Effect of orthodontic forces on levels of enzymes in gingival crevicular fluid (GCF): A systematic review

Abstract Objective: Orthodontic force application releases multiple enzymes in gingival crevicular fluid (GCF) for activation, resorption, reversal, deposition of osseous elements and extracellular matrix degradation. The current systematic review critically evaluated all existing evidence on enzymes in orthodontic tooth movement. Methods: Literature was searched with predetermined search strategy on electronic databases (PubMed, Scopus, Embase), along with hand search. Results: Initial search identified 652 studies, shortlisted to 52 studies based on PRISMA. Quality assessment further led to final inclusion of 48 studies (13 moderately and 35 highly sensitive studies). Primary outcomes are significant upregulation in GCF levels of enzymes-aspartate aminotransferase (AST), alkaline phosphatase (ALP), matrix metalloproteinases (MMPs), lactate dehydrogenase (LDH), β-glucuronidase (βG), tartrate resistant acid phosphatase (TRAP), acid phosphatase (ACP) and down regulation in cathepsin B (Cb). Site specificity is shown by ALP, TRAP, AST, LDH, MMP9 with levels at compression site increasing earlier and in higher quantities compared with tension site. ALP levels are higher at tension site only in retention. A positive correlation of LDH, ALP and AST is also observed with increasing orthodontic force magnitude. Conclusions: A strong evidence of variation in enzymes (ALP, AST, ACP TRAP, LDH, MMPs, Cb) in GCF is found in association with different magnitude, stages and sites of orthodontic force application.


INTRODUCTION
Orthodontic forces cause an initial inflammatory response followed by alterations in the vascular and neural envelope and perpetual bone and tissue remodelling accompanied by paracrine release of bioactive mediators. 1-3 During orthodontic tooth movement (OTM), host-derived enzymes are released at various stages of activation, resorption, reversal and deposition of osseous elements and degradation of the extracellular matrix. 4 Some of these enzymes have been identified in the periodontal (pdl) tissue of orthodontically moved teeth. 5 Gingival crevicular fluid (GCF) is however a better choice for assessing biomolecules or mediators as sample collection is simple, sensitive, convenient, repetitive and noninvasive. 6 Thus, the quantitative estimations of mediators in GCF reflect biochemical mechanisms associated with OTM. A systematic review (SR) by Kapoor et al 6 in 2014 studied variation in GCF level of cytokines with type and magnitude of orthodontic forces and growth status of patients. It established a positive correlation of GCF activity index IL1RA (interleukin receptor antagonist)/ IL-1β) with intensity of pain and velocity of OTM and a negative correlation with growth status of patients. Besides cytokines, numerous other mediators also alter GCF during OTM, comprehensively reviewed in SR by Alhadlaq 3 in 2015. This SR highlighted working mechanisms of multiple mediators but heterogeneity of studies precluded attainment of concrete conclusions. Hence, the present SR aims to assess only a single family of mediators, enzymes, to establish their clinical correlations on sequential release in different phases of OTM and varying magnitude of orthodontic forces.
Soluble enzymes like lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) present in cytoplasm are known to release in GCF only after cellular necrosis or hyalinization with heavy orthodontic forces. 4 Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) exhibit osteoclastic and osteoblastic activity, respectively, 1 and are identified in areas of tension (TS) or compression (CS) of teeth undergoing OTM. Heavy orthopedic forces of rapid maxillary expansion show an increase of ß-glucuronidase (ßG) lysosomal enzyme upon release from polymorphonuclear (PMN) leukocytes. 7 Rise in PMN granules in surrounding tissues after fixed orthodontic appliance activation also show increase in myeloperoxidase (MPO) 2 hours (hr) after activation, traced both in GCF and saliva. 8 The evidence on enzymes in OTM is plenty but scattered and lacks critical appraisal. Hence, the current SR is conducted to establish associations of enzymes in GCF to the site of application, magnitude and type of force, patient's growth status and the type of archwire ligation.

MATERIAL AND METHODS Protocol and registration
The protocol for SR was registered in PROSPERO (www.crd.york.ac.uk/prospero, CRD42015017496) with a predetermined search strategy (Fig 1). It comprised of MeSH terms, Boolean terminology and free text terms with the keywords "enzyme" "protease", "orthodontic tooth movement" and "gingival crevicular fluid", together with several key enzymes. This search strategy was applied to key databases PubMed, Scopus and Embase in February 2018 with no language restrictions. Additional publications were identified through reference tracking and hand search of journals (Sains Malaysiana, Orthodontic Waves, Journal of Applied Sciences, APMC). The search was performed by two reviewers, followed by a cross-check by a third reviewer, in conformity with PRISMA, as shown in Figure 2.

