Electrical stimulation in bone tissue engineering treatments

Bone is one of the few tissues in mammals, that when fractured “regenerates” on its own. However, in cases where large volumes of bone are missing, like in severe injury, surgical extirpation of large amounts of infected bone or tumors, and congenital skeletal abnormalities, these regenerative capabilities are overwhelmed and complex and costly treatments must be employed to close the defect. Among conventional treatment options, bone autografts are considered to be the gold standard. However, in spite of the success they enjoy, autografts are associated with drawbacks, like donor site morbidity, limited availability in overly large defects, and the risk of infection (reviewed in [1]), which continue to fuel the search for better, alternative treatments. Bone tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments, for large non-healing bone defects, and holds great promise for promoting bone healing and regeneration without the associated drawbacks [2]. BTE approaches, in many ways, simulate bone autografts, in that they fill the defect with bone-forming stem/progenitor cells, scaffolds that restore missing bone volume, and growth factors that control cell–cell and cell–scaffold interactions [3]. Success of BTE approaches, in clinical settings, depends largely on the choice of cells, scaffold material, and signaling stimuli added to the cell–scaffold mix, and/or present in the microenvironment of the healing defect. While pre-clinical and clinical BTE treatments have demonstrated encouraging early outcomes [4, 5], the logistics associated with harvesting, isolating and amplifying the cells, and the time required to do so, are not optimal and continue to stimulate the search for strategies to manipulate/fine-tune the type, quantity, and composition of stem cells, scaffolds, and stimuli (reviewed in [3]).

For decades, electrical stimulation (EStim) has been studied and used successfully in clinical practice to stimulate bone healing (reviewed in [6]). While the detailed mechanisms by which EStim promotes healing are poorly understood, several recently published in vitro studies suggest that EStim’s pro-healing effect is due to its influence on the behavior and/or function of bone-forming stem cells, such as migration [7, 8], proliferation [9], differentiation [10, 11], mineralization [12], extracellular matrix deposition [13], and attachment to scaffold materials [14]. Importantly, all these cell behaviors/functions that are central to healing could potentially be used to optimize outcomes in BTE treatments. In this review, we provide an overview of different methods and results using EStim to treat cells, scaffolds, and tissues in in vitro and in vivo model systems, with an eye toward its potential use in BTE treatments. We discuss mechanisms by which EStim acts at cellular and molecular levels and discuss limitations and technical aspects of delivering EStim both in experimental and clinical settings. This knowledge could assist in the development of future clinical strategies for combining EStim and BTE treatments.

EStim could potentially be added in clinical BTE treatments either ex vivo, when the cell–scaffold construct is prepared, or in vivo, after the cell–scaffold construct is delivered into the bone defect.

Previous in vitro experiments that exposed cells and/or scaffolds to EStim, demonstrated its ability to influence cell functions associated with enhanced bone healing [7‐14]. These experiments were conducted on a variety of different cell types; bone marrow-derived mesenchymal stem cells (BM-MSC), from human and animal origin [10, 11, 15‐25]; adipose-derived mesenchymal stem cells (AT-MSC) [11, 20, 26‐30], mouse osteoblast-like cells [31‐33] and more recently, human dental pulp stem cells (DPSC) [34]. The above cell types are commonly studied for use in BTE applications. Based on these findings, one can speculate that treating cell–scaffold constructs with EStim ex vivo, prior to placing the mix in a bone defect, would greatly improve outcomes in BTE in treatments. How DC EStim affects these cells is summarized in the following paragraphs and Fig. 1.

Exposing cells to exogenous EStim generates a response called electrocoupling, caused by high resistance of the plasma membrane, which prevents the penetration of electric stimuli, independent of the cytoplasm conductive capacity [62]. One of the possible electrocoupling mechanisms involves asymmetric redistribution/diffusion of electrically charged cell membrane receptors in response to electric fields, which further activates numerous downstream signaling cascades. Another possible mode of action is related to the cell membrane depolarization due to direct activation of voltage-gated Ca²⁺ channels, which leads to increase in intracellular calcium ion concentration, a cellular response consistently reported after electric stimuli. These and other mechanisms are discussed with more details below.

