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Background: Electrochemical signals play an important role in cell communication and behavior. Electrically charged ions transported across cell membranes maintain an electrochemical imbalance that gives rise to bioelectric signaling, called membrane potential or Vmem. Vmem plays a key role in numerous inter- and intracellular functions that regulate cell behaviors like proliferation, differentiation and migration, all playing a critical role in embryonic development, healing, and regeneration.
Methods: With the goal of analyzing the changes in Vmem during cell proliferation and differentiation, here we used direct current electrical stimulation (EStim) to promote cell proliferation and differentiation and simultaneously tracked the corresponding changes in Vmem in adipose derived mesenchymal stem cells (AT-MSC).
Results: We found that EStim caused increased AT-MSC proliferation that corresponded to Vmem depolarization and increased osteogenic differentiation that corresponded to Vmem hyperpolarization. Taken together, this shows that Vmem changes associated with EStim induced cell proliferation and differentiation can be accurately tracked during these important cell functions. Using this tool to monitor Vmem changes associated with these important cell behaviors we hope to learn more about how these electrochemical cues regulate cell function with the ultimate goal of developing new EStim based treatments capable of controlling healing and regeneration.
Most living organisms possess varying degrees of regenerative capabilities but how these regenerative processes are controlled is still poorly understood. Naturally occurring bioelectric voltages (like Vmem) are thought to be playing instructive role in tissue regeneration, as well as embryonic development. The different distribution of ions on the either side of the cell membrane results in intra- and extra-cellular voltage differences, known as membrane potential or Vmem. The relationship between Vmem and cell physiology is conserved in a wide range of cell types and suggests that Vmem regulation is a fundamental control mechanism for regeneration related processes e.g., proliferation and differentiation. In the present study we measured Vmem in three different cell types (human osteogenic sarcoma cell line (OSC), rat bone marrow derived mesenchymal stem cells (BM-MSC), and rat dermal fibroblasts) and characterized the relationship between their Vmem and proliferation. In order to find out if Vmem controls proliferation, or visa-versa, we blocked and then unblocked Na+/K+-exchanging ATPase using ouabain and measured the proliferation. Our results demonstrate that Vmem can be pharmacologically manipulated to control proliferation in certain cell types like BM-MSC. Taken together, it is clear that control of bioelectrical properties in non-excitable cells could prove to be potentially a useful tool in regenerative medicine efforts.
Electrical stimulation shifts healing/scarring towards regeneration in a rat limb amputation model
(2019)
Different species respond differently to severe injury, such as limb loss. In species that regenerate, limb loss is met with complete restoration of the limbs’ form and function, whereas in mammals the amputated limb’s stump heals and scars. In in vitro studies, electrical stimulation (EStim) has been shown to promote cell migration, and osteo- and chondrogenesis. In in vivo studies, after limb amputation, EStim causes significant new bone, cartilage and vessel growth. Here, in a rat model, the stumps of amputated rat limbs were exposed to EStim, and we measured extracellular matrix (ECM) deposition, macrophage distribution, cell proliferation and gene expression changes at early (3 and 7 days) and later stages (28 days). We found that EStim caused differences in ECM deposition, with less condensed collagen fibrils, and modified macrophage response by changing M1 to M2 macrophage ratio. The number of proliferating cells was increased in EStim treated stumps 7 days after amputation, and transcriptome data strongly supported our histological findings, with activated gene pathways known to play key roles in embryonic development and regeneration. In conclusion, our findings support the hypothesis that EStim shifts injury response from healing/scarring towards regeneration. A better understanding of if and how EStim controls these changes, could lead to strategies that replace scarring with regeneration.
This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA.
Background: Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES’s demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect.
Methods: Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression.
Results: We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure.
Discussion: This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential.
Introduction. Cancellous bone is frequently used for filling bone defects in a clinical setting. It provides favourable conditions for regenerative cells such as MSC and early EPC. The combination of MSC and EPC results in superior bone healing in experimental bone healing models. Materials and Methods. We investigated the influence of osteogenic culture conditions on the endothelial properties of early EPC and the osteogenic properties of MSC when cocultured on cancellous bone. Additionally, cell adhesion, metabolic activity, and differentiation were assessed 2, 6, and 10 days after seeding.
Results. The number of adhering EPC and MSC decreased over time; however the cells remained metabolically active over the 10-day measurement period. In spite of a decline of lineage specific markers, cells maintained their differentiation to a reduced level. Osteogenic stimulation of EPC caused a decline but not abolishment of endothelial characteristics and did not induce osteogenic gene expression. Osteogenic stimulation of MSC significantly increased their metabolic activity whereas collagen-1α and alkaline phosphatase gene expressions declined. When cocultured with EPC, MSC’s collagen-1α gene expression increased significantly. Conclusion. EPC and MSC can be cocultured in vitro on cancellous bone under osteogenic conditions, and coculturing EPC with MSC stabilizes the latter’s collagen-1α gene expression.
