Cellular response within the periodontal ligament on application of orthodontic forces

Abstract

During application of controlled orthodontic force on teeth, remodeling of the periodontal ligament (PDL) and the alveolar bone takes place. Orthodontic forces induce a multifaceted bone remodeling response. Osteoclasts responsible for bone resorption are mainly derived from the macrophages and osteoblasts are produced by proliferations of the cells of the periodontal ligament. Orthodontic force produces local alterations in vascularity, as well as cellular and extracellular matrix reorganization, leading to the synthesis and release of various neurotransmitters, cytokines, growth factors, colony-stimulating factors, and metabolites of arachidonic acid. Although many studies have been reported in the orthodontic and related scientific literature, research is constantly being done in this field resulting in numerous current updates in the biology of tooth movement, in response to orthodontic force. Therefore, the aim of this review is to describe the mechanical and biological processes taking place at the cellular level during orthodontic tooth movement.

INTRODUCTION

The application of orthodontic force results in tooth movement within the alveolar bone. This is due to the fact that any alteration in the biological system as a result of mechanical loading results in strain within the biological system. This in turn, leads to release of various neurotransmitters resulting in remodeling and adaptation of the biological system to the newer environment. As a result of this principle, remodeling of the periodontal ligament (PDL) and the alveolar bone around a tooth takes place during orthodontic force application. Research is constantly being done and updated regularly on the biology of orthodontic tooth movement and the tissue level response within the cellular level on application of orthodontic force. Orthodontic tooth movement may broadly be divided into three phases: The initial phase, the lag phase, and the post-lag phase.[1] During the initial phase there is immediate and rapid tooth movement occurring within 24 to 48 hrs after force application to the tooth. This is mainly attributed to the displacement of the tooth in the PDL space. The lag phase lasts approximately 20 to 30 days, showing relatively little to no tooth displacement. This phase is marked by PDL hyalinization in the region of compression. No subsequent tooth movement occurs until the cells complete the removal of all of the necrotic tissues. The post-lag phase follows the lag phase, during which the rate of movement again increases.

INFLAMMATORY MEDIATORS AND MARKERS OF ALVEOLAR BONE REMODELLING IN THE PERIODONTUM

Prostaglandins

Prostaglandins (PG’- s) are local hormone-like chemical agents produced by mammalian cells, including osteoblasts and are derivatives of arachidonic acid. They are synthesized immediately in response to tissue injury.[2,3] PGs exist in two different iso-forms: The constitutive isoform or cyclooxygenase-1 (COX-1) and the inducible isoform or cyclooxygenase-2 (COX-2). PGs are produced in large amounts during inflammatory processes occurring due to cell injury.[4] Application of orthodontic force results in stress concentration in the extracellular matrix and the deformation of the osteocytes of alveolar bone. This deformation, in turn opens the hemi-channels in the strained osteocytes allowing the release of prostaglandins, which are responsible for orthodontic tooth movement.[5]

It has been proved that COX is also closely associated with periodontitis and that PGs are mediators of gingival inflammation and alveolar bone resorption.[6,7] In addition, numerous studies have confirmed PGE2 levels in periodontal tissues and gingival crevicular fluid are highly correlated with periodontal tissue destruction.[8] COX-2 has been proved to play an important role in gingival inflammation and alveolar bone loss during the progress of periodontitis.[9] In vitro studies have proved that the expression and production of PGE2 is promoted by mechanical stimulation of the periodontal ligament.[10,11] Prostaglandins of the E series also play an important role in the pathogenesis of chronic periodontitis by regulating production of osteoclast activating factor in activated lymphocytes.[12] Application of orthodontic force results in physical distortion of PDL and alveolar bone cells. They can also trigger a multilevel cascade of signal transduction pathways, including the prostaglandin E2 (PGE2) pathway, which in turn initiates structural and functional changes in extracellular, cell membrane, and cyto-skeletal proteins.[13] Subsequent changes in cyto-skeletal protein structure and function lead to the creation of new cells and bone matrix formation.[14] PGE2 is one of the earliest biomarker for bone resorption, which can be used for monitoring orthodontic tooth movement (OTM).[13,14]

Cytokines and chemokines

The principal trigger factor responsible for orthodontic tooth movement (OTM) is the strain experienced by the PDL cells and the extracellular matrix. This strain results in alteration in the gene expression within the cells and the extracellular matrix. This in turn results in expression of various cytokines and chemokines. The cytokines and chemokines regulate alveolar bone remodeling in response to mechanical loading. Orthodontic force causes capillary vasodilatation within the blood vessels periodontal ligament, resulting in migration of inflammatory cells and cytokine production. This helps in the process of bone remodeling.[15] These cytokines are actually proteins, acting as signals between the cells of the immune system, produced during the activation of immune cells. They usually act locally, although some might act systemically with overlapping functions. Cytokines like IL-1, IL-6, IL-8 and TNF-α have been proved to be associated with bone remodeling.[15]

