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RESEARCH REPORT |
1 Department of Orthodontics and 2 Department of Oral Pathology, School of Dentistry, Showa University, 2-1-2 Kitasenzoku, Ohta-ku, Tokyo, 145-8515, Japan;
* corresponding author, junwata2000{at}ybb.ne.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: laser microdissection • cDNA array • mastication • condylar cartilage
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, reports have documented the influence of various genes on mandibular condylar cartilage growth. These genes are known to play a role in cartilage growth regulation. Further reports have focused on the expression of specific gene products in the mandibular condylar cartilage. However, before the introduction of cDNA arrays, it was impossible to obtain a single, comprehensive profile of the changes in gene expression that occur during condylar growth (Huang et al., 2001). Since the mandibular condylar cartilage is greatly influenced by surrounding environmental changes such as mechanical loading, obtaining cartilage tissue samples that reflect the living organism was previously extremely difficult.
In previous research, a cDNA array and the laser microdissection (laser pressure cell transfer-type) of soft tissue were used to monitor the expression of various genes in the mandibular condylar cartilage of mice (Irie et al., 2001). In this study, we used a cDNA array and the laser microdissection technique to investigate the patterns of gene expression linked to functional changes in young post-natal mice from weaning to mastication.
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MATERIALS & METHODS |
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Tissue Preparation and Microdissection
The dissected tissues were embedded in an OCT compound (SAKURA, Torrance, CA, USA) and frozen in isopentane cooled in liquid nitrogen. Seven-µm-thick sections were sliced from the frozen block of tissue with the use of a cryomicrotome, affixed to slides, and stored at -40°C. These samples were then immediately fixed in 100% methanol for 3 min, after which they were returned to room temperature and stained in 1% toluidine blue. Laser Scissors Pro 300 (Cell Robotics, NE Albuquerque, NM, USA) were used for laser microdissection of the mandibular condylar cartilage (Figs. 1A
, 1B, 1C).
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cDNA Probe Labeling and Array Hybridization
Isolated total RNA was amplified with the use of an Atlas SMART Probe Amplification Kit (Clontech Laboratories Inc., Palo Alto, CA, USA), and the amplification products were labeled with [
32P]dATP, and hybridized to Atlas Mouse 1.2 Arrays (Clontech) overnight. The array membranes contained 1176 mouse cDNA fragments and were purchased from Clontech. After hybridization, the membranes were exposed to Fuji BAS-MS image plates (Fuji, Nakanuma, Japan) with an intensifying screen, at room temperature for three days.
cDNA Array Data Analysis
The array image plates were scanned by means of Storm 830 (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ, USA) and analyzed with Atlas Image 2.01 (Clontech). Using global normalization (sum method), which is based on the values of all genes, we compared gene expression levels at three intervals: 1–7 days, 7–21 days, and 21–35 days. In the image data in Fig. 3, genes showing a more than two-fold expression increase are shown in red (up-regulated genes), those showing a more than 0.5-fold expression decrease are shown in blue (down-regulated genes), and those showing an intermediate expression level are shown in green (uniformly regulated genes).
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Hematoxylin and Eosin Staining
Deparaffinized paraffin sections were stained with hematoxylin and eosin (H&E).
Immunohistochemical Staining
The diluted mouse antiproliferating cell nuclear antigen (PCNA) (Oncogene, Boston MA, USA) antibodies and EnVision + HRP Mouse (DAKO, Copenhagen, Denmark) were incubated for 1 hr at room temperature. A blocking reagent containing normal mouse serum was then added to the mixture, which was left to stand for a further 1 hr at room temperature. Deparaffinized paraffin sections were reacted with the HRP-labeled primary antibodies, with diaminobenzidine (DAB) as the substrate.
Analysis of Proliferative Activity
The percentages of cells in the total condylar cartilage area were labeled by immunostaining for PCNA.
