Soluble epoxide hydrolase disruption as therapeutic target for wound healing.

BACKGROUND: Cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) possess angiogenic effects. However, the effect of CYP-derived EETs and soluble epoxide hydrolase (sEH) deletion on wound healing in vivo has not been rigorously investigated. In this study, we measured the effect of exogenous CYP-derived EETs and targeted disruption of sEH in an in vivo wound model. MATERIALS AND METHODS: Standardized full-thickness dermal wounds were created on the dorsum of mouse ears. Wound epithelialization was directly viewed and measured using intravital microscopy and computerized planimetry every second day until healing was complete. Wound sections were analyzed by immunostaining for metalloproteinase (MMP) 2, MMP7, MMP9, tissue inhibitor of metalloproteinases (TIMP) 1, and tumor necrosis factor (TNF) α on days 2, 4, and 12. RESULTS: Treatment with 11,12-EETs, 14,15-EETs, and sEH deletion significantly accelerated wound closure. This effect was attenuated by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) in sEH(-/-) mice. Neither 11,12- nor 14,15-EETs caused significant alterations in MMP9 expression in wounds. In contrast, MMP2 and MMP7 were significantly upregulated in the EET-treated groups, whereas TIMP1 and TNF-α were downregulated. CONCLUSIONS: Collectively, these data demonstrated that potentiation of the CYP epoxy-genase pathway by either exogenous CYP-derived EETs or sEH deletion significantly accelerated wound epithelialization in vivo. This beneficial effect might be due to downregulation of TNF-α production and, to a lesser degree, to the release of MMPs and could be used as a viable angiogenic therapeutic strategy.

Intensive hands-on microsurgery course provides a solid foundation for performing clinical microvascular surgery.

PURPOSE: Microvascular surgery requires highly specialized and individualized training; most surgical residency training programs are not equipped with microsurgery teaching expertise and/or facilities. The aim of this manuscript was to describe the methodology and clinical effectiveness of an international microsurgery course, currently taught year-round in eight countries. METHODS: In the 5-day microsurgery course trainees perform arterial and venous end-to-end, end-to-side, one-way-up, and continuous suture anastomoses and vein graft techniques in live animals, supported by video demonstrations and hands-on guidance by a full-time instructor. To assess and monitor each trainee's progress, the course's effectiveness is evaluated using "in-course" evaluations, and participant satisfaction and clinical relevance are assessed using a "post-course" survey. RESULTS: Between 2007 and 2017, more than 600 trainees participated in the microsurgery course. "In-course" evaluations of patency rates revealed 80.3% (arterial) and 39% (venous) performed in end-to-end, 82.7% in end-to-side, 72.6% in continuous suture, and 89.5% (arterial) and 62.5% (venous) one-way-up anastomoses, and 58.1% in vein graft technique. "Post-course" survey results indicated that participants considered the most important components of the microcourse to be "practicing on live animals", followed by "the presence of a full-time instructor". In addition, almost all respondents indicated that they were more confident performing clinical microsurgery cases after completing the course. CONCLUSIONS: Microvascular surgery requires highly specialized and individualized training to achieve the competences required to perform and master the delicate fine motor skills necessary to successfully handle and anastomose very small and delicate microvascular structures. The ever-expanding clinical applications of microvascular procedures has led to an increased demand for training opportunities. By teaching time-tested basic motor skills that form the foundation of microsurgical technique this international microsurgery-teaching course is helping to meet this demand.

A novel liposome-based therapy to reduce complement-mediated injury in revascularized tissues.

BACKGROUND: Ischemia/reperfusion (IR) injury is an unavoidable consequence of tissue transplantation or replantation that often leads to inflammation and cell death. Excessive complement activation following IR induces endothelial cell injury, altering vascular and endothelial barrier function causing tissue dysfunction. To mitigate the IR response, various systemic anti-complement therapies have been tried. Recently, we developed a localized therapy that uses biotinylated fusogenic lipid vesicles (BioFLVs) to first incorporate biotin tethers onto cell membranes, which are then used to bind therapeutic fusion proteins containing streptavidin (SA) resulting in the decoration of cell membranes. The therapy is applied in two steps using solutions delivered intra-arterially. MATERIALS AND METHODS: Alteration of formulation, concentration and duration of incubation of BioFLVs were conducted to demonstrate the ability of the system to modulate biotin tether incorporation in cultured cells. Using a rat hind limb model, the ability of BioFLVs to decorate endothelium of femoral vessels with FITC-labeled SA for 48 h of reperfusion was demonstrated. The feasibility of a BioFLV-based anti-complement therapy was tested in cultured cells using SA fused with vaccinia virus complement control protein (SA-VCP), a C3 convertase inhibitor. Human ovarian carcinoma (SKOV-3) cells were incubated with BioFLVs first and then with SA-VCP. To activate complement the cells were treated with a SKOV-3-specific antibody (trastuzumab) and incubated in human serum. RESULTS: Decoration of cells with SA-VCP effectively reduced complement deposition. CONCLUSIONS: We conclude that BioFLV-mediated decoration of cell membranes with anti-complement proteins reduces complement activation and deposition in vitro and has the potential for application against inappropropriate complement activation in vivo.

