Mitchell S. Fourman

In Vivo Cardiac Cellular Reprogramming Efficacy Is Enhanced by Angiogenic Preconditioning of the Infarcted Myocardium With Vascular Endothelial Growth Factor

Background In situ cellular reprogramming offers the possibility of regenerating functional cardiomyocytes directly from scar fibroblasts, obviating the challenges of cell implantation. We hypothesized that pretreating scar with gene transfer of the angiogenic vascular endothelial growth factor (VEGF) would enhance the efficacy of this strategy. Methods and Results Gata4, Mef2c, and Tbx5 (GMT) administration via lentiviral transduction was demonstrated to transdifferentiate rat fibroblasts into (induced) cardiomyocytes in vitro by cardiomyocyte marker studies. Fisher 344 rats underwent coronary ligation and intramyocardial administration of an adenovirus encoding all 3 major isoforms of VEGF (AdVEGF‐All6A+) or an AdNull control vector (n=12/group). Lentivirus encoding GMT or a GFP control was administered to each animal 3 weeks later, followed by histologic and echocardiographic analyses. GMT administration reduced the extent of fibrosis by half compared with GFP controls (12±2% vs 24±3%, P<0.01) and reduced the number of myofibroblasts detected in the infarct zone by 4‐fold. GMT‐treated animals also demonstrated greater density of cardiomyocyte‐specific marker beta myosin heavy chain 7+ cells compared with animals receiving GFP with or without VEGF (P<0.01). Ejection fraction was significantly improved after GMT vs GFP administration (12±3% vs −7±3%, P<0.01). Eight (73%) GFP animals but no GMT animals demonstrated decreased ejection fraction during this interval (P<0.01). Also, improvement in ejection fraction was 4‐fold greater in GMT/VEGF vs GMT/null animals (17±2% vs 4±1%, P<0.05). Conclusions VEGF administration to infarcted myocardium enhances the efficacy of GMT‐mediated cellular reprogramming in improving myocardial function and reducing the extent of myocardial fibrosis compared with the use of GMT or VEGF alone.

Recombinant Human BMP-2 Increases the Incidence and Rate of Healing in Complex Ankle Arthrodesis

BackgroundAlthough its FDA-approved applications are limited, the pro-osteogenic benefits of recombinant human BMP-2 (rhBMP-2) administration have been shown in off-label surgical applications. However, the effects of rhBMP-2 on ankle fusions are insufficiently addressed in the literature, which fails to include a case-control study of adequate sample size to evaluate the efficacy of rhBMP-2 treatment.Questions/purposesIn this study we asked whether rhBMP-2 treatment (1) would increase the rate of successful ankle fusion in complex patients (patients with comorbidities associated with poor surgical healing) compared with a control group of patients undergoing ankle fusion who did not receive rhBMP-2; (2) would reduce total time wearing a frame when compared with the control group; (3) would result in a difference in the percentage of bone bridging between the group treated with rhBMP-2 and the control group, as determined by CT scans 3 months after surgery; and (4) would encounter an equal rate of complications different from untreated patients.MethodsA retrospective chart study was performed on 82 patients who, because of a host of comorbidities associated with poor healing, required a complex ankle arthrodesis with the Ilizarov technique. The first 40 patients did not receive rhBMP-2, whereas the subsequent 42 patients received intraoperative rhBMP-2. Time wearing the frame was determined by chart review; decision to remove the frame was made by the surgeon based on quantitative bone bridging measured using a CT scan taken 3 months after fusion.ResultsPatients treated with rhBMP-2 were more likely to obtain fusion after the initial surgery (93% versus 53%, p < 0.001; OR, 11.76; 95% CI, 3.12–44.41), spent less total time wearing the frame (124 versus 161 days, p < 0.01), and showed more bone bridging on CT scans (48% versus 32%, p < 0.05). All patients with greater than 30% bone bridging observed on CT scans 3 months postoperatively achieved successful union without further intervention.ConclusionsOur findings suggest that rhBMP-2 is a beneficial adjunct for selected groups of patients undergoing complex ankle arthrodesis. CT is a promising modality in the assessment of bone healing in ankle fusion. A proper randomized controlled trial remains necessary to fully describe the efficacy of rhBMP-2 in accelerating bone healing.Level of EvidenceLevel III, therapeutic study. See the Instructions for Authors for a complete description of levels of evidence.