Reverse Transcription and Real-time PCR
First-strand cDNA was synthesized from 5 µg of total RNA with the use of Super Script II RT (Gibco, Grand Island, NY, USA) and random hexamers (Gibco). Real-time quantitative PCR was performed with the use of a Gene Amp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with a QuantiTect SYBR Green PCR Kit (QIAGEN). The reaction mixture (30 µL) contained 5 µL of cDNA sample and 0.25 µM of the appropriate PCR primer. The cycle profile was as follows: a hold at 95°C for 15 min, 45 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 45 sec. After the final cycle, a dissociation protocol was performed as follows: a hold at 95°C for 15 sec, a hold at 60°C for 20 sec, and a slow ramp from 60 to 95°C for 20 min. Values were normalized against GAPDH, and all data are expressed as the mean ± SEM taken from three experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The cartilage of the seven-day-old condyle was larger but narrower than the one-day-old condyle, although its general shape was unchanged (Fig. 2B). The 21-day-old condyle was enlarged, and the condylar cartilage was clearly divided into three layers. The shape of the condyle, or the superior surface, was fatter as a result of the formation of medial and lateral poles, and a large bone marrow cavity was observable between the bony trabeculae (Fig. 2C). In the 35-day-old condyle, the cartilage was narrower than the early-stage condyles (Fig. 2D).
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Gene Expression Patterns of the Mandibular Condylar Cartilage during Condylar Growth
Comparisons of the array-based expression levels of the 1-7-, 7-21-, and 21-35-day-old groups showed growth-stage-dependent changes in expression (Fig. 3). Insulin-like growth factor-1 (IGF-I) expression levels in the mandibular condylar cartilage progressively increased in one- to 21-day-old mice, reaching a peak at 21 days and thereafter progressively decreasing (Fig. 4A). Insulin-like growth factor-2 (IGF-II) expression levels started to decrease gradually immediately after the animals’ birth (Fig. 4B). In this study, fibroblast growth factor-2 (FGF2) expression levels were relatively well maintained until animals reached 35 days of age, although there was some variation (Fig. 4C). Unlike the levels shown in the microarray analyses, transforming growth factor beta-2 (TGFbeta2) expression levels peaked at 21 days, and rapidly decreased at 35 days (Fig. 4D). In contrast to the results from this microarray analysis, Indian hedgehog (Ihh) gene expression levels also peaked at 7 days and rapidly decreased from 21 days onward (Fig. 4E). Parathyroid-hormone-related protein (PTHrP) gene expression levels were constant between 1 and 7 days old, and tended to decrease markedly from 21 days onward (Fig. 4F). The expression levels of the aggrecan gene markedly increased at 21 days (Fig. 4G). The expression levels of the osteopontin gene increased gradually over 7–35 days (Fig. 4H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, laser microdissection was used for the first time on hard tissues. Using this method, along with cDNA array and real-time PCR techniques, we were able to quantify gene expression levels in vivo accurately by selectively obtaining cells from the mandibular condylar cartilage.
Using laser microdissection and real-time PCR techniques, it was also possible for us to conduct time-dependent studies of further accurate expression levels of the following genes from among those showing changes dependent on mandibular condylar cartilage growth: the genes IGF-1, IGF-2, TGFbeta2, Indian hedgehog, aggrecan, and osteopontin, and the genes FGF2 and PTHrp, which were not spotted on the arrays used but are thought to be closely related to the above genes.
Numerous studies have been carried out on exogenous IGF-1 (Tsukazaki et al., 1994), which is closely involved in promoting the proliferation and differentiation of mandibular condylar chondrocytes (Maor et al., 1993a) and is induced by growth hormones, and on endogenous IGF-1 (Maor et al., 1993b), which is involved in growth immediately after birth. It was discovered that IGF-1 gradually increases after birth, peaks at 21 days old, and decreases gradually thereafter. This finding suggests that, compared with other growth factors, endogenous IGF-I is expressed in the mandibular condylar cartilage for relatively longer periods, and its peak coincides with the initiation of mastication (21 days old). On the other hand, the expression of IGF-II, which was reported to be more important for embryonic chondrocytes (Baker et al., 1993), decreases at a roughly constant rate after birth, suggesting that, in the mandibular condylar cartilage, IGF-II expression peaks are prenatally different from IGF-I.
