The Matrix Metalloproteinases and their Inhibitors in Orthodontics and Periodontitis
A scientific essay in Medical Sciences
DOCTORAL THESIS defended in public on 30th of September 2009
Orthodontic tooth movement requires extensive remodelling of the tissues that surround the tooth (the ‘periodontium’), in which MMPs and TIMPs are strongly involved. MMPs degrade the extracellular matrix during remodelling, while their activity is regulated by TIMPs. They are not only thought to play a role in orthodontic tooth movement but also in negative side effects such as relapse and root resorption. For clinicians as well as researchers, it is important to gain more insight into the role of MMPs and TIMPs in tooth movement. This might promote the development of more effective orthodontic therapies. Synthetic inhibitors that reduce MMP activity, might have therapeutic value in reducing the side effects of treatment. The purpose of this research was to investigate the role of MMPs and TIMPs in periodontal remodelling, especially in orthodontic tooth movement. The effects of synthetic MMP inhibitors were also studied. These backgrounds are presented in a general introduction that precedes the chapters of this PhD thesis in which the studies have been described.
Firstly, the effect of CMTs on gelatinase production by PDL cells in two-dimensional cultures was investigated. Remarkably, some CMTs stimulated MMP-2 production at relatively low concentrations. A much stronger inhibitory effect on gelatinase activity was found at higher concentrations. At these higher concentrations, the direct inhibition of activity compensates the increased production, resulting in a net decrease of MMP activity. Nevertheless, a possible stimulatory effect of CMTs on MMP production should be considered in their clinical use.
Next, a three-dimensional culture system for PDL cells was used, which is a better representation of the physiological situation. It consists of a collagen gel in which PDL cells are seeded. This culture model reflects certain aspects of increased matrix remodelling during tooth movement. Alpha smooth muscle actin (ASMA) is thought to be involved in the contractile properties of these cells. Therefore, the expression of ASMA was also analysed beside that of MMPs. In this 3-D environment, the PDL cells also produced MMP-9 next to MMP-2. The synthetic MMP-inhibitors BB94, CMT-3, Ilomastat and doxycycline all reduced MMP activity and gel contraction, but CMT-3 and doxycycline had the strongest effect. These inhibitors also reduced MMP-2, -3, and ASMA mRNA expression. The inhibitory effect on gel contraction was primarily due to inhibition of MMP activity. In addition, ASMA expression was reduced by CMT-3 and doxycycline, which might limit the contractile activity of the fibroblasts.
In the next study, MMPs and TIMPs in samples of gingiva, PDL and GCF from healthy controls and periodontitis patients were analysed by zymography, and total MMP activity was determined. TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained much higher amounts of MMPs as reported by others. Surprisingly, in healthy gingiva and PDL some MMPs were more abundant than in diseased tissue. These specific MMPs might be involved in physiological rather than pathological remodelling processes. In all samples active MMP-2 complexes and collagenolytic fragments were found, but much more in the PDL and GCF samples from periodontitis patients than in those from healthy subjects. These forms of MMPs are generally overlooked, but their proteolytic activity might contribute considerably to periodontal destruction.
A rat model was used to study the effect of CMT-3 on orthodontic tooth movement. It was hypothesized that CMT-3 inhibits tooth movement due to its inhibitory effect on MMPs. During 14 days, three groups of rats received different dosages of CMT-3, and tooth displacement was measured. CMT-3 significantly inhibited tooth movement and also decreased the number of osteoclasts at the resorption sides. The latter phenomenon might be due to the induction of apoptosis in activated osteoclasts or reduced osteoclast migration. CMT-3 might also directly inhibit MMP-mediated degradation of the organic bone matrix.
MMPs and TIMPs in GCF from orthodontic patients were analyzed by zymography. In general, a larger amount of MMPs and TIMPs was found at both the resorption side and the apposition side compared to the control teeth. The general increase in MMPs and TIMPs around the experimental teeth probably results from increased remodelling induced by tooth movement. Especially MMP-1 seems to be involved in this increased remodelling, since partially active MMP-1 was found in GCF from both experimental sides, but not in that from control teeth. MMP-9, which is known to be involved in bone degradation, and a 48 kDa gelatinase were higher at the resorption side. The small increase in TIMP-1 at the resorption side might stimulate bone resorption, whereas the high increase at the resorption side reduces bone resorption. The analysis of MMPs and TIMPs may contribute to more predictable treatment outcomes in orthodontics.
In the last chapter of the thesis, the previous chapters are discussed. Suggestions for future research and possible applications are given.