Quantitative Analysis and Localization of mRNA Transcripts of Type I Collagen Osteocalcin MMP 2 MMP 8 and MMP 13 During Bone Healing in a Rat Calvarial Experimental Defect Model.код для вставкиСкачать
THE ANATOMICAL RECORD 291:1038–1046 (2008) Quantitative Analysis and Localization of mRNA Transcripts of Type I Collagen, Osteocalcin, MMP 2, MMP 8, and MMP 13 During Bone Healing in a Rat Calvarial Experimental Defect Model TOMOKO ITAGAKI,1,2 TAKAHIRO HONMA,1,2 ICHIRO TAKAHASHI,3 SEISHI ECHIGO,1 AND YASUYUKI SASANO2* 1 Division of Oral Surgery, Tohoku University Graduate School of Dentistry, Sendai, Japan 2 Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, Sendai, Japan 3 Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Sendai, Japan ABSTRACT The study examined the expression of matrix metalloproteinases (MMPs), type I collagen and osteocalcin during bone healing in a rat calvarial experimental defect model. Twelve-week-old male Wistar rats were used. A full-thickness standardized trephine defect was made in the parietal bone, with the rat under anesthesia. RNA was extracted from tissue that ﬁlled the original bone defect on days 1 and 3 and in weeks 1, 2, 3, 5, 8, 10, 12, 18, and 24 and processed for quantitative analysis of expression of type I collagen, osteocalcin and matrix metalloproteinases (MMPs) 2, 8, and 13 by using real-time polymerase chain reaction. Alternatively, the rats were ﬁxed by perfusion through the aorta and resected calvaria were processed for in situ hybridization for these molecules. The expression of type I collagen, osteocalcin and MMPs 2 and 13 increased toward week 2 and decreased thereafter, whereas the expression of MMP 8 was the highest on day 1. The mRNA transcripts of type I collagen and osteocalcin were localized in osteoblasts and osteocytes, some of which expressed MMPs 2, 8, and 13. Osteoblasts and osteocytes may play a role in the remodeling of extracellular matrices with MMPs during healing of a defect in bone. Anat Rec, 291:1038–1046, 2008. Ó 2008 Wiley-Liss, Inc. Key words: bone healing; rat calvarium; MMP; type I collagen; osteocalcin There have been few reports on healing of bone defects, whereas numerous studies have investigated that of bone fractures (Precious and Hall, 1994; Dimitriou et al., 2005; Einhorn, 2005). It has been suggested that a bone defect larger than a critical size cannot be healed with bone, and the remaining defect is ﬁlled with ﬁbrous connective tissue, resulting in a nonunion repair (Schmitz and Hollinger, 1986; Schmitz et al., 1990). Our recent study using in situ hybridization reported that osteoblasts and osteocytes cease production of bone matrix proteins, that is, type I collagen and osteocalcin, within a limited period during repair of the critical-size Ó 2008 WILEY-LISS, INC. Grant sponsor: the Ministry of Education, Science, Sports, and Culture of Japan; Grant numbers: 15390550, 17659573 and 18390532. *Correspondence to: Yasuyuki Sasano, Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 9808575, Japan. Fax: 81-22-717-8288. E-mail: firstname.lastname@example.org Received 20 January 2008; Accepted 18 March 2008 DOI 10.1002/ar.20717 Published online in Wiley InterScience (www.interscience.wiley. com). 1039 MMP EXPRESSION DURING RAT BONE DEFECT HEALING TABLE 1. Real-time PCR primer sequences (50 ?30 ) Type I collagen Osteocalcin MMP-2 MMP-8 MMP-13 GAPDH Forward primer Reverse primer GTCGATTCACCTACAGCACG AAGCCTTCATGTCCAAGCAG CCTACACCAAGAACTTCCG CTATGGATTCCCAAGGAGT GTGTGGAGTTATGATGATGC TGAACGGGAAGCTCACTGG GTCGATTCACCTACAGCACG TCGTCACATTGGGGTTGAG GAGTGACAGGTCCCAAT GGATCTTCTTTGATTGTCG CAATGCGATTACTCCAGAT TCCACCACCCTGTTGCAGTA Fig. 1. a,b: Radiographs of the defect in weeks 2 (a) and 12 (b). Bone healing proceeds gradually and almost ceases in week 12, leaving approximately 60% of the defect unrepaired. Scale bars 5 2.2 mm. Fig. 2. Expression of type I collagen examined with real-time polymerase chain reaction. The expression of type I collagen increases toward week 2 and decreases thereafter. The expression in week 2 is signiﬁcantly higher than any time before or after (P < 0.01). The expression levels are normalized to the mean value found on day 1. calvarial bone defect (Honma et al., 2008). However, when and how these cells increase and decrease production of these proteins in the course of the healing process is not known. Bone formation