Electric stimulation may be able to help blood vessels carry white blood cells and oxygen to wounds, speeding healing, a new study suggests.
The study, published in the Royal Society of Chemistry journal Lab on a Chip, found that steady electrical stimulation generates increased permeability across blood vessels, providing new insight into the ways new blood vessels might grow.
The electrical stimulation provided a constant voltage with an accompanying electric current in the presence of fluid flow. The findings indicate that stimulation increases permeability of the blood vessel – an important characteristic that can help wound-healing substances in the blood reach injuries more efficiently.
“There was this speculation that blood vessels could grow better if you stimulated them electrically,” said Shaurya Prakash, senior author of the study and associate professor of mechanical and aerospace engineering at The Ohio State University.
“And we found that the response of the cells in our blood vessel models shows significant promise towards changing the permeability of the vessels that can have positive outcomes for our ongoing work in wound healing.”
Blood vessels are crucial for wound healing: They thread throughout your body, carrying nutrients, cells and chemicals that can help control inflammation caused by an injury. Oxygen and white blood cells – which protect the body from foreign invaders – are two key components delivered by blood vessels.
But when there is an injury – for example, a cut on your finger – the architecture of the blood vessels at the wound site are disrupted. That also interrupts the vessels’ ability to help the wound heal.
Blood vessels regrow on their own, almost like the branches of trees, without external sources of electricity, as part of the healing process.
“And as the blood vessels begin to grow, they replenish the skin and cells and establish a healing barrier again,” Prakash said. “But our question was: How do you make this process better and faster, and is there any benefit to doing that?”
What they found, in laboratory tests performed using human cells, is that stimulating blood vessels with electricity showed a marked increase in blood vessel permeability, which is a physical marker suggestive of possible new vessel growth.
“These initial findings are exciting, and the next phase of the work will require us to study if and how we can actually grow new vessels,” Prakash said.
Jon Song, co-author of the paper and associate professor of mechanical and aerospace engineering at Ohio State, said the results imply that one of the primary ways blood vessels work to heal injuries is by allowing molecules and cells to move across the vessels’ walls.
“And now we have better understanding for how electric stimulation can change the permeability across the vessel walls,” Song said. “Let’s say you have a cutaneous wound, like a paper cut, and your blood vessels are severed and that’s why you have blood leaking out. What you need is a bunch of bloodborne cells to come to that place and exit out the blood vessel to initiate the wound repair.”
The study suggested that changes in blood vessel permeability could get those bloodborne cells to a wound site more quickly, though it did not explain the reasons why that happened. The study seemed to indicate that electricity affected the proteins that hold blood vessel cells together, but those results were not conclusive.
The study is an extension of work by a broader team, led by Prakash, that previously showed electric bandages could help stimulate healing in wounded dogs. That work indicated that electrical stimulation might also help manage infections at wound sites – a phenomenon the researchers also hope to research further.
Tissue engineering is a significant field emerging in the world of health care research. This discipline aims to provide new and efficient ways to treat a multitude of clinical injuries, ranging from bone non-unions to nerve transections. Modern treatments for common tissue defects include autografts, using harvested tissue from elsewhere in the patient’s body; allografts, using harvested tissue from other patients; or on rare occasions xenografts, using tissues harvested from other species [, , , , , ].
There are many downsides with these approaches, including donor site morbidity, risk of infection, and scarcity of donor material [, , , , , , , ]. For these reasons, tissue engineering efforts continue to look towards the development of alternative therapeutic options including those summarized in Fig. 1.
A common goal of tissue engineering is to develop a scaffold suitable for cell growth, which can then be stimulated and manipulated towards a desired tissue type. The scaffold used must be mechanically competent for the forces present in the relevant tissue, non-immunogenic, biocompatible with the endogenous tissue, and must degrade in the body over time.
A variety of cells can be used, including cells derived from natural tissues, specific progenitor cells, as well as various potencies of stem cells [, , ]. An array of stimuli has been used to cause the differentiation and integration of the tissue engineered system into an implantation site.
Two examples of external stimuli that remain the focus of ongoing investigations are mechanical and electrical stimulation (ES) [14,15,, , , ]. These methods of external stimuli result in differentiation by promoting the release of growth factors and bimolecular signals [16,21].
Growth factors play an important role in tissue regeneration. It has been shown that the independent and direct use of exogenous growth factors is an effective means to induce cellular healing and regeneration . While the exogenous use of growth factors has its advantages, adjunctive use of ES for tissue regeneration is perhaps a superior modality, given that side effects are markedly limited for ES.
