Mesenchymal stem cells correct impaired diabetic wound healing by decreasing ECM proteolysis.

Impaired diabetic wound healing is associated with a dermal extracellular matrix protein profile favoring proteolysis; within the healing diabetic wound, this is represented by an increase in activated matrix metalloproteinase (MMPs). Treatment of diabetic wounds with mesenchymal stem cells (MSCs) has been shown to improve wound healing; however, there has not yet been an assessment of their ability to correct dysregulation of MMPs in diabetic wounds. Furthermore, there has been no prior assessment of the role of microRNA29b (miR-29b), an inhibitory regulatory molecule that targets MMP-9 mRNA. Using in vitro models of fibroblast coculture with MSCs and in vivo murine wound healing models, we tested the hypothesis that MSCs correct dysregulation of MMPs in a microRNA-29b-dependent mechanism. In this study, we first demonstrated that collagen I and III protein content is significantly reduced in diabetic wounds, and treatment with MSCs significantly improves collagen I content in both nondiabetic and diabetic wounds. We then found that MMP-9 gene expression and protein content were significantly upregulated in diabetic wounds, indicating elevated proteolysis. Treatment with MSCs resulted in a decrease in MMP-9 gene expression and protein content level in diabetic wounds 3 and 7 days after wounding. Zymographic analysis indicated that MSC treatment also decreased the amount of activated MMP-9 present in diabetic wounds. Furthermore, miR-29b expression was inversely associated with MMP-9 gene expression; miR-29b expression was decreased in diabetic wounds and diabetic fibroblast. Following treatment of diabetic wounds with MSCs, as well as in diabetic fibroblasts cocultured with MSCs, miR-29b was significantly increased. These findings suggest a potential mechanism through which MSCs enhance diabetic wound healing by improving collagen I content in diabetic wounds through decreasing MMP-9 expression and increasing miR-29b expression.

SCF increases in utero-labeled stem cells migration and improves wound healing.

Diabetic skin wounds lack the ability to heal properly and constitute a major and significant complication of diabetes. Nontraumatic lower extremity amputations are the number one complication of diabetic skin wounds. The complexity of their pathophysiology requires an intervention at many levels to enhance healing and wound closure. Stem cells are a promising treatment for diabetic skin wounds as they have the ability to correct abnormal healing. Stem cell factor (SCF), a chemokine expressed in the skin, can induce stem cells migration, however the role of SCF in diabetic skin wound healing is still unknown. We hypothesize that SCF would correct the impairment and promote the healing of diabetic skin wounds. Our results show that SCF improved wound closure in diabetic mice and increased HIF-1α and vascular endothelial growth factor (VEGF) expression levels in these wounds. SCF treatment also enhanced the migration of red fluorescent protein (RFP)-labeled skin stem cells via in utero intra-amniotic injection of lenti-RFP at E8. Interestingly these RFP+ cells are present in the epidermis, stain negative for K15, and appear to be distinct from the already known hair follicle stem cells. These results demonstrate that SCF improves diabetic wound healing in part by increasing the recruitment of a unique stem cell population present in the skin.

Dysregulation of collagen production in diabetes following recurrent skin injury: contribution to the development of a chronic wound.

Recurrent injury has been implicated in the development of chronic diabetic wounds. We have developed a chronic diabetic wound model based upon recurrent injury in diabetic mice. We hypothesized that dysregulation of collagen production at both the mRNA and microRNA levels contributes to the development of chronic diabetic wounds. To test this, both diabetic and nondiabetic mice were made to undergo recurrent injury. Real-time PCR for TGF-ß1, SMAD-3, Col1α1, Col3α1, microRNA-25, and microRNA-29a and Western blot for collagen I and III were performed 7 days following each injury. Diabetic wounds displayed decreased collagen at all time points. This was associated with dysregulated collagen production at both the gene and microRNA levels at all time points. Following the final injury, however, diabetic collagen production significantly improved. This appeared to be due to a substantial decrease in both microRNAs as well as an increase in the expression of collagen pathway genes. That dysregulated collagen production progressed throughout the course of wounding suggests that this is one factor contributing to the development of chronic diabetic wounds. Future studies using this model will allow for the determination of other factors that may also contribute to the development and/or persistence of chronic diabetic wounds.

Modulation of the inflammatory response by increasing fetal wound size or interleukin-10 overexpression determines wound phenotype and scar formation.

Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro-inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin-10 (IL-10). To test this hypothesis, 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep. A subset of 8-mm wounds were injected with a lentiviral vector containing the IL-10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real-time polymerase chain reaction. In contrast to the scarless 2-mm wounds, 8-mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral-mediated overexpression of the IL-10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL-10 in the transition from fibroplasia to fetal regenerative healing.

