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MMP-3 Response to Compressive Forces in vitro and in vivo
1 School of Dentistry, National Taiwan University, Taiwan; Correspondence: * corresponding author, janeyao{at}ntu.edu.tw
During orthodontic tooth movement, bone resorption occurs at the compression site. However, the mechanism underlying resorption remains unclear. Applying compressive force to human osteoblast-like cells grown in a 3D collagen gel, we examined gene induction by using microarray and RT-PCR analysis. Among 43 genes exhibiting significant changes, cyclo-oxygenase-2, ornithine decarboxylase, and matrix metalloproteinase-3 (MMP-3) were up-regulated, whereas membrane-bound interleukin-1 receptor accessory protein was down-regulated. The MMP-3 protein increases were further confirmed by Western blot. To ascertain whether MMP-3 is up-regulated in vivo by orthodontic force, we examined human bone samples at the compressive site by realigning the angulated molars. Immunohistochemical staining revealed MMP-3 distributed along the compressive site of the bony region within 3 days of compression. Since MMP-3 participates in degradation of a wide range of extracellular matrix molecules, we propose that MMP-3 plays an important role in bone resorption during orthodontic tooth movement.
Key Words: MMP • compressive force • osteoblasts • tooth movement
Mechanical stress is an important factor in regulating bone remodeling. Disuse or a prolonged period of microgravity, such as in space flight, can lead to loss of bone mass. Increased loading, as experienced by gymnasts, may increase skeletal muscle and bone mass. To simulate the mechanical/functional environment, whether during in vivo or in vitro study, bones or osteoblasts are subjected to a variety of mechanical forces, including fluid shear, uni-axial or bi-axial stretching, hydrostatic compression, or a combination of two or more of these forces (Basso and Heersche, 2002). During orthodontic tooth movement, tissue tension and compression are associated with bone apposition and resorption (Katona et al., 1995). For the compression effect, several studies with intermittent compressive force on osteoblasts showed increased calcium signaling and expression of c-fos, osteopontin, alkaline phosphatase mRNA, and stimulated release of tumor growth factor (TGF)-β (Klein-Nulend et al., 1986, 1995, 1997; Roelofsen et al., 1995). Most previous studies were performed in two-dimensional monolayer cell culture systems. In contrast, to simulate a more physiological environment, we constructed three-dimensional collagen matrices (Riikonen et al., 1995; Akhouayri et al., 1999). MG63 osteoblast-like cells were seeded and grew on the matrices before being subjected to stimuli by compressive force. The magnitude of the compressive pressure used was within the range used in clinical orthodontics. We used cDNA microarray analysis to identify genes affected by compressive forces, and then verified changes in the expressions of certain candidate genes using reverse-transcription polymerase chain-reaction (RT-PCR). The increased gene expression was further confirmed at the protein level by Western blot analysis. Therefore, in the present study, we examined whether the genes of interest obtained from a high-throughput approach could survive in the physiological setting. The change in gene expression under both in vitro and in vivo compressive loading can provide a consistent and reliable model for the study of the mechanisms of bone remodeling and orthodontic tooth movement.
Preparation of Cell-loaded Collagen Gels We prepared cell-loaded collagen gels by mixing a type I collagen solution with human osteoblast-like MG-63 cells. Collagen gels were prepared with 2.4 mg/mL PureCol collagen (Inamed Biomaterials, Fremont, CA, USA), and 2 x 106 cells. After gelation, 10% FCS was added for 1 day and then replaced with 2% FCS for 1 day.
Compression Experiments
Complementary (c)DNA Microarray
Total RNA was extracted from cells in the collagen gel by means of the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Total RNA was reverse-transcribed with the ImProm-IITM Reverse Transcription System (Promega, Madison, WI, USA). For semiquantitative (sq) analysis, reverse-transcribed cDNA was amplified with pairs of PCR primers (listed in the Appendix). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for the RT-PCR control. cDNAs amplified by 30 ~ 35 cycles of sq PCR were analyzed by gel electrophoresis as described previously (Yang et al., 2004). For real-time PCR, the mRNA expression of MMP-3 and COX-2 was assessed by TaqMan gene expression assays (Hs00233962 and Hs00153133; Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. Experimental details are given in the Appendix.