Signal transduction pathways Electrical signals are sensed and converted into biochemical cues by multiple pathways within cells, resulting in various biological responses. The activation of the MAPK (mitogen-activated protein kinase) cascades represents a major signal transduction pathway, which regulates specific mRNAs transcription as consequence of external stimuli [63]. This leads to the activation of extracellular signal-regulated kinase ERK1/2 and 5, JNK and p38MAPK, that consecutively intervenes in important cell activities, such as proliferation, differentiation, apoptosis and others, depending on the type of cell and stimuli [64, 65]. Fast and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated kinase (MAPK), Src and Akt, was demonstrated by Zhao et al. in cells migrating under the influence of electrical fields [66, 67]. EStim was shown to induce direction and movement of adult stromal cells through the activation of PI3K and ROCK signaling pathways [59].

Ca²⁺ transients Increase in intracellular Ca²⁺ is one of the prompt effects of EStim on cellular response. Calcium ions are important cellular mediators, which play a role in many important vital activities such as proliferation, differentiation, and apoptosis [68]. Intracellular Ca²⁺ could be increased via two essential events; by the passage of extracellular Ca²⁺ into intracellular space through plasma membrane ion channels, or by activation of specialized receptor/channels on the surface of the endoplasmic reticulum (ER), which release Ca²⁺ from internal stores in the ER [69]. Calcium oscillations can increase the efficiency and specificity of gene expression, which guides the direction of cell differentiation [70, 71]. EStim was shown to facilitate differentiation of hMSC by changing Ca²⁺ oscillation patterns to patterns similar to those seen in osteoblasts [72]. Of note, EStim exposure can directly stimulate L-type voltage-gated Ca²⁺ channels (VGCCs) in the plasma membrane [73] that can elicit many regulatory responses through the enzymatic action of the Ca²⁺/calmodulin-dependent nitric oxide synthases [74].

Mechanotransduction—cytoskeletal reorganization and actin distribution Mechanotransduction is the conversion of external mechanical stimuli into intracellular electrical or chemical signals [75]. The inverse effect of mechanotransduction is the transformation of electrical stimuli into mechanical activity that causes tension in the cytoskeleton due to reorganization of the cytoskeletal filaments and actin redistribution. Changes in the actin structure could occur as consequence of interactions between plasma membrane and electrical stimuli [44]. Hereof, EStim has been shown to cause either direct effects on the cytoskeleton, or intervene on cellular processes regulated by the cytoskeleton [76].

Surface receptor redistribution Most likely, DC EStim is restrained by cellular plasma membrane, which holds high electrical resistance, and events take place at the cell surface rather than penetrating inside the cell. As a result, most of the biochemical signal transduction cascades in response to EStim, arise due to the redistribution of charged cell surface receptors (CSRs) at the external space of cell membrane [77]. It is reported that the exposure to 100–3000 mV/mm of external EStim results in redistribution membrane proteins and lipids on external site of the cell due to induction of relative electrophoretic movement these components on the cell exterior [78]. Specifically, epidermal growth factor receptor (EGFR) was shown to be up-regulated by the application of low levels of EStim, which also induces EGFR redistribution and accumulation at the cathode side of the cell [79]. In addition to promoting an asymmetric distribution of EGFR, colocalization of membrane lipids and second-messenger signaling molecule ERK ½ could also occur due to influence of small amounts of EStim, resulting on the triggering of MAPK signaling cascade [49, 79].

