MMPs and TIMPs Expression Levels in the Periodontal Ligament during Orthodontic Tooth Movement: A Systematic Review of In Vitro and In Vivo Studies

Background: During orthodontic tooth movement (OTM), applied orthodontic forces cause an extensive remodeling of the extracellular matrix (ECM) in the periodontal ligament (PDL). This is mainly orchestrated by different types of matrix metalloproteinases (MMPs) and their tissue inhibitors of matrix metalloproteinases (TIMPs), which are both secreted by periodontal ligament (PDL) fibroblasts. Multiple in vitro and in vivo studies already investigated the influence of applied orthodontic forces on the expression of MMPs and TIMPs. The aim of this systematic review was to explore the expression levels of MMPs and TIMPs during OTM and the influence of specific orthodontic force-related parameters. Methods: Electronic article search was performed on PubMed and Web of Science until 31 January 2021. Screenings of titles, abstracts and full texts were performed according to PRISMA, whereas eligibility criteria were defined for in vitro and in vivo studies, respectively, according to the PICO schema. Risk of bias assessment for in vitro studies was verified by specific methodological and reporting criteria. For in vivo studies, risk of bias assessment was adapted from the Joanna Briggs Institute Critical Appraisal Checklist for analytical cross-sectional study. Results: Electronic article search identified 3266 records, from which 28 in vitro and 12 in vivo studies were included. The studies showed that orthodontic forces mainly caused increased MMPs and TIMPs expression levels, whereas the exact effect may depend on various intervention and sample parameters and subject characteristics. Conclusion: This systematic review revealed that orthodontic forces induce a significant effect on MMPs and TIMPs in the PDL. This connection may contribute to the controlled depletion and formation of the PDLs’ ECM at the compression and tension site, respectively, and finally to the highly regulated OTM.


Introduction
The periodontal ligament (PDL) is a highly specialized connective tissue, including a heterogenous cell population (PDL cells) [1] and a fibrous extracellular matrix (ECM) [2]. The PDL is essential in sensing and transmitting mechanical forces from the teeth to the alveolar bone, such as during orthodontic tooth movement (OTM) [3]. In addition to bone remodeling, orthodontic tension as well as compression forces cause a continuous re-organization of the PDL's ECM. This ECM remodeling is mainly achieved by PDL cells [3][4][5][6][7], which contribute to ECM deposition by secreting matrix proteins [8,9], but the remodeling is also related to ECM protein degradation by the expression of various proteolytic enzymes, such as matrix-metalloproteinases (MMPs) [3,5,10].
MMPs are a growing family of calcium-dependent and zinc-dependent endopeptidases, encompassing at least 23 different types which are expressed in human tissues. The various MMP types are divided into six main groups based on the arrangement of their structural domains and their substrate preferences: collagenases (MMP-1, -8, -13 and -18), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), matrilysins (MMP-7 and -26), membrane-type MMPs  and others . In addition, several other MMP types exist, which are distinguished from typical MMPs by their unique structural features. All these MMPs are released as inactive zymogen (pro-MMPs), which have to be proteolytically processed for activation [11]. For sustaining homeostatic ECM conditions, MMPs are regulated via their expression, the processing of their zymogens to active MMPs and via endogenous tissue inhibitors of matrix metalloproteinases (TIMPs) [11], which are also expressed by PDL cells [12][13][14][15]. This family of MMPs inhibitors comprises four different types (TIMP-1, -2, -3 and -4), which bind MMPs unspecifically in a 1:1 stoichiometry. Alterations on the MMPs or TIMPs levels change the MMP/TIMP ratio, resulting in a particular net MMP activity [11].
In the last two decades multiple human studies have investigated the MMPs and TIMPs expression levels during OTM in vitro [3,5,9, and in vivo [10,[37][38][39][40][41][42][43][44][45][46][47]. All these experimental studies investigated expression levels of single or various combinations of MMPs/TIMPs types under different in vitro (force type, magnitude, application mode and force duration) [3,5,9,48] and in vivo (type of appliance, investigated teeth, force magnitude and observation time) [10,[37][38][39][40][41][42][43][44][45][46][47] conditions. However, the differences in MMPs and TIMPs expression between tension and compression areas in the PDL and between different orthodontic appliances have never been systematically documented. Hence, the main aim of this systematic review is to review the literature on MMPs/TIMPs and OTM, with a focus on the changes in expression levels of certain MMPs or TIMPs types in the PDL and on possible associations between specific orthodontic force-related parameters (e.g., tension/compression and cyclic/static). In order to achieve this aim, PubMed and Web of Science were systematically scanned for in vitro and in vivo studies with a focus on the expression levels of MMPs and TIMPs in the human PDL during the application of orthodontic forces. For a clearer view, in vitro and in vivo studies were separately analysed with respect to the changes in MMPs and TIMPs expression levels during the application of mechanical forces or during orthodontic tooth movement, respectively. In the discussion section, results from the in vitro and in vivo part were merged and interpreted together.