during development involves extensive remodeling of extracellular matrices (ECM), which is achieved by both production and degradation of ECM proteins (Werb and Chin, 1998; Sasano et al., 2002; Ortega et al., 2003; Nakamura et al., 2005). The matrix metalloproteinases (MMPs) play a central role in the breakdown of ECM, which is essential for embryonic development, morphogenesis and tissue remodeling (Birkedal-Hansen et al., 1993; Nagase and Woessner, 1999). Product size 155 145 126 112 175 307 bp bp bp bp bp bp Accession no. Z78279 X04141 X71466 AJ0072KK M6G616 NM_017008 Fig. 3. Expression of osteocalcin examined with real-time PCR. The expression of osteocalcin increases toward week 2 and decreases thereafter. The expression in week 2 is signiﬁcantly higher than any time before or after (P < 0.01). The expression levels are normalized to the mean value found on day 1. MMPs 2, 8, and 13 have been implicated in the degradation of ECM during bone development (Gack et al., 1995; Mattot et al., 1995; Chin and Werb, 1997; Johansson et al., 1997; Sasano et al., 2002; Maruya et al., 2003). MMPs 2, 8, 9, 13, and 14 have been shown to participate in the healing of bone fracture (Yamagiwa et al., 1999; Gerstenfeld et al., 2003; Henle et al., 2005; Wang et al., 2006). In contrast, information about involvement of MMPs in healing of bone defects is not available. We hypothesized that osteoblasts and osteocytes increase and then decrease production of bone matrix proteins, such as type I collagen and osteocalcin, within a limited period during healing of critical-size bone defects and produce MMPs to remodel bone ECM. To test the hypothesis, this study was designed, ﬁrst, to examine expression of type I collagen, osteocalcin, MMP 2, MMP 8, and MMP 13 in bone healing of a critical calvarial defect in the rat, by using real-time polymerase chain reaction (PCR) quantitatively and, second, to localize their mRNA transcripts using in situ hybridization qualitatively. MATERIALS AND METHODS Animals Twelve-week-old male Wistar rats weighing 260–280 g were used. They were obtained from SLC corporation (Hamamatsu, Shizuoka, Japan) and kept under a standard light–dark schedule and standard relative humidity. Stock diet and tap water were available ad libitum. All 1040 ITAGAKI ET AL. Fig. 4. Expression of MMP 2 examined with real-time PCR. The expression of MMP 2 increases toward week 2 and decreases gradually. The expression in week 2 is signiﬁcantly higher than any time before or after (P < 0.05). The expression levels are normalized to the mean value found on day 1. Fig. 6. Expression of MMP 8 examined with real-time PCR. The expression of MMP 8 is the highest on day 1 (P < 0.05) and decreases. The expression levels are normalized to the mean value found on day 1. RNA Extraction The rats were killed by ether inhalation on days 1 and 3 and in weeks 1, 2, 3, 5, 8, 10, 12, 18, and 24. The tissue including newly formed bone that ﬁlled the 8.8-mm diameter of the original bone defect was homogenized mechanically and the total RNA was extracted with RNeasy Lipid Tissue Kits (QIAGEN, Hilden, Germany). The RNA samples were collected from ﬁve to seven rats at each time point. cDNA was synthesized by using 1.0 mg of the extracted total RNA primed with 1.5 mg of random primers (Invitrogen; Carlsbad, CA) in the presence of reverse transcriptase at 100 U/mg RNA in the reverse transcription buffer supplied by Invitrogen (Nakamura et al., 2005). Fig. 5. Expression of MMP 13 examined with real-time PCR. The expression of MMP 13 increases toward week 2 and decreases gradually. The expression in week 2 is signiﬁcantly higher than any time before or after (P < 0.05). The expression levels are normalized to the mean value found on day 1. procedures were approved by the Animal Research Committee of Tohoku University. Experimental Procedure Eighty-ﬁve rats were used. The experimental rats were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg) supplemented by ether inhalation. A skin incision was made aseptically along the temporal line bilaterally and the middle of the forehead, an incision was made on the periosteum, and the ﬂap was gently turned over to expose the calvarial bone. A fullthickness standardized trephine defect, 8.8 mm in diameter, was made in the parietal bone under continuous saline buffer irrigation. Extreme care was exercised to