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair.

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.

Interprofessional Simulations to Inform Perioperative Facility Planning and Design.

OBJECTIVE: The purpose of this study is to report our experience with interprofessional simulations, executed in a mock-up of a proposed perioperative space, that were designed to elicit valuable end-user feedback on the design of the perioperative space. DESIGN: A styrofoam, life-sized model of a perioperative unit was constructed. Various medical professionals and support staff participated in interactive sessions, including workflow simulations, and provided feedback on the perioperative design. Based on participant feedback, the perioperative design was modified, and the styrofoam model was re-constructed. A second round of sessions was conducted, and the change in participant feedback was analyzed. SETTING: This study took place under the auspices of the University of Pennsylvania in Philadelphia, Pennsylvania, within Penn Medicine. PARTICIPANTS: One-hundred and ninety-three medical professionals and front line operating room staff participated in the initial round of interactive sessions, and 134 participated in the second round (after re-construction). RESULTS: In the first round of simulations and interactive sessions, participants spent 560 hours engaging with the space. Modifications were then made to the perioperative design based on participant feedback, and a second round was conducted, in which participants spent 403 hours in the space. Floor plans for round 2 show significant changes compared with round 1, and mean design satisfaction scores for round 2 (3.78 ± 0.41) were significantly higher than for round 1 (3.61 ± 0.49) (p = 0.02). The quality of feedback was associated with the type of interactive session the user participated in. CONCLUSIONS: This study suggests that simulations and other interactive sessions, when executed in a mock-up of a proposed perioperative space, can elicit valuable end-user feedback that impacts the final design of the perioperative space and that would traditionally be difficult to obtain until after construction and move-in.

"How To" Videos Improve Residents Performance of Essential Perioperative Electronic Medical Records and Clinical Tasks.

OBJECTIVE: The ability to use electronic medical records (EMR) is an essential skill for surgical residents. However, frustration and anxiety surrounding EMR tasks may detract from clinical performance. We created a series of brief, 1-3 minutes "how to" videos demonstrating 7 key perioperative EMR tasks: booking OR cases, placing preprocedure orders, ordering negative-pressure wound dressing supplies, updating day-of-surgery history and physical notes, writing brief operative notes, discharging patients from the postanesthesia care unit, and checking vital signs. Additionally, we used "Cutting Insights"-a locally developed responsive mobile application for surgical trainee education-as a platform for providing interns with easy access to these videos. We hypothesized that exposure to these videos would lead to increased resident efficiency and confidence in performing essential perioperative tasks, ultimately leading to improved clinical performance. METHODS: Eleven surgery interns participated in this initiative. Before watching the "how to" videos, each intern was timed performing the aforementioned 7 key perioperative EMR tasks. They also underwent a simulated perioperative emergency requiring the performance of 3 of these EMR tasks in conjunction with 5 other required interventions (including notifying the chief resident, the anesthesia team, and the OR coordinator; and ordering fluid boluses, appropriate laboratories, and blood products). These simulations were scored on a scale from 0 to 8. The interns were then directed to watch the videos. Two days later, their times for performing the 7 tasks and their scores for a similar perioperative emergency simulation were once again recorded. Before and after watching the videos, participants were surveyed to assess their confidence in performing each EMR task using a 5-point Likert scale. We also elicited their opinions of the videos and web-based mobile application using a 5-point scale. Statistical analyses to assess for statistical significance (p ≤ 0.05) were conducted using paired t-test for parametric variables and a Wilcoxon matched-pair test for nonparametric variables. SETTING: Hospital of the University of Pennsylvania, Philadelphia, PA (a quaternary teaching hospital within the University of Pennsylvania Health System). PARTICIPANTS: Eleven out of 15 interns (12 entered and 11 completed the study) from our categorical and preliminary general surgery residency programs during the 2016 academic year. RESULTS: Before exposure to the brief "how to" videos, 6 of 11 interns were unable to complete all 7 EMR tasks; after exposure, all 11 interns were able to complete all 7 EMR tasks. Moreover, interns' times for each task improved following exposure. Interns self-reported improved confidence in booking an OR case (4 ± 0.9 vs. 4.7 ± 0.6, p = 0.05), ordering negative-pressure wound therapy supplies (3.1 ± 1.6 vs. 4.5 ± 0.7, p < 0.05), writing a brief operative note (3.7 ± 1.2 vs. 4.6 ± 0.7, p = 0.05), discharging patients from the postanesthesia care unit (3.3 ± 1.0 vs. 4.4 ± 0.8, p < 0.05), checking vital signs (2.5 ± 1.4 vs. 4.5 ± 0.8, p ≤ 0.01), and performing necessary EMR tasks during an emergency situation (2.4 ± 0.8 vs. 4.6 ± 0.7, p ≤ 0.0001). Participants also demonstrated a significant improvement in average clinical score on the emergency simulations (5.2 ± 1.7 vs. 6.6 ± 0.9, p < 0.05). Interns' opinions of the videos and the mobile phone application were favorable. CONCLUSIONS: In our group of 11 surgery interns, exposure to a series of short "how to" videos led to increased confidence and shortened times in performing 7 essential EMR tasks. Additionally, during a simulated perioperative emergency, EMR tasks were performed significantly faster. Clinical performance also improved significantly following exposure to the videos. This just-in-time educational intervention could improve workflow efficiency and clinical performance, both of which may ultimately enhance perioperative patient safety.