Systematic Search Results
The systematic search and eligibility process, which is presented as PRISMA Flow Diagram in Figure 1, identified 28 in vitro [3,5,9, and 12 in vivo [10,[37][38][39][40][41][42][43][44][45][46][47] studies, which were included in this systematic review. Overall, 9 in vitro studies were excluded: one study due to the use of an unclear cell type [49]; one study due to the use of cells isolated from tissues other than the PDL [50]; one study due to the use of a non-human cell line [51]; four studies due to the use of 3D cell culture models [41,48,52,53]; one study which did not use force application [54]; and one study due the use of cells isolated from the gingiva in a 3D cell culture model [55]. Concerning in vivo studies, 6 articles were excluded: three studies did not use untreated healthy patients as control [56][57][58]; one study which investigated expression levels in the PDL tissue [59]; one study which used only periodontitis subjects [60]; and one study due to an unclear outcome [61]. Due to a large heterogeneity of the intervention parameters of the included studies, no quantitative meta-analysis was feasible.

Intervention Parameters
The intervention parameters of the included in vitro studies are summarised in Table 2. There were 17 studies that applied tensile mechanical strain, from which seven [5,15,17,23,28,33,34] used a static application mode and 10 [3,9,12,18,22,24,27,29,30,35] used a cyclic application mode. The frequency of the applied cyclic tensile strain varied between 0.005 and 0.5 Hertz (Hz). One study did not specify the used frequency [18]. All 17 studies applied mechanical tensile strain by stretching flexible-bottomed culture dishes and specifying the force magnitude by the percentage of elongation, which ranged from 1% to 110%. One study indicated the used force magnitude in kilopascal (20 kPa = 2 N/cm 2 ) [15]. In most cases, the duration of force application ranged between 0.25 to 48 h. Only one study used an extended period of force application (from one to 7 days) [24]. The 15 studies evaluated MMPs/TIMPs expression levels immediately after force application, whereas two studies analysed expression levels several hours after force application was stopped (0-12 h [5] and 2-48 h [3] after treatment was finished). Nine included studies used compressive mechanical load. In 8 studies, the static application mode was applied [13,14,16,[19][20][21]25,26]. Only one study investigated, in addition to the static application form, the cyclic application form, using two different frequencies (30 min or 15 min with 5 min intervals) [36]. Seven studies applied compressive forces by centrifugation [13,16,[19][20][21]26,36] and one study by compressive plates [14]. One study only stated that a "static weight application" was used [25]. The used force magnitudes were indicated as g/cm 2 , varying from two to 36.3 g/cm 2 . Three studies specified applied force magnitudes in g (141 g) [19][20][21] and one study in cN/mm 2 (2-4 cN/mm 2 ) [14]. The duration of force application ranged between 10 min to 24 h. Five studies verified MMPs/TIMPs expression levels immediately after force application was stopped [13,14,16,25,26], whereas three studies used a 24 h post-treatment incubation period [20,21,36]. One study extended this period to 72 h [19]. Two studies used a steady laminar shear flow in a static mode [31,32]. The force magnitude varied between 6 to 12 dyn/cm 2 and the force duration between 4 to 12 h. Expression level evaluation was carried out immediately after force application was completed.   Figure 2 and Supplementary Materials Tables S1 and S2 show the methodological and reporting quality of all included in vitro studies. All in vitro studies showed high methodological quality (Figure 2a and Supplementary Materials Table S2). An appropriate control group selection, complete outcome data, a controlled exposure, a valid test system and a detailed description of the treatment were found in all 28 studies [3,5,9,. No selective outcome reporting was observed in any of these studies. One study did not accurately describe the statistical analysis [20]. The "Conflicts of Interest" statement and funding source were stated in 15 studies [5,[12][13][14]17,18,20,21,24,25,28,31,32,34,62]. Sample size determination was not observed in any of the investigated studies [3,5,9,.
The overall reporting quality of included in vitro studies was sufficient (Figure 2b and Supplementary Materials Table S1). The description of the scientific background and of the objectives defined the experimental outcomes and cell maintenance conditions were sufficiently stated in all in vitro studies [3,5,9,. The justification for the used model was inaccurately described or missing in five studies [13,20,21,23,35], whereas one study contained a deficient description of the study design [18]. The ethical statement was missing in 7 studies [9,17,22,23,26,31,35]. One study did not mention statistical analysis in the material and methods section [20], whereas none of the included in vitro studies contained a description of measurement precision [3,5,9,].
MMPs/TIMPs expression levels were measured during orthodontic treatment in various intervals. The observation time points ranged from several hours to several months after orthodontic force application. One study observed MMPs/TIMPs expression levels even 12 months after bracket bonding [47]. A huge variability was observed concerning the used control groups. Four studies compared orthodontic treatment affected MMPs/TIMPs expression levels with GCF samples collected from the same teeth immediately to seven days before starting the treatment [37,42,45,47] or they used orthodontic untreated teeth as control [10,38,41,46], respectively. Grant et al. [43] and Zhang et al. [39] used both control types. For orthodontic untreated controls, studies used antagonistic teeth [10], second molars [43], same tooth from the other jaw [46] or contralateral teeth [38,39]. One study did not specify the used control teeth [41]. A non-treated control cohort with systemically healthy subjects was used in two other studies [40,44]. Figure 3 and Table 3 showed the risk of bias assessment of all included in vivo studies. The overall risk of bias assessment of the included in vivo studies was good [10,[37][38][39][40][41][42][43][44][45][46][47]. The use of standard criteria for measurement of the conditions and the outcome measurements in a valid and reliable manner were assessed with a lower bias risk for all included in vivo studies [10,[37][38][39][40][41][42][43][44][45][46][47]. Two studies did not clearly define the criteria for inclusion [40,44]. Only one study contained a sufficient description of the study subjects and the utilised settings [41]. Co-founding factors were identified in three studies [37,43,47], in which two of these studies stated strategies to deal with these co-founding factors [37,43]. Appropriate statistical analysis was not mentioned in one study [44].