RESULTS
Were identified 102 articles in Pubmed, 460 in Scopus, 84 in Embase and 6 from hand search, in the initial search. Strict inclusion and exclusion criteria ( Table 2) were applied after removing duplicates, resulting in 41 relevant articles. Five studies were further excluded: three studies whose full texts were not retrieved despite contacting the authors repeatedly through mail and academic social networking sites; one was a review on MMPs, and one had sample size smaller than inclusion criteria. Additional exclusion of three studies was done: two with QAI score smaller than 13, and one with a cross-sectional study design (Fig 2).    PRISMA finally resulted in 48 publications in total, with consensus among all reviewers. The QAI of these studies indicated 13 moderately sensitive and 35 highly sensitive studies.
» Study duration: The duration of studies ranged from 8 hr to 24 weeks (wk) to the maximum of one year (y). One study each was done for 8hr, 1wk, 5month (m) and 1y duration, two studies for 6m, three for 2m, five each for 2wk and 3m, eight for 3wk, 15 for approximately 1m. One study did not specify duration -only completion of alignment.
» Observation intervals for GCF collection: Studies had GCF collection at repeated observation time points (OTP) ranging from 2 times 28 to 31 times (each day of the month). 27 Six OTPs were taken in 16 studies, closely followed by 4 OTPs in 15 studies, 9 OTPs in nine studies, 3 and 10 OTPs in two studies each, 2, 7, 8 and 31 OTPs in single study each.
» Site for GCF collection: Forty one studies specified mesial or distal or buccal site for GCF collection while seven studies mentioned the tooth but not the site for sample retrieval. The technique by Lamster et al. 55 utilizing six sites was used in four studies. 10,19,33,44,47 » Mechanics of force: Studies used continuous force both for tooth retraction (26 studies) and leveling of arches (13 studies). Retraction involved 19 studies using NiTi coil spring, two using steel ligature lacebacks, three using NiTi push coil spring, and one study each for V loop and NiTi open coil spring. Besides, nine studies used intermittent orthodontic/orthopaedic forces, employing elastomeric chain for retraction in five, Hyrax for expansion in three, and TMA spring for intrusion in one study.
» The level of force: Only 33 studies mentioned force levels for OTM. The level of forces ranged from 50g, 50-75g, 100-150g, 16N/turn, 1-1.5N, 200cN, 400g in one study each, 125g in three, 100g in six, 250g in eight and 150g in seven studies. Few studies had different treatment groups employing variable magnitudes of force. 9,11,34,35,36 Oral hygiene regimen and gingival health assessment (Table 4) Professional oral prophylaxis was done before treatment in 34 studies and at every OTP in 16 studies, but was not mentioned in 12 studies. Verbal edification for oral hygiene maintenance was done in 33 studies.
Nine studies advocated chlorhexidine mouthwash and two studies, benzydamine hydrochloride; but six studies refrained the use of any mouthwash during study period. Gingival and pdl health evaluation was done before treatment in 31 studies and at every OTP in 24 studies using "Quigley Hein Index" for visual plaque or its Turesky modification, Eastman interdental bleeding score, generalized probing depths <3 mm, radiographic evidence of pdl bone loss, gingival recession, full-mouth plaque score or full-mouth bleeding score (<20%).
GCF characteristics (Table 5) » GCF collection: GCF was collected by Periopaper (OraFlow, Plainview, New York, NY, USA) in 32 studies, micropipette in seven, filter paper in two, paper point in two and endodontic paper strip in five studies. Time of sample collection, room temperature and humidity conditions were specified in three studies each.