Limitations of growth factors include cost, complications of drug delivery such as avoiding growth factor delivery bolus, and questions of safety . The benefits of ES on excitable tissues, including neurons and muscle tissue, have been studied . ES possesses the ability to promote and direct neurite outgrowth, incite muscle contractions, and promote bone growth [18,, , ]. Moreover, ES has the ability to enhance the maturation and differentiation of cells [17,18].
Numerous methods for delivering ES exist, including surface electrodes and direct tissue stimulation; however, none provide researchers with the ability to deliver ES with high specificity. For this reason, a suitable graft material has been sought that has the ability to conduct and distribute ES to targeted cells while also fulfilling the requirements of a tissue-engineered construct.
Electroactive materials that conduct applied electric potential are appropriate materials for the delivery of ES. Conducting polymers with conjugated pi-bond systems on their backbones lead to a large chain of loosely bound electrons . Once reduced or oxidized by a dopant molecule, this system allows polymers to have high electron mobility [, , ].
As described in Shirakawa et al. iodine vapor was used as a doping agent to oxidize polyacetylene and increase its conductivity 10 million-fold [31,32]. Alternatively, ionically conductive (IC) polymers can be generated on any polymeric biomaterial by introducing ionic functional groups such as carboxylic, sulfonic, phosphonic, and amine, to name a few. This class of materials conducts electricity in the physiologic environment via counter flow of ions upon application of ES [33,34]. Unlike electronically conducting thermoset polymers like polyaniline, IC polymers exhibit properties of the parent polymers chosen for ionic functionalization. Polymers can be chosen to improve overall physicochemical properties leading to improved biocompatibility and degradation profiles .
In our previous work, we showed the use of IC polymers and their conductive properties for nerve regeneration . The purpose of this review will be to analyze the current state of conductive materials used in conjunction with ES for tissue regeneration.
In addition, this review will provide an in-depth assessment on the effects of ES on major tissues in the body, including how electroactive materials have been used to implement these effects. An analysis of the cellular mechanisms and theories as to how ES works will be discussed, and both in vitro and in vivo studies will be reviewed. It is important to note that other ongoing tissue engineering strategies exist, including ultrasound and laser therapies, which are beyond the scope of this review .
The mechanistic effects of stimulation
Effects of electrical stimulation on cell alignment
ES direction plays a significant role in aligning and redirecting random cells to the direction in relation to the electric field direction applied. There are various types of cells that align perpendicularly and parallel to electric field vector direction to minimize the field gradient across the cells. Cardiac adipose tissue-derived progenitor cells, endothelial progenitor cells, vascular ECs, BMSCs, adipose-derived stromal cells are the different types of cells that line up themselves perpendicularly. The cells which align parallel to the applied effects of electrical stimulation are the ventricular myocytes, cardiomyocytes, myoblasts, PC-12 cells, and osteoblasts. Typically, the intensity of the ES is < 10V/cm, and the cells aligned better with the intensity of the ES, but the activity of the cells is decreased comparatively .
Effects of electrical stimulation on cell migration
The cell migration also relies on the electrical field applied along with the cell alignment. ES′ guiding effect on cell migration often depends on the type of cell, and electrotaxis is called the method of directing the cells . The following cells are drawn to the cathode and are NSCs, macrophages, mouse neural precursor cells (NPCs), osteoblasts, and endothelial progenitor cells, while the cell types such as BMSCs, human dermal fibroblasts, and SCs are at the anode.
The ES intensity can induce cell migration from a minimum of 0.1V/cm to a maximum of 12V/cm and did not result in significant cell damage, did not affect the cell phenotype or the potential for differentiation. At the same time, cells with higher ES intensity showed increasingly increased migration rate and size. It is still uncertain what mechanism contributes to electrotaxis. Other factors may be considered, such as endogenous microenvironment, ion channels, membrane receptors, transportation proteins and competing signal pathways such as Wnt/GSK3β and TGFβ1/ERK/NF-ÿB .
In addition to signal pathways, ion channels such as voltage gated Ca2+ channels, during ES, are a critical part of membrane polarization and cell response. The current causes a flood of ions through ion channels and transporters (Na+, Cl−, K+, Ca2+, etc.) In response to ES, intracellular molecular polarization and transport channel polarization, then ion flow occurs, and cytoskeleton changes cause direct cell migration, thus leading to persistent cell migration to cathode.