Discussion
The PDL undergoes a constant physiological turnover, which is partly executed by the ECM protein degrading MMPs and their local inhibitors TIMPs [59]. Since this ECM remodelling is affected by applied orthodontic forces [3][4][5][6][7], a plurality of in vitro [3,5,9, studies have already investigated the impact of orthodontic forces on MMPs and TIMPs expression levels in PDL cells. The importance of changes in MMPs and TIMPs expression levels in the PDL during OTM was verified by various in vivo studies [10,[37][38][39][40][41][42][43][44][45][46][47] and also by several animal studies, which reported decreased OTM after the inhibition of MMPs with synthetic MMPs inhibitors [64,65]. However, all of these studies show a huge variability in their outcomes and, hence, this systematic review aimed to analyse the literature on MMPs and TIMPs during OTM and examine their potential association with specific orthodontic force-related parameters. This may result in a clearer understanding on the potential differences in MMPs and TIMPs expression levels between compression and tension areas of the PDL and between the usage of different orthodontic appliances during orthodontic treatment. It may cause an overall clearer picture on the role of the different MMPs and TIMPs types on PDL remodelling during OTM.
Most of the included in vitro studies [3,5,9, applied simulated orthodontic forces to cells isolated from the human PDL, which are known to be mechano-sensitive. These studies investigated a huge number of different MMPs and TIMPs, verifying a significant influence of mechanical forces on MMPs and TIMPs expression levels in PDL cells. Since the ECM of the PDL consist mainly of type I and III collagens [9], these in vitro studies mainly focused on the expression levels of the collagenases MMP-1 and MMP-8. Additionally, the expression of the gelatinase MMP-2 was often investigated [3,5,9,. The expression levels of these three and also of the other MMPs were increased by orthodontic forces. This increasing effect was observed by various combinations of the used treatment parameters: By using tensile or compressive forces in combination with static or cyclic application mode at high and low force magnitudes and during a broad treatment period (30 min to 7 days). However, certain studies also observed no impact [3,[20][21][22][28][29][30]34] or even a decrease [12,19,24,27,32,33] in the expression levels of specific MMPs. The heterogeneity in results may be explained by the use of different combinations of force type, application mode, force magnitude and duration. Additionally, the inconsistency in the used teeth for cell isolation in donors' gender and age may also contribute to this variable influence. In order to overcome this inter-study heterogeneity, future studies in this field should test and directly compare MMPs expression levels under different combinations of force type, application mode, force magnitude and duration. The usage of cells from the same passage and donor will reduce donor variability.
Several in vitro studies [3,15,21,22,24,27,[29][30][31]33,34] determined the expression levels of multiple MMPs in parallel. The applied mechanical forces caused mainly similar effects on the expression levels of different MMPs within one study. This indicates that mechanical forces with specific parameters regulate different MMP types in PDL cells via presumably the same methods, e.g., their expression, the processing of their zymogens to active MMPs or influencing TIMPs [11]. However, Nemoto et al. [24] observed increased MMP-1 and decreased MMP-2 expression levels. Tantilertanant et al. [3] showed no effect of applied mechanical forces on MMP-8 expression, whereas MMP-1, MMP-2 and MMP-3 expression levels were significantly increased.
Since TIMPs are important endogenous inhibitors of MMPs [11], it is not surprisingly, that mechanical forces also affect TIMPs expression levels in PDL cells, which was proven in several in vitro studies [9,[12][13][14][15]17,18,26,29,31]. Multiple studies, using mainly tensile forces, observed a significant increase in the expression levels of TIMP-1 and TIMP-2 [12,15,17,18,29,31]. Two studies revealed a decrease in TIMP-1 expression when applying compressive mechanical forces between 3 and 12 h [13,14]. In contrast, Redlich et al. [26] revealed a significant increase in TIMP-1 and TIMP-2 expression levels, however, after applying compressive mechanical forces only for 10 to 60 min. This indicates that upregulated TIMP levels in the tension area may be essential for inhibiting MMPs to stop ECM degradation, which may indirectly contribute to new ECM formation. At the compression site, a delayed decrease in TIMPs expression levels may favour MMPs' enzymatic activity and consequently ECM degradation.