DISCUSSION
The findings of the current review are presented in Table 6. It depicts various enzymes released in GCF in a time-dependent manner and also establishes correlations (if any) with levels or type of force applied. In this review, we have tried to establish associations of enzyme levels to magnitude or type of force in each phase of OTM, given by Burstone 56 in his classic model or four phase time/displacement modification model. 57,58 An initial upregulation in enzymes for bone resorption and matrix degradation like TRAP, ACP or MMPs and an immediate decrease in bone formative ALP corresponded with Burstone's initial phases of OTM. Different MMPs responsible for extracellular matrix (ECM) breakdown are increased at variable times in OTM, 13,15,17,18,20,22,27,50 as early as 1hr or till completion of alignment. 50 MMP-9 increased in 4hr, peaked at 8hr using stainless steel ligatures for canine retraction in one study, while MMP9/NGAL ratio peaked in 72hr in another study. 13 MMPs also varied with different magnitudes of force as MMP-9 peaked in 4hr in a study using 100g force for canine retraction, 18 compared to another study using 150g force in which MMP3, 9 and 13 peaked in 24hr. 20 The difference in peaks of various MMPs can be explained on the basis of difference in their roles in bone turnover and remodeling with orthodontic forces. 59 MMP-9 is responsible for cleavage of denatured collagen, i.e gelatin; 60 MMP-13 dissolves native fibrillar collagen; MMP-1 is an in-terstitial collagenase hydrolyzing mainly type III collagen, 61 and MMP-3 is responsible for activation of MMPs 8 and 9. 62 Hence peaks of MMP8 and MMP9/ NGAL ratio at 14d 17 and 72hr, 13 respectively, occur subsequent to peak of MMP-3 in 1hr/24hr. 17,20 In vitro studies also support rise in MMPs in orthodontic forces, specifically MMP-1,2 mRNA and protein production in human gingival and pdl fibroblasts 63,64 and MMP-1,2, 9 in gingival tissue of dogs. 60 On the other hand, no significant change in MMP levels were seen in control teeth where no orthodontic force was applied. 17,22 This clearly supports MMPs as key mediators of remodeling in OTM.
MMPs are also shown to vary with site (tension and compression) in a time-dependent manner, as supported by in vitro models on pdl fibroblasts. 65,66 Current review showed an increase in MMP1,2 in 1-3hr on tension site (TS) of maxillary canine after activation of NiTi spring while in compression (CS), MMP1 increased at 1hr and MMP2 later, at 8hr. 22 MMP-9 also increased from 4hr to 7d on compression site in another study. 13 This upsurge in levels indicate initial collagen turnover and disintegration of ECM on both tension and compression sites in initial phases of OTM.
Contrary to the MMPs, CS showed a significant increase in GCF levels of MMP inhibitors, TIMP-1 at 4hr and TIMP-2 after 7d during retraction of canines, coinciding with lag phase where tooth movement slows down. 18,50 At TS, a significant increase in TIMP1 and 2 levels was seen at 4hr, 7d and 42d. This finding is in agreement with the results of a study by Bildt et al 67 where a continuous force with NiTi spring of 150cN was applied for retraction and an increase in MMP1 and TIMP1 was seen on pooled samples from resorption (corresponding to compression) and apposition side (tension) but no trace of TIMP2 was found. The mechanism of action of TIMP-1 stimulates release of MMP1, 68 an interstitial collagenase, associated with normal tissue remodeling or stretch of pdl fibers, hydrolysing mainly type III collagen. 64 Also, TIMP-1 increases in smaller amounts on the site of compression, while retraction due to stimulation of bone resorption but in higher amounts on tension, it decreases bone resorption. 67 A study by Garlet et al. 69 provided evidence of greater expression of TIMP-1 mRNA on TS and MMP-1 mRNA on CS and TS of experimental teeth compared with the control.
