A prospective clinical trial evaluating changes in the wound microenvironment in patients with chronic venous leg ulcers treated with a hypothermically stored amniotic membrane.

Amniotic tissues have been long utilised to treat chronic wounds; however, there are few studies evaluating how the wound microenvironment responds to these therapies. The goal of this study was to evaluate the changes in wounds treated with a hypothermically stored amniotic membrane (HSAM). In this prospective single-arm study, 15 female patients with venous leg ulcers were treated with HSAM from male donors and standard of care for 12 weeks. Over the course of the study, wound exudate was collected and evaluated using proteomic microarrays. Biopsies were collected during the course of treatment to detect the presence of HSAM tissue. By 4 weeks, 60% of subjects achieved 50% or greater reduction in wound size, and by 12 weeks, 53% of subjects achieved 100% re-epithelialization. HSAM DNA was detected in 20% of biopsies as determined by the detection TSPY4, indicating HSAM was no longer present within the wound bed approximately 7 days from the last treatment for the majority of wounds. Proteomic analysis of wound exudate found that wounds on a healing trajectory had significantly higher levels of MMP-10, MMP-7, and TIMP-4 and significantly lower levels of CX3CL1, FLT-3 L, IL-1ra, IL-1a, IL-9, IL-2, IL-3, MCP-1, and TNF-b compared with other wounds.

Controlled Delivery of Slit3 Proteins from Alginate Microbeads Inhibits In Vitro Angiogenesis.

BACKGROUND: The Slit-Robo pathway is a key regulator of angiogenesis and cellular function in experimental models. Slit3 proteins exhibit both proangiogenic and antiangiogenic properties, but the exact mechanism remains unclear. It is theorized that Slit3 may be a potential treatment for vascular diseases and cancer. METHODS: Slit3 labeled with I-125 was encapsulated in microbeads composed of low-viscosity alginate of high-glucuronic acid content, first coated with poly-L-ornithine for various durations and finally with low-viscosity high mannuronic acid. Gamma counter was used to measure microbead encapsulation efficiency and Slit3 release. Markers of angiogenesis were assessed with Boyden chamber, scratch wound, and Matrigel tube formation assays using human umbilical vein and mouse endothelial cells. RESULTS: On incubation of Slit3-loaded microbeads, there was an initial burst phase release of Slit3 for the first 24 h followed by sustained release for 6 to 12 d. Microbead composition determined encapsulation efficiency and rate of release; Slit3 encapsulation was most efficient in microbeads with lower low-viscosity alginate of high-glucuronic acid content concentrations (1.5%) and no poly-L-ornithine coating. Compared with controls (media alone), Slit3 microbeads significantly inhibited in vitro cellular migration, endothelial cell migration for wound closure at 24 and 48 h and endothelial tube formation (P < 0.001, respectively). CONCLUSIONS: Slit3 can be effectively encapsulated and delivered via a controlled release pattern using alginate microbeads. Microbead encapsulation reduces in vitro endothelial tube formation and inhibits cellular migration to impair angiogenesis. Thus, Slit3 microparticles may be explored as a therapeutic option to mitigate tumor proliferation.

A mechanistic evaluation of the angiogenic properties of a dehydrated amnion chorion membrane in vitro and in vivo.

Angiogenesis is essential for the successful repair of tissues; however, in many chronic conditions, angiogenesis is inhibited. Placental tissues have been shown to illicit an angiogenic response both in vitro and in vivo, and the angiogenic properties of these tissues likely contribute to observed clinical outcomes. Although there is some work describing the angiogenic effects of these tissues, comparatively little has been done to determine the possible mechanisms responsible for this effect. The purpose of this study was to conduct a thorough evaluation of a commercially available dehydrated amnion chorion membrane to better understand how these tissues may promote angiogenesis. The proteomic content of this tissue was evaluated using a high throughput proteomic microarray, and then the effects of these grafts were evaluated in vivo using subcutaneous gelfoam sponge implants containing conditioned media (CM) from the graft. Human microvascular endothelial cells were then used to determine how released factors effect migration, proliferation, gene expression, and protein production in vitro. Finally, to elucidate potential signaling-pathways through which tissue-derived factors act to induce pro-angiogenetic phenotypes in endothelial cells in vitro, we performed a global analysis of both serine/threonine and tyrosine kinase activity. Kinomic and proteomic data were then combined to generate protein-protein interaction networks that enabled the identification of multiple growth factors and cytokines with both pro- and anti-angiogenetic properties. In vivo, the addition of CM resulted in increased CD31 and αSMA staining and increases in pro-angiogenic gene expression. In vitro, CM resulted in significant increases in endothelial proliferation, migration, and the expression of granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, and transforming growth factor beta-3. Integrated kinomic analysis implicated ERK1/2 signaling as the primary pathway activated following culture of endothelial cells with dehydrated amnion/chorion membrane (dACM) CM. In conclusion, dACM grafts triggered pro-angiogenic responses both in vitro and in vivo that are likely at least partially mediated by ERK1/2 signaling.