The results of in vitro studies are mainly supported by the outcome of included in vivo studies. All in vivo studies [10,[37][38][39][40][41][42][43][44][45][46][47] used GCF samples from healthy human orthodontic patients to investigate the influence of orthodontic forces on the expression levels of a broad range of MMPs, including MMP-1, MMP-8 and MMP-13 collagenases. These studies showed a high variability in investigated teeth, used appliance types, used force durations and used controls. Nevertheless, the expression levels of MMPs were mainly increased by applying orthodontic forces within a broad time range after orthodontic treatment initiation at both the compression and tension areas. The increased MMPs' expression levels were partially higher at the compression zone. These results are in accordance with Garlet et al. [59], who showed significantly increased MMP-1 expression levels in the PDL tissue at both the tension and compression areas with a significant higher expression level at the compression zone. This indicates a potential higher importance of MMPs-driven ECM protein degradation at the compression site. Different appliance forms are known to cause different types of forces. In our systematic review, three studies used nickel-titanium coil springs [10,37,41], which results in static forces during OTM [66,67]. Four studies used multibracket appliances [38,44,46,47], which causeed, together with occlusal forces, a cyclic orthodontic load [17,68]. Our systematic review revealed no definitive differences in MMPs expression levels concerning the two different force application forms in vivo [10,[37][38][39][40][41][42][43][44][45][46][47]. This conclusion is also supported by in vitro studies [3,5,9,. Only one in vivo study demonstrated a significant decrease in MMP-8 expression levels. However, this was observed only after 1 week of orthodontic treatment and was followed by a significant increase in the expression level [45]. Rody et al. [46] and Shirozaki et al. [47] showed no significant influence of orthodontic treatment on MMP-8 or MMP-9 expression levels, respectively. It should be noted that, Rody et al. [46] used orthodontic treated teeth from the upper arch and untreated control teeth from the lower jaw. Since orthodontic treatment causes differences in OTM between the upper and lower jaw, this comparison may cause a bias [69].
Only two included in vivo studies investigated TIMP-1 and TIMP-2 expression levels, showing a significant increase at both the tension and compression area up to 4 weeks after beginning the orthodontic treatment [41,43]. Directly in the PDL tissue, Garlet et al. [59] showed a significant increase in TIMP-1 expression at the tension site but no changes in the compression area.
Taken together, it could be possible that mechanical forces mainly upregulate the expression of MMPs in the PDL directly at both the tension and compression site, however, with higher local MMPs concentrations at the compression site. This may be essential for the required depletion of ECM proteins at the compression site. A potential decrease in TIMPs' concentration by orthodontic compressive forces might further facilitate this process. In contrast, the necessary downregulation of MMPs activity at the tension area may occur predominantly via increased TIMPs' production and not via the inhibition of MMPs' gene expression. This may contribute to the essential formation of the PDL's ECM in the tension area.
The outcome and assumptions of this systematic review have to be handled with caution, since the included in vitro and in vivo studies have several limitations. TIMP-1 and TIMP-2 were analysed only in two in vivo studies [41,43]. Only a few in vitro and in vivo studies have discriminated between active and latent forms of MMP types [41]. All in vitro studies [3,5,9, applied orthodontic forces on cells isolated from the human PDL. However, only three studies verified the cell type [22,25,32] and only one study checked the mesenchymal stromal cell character of isolated cells [34]. All included in vivo studies [10,[37][38][39][40][41][42][43][44][45][46][47] measured MMPs and TIMPs expression levels in the GCF, which does not necessarily reflect their levels in the PDL. Only one excluded study determined expression levels directly in the PDL [59]. Hence further in vivo studies should investigate MMPs and TIMPs expression levels and activities directly in the PDL. Since applied orthodontic forces markedly differ between the upper and lower jaw [69], there is a bias by comparing the included in vivo studies that used various treated teeth in the upper and lower jaw for sample taking. One study even used tested and control teeth from the upper and lower jaw, respectively [46]. Additionally, different controls (untreated teeth from the same patient versus untreated before OTM) were used in in vivo studies. Hence, future studies should precisely describe their control groups and substantiate the choice of their control type. Due to these inconsistencies, performing a meta-analysis was not possible. Lastly, the risk of bias in in vivo studies could have been reduced by consistently identifying confounding factors and possible strategies to deal with those factors and by describing the study subjects and settings in more detail. This systematic review is further limited by the inclusion of only English written papers and the exclusion of grey literature, such as conference abstracts and dissertations.