Enhancing complement control on endothelial barrier reduces renal post-ischemia dysfunction.

BACKGROUND: Excessive complement activation is an integral part of ischemia and reperfusion (IR) injury (IRI) of organs. In kidney transplantation, the pathologic consequence of IRI and complement activation can lead to delayed graft function, which in turn is associated with acute rejection. Previous strategies to reduce complement-induced IRI required systemic administration of agents, which can lead to increased susceptibility to infections/immune diseases. The objective of this study was to determine whether an increase in complement control defenses of rat kidney endothelium reduces IRI. We hypothesized that increased complement control on the endothelial barrier reduces IR-mediated complement activation and reduces kidney dysfunction. MATERIALS AND METHODS: Fischer 344 rats underwent left kidney ischemia for 45 min and treatment with a novel fusogenic lipid vesicle (FLVs) delivery system to decorate endothelial cells with vaccinia virus complement control protein (VCP), followed by reperfusion for 24 h. Assessment included renal function by serum creatinine and urea, myeloperoxidase assay for neutrophil infiltration, histopathology, and quantification of C3 production in kidneys. RESULTS: Animals in which the kidney endothelium was bolstered by FLVs+VCP treatment had better renal function with a significant reduction in serum creatinine compared with vehicle controls (P < 0.05). Also, C3 production was significantly reduced (P < 0.05) in treated animals compared with vehicle controls. CONCLUSION: Increasing complement control at the endothelial barrier with FLVs+VCP modulates complement activation/production during the first 24 h, reducing renal dysfunction following IRI.

Bone Fracture Sensing Using Ultrasound Pitch-Catch Measurements: A Proof-of-Principle Study.

The most common imaging method used to diagnose and monitor bone fractures and healing is multiple radiographic images performed by highly trained professionals with expensive equipment that can expose patients to high levels of ionizing radiation. Here we introduce and illustrate proof-of-concept of a potential alternative method for measuring bone fractures: ultrasound pitch-catch measurement technique. Measurements are performed with two piezoelectric transducers, housed in standard orthopedic screws and fixed on either side of simulated fractures, with and without an orthopedic plate, in ex vivo pig limb bones. Using this measurement method, we were able to detect significant differences between uncut and 2-, 5- and 10-mm-deep bone cuts using a two-sided t-test with an α level of 5%.

Effects of Electrical Stimulation on Stem Cells.

Recent interest in developing new regenerative medicine- and tissue engineering-based treatments has motivated researchers to develop strategies for manipulating stem cells to optimize outcomes in these potentially, game-changing treatments. Cells communicate with each other, and with their surrounding tissues and organs via electrochemical signals. These signals originate from ions passing back and forth through cell membranes and play a key role in regulating cell function during embryonic development, healing, and regeneration. To study the effects of electrical signals on cell function, investigators have exposed cells to exogenous electrical stimulation and have been able to increase, decrease and entirely block cell proliferation, differentiation, migration, alignment, and adherence to scaffold materials. In this review, we discuss research focused on the use of electrical stimulation to manipulate stem cell function with a focus on its incorporation in tissue engineering-based treatments.

Role of Bioelectricity During Cell Proliferation in Different Cell Types.

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 Decreases Dental Pulp Stem Cell Osteo-/Odontogenic Differentiation.

Dental pulp stem cells (DPSCs) have great potential for use in tissue engineering (TE)-based dental treatments. Electrical stimulation (EStim) has been shown to influence cellular functions that could play an important role in the success of TE treatments. Despite many recent studies focused on DPSCs, few have investigated the effect EStim has on these cells. The aim of this research was to investigate the effects of direct current (DC) EStim on osteo-/odontogenic differentiation of DPSCs. To do so cells were isolated from male Sprague Dawley rats (7-8 weeks old), and phenotype characterization and multilineage differentiation analysis were conducted to verify their "stemness." Different voltages of DC EStim were administrated 1 h/day for 7 days, and the effect of EStim on DPSC osteo-/odontogenic differentiation was assessed by measuring calcium and collagen deposition, alkaline phosphatase (ALP) activity, and expression of osteo- and odontogenic marker genes at days 7 and 14 of culture. We found that while 10 and 50 mV/mm of EStim had no effect on cell number or metabolic activity, 100 mV/mm caused a significant reduction in cell number, and 150 mV/mm resulted in cell death. Despite increased gene expression of osteo-/odontogenic gene markers, Osteocalcin, RunX2, BSP, and DMP1, at day 7 in EStim treated cells, 50 mV/mm of EStim decreased collagen deposition and ALP activity at both time points, and calcium deposition was found to be lower at day 14. In conclusion, under the conditions tested, EStim appears to impair DPSC osteo-/odontogenic differentiation. Additional studies are needed to further characterize and understand the mechanisms involved in DPSC response to EStim, with an eye toward its potential use in TE-based dental treatments.