Characterisation of dehydrated amnion chorion membranes and evaluation of fibroblast and keratinocyte responses in vitro.

The purpose of this study is to characterise the composition of a dehydrated amnion and chorion graft and investigate how factors released from this graft interact with cells important to the wound microenvironment using in vitro models. Characterisation was completed by proteomic analysis of growth factors and cytokines, evaluation of matrix components and protease inhibition, immunohistochemistry, and in vitro release of key growth factors and cytokines. To evaluate the effect of released factors on cells found within the microenvironment, in vitro assays including: cell proliferation, migration, gene expression, protein production, and intracellular pathway activation were used; additionally, responses of fibroblasts in the context of inflammation were measured. We found that released factors from dehydrated amnion/chorion membranes (dACM) stimulated cell proliferation, migration, and altered gene and protein expression profiles of cells important for wound repair in vitro. When cells were cultured in the presence of pro-inflammatory cytokines, the addition of releasate from dACM resulted in an altered production of cytokines, including a reduction of pro-inflammatory regulated on activation, normal T cell expressed and secreted (RANTES). In sum, the results presented here characterise the components of dACM, and in vitro studies were used to evaluate interactions of dACM with cell types important in wound healing.

Ethical considerations in tissue engineering research: Case studies in translation.

Tissue engineering research is a complex process that requires investigators to focus on the relationship between their research and anticipated gains in both knowledge and treatment improvements. The ethical considerations arising from tissue engineering research are similarly complex when addressing the translational progression from bench to bedside, and investigators in the field of tissue engineering act as moral agents at each step of their research along the translational pathway, from early benchwork and preclinical studies to clinical research. This review highlights the ethical considerations and challenges at each stage of research, by comparing issues surrounding two translational tissue engineering technologies: the bioartificial pancreas and a tissue engineered skeletal muscle construct. We present relevant ethical issues and questions to consider at each step along the translational pathway, from the basic science bench to preclinical research to first-in-human clinical trials. Topics at the bench level include maintaining data integrity, appropriate reporting and dissemination of results, and ensuring that studies are designed to yield results suitable for advancing research. Topics in preclinical research include the principle of "modest translational distance" and appropriate animal models. Topics in clinical research include key issues that arise in early-stage clinical trials, including selection of patient-subjects, disclosure of uncertainty, and defining success. The comparison of these two technologies and their ethical issues brings to light many challenges for translational tissue engineering research and provides guidance for investigators engaged in development of any tissue engineering technology.

In vitro assessment of a novel, hypothermically stored amniotic membrane for use in a chronic wound environment.

Chronic wounds require extensive healing time and place patients at risk of infection and amputation. Recently, a fresh hypothermically stored amniotic membrane (HSAM) was developed and has subsequently shown promise in its ability to effectively heal chronic wounds. The purpose of this study is to investigate the mechanisms of action that contribute to wound-healing responses observed with HSAM. A proteomic analysis was conducted on HSAM, measuring 25 growth factors specific to wound healing within the grafts. The rate of release of these cytokines from HSAMs was also measured. To model the effect of these cytokines and their role in wound healing, proliferation and migration assays with human fibroblasts and keratinocytes were conducted, along with tube formation assays measuring angiogenesis using media conditioned from HSAM. Additionally, the cell-matrix interactions between fibroblasts and HSAM were investigated. Conditioned media from HSAM significantly increased both fibroblast and keratinocyte proliferation and migration and induced more robust tube formation in angiogenesis assays. Fibroblasts cultured on HSAMs were found to migrate into and deposit matrix molecules within the HSAM graft. These collective results suggest that HSAM positively affects various critical pathways in chronic wound healing, lending further support to promising qualitative results seen clinically and providing further validation for ongoing clinical trials.