Knee Arthrodesis as Limb Salvage for Complex Failures of Total Knee Arthroplasty

Patients with multiple failures of total knee arthroplasty (TKA) are challenging limb salvage cases. Twenty one patients over the last 10 years were referred to our service for knee fusion by arthroplasty surgeons who felt they were not candidates for revision TKA. Active infection was present in 76.2% and total bone loss averaged 6.6 cm. Lengthening was performed in 7/22 patients. Total time in Ilizarov frames was 9 months, with 93.3% union. Patients treated with IM fusion nails had 100% union. Average LLD increased from 3.6 to 4.5 cm following intervention, while those with concurrent lengthening improved to 1.6 cm. Findings suggest that bone loss and the soft-tissue envelope dictate knee fusion method, and multiple techniques may be needed. A treatment algorithm is presented.

Indocyanine green dye angiography accurately predicts survival in the zone of ischemia in a burn comb model

INTRODUCTION Surgical evaluation of burn depth is performed via clinical observation, with only moderate reliability. While perfusion analysis has been proposed to enhance accuracy, no perfusion study has attempted to predict burn extension into the area of ischemia surrounding the original insult. We examined whether laser Doppler imaging (LDI) and indocyanine green (ICG) angiography predicted survival in the zone of ischemia in a porcine hot comb burn model. METHODOLOGY Six full-thickness wounds were created on 5 female Yorkshire swine using a validated porcine hot comb burn model. 4 full-thickness burns were created separated by 3 unburned interspaces that represent the zone of ischemia. The interspaces between each comb burn were monitored using LDI and ICG Angiography at 1, 4, 24, and 48 h after burn. Interspace survival was assessed via gross observation and blinded histological readings 7 days after injury. RESULTS ICG Angiographic assessments of burn perfusion were significantly different in viable vs. non-viable interspace perfusion at 1 h, 4 h, and 48 h. Temporal plotting of a trend-line derived from quantitative perfusion measurements rendered two distinct graphs, allowing for the derivation of a predictive algorithm to separate viable and non-viable interspaces. LDI revealed no such prognostic trend. CONCLUSION Results from a validated porcine burn comb model suggest that ICG angiography has significant potential in the prediction of burn progression early after burn. However, the full potential of this technology cannot be determined until completion of clinical trials.

Modeling fade patterns of nipple areola complex tattoos following breast reconstruction.

BackgroundNipple-areolar complex (NAC) tattoos are an effective cosmetic solution for creating a finished look following breast reconstruction procedures. NAC tattoos are prone to significant fading, leading patients to seek revisions. This study was designed to quantify changes in NAC tattoo appearance over time. MethodsA total of 71 images of 39 patients were analyzed for NAC tattoo color and shape by 5 blinded medical student graders using a customized scoring system. Subsequently, each image was analyzed using ColorPic software (Iconico, New York, NY). Red/green/blue and hue/saturation/value color parameters were collected. Color quantities were normalized to the individual patient’s skin tone to control for variability in lighting. Spearman correlations and nonlinear regressions were calculated utilizing GraphPad Prism 6.0 (GraphPad, La Jolla, CA). ResultsThe length of time after tattoo placement inversely correlated with color score (P < 0.0001) and shape score (P = 0.0007). The time following tattoo placement was also inversely correlated with all quantitative color parameters. Each color parameter fit a 1-phase exponential decay model. ConclusionsThe decline in qualitative color and shape score agrees with clinical experience of tattoo quality declining over time. The color qualities of the tattoo approach those of the patient’s skin over time, ultimately reaching a plateau. This can be modeled using a 1-phase decay equation. In practice, tattoo colors may be selected that compensate for the predictable changes that will occur. The results of this study will help optimize tattoo color and may alleviate the need for NAC tattoo revisions.

Utilizing Indocyanine Green Dye Angiography to Detect Simulated Flap Venous Congestion in a Novel Experimental Rat Model.

BACKGROUND Venous congestion is a leading cause for free flap failure and still relies on clinical observation as the diagnostic gold standard. We sought to characterize blood flow in a variable venous congestion murine hind limb model using indocyanine green (ICG, SPY Pack, LifeCell, Branchburg, NJ) angiography. METHODS Male Sprague-Dawley rats (Charles River, Hudson, NY) underwent bilateral partial amputation at the inguinal ligament, leaving only the femoral vessels and femur intact. Complete unilateral venous occlusion was achieved via suture ligation, while partial occlusion was achieved by surrounding the femoral vein with a synthetic microtube to achieve 25, 75, 85, or 92% occlusion. Relative blood flow of occluded and control limbs was tracked with ICG angiography throughout a 90-minute time course. RESULTS ICG angiography detected statistically significant (p < 0.05) reductions in limb blood flow 1 and 2 minutes following ICG injection in the 100, 92, and 85% occluded limbs when compared with contralateral control limbs. Dynamic tracking using the slope of ICG inflow for 45 seconds postinjection reflected this same significant difference. No statistically significant change in limb blood flow or dye influx rate was observed in the 25 and 75% occlusion groups. CONCLUSIONS ICG angiography can detect venous congestion in a rat lower extremity model reliably at occlusion rates ≥ 85%. This method may offer surgeons an intraoperative diagnostic tool to identify venous congestion at extremely early time points, allowing for immediate intervention. Further investigation and characterization is warranted in a larger animal model before clinical adaptation.