avoid injury to the midsagittal blood sinus and dura mater. The periosteum and skin ﬂaps were returned and sutured (Honma et al., 2008). Real-time Quantitative PCR Real-time quantitative PCR analysis was performed by using LightCycler 1.5 (Roche; Mannheim, Germany) associated with LightCycler Software Version 3.5 for type I collagen, osteocalcin, MMP2, MMP8, and MMP13, according to the manufacturer’s instructions, with LightCycler-FastStart DNA Master SYBR Green I (Roche). Relative standard curves were constructed for type I collagen, osteocalcin, MMP2, MMP8, MMP13, and an endogenous reference (GAPDH), using serial dilutions of cDNA, and these standards were included in each run. Samples of unknown concentration were quantiﬁed relative to these standard curves. Type I collagen, osteocalcin, MMP2, MMP8, and MMP13 mRNA levels were normalized to the reference gene (GAPDH) to account for cDNA loading differences. Samples were run three times. Average values were used for subsequent statistical analysis. The PCR conditions used a 10-min initial enzyme activation step followed by 40 cycles of 10 sec at 958C, 10 sec at 628C, and 7 sec at 728C for type I collagen, MMP2 and MMP8, 10 sec at 958C, 10 sec at 628C, and 6 sec at 728C for osteocalcin, 10 sec at 958C, 10 sec at 628C, and 8 sec at 728C for MMP13, and 40 cycles of 10 sec at 958C, 10 sec at 628C, and 9 sec at 728C for GAPDH (Pfafﬂ, 2001; Zaman et al., 2006). Primer sequences, the size of the expected real-time PCR prod- MMP EXPRESSION DURING RAT BONE DEFECT HEALING 1041 Fig. 7. Day 1. Hematoxylin-eosin staining (a,d) and in situ hybridization for MMP 8 (b,c,e). Periosteal cells (arrowheads) on the parietal bone surface near the defect expressed MMP8 (b). No hybridization signal is identiﬁed in the adjacent section processed with sense probes (c). Endothelial cells (arrowheads) in connective tissue that ﬁll the defect express MMP8 (d,e). Scale bars 5 50 mm in a–c; 75 mm in d; 25 mm in e. ucts and the accession number for each molecule are summarized in Table 1. through the aorta. Calvaria were resected and kept in the same ﬁxative overnight at 48C. Selected specimens were radiographed by means of a microradiography unit (Softex CMR Unit; Softex, Tokyo, Japan) with X-ray ﬁlm (FR; Fuji photo ﬁlm, Tokyo, Japan) under standardized conditions (20 kV, 5 mA, 1 min). The ﬁxed specimens were decalciﬁed in autoclaved 10% EDTA in 0.01 M phosphate buffer, pH 7.4, for 4–6 weeks at 48C. After dehydration through a graded series of ethanol solutions, the tissues were embedded in parafﬁn. Serial sections 5 mm thick were cut, and selected sections were processed for hematoxylin-eosin staining or in situ hybridization. Statistical Analysis All data are expressed as the mean 6 SD for each group. Statistical differences were analyzed using Scheffe’s test. Values of P < 0.05 were considered statistically signiﬁcant. Tissue Preparation On days 1 and 3 and weeks 1, 2, 3, 5, 8, 10, 12, 18, and 24 after the defects were made, the rats were anesthetized intraperitoneally with sodium pentobarbital and were perfusion ﬁxed. For this, a ﬁxative containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4, was perfused In Situ Hybridization The protocol used has been reported elsewhere (Nakamura et al., 2005) and is only brieﬂy described as follows. 1042 ITAGAKI ET AL. Fig. 8. a–e: Weeks 1 (b,c) and 2 (a,d,e); hematoxylin-eosin staining (a) and in situ hybridization for type I collagen (b,d) and osteocalcin (c,e). The mRNA transcripts of collagen and osteocalcin are localized in osteoblasts and osteocytes (arrowheads). O, outer, and I, inner, surfaces of the parietal bone; E, the edge of the original defect in the parietal bone. Scale bars 5 50 mm. MMP EXPRESSION DURING RAT BONE DEFECT HEALING 1043 Fig. 9. a–d: Weeks 1 (a,c) and 2 (b,d); in situ hybridization for MMP 2 (a,b), MMP 8 (c), and MMP 13 (d). Some of osteoblasts and osteocytes (arrowheads) express MMPs 2, 8, and 13. Scale bars 5 25 mm in a,c; 50 mm in b,d). The sections were deparafﬁnized and washed in PBS, pH 7.4, and then immersed in 0.2 N HCl for 20 min. After washing, the sections were incubated in proteinase K (20 mg/mL; Roche) in PBS for 30 min at 378C. The sections were then dipped in 100% ethanol and dried in air and incubated with the antisense probe or the sense control probe (1.0–4.0 mg/mL) in a hybridization mixture for 16 hr