High ratio plasma resuscitation does not improve survival in pediatric trauma patients.

BACKGROUND: Damage control resuscitation including balanced resuscitation with high ratios of plasma (PLAS) and platelets (PLT) to packed red blood cells (PRBC) improves survival in adult patients. We sought to evaluate the effect of a high ratio PLAS to PRBC resuscitation strategy in massively transfused pediatric patients with combat injuries. METHODS: The Department of Defense Trauma Registry was queried from 2001 to 2013 for pediatric trauma patients (<18 years). Burns, drowning, isolated head trauma, and older teens were excluded. Those who received massive transfusion (≥40 mL/kg total blood products in 24 hours) and early deaths who received any blood products were then evaluated. Primary outcomes were mortality at 24 hours and in-hospital. Secondary outcomes included blood product utilization over 24 hours, ventilator-free days, intensive care unit-free days, and hospital length of stay. RESULTS: The Department of Defense Trauma Registry yielded 4,980 combat-injured pediatric trauma patients, of whom 364 met inclusion criteria. Analysis of PLAS/PRBC ratios across the entire spectrum of possible ratios in these patients demonstrated no clear inflection point for mortality. Using a division between low (LO) and high (HI) ratios of PLAS/PRBC 1:2, there was no difference in all-cause mortality at 24 hours (LO, 9.2% vs. HI, 8.0%; p = 0.75) and hospital discharge (LO, 21.5% vs. HI, 17.1%; p = 0.39). HI ratio patients received less PRBC but more PLAS and PLT and more total blood products. Those in the HI ratio group also had longer hospital length of stay. Regression analysis demonstrated no associated mortality benefit with a HI ratio (hazards ratio, 2.04; 95% confidence interval, 0.48-8.73; p = 0.34). CONCLUSION: In combat-injured children undergoing a massive transfusion, a high ratio of PLAS/PRBC was not associated with improved survival. Further prospective studies should be performed to determine the optimal resuscitation strategy in critically injured pediatric patients. LEVEL OF EVIDENCE: Therapeutic study, level III.

Integration of Hands-On Team Training into Existing Curriculum Improves Both Technical and Nontechnical Skills in Laparoscopic Cholecystectomy.

OBJECTIVE: Nontechnical skills are an essential component of surgical education and a major competency assessed by the ACGME milestones project. However, the optimal way to integrate nontechnical skills training into existing curricula and then objectively evaluate the outcome is still unknown. The aim of this study was to determine the effect laparoscopic team-based task training would have on the nontechnical skills needed for laparoscopic surgery. DESIGN: 9 PGY-1 residents underwent an established training curriculum for teaching the knowledge and technical skills involved in laparoscopic cholecystectomy. Initial training involved a didactic session, expert-led practice on a porcine model in a simulated operating room and laparoscopic skills practice on a virtual reality trainer. Residents then performed a laparoscopic cholecystectomy on the same porcine model as a preintervention test. Three to four months following this, residents were subjected to specific nontechnical skills training involving 2 simple team-based laparoscopic tasks. They then practiced a further 4 to 6 hours on the virtual reality trainer. A repeat postintervention laparoscopic cholecystectomy was then performed 3 to 4 months after nontechnical skills training. Both the preintervention and postintervention laparoscopic cholecystectomies were audiovisually recorded and then evaluated by 2 independent surgeons in a blinded fashion. Technical skills were assessed using objective structured assessment of technical skills (OSATS) and a technique specific rating scale (TRS) that we developed for laparoscopic cholecystectomy. Nontechnical skills were assessed using nontechnical skills for surgeons (NOTSS). Residents also completed a survey at the beginning and end of the training. SETTING: Tertiary care, university based teaching institution. PARTICIPANTS: A total of 9 general surgery residents at the intern level. RESULTS: The mean OSATS score improved from 13.7 ± 1.24 to 26.7 ± 0.31 (p < 0.001), the mean TRS score improved from 6 ± 0.46 to 13.1 ± 0.36 (p < 0.001) and the mean NOTSS score improved from 21.7 ± 1.83 to 36.3 ± 0.87 (p < 0.001) following the training. There was a strong correlation between OSATS and NOTSS scores (Pearson's R = 0.98) and TRS and NOTSS (R = 0.94). The inter-rater agreement was 0.79 for NOTSS, 0.9 for OSATS, and 0.82 for TRS. Following completion of the training, residents self-reported improvements in exchanging information (p < 0.01), coordinating activities (p < 0.01) and coping with pressure in the operating room (p < 0.001). CONCLUSION: Simple, team-based nontechnical skills training for laparoscopic cholecystectomy that was separate from technical skills training led to a sustained increase in residents' nontechnical skills 3 to 4 months after training. This was associated with a self-reported improvement in many nontechnical skills based on resident survey. Based on these results, we recommend that such designated nontechnical skills training is a valid alternative to other methods such as coaching and debriefing. We, therefore, plan to continue our efforts to develop team-based simulation tasks aimed at improving nontechnical skills for multiple surgical modalities.