Besides MMPs, histological studies on rats provide evidence of other enzymes for bone resorption predominant in CS in early phases of OTM followed by bone deposition in TS. 70,71 In accordance, the current review also shows resorptive enzyme -ACP in initial 3-5d of tooth movement. 14 Few studies on retraction with continuous forces document an initial rise in ACP both on TS and CS with a peak in 14d 42 and 21d. 46 Initial resorption is followed by a late phase of bone deposition (7-14d) marked by an increase in bone formative ALP levels, 37,45 seen both in TS and CS of alveolar wall. Increase in ALP occurs by increasing the local concentration of phosphate ions after hydrolysis of phosphomonoester bonds, thus bone mineralisation. Highest serum ALP activity in humans has been correlated with greatest osteoblastic activity during growth spurts. 72,73 The current review has 17 studies evaluating ALP in association with type, site and magnitude of force. ALP levels increased at TS in continuous retraction forces by NiTi spring as well as in gradually increasing force from 50 cN to 150cN at 2wk, showing a predisposition towards bone deposition. 9 A study in rats supported osteoid deposition in the lacunae on TS in 80-120d. 74 The current review shows peak in ALP levels at 2wk on continuous force application of 150cN, 100g or 150g force 9,10,14,24,28,45 , with greater levels on TS compared to CS. This is followed by fall in ALP levels corresponding to hyalinised tissue removal and initiation of post lag phase. 9,24 Magnitude of force was another determinant of variation in ALP. Decrease in ALP levels seen at 1hr, 1d after intrusion by TMA spring is believed to be caused by heavy forces leading to a hyalinised zone. 25 Conversely, distalisation of molars with heavy cF of 250g 31 showing high ALP levels at both TS and CS and ALP levels greater in 150g than 100g force, 34 were attributed to extensive osteoblast recruitment on application of heavy forces. 9 One study showing decreased ALP levels on both TS and CS of canine retraction with push coil spring was probably due to combination of bodily and tipping movement, which precludes pure compression and tension areas. 38 ALP also varied with type of force: one study compared levels in Hycon ® screw with active tie-backs for retraction. A significant difference was seen at 3 and 4 wk of retraction with levels in Hycon screw group 260% higher after one half turn twice weekly activation, compared with 200% increase in active tie-back group. 49 This may be ascribed to elas-tomeric force decay to 30-40% of original force in 3 weeks. Another study on maxillary expansion by hyrax followed by retention noticed fall in ALP levels on CS and TS till four weeks of activation, followed by peak at 1yr on TS, thus indicating bone apposition during retention period. 51 Contrary to ALP, TRAP or ACP facilitates dissolution of bone minerals by forming a highly acidic extracellular environment and are potent osteoclast biomarkers expressed in areas of compression. 74 The present review supports rise in TRAP levels at CS more than TS to reach peak at 1wk, 33 2wk 11 and 4-5wk. 34,36 This is supported by histochemical study by Casa et al, 75 suggestive of appearance of mononuclear TRAP positive cells on application of forces at 2wk and multinucleated TRAP positive cells at 3 and 4wk. Even ACP activity was maximum at 3d, followed by its reversal, explained by natal release of enzymes from surface of osteoclasts. 14 A secondary outcome of faster rate of OTM with minimal lateral and apical root resorption was noticed with higher levels of TRAP in 150g, compared with 100g force. 34,36 The consummation of bone resorption occurs by resolution of organic matrix mediated by lysosomal cysteine protease cathepsin B that is increased 1d after application of 100-150g or 250g retraction force by E chain, 21,30 while levels of inhibitor cystatin decreases in 1d. 21 In association, plasminogen activator (t-PA) and its inhibitor (PAI) responsible for extravascular fibrinolysis, reach peak at 24hr only to fall later at 7d. 23 AST is another cytoplasmic enzyme released in extracellular environment after cell membrane lysis following necrosis 76 and has been evaluated in 10 studies in the current SR. Peak levels of AST were seen at 1wk, 11,40,41,54 2wk, 14,48 and 4wk. 28,39 This may be explained on the basis of increase in AST activity for 14d due to hyalinization of pdl in compression zone, decreased later upon resolution of hyalinized area by macrophages. 14 The formation of hyalinised zone and cellular necrosis may cause higher levels on CS than TS in retraction cases 39,48 and also in 150g force, compared to 100g. 11,34 But, such sporadic evidence could not be definitive for site predilection. Rather this enzyme has been associated more with destruction of gingival tissues in experimental and chronic periodontitis 77 and subgingival colonization with arch wire ligation 41 than orthodontic force application.
The current review has also monitored LDH, an enzyme released from cytoplasm to extracellular space after cell death in gingivitis or periodontitis 78 as well as in orthodontic treatment. 16,26 Variation in LDH levels were recorded with type, magnitude and direction of application of force. Continuous force of 125g with NiTi spring showed increase in levels at 7d to peak at 14d, 26 21d 19 and 28d, 48 but remained higher in CS than TS at 1.5 N, 35 thus favouring its release after cell death. Timing of increase varied with force level, with an early increase seen at 2wk in heavy force of 250g applied for molar distalisation. 26 compared with rise in 3wk in 125g force. 19,47 However no significant difference in LDH levels could be correlated to high friction between selfligating brackets and thermoelastic or superelastic Nitinol wires, as the forces produced by frictional resistance are insufficient for LDH release. 16 One study supporting greater LDH levels in teeth undergoing retraction compared with controls was excluded from this review because of its cross-sectional study design. 79 It supported LDH as a sensitive marker of the pdl metabolism changes during OTM.