Materials and Methods
This systematic review was conducted in accordance to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) [70]. Due to the in vitro and in vivo characteristics of included studies, this systematic review was not registered in the PROSPERO database. The whole protocol was independently conducted by two different researchers. The results were compared and discrepancies were discussed until differences were resolved.

Database Search and Screening Strategy
An electronic article search was performed in the PubMed and Web of Science databases. All articles which were indexed in PubMed and Web of Science until 31 January 2021 were included in this review. The search strategy, including only MeSH terms, was cre-ated specifically for each database. Pubmed: ("Tooth Movement" OR tooth movement* OR "Tooth Migration" OR tooth migration OR tooth drift* OR tooth displacement OR "Tooth Mobility" OR tooth mobility OR tooth mobilities OR "Orthodontics" OR orthodontic* OR Mechanical force OR orthodontic force) AND  All found studies were imported into the Mendeley reference manager (Elsevier, The Netherlands) and screened on the basis of the title and abstract. Studies that dealt with the influence of mechanical forces on MMPs and TIMPs in the PDL in vitro and in vivo were included. In a second step, full-texts of all included studies were screened for eligibility on the basis of defined inclusion and exclusion criteria. Additionally, the reference lists of all included studies were manually screened for further relevant literature, which were not recorded during the initial database search. These studies were only included when they met the eligibility criteria. All included studies were divided into in vitro and in vivo for separate qualitative examination. Due to a large heterogeneity within the included in vitro studies concerning the applied force type, mode, magnitude and duration and within included in vivo studies concerning sampling point, appliance type, duration of treatment and observation time, no quantitative meta-analysis was possible.