at 458C. The sections were washed and treated with RNase (Type 1A, 20 mg/ml; Sigma, St Louis, MO, USA) for 30 min at 378C. After washing, the hybridized probes were detected immunologically with the Nucleic Acid Detection Kit (Roche), counterstained with methyl green, and mounted with a mounting medium. At least two sections from each of three specimens at each stage were examined using the same probe. The intensity of hybridization signals was evaluated by observing at least three ﬁelds of every section. Real-time PCR Expression of type I collagen and osteocalcin. The expression of type I collagen increased toward week 2 and decreased thereafter (Fig. 2). The expression of osteocalcin increased, with a peak in week 2, and then decreased sharply (Fig. 3). The expression in week 2 was signiﬁcantly higher than any time before or after, for both collagen and osteocalcin. Expression of MMPs. Expression of MMP 2 increased toward week 2 and decreased gradually (Fig. 4). The expression in week 2 was signiﬁcantly higher than any time before or after. Similarly, expression of MMP 13 increased toward week 2 and declined thereafter (Fig. 5), but expression of MMP8 was the highest on day 1 and then decreased (Fig. 6). Histology and In Situ Hybridization RESULTS Radiographs Bone healing proceeded gradually and almost ceased in week 12, leaving approximately 60% of the defect unrepaired (Fig. 1). Day 1. The defect was ﬁlled with blood clots, connective tissue, and blood vessels. In situ hybridization showed that periosteal cells on the inner surface of the parietal bone facing the dura mater and the outer bone surface toward the skin of the parietal bone near the defect expressed MMP8 (Fig. 7a–c). Endothelial cells in 1044 ITAGAKI ET AL. Fig. 10. a–f: Week 12; hematoxylin-eosin staining (a) and in situ hybridization for type I collagen (b), osteocalcin (c), MMP 2 (d), MMP 8 (e), and MMP 13 (f). The bone is covered with bone lining cells (arrowheads). Only weak expression of type I collagen is seen on the bone surface, whereas expression of osteocalcin and MMPs is hardly identiﬁed in healed bone in the defect. H, healed bone; O, outer and I, inner surfaces of the healed bone in the defect. Scale bars 5 50 mm in a, 100 mm in b–f. connective tissue that ﬁlled the defect expressed MMP8 (Fig. 7d,e). Week 12. Bone healing proceeded, but the defect was not ﬁlled with bone completely. The remaining defect was ﬁlled with ﬁbrous connective tissues. The healed bone was covered with bone lining cells (Fig. 10a). Weak expression of type I collagen was seen on the bone surface (Fig. 10b), whereas expression of osteocalcin (Fig. 10c) and MMPs (Fig. 10d–f) was hardly seen in week 12. Weeks 1 and 2. The defect was ﬁlled with connective tissue, and a few inﬂammatory cells were seen. New bone formation was formed into the defect from the inner and outer surfaces of the parietal bone. Cuboidal osteoblasts lined the new bone, and osteocytes were embedded in the bone matrix (Fig. 8a). The mRNA transcripts of type I collagen (Fig. 8b,d) and osteocalcin (Fig. 8c,e) were localized in osteoblasts and osteocytes. Some of those cells expressed MMPs 2 (Fig. 9a,b), 8 (Fig. 9c), and 13 (Fig. 9d). Controls No hybridization signal was identiﬁed in the sections processed with the sense RNA probes. MMP EXPRESSION DURING RAT BONE DEFECT HEALING DISCUSSION Our previous study using in situ hybridization demonstrated that osteoblasts and osteocytes no longer express bone matrix proteins in the repairing process of rat calvarial bone defects, and then bone healing ceases within a limited period, regardless of completion of the defect repair (Honma et al., 2008). However, how long osteoblasts and osteocytes keep producing bone matrix proteins, such as type I collagen and osteocalcin during the healing process, was not known. The present study has shown that the expression of type I collagen and osteocalcin increases signiﬁcantly toward week 2, and then decreases sharply toward week 24. The activity of osteoblasts and osteocytes for producing bone matrix proteins during the defect repair may be highest around week 2 and then decline sharply. This transient increase in the production of type I collagen and osteocalcin is likely associated with the stimulation of osteoblast differentiation by the surgical procedure. Our previous study showed that the rate of bone matrix