ATP synthesis Direct current EStim, ranging from 10 to 1000 µA, is known to stimulate membrane-bound ATP synthesis [80]. This is thought to be due to EStim guiding migrating protons to reach the mitochondrial membrane-bound H1-ATPases, to generate ATP. This is supported by the observation of high levels of released ATP measured in electrically stimulated cells [81]. The relationship between ATP synthesis and actin cytoskeleton is one of the intriguing mechanisms to explain how cells sense electrical fields. It has been well documented that intracellular ATP is consumed for the conversion of monomeric G-actin to polymeric F-actin [82] and that EStim-induced ATP depletion is implicated in the reorganization of actin cytoskeleton in electrically stimulated hMSC [76].

Heat shock proteins It has been generally hypothesized that cells’ response to EStim (especially at levels higher that those occurring naturally) could follow physiological stress response and function through activation of stress heat shock proteins [83]. The involvement of heat shock proteins (hsp 27 and hsp 70) in the upregulation of some of the transcription factors was previously reported in hMSC osteogenic differentiation [84].

Reactive oxygen species Participating in crucial signaling pathways, reactive oxygen species (ROS) is considered another important mechanism involved in stem cell response to EStim [85]. Controlled induction of ROS at physiologic levels can benefit interactions of other signaling molecules influencing differentiation. Numerous studies have demonstrated that MAPK pathways and the subsequent signaling cascades of ERK1,2, JHK, and p38 are activated by moderate levels of ROS [86]. Proliferation and differentiation of MSC were shown to be mediated by a mild rise in hypoxia-induced ROS [87‐89].

Lipid rafts Recent research has shown that in addition to proteins, lipids in the cellular membrane also participate in the response to externally applied EStim. It was shown that due to externally applied EStim, plasma membrane glycolipids could redistribute and congregate into nanodomain structures, known as lipid rafts [90]. Acting as the initial sensor of electric fields, these nanodomain structures polarize, coalesce, and segregate membrane proteins, which in consequence trigger intracellular signaling events to guide cell migration [91].

All these cellular mechanisms are involved in a complex and finely orchestrated network of signaling and responses. Biological processes are programmed as a chain reaction starting from a cellular activity, which normally leads to implications in the tissues they compose. Therefore, it follows that external interferences in the cell response, promoted by the application of EStim, influence not only cells but also tissues as well.

Bone healing is a complex and well-orchestrated process, both in time and space, requiring coordinated function of different cell types and systems [92]. EStim’s ability to promote bone healing has been demonstrated both in animal experiments and clinical settings (reviewed in [6]). When EStim is applied to a bone defect, alone or in combination with BTE constructs, its effect on all the resident cells needs to be considered. In the following lines we discuss how EStim, when added to BTE treatment might influence key bone healing parameters, like osteogenesis, vascularization, and inflammation.

There are a growing number of studies in the literature that focus on combining EStim and BTE treatments [117]. In addition to studying how best to use EStim to manipulate cell behavior, it is also important to consider technical aspects of delivering EStim to cell + scaffold constructs and/or tissues in clinical treatments. EStim devices for use in treating ex vivo cells prior to transplantation will have to be developed, while commercially available DC bone growth stimulators (OsteoGen® from Biomet EBI and Zimmer direct current bone growth stimulator, reviewed in [118, 119]) could be adapted for treating transplanted BTE constructs in clinical settings.

In most in vitro applications, cells grown in 2-D or 3-D culture can be treated with specific regimens of EStim in purpose-built chambers. As these chambers are not commercially available, different laboratories have developed their own to satisfy their specific needs. Below, we describe a few of the most commonly used setups (Fig. 2).

There are a number of commercially available clinical EStim devices that could be used/adapted to treat BTE constructs with EStim, before they are loaded into bone defects. Devices used for EStim bone treatment in clinical settings, can be categorized into external and internal stimulators, that deliver EStim via an external field or percutaneously, and via internal surgically implanted electrodes. The external stimulators deliver capacitive coupling (CC) and inductive coupling (Pulsed ElectroMagnetic Field—PEMF) EStim, and the internal stimulators deliver direct current (DC) EStim [137, 138] (Fig. 3).

