Optimal delivery of endothelial progenitor cells in a rat model of critical-size bone defects.

Nonunion and segmental bone defects are complex issues in orthopedic trauma. The use of endothelial progenitor cells (EPCs), as part of a cell-based therapy for bone healing is a promising approach. In preclinical studies, culture medium (CM) is commonly used to deliver EPCs to the defect site, which has the potential for immunogenicity in humans. The goal of this study was to find an effective and clinically translatable delivery medium for EPCs. Accordingly, this study compared EPCs delivered in CM, phosphate-buffered saline (PBS), platelet-poor plasma (PPP), and platelet-rich plasma (PRP) in a rat model of femoral critical-size defects. Fischer 344 rats (n = 35) were divided into six groups: EPC+CM, EPC+PBS, EPC+PPP, EPC+PRP, PPP alone, and PRP alone. A 5 mm mid-diaphyseal defect was created in the right femur and stabilized with a miniplate. The defect was filled with a gelatin scaffold impregnated with the corresponding treatment. Radiographic, microcomputed tomography and biomechanical analyses were performed. Overall, regardless of the delivery medium, groups that received EPCs had higher radiographic scores and union rates, higher bone volume, and superior biomechanical properties compared to groups treated with PPP or PRP alone. There were no significant differences in any outcomes between EPC subgroups or between PPP and PRP alone. These results suggest that EPCs are effective in treating segmental defects in a rat model of critical-size defects regardless of the delivery medium used. Consequently, PBS could be the optimal medium for delivering EPCs, given its low cost, ease of preparation, accessibility, noninvasiveness, and nonimmunogenic properties.

Chronic noncancer pain management: Integration of a nurse-led program in primary care.

PROBLEM ADDRESSED: Chronic noncancer pain is often excessively managed with medications (most notably opioids) instead of nonpharmacologic options or multidisciplinary care-the gold standards. OBJECTIVE OF PROGRAM: To offer an effective alternative to pharmacologic management of chronic noncancer pain in primary care. PROGRAM DESCRIPTION: Patients 18 years of age or older with chronic noncancer pain were referred by family physicians or nurse practitioners in a family health team (outpatient, multidisciplinary clinic) in Ottawa, Ont. A registered nurse used the Pain Explanation and Treatment Diagram with patients, taught self-management skills (related to habits [smoking, consumption of alcohol, diet], exercise, sleep, ergonomics, and psychosocial factors), and referred patients to relevant resources. CONCLUSION: A nurse-led chronic pain program, initiated without extra funding, was successfully integrated into a primary care setting. Among the participating patients in the pilot project, outcomes related to pain intensity, pain interference with daily living, and opioid use were encouraging. This program could serve as a model for improving chronic noncancer pain management in primary care.

The induced membrane technique: Optimization of bone grafting in a rat model of segmental bone defect.

INTRODUCTION: The induced membrane technique (IMT) is a two-stage surgical procedure used to treat fracture nonunion and bone defects. Although there is an increasing number of animal studies investigating the IMT, few have examined the outcomes of bone healing after a second stage grafting procedure. This study aimed at comparing two bone grafting procedures, as part of the IMT, in order to establish a rat model providing consistent healing outcomes. METHODS: In male Fischer 344 rats, we created a 5 mm defect in the right femur, stabilized the bone with a plate and screws, and inserted a polymethylmethacrylate spacer into the defect. Four weeks later, the spacer was removed. Bone graft was harvested from a donor rat and placed into the defect, followed by membrane and wound closure. Experiments were conducted in two groups. In group 1 (n = 11), the bone graft contained a variable amount of cortical and cancellous bone, the time from donor euthanasia to grafting was up to 240 min, and one donor rat provided graft for 5-6 recipients. In group 2 (n = 12), we reduced the contribution of cortical bone to the graft, included bone marrow, and kept donor euthanasia to grafting time under 150 min. One donor was used per 3-4 recipients. The volume of graft per recipient and all other elements of the protocol were the same across groups. Bone healing at 12 weeks post grafting was compared radiographically by two orthopaedic surgeons in a blinded fashion, based on union status and a modified Lane & Sandhu score. RESULTS: Healing rates improved from 36.4% in Group 1 to 91.6% in Group 2. There was a significant relationship between the methods and resulting union status (p = 0.004). The odds of achieving full union were significantly higher in group 2 compared to group 1 (odds ratio=19.25, 95% confidence interval [1.77-209.55]; p = 0.009). The average radiographic score was also significantly higher in group 2 (p = 0.005). CONCLUSION: The revised bone grafting method significantly improved the healing outcomes and contributed to establishing a consistent rat model of the IMT. This model can benefit preclinical investigations by allowing for reliable and clinically-relevant comparisons.