Curriculum Using the In-Situ Operating Room Setting.

OBJECTIVE: The American College of Surgeons/Association of Program Directors in Surgery is a comprehensive, simulation-based curriculum for General Surgery residents which exists in 3 phases. While phases 1 and 2 deal with core skills and advanced procedures respectively, phase 3 targets team-based skills. To date, the 3rd phase of this curriculum has not seen wide scale implementation. This is a pilot study to verify the feasibility of implementing the phase 3 curriculum in the in-situ setting. DESIGN: In our initial attempt to implement Phase 3 at our institution, we chose to perform the training in an in-situ setting within an operating room (OR) at our main hospital, despite our having a separate simulation center. By choosing the in-situ OR environment for this training we were able to minimize concerns regarding resident and faculty availability and able to successfully complete 8 separate sessions during the academic year. During 7 sessions, 2 separate scenarios were performed while a single scenario was performed in 1 session. This single session was excluded from analysis, leaving a total of 14 scenarios to evaluate. The unique scenarios included laparoscopic crisis, postoperative myocardial infarction, anaphylaxis, and postoperative hypotension. All sessions were audiovisually recorded. In order to evaluate the effect of the training, the videos were viewed by 3 independent reviewers and all surgery, anesthesia and nursing participants were rated using the NOTECHs II scale. Degree of inter-rater agreement was established. The difference between the first and second simulations on the same day was then assessed. In addition, participant opinions of the simulations were assessed through electronic surveys following the training. SETTING: Tertiary Care University Hospital. PARTICIPANTS: We performed a total of 8 sessions, for a total of 15 scenarios. Eight surgery residents at the postgraduate year 1 (PGY1)-PGY3 level, 16 anesthesia residents at the PGY3-PGY4 level, 16 nurses and 13 ancillary staff participated. RESULTS: From the first to the second scenario, the total team NOTECHs II score increased from 69.4 ± 1.4 to 77.3 ± 0.5 (p = 0.007). The NOTECHs II scores for each subteam also improved, from 24.2 ± 0.6 to 26.4 ± 0.5 (p = 0.007) for surgery residents, 23.7 ± 0.9 to 26.7 ± 0.4 (p = 0.03) for anesthesia, and 21.6 ± 0.3 to 24.3 ± 0.5 (p = 0.01) for nursing. The inter-rater reliability as measured by Kendall's coefficient of concordance was modest for the whole team score. Most of the participant responses were either favorable or strongly favorable. CONCLUSION: The in-situ OR environment is both a unique and effective setting to perform team-based training. Furthermore, training in the in-situ setting minimizes or removes many of the logistic issues involved in designing and implementing team-based training curricula for general surgery residency programs. However, we found that administrative and departmental (surgery, anesthesia, and nursing) "buy in" as well as protected faculty time for education were all necessary for in-situ training to be successful. NOTECHs II is an established scale for the evaluation of teams in this simulation setting and appears to be a valid tool based on the results of this study. However, further assessment of inter-rater reliability as well as improved training of evaluators are necessary to determine if inter-rater reliability can improve.

In Situ Operating Room-Based Simulation: A Review.