Capacitive coupling stimulators are small, lightweight devices, which use an external power source. Despite the obvious advantages of not having to be surgically implanted, and ease of use, disadvantages include, patients must change batteries daily, skin irritation, and patient non-compliance are common problems [138]. There are only a few clinical studies available that support the effectiveness of CC devices (reviewed in [6]).

Inductive coupling or pulsed electromagnetic field (PEMF): The electrodes of these devices can be placed under casting material or used through a cast. These devices create low-level electromagnetic signals, which after reaching the fracture site, are converted into electric currents and are said to mimic the body’s normal physiologic processes. The primary advantage of PEMF bone stimulators is their noninvasive application; however, drawbacks include, the heavy weight of these devices, difficulty assessing treatment dosage, and patient non-compliance [139].

DC electrical stimulators have the benefits of providing constant and uniform current delivery, the EStim is focused at the bone defect, and elimination of patient non-compliance. Surgical implantation consists of placing a cathode at the fracture site and an anode in the nearby subcutaneous tissue, that deliver electric current flow between them. The electrodes are connected to a stimulator device, which is implanted subdermally [140, 141]. The power source of these devices typically last from 6 to 8 months, at which time the implanted device and electrodes must be removed in a second procedure. Over the last 3 decades, numerous studies support the clinical efficacy of these DC EStim stimulators [138, 142, 143]. Recent clinical studies using implantable DC EStim stimulators, alone [144], or in combination with bone grafts [145] have reported increased bone healing rates.

Other physical stimulation techniques, like magnetic and vibration stimulations, have been tried and found to promote bone healing [146], in general their administration in patients in clinical settings have come up against serious limitations. The wearable devices used for their administration are cumbersome, thus when used for prolonged periods tends to interfere with patients’ daily activities leading to decreased compliance [6]. In addition, these units have been reported to give inconsistent results, and one of the reasons for this has been attributed to the fact that the stimulation energy they generate is not focused at the fracture site. In contrast, when DC EStim is administered with surgically implanted device compliance is not an issue and the electrical energy is focused at the fracture site, which has led to the reporting of more consistent treatment outcomes.

Despite EStim’s demonstrated effectiveness improving bone healing, both in pre-clinical animal studies and in clinical settings, few studies have investigated the effectiveness of combining EStim with BTE treatments in vivo [27, 29, 94, 147, 148]. In one of these studies, our group treated large defects in rat femurs with AT-MSC + Scaffold + EStim. We found that the rate and quality of bone healing at 8 weeks, in defects treated with AT-MSC + Scaffold + EStim, was significantly better than controls [29]. Our own experience from this study, together with reports in the literature [141, 149], suggests that the problems of combining EStim with BTE in clinical settings would be similar to those experienced in current clinical EStim bone treatments. Namely, complications associated with the surgical procedures used to implant and explant the EStim device, electrode breakage or dislodgement, and infection.

Summarizing, in vitro experiments that expose cells and/or scaffolds to EStim, generally show positive results, although, a lack of standardization of cell types, models and protocols make it difficult to draw definitive conclusions. Additional studies are needed to develop strategies for transferring these encouraging in vitro findings into meaningful in vivo BTE applications. In addition, the logistics of combining EStim and BTE treatments in practical, cost effective ways in clinical settings must be considered.

Some exciting new developments that could be incorporated into these strategies include the use of electroactive smart polymeric biomaterials that could potentially combine scaffold and EStim into one. Recent advancements in polymer science, using “smart” biomaterials, that enable built-in stimulus/response behavior capabilities, have tremendous potential [150]. Electroactive smart polymeric biomaterials could be used to build scaffolds that offer precise control over the amount, duration, and localization of the electrical stimulus, thus obviating the need for bone stimulators. These materials have already been tested in vitro, where they have demonstrated the ability to improve cell proliferation and differentiation [151‐153]. If in addition to promoting these pro-osteogenic activities, these smart biomaterials can also be designed to biodegrade after a given useful time period, this would be yet another benefit [154].