apposition per week during healing of the bone defect was highest in week 4 and decreased thereafter (Honma et al., 2008). A period of time that it takes for translation from mRNA to proteins and calciﬁcation of the bone matrix to be identiﬁed on radiographs may be involved in the differences in timing between the highest mRNA expression of the bone matrix proteins in week 2 and the highest rate of bone matrix apposition in week 4. This study demonstrated for the ﬁrst time that MMPs 2, 8, and 13 are expressed in the process of bone healing in the defect. Expression of MMP 2 increases toward week 2 and decreases gradually. Similarly, expression of MMP 13 increases toward week 2 and declines thereafter. The expressions of MMPs 2 and 13 are highest around week 2. These MMPs are expressed by ostoblasts and osteocytes, which also express type I collagen and osteocalcin. This study suggests that osteoblasts and osteocytes degrade ECM proteins during bone healing by using enzymes such as MMPs 2 and 13. Osteoblasts and osteocytes may play a role in remodeling of the ECM during bone healing by coordinating the production and degradation of ECM proteins. Similar observations have been reported during bone development (Sasano et al., 2002; Tsubota et al., 2002; Maruya et al., 2003; Nakamura et al., 2005; Sone et al., 2005) and during appositional bone formation (Hatori et al., 2004). In contrast to the results obtained for MMPs 2 and 8, the expression of MMP 8 was highest on day 1, and then decreased. Previous studies reported that MMP 8 is expressed by mononuclear phagocytes, smooth muscle cells, and endothelial cells (Herman et al., 2001). Involvement of MMP 8 in skin wound healing has been demonstrated in mice deﬁcient in MMP 8 (GutiérrezFernández et al., 2007). Our study has shown, by in situ hybridization, that endothelial cells, osteoblasts, and osteocytes express MMP 8 during bone healing. MMP 8 may participate in two distinct phases of bone healing. The early phase, around day 1, may be characterized by recruitment of connective tissue to the defect site, a process that is required for wound healing. The later phase, around week 1, may involve MMP 8 expressed by osteoblasts and osteocytes, which degrade ECM proteins, perhaps in conjunction with MMPs 2 and 13 as discussed above. 1045 It has been suggested that inﬂammation promotes osteoblast differentiation directly or indirectly (Rifas et al., 2003; Gortz et al., 2004; Shen et al., 2005). Inﬂammation caused by the surgical procedure may have initiated differentiation of osteoblasts and enhanced their production and degradation of ECM proteins. Meanwhile, the functional activity of osteoblasts may decline as the inﬂammation calms down. Further studies are required for understanding how inﬂammation may regulate differentiation and the functional activity of osteoblasts. There was little, if any, expression of MMPs and bone matrix proteins, measured by in situ hybridization, at week 12. Osteoblasts and osteocytes may cease degradation of ECM within a limited period, regardless of completion of the defect repair in the rat model, as we previously reported for production of ECM (Honma et al., 2008). Further investigation with this experimental model may help provide a greater understanding of the cellular and molecular events associated with the remodeling of the ECM during bone healing. ACKNOWLEDGMENTS The authors thank Dr. Megumi Nakamura, Mr. Masami Eguchi, and Mr. Yasuto Mikami, Division of Craniofacial Development and Regeneration, Tohoku University Graduate School of Dentistry, for their excellent advice and assistance in this study. LITERATURE CITED Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, BirkedalHansen B, Decarlo A, Engler JA. 1993. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4:197–250. Chin JR, Werb Z. 1997. Matrix metalloproteinases regulate morphogenesis, migration and remodeling of epithelium, tongue skeletal muscle and cartilage in the mandibular arch. Development 124:1519–1530. Dimitriou R, Tsiridis E, Giannoudis PV. 2005. Current concepts of molecular aspects of bone healing. Injury 36:1392–1404. Einhorn TA. 2005. The science of fracture healing. J Orthop Trauma 19(suppl):S4–S6. Gack S, vallon R, Schmidt J, Grigoriadis A, Tuckermann J, Schenkel J, Weiher H, Wagner EF, Angel P. 1995. 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