Electrical stimulation-based bone fracture treatment, if it works so well why do not more surgeons use it?

BACKGROUND: Electrical stimulation (EStim) has been proven to promote bone healing in experimental settings and has been used clinically for many years and yet it has not become a mainstream clinical treatment. METHODS: To better understand this discrepancy we reviewed 72 animal and 69 clinical studies published between 1978 and 2017, and separately asked 161 orthopedic surgeons worldwide about their awareness, experience, and acceptance of EStim for treating fracture patients. RESULTS: Of the 72 animal studies, 77% reported positive outcomes, and the most common model, bone, fracture type, and method of administering EStim were dog, tibia, large bone defects, and DC, respectively. Of the 69 clinical studies, 73% reported positive outcomes, and the most common bone treated, fracture type, and method of administration were tibia, delayed/non-unions, and PEMF, respectively. Of the 161 survey respondents, most (73%) were aware of the positive outcomes reported in the literature, yet only 32% used EStim in their patients. The most common fracture they treated was delayed/non-unions, and the greatest problems with EStim were high costs and inconsistent results. CONCLUSION: Despite their awareness of EStim's pro-fracture healing effects few orthopedic surgeons use it in their patients. Our review of the literature and survey indicate that this is due to confusion in the literature due to the great variation in methods reported, and the inconsistent results associated with this treatment approach. In spite of this surgeons seem to be open to using this treatment if advancements in the technology were able to provide an easy to use, cost-effective method to deliver EStim in their fracture patients.

In vivo effect of hyperbaric oxygen on wound angiogenesis and epithelialization.

Hyperbaric oxygen (HBO) therapy is increasingly being used in different areas of medical practice. While demonstrated to be effective in several settings, its mechanism of action is not well understood. In the present study, we determined the effects of HBO on wound epithelialization and neovascularization in an in vivo hairless mouse ear "impaired" wound model. To impair wound healing, macrophages were depleted by pretreatment with iota-carrageenan. Wound epithelialization and neovascularization were measured using intravital microscopy and computerized planimetry. Metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and tumor necrosis factor-alpha (TNF-alpha) were measured on days 2 and 7 using immunohistochemistry. In nonimpaired healing wounds, the rate of epithelialization and neovascularization was significantly accelerated in the groups treated with HBO. Time to wound closure was significantly delayed in impaired compared with nonimpaired healing wounds and HBO treatment completely reversed this delay. Neither HBO treatment nor macrophage depletion caused significant alterations in MMP-2 expression in wounds. In contrast, TNF-alpha, MMP-9, and TIMP-1 were significantly up-regulated in the impaired healing group receiving HBO treatment. These results show that HBO therapy effectively reversed the negative effect exerted by macrophage reduction on wound epithelialization and neovascularization. This beneficial effect could be due to stimulation of TNF-alpha production and, to a lesser degree due to release of metalloproteinases.

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Patient expectations in facial transplantation.

Meeting patients' expectations is essential for successful outcomes in reconstructive surgery. In the case of new procedures like facial transplantation that carry with them many unknowns and real, or potential, risk of toxic side effects this becomes especially important. In this study we assessed patient expectations in facial transplantation by surveying individuals with facial disfigurement (n = 34), reconstructive surgeons (n = 45), and controls from the general population (n = 148). Questions focused on quality of life improvement, esthetic and functional outcomes. Student t test was used to compare means of the 3 study groups. All groups projected low quality of life for nontreated disfigured persons, controls responding the most negatively (M = 1.91), followed by disfigured persons (M = 2.91; t = 2.14, P

Histological Scoring Method to Assess Bone Healing in Critical Size Bone Defect Models.