Comparison of Laser Doppler and Laser-Assisted Indocyanine Green Angiography Prediction of Flap Survival in a Novel Modification of the McFarlane Flap.

BackgroundThe McFarlane rat ischemic dorsal skin flap model has been commonly used for clinical vector studies, as well as the testing of noninvasive diagnostics. However, variability of this model secondary to flap contact with the wound bed has led many to question its validity. Here we present a novel modification to the McFarlane skin flap using sterile silicone. We also use this model to test the prognostic efficacy of laser-assisted indocyanine green (ICG) angiography and laser Doppler imaging (LDI). MethodologyA 3 × 9-cm dorsal skin flap with a cranially based pedicle was created, centered 1 cm distal to the scapulae. The flap was undermined, and in one of the 2 groups, a sterile silicone sheet was placed onto the wound bed. All flaps were then reapproximated with sutures 1-cm intervals. Clinical assessment and perfusion imaging was performed immediately postoperative, and at 24, 48, and 72 hours postsurgery. Postoperative day 7 clinical assessment was obtained before euthanasia. ResultsA comparative study using silicone blocked versus unblocked models (n = 6 per group) showed that, clinically, both models had equivalent flap survival [8.5 (0.913) vs 9.5 (1.01) cm2]. However, a statistically significant increase in perfusion in the mid-third of unblocked models was observed on POD3 [20.28% (2.7%) vs blocked 13.45% (2.5%), P < 0.05], with a similar increase in the distal third on POD7 [18.73% (2.064%) vs 10.91% (4.19%), P < 0.05]. A prognostic study comparing LDI and ICG angiography prediction of POD7 survival at early time points (n = 10) found that LDI underpredicted flap survival at early time points [84.2% (12.03%) on POD0, 87.35% (16.11%) on POD1]. In contrast, ICG was more proficient [100.1% (10.1%) on POD0]. ConclusionsWe present a modification of the McFarlane skin flap model that results in similar clinical results, but with a noted reduction in perfusion inconsistencies noted in unblocked models. The ICG angiography is superior to LDI in predicting POD7 flap necrosis within the first 48 hours postinjury. Future work will focus on histologic validation of our model, and vector efficacy testing.

Comparison of native porcine skin and a dermal substitute using tensiometry and digital image speckle correlation.

AbstractDermal substitutes are currently used in plastic surgery to cover various soft tissue defects caused by trauma, burns, or ablative cancer surgery. Little information is available on the biomechanical properties of these dermal substitutes after adequate incorporation as compared to normal skin. Determining parameters such as tensile strength in these skin substitutes will help us further understand their wound healing properties and potential in developing artificial tissue constructs. We hypothesize that a dermal substitute has a lower stress-strain curve and altered stress-induced deformation quantified with tensiometry and digital image speckle correlation (DISC) analysis. Two separate 5 × 10-cm full-thickness wounds were created on the dorsum of 3 female swine. Fibrin glue was applied before either a full-thickness skin graft (FTSG) or application of artificial dermal matrix. On day 42, cultured autologous keratinocytes were applied as a cell sheet to the wound covered with Integra. On day 56, the wounds were fully excised and fresh tissue specimens, including normal skin, were stored in a physiological solution and prepared for analysis. Rectangular samples were excised from the center of each specimen measuring 4 × 4 × 30 mm. Using a tensiometer and DISC analysis, we evaluated the tensile strength of 3 different groups of skin, namely, normal, FTSG, and Integra. There is a significant difference between the Integra specimen when compared to normal skin and FTSG. We found a minimal difference in the stress-strain curves of the latter two. Integra alone shows plastic deformation with continued stretching before ultimate midline fracture. There is significant change between the Young’s moduli of the normal skin and the Integra, whereas there is little difference between the FTSG and the normal skin; DISC confirms this analysis. The normal skin and FTSG show a convergence of vectors to a linear plane, whereas Integra shows very little organization. Using 2 different methods of analysis, we have shown a dermal substitute does not display similar biomechanical properties after adequate incorporation. These major tensile strength differences are shown between normal, grafted, and Integra constructs under physiological conditions. These properties will lead to further understanding of artificial tissue and engineered constructs in laboratory and clinical applications.