Other inflammatory mediators like MPO and βG were also evaluated in this review. MPO released from PMNLs (polymorphonuclear leukocytes) is a sensitive marker for inflammation and pain associated to OTM and showed an early increase at 2hr. 8,12,50,52,53 In cases of alignment, the levels of MPO increase from baseline to 1hr to 1d till completion of alignment, correlating it with inflammation caused by NiTi wire alignment. 50 Studies on MPO also supported superelastic NiTi wires as best alignment wires, giving low continuous force and rapid tooth movement, showing higher MPO levels at 2hr, compared with heat-activated NiTi or multistranded NiTi or stainless steel wires. 52,53 Studies also mentioned increase in lysosomal enzyme, βG released from PMNLs after 14d of heavy interrupted force for mid-palatal hyrax expansion in adolescents. 7,31 However, the levels remained high till 28d in retention, probably due to elastic recoil of stretched supracrestal gingival fibers. 7,31 The risk of bias assessment in QAI though indicated all studies as moderately or highly sensitive, revealed certain strengths and weaknesses of variable study designs (Table 7). While the objectives of the studies, selection criteria and orthodontic mechanics were generally clear, they strikingly lacked sample size calculation with only one study indicating the same. 9 The authors took 5 as the sample size for inclusion, based on statistician's advice. Randomization of experimental teeth/ side / patients falling into study and control group have been clearly stated in only 21 out of 48 studies, suggesting substantial bias in all studies. The present SR deals with biomarker evaluation in GCF, hence the GCF handling characteristics have been adequate in all studies. However, the specification of time, temperature and humidity at the time of GCF collection was a major shortfall, with only four studies mentioning it. The statistical significance of the results, wherever applicable, have been stated in all the studies, but none of the studies mentioned dropouts or confounders, which might influence the results.
Despite the various shortcomings noticed in the study designs, the current evidence has generated ample evidence related to enzymes in OTM and has also opened new arena for future research in this direction.
Perhaps a most exciting area of research will involve biological basis of tooth movement with different ligation modes of brackets. Further studies could be conducted with LDH as marker for high frictional resistance in different combinations of brackets and wires, as only single study in this SR found no significant change in LDH in initial OTM with self-ligating brackets and superelastic or thermoactive archwire. Another splitmouth study correlating biomarker level with microbial colonization in different ligation modes showed a significantly greater level of AST in arch wire ligation than self-ligation, associated with greater microbial count.
An interesting correlation of MPO with pain was established with an early increase in MPO within 2hr of force application, coinciding with initial pain incidence in orthodontic patients. βG has been explored for its association with the most suitable wires for alignment and could be explored further in different types and magnitudes of forces.
Based on similarity between peri-implant fluid (PIMF) and GCF, the mediators studied in GCF could also be evaluated in PICF to assess stability of contemporary orthodontic anchorage devices, microimplants, as has been suggested by study of interleukin 1β in PIMF. 80 Despite the heterogeneity in study design and categories of enzymes studied in literature, this SR provides an essential overview of the mechanism by which en-zymes play a role in bone apposition, resorption as well as ECM degradation. The current SR also correlates mediator levels in GCF with phases of OTM at different magnitudes and types of forces and also ligation modes. It goes a step further in suggesting the potential areas of research in this field, based on individual studies designed for associations of mediator levels with ideal orthodontic force magnitudes, method of ligation and periodontal status, thus setting a direct implication in clinical practice.
4. Distinction between TS and CS could be made with levels of TRAP, AST, LDH, MMP9, being greater on CS than TS, and ALP greater on TS.
5. ALP, TRAP levels were greater in 150g force than 100g force. An early rise in AST levels was seen in 150g force at 3 and 4wk, as compared to 100g force at 4 and 5 wk.
6. Mechanical stress with continuous force of NiTi spring causes increase in MMPs 1, 3 in 24hr in CS and of ALP as early as 7d in TS.
7. No significant association between levels of MMP-9 or AST and growth status could be established as adult and adolescents, gave no significant difference in levels.