The induced membrane technique in animal models: a systematic review.

Objectives: The induced membrane technique (IMT) is a 2-stage surgical approach that has become increasingly popular to manage bone defects. Preclinical investigations have been conducted to better understand and define several aspects of this technique. This review summarizes the literature regarding the IMT performed in animal models and identifies potential future directions. Data Sources: Biosis Citation Index, Ovid Embase, and Ovid MEDLINE databases were searched from inception up to June 23, 2021 for articles related to the IMT. Study Selection: Animal studies involving the use of the IMT for segmental defects in long bones were selected. Only full-length original research articles published in English or French were included. Data Extraction: Two authors extracted the data from the selected studies and a third author verified the accuracy of the information. Data Synthesis: Information concerning the animal model, the surgical procedures, and the outcome measures were recorded for each study and compiled. Conclusions: Forty-seven studies were included in this review. Twenty-nine studies (62%) performed both stages of the technique, but only 8 (17%) reported on radiographic union rates explicitly and 5 (11%) included biomechanical testing. A large proportion of the preclinical literature on the IMT has failed to report on radiographic union as an outcome. While studies reporting membrane properties are valuable, they may not provide information that translates into clinical practice or further clinical research if the ultimate outcome of bony healing is not considered. Future animal studies of the IMT should consider this in their study design.

Systemically impaired fracture healing in small animal research: A review of fracture repair models.

Fracture healing is a complex process requiring mechanical stability, an osteoconductive matrix, and osteoinductive and osteogenic biology. This intricate process is easily disrupted by various patient factors such as chronic disease and lifestyle. As the medical complexity and age of patients with fractures continue to increase, the importance of developing relevant experimental models is becoming paramount in preclinical research. The objective of this review is to describe the most common small animal models of systemically impaired fracture healing used in the orthopedic literature including osteoporosis, diabetes mellitus, smoking, alcohol use, obesity, and ageing. This review will provide orthopedic researchers with a summary of current models of systemically impaired fracture healing used in small animals and present an overview of the methods of induction for each condition.

The induced membrane technique for the management of long bone defects.

AIMS: The purpose of this study was to: review the efficacy of the induced membrane technique (IMT), also known as the Masquelet technique; and investigate the relationship between patient factors and technique variations on the outcomes of the IMT. METHODS: A systematic search was performed in CINAHL, The Cochrane Library, Embase, Ovid MEDLINE, and PubMed. We included articles from 1 January 1980 to 30 September 2019. Studies with a minimum sample size of five cases, where the IMT was performed primarily in adult patients (≥ 18 years old), in a long bone were included. Multivariate regression models were performed on patient-level data to determine variables associated with nonunion, postoperative infection, and the need for additional procedures. RESULTS: A total of 48 studies were included, with 1,386 cases treated with the IMT. Patients had a mean age of 40.7 years (4 to 88), and the mean defect size was 5.9 cm (0.5 to 26). In total, 82.3% of cases achieved union after the index second stage procedure. The mean time to union was 6.6 months (1.4 to 58.7) after the second stage. Our multivariate analysis of 450 individual patients showed that the odds of developing a nonunion were significantly increased in those with preoperative infection. Patients with tibial defects, and those with larger defects, were at significantly higher odds of developing a postoperative infection. Our analysis also demonstrated a trend towards the inclusion of antibiotics in the cement spacer having a protective effect against the need for additional procedures. CONCLUSION: The IMT is an effective management strategy for complex segmental bone defects. Standardized reporting of individual patient data or larger prospective trials is required to determine the optimal implementation of this technique. This is the most comprehensive review of the IMT, and the first to compile individual patient data and use regression models to determine predictors of outcomes. Cite this article: Bone Joint J 2020;102-B(12):1723-1734.

