Numerous approaches have been proposed to enhance the healing capability of the anterior cruciate ligament (ACL). Electrical stimulation is widely used for the treatment of pain and to promote wound healing [1]. In orthopaedic practices, applied electric fields (EFs) have been used clinically to promote bone healing [2]. EF has also been shown to improve lapine ligament repair in vivo [3]. We have previously
demonstrated that applied EF enhanced ligament fibroblast migration and collagen production, depending on the applied EF parameters[4]. Electric stimulation of various types has been found to be an effective treatment technique for soft and hard tissue healing. Besides the long known, low-frequency currents (galvanization, impulse galvanization, diadynamic currents, TENS, faradic current, HVPGS) are increasingly applied today, as they cause patients less discomfort. Low intensity direct current can accelerate the healing rate of both hard and soft tissue injuries [5]. Electrical
stimulation of osteogenesis involves the use of faradic waveform to promote the growth of new bone [6]. Faradic stimulation maximizes the restoration of elastic properties of the muscle by encouraging the correct parallel configurational
arrangement of the collagen fibres within the cellular matrix [7]. Diadynamic current is a low frequency and biphasic sinusoidal-like waveform. The phase duration (10~20
ms) is longer than faradic current. Clinical usage of diadynamic current (fluctuation current) is for pain [8], analgesia [9], electro-myo stimulation [10], throboembolism (circulating) [11], and suppurating wounds healing [12]. In particular, several recent reports that high-voltage, pulsed galvanic stimulation (HVPGS) significantly
increases proliferation, collagen synthesis and healing rate of skin wounds. Similarly, in animal models HVPGS have shown an accelerated rate of full-thickness burns healing [13, 14]. In the current study, several stimulation waveforms used in physical therapy for promoting tissue repair were adapted to examine their effects on ACL fibroblast migration. Here, we focused on clinical electro-therapy on cell migration behavior.
Integrin: important role for mediating cell migration
Cell adhesion underlies several important physiological processes, such as cell migration, spreading, polarity, anchoring, differentiation, morphogenesis and wound healing. Focal adhesions are provided by adhesion molecules and expressed at the cell surface of all nucleated cells, and they mediate extracellular binding to cell and tissue substrates and transmit mechanical docking to the intracellular actomyosin or
intermediate filament cytoskeleton.
During cell migration, many classes of membrane receptors fulfill adhesion and
cytoskeletal coupling functions and provide a range of adhesion strength, specificity and turn-over rates. High affinity adhesion to ECM ligands is predominantly provided by the integrin family [15].The integrin-mediated cell substrate interactions and
linkages to the actin cytoskeleton, which is mediating cell movement, and its form and turn-over dynamics and polarity determine cell speed and directionality.
EF-induced integrin redistribution
The electrocoupling mechanisms are confined to the cell surface. Because cell membrane receptors such as integrins have been demonstrated to laterally diffuse [16]
and redistributed by ES, the mechanism for altered receptor distribution patterns (e.g., clustering) could be offered as at least one of the electrocoupling pathways. In recent
years, laboratories use several ways to change the distribution of integrin to find out the linkage between integrin redistribution and cell migration. The DC electrical stimulus (0.1V/cm) facilitates the cellular focal adhesion formation of human fibroblast in three-dimensional collagen gel by clustering of integrins on the membrane [17]. Cell migration may be extended to postulate that application of ES triggers the initial event of integrin clustering [18, 19] that serves as the site for formation of a focal adhesion by recruiting other molecules such as actin-binding proteins and signaling molecules, and integrin ligation mediating its activation and/or
the clustering of adhesion components to initiate a nascent adhesion [20, 21].
Members of the Rho Family of GTPases are regulated by
Integrin-mediated Signals
Integrin engagement and subsequent clustering of these receptors in focal adhesions lead to the generation of intracellular macromolecular complexes[22]. Numerous enzymatic activity (GTPases) signaling proteins are also structural components of focal adhesions. The Rho family of GTPases contributes to these integrin-mediated signals; in particular, signals that control cytoskeletal organization involved in changes in cell motility. RhoA are part of the Ras superfamily of proteins that cycle between an active, GTP-bound state and an inactive, GDP-bound state.
Activated RhoA is capable of stimulating microfilament bundling in serum-starved cells that are already adherent [23]. Rho is also essential for the formation of focal complexes [24]
Studies devoted to how Ras superfamily protein induces integrin clustering into FAs, and much less are known about the clustering of integrins into focal complexes influences RhoA, Rac and Cdc42. Many studies have also demonstrated that
shear-induced RhoA activation is secondary to integrin activation [25] . With
increasing numbers of GEFs being identified [26]Rho can be activated by integrin ligation [27, 28], leading to the formation of stress fibers and FAs. Many studies have also indicated that RhoA is activatedupon integrin-mediated adhesion [29, 30].
Development in the field has been the recognition that integrin-mediated adhesion stimulates activation of RhoA. GTP-bound RhoA measurement reveals that integrin-mediated adhesion causes modest increase in RhoGTP levels [27].
Integrins not only control the activation of Rho family but also separately regulate the translocation of activated RhoA, Rac1 and Cdc42 to the plasma membrane [31]. Integrin signals regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in
anchorage-dependent cells. RhoA is also thought to be concentrated in lipid rafts [32], and GTP-Rac1 binds more effectively to membranes from adherent than from suspended fibroblasts, indicating that integrins regulate RhoA and Rac1 membrane binding sites at the cell surface [31]
We anticipate that adhesion-mediated regulation of Rho family GTPases will play an important role in the complex process of cell migration behavior
In this experiment, we look further into the hypothesis that (1) different
waveform (direct current, diadynamic current, faradic current, sinusoidal current, and high voltage pulsed galvanic stimulation) caused different integrin distribution and
induced different fibroblast cell behavior (cell migration, orientation) (2) Electrical stimulation waveforms affects integrin redistribution which mediates EF-induced directionality. (3) integrin clustering and redistribution serves a mediator to activate and redistribute RhoA then induces cell migration directionality.