Electrical stimulation (EStim) has been shown to promote bone healing and regeneration both in animal experiments and clinical treatments. Therefore, incorporating EStim into promising new bone tissue engineering (BTE) therapies is a logical next step. The goal of current BTE research is to develop combinations of cells, scaffolds, and chemical and physical stimuli that optimize treatment outcomes. Recent studies demonstrating EStim’s positive osteogenic effects at the cellular and molecular level provide intriguing clues to the underlying mechanisms by which it promotes bone healing. In this review, we discuss results of recent in vitro and in vivo research focused on using EStim to promote bone healing and regeneration and consider possible strategies for its application to improve outcomes in BTE treatments. Technical aspects of exposing cells and tissues to EStim in in vitro and in vivo model systems are also discussed.
Limb loss is a devastating disability and while current treatments provide aesthetic and functional restoration, they are associated with complications and risks. The optimal solution would be to harness the body's regenerative capabilities to regrow new limbs. Several methods have been tried to regrow limbs in mammals, but none have succeeded. One such attempt, in the early 1970s, used electrical stimulation and demonstrated partial limb regeneration. Several researchers reproduced these findings, applying low voltage DC electrical stimulation to the stumps of amputated rat forelimbs reporting "blastema, and new bone, bone marrow, cartilage, nerve, skin, muscle and epiphyseal plate formation". In spite of these encouraging results this research was discontinued. Recently there has been renewed interest in studying electrical stimulation, primarily at a cellular and subcellular level, and studies have demonstrated changes in stem cell behavior with increased proliferation, differentiation, matrix formation and migration, all important in tissue regeneration. We applied electrical stimulation, in vivo, to the stumps of amputated rat limbs and observed significant new bone, cartilage and vessel formation and prevention of neuroma formation. These findings demonstrate that electricity stimulates tissue regeneration and form the basis for further research leading to possible new treatments for regenerating limbs.
Background: Electrical stimulation (ES) has been successfully used to treat bone defects clinically. Recently, both cellular and molecular approaches have demonstrated that ES can change cell behavior such as migration, proliferation and differentiation.
Methods: In the present study we exposed rat bone marrow- (BM-) and adipose tissue- (AT-) derived mesenchymal stem cells (MSCs) to direct current electrical stimulation (DC ES) and assessed temporal changes in osteogenic differentiation. We applied 100 mV/mm of DC ES for 1 h per day for three, seven and 14 days to cells cultivated in osteogenic differentiation medium and assessed viability and calcium deposition at the different time points. In addition, expression of osteogenic genes, Runx2, Osteopontin, and Col1A2 was assessed in BM- and AT-derived MSCs at the different time points.
Results: Results showed that ES changed osteogenic gene expression patterns in both BM- and AT-MSCs, and these changes differed between the two groups. In BM-MSCs, ES caused a significant increase in mRNA levels of Runx2, Osteopontin and Col1A2 at day 7, while in AT-MSCs, the increase in Runx2 and Osteopontin expression were observed after 14 days of ES.
Discussion: This study shows that rat bone marrow- and adipose tissue-derived stem cells react differently to electrical stimuli, an observation that could be important for application of electrical stimulation in tissue engineering.
Introduction: Microsurgery courses, taught external to surgical training programs, are essential for acquiring the high level of technical skill required for clinical proficiency.
Methods: The Frankfurt microsurgery course is a 5-day, intensive course that teaches arterial and venous anastomosis using end-to-end, end-to-side, one-way-up, continuous-suture, and vessel graft techniques. During the course, the instructor records the level of skill (in-course data) achieved by each trainee by assessing anastomosis completion and patency. Demographic information is also collected. Post-course trainees are invited to complete an online survey (post-course data) to get their opinions of the courses’ effectiveness.
Results: The in-course “skill achievement” and post-course “course effectiveness” data are presented below. In-course data: 94.8 and 59.9% of participants completed patent end-to-end arterial and venous anastomoses, respectively, while 85.4% performed a patent end-to-side anastomosis. 96.1 and 57.1% of participants who attempted arterial and venous anastomoses using the one-way-up technique were successful, as were 90.9% of those attempting continuous-suture technique. Patent venous grafts were performed by 54.7% of participants.
Post-course data: All respondents indicated significant improvement of their microsurgical skills after taking the course. 66.7% of respondents considered the full-time presence of the instructor to be the most valuable aspect of the course. All respondents would highly recommend the course to colleagues.
Conclusion: The microcourse significantly increased trainees’ clinical microsurgery skills, confidence, and the number of clinical cases they perform. Of all the anastomosis techniques taught, venous anastomosis and grafting were the most difficult to learn. The presence of a full-time experienced instructor was most important.