aq RNA was included as a positive control. The cDNA probes Ž1 ; 5 = 10 9 cpmrg. used were 0.47 kb of rat OC cDNA ŽHirakawa et al., 1994., 1.2 kb of mouse OPN cDNA ŽIslam et al., 1996., 1.0 kb of mouse ON cDNA ŽNomura et al., 1988., and 0.47 kb of rat DSPP PCR product ŽRitchie and Wang, 1996., and 0.9 kb of human glyceraldehyde-3-phosphate dehydrogenase ŽGAPDH. cDNA was used as a control of RNA loading. PCR amplification was conducted on a portion of the reverse transcription reaction of poly Aq RNA from rat incisor odontoblasts with 5⬘-CAAGCCCCAAGGGCTGCCGA-3⬘ Žpositions q1 to q20. as 5 ⬘-p rim e r a n d 5 ⬘-C G T G T C G C T A C T G T CACTGCTG-3⬘ Žpositions q474 to q453. as 3⬘primer to generate a 474-bp PCR product for DSPP cDNA ŽRitchie and Wang, 1996.. 4.8. Statistical analysis Unless otherwise specified, results were compared by Student’s t-test and a P value of less than 0.05 was regarded as significant.
Acknowledgements The authors would like to thank Dr Keiko Miyoshi for her helpful comments and assistance in performing the experiments described in this report.
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