On application of orthodontic force, the compression region within the PDL shows increased osteoclastic activity, whereas in the tension region, there is proliferation of osteoblasts and mineralization of the extracellular matrix.[16] The Osteoclastic cells involved in bone resorption are multinucleated giant cells originating from hematopoietic stem cells.[17] Interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and other inflammatory cytokines facilitate osteoclastic bone resorption processes and have the potential to serve as one of the earliest biomarkers for monitoring and validating orthodontic tooth movement.[18]

These proteins regulate osteoclastic activity through the activation of the nuclear factor kappa B (RANK) and of the nuclear factor kappa B ligand (RANKL). CC chemokines Ligand 2 (CCL2) has been found to be involved in osteoclast activity and its expression is increased within the PDL on orthodontic force application.[19] There is a reduction of osteoclast and osteoblast activities in the absence of CCL2. Similarly CCR5 has been suggested to be a down regulator of alveolar bone resorption during orthodontic tooth movement.[20]

Matrix metalloproteinases (MMPs) help in bone remodeling by breaking down the extracellular matrix. It has been found that, compression of PDL induces an increase in MMP-1 levels 1hr after mechanical loading. This increase lasted for 2hrs and subsequently disappeared. Tension within the PDL too resulted in significantly increased levels of MMP-1 protein after 1hr of force application which also subsequently disappeared.[21]

MMP-2 protein was induced by PDL compression, which increased significantly in a time-dependent fashion, reaching a peak after 8 hrs after mechanical loading. MMP-2 was significantly increased on the tension side 1 hr after force application, but gradually returned to basal levels within 8 hrs.[22] This indicates that MMP-2 could be used as a biomarker for monitoring active tooth movement during the early stages of orthodontic treatment.

Type I procollagen is a bone formation biomarker secreted by osteoblast cells. The cleavages of procollagen produces procollagen type I C-terminal pro-peptide (PICP) and procollagen type I N-terminal pro-peptide (PINP) and were proposed to be measured as bone formation markers.[23] However, both PICP and PINP are markers that can only indicate the formation of type I collagen and not totally bone specific.[23] Therefore, they cannot be used to monitor OTM.

Bone morphogenetic proteins (BMPs), transforming growth factor-beta (TGF-β) and growth-factor- (GFs) associated internal signaling molecules are other bone-forming genes that encode proteins for GFs.[16] BMPs bind to the surface receptors on progenitor and mature osteoblasts and subsequently trigger a signaling pathway which promotes the differentiation of osteoprogenitor cells and the up-regulation of osteoblast activity.

Osteoblastic activity can also be promoted by the interaction of Growth factors with specific surface receptors on osteoblasts, thereby stimulating insulin-like GF-1. They have growth-promoting effects on bone, in addition to regulating cell growth and development. Studies have found that Msx 1 and Msx 2 are potential regulators of osteoblastic activity.[24,25] The Msx 1, protein is known as a critical modulator of bone development and remodeling, and Msx 2 is an alternative regulator protein of Cbfa1 expression in bone formation during OTM. Msx 1 and Msx 2 can be used as potential biomarkers during the development of stem cells into osteoblasts during OTM.[24,25]

TNF-α is a pro-inflammatory cytokine that is expressed during periodontal disease and is responsible for alveolar bone resorption during periodontal breakdown.[26,27] TNF-α has been found to play a major role in osteo-clastogenesis.[28] RANKL and its receptor RANK, which are present on osteoblasts and precursor osteoclasts, respectively, have been recognized as the key factors that stimulate osteoclast formation.[29]

Recent studies analyzed the cytokine expression pattern in compression and tension sides of the PDL during orthodontic tooth movement in humans by means of real-time polymerase chain reaction (PCR). It was found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the PDL of normal teeth. The compression side exhibited higher expression of TNF-α, matrix metalloproteinase I (MMP-1) and RANKL, whereas the tension side presented higher expression of type I collagen, IL-10, Tissue Inhibitor of Matrix Metalloproteinase I (TIMP-1), Osteoprotegerin (OPG) and Osteocalcin (OCN). The expression of TGF-β was found to be similar in both pressure and tension sides.[30]

It is now clear that RANKL, together with macrophage-colony stimulating factor, is required for osteoclast formation from precursor monocytes and macrophages.[31] The natural inhibitor of RANK-RANKL interactions is the soluble TNF receptor-like molecule osteoprotegerin (OPG), which binds to RANKL and prevents its ligation, thereby preventing osteoclast differentiation and activation. RANKL protein was found to be predominant in inflammatory cells adjacent to areas of pathological bone loss in periodontal disease[32,33] and is associated with the progress of periodontal disease.