Western Immunoblot Analysis
Tissue Sample Collection and Immunohistochemistry
Comparison of Gene Expression between Compression and Control Groups To identify genes that may be affected by the compressive force, we applied a cyclic compressive force to MG63 osteoblast-like cells in a 3D collagen gel for 1 day, then analyzed the differences in overall gene transcription levels between the compressive and control groups using a microarray containing 7680 cDNA elements. We filtered the data with a threshold of at least a two-fold difference, to ensure biological significance. We found 43 genes that fit our requirement of twofold differential expression (Table  ). Globally, the number of up-regulated genes was higher than those down-regulated. Among them, 30 genes were found to be more up-regulated (the intensity of Cy5/Cy3 > 2) in the compression group, whereas 13 genes were down-regulated (the intensity of Cy5/Cy3 < 0.5).
Confirmation of Differential Expression with RT-PCR To verify the results of the differential expression of the genes of interest, we selected COX-2, MMP-3, ODC, and membrane-bound IL-1 receptor accessory protein (mIL-1RAcP) for further analysis, since they were differentially expressed to an appreciable extent between the stress (S) and control (C) groups. The levels of the respective transcripts in the RNA samples were determined by sq RT-PCR. The relative expression of COX-2 to GAPDH was 1.94-fold higher in the compression group than in the control group; ODC was 2.08-fold higher; and MMP-3 was 1.9-fold higher (Fig. 1 ). The expression of mIL-1RAcP relative to GAPDH showed a 0.58-fold decrease in the compression group, compared with the control group. Using real-time RT-PCR for COX-2 and MMP-3, we found the ratios of COX-2 to GAPDH and MMP-3 to GAPDH to be 2.27 and 2.3, respectively. This increased ratio of transcripts of COX-2 or MMP-3 in the compression group, with real-time PCR, was compatible with findings of the cDNA array and RT-PCR.
Effects of a Cyclic Compressive Force on MMP-3 Protein Expression To determine the effects of a compressive force on MMP-3 protein expression by MG-63 cells in 3D collagen gels, we performed Western blotting. Both the latent (57-kDa) and active (47-kDa) forms were detected with higher intensity in the compression group than in the control group, in both cell lysates and the conditioned medium (Fig. 2 ). The increased protein expression of secreted MMP-3 was more evident in the compressed 3D collagen gel of MG-63 osteoblast-like cells.
Increased MMP-3 during Orthodontic Movement in Compressed Tissues To verify that the increased expression of MMP-3 indeed occurs in physiologically compressed bone tissues, we recruited persons to place partial fixed orthodontic appliances for pushing the mesially impacted mandibular third molars for various time periods (Fig. 3A ). Distal covering bone, with or without compression, was retrieved and collected during removal of the impacted molars. Immunohistochemistry with an anti-MMP-3 antibody revealed that its expression was abundant where the soft tissue attached to the bone for partially impacted teeth in both the control (Fig. 3B) and experimental groups (Figs. 3C, 3D). Nevertheless, the bone edge under compressive force with direct contact with soft tissues showed strong staining for MMP-3 after 3 or 7 days of compression in the majority of experimental groups, but not in the control group (Figs. 3C, 3D). The specific staining on the bone edge was detected in two of four people in the three-day-duration group, and in three of four people in the seven-day-duration group. Although the differences did not reach a statistically significant level, positive staining on the bone edge was never seen in the control group or in samples of compression for 1 day.