Comparison of Diabetic Charcot Patients With and Without Foot Wounds

Background: The primary aim of this study was to evaluate the outcomes of a series of patients with Charcot neuroarthropathy (CN) who were evaluated in a tertiary care setting. We hypothesized that those patients with CN who presented with a Charcot-related foot wound would have lower rates of successful limb salvage than patients who presented without a wound. Methods: Two hundred forty-five patients (280 feet) were identified with diabetic CN during the time period from January 1, 2005, to June 1, 2015. This consecutive cohort of patients was treated by a single surgeon and had a mean age of 57.9 ± 10.0 years. Our CN patients were divided into 2 groups for the purpose of our analysis. Our study group included those patients who presented to our clinic with a Charcot-related foot wound. Our control group was composed of CN patients who presented without a Charcot-related foot wound. Results: Overall, 78 feet (27.9%) were successfully treated nonoperatively and 202 feet (72.1%) required some type of surgery. Of the 202 feet that received surgery, 22 (10.9%) were not felt to be suitable for reconstruction and underwent a definitive transtibial amputation without an attempt at reconstruction. An additional 18 patients underwent soft tissue surgery, which included drainage of infection, posterior muscle group lengthening, or soft tissue reconstructive flap surgery. The remaining 162 feet underwent osseous surgery, which included ostectomies for osteomyelitis, exostectomies, osteotomies, and arthrodesis. Eighteen of the 180 limbs (10.0%) that underwent soft tissue or osseous reconstruction ultimately required a transtibial amputation, resulting in a successful limb salvage rate of 90.0%. Thirty-five amputations were performed in 164 feet (21.3%) with Charcot-related foot wounds compared with 5 amputations in 116 feet (4.5%) without Charcot-related foot wounds (OR 6.02, 95% CI 2.28-15.91, P < .0001). Conclusion: The presence of a Charcot-related foot wound at presentation increased the likelihood of a major lower extremity amputation by a factor of 6. Other risk factors that were associated with major amputation in patients included active infection at presentation, nonunion/instability after reconstruction, and a postoperative wound problem. The overall rate of successful limb salvage in patients deemed reconstructive candidates was 90%. Level of Clinical Evidence: Level III, retrospective, case-control study.

Laser-assisted indocyanine green dye angiography accurately predicts the split-thickness graft timing of integra artificial dermis.

BackgroundThe use of an artificial dermal substitute such as Integra—a bilaminate combination of thin silicone and cross-linked bovine tendon collagen and chondroitin-6-sulfate—has become a popular method to address large surface area wounds or smaller, complex wounds devoid of a vascular bed. The incorporation of Integra depends on a vascular wound bed or periphery and can take 4 weeks or longer to occur. If the Integra has not fully incorporated at the time of placement of the split-thickness graft, complete graft loss may result. The availability of a minimally invasive method to assess the incorporation of Integra would be of great value. MethodsTwo 5 × 10-cm paraspinal full-thickness wounds were created on 3 female swine. Wounds were randomly assigned full-thickness skin graft or Integra (Plainsboro, NJ) treatment. Both types of grafts were placed after the application of fibrin glue (Tisseel, Deerfield, Ill) to the wound bed. Laser Doppler imaging (LDI) (Moor), indocyanine green dye (ICG) angiography (LifeCell SPY), and clinical scoring were performed weekly for a period of 8 weeks after grafting. At 4 weeks, the silicone layer of the Integra was removed, and a culture of autologous keratinocytes was applied. A 4-mm punch biopsy sample of each graft was taken 1, 2, 4, 6, 7, and 8 weeks postoperatively for histologic analysis. ResultsBoth ICG angiography and LDI perfusion measurements noted an increase in perfusion at the Integra graft site that peaked 3 weeks after grafting, corresponding with the start of neovascularization and the optimal time for the application of a split-thickness skin graft. indocyanine green dye angiography measurements exhibit greater reproducibility between animals at late time points as compared with LDI. This decrease in LDI precision is directly related to increases in scar tissue thickness of greater than 5 mm as determined via histologic analysis and corresponds with the accepted maximum penetration depth of the LDI laser. ConclusionsIndocyanine green dye angiography may provide valuable information as to graft integrity and split-thickness skin graft timing at late time points. Range of LDI seems to be insufficient for split-thickness graft timing or late time point accuracy. Future exploration of ICG angiography potential will involve tracking Integra graft delay in porcine models.