Large bone defects are a major problem in trauma and orthopedic surgery. Tissue engineering based treatments have emerged as promising alternatives to traditional bone grafting techniques. Critical size bone defect animal models have been developed and widely used to evaluate and compare therapeutic effectiveness in bone tissue engineering treatments. To measure healing in a given defect after treatment, histological assessment methods are commonly used. These histological methods are typically qualitative and only measure the amount of newly formed bone. In this study, we introduce a new histological scoring method that in addition to new bone formation also measures newly formed "cartilage," "fibrous tissue," and "remnant bone defect size." Using Kappa analysis and interclass correlation analysis, we verified the reliability of our new scoring method. These additional parameters make it possible to differentiate between the hard callus and soft callus phases of healing and, thus, derive more valuable information about the effect different tissue-engineering treatments have on the healing process.

Time course of traumatic neuroma development.

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.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.

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.

Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model.

Bone Tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments for large non-healing bone defects. BTE approaches mimic autologous bone grafts, by combining cells, scaffold, and growth factors, and have the added benefit of being able to manipulate these constituents to optimize healing. Electrical stimulation (ES) has long been used to successfully treat non-healing fractures and has recently been shown to stimulate bone cells to migrate, proliferate, align, differentiate, and adhere to bio compatible scaffolds, all cell behaviors that could improve BTE treatment outcomes. With the above in mind we performed in vitro experiments and demonstrated that exposing Mesenchymal Stem Cells (MSC) + scaffold to ES for 3 weeks resulted in significant increases in osteogenic differentiation. Then in in vivo experiments, for the first time, we demonstrated that exposing BTE treated rat femur large defects to ES for 8 weeks, caused improved healing, as indicated by increased bone formation, strength, vessel density, and osteogenic gene expression. Our results demonstrate that ES significantly increases osteogenic differentiation in vitro and that this effect is translated into improved healing in vivo. These findings support the use of ES to help BTE treatments achieve their full therapeutic potential.

Research and events leading to facial transplantation.

Facial transplantation has long captured the interest and imagination of scientists, the media, and the lay public. Facial transplantation could provide an excellent alternative to current treatments for facial disfigurement caused by burns, trauma, cancer extirpation, or congenital birth defects. This article discusses the major technical, immunologic, psychosocial and ethical hurdles that have been overcome to bring facial transplantation from an idea to a clinical reality by providing the reader with a chronologic overview of the research and events that have led this exciting new treatment into the clinical arena.

The immunologic considerations in human head transplantation.

The idea of head transplantation appears at first as unrealistic, unethical, and futile. Here we discuss immunological considerations in human head transplantation. In a separate accompanying article we discuss surgical, ethical, and psychosocial issues concerned in body-to-head transplantation (BHT) [1]. The success of such an unusual allograft, where the donor and the recipient can reject each other, depends on prevention of complex immunologic reactions, especially rejection of the head by the body (graft-vs-host) or probably less likely, the possibility of the head rejecting the total body allograft (host-vs-graft). The technical and immunologic difficulties are enormous, especially since rapid nerve and cord connections and regeneration have not yet been possible to achieve. In this article we begin by briefly reviewing neuro-immunologic issues that may favor BHT such as the blood brain barrier (BBB) and point out its shortcomings. And we touch on the cellular and humoral elements in the brain proper that differ in some respects from those in other organs and in the periphery. Based on recent successes in vascular composite allografts (VCAs), we will elaborate on potential specific advantages and difficulties in BHT of various available immunosuppressive medications already utilized in VCAs. The risk/benefit ratio of these drugs will be emphasized in relation to direct brain toxicity such as seizure disorders, interference, or promotion of nerve regeneration, and potentiation of cerebral viral infections. The final portion of this article will focus on pre-transplant immunologic manipulation of the deceased donor body along with pretreatment of the recipient.

Surgical, ethical, and psychosocial considerations in human head transplantation.

Transplanting a head and brain is perhaps the final frontier of organ transplantation. The goal of body-to-head transplantation (BHT) is to sustain the life of individuals who suffer from terminal disease, but whose head and brain are healthy. Ideally BHT could provide a lifesaving treatment for several conditions where none currently exists. BHT is no ordinary experiment, to transfer a head to another body involves extraordinarily complex medical challenges as well as ethical and existential dilemmas that were previously confined to the imagination of writers of fiction. The possibility of replacing an incurably ill body with a healthy one tests not only our surgical limits, but also the social and psychological boundaries of physical life and alters what we recognize life to be. The purpose of this target article, the complementary manuscript focused on immunological issues in BHT, and the accompanying Commentaries by scholars and practitioners in medicine, immunology, and bioethics is to review major surgical and psychosocial-ethical and immunological considerations surrounding body-to-head transplantation. We hope that together these ideas will provide readers with a comprehensive overview of the possibilities and challenges associated with BHT and initiate professional discussion and debate through which this new frontier in medicine is considered and approached.