The local RANKL gene transfer to the periodontal tissue using a Hemagglutinating Virus of Japan (HVJ) envelope vector was reported to accelerate OTM in six-week-old male Wistar rats. The activation of transferred RANKL gene in the periodontal tissue indicates that RANKL is involved during active OTM especially in periodontal area.[34] However, the activation of the OPG gene through gene transfer to periodontal tissues neutralized RANKL activity and hence inhibited osteo-clastogenesis. Thus, activation of the OPG gene inhibits bone remodeling.

Alkaline phosphatase activity in the GCF is known to decrease during active tooth movement. This decrease was found to be significant during the first month of active tooth movement and started to stabilize later.[35] Batra et al.,[36] found that the alkaline phosphatase activity in the GCF showed an increase during canine retraction which peaked during the 14^th day. There was a significant fall in the level of activity around the 21^st day. It is one of the important enzymes responsible for initiating orthodontic tooth movement and then sustaining it for a two week period. Any disturbance in this enzyme activity could delay orthodontic tooth movement.

Nitric oxide (NO) is produced through the activity of constitutive endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) and plays an important role in regulating bone response to mechanical stress. NO mediates adaptive bone formation, protects osteocytes against apoptosis and mediates osteoclastic activity. High levels of NO reduce the osteoclastic activity, while the inhibition of NO production increases osteoclastogenesis and osteoclastic activity.[37]

The potential of tartrate-resistant acid phosphatase (TRAP) as a biomarker of bone resorption has been long recognized.[38] TRAP activity was very strong in in-vitro osteoclast cultures. Refinement of the activity assay to primarily measure TRAP 5b at a pH level of 6.1 was suggested for investigation of osteoclastic activity and bone resorption rates.[38] However, controlled experimental studies are required to validate the use of TRAP as a biomarker to monitor OTM.

Osteocalcin is the most abundant non-collagenous matrix protein found in bone. It is expressed by highly differentiated osteoblasts and is incorporated into the bony matrix during alveolar bone remodeling. Smaller osteocalcin fragments are found in areas of bone remodeling and are actually degradation products of the bone matrix. This suggests its potential as a bone resorption marker during OTM.[23]

Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) are inflammatory biomarkers found outside cells during inflammation. Increased levels of lactate dehydrogenase[39,40] and aspartate aminotransferase[41–43] were detected in human GCF samples obtained during OTM. They are also found in higher levels in the presence of periodontal disease. They have the potential to be used as a biomarker to monitor OTM.

Myeloperoxidase (MPO) is an enzyme found in polymorphonuclear neutrophil (PMN) granules and can be used to estimate the number of PMN granules in tissues. Mean MPO activity was increased in both the GCF and saliva of orthodontic patients at 2 hrs after appliance activation. MPO might be a good biomarker to assess inflammation in orthodontic movement.[44]

Cathepsin B is an intracellular lysosomal cysteine proteinase, capable of degrading extracellular components including collagen. It also increases protein turnover in the lysosomal system. It is also known to play an important role in the initiation and perpetuation of inflammatory processes. The accumulation of cathepsin B in the gingival crevicular fluid has been shown to increase on mechanical loading. Cathepsin B is present in higher concentrations around osteoclasts and plays a major role in alveolar bone remodeling.[45]

Leptins are polypeptide hormones mainly secreted from the adipose tissue in humans. They have been classified as cytokines.[46] Leptin and its receptor share structural and functional similarities with members of the long-chain helical cytokines including IL-6, IL-11, IL-12 and oncostatin M.

Leptin stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages. There is an overall increase in leptin levels during inflammation and infection.[47] Previous studies have suggested a strong relationship between leptin levels and periodontal disease.[48] Several studies have observed that the levels of GCF leptin activity may play an important role in the development of periodontal disease. Karthikeyan[49] reported that leptin levels in the gingival crevicular fluid decreased progressively as periodontal disease progressed. Leptins might have the potential to serve as a biomarker for OTM. However, controlled experimental studies are required to validate its use as a biomarker.

CONCLUSION

Bone remodeling that occurs during orthodontic tooth movement is a biological process involving an acute inflammatory response in the periodontal tissues. Application of a mechanical stimulus in the form of orthodontic forces cause an inflammatory reaction within periodontal tissues, which in turn may trigger the biological processes associated with bone remodeling. The various neurotransmitters, growth factors, interleukins, leptins and enzymes like aspartate aminotransferase, cathepsin K and matrix metalloproteinases have the potential to serve as biological markers in order to monitor and validate orthodontic tooth movement. Tooth movement is a highly conserved physiological mechanism for continuous adaptation of the dentition. Orthodontic tooth movement is a biomechanical exploitation of the physiologic mechanisms for developing and maintaining optimal occlusal function.