OBJECTIVE: To systematically review the literature surrounding operating room-based in situ training in surgery. METHODS: A systematic review was conducted of MEDLINE. The review was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology, and employed the Population, Intervention, Comparator, Outcome (PICO) structure to define inclusion/exclusion criteria. The Kirkpatrick model was used to further classify the outcome of in situ training when possible. RESULTS: The search returned 308 database hits, and ultimately 19 articles were identified that met the stated PICO inclusion criteria. Operating room-based in situ simulation is used for a variety of purposes and in a variety of settings, and it has the potential to offer unique advantages over other types of simulation. Only one randomized controlled trial was conducted comparing in situ simulation to off-site simulation, which found few significant differences. One large-scale outcome study showed improved perinatal outcomes in obstetrics. CONCLUSIONS: Although in situ simulation theoretically offers certain advantages over other types of simulation, especially in addressing system-wide or environmental threats, its efficacy has yet to be clearly demonstrated.

An extra-uterine system to physiologically support the extreme premature lamb.

In the developed world, extreme prematurity is the leading cause of neonatal mortality and morbidity due to a combination of organ immaturity and iatrogenic injury. Until now, efforts to extend gestation using extracorporeal systems have achieved limited success. Here we report the development of a system that incorporates a pumpless oxygenator circuit connected to the fetus of a lamb via an umbilical cord interface that is maintained within a closed 'amniotic fluid' circuit that closely reproduces the environment of the womb. We show that fetal lambs that are developmentally equivalent to the extreme premature human infant can be physiologically supported in this extra-uterine device for up to 4 weeks. Lambs on support maintain stable haemodynamics, have normal blood gas and oxygenation parameters and maintain patency of the fetal circulation. With appropriate nutritional support, lambs on the system demonstrate normal somatic growth, lung maturation and brain growth and myelination.

Corrigendum: An extra-uterine system to physiologically support the extreme premature lamb.

This corrects the article DOI: 10.1038/ncomms15112.

Management of pediatric mild traumatic brain injury.

mTBI is a very common and underdiagnosed problem in the pediatric population. While most children who receive appropriate management recover uneventfully (80%-90%), referral to a concussion specialist is necessary for those with a complicated course. Fortunately, further improvements in identifying those children who have sustained an mTBI, coupled with strict adherence to the Return to Learn and Return to Play protocols, will ultimately lead to better long-term outcomes.

Mammalian fetal cardiac regeneration after myocardial infarction is associated with differential gene expression compared with the adult.

BACKGROUND: In adults, myocardial infarction (MI) results in a brisk inflammatory response, myocardium loss, and scar formation. We have recently reported the first mammalian large-animal model of cardiac regeneration after MI in fetal sheep. We hypothesize that the ability of the fetus to regenerate functional myocardium after MI is owing to differential gene expression regulating the response to MI in the fetus compared with the adult. METHODS: Myocardial infarction was created in adult (n=4) or early gestation fetal (n=4) sheep. Tissue was harvested after 3 or 30 days, and RNA was extracted for microarray, followed by principal component analysis and global gene expression analysis for the following gene ontology terms: response to wounding, inflammatory response, extracellular matrix, cell cycle, cell migration, cell proliferation, and apoptosis. RESULTS: Principal component analysis demonstrated that the global gene expression pattern in adult infarcts was distinctly different from the uninfarcted region at 3 days and remained different at 30 days after MI. In contrast, gene expression in the fetal infarct was different from the uninfarcted region at 3 days, but by 30 days it returned to a baseline expression pattern similar to the uninfarcted region. Three days after MI there was an increase in the expression of genes related to all gene ontology terms in fetal and adult infarcts, but this increase was much more pronounced in adults. By 30 days, the fetal gene expression returned to baseline, whereas in the adult it remained significantly elevated. CONCLUSIONS: These data demonstrate that the global gene expression pattern is dramatically different in the fetal regenerative response to MI compared with the adult response and may partly be responsible for the regeneration.

Novel animal models for tracking the fate and contributions of bone marrow derived cells in diabetic healing.

There is a vast wealth of information to be gained by tracking both the fate and contribution of individual cell types to the wound healing response. This is particularly important in research focused on impaired healing, such as diabetic wound healing, where the number or function of one or more specific cell types may be abnormal and contribute to the observed healing derangements. Specifically, diabetic wounds have been shown to have an overactive inflammatory response and decreased angiogenesis. The ability to track specific cell types participating in these responses would dramatically improve our understanding of the cellular derangements in diabetic healing. In this chapter, we review two novel chimeric models based on the leptin deficient Db/Db mouse. The use of these models allows for the tracking of bone marrow derived inflammatory and progenitor cell populations as well as the determination of the molecular contributions of these cell populations to the wound healing response.