Effects of electrical stimulation on cell proliferation
The cell proliferation increases with applied proper electrical stimulation, usually under < 1V/cm of continuous stimulation. Within the range of ES intensity, the rate of cell proliferation increases as the intensity increases. For instance, preosteoblasts, osteoblasts, unregulated human somatic stem cells, human umbilical vein ECs, NSCs, human dermal fibroblasts display 0.2 to 1.5 times proliferation, with cellular metabolic activity increasing, and do not affect cell phenotype. High-intensity ES of >100V/cm is also beneficial for single-stimulation cell proliferation in a short time (<1 ms), but extremely high-intensity results in cell death .
Effects of electrical stimulation on cell differentiation
Stem cell therapy provides a promising approach for regenerative medicine. The cardiac differentiation of pluripotent stem cells and muscle cells induced by humans should encourage electrical stimulation factors of low intensity (several minutes to 0.06~6V/cm) by the short term . Neurons are separated from neural progenitor cells with the ES influence, neural precursor cells rather than being divided into glial cells.
The stimulation intensity is basically <2 V/cm and sustain more than 7days. The differential medium can appropriately add with FBS, retinoic acid, and nerve growth factor. ES can induce bone marrow stromal cells, BMSCs, MC3T3-E1 cells osteogenic differentiation instead of cartilage, stimulation intensity should be < 2 V/cm and sustain 14–28 days, the medium need to add dexamethasone in most cases .
Therefore, the application of ES could provide a valid approach to induce cell differentiation in tissue engineering. The following sections detail the use of these electroactive materials in the form of scaffolds for a variety of tissue regeneration applications.
Effects of ES on skin/wound healing
Skin establishes an endogenous electric field following injury. Zhao et al. displayed that following wounds of both a rat cornea and human skin, an outward current from the wound with a magnitude of 1–10 μA/cm2 could be measured at various stages of healing .
The electric field variation changed to positive charges at the wound center. Reversing the applied field with an external electric field caused wound opening while the opposite of this significantly increased epithelial cell movement into the wound . When directed towards skin wounds, ES has been shown to accelerate the wound healing process .
ES promotes accelerated healing of wounds, ulcers, and breaks in the epidermis [70,71]. It has been shown to increase the presence and activity of human skin fibroblasts [, , ]. The ability of fibroblasts to respond to ES in the dermis is thought to be via an opening of voltage-gated Ca2+ channels found on the surface of fibroblasts, with a subsequent increase in their activity and presence at the site of a wound . A second mechanism by which ES is thought to accelerate wound healing is via ionic attraction.
ES can serve to attract cells necessary for the healing process including neutrophils, macrophages, fibroblasts, and epidermal cells, as these all carry a negative charge . Studies have also shown ES promotes accelerated healing, and regenerated tissue exhibits increased tensile properties compared with control .
Furthermore, ES is beneficial in providing an increase in temperature to the affected skin, resulting in increased perfusion to the area, promoting tissue healing . ES also has been shown to promote angiogenesis, another important mediator of wound healing [70,76].
Of note, emerging evidence suggests that low frequency ES (<10 Hz) has higher efficacy and faster healing rates than higher frequencies of ES (>50 Hz). The benefits of wound healing and associated changes at the cellular/molecular level under the influence of ES are summarized in Fig. 5. Though initial findings are encouraging, further studies are warranted to fine tune ES parameters for specific wound size and thickness to harness benefits . Specifically, future studies should investigate different voltage intensities, frequencies, and delays from initial injury to adequately assess the most beneficial way that ES could be used in a clinical setting.
Role of conducting polymers with ES
While many studies have investigated the effects of ES on skin healing, very few have done so with a conductive material. An in vitro study by Rouabhia et al. evaluated the viability of this approach through its effects on fibroblasts in culture . The study used a PPY/poly (l-lactic acid) (PLA) membrane supplemented with bioactive heparin. ES was applied to fibroblasts through the conductive membrane they were seeded on. No toxic effects were noted as measured by LDH levels.
Further, ES applied to a scratched defect in this culture increased the rate of fibroblast healing . Briefly, after fibroblasts were electrically stimulated on the membrane, they were grown to confluence in 6-well plates at which point a sterile pipette tip was used to create a 0.5 mm defect. Cells initially stimulated through the membrane closed the wound in a 24 h period, outperforming the non-stimulated cells which remained with 30–40% of their initial wound left to heal in the same time frame .
reference link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139146/
More information: Prashanth Mohana Sundaram et al. Direct current electric field regulates endothelial permeability under physiologically relevant fluid forces in a microfluidic vessel bifurcation model, Lab on a Chip (2020). DOI: 10.1039/D0LC00507J