Understanding Articular Cartilage Injury and Potential Treatments.

The goals of all orthopaedic surgeons treating articular cartilage injuries have been anatomic reduction and stable fixation of the articular cartilage surface with restoration of limb alignment and/or reestablishment of the joint stability, all while minimizing the risk of surgical complications. Recent developments in the study of articular cartilage injury have shown that there is a robust cellular response to joint injury. This response has been shown to involve the synoviocytes, chondrocytes, and osteocytes in and around the injured joint and if these responses are left unchecked, they can lead to the development of posttraumatic osteoarthritis (PTOA). Therefore, to predictably and successfully treat articular cartilage injuries, it is not sufficient to just restore articular congruity, limb alignment, and joint stability, but we must also recognize and attempt to mitigate this associated cellular response. Understanding not only the mechanical aspects of these joint injuries but also the biological aspects is paramount to giving our patients the best opportunity to heal their injuries, recover full function, and avoid the potential devastating development of PTOA. Gone is the simplistic view that if one can achieve articular congruity after intraarticular fracture, as well as joint stability after ligamentous injury, that our patients will do just fine. This review sheds new light on the molecular response to cartilage injury, how residual joint incongruity and instability affect the joint's ability to recover from injury, and how chondrocyte apoptosis in response to injury can influence joint. This article then briefly reviews how cellular and growth factors may be beneficial to the treatment of articular cartilage injury and how ultimately cartilage regeneration may be used in the future to salvage the joints ravaged by PTOA in response to injury.

Experimental Validation of the Radiographic Union Score for Tibial Fractures (RUST) Using Micro-Computed Tomography Scanning and Biomechanical Testing in an in-Vivo Rat Model.

BACKGROUND: The Radiographic Union Score for Tibial fractures (RUST) and the modified version of the system, mRUST, are popular standards for assessing fracture-healing progress with use of radiographs. To our knowledge, this is the first study to experimentally validate the ability of RUST and mRUST to accurately assess bone-healing progression with use of both micro-computed tomography (micro-CT) scanning and biomechanical testing. METHODS: Adult male rats (n = 29) underwent osteotomy with a midshaft fracture gap repaired with use of a polyetheretherketone plate. Anteroposterior and lateral radiographs were made of the repaired femora prior to rat death at end points of 5, 6, 7, 8, 9, and 17 weeks, and 2 fellowship-trained orthopaedic trauma surgeons independently assigned RUST and mRUST scores to repaired femora. The repaired and intact contralateral femora were then dissected. Bones underwent dissection, micro-CT scanning, and biomechanical torsion testing at the end points. RESULTS: RUST scores ranged from 5 to 12 and mRUST scores ranged from 5 to 16. Intraclass correlation coefficients (ICCs) were 0.89 (95% confidence interval [CI]: 0.78 to 0.94) for RUST and 0.86 (95% CI: 0.74 to 0.93) for mRUST, which fall within the "almost perfect agreement" category for ICCs. Spearman rank correlation coefficients (RS) showed correlation of RUST (RS range, 0.456 to 0.818) and mRUST (RS range, 0.519 to 0.862) with micro-CT measurements of mineralized callus volume (BV), total callus volume (TV), and BV/TV ratio, but less so with bone mineral density (BMD). Additionally, RUST (RS range, 0.524 to 0.863) and mRUST (RS range, 0.434 to 0.850) were correlated with some biomechanical properties. A RUST score of 10 or an mRUST score of 15 may be considered the threshold above which a plated bone is "healed" because, at these scores, 120% or 140% of failure torque, respectively, was achieved by the repaired femora as compared with the intact contralateral femora. CONCLUSIONS: RUST and mRUST both show strong statistical correlations with micro-CT and biomechanical parameters. CLINICAL RELEVANCE: RUST and mRUST scoring systems provide clinicians with validated, reliable, and available tools to assess the progress of fracture-healing.

Diabetes and Healing Outcomes in Lower Extremity Fractures: A Systematic Review.

