Microporous Polylactic Acid Scaffolds Enable Fluorescence-Based Perfusion Imaging of Intrinsic In Vivo Vascularization.

In vivo tissue engineering (TE) techniques like the AV loop model provide an isolated and well-defined microenvironment to study angiogenesis-related cell interactions. Functional visualization of the microvascular network within these artificial tissue constructs is crucial for the fundamental understanding of vessel network formation and to identify the underlying key regulatory mechanisms. To facilitate microvascular tracking advanced fluorescence imaging techniques are required. We studied the suitability of microporous polylactic acid (PLA) scaffolds with known low autofluorescence to form axial vascularized tissue constructs in the AV loop model and to validate these scaffolds for fluorescence-based perfusion imaging. Compared to commonly used collagen elastin (CE) scaffolds, the total number of vessels and cells in PLA scaffolds was lower. In detail, CE-based constructs exhibited significantly higher vessel numbers on day 14 and 28 (d14: 316 ± 53; d28: 610 ± 74) compared to the respective time points in PLA-based constructs (d14: 144 ± 18; d28: 327 ± 34; each p < 0.05). Analogously, cell counts in CE scaffolds were higher compared to corresponding PLA constructs (d14: 7661.25 ± 505.93 and 5804.04 ± 716.59; d28: 11211.75 + 1278.97 and 6045.71 ± 572.72, p < 0.05). CE scaffolds showed significantly higher vessel densities in proximity to the main vessel axis compared to PLA scaffolds (200-400 µm and 600-800 µm on day 14; 400-1000 µm and 1400-1600 µm on day 28). CE scaffolds had significantly higher cell counts on day 14 at distances from 800 to 2000 µm and at distances from 400 to 1600 µm on day 28. While the total number of vessels and cells in PLA scaffolds were lower, both scaffold types were ideally suited for axial vascularization techniques. The intravascular perfusion of PLA-based constructs with fluorescence dye MHI148-PEI demonstrated dye specificity against vascular walls of low- and high-order branches as well as capillaries and facilitated the fluorescence-based visualization of microcirculatory networks. Fluorophore tracking may contribute to the development of automated quantification methods after 3D reconstruction and image segmentation. These technologies may facilitate the characterization of key regulators within specific subdomains and add to the current understanding of vessel formation in axially vascularized tissue constructs.

Expression of Connexin43 Stimulates Endothelial Angiogenesis Independently of Gap Junctional Communication In Vitro.

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.

A Structured, Microsurgical Training Curriculum Improves the Outcome in Lower Extremity Reconstruction Free Flap Residency Training: The Ludwigshafen Concept.

BACKGROUND: Risk stratification, economic pressure, and a flat learning curve make the realization and development of proper microsurgical skills and competences a challenging task in the daily clinical practice. In previous studies, we were able to show that microsurgical procedures, e.g., free flaps and replantations, are safe training procedures and teachable in daily clinical practice in view of certain issues of risk stratification. The present study aims to evaluate further improvements in terms of safety and complication rates for free flaps as a training procedure after introduction and continuous implementation of a structured in-house training curriculum for microsurgical skills and competences and a 24-hour free accessible microsurgical training facility for the plastic surgery resident. METHODS: This retrospective comparative cohort study was conducted to review whether microsurgical skills for free flaps to the lower extremity can further be improved after implementation of the curriculum and a 24-hour accessible training facility. Therefore, we compared cohort A before (2009-2012) and B after (2014-2017) implementation. Patient demographics, procedural characteristics, and outcome parameters for free tissue transfer of the lower extremity were evaluated. RESULTS: The comparison of both cohorts showed a significantly reduced postoperative complication rate for cohort B (p <0.05). Furthermore, operation time was shorter, and the hospital stay could be significantly decreased (p <0.01). Workhorse flaps for plastic surgical training were the anterior lateral thigh (ALT) flap or the musculus latissimus dorsi (LD) flap. However, even more complex procedures with arteriovenous loops could be safely performed by plastic surgery residents under the supervision of the senior surgeon in exceptional cases. CONCLUSION: The implementation of a regularly held, microsurgical in-house training curriculum with 24-hour accessible training facility improves procedural and outcome parameters for free flaps to the lower extremity for surgical residents and is an elementary part of skills and competency training. However, risk stratification, repeated surgical exposure, expertise, and institutional infrastructures are essential and must be taken into consideration.

Dual Mechanism for Inhibition of Inwardly Rectifying Kir2.x Channels by Quinidine Involving Direct Pore Block and PIP2-interference.

Class IA antiarrhythmic drug quinidine was one of the first clinically used compounds to terminate atrial fibrillation and acts as multichannel inhibitor with well-documented inhibitory effects on several cardiac potassium channels. In the mammalian heart, heteromeric assembly of Kir2.1-2.3 channels underlies IK1 current. Although a low-affinity block of quinidine on Kir2.1 has already been described, a comparative analysis of effects on other Kir2.x channels has not been performed to date. Therefore, we analyzed the effects of quinidine on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Quinidine exerted differential inhibitory effects on Kir2.x channels with the highest affinity toward Kir2.3 subunits. Onset of block was slow and solely reversible in Kir2.2 subunits. Quinidine inhibited Kir2.x currents in a voltage-independent manner. By means of comparative Ala-scanning mutagenesis, we further found that residues E224, F254, D259, and E299 are essential for quinidine block in Kir2.1 subunits. Analogously, quinidine mediated Kir2.3 inhibition by binding corresponding residues E216, D247, D251, and E291. In contrast, Kir2.2 current block merely involved corresponding residue D260. Using channel mutants with altered (phosphatidylinositol 4,5-bisphosphate PIP2) affinities, we were able to demonstrate that high PIP2 affinities (i.e., Kir2.3 I214L) correlate with low quinidine sensitivity. Inversely, mutant channels interacting only weakly with PIP2 (i.e., Kir2.1 K182Q, and L221I) are prone to a higher inhibitory effect. Thus, we conclude that inhibition of Kir2.x channels by quinidine is mediated by joint modes of action involving direct cytoplasmic pore block and an impaired channel stabilization via interference with PIP2.

