Anil Sebastian

Acceleration of cutaneous healing by electrical stimulation: degenerate electrical waveform down-regulates inflammation, up-regulates angiogenesis and advances remodeling in temporal punch biopsies in a human volunteer study.

We previously demonstrated the beneficial effect of a novel electrical stimulation (ES) waveform, degenerate wave (DW) on skin fibroblasts, and now hypothesize that DW can enhance cutaneous wound healing in vivo. Therefore, a punch biopsy was taken from the upper arm of 20 volunteers on day 0 and repeated on day 14 (NSD14). A contralateral upper arm biopsy was taken on day 0 and treated with DW for 14 days prior to a repeat biopsy on day 14 (ESD14). A near‐completed inflammatory stage of wound healing in ESD14, compared to NSD14 was demonstrated by up‐regulation of interleukin‐10 and vasoactive intestinal peptide using quantitative real time polymerase chain reaction and down‐regulation of CD3 by immunohistochemistry (IHC) (p < 0.05). In addition to up‐regulation (p < 0.05) of mRNA transcripts for re‐epithelialization and angiogenesis, IHC showed significant overexpression (p < 0.05) of CD31 (15.5%), vascular endothelial growth factor (66%), and Melan A (8.6 cells/0.95 mm2) in ESD14 compared to NSD14 (9.5%, 38% and 4.3 cells/0.95 mm2, respectively). Furthermore, granulation tissue formation (by hematoxylin and eosin staining), and myofibroblastic proliferation demonstrated by alpha‐smooth muscle actin (62.7%) plus CD3+ T lymphocytes (8.1%) showed significant up‐regulation (p < 0.05) in NSD14. In the remodeling stage, mRNA transcripts for fibronectin, collagen IV (by IHC, 14.1%) and mature collagen synthesis (by Herovici staining, 71.44%) were significantly up‐regulated (p < 0.05) in ESD14. Apoptotic (TUNEL assay) and proliferative cells (Ki67) were significantly up‐regulated (p < 0.05) in NSD14 (5.34 and 11.9 cells/0.95 mm2) while the proliferation index of ESD14 was similar to normal skin. In summary, cutaneous wounds receiving DW electrical stimulation display accelerated healing seen by reduced inflammation, enhanced angiogenesis and advanced remodeling phase.

Angiogenesis is induced and wound size is reduced by electrical stimulation in an acute wound healing model in human skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model

Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G2/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site.

Formation of multilayer aggregates of mammalian cells by dielectrophoresis

The formation of aggregates of mammalian cells at interdigitated oppositely castellated electrodes by positive dielectrophoresis was investigated. It is shown that, by using a constant small flow of fresh sorbitol iso-osmotic buffer through the chamber to remove ions leaking from the cells, a high positive DEP force can be maintained throughout the formation of the aggregates. Flow-rate dependent optima were found in the aggregate height as a function of the electrode size. It is shown that at low flow rates the creation of aggregates of mammalian cells with heights over 150 µm is feasible using relatively low voltages (20 Vpk–pk, 1 MHz). The formation of layered aggregates of two specialized cell types—stromal cells and Jurkat T lymphocytes—is demonstrated. The work confirms that dielectrophoresis can be reliably used for the formation of aggregates with three-dimensional architectures, which could be used as artificial microniches for the study of interactions between cells.

A novel in vitro assay for electrophysiological research on human skin fibroblasts: Degenerate electrical waves downregulate collagen I expression in keloid fibroblasts

Abstract:  Electrical stimulation (ES) has been used for the treatment of wounds and has been shown to alter gene expression and protein synthesis in skin fibroblasts in vitro. Here, we have developed a new in vitro model system for testing the effects of precisely defined, different types of ES on the collagen expression of normal and keloid human skin fibroblasts. Keloid fibroblasts were studied because they show excessive collagen production. Both types of fibroblasts were electrically stimulated with alternating current (AC), direct current (DC) or degenerate waves (DW). Cells were subjected to 20, 75 and 150 mV/mm electric field strengths at 10 and 60 Hz frequencies. At lower electric fields, all types of ES upregulated collagen I in both cell types compared to controls. However, at higher electric field strength (150 mV/mm) and frequency (60 Hz), DW maximally downregulated collagen I in keloid fibroblasts, yet had significantly lower cytotoxic effects on normal fibroblasts than AC and DC. Compared to unstimulated cells, both normal skin and keloid fibroblasts showed a significant decrease in collagen I expression after 12 h of DW and AC stimulation. In contrast, increasing amplitude of DC upregulated collagen I and PAI‐1 gene transcription in normal and keloid fibroblasts, along with increased cytotoxicity effects. Thus, our new preclinical assay system shows highly differential effects of specific types of ES on human fibroblast collagen expression and cytotoxicity and identifies DW of electrical current (DW) as a promising, novel therapeutic strategy for suppressing excessive collagen I formation in keloid disease.

Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: first report of a potential combination therapy.

BACKGROUND We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts. OBJECTIVE In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts. METHODS The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed. RESULTS PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05). CONCLUSION The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.

Formation of embryoid bodies using dielectrophoresis

Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs.

Optimization of an ex vivo wound healing model in the adult human skin: Functional evaluation using photodynamic therapy: functional evaluation using photodynamic therapy

Limited utility of in vitro tests and animal models of human repair, create a demand for alternative models of cutaneous healing capable of functional testing. The adult human skin Wound Healing Organ Culture (WHOC) provides a useful model, to study repair and enable evaluation of therapies such as the photodynamic therapy (PDT). Thus, the aim here was to identify the optimal WHOC model and to evaluate the role of PDT in repair. Wound geometry, system of support, and growth media, cellular and matrix biomarkers were investigated in WHOC models. Subsequently, cellular activity, extracellular matrix remodeling, and oxidative stress plus gene and protein levels of makers of wound repair measured the effect of PDT on the optimized WHOC. WHOCs embedded in collagen and supplemented DMEM were better organized showing stratified epidermis and compact dermis with developing neo‐epidermis. Post‐PDT, the advancing reepithelialization tongue was 3.5 folds longer, and was highly proliferative with CK‐14 plus p16 increased (p < 0.05) compared to controls. The neo‐epidermis was fully differentiated forming neo‐collagen. Proliferating nuclear antigen, p16, COLI, COLIII, MMP3, MMP19, and α‐SMA were significantly more expressed (p < 0.05) in dermis surrounding the healing wound. In conclusion, an optimal model of WHOC treated with PDT shows increased reepithelialization and extracellular matrix reconstruction and remodeling, supporting evidence toward development of an optimal ex vivo wound healing model.

Optimization of an ex vivo wound healing model in the adult human skin: Functional evaluation using photodynamic therapy

Limited utility of in vitro tests and animal models of human repair, create a demand for alternative models of cutaneous healing capable of functional testing. The adult human skin Wound Healing Organ Culture (WHOC) provides a useful model, to study repair and enable evaluation of therapies such as the photodynamic therapy (PDT). Thus, the aim here was to identify the optimal WHOC model and to evaluate the role of PDT in repair. Wound geometry, system of support, and growth media, cellular and matrix biomarkers were investigated in WHOC models. Subsequently, cellular activity, extracellular matrix remodeling, and oxidative stress plus gene and protein levels of makers of wound repair measured the effect of PDT on the optimized WHOC. WHOCs embedded in collagen and supplemented DMEM were better organized showing stratified epidermis and compact dermis with developing neo‐epidermis. Post‐PDT, the advancing reepithelialization tongue was 3.5 folds longer, and was highly proliferative with CK‐14 plus p16 increased (p < 0.05) compared to controls. The neo‐epidermis was fully differentiated forming neo‐collagen. Proliferating nuclear antigen, p16, COLI, COLIII, MMP3, MMP19, and α‐SMA were significantly more expressed (p < 0.05) in dermis surrounding the healing wound. In conclusion, an optimal model of WHOC treated with PDT shows increased reepithelialization and extracellular matrix reconstruction and remodeling, supporting evidence toward development of an optimal ex vivo wound healing model.

Enhancement of Differentiation and Mineralisation of Osteoblast-like Cells by Degenerate Electrical Waveform in an In Vitro Electrical Stimulation Model Compared to Capacitive Coupling

Electrical stimulation (ES) is effective in enhancing bone healing, however the best electrical waveform, mode of application and mechanisms remains unclear. We recently reported the in vitro differential healing response of a novel electrical waveform called degenerate sine wave (DW) compared to other forms of ES. This study further explores this original observation on osteoblast cells. Here, we electrically stimulated SaOS-2 osteoblast-like cells with DW in an in vitro ES chamber (referred to as ‘DW stimulation’) and compared the intracellular effects to capacitive coupling (CC) stimulation. ES lasted for 4 h, followed by an incubation period of 20 h and subsequent ES for 4 additional hours. Cytotoxicity, proliferation, differentiation and mineralisation of the osteoblast-like cells were evaluated to determine the cell maturation process. DW significantly enhanced the differentiation of cells when compared to CC stimulation with increased alkaline phosphatase and collagen I gene expression by quantitative real time- polymerase chain reaction analysis (p<0.01). Moreover, DW significantly increased the mineralisation of cells compared to CC stimulation. Furthermore the transcription of osteocalcin, osteonectin, osteopontin and bone sialoprotein (p<0.05) was also up regulated by DW. However, ES did not augment the proliferation of cells. Translational analysis by immunocytochemistry and Western blotting showed increased collagen I, osteocalcin and osteonectin expression after DW than CC stimulation. In summary, we have demonstrated for the first time that DW stimulation in an in vitro ES chamber has a significant effect on maturation of osteoblast-like cells compared to CC stimulation of the same magnitude.