OBJECTIVE: The purpose of this study was to review the rates of adverse healing outcomes following surgical fixation of lower extremity fractures in diabetic patients and matched controls. MATERIALS AND METHODS: Searches of PubMed, MEDLINE, CINAHL and Embase were performed for studies published between the date of database inception and July 6, 2015. Patient characteristics and the incidence of adverse healing outcomes (nonunion, malunion, delayed union, infection and reoperation) were extracted from each study. The occurrence of each fracture healing complication was pooled and analyzed for comparisons between diabetic and non-diabetic patients. An odds ratio with a 95% confidence interval for each healing outcome was calculated between the diabetic and non-diabetic groups. RESULTS: Diabetes was found to significantly increase rates of malunion, infection and reoperation in patients with surgically treated lower extremity fractures. In addition, when only peripheral lower extremity fractures (i.e. below the knee) were examined, diabetes significantly increased the rates of nonunion. CONCLUSION: Diabetes substantially alters bone metabolism and soft tissue healing, posing a risk of adverse fracture healing and other complications. This systematic review provides evidence that the presence of diabetes significantly increases the risks of infection, malunion, nonunion and re-operation across a wide variety of surgically treated lower extremity fractures. This study provides prognostic information for clinicians and may aid in guiding treatment for this population.

Subtypes of endothelial progenitor cells affect healing of segmental bone defects differently.

PURPOSE: Treating fracture nonunion with endothelial progenitor cells (EPCs) is a promising approach. Nevertheless, the effect of different EPC-related cell populations remains unclear. In this study, we compared the therapeutic potential of early (E-EPCs) and late EPCs (L-EPCs). METHODS: Male Fischer 344 rats were used for cell isolation and in vivo experiments. Bone marrow-derived E-EPCs and L-EPCs were kept in culture for seven to ten days and four weeks, respectively. For each treatment group, we seeded one million cells on a gelatin scaffold before implantation in a segmental defect created in a rat femur; control animals received a cell-free scaffold. Bone healing was monitored via radiographs for up to ten weeks after surgery. In vitro, secretion of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 was quantified by ELISA for both cell populations. Tube formation assays were also performed. RESULTS: Final radiographs showed complete (four out of five rats) or partial (one out of five rats) union with E-EPC treatment. In contrast, complete healing was achieved in only one of five animals after L-EPC implantation, while control treatment resulted in nonunion in all animals. In vitro, E-EPCs released more VEGF, but less BMP-2 than L-EPCs. In addition, L-EPCs formed longer and more mature tubules on basement membrane matrix than E-EPCs. However, co-culture with primary osteoblasts stimulated tubulogenesis of E-EPCs while inhibiting that of L-EPCs. CONCLUSIONS: We demonstrated that bone marrow-derived E-EPCs are a better alternative than L-EPCs for treatment of nonunion. We hypothesize that the expression profile of E-EPCs and their adaptation to the local environment contribute to superior bone healing.

Delayed Endothelial Progenitor Cell Therapy Promotes Bone Defect Repair in a Clinically Relevant Rat Model.

The repair of segmental bone defects remains a significant challenge for orthopaedic surgeons. Endothelial progenitor cells (EPCs) have successfully promoted the repair of acute defects in animal models; however, the ability of EPCs to induce the repair of chronic nonhealing defects, such as those often encountered clinically, has not been investigated. Therefore, the purpose of this study was to investigate the ability of EPCs delivered in delayed fashion to induce the repair of nonhealing defects in a clinically relevant model. In order to simulate delayed treatment, 5 mm segmental defects in Fischer 344 rat femora were treated with bone marrow-derived EPCs on a Gelfoam scaffold at 3 weeks post creation of the defect. At ten weeks posttreatment, 100% of EPC-treated defects achieved union, whereas complete union was only achieved in 37.5% of defects treated with Gelfoam alone. Furthermore, significant increases in ultimate torque (p = 0.022) and torsional stiffness (p = 0.003) were found in EPC-treated defects compared to controls. Critically, no differences in outcomes were observed between acute and delayed EPC treatments. These results suggest that EPCs can enhance bone healing when applied in an acute or delayed fashion and that their use may represent a clinically translatable therapy for bone healing in humans.

Differences between the cell populations from the peritenon and the tendon core with regard to their potential implication in tendon repair.