Flow Induced Microvascular Network Formation of Therapeutic Relevant Arteriovenous (AV) Loop-Based Constructs in Response to Ionizing Radiation.

BACKGROUND The arteriovenous (AV) loop model enables axial vascularization to gain a functional microcirculatory system in tissue engineering constructs in vivo. These constructs might replace surgical flaps for the treatment of complex wounds in the future. Today, free flaps are often exposed to high-dose radiation after defect coverage, according to guideline-oriented treatment plans. Vascular response of AV loop-based constructs has not been evaluated after radiation, although it is of particular importance. It is further unclear whether the interposed venous AV loop graft is crucial for the induction of angiogenesis. MATERIAL AND METHODS We exposed the grafted vein to a single radiation dose of 2 Gy prior to loop construction to alter intrinsic and angio-inductive properties specifically within the graft. Vessel loops were embedded in a fibrin-filled chamber for 15 days and radiation-induced effects on flow-mediated vascularization were assessed by micro-CT and two-dimensional histological analysis. RESULTS Vessel amount was significantly impaired when an irradiated vein graft was used for AV loop construction. However, vessel growth and differentiation were still present. In contrast to vessel density, which was homogeneously diminished in constructs containing irradiated veins, vessel diameter was primarily decreased in the more peripheral regions. CONCLUSIONS Vascular luminal sprouts were significantly diminished in irradiated venous grafts, suggesting that the interposing vein constitutes a vital part of the AV loop model and is essential to initiate flow-mediate angiogenesis. These results add to the current understanding of AV loop-based neovascularization and suggest clinical implications for patients requiring combined AV loop-based tissue transfer and adjuvant radiotherapy.

Comparison of Fasciocutaneous and Muscle-based Free Flaps for Soft Tissue Reconstruction of the Upper Extremity.

Soft tissue free flap reconstruction of upper extremities has proven to be reliable and essential for limb salvage and function. Nevertheless, comparative data regarding flap outcome are still lacking. The present study aimed to compare procedural features and individual complication rates of different free flaps used for upper extremity reconstruction. METHODS: The authors evaluated retrospectively the results of 164 free flaps in 149 patients with upper extremity defects. Chart reviews were performed from April 2000 to June 2014, analyzing flap choices, complication, and success rate assessment for patients >18 years old, with a soft tissue defect of the upper extremity. Chosen flap types were classified as fasciocutaneous (including adipocutaneous) and muscle-based, respectively. We comparatively analyzed total flap loss, flap survival after microsurgical revisions, and susceptibility rates for thromboses rates and partial flap necrosis. RESULTS: Defect size was larger when muscle-based flaps were used (231 ± 38.6 versus 164 ± 13.7 cm2, P < 0.05). Outcome analysis revealed a tendency towards higher arterial thrombosis rates for muscle flaps (10.2% versus 4.3%) and venous thrombosis rates for fasciocutaneous flaps (2% versus 7%). Total flap loss (6.1% versus 7.8%) and flap survival after vascular revisions (75% versus 70.6%) showed comparable rates. Partial flap necrosis was generally higher in muscle-based flaps (22.4% versus 8.6%, P = 0.02) with impact on patients' hospital stay (37.2 ± 4.69 and 27.11 ± 1.62 days, n = 115, P = 0.01), while no differences in partial necrosis rates were noted in flaps larger than 300 cm2 (25% versus 10%, P = 0.55). There was a trend over time towards using fasciocutaneous-based flaps more frequently with a final overall percentage of 83.7% between 2012 and 2014. CONCLUSIONS: Microsurgical tissue transfer to the upper extremity is safe and reliable, but flap-type specific procedural and measures should be taken into consideration. Total flap loss as well as flap survival after microsurgical revisions are not altered between these flaps. They differ, however, in their susceptibilities for thromboses rates, partial flap necrosis and thus require individual risk stratification and flap placement.

Class III antiarrhythmic drug dronedarone inhibits cardiac inwardly rectifying Kir2.1 channels through binding at residue E224.

Dronedarone is a novel class III antiarrhythmic drug that is widely used in atrial fibrillation. It has been shown in native cardiomyocytes that dronedarone inhibits cardiac inwardly rectifying current IK1 at high concentrations, which may contribute both its antifibrillatory efficacy and its potential proarrhythmic side effects. However, the underlying mechanism has not been studied in further detail to date. In the mammalian heart, heterotetrameric assembly of Kir2.x channels is the molecular basis of IK1 current. Therefore, we studied the effects of dronedarone on wild-type and mutant Kir2.x channels in the Xenopus oocyte expression system. Dronedarone inhibited Kir2.1 currents but had no effect on Kir2.2 or Kir2.3 currents. Onset of block was slow but completely reversible upon washout. Blockade of Kir2.1 channels did not exhibit strong voltage dependence or frequency dependence. In a screening with different Kir2.1 mutants lacking specific binding sites within the cytoplasmic pore region, we found that residue E224 is essential for binding of dronedarone to Kir2.1 channels. In conclusion, direct block of Kir2.1 channel subunits by dronedarone through binding at E224 may underlie its inhibitory effects on cardiac IK1 current.