The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas.

OBJECTIVES: Our study aims at producing acellular extracellular matrix scaffolds from the human pancreas (hpaECMs) as a first critical step toward the production of a new-generation, fully human-derived bioartificial endocrine pancreas. In this bioartificial endocrine pancreas, the hardware will be represented by hpaECMs, whereas the software will consist in the cellular compartment generated from patient's own cells. BACKGROUND: Extracellular matrix (ECM)-based scaffolds obtained through the decellularization of native organs have become the favored platform in the field of complex organ bioengineering. However, the paradigm is now switching from the porcine to the human model. METHODS: To achieve our goal, human pancreata were decellularized with Triton-based solution and thoroughly characterized. Primary endpoints were complete cell and DNA clearance, preservation of ECM components, growth factors and stiffness, ability to induce angiogenesis, conservation of the framework of the innate vasculature, and immunogenicity. Secondary endpoint was hpaECMs' ability to sustain growth and function of human islet and human primary pancreatic endothelial cells. RESULTS: Results show that hpaECMs can be successfully and consistently produced from human pancreata and maintain their innate molecular and spatial framework and stiffness, and vital growth factors. Importantly, hpaECMs inhibit human naïve CD4 T-cell expansion in response to polyclonal stimuli by inducing their apoptosis and promoting their conversion into regulatory T cells. hpaECMs are cytocompatible and supportive of representative pancreatic cell types. DISCUSSION: We, therefore, conclude that hpaECMs has the potential to become an ideal platform for investigations aiming at the manufacturing of a regenerative medicine-inspired bioartificial endocrine pancreas.

Evaluation of two distinct placental-derived membranes and their effect on tenocyte responses in vitro.

Tendon healing is a complex, multiphase process that results in increased scar tissue formation, leading to weaker tendons. The purpose of this study was to evaluate the response of tenocytes to both hypothermically stored amniotic membrane (HSAM) and dehydrated amnion/chorion membrane (dACM). Composition and growth factor release from HSAM and dACM were evaluated using proteomics microarrays. HSAM and dACM releasate was used to assess tenocyte proliferation, migration, gene expression, extracellular matrix (ECM) protein deposition, and response to inflammation. Additionally, tenocyte-ECM interactions were evaluated. HSAM and dACM contain and release growth factors relevant to tendon healing, including insulin-like growth factor I, platelet-derived growth factor, and basic fibroblast growth factor. Both dACM and HSAM promoted increased tenocyte proliferation and migration; tenocytes treated with dACM proliferated more robustly, whereas treatment with HSAM resulted in higher migration. Both dACM and HSAM resulted in altered ECM gene expression; dACM grafts alone resulted in increases in collagen deposition. Furthermore, both allografts resulted in altered tenocyte responses to inflammation with reduced transforming growth factor beta levels. Additionally, dACM treatment resulted in increased expression and production of matrix metalloprotease-1 (MMP-1), whereas HSAM treatment resulted in decreased production of MMP-1. Tenocytes migrated into and remodeled HSAM only. These results indicate that both grafts have properties that support tendon healing; however, the results presented here suggest that the responses to each type of graft may be different. Due to the complex environment during tendon repair, additional work is needed to evaluate these effects using in vivo models.

Tenocyte cell density, migration, and extracellular matrix deposition with amniotic suspension allograft.

Amniotic suspension allografts (ASA), derived from placental tissues, contain particulated amniotic membrane and amniotic fluid cells. Recently, ASA and other placental-derived allografts have been used in orthopaedic applications, including tendinopathies and tendon injuries. The purpose of this study was to determine the potential effects of ASA on tenocyte cell density, migration, and responses to inflammatory stimuli. Tenocyte cell density was measured using AlamarBlue over multiple time points, while migration was determined using a Boyden chamber assay. Deposition of ECM markers were measured using BioColor kits. Gene expression and protein production of cytokines and growth factors following stimulus with pro-inflammatory IL-1ß and TNF-α was measured using qPCR and ELISAs. Conditioned media (CM) was made from ASA and used for all assays in this study. In vitro, ASA CM treatment significantly promoted tenocyte increases in cell density and migration compared to assay media controls. ASA CM also increased the deposition of extracellular matrix (ECM) proteins, including collagen, elastin, and sGAG. Following inflammatory stimulation and treatment with ASA CM, tenocytes downregulated IL-8 gene expression, a pro-inflammatory cytokine normally elevated during the inflammatory phase of tendon healing. Additionally, tenocytes treated with ASA CM had significantly lower protein levels of TGF-ß1 compared to controls. This study evaluated ASA and its effect on tenocytes; specifically, treatment with ASA resulted in increased cell density, more robust migration and matrix deposition, and some alteration of inflammatory targets. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:412-420, 2019.

Dehydrated Amnion/Chorion Improves Achilles Tendon Repair in a Diabetic Animal Model.

INTRODUCTION: Healing of tendon injuries is often plagued by significant scar formation and compromised biomechanical function. For those with diabetes, these injuries are further complicated by alterations to the extracellular matrix of the tendon, poor circulation, and delayed wound healing; consequently, complications and re-rupture rates for patients with diabetes are reported higher than the typical patient population. Placental derived membranes, specifically dehydrated human amnion/chorion membranes (dACMs), have been utilized clinically as an adhesion barrier, and these membranes have been shown to reduce scarring and aid in tissue repair. OBJECTIVE: The purpose of this study was to evaluate the effect of dACMs on tendon repair in a diabetic model with impaired healing. MATERIALS AND METHODS: Using a type II diabetic model (BBZDR/WOR rats), a full-thickness injury was made through the Achilles tendon and repaired using a modified Kessler method. Repaired tendons were wrapped with dACM or left unwrapped as a control (n = 15/group; n = 30 total). Tendons were retrieved at 14 (n = 5/group; n = 10 total) or 28 days (n = 10/group; n = 20 total) and evaluated using histology, immunofluorescence, and biomechanical testing. RESULTS: Treatment of tendons with dACM resulted in reduced failure rates, increased cell migration, and improved mechanical properties (compared with unwrapped controls). The dACM-treated tendons also showed changes in the production of several important biomarkers to tendon healing at both 14 and 28 days; most notably, Scleraxis was found to be upregulated in dACM-treated tendons. CONCLUSIONS: This study highlights a promising treatment option for this challenging clinical population.

Treatment with Human Amniotic Suspension Allograft Improves Tendon Healing in a Rat Model of Collagenase-Induced Tendinopathy.

Treatment of tendon injuries is challenging, with neither conservative nor surgical approaches providing full recovery. Placental-derived tissues represent a promising tool for the treatment of tendon injuries. In this study, human amniotic suspension allograft (ASA) was investigated in a pre-clinical model of Achilles tendinopathy. Collagenase type I was injected in the right hind limb of Sprague Dawley rats to induce disease. Contralateral tendons were either left untreated or injected with saline as controls. Seven days following induction, tendons were injected with saline, ASA, or left untreated. Rats were sacrificed 14 and 28 days post-treatment. Histological and biomechanical analysis of tendons was completed. Fourteen days after ASA injection, improved fiber alignment and reduced cell density demonstrated improvement in degenerated tendons. Twenty-eight days post-treatment, tendons in all treatment groups showed fewer signs of degeneration, which is consistent with normal tendon healing. No statistically significant differences in histological or biomechanical analyses were observed between treatment groups at 28 days independent of the treatment they received. In this study, ASA treatment was safe, well-tolerated, and resulted in a widespread improvement of the tissue. The results of this study provide preliminary insights regarding the potential use of ASA for the treatment of Achilles tendinopathy.

Proteomic Comparison of Amnion and Chorion and Evaluation of the Effects of Processing on Placental Membranes.

OBJECTIVE: The purpose of this study is to compare the growth factor and cytokine content found within the amnion and chorion layers and to determine the effects of dehydration on them. MATERIALS AND METHODS: Placentas were collected from 5 to 6 consented donors following elective cesarean section, and 1-cm2 sections of either amnion or chorion were immediately stored at -80°C or dehydrated prior to -80°C storage until proteomic analysis. Signaling molecules from tissue samples were evaluated using quantitative multiplex proteomics microarrays, and data were analyzed based on a per cm2 basis and also on pg/mg of extracted protein for potency. RESULTS: Fresh chorion contained more of some signaling molecules per cm2 compared with amnion. Specifically, the chorion contained significantly higher levels of adiponectin, APN, ANG-2, bFGF, EG-VEGF, HGF, IGF-1, PDGF-AA, PDGF-BB, TIMP-2, and TIMP-4. When samples were dehydrated, a significant drop in total growth factor and cytokine content was observed in both amnion and chorion samples with a loss of 51.1% ± 20.2% and 55.5% ± 37.3%, respectively. When evaluating the potency of fresh amnion and fresh chorion, there were similar levels of signaling molecules found with some exceptions. Amnion had significantly higher GAL-7, TGF-ß1, and IL-1F5, and chorion had significantly more EG-VEGF, PDGF-BB, and TIMP-2. CONCLUSION: The processing of placental membranes can have a dramatic effect on the total growth factor and cytokine load found within these tissues.

Applications of particulate oxygen-generating substances (POGS) in the bioartificial pancreas.

Type-1 Diabetes (T1D) is a devastating autoimmune disorder which results in the destruction of beta cells within the pancreas. A promising treatment strategy for T1D is the replacement of the lost beta cell mass through implantation of immune-isolated microencapsulated islets referred to as the bioartificial pancreas. The goal of this approach is to restore blood glucose regulation and prevent the long-term comorbidities of T1D without the need for immunosuppressants. A major requirement in the quest to achieve this goal is to address the oxygen needs of islet cells. Islets are highly metabolically active and require a significant amount of oxygen for normal function. During the process of isolation, microencapsulation, and processing prior to transplantation, the islets' oxygen supply is disrupted, and a large amount of islet cells are therefore lost due to extended hypoxia, thus creating a major barrier to clinical success with this treatment. In this work, we have investigated the oxygen generating compounds, sodium percarbonate (SPO) and calcium peroxide (CPO) as potential supplemental oxygen sources for islets during isolation and encapsulation before and immediately after transplantation. First, SPO particles were used as an oxygen source for islets during isolation. Secondly, silicone films containing SPO were used to provide supplemental oxygen to islets for up to 4 days in culture. Finally, CPO was used as an oxygen source for encapsulated cells by co-encapsulating CPO particles with islets in permselective alginate microspheres. These studies provide an important proof of concept for the utilization of these oxygen generating materials to prevent beta cell death caused by hypoxia.

In vivo transplantation of 3D encapsulated ovarian constructs in rats corrects abnormalities of ovarian failure.

Safe clinical hormone replacement (HR) will likely become increasingly important in the growing populations of aged women and cancer patients undergoing treatments that ablate the ovaries. Cell-based HRT (cHRT) is an alternative approach that may allow certain physiological outcomes to be achieved with lower circulating hormone levels than pharmacological means due to participation of cells in the hypothalamus-pituitary-ovary feedback control loop. Here we describe the in vivo performance of 3D bioengineered ovarian constructs that recapitulate native cell-cell interactions between ovarian granulosa and theca cells as an approach to cHRT. The constructs are fabricated using either Ca++ or Sr++ to crosslink alginate. Following implantation in ovariectomized (ovx) rats, the Sr++-cross-linked constructs achieve stable secretion of hormones during 90 days of study. Further, we show these constructs with isogeneic cells to be effective in ameliorating adverse effects of hormone deficiency, including bone health, uterine health, and body composition in this rat model.

In Vitro Proliferation of Porcine Pancreatic Islet Cells for ß-Cell Therapy Applications.

ß-Cell replacement through transplantation is the only curative treatment to establish a long-term stable euglycemia in diabetic patients. Owing to the shortage of donor tissue, attempts are being made to develop alternative sources of insulin-secreting cells. Stem cells differentiation and reprograming as well as isolating pancreatic progenitors from different sources are some examples; however, no approach has yet yielded a clinically relevant solution. Dissociated islet cells that are cultured in cell numbers by in vitro proliferation provide a promising platform for redifferentiation towards ß-cells phenotype. In this study, we cultured islet-derived cells in vitro and examined the expression of ß-cell genes during the proliferation. Islets were isolated from porcine pancreases and enzymatically digested to dissociate the component cells. The cells proliferated well in tissue culture plates and were subcultured for no more than 5 passages. Only 10% of insulin expression, as measured by PCR, was preserved in each passage. High glucose media enhanced insulin expression by about 4-18 fold, suggesting a glucose-dependent effect in the proliferated islet-derived cells. The islet-derived cells also expressed other pancreatic genes such as Pdx1, NeuroD, glucagon, and somatostatin. Taken together, these results indicate that pancreatic islet-derived cells, proliferated in vitro, retained the expression capacity for key pancreatic genes, thus suggesting that the cells may be redifferentiated into insulin-secreting ß-like cells.

Long-term function of islets encapsulated in a redesigned alginate microcapsule construct in omentum pouches of immune-competent diabetic rats.

OBJECTIVE: Our study aim was to determine encapsulated islet graft viability in an omentum pouch and the effect of fibroblast growth factor 1 (FGF-1) released from our redesigned alginate microcapsules on the function of the graft. METHODS: Isolated rat islets were encapsulated in an inner core made with 1.5% low-viscosity-high-mannuronic-acid alginate followed by an external layer made with 1.25% low-viscosity high-guluronic acid alginate with or without FGF-1, in microcapsules measuring 300 to 400 µm in diameter. The 2 alginate layers were separated by a perm-selective membrane made with 0.1% poly-L-ornithine, and the inner low-viscosity-high-mannuronic-acid core was partially chelated using 55 mM sodium citrate for 2 minutes. RESULTS: A marginal mass of encapsulated islet allografts (∼2000 islets/kg) in streptozotocin-diabetic Lewis rats caused significant reduction in blood glucose levels similar to the effect observed with encapsulated islet isografts. Transplantation of alloislets coencapsulated with FGF-1 did not result in better glycemic control, but induced greater body weight maintenance in transplant recipients compared with those that received only alloislets. Histological examination of the retrieved tissue demonstrated morphologically and functionally intact islets in the microcapsules, with no signs of fibrosis. CONCLUSIONS: We conclude that the omentum is a viable site for encapsulated islet transplantation.

Microencapsulation of pancreatic islets for use in a bioartificial pancreas.

Islet transplantation is the most exciting treatment option for individuals afflicted with Type 1 diabetes. However, the severe shortage of human pancreas and the need to use risky immunosuppressive drugs to prevent transplant rejection remain two major obstacles for the routine use of islet transplantation in diabetic patients. Successful development of a bioartificial pancreas using the approach of microencapsulation with perm-selective coating of islets with biopolymers for graft immunoisolation holds tremendous promise for diabetic patients because it has great potential to overcome these two barriers. In this chapter, we provide a detailed description of the microencapsulation process.

Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering.

Emergent technologies of regenerative medicine have the potential to overcome the limitations of organ transplantation by supplying tissues and organs bioengineered in the laboratory. Pancreas bioengineering requires a scaffold that approximates the biochemical, spatial and vascular relationships of the native extracellular matrix (ECM). We describe the generation of a whole organ, three-dimensional pancreas scaffold using acellular porcine pancreas. Imaging studies confirm that our protocol effectively removes cellular material while preserving ECM proteins and the native vascular tree. The scaffold was seeded with human stem cells and porcine pancreatic islets, demonstrating that the decellularized pancreas can support cellular adhesion and maintenance of cell functions. These findings advance the field of regenerative medicine towards the development of a fully functional, bioengineered pancreas capable of establishing and sustaining euglycemia and may be used for transplantation to cure diabetes mellitus.

Application of low-frequency alternating current electric fields via interdigitated electrodes: effects on cellular viability, cytoplasmic calcium, and osteogenic differentiation of human adipose-derived stem cells.

Electric stimulation is known to initiate signaling pathways and provides a technique to enhance osteogenic differentiation of stem and/or progenitor cells. There are a variety of in vitro stimulation devices to apply electric fields to such cells. Herein, we describe and highlight the use of interdigitated electrodes to characterize signaling pathways and the effect of electric fields on the proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). The advantage of the interdigitated electrode configuration is that cells can be easily imaged during short-term (acute) stimulation, and this identical configuration can be utilized for long-term (chronic) studies. Acute exposure of hASCs to alternating current (AC) sinusoidal electric fields of 1 Hz induced a dose-dependent increase in cytoplasmic calcium in response to electric field magnitude, as observed by fluorescence microscopy. hASCs that were chronically exposed to AC electric field treatment of 1 V/cm (4 h/day for 14 days, cultured in the osteogenic differentiation medium containing dexamethasone, ascorbic acid, and ß-glycerol phosphate) displayed a significant increase in mineral deposition relative to unstimulated controls. This is the first study to evaluate the effects of sinusoidal AC electric fields on hASCs and to demonstrate that acute and chronic electric field exposure can significantly increase intracellular calcium signaling and the deposition of accreted calcium under osteogenic stimulation, respectively.