In this study, we used a cDNA microarray approach to explore the expression of numerous genes regulated by compressive forces in human osteoblast-like cells. Genes of interest, either up- or down-regulated more than two-fold, were simultaneously identified and further verified by quantitative and real-time RT-PCR analyses. Despite different origins of force stimulation in many studies, we found that the COX-2 mRNA increased with compressive forces. This indicated that the compressive force indeed induced a response by bone cells experiencing mechanical stimulation. As bone cells mature, the capacity to produce prostaglandins also increases. Increased expression of COX-2 in response to a pulsating flow has been observed previously, and thereby production of prostaglandin E2 was also detected (Westbroek et al., 2000). Inhibitors of PGE2 synthesis inhibit bony synthesis, implying a requirement for PGE2 in bone repair. Periodontal ligament cells under compressive or tension force also exhibited significant increases of COX-2 mRNA and PGE2 production (Shimizu et al., 1998; Ohzeki et al., 1999; Kanzaki et al., 2002). In addition to COX-2 gene regulation by mechanical stimulation, 3 other genes—ODC, mIL-1RAcP, and MMP-3—with significant differential expression were selected, since they are related to inflammation and tissue degradation. However, other inflammation- or tissue-degradation-associated genes—for example, IL-1, IL-1 receptor, IL-2 receptor, IL-8, MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, TIMP2, and TIMP-3—appeared not to be affected by compressive stimulation (data not shown). ODC converts ornithine to putrescine, which is the initial and often rate-limiting step in polyamine biosynthesis (Klein and Carlos, 1995). Polyamines are important for cell growth and division. However, little is known about the role of ODC in mechanically induced bone remodeling. The down-regulated gene selected was mIL-1RAcP. IL-1 and its receptors are crucial for controlling immunologic and inflammatory responses. When IL-1 binds to IL-1RI, a complex is formed that then binds to IL-1RAcP, resulting in high-affinity binding, after which IL-1 cellular responses begin (Jensen and Whitehead, 2004). In other words, mIL-1RAcP plays an important role in triggering IL-1 signal transduction. IL-1 induces transcription of COX-2. Once triggered, COX-2 and PGE2 are elevated for several hours in cells stimulated with IL-1 (Dinarello, 1996). We found that the compressive force inhibited the expression of mIL-1RAcP mRNA. Potentially, this mechanism can modulate PGE2 production during mechanical stimulation. The up-regulation of MMP-3 in MG-63 cells by compressive forces was detected at both the mRNA and protein levels. MMP-3, a stromelysin, has broad activity on a variety of extracellular matrix molecules, including proteoglycans, fibronectin, gelatin, collagen types IV and IX, and laminin (Nagase et al., 1990; Lapp et al., 2003). Recent studies have shown that MMP-3 increases in loaded cartilage in vitro and in vivo (Patwari et al., 2001; Lin et al., 2004), in synovial fluid from persons after traumatic knee injury (Lohmander et al., 1999), in MG-63 cells exposed to intermittent cyclic or static hydrostatic pressures (Tasevski et al., 2005), and in synovium-derived cells embedded in 3D gels (Muroi et al., 2007). But most of those findings were either limited to the transcriptional level or in laboratory settings, while the current study extends the MMP-3 investigation to the protein level by both Western blotting in vitro and immunohistochemical staining in vivo, for further verification of its physiological significance. Moreover, this is also the first registered clinical trial for collecting human bone samples involved in orthodontic tooth movement to validate laboratory findings. Since the study focused on gene expression occurring 24 hrs after mechanical loading, the earlier responses, which may have occurred within minutes or hours, were not addressed. In summary, our results showed that a compressive force induces COX-2, MMP-3, and ODC mRNA expression in bone cells, and the increased MMP-3 protein levels reflect the gene induction. The MMP-3 up-regulation by compressive force, possibly mimicking bone metabolic changes at the compression side of orthodontic tooth movement, was further verified by the examination of bone samples collected from individuals undergoing orthodontic treatment. This up-regulation of MMP-3 provides a way to investigate the basic mechanisms of transcriptional regulation during the application of compressive forces, and possible signaling pathways are currently being characterized.
This work was funded by grants NSC95-2314-B-002-212, NTUH95-000299, and NTUH96-S585 (to CCY). The paper is based on theses submitted to the Graduate Institute of Clinical Dentistry, National Taiwan University, in partial fulfillment of the requirements for a master’s degree. Preliminary reports were presented at the 18th and 19th Annual Meetings of the Taiwan Orthodontists Association, and the 35th Annual Meeting of the American Association for Dental Research (Orlando, FL, USA, 2006).
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/7/692/DC1. Received for publication September 28, 2007. Revision received January 21, 2008. Accepted for publication February 12, 2008.
Journal of Dental Research, Vol. 87, No. 7,
692-696 (2008) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
0.5), When Compressive Forces were Applied to 3D Collagen Gels of MG-63 Osteoblast-like Cells