Eligibility Criteria
Inclusion and exclusion criteria were defined separately for in vitro and in vivo studies and followed the PICO schema. Inclusion criteria for in vitro studies were specified as follows: (P) human PDL cells in a conventional 2D cell culture; (I) in vitro static or cyclic mechanical load; (C) human PDL cells not exposed to mechanical load; (O) evaluation of expression levels and/or enzymatic activities of MMPs and/or TIMPs. In vitro studies were excluded if they fulfilled one of the following exclusion criteria: using 3D cell culture models; usage of non-human PDL cells and/or PDL cells isolated from inflamed PDL tissue (e.g., periodontitis); describing expression of MMPs and/or TIMPs ex vivo after a preceding orthodontic treatment in vivo; reviews, expert opinions, letters and papers not written in English. No limitations were set concerning used teeth for PDL cell isolation, donor age and gender, force type, mode and frequency and screening methods. Inclusion criteria for in vivo studies were defined as follows: (P) patients undergoing orthodontic treatment; (I) applying orthodontic forces to achieve OTM; (C) teeth not exposed to orthodontic forces and/or samples which were taken before orthodontic forces were applied; (O) evaluation of expression levels and/or enzymatic activities of MMPs and/or TIMPs in the GCF which surrounds teeth exposed to orthodontic forces. In vivo studies were excluded if they met one of the following exclusion criteria: animal studies; studies investigating tooth movement acceleration and/or studies which investigate additional intervention during OTM; articles focusing on diseased patients (e.g., periodontitis and obesity) with orthodontic treatments that had no untreated healthy control group; expert opinions, reviews, letters and studies not written in English. No restrictions were made concerning patient age and gender, location of GCF sampling, necessity of orthodontic treatment, appliance type and the duration of treatment.

Data Synthesis
The included studies were screened for predefined parameters which were summarised and organized in tabularized form. In each table, studies can be identified by their listed study details (first-author name and year of publication). For in vitro and in vivo studies three different table types were created, respectively, with each of them summarising specific data points.

Risk of Bias Assessment
The appraisal of quality and risk of bias was conducted separately of included in vitro and in vivo studies. The risk of bias of in vitro studies was assessed on the basis of the modified guidelines depicted from Samuel et al. [71]. Methodological and reporting qualities were evaluated by nine discrete criteria, respectively. These methodological and reporting criteria are listed in detail in Supplementary Materials Tables S1 and S2. Each individual criterion was rated with yes or no, implying a lower or a higher bias risk, respectively. The risk of bias of relative to in vivo studies was appraised by adapting the Joanna Briggs Institute Critical Appraisal Checklist for analytical cross-sectional study [72]. The quality of studies was assessed by answering seven questions with yes or no, implying a lower or a higher bias risk, respectively. All quality reporting questions are listed in detail in Table 3.

Conclusions
In conclusion, this systematic review revealed that orthodontic forces have a significant influence on MMPs and TIMPs in the PDL. It is possible that the exact effect of these mechanical forces on different MMP and TIMP types depend on various treatment parameters, such as the appliance type, the force magnitude, treatment duration, and also on tooth localization, the time point of sample collection and the compression versus tension area. Due to a very high variability in the combination of used intervention parameters, it was not possible to verify the influence of a certain combination on specific MMPs/TIMPs within this systematic review by meta-analysis. While increased MMP concentrations at the compression and tension sites are mainly caused by mechanical force-induced MMP expression, the force-induced TIMP expressions seem to be mainly responsible for downregulating MMP activities at the tension site. This mechanism may contribute to the controlled depletion and formation of the PDL's ECM at the compression and tension zone, respectively, and finally to the highly regulated OTM.