Indian hedgehog is important for the proliferation of chondrocytes and their differentiation into hypertrophied chondrocytes. Indian hedgehog relationships with PTHrP and TGFbeta2 have recently been reported (Alvarez et al., 2002). This study showed that Ihh, PTHrP, and TGFbeta2 are highly expressed in relatively early post-natal stages, and then rapidly decrease from 21 days old. These results are similar to those reported by Kindlom et al.(2002) on the human growth plate, where Ihh and PTHrP expression levels were high during early puberty and decreased thereafter. These results indicate that, in the mandibular condylar cartilage, expression levels of Ihh and PTHrP are high in relatively early stages before animals are weaned, and these results indicate that, in the mandibular condylar cartilage, expression levels of Ihh and PTHrP are high in relatively early stages before animals are weaned. Interestingly, expression levels decreased with the initiation of mastication.
Like IGF-I, PTHrP, and Ihh, FGF-2 is also an important growth factor for the mandibular condylar cartilage. Although not spotted on the arrays, its expression levels were analyzed by real-time PCR and were found to be relatively stable immediately after birth. This finding suggests that although FGF-2 is regulated by other complex factors, its expression level remains constant.
FGF-2 thus plays an important role in the proliferation of mandibular condylar chondrocytes. The above findings suggest that the gene expression patterns of various growth factors involved in the proliferation of mandibular condylar chondrocytes characteristically differ depending on growth stage.
This study supports that the mandibular condyle undergoes marked morphological changes in 21-day-old mice, with the cartilage layer thinning and increasing in width. It can be speculated, therefore, that since the age of 21 days coincides with weaning and the initiation of mastication, these morphologic changes might reflect the effects of masticatory mechanical stress on the mandibular condylar cartilage. These findings indicate that although cell proliferation in the whole mandibular condylar cartilage decreases post-natally, the expression of various genes related to cell proliferation, with the exception of FGF2, does not decrease uniformly after birth, but instead exhibits a distinct pattern, changing markedly on and around the day that mastication is initiated.
Aggrecan and osteopontin, typical genes of the cartilage matrix as confirmed by the cDNA array technique, were quantitatively analyzed by real-time PCR to determine their expression levels. The expression levels of aggrecan, one of the main cartilage-composing proteoglycans, rapidly increased transiently at 21 days old from the early post-natal period levels, and decreased thereafter. In addition, type II collagen showed similar changes (data not shown). It was speculated from these results that, in the mandibular condylar cartilage, the mechanical stress stimuli of the masticatory function increase the gene expression of aggrecan and type II collagen. These results agree with those reported previously (Ragan et al., 1999), where the expression levels of aggrecan and type II collagen increased transiently soon after the in vitro application of mechanical stress, and decreased thereafter. According to the immunostaining results reported by Sugiyama et al.(2001), the gene expression levels of osteopontin, a major non-collagenous bone matrix protein involved in calcification in endochondral ossification, increased from 21 days old onward. Presumably, mechanical stress caused by the masticatory function increased the expression of the osteopontin gene, one of the mechanosensitive genes. These results support the histological findings, which showed a rapid thinning of the cartilage layer with the progression of ossification from 21 days old (the initiation of mastication) onward.
Other genes that showed changes on cDNA arrays included genes of the heat-shock protein (HSP) family, which are stress-response proteins and whose expression has been shown to increase with many kinds of stress. Recently, Sironen et al.(2002) reported on a group of genes whose expression is changed by hydrostatic pressure stimuli, including genes for heat-shock proteins showing a marked increase in expression, while Roth et al.(1984) reported that the masticatory function exerted mechanical stress, such as hydrostatic pressure, on the mandibular condyle. These observations support the postulation that the increase in heat-shock proteins, excluding HSP-60, at 21 and 35 days old resulted from mechanical stress due to hydrostatic pressure generated by the masticatory function.
In summary, this experiment monitored in vivo changes in gene expression levels in normal growing mandibular condylar cartilages before, during, and after the initiation of mastication, revealing that the expression levels of several genes change markedly with the initiation of mastication.
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ACKNOWLEDGMENTS |
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Received March 13, 2003; Last revision December 5, 2003; Accepted December 8, 2003
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REFERENCES |
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