The role of intrinsic and extrinsic healing in injured tendons is still debated. In this study, we characterized cell plasticity, proliferative capacity, and migration characteristics as proxy measures of healing potential in cells derived from the peritenon (extrinsic healing) and compared these to cells from the tendon core (intrinsic healing). Both cell populations were extracted from horse superficial digital flexor tendon and characterized for tenogenic and matrix remodeling markers as well as for rates of migration and replication. Furthermore, colony-forming unit assays, multipotency assays, and real-time quantitative polymerase chain reaction analyses of markers of osteogenic and adipogenic differentiation after culture in induction media were performed. Finally, cellular capacity for differentiation towards a myofibroblastic phenotype was assessed. Our results demonstrate that both tendon- and peritenon-derived cell populations are capable of adipogenic and osteogenic differentiation, with higher expression of progenitor cell markers in peritenon cells. Cells from the peritenon also migrated faster, replicate more quickly, and show higher differentiation potential toward a myofibroblastic phenotype when compared to cells from the tendon core. Based on these data, we suggest that cells from the peritenon have substantial potential to influence tendon-healing outcome, warranting further scrutiny of their role.

The mechanical environment modulates intracellular calcium oscillation activities of myofibroblasts.

Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.

Hypoxia impairs skin myofibroblast differentiation and function.

Ischemic wounds are characterized by oxygen levels lower than that of healthy skin (hypoxia) and poor healing. To better understand the pathophysiology of impaired wound healing, we investigated how switching from high (21%) to low (2%) oxygen levels directly affects cultured skin myofibroblasts, essential cells for the normal wound repair process. Myofibroblast differentiation and function were assessed by quantifying α-smooth muscle actin expression and cell contraction in collagen gels and on wrinkling silicone substrates. Culture for 5 days at 2% oxygen is perceived as hypoxia and significantly reduced myofibroblast differentiation and contraction despite high levels of the profibrotic transforming growth factor-ß1. Analysis of α-smooth muscle actin expression on wrinkling substrates over time showed that reduced myofibroblast contraction preceded α-smooth muscle actin disassembly from stress fibers after switching from 21 to 2% oxygen. These effects were reversible by restoring high oxygen conditions and by applying mechanical stress. We suggest that mechanical challenge is a clinical relevant strategy to improve ischemic and chronic wound healing by supporting myofibroblast formation.

A new lock-step mechanism of matrix remodelling based on subcellular contractile events.

Myofibroblasts promote tissue contractures during fibrotic diseases. To understand how spontaneous changes in the intracellular calcium concentration, [Ca(2+)](i), contribute to myofibroblast contraction, we analysed both [Ca(2+)](i) and subcellular contractions. Contractile events were assessed by tracking stress-fibre-linked microbeads and measured by atomic force microscopy. Myofibroblasts exhibit periodic (approximately 100 seconds) [Ca(2+)](i) oscillations that control small (approximately 400 nm) and weak (approximately 100 pN) contractions. Whereas depletion of [Ca(2+)](i) reduces these microcontractions, cell isometric tension is unaffected, as shown by growing cells on deformable substrates. Inhibition of Rho- and ROCK-mediated Ca(2+)-independent contraction has no effect on microcontractions, but abolishes cell tension. On the basis of this two-level regulation of myofibroblast contraction, we propose a single-cell lock-step model. Rho- and ROCK-dependent isometric tension generates slack in extracellular matrix fibrils, which are then accessible for the low-amplitude and high-frequency contractions mediated by [Ca(2+)](i). The joint action of both contraction modes can result in macroscopic tissue contractures of approximately 1 cm per month.

Transient neutropenia increases macrophage accumulation and cell proliferation but does not improve repair following intratendinous rupture of Achilles tendon.

Neutrophils are the first leukocytes to invade tendons after an acute injury. They could modulate both the inflammatory response and early repair processes through the release of reactive species, cytokines, growth factors, and proteinases. However, the exact role of these cells in damaged tendons remains unclear. We investigated their role by inducing a transient neutropenia in C57BL/6 male mice using an anti-Ly6C/Ly6G antibody. Placebo mice received only serum. The right Achilles tendon was sectioned and sutured using the 8-strand technique, which allowed immediate weight bearing. A significant increase in macrophage accumulation and cell proliferation was observed in tendons from neutropenic animals compared to the placebo group at days 3 and/or 7 postinjury. However, there was a reduction in cell proliferation in a group of mice depleted in macrophages, indicating that macrophages play a role in cell replication in injured tendons. Lastly, the tendons of neutropenic and placebo mice had similar collagen content and mechanical properties at days 7, 14, and/or 28 postinjury. Our findings demonstrate that neutropenia modulates macrophage accumulation and cell proliferation, but overall, a reduction in neutrophil number has no significant effect on tendon repair.

Thrombocytopenia alters early but not late repair in a mouse model of Achilles tendon injury.

Besides their hemostatic function, platelets can express key factors involved in tissue healing. However, the role of platelets in tendon healing following acute injury is poorly understood. We investigated this role by injecting male C57BL/6 mice with an antiplatelet antibody to induce thrombocytopenia. Placebo animals received serum only. The right Achilles tendon was sectioned and sutured using the 8-strand technique that allows immediate weight bearing. Platelet depletion did not alter the accumulation of neutrophils and macrophages or cell proliferation. A slight increase in vascularization was observed 7 days postinjury in tendons from thrombocytopenic mice relative to placebo animals, but the effect had disappeared by day 14. Furthermore, collagen content had a tendency to decrease in Achilles tendons under thrombocytopenia when compared with placebo treatment at 7 days posttrauma. This was correlated with a decline in maximal stress sustained by tendons at day 14 but not after 28 days. The impact of thrombocytopenia was otherwise negligible, as force relaxation and stiffness were similar in the two groups. Our findings demonstrate that platelets modulate early tendon repair following rupture, although the effect is limited over time. Nevertheless, platelets are not essential for the recruitment of inflammatory cells, proliferation, angiogenesis, and tendon maturation.

Early voluntary exercise does not promote healing in a rat model of Achilles tendon injury.

Mechanical stress is an important modulator of connective tissue repair. However, the effects on tendon healing are very poorly defined, preventing optimal use of mechanical stress. We hypothesized that early voluntary exercise initially retards tendon repair but results in a faster recovery rate at longer term. Male Wistar rats were injured by a collagenase injection in the Achilles tendon, and exercise was voluntarily performed on a running wheel. We observed the persistent presence of neutrophils in injured tendons of rats that began exercise immediately after the trauma [injured + early exercise (Inj+EEx)]. Early exercise also increased the concentration of ED1(+) macrophages in injured tendons after 3 and 7 days compared with ambulatory injured rats (Inj). Similar results were obtained with the subset of ED2(+) macrophages in the tendon core 3 days after the collagenase injection. Furthermore, collagen content returned to normal values more rapidly in the Inj+EEx tendons than in the Inj group, but this was not associated with an increase in cell proliferation. Surprisingly, Inj+EEx tendons roughly displayed lower stiffness and force at rupture point relative to Inj tendons at day 28. Injured tendons of rats that began exercise only from day 7 had better mechanical properties than those of early-exercised rats 28 days postinjury. We speculate that the persistence of the inflammatory response and undue mechanical loading in the Inj+EEx tendons led to fibrosis and a loss of tendon function.

Periodic direct current does not promote wound closure in an in vitro dynamic model of cell migration.

BACKGROUND AND PURPOSE: A prevailing paradigm is that electrical fields can promote cell migration and tissue healing. To further validate this paradigm, we tested the hypothesis that periodic direct current (DC) can enhance wound closure using an in vitro dynamic model of cell migration. METHODS AND RESULTS: Layers of primary fibroblasts were wounded and treated with DC under various voltages. Repair area, cell velocity, and directionality as well as lamellipodium area were evaluated at different times. Direct current had no beneficial effect on cell migration. Moreover, prolonged stimulation under the highest voltage led to significant reduction in wound closure and cell velocity. The reduction of membrane protusions in stimulated cells may be associated with the deleterious effect of DC. DISCUSSION AND CONCLUSION: Contrary to the authors' expectations, they found that periodic DC did not promote wound closure, a finding that emphasizes the need to clarify